Pharmaceutics 15 01850

pharmaceutics
Review
Practical Considerations for Next-Generation Adjuvant
Development and Translation
William R. Lykins and Christopher B. Fox *
Access to Advanced Health Institute, Seattle, WA 98102, USA

* Correspondence: christopher.fox@aahi.org
Abstract: Over the last several years, there has been increased interest from academia and the
pharmaceutical/biotech industry in the development of vaccine adjuvants for new and emerging
vaccine modalities. Despite this, vaccine adjuvant development still has some of the longest timelines
in the pharmaceutical space, from discovery to clinical approval. The reasons for this are manyfold
and range from complexities in translation from animal to human models, concerns about safety
or reactogenicity, to challenges in sourcing the necessary raw materials at scale. In this review, we
will describe the current state of the art for many adjuvant technologies and how they should be
approached or applied in the development of new vaccine products. We postulate that there are many
factors to be considered and tools to be applied earlier on in the vaccine development pipeline to
improve the likelihood of clinical success. These recommendations may require a modified approach
to some of the common practices in new product development but would result in more accessible
and practical adjuvant-containing products.
Keywords: adjuvants; formulation; future perspectives; vaccine development; natural product discovery;
route of administration; design of experiments; systems immunology; mucosal vaccine; sustainability
1. Introduction
Citation: Lykins, W.R.; Fox, C.B.
Throughout the last two decades, there has been increasing focus on the discovery
Practical Considerations for
and translation of new immune-stimulating agents [1,2]. These compounds are often
Next-Generation Adjuvant
Development and Translation.
collectively referred to as adjuvants due to their precedent of use in vaccine development.
Pharmaceutics 2023, 15, 1850.
In recent years, there has been an expansion in the application of adjuvants in oncology
https://doi.org/10.3390/ and other areas as our understanding and definition of adjuvants continue to grow [3].
pharmaceutics15071850 Adjuvants stimulate key cell types in the innate immune system and can influence the
scale and class of immune response directed towards a given antigen or antigens [4]. These
Academic Editors: Derek T. O’Hagan
innate immune cell subsets can then engage with effector cells in the adaptive immune
and Rushit N. Lodaya
system—e.g., T and B cells—which then mediate the development of cellular and humoral
Received: 1 June 2023 immunity, respectively. Adjuvants are also critical in the development of long-term memory
Revised: 21 June 2023 cell populations, which in turn can lead to years-long protection [5,6]. In existing clinical
Accepted: 27 June 2023 products, adjuvants are often combined with recombinant subunit antigens, which are
Published: 29 June 2023 generally not sufficiently immunogenic to generate protective immune responses on their
own [7].
In recent years, there have been a handful of successful FDA approvals for vaccines
containing new-to-market adjuvant systems, such as AS01, developed by GSK and included
Copyright: © 2023 by the authors.
in Shingrix, Mosquirix, and Arexvy [8]. However, despite its success, AS01 is based on
Licensee MDPI, Basel, Switzerland.
techniques and technologies that were developed decades ago, and many reports note
This article is an open access article
that vaccine adjuvants have some of the longest times to commercialization of any clinical
distributed under the terms and
conditions of the Creative Commons
product [1]. A marked need exists for new adjuvant and immunostimulatory systems;
Attribution (CC BY) license (https://
however, there is also a need to better understand and plan for the barriers to translation
creativecommons.org/licenses/by/
earlier on in the adjuvant development pipeline.
4.0/).
Pharmaceutics 2023, 15, 1850. https://doi.org/10.3390/pharmaceutics15071850 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2023, 15, 1850 2 of 18
The most widely used class of adjuvants is Alum, a generic term applied to aluminum
salts, such as aluminum phosphate, aluminum hydroxide, aluminum hydroxyphosphate
sulfate, and aluminum oxyhydroxide. Alum often serves the useful functions of adsorbing
the protein antigen and mediating its presentation to innate immune cells in draining lymph
nodes [4,9]. Aluminum-based adjuvants are present in many common vaccines, including
the 9-valent HPV vaccine Gardasil and the Tdap vaccines (among many others) [10].
Another common class of adjuvants is oil-in-water emulsions (e.g., MF59 and AS03), which
typically include squalene in the oil phase, emulsifying agent(s), and other excipients [4,11].
Much like Alum adjuvants, squalene emulsions do not appear to engage a specific pattern
recognition receptor (PRR) but instead mediate a local inflammatory response and increase
the trafficking of antigens to draining lymph nodes [12]. Notably, the tetravalent influenza
vaccine Fluad contains MF59 [10]. Similarly, saponin-based adjuvants, such as Matrix-M,
offer pleiotropic modes of action, which tend to augment humoral and cellular immune
responses. Likewise, the saponin QS-21 functions through an unknown receptor(s) but is
known to trigger the inflammasome in model systems of immunogenicity [13].
In contrast to the particulate adjuvant formulations described above, another class of
clinical adjuvants includes specific chemical triggers of defined pathways. Most of these
agents signal through PRRs, which are a critical part of the innate immune system and
are involved in the detection of pathogen-associated molecular patterns (PAMPs) [14].
The vaccine adjuvants monophosphoryl lipid A (MPL) and CpG 1018 are both highly
characterized agonists of Toll-like receptors (TLRs) and help to shape the immune response
towards a Th1-type quality [10]. In many cases, adjuvant products comprise combinations
that may include TLR ligands, saponins, and particulate formulations, such as AS01 in
Shingrix and Arexvy (liposomal TLR4 agonist and QS-21), CpG 1018-Alum in Corbevax
(TLR9 agonist adsorbed to Alum), and Alhydroxiquim-II in Covaxin (TLR7/8 agonist
adsorbed to Alum) [10].
Many unique adjuvants and adjuvant systems exist in preclinical development, as has
been the case for many years. However, there continues to be a disconnect between the
state of our understanding of adjuvants and our ability to deliver on that understanding
in terms of clinical translation. At the same time, it is widely appreciated that adjuvants
play a critical role in the development of long-lasting immunity and protection against
target pathogens. This is most recently highlighted by the first generation of mRNA
vaccines against SARS-CoV-2, which induced impressive initial antibody titers that rapidly
waned [15], a liability that adjuvanted vaccine candidates can improve upon [16]. Moreover,
much work is needed to establish adjuvant compatibility with next-generation vaccine
platforms, including mRNA, DNA, and adeno-associated virus (AAV)-based vaccines. The
gap between our understanding of the need for adjuvants and our ability to capitalize on
their use in clinical products is related to multiple factors, including a lack of appreciation
for the nuanced formulation considerations in vaccine adjuvant development, unclear
rationale or parameters to select one adjuvant over another, and a failure to consider
adjuvants with product potential until too late in the development process. At the same
time, there are avenues for further development and discovery in adjuvant systems that will
make them more accessible and simplify their integration into emerging vaccine systems.
From this perspective, we attempt to summarize what steps can be taken and what
factors can be considered in the early stage of vaccine adjuvant design and development.
We overview the importance of sustainable material sourcing, storage stability, alternate
routes of administration, controlled release kinetics, and methods the vaccinologist can
use to weigh these options while positioning their product toward clinical translation
(Figure 1).
Pharmaceutics 2023, 15, x FOR PEER REVIEW 3 of 18

Figure 1. Where do novel adjuvant systems come from, and why are they important? Figure was
Figure 1. Where do novel adjuvant systems come from, and why are they important? Figure was
created with Biorender.com (accessed on 26 June 2023).
created with Biorender.com (accessed on 26 June 2023).
2. Sustainable Raw Materials
2. Sustainable Raw Materials
Natural product discovery generally refers to the practice of identifying compounds
Natural product discovery generally refers to the practice of identifying compounds
in existing natural reservoirs, usually plants, fungi, or bacteria [17,18]. In an idealized pro-
in existing natural reservoirs, usually plants, fungi, or bacteria [17,18]. In an idealized
cess, the specific active compound is identified via fractionation and a high-throughput
process, the specific active compound is identified via fractionation and a high-throughput
bioassay [17]. Next, a highly refined purified compound is generated, then physical and
bioassay [17]. Next, a highly refined purified compound is generated, then physical and
analytical chemistry techniques are employed to elucidate its active structure (e.g., nuclear
analytical chemistry techniques are employed to elucidate its active structure (e.g., nuclear
magnetic resonance, time-of-flight mass spectrometry, and Raman spectroscopy). The ac-
magnetic resonance, time-of-flight mass spectrometry, and Raman spectroscopy). The
tive compound
active compoundis issynthetically
syntheticallyproduced
producedvia
viatraditional
traditional organic
organic chemistry techniques;
chemistry techniques;
then, medicinal chemistry approaches can be employed to refine the drug characteristics
then, medicinal chemistry approaches can be employed to refine the drug characteristics
of the final active pharmaceutical ingredient (API). This approach generally relies on the
of the final active pharmaceutical ingredient (API). This approach generally relies on the
eventual ability
eventual ability to
to fully
fully synthesize
synthesize the
the compound
compound of of interest,
interest, as
as chemical
chemical extraction
extraction of
of
refined compounds is challenging and costly to scale.
refined compounds is challenging and costly to scale.
Despite the associated costs and challenges, many existing adjuvants are derived di-
rectly from refined natural products. The saponin pool that QS-21 is refined from comes

Despite the associated costs and challenges, many existing adjuvants are derived
directly from refined natural products. The saponin pool that QS-21 is refined from comes
from Quillaja saponaria, the soap bark tree, which is native to a relatively small area of central
Chile [13]. Saponins from soap bark trees were first studied as immunostimulants in the first
half of the 20th century and were later refined into Quil A, a heterogeneous saponin mixture
that is presently used as an adjuvant in veterinary vaccines. In an effort to reduce the
reactogenicity of Quil A, scientists identified and isolated the particularly active component
QS-21 (named as the 21st peak on a chromatogram of Quil A) [13]. The resulting QS-21
is a blend of two isomeric compounds differentiated by a terminal sugar structure [19].
Despite the relatively low-yield industrial process, QS-21 is presently included in several
vaccine products, including Shingrix, Mosquirix, and Arexvy, and is one of the multiple
saponins in Nuvavoxid. As the number of QS-21-containing vaccines increases, alternative
industrial processes to obtain QS-21 or suitable analog molecules could be required due
to the limited availability of Quillaja saponaria and the poor extraction yields [20,21]. For
example, synthetic biology approaches, such as expressing saponins in bioengineered
organisms, may provide a more sustainable avenue to saponin production [22]. Synthetic or
semi-synthetic approaches to generating QS-21 analogs have also been demonstrated [23].
Squalene provides another example of a natural product with a long history of use as
an adjuvant vaccine component. All current squalene-based pharmaceutical products use
refined oil derived from shark livers, including those containing MF59 or AS03 [24]. While
it is difficult to quantify how an increase in squalene-based vaccine adjuvant demand would
impact shark populations, a more sustainable source of squalene or alternative analogs
are desirable objectives. In recent years, many cosmetic companies have replaced shark
squalene with olive-derived squalene. Olive squalene is concentrated in the skin and seeds
of the fruit, meaning that it can be a byproduct of the global olive oil industry. Emulsified
olive-derived squalene appears to maintain adjuvant properties in preclinical testing [25,26].
Other companies are working to distill squalene from the amaranth grain, which can be
selectively cultivated and processed to increase squalene production [27]. However, due
in part to the difficulty and cost associated with extracting highly purified squalene from
plants, plant-derived squalene has not yet been employed in any FDA-approved vaccine
product [24]. Other plant-derived terpenoid structures have also demonstrated adjuvant
properties and merit further investigation [28,29]. Alternative routes to a more sustain-
able source of squalene-like molecules include synthetic biology and synthetic chemistry
approaches. For example, non-shark squalene or squalene analog emulsions obtained
using bioengineered yeast and/or synthetic chemistry have demonstrated equivalent
or improved adjuvant activity compared to shark squalene emulsions [28,30,31]. These
approaches have the advantage of existing infrastructure in microbial fermentation and
industrial chemical engineering. An added benefit of generating analog structures is
that structure-function relationships can help elucidate features important for improved
adjuvant activity in emulsions containing squalene-like molecules [28].
MPL, a TLR4 agonist derived from Salmonella minnesota, was the first TLR agonist to be
used in approved vaccines. The manufacturing process of MPL results in a heterogeneous
mixture of compounds with different numbers of acyl chains, each of which may have
different adjuvant properties [32]. In contrast, synthetic TLR4 agonists offer highly pure
single constructs with structures optimized for human TLR4 activity. Several synthetic
TLR4 ligands as components of vaccine adjuvant formulations are undergoing clinical
testing [33]. However, other synthetic TLR ligands have already reached commercial
product approval, including CpG 1018 (TLR9) in the hepatitis B vaccine Heplisav-B and,
in some geographies, Alhydroxiquim-II (TLR7/8) in the SARS-CoV-2 vaccine Covaxin. In
addition to TLR ligands, other synthetic PRR ligands, such as trehalose dibehenate (TDB), a
ligand of the C-type lectin receptor Mincle, have advanced to clinical testing [34]. Synthetic
ligands for TLRs and other PRRs offer well-defined structures for precisely engineering the
adjuvant properties of next-generation vaccines.
The above examples represent a range of adjuvant types and sources but are not
intended to be comprehensive. Many other types of molecular and particulate adju-
vants are in development. Nevertheless, regardless of the type of adjuvant and whether
it is obtained from natural or synthetic sources, raw material sustainability must be a
primary consideration.
3. Thermostability
Much has been written elsewhere about vaccine cold-chain storage and the need for
thermostable vaccine products to meet global vaccine needs [35,36], which is highlighted
by the poor stability profile of mRNA-based COVID-19 vaccines that made them challeng-
ing to access in many areas [37,38]. Thermostability generally refers to the ability of the
product to remain stable at ambient temperatures for sufficient amounts of time. Because
of the logistical challenges of maintaining cold-chain transport, as much as 50% of vac-
cine doses are rendered unusable before administration in many low- and middle-income
countries [39]. In particular, more effort is needed in the development of thermostable
adjuvant-containing vaccines [39,40]. Generating a thermostabilized product often neces-
sitates reducing the water activity of a product to slow the rate of hydrolysis and other
aqueous-phase decomposing processes, which are the major driving forces in antigen and
adjuvant degradation [41,42]. This is often accomplished by generating a dried product,
generally either through lyophilization or spray drying [42]. However, transforming liquid
adjuvant formulations into solid-state formulations with enhanced thermostability is not a
trivial endeavor due to the complexities of adjuvant particulate structures. It is, therefore,
worthwhile to consider optimizing formulations for enhanced thermostability early in
development. The generation of solid-state materials is generally achieved via some form
of drying technology, such as lyophilization or spray drying [43–45]. Recent progress in
applying such techniques to generate thermostable adjuvant formulations has been en-
couraging. For instance, a thermostable lyophilized presentation of a vaccine composition
consisting of a protein antigen and an oil-in-water emulsion containing a synthetic TLR4
ligand was shown to maintain or even improve the safety and immunogenicity profile of
the non-thermostable vaccine in a Phase 1 clinical trial of a GLA-SE adjuvanted subunit
tuberculosis vaccine [46]. The emerging class of nucleic acid-based adjuvants, such as
TLR3 and RIG-I agonists, is likely to benefit greatly from dried preparations due to its
relatively poor liquid stability compared to its improved stability in a dried state [47,48].
Alternative strategies to generate solid formulations include entrapment in an implant
or microneedle patch, which may in turn provide controlled release benefits [49,50]. In
any case, by prioritizing thermostability in new adjuvant-containing vaccines, more global
markets may become accessible, which has clear benefits from economic, ethical, and global
health perspectives.
4. Alternative Routes of Delivery

Mucosal immunology is a somewhat less-explored field than systemic immunity,
despite the mucosal route of infection for many infectious diseases. Each major mucosal
system, including the urogenital, respiratory, and gastrointestinal tracts, has its own unique
combination of defensive structures and effector cell populations. A large body of evidence
demonstrates that vaccines delivered mucosally, such as the pulmonary or oral route, lead
to immunological outcomes that are unique from systemically delivered products [51–53].
Moreover, mucosal immune responses generated by mucosally delivered vaccines may
be more effective in preventing infection for some indications [53,54]. Currently, there are
some approved vaccines delivered by the oral or nasal route, but these are usually live-
attenuated pathogens that do not contain extrinsic adjuvants. SARS-CoV-2 has generated
even greater interest in the potential for mucosal delivery of vaccines [55,56]. However,
several challenges must be overcome for more widespread adoption of mucosally delivered
adjuvant-containing vaccines, including issues associated with absorption, toxicity, and
administration technique.
The absorption of intact biomolecules across mucous membranes has been a clinical
challenge for decades. Compared to parenterally dosed vaccine products that offer imme-
diate bioavailability, only a fraction of mucosally administered material may successfully
pass through epithelial tissue to generate a productive interaction with the effector cell
populations residing below. This is especially true for mucosally delivered biologics, such
as protein antigens or nucleic acids, which often have single-digit bioavailability percent-
ages, even in advantageous mouse models [57–59]. This low permeability stems from the
relative lack of diffusion of large biomolecules across intact epithelial tissue, in addition to
ubiquitous chemical and enzymatic proteolysis. In this regard, pulmonary dosage forms
may behave more favorably due to a large surface area, a high degree of vascularization,
and relatively thin mucosal barriers [60]. In any case, mucosal adjuvant formulation devel-
opment efforts may be needed to target cell uptake by resident antigen-presenting cells,
which can sample antigens from across the epithelial barrier and then present them to
effector subsets in the draining lymph node. Optimization of particle size and material
properties are often exploited to achieve the desired tissue deposition [61,62].
Due to the issue of potential toxicity of mucosally delivered vaccine adjuvants, addi-
tional considerations must be taken into account compared to parenteral delivery. Most
adjuvants generate some kind of local inflammation, which, depending on the individual
and the product, could result in some short-term local or systemic reactogenicity [63]. This
disruption to tissue homeostasis is more pronounced in mucosal tissues, such as the lung
or nose, where there might be more acutely undesirable side effects due to epithelial tissue
inflammation. For this reason, the inclusion of adjuvants in mucosal vaccines is challenging
because, while there is a need to elicit potent localized immune responses, there is also a
more overt risk to doing so. The widely publicized experience with a nasally delivered
inactivated influenza vaccine containing a bacterial toxoid adjuvant that caused Bell’s palsy
in some recipients provides an unfortunate warning lesson in this regard [64].
Finally, mucosally delivered vaccine and adjuvant preparations need to take the ad-
ministration technique/device into account. Because of their limited application in current
clinical products, new vaccine dosing methods will pose an adoption challenge. Intramus-
cular injections are a well-defined and ubiquitous means of administering vaccines, which
offers clarity to both the patient and provider that the vaccine was administered success-
fully. In contrast, correct and incorrect user techniques may occur with equal frequency
in nasal or pulmonary delivery [65]. The absorption of oral products is similarly challeng-
ing and may be affected by a host of factors, including an individual’s diet before/after
vaccination, microbiome status, enteropathy, stress, or presence of enteric pathogens or
other conditions, such as inflammatory bowel disease [66,67]. In any case, it is essential
that device compatibility with the adjuvant formulation is comprehensively assessed so
that optimal delivery performance can be achieved [62].
In summary, emerging adjuvant systems hoping to capitalize on the benefits of mu-
cosal administration need to be evaluated thoroughly by taking the above factors into
account. Thus, it is unlikely that an adjuvant formulation that has been designed for
intramuscular use will automatically be optimized for mucosal delivery without some
modifications to the composition. Furthermore, there must be a clear benefit demonstrated
compared to more traditional delivery approaches that justifies the need for investing in
mucosal delivery development efforts. For example, for vaccines against highly contagious
respiratory pathogens, such as SARS-CoV-2 or influenza, an obvious added value would
be if an adjuvanted vaccine could induce sterilizing immunity at the site of infection rather
than being limited to primarily reducing disease severity.
5. Control of Exposure Duration and Kinetics

In recent years, a number of reports have looked at the use of implantable controlled-
release systems as a means to control the kinetics of vaccine antigen and adjuvant exposure.
In general, extended, sustained, or pulsatile exposure to vaccine components over the
course of weeks to months can improve the potency of an immune response and support the
development of a broadly neutralizing antibody response [68,69]. This is especially relevant

when developing vaccines against highly challenging pathogens, such as HIV [70,71].
These implantable systems also have the potential to enable single-intervention vaccine
regimens, eliminating the need for booster doses [72]. There has also been extensive
preclinical research into the use of intertumoral implantation of extended delivery systems
for immune-oncology agents, with and without surgical resection, as a means to improve
the efficacy of cancer immunotherapies [73]. While our mechanistic understanding of these
controlled-release systems is still evolving, it is generally thought that prolonged exposure
to an antigen at draining lymph nodes leads to the development of more mature T and B
effector cell responses [74].
Controlled-release systems are generally designed for use with protein subunit anti-
gens, and many have incorporated adjuvants to enhance the immunogenicity of these
vaccines. To this end, many adjuvants have been included in a number of controlled-release
vaccine systems, but most are off-the-shelf materials as opposed to fit-for-purpose composi-
tions, including MPL, Alum, and CpG (among others) [75]. These adjuvants, while effective,
are unlikely to be optimal in the unique kinetics of a controlled-release system without
additional modification. It is, therefore, worthwhile to test adjuvant platforms in the context
of extended-release systems now so that, once the technology reaches clinical maturity,
there will be front-runner adjuvants that could be applied in a second-generation product.
From a design perspective, implantable vaccines that have an active lifespan of weeks
to months would be best served by a biodegradable material; otherwise, the requirement
for removal of the device defeats many potential patient benefits. To this end, many
biodegradable materials have been explored, including poly(D,L-lactic acid-co-glycolic
acid) and polyanhydride as implants and various materials in gels, such as a variety of
polyester and polyethylene block-copolymers, polysaccharide polymers like alginate or
chitosan, synthetic peptide scaffolds, and cellulose derivatives (among others) [75]. From
a use standpoint, vaccine products, in general, are dependent on high uptake in relevant
populations, meaning that dosing procedures that are more complex than a standard
percutaneous injection will be more challenging to market [76,77]. For this reason, shear-
thinning or in situ crosslinking materials are likely to have a market advantage over solid
form factors that will require more complex outpatient procedures to place correctly [75].
A potential advantage of these novel polymer-gel systems is that adjuvants can interact
directly with the chemistry of the material to better synchronize release with a protein
antigen [78,79]. Another approach is to use aluminum oxyhydroxide as a carrier for both
the protein antigen and an anionic adjuvant, linking the release of both components [72,80].
Lastly, the largest hurdle adjuvant-containing systems will face is the regulatory challenge
posed by establishing safety profiles for long-term exposure to inflammatory compounds
in healthy populations. Extended exposure to adjuvants is likely to be a concern for both
regulatory bodies and patients, so a very high bar for safety must be achieved in preclinical
and early clinical studies. Indeed, long-term exposure to even sub-therapeutic amounts of
pro-inflammatory compounds might have unintended risks [68,69]. These concerns are of
course different in the field of oncology, where sustained systemic exposure to one or even
multiple pro-inflammatory immune oncology drugs is becoming a standard of care for
many indications [81,82]. So, there is a reason to anticipate that a tumor-targeting vaccine
implant may be the first to market, with subsequent prophylactic vaccination efforts for
HIV or other difficult-to-target pathogens following.
6. Combining Multiple Adjuvants and Vaccine Platforms

The success of the AS01 adjuvant system has demonstrated the potential clinical value
to be realized with combinations of immunostimulants. AS01 contains the TLR4 agonist
MPL and the fractionated saponin QS-21, formulated in a liposome that is admixed with
or used to reconstitute the vaccine antigen [83]. Studies have shown that AS01 benefits
from an inherent synergy between the two agonists, leading to enhancements in both
cellular and humoral immunity [84,85]. Interestingly, AS01 has often outperformed AS02 in
clinical testing, notably in the development process of the now WHO-prequalified malaria
vaccine Mosquirix, even though AS02 also contains MPL and QS-21 but is formulated in
an oil-in-water emulsion [86]. Thus, it is critical to consider not only the activity of each
immunostimulant separately but also how they function together, how best to formulate
them, and how the adjuvant formulation interacts with the vaccine antigen. Regardless
of the actual components, the goal of adjuvant systems that combine multiple immunos-
timulants is to generate a complementary immune response without approaching the
reactogenicity that might arise from using higher doses of a single immunostimulant. This
requires identifying a synergistic effect between two agonists, which is most likely to occur
between agonists that act in at least a partially orthogonal manner or target different cell
types, as opposed to the additive effect that would be observed between two agonists that
act along identical pathways. For instance, MPL and QS-21 are thought to act through
complementary immunomodulatory pathways, resulting in a synergistic combination.
Many other multi-agonist combinations have been trialed in preclinical models, notably
the nucleic acid-based adjuvants CpG and 20 30 -cGAMP and the TLR4 agonist GLA with the
TLR7/8 agonist 3M-052 [87,88]. Formulations of particulate adjuvants (such as Alum or
squalene emulsion) with TLR ligands should also be considered combination adjuvants, as
well as delivery vehicles for adsorbed agonists and/or antigens (e.g., AS04 and GLA-SE).
Alum, in particular, is effective at binding protein antigens and molecular agonists and
facilitates the codelivery of both to relevant immune populations in the draining lymph
node, such as in the HPV vaccine Cervarix [89].
Until recently, any given infectious disease had only a few commercial vaccines, and
at any given time, most of the available vaccines for that disease used similar modalities
(live attenuated, whole killed virus, subunit, virus-like particle, etc.). In the wake of
the 2019 coronavirus pandemic, where nucleic acid and adenoviral vector vaccines both
reached global markets alongside more traditional protein subunit or inactivated virus
vaccines, there were expanded opportunities to consider how different vaccine modalities
may interact with each other. Similar to how components of combination adjuvants, such as
AS01, have an innate synergy, leading to more effective immune responses, it is reasonable
that different vaccine modalities might also demonstrate beneficial complementarity. These
kinds of vaccine schedules, where an individual receives different products between the
prime and subsequent boost immunizations, are generally referred to as heterologous
combinations [90,91]. The data obtained from heterologous COVID-19 vaccine regimens
have generally focused on the near-term immunogenic profile, where the highly potent
mRNA vaccines appear to be the top performers. However, it has become clear, in the
context of SARS-CoV-2, that long-lasting protective immune memory has been more elusive
to achieve with the current vaccines, particularly in the presence of rapidly mutating virus
strains [92]. In some cases, other vaccine platforms, such as appropriately adjuvanted
subunit proteins or nanoparticle presentations, may provide distinct advantages, such as
increased durability or breadth [16,93,94]. By presenting a combination of antigen types and
structures in different vaccine platforms, including adjuvants where appropriate, it may be
possible to better shape the quality of the final immune response [95]. Even simultaneous
administration of multiple vaccine modalities may be worth considering [96]. However, the
efficacy of mRNA-based vaccines may be suppressed when dosed along potent activators
of type-one interferons (e.g., TLR3, TLR7/8, TLR9, STING, and RIG-I agonists) due to a
reduction in protein expression [97–99]. Nevertheless, there is potential that orthogonal
immune potentiators might improve the limited durability of existing mRNA vaccines. To
this end, an increased understanding of the interaction between different adjuvants, vaccine
modalities, and routes of delivery might improve our ability to respond to pandemics.
Another approach to heterologous vaccination is alternating the route of administra-
tion to generate more effective mucosal immunity. This kind of “prime and pull” strategy
was first described as a means to improve T cell responses in the lower female genital
tract to a herpes simplex virus 2 (HSV2) subunit vaccine [100]. The theory is that by first
vaccinating via a conventional route (i.e., intramuscular) to elicit a mature systemic T cell
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Pharmaceutics 2023, 15, 1850

to a herpes simplex virus 2 (HSV2) subunit vaccine [100]. The theory is that by first vac- 9 of 18
cinating via a conventional route (i.e., intramuscular) to elicit a mature systemic T cell
response, peripheral T cell populations can then be recruited to mucosal sites using the
local delivery of chemokines or other adjuvants. This approach has recently been applied
response, peripheral T cell populations can then be recruited to mucosal sites using the
as a therapeutic vaccine candidate in an animal model of HSV2 and demonstrated that the
local delivery of chemokines or other adjuvants. This approach has recently been applied
prime and pull strategy was more effective than either the systemic or mucosal approach
as a therapeutic vaccine candidate in an animal model of HSV2 and demonstrated that
in isolation [101]. In addition to providing protection in the urogenital tract, prime and
the prime and pull strategy was more effective than either the systemic or mucosal ap-
pull strategies have been effective in animal models of nasal, pulmonary, and ocular in-
proach in isolation [101]. In addition to providing protection in the urogenital tract, prime
fection [102–105].
and pull strategies have been effective in animal models of nasal, pulmonary, and ocular
The necessary collaborations between vaccine manufacturers required to clinically
infection [102–105].
evaluate these combinations could present substantial complexities; however, similar
The necessary collaborations between vaccine manufacturers required to clinically
cross-branded combinations are commonly employed in oncology and are often beneficial
evaluate these combinations could present substantial complexities; however, similar cross-
to both manufacturers and patients [106]. Given that some technologies will be first to
branded combinations are commonly employed in oncology and are often beneficial to both
market, it could be advantageous to design later products to provide complementary
manufacturers and patients [106]. Given that some technologies will be first to market, it
boost responses to the initial products. On the other hand, there is a risk that heterologous
could be advantageous to design later products to provide complementary boost responses
combinations might be ineffectual. In the context of the COVID-19 pandemic, it was
to the initial products. On the other hand, there is a risk that heterologous combinations
shown in a small clinical cohort that individuals who received the BNT162b2 mRNA vac-
might be ineffectual. In the context of the COVID-19 pandemic, it was shown in a small
cine followed by the Novavax subunit boost generated a less substantial immune response
clinical cohort that individuals who received the BNT162b2 mRNA vaccine followed by the
than patients who received the BNT162b2 vaccine as a prime and boost. However, patients
Novavax subunit boost generated a less substantial immune response than patients who
who first received the Oxford adenovirus vaccine, followed by the Novavax vaccine, out-
received the BNT162b2 vaccine as a prime and boost. However, patients who first received
performed groups who only received the BNT162b2 and/or Moderna mRNA vaccines in
the Oxford adenovirus vaccine, followed by the Novavax vaccine, outperformed groups
terms of T cell activity [91]. This suggests that for difficult indications, considering heter-
who only received the BNT162b2 and/or Moderna mRNA vaccines in terms of T cell activ-
ologous combination products earlier on in vaccine development would allow for opti-
ity [91]. This suggests that for difficult indications, considering heterologous combination
mizing each individual product to achieve a subset of clinical endpoints rather than ex-
products earlier on in vaccine development would allow for optimizing each individual
pecting comprehensive immune protection from each product on its own. In the context
product to achieve a subset of clinical endpoints rather than expecting comprehensive
of pandemic preparedness, this approach might mean that early-stage products are fo-
immune protection from each product on its own. In the context of pandemic preparedness,
cused on generating
this approach strong
might mean systemic
that early-stagehumoral immunity
products to reduce
are focused disease severity,
on generating strong
whereas subsequent products can be designed to emphasize enhanced durability or mu-
systemic humoral immunity to reduce disease severity, whereas subsequent products can
cosal responses (Figure 2).
be designed to emphasize enhanced durability or mucosal responses (Figure 2).

Figure 2. Careful selection of adjuvants streamlines future pandemic responses. Figure was created
Figure 2. Careful selection of adjuvants streamlines future pandemic responses. Figure was cre-
ated with Biorender.com (accessed on 26 June 2023).
with Biorender.com (accessed on 26 June 2023).
7. Optimization via Aggregate Metrics

The increased efficacy of AS01-based adjuvants compared to AS02 is somewhat sur-
prising considering the well-accepted adjuvant properties of squalene emulsions, even
without the addition of receptor agonists, and the generally immune inert nature of con-
ventional liposomes [86,107,108]. This suggests that the immune response generated from
multiple immune stimulants may not be well modeled by a simple linear combination of
the inputs. In practice, the mechanistic interplay between different immunostimulants
is often challenging to define fully, and empirical studies remain the typical approach
for adjuvant selection. However, characterizing immune responses to a combination of
immunostimulants might be better achieved using tools and models built for complex
physical systems, such as those found in digital signal processing or microscopy, where
the specific underlying features that dictate the change from a signal input to output are
governed by an empirical state-space model [109]. State-space models and similar control
theory techniques, such as transfer function analysis, are used across multiple engineering
fields in the development of new processes or products as a way to predict performance
during the design process [110,111]. Alternatively, these models can be derived empirically
from systems using specific test signals [112]. This approach is most useful when systems
can be approximated as linear time-invariant (LTI) systems, meaning that multiple inputs
can be condensed into a linear combination of inputs and that the system state does not
vary as a function of time [113]. In the case of immune responses to adjuvants, the immune
system is known to be highly non-linear, as exemplified by synergies and anti-synergies
between different immunostimulants, and immune responses are highly dynamic and
time-variant, as exemplified by immune memory as well as T cell exhaustion and other
metabolic phenomena. These complications mean that modeling of the immune system
is unlikely to completely replace empirical adjuvant screening; however, techniques such
as systems immunology and statistical design of experiments (DoE) might improve the
efficiency of the adjuvant selection process.
Systems immunology, the study of immune interactions on a pathway and cell/tissue
level in response to different stimuli, has been a remarkable tool to discern how signals
are propagated through the immune system and uncover unknown mechanisms of action
for immune agonists [114,115]. Like other clinical systems biology approaches, systems
immunology seeks to maximize the amount of data that can be generated from minimal
biological samples, most often peripheral blood. Techniques vary depending on the goals
of the study but often include mass cytometry, single-cell transcriptomics and proteomics,
bead-based analysis of soluble factors (i.e., Luminex assays), and more conventional read-
outs, such as antibody titer and breadth. These high-dimensional datasets are then analyzed
using bioinformatics techniques and machine learning algorithms to build models that
enable hypothesis testing and forecasting [115,116]. Systems immunology has provided
insights on a range of products, from predicting clinical responses to the yellow fever
vaccine to clarifying cellular responses to the BNT162b2 mRNA vaccine [117,118]. For
example, the B cell responses to the yellow fever vaccine can be correlated almost 1:1 to the
expression of the TNFRS17 B cell growth factor receptor, which can be measured as soon as
7 days after vaccination as a more accelerated means of determining successful vaccination
than traditional antibody titers [117]. Systems analysis has also been applied clinically to
better understand the mechanism of action and underlying kinetic differences between
seasonal influenza vaccines with or without the addition of MF59 [119]. These approaches
provide an unprecedented ability to characterize immune responses and even confirm the
immunogenicity of vaccines weeks to months before more conventional methods, such as
antibody titer. Such systems vaccinology approaches will likely have an increasing impact
on assessing immune response profiles in clinical studies of experimental vaccines and
adjuvants [1,120].
Because of the large amount of data output that can be measured via systems immunol-
ogy methods, and the large number of input factors that can be considered in adjuvant
design and dosing, techniques are needed that can simultaneously account for and opti-
mize multiple parameters at once. These techniques, especially ones that would be useful
in early measurements of human immune responses (i.e., a Phase 0 study), could derisk
future clinical studies [121]. A method commonly employed by formulation scientists
and engineers is the DoE approach [122,123]. DoE is a method to optimize experimental
efficiency and multiple input parameters simultaneously. By applying a subset of the
input space, researchers can use their empirical results to extrapolate toward the larger
output space, often referred to as a response surface [124,125]. Optimized combinations
of inputs are determined by applying desirability weights to each output and running an
optimization algorithm on the resulting response surface to identify a set of inputs that will
yield a desired set of outputs. DoE is a prospective method, meaning that the experimental
groups are designed via a statistical tool set before the study is carried out to minimize
the total number of replicates and groups needed to capture the system response to the
input parameters to the desired degree. Once the inputs and outputs are programed into
the statistical software, a set of experimental conditions and sample sizes are generated
that will enable a model of each output as a function of each input and potentially higher
order combinations of inputs as a measurement of interactions [122]. Depending on the
needs of the researcher, this can be used to determine which input factors have an effect
on the desired output and which input variables can be simplified in future studies [126].
Alternatively, once the input set is refined, the output models can be used to identify
the optimal conditions of each relevant input parameter. Because of the group-reduction
nature of DoE, the optimal combination of input parameters might not be a group that was
physically tested but rather inferred from the model of the system [88,127].
In the context of preclinical adjuvant development, DoE inputs could include the
dose of one or more PRR agonists, the composition of formulation active ingredients and
excipients (such as Alum, squalene emulsion, or liposomes), the frequency of booster
administration, the route of delivery, etc. The outputs for this kind of system could be
maximizing serum antibodies and cytokines, peripheral cellular activation via flow cy-
tometric measurements, measurements derived from tissues sampled at the conclusion
of the study (i.e., long-lived antibody-secreting cells in bone marrow, splenocyte/lymph
node activation, mucosal antibodies), neutralizing antibody titers, upregulation of genes
in systems immunology datasets, or even physicochemical stability of the adjuvant for-
mulation. Other outputs could include minimizing reactogenicity or undesirable immune
response signals (e.g., Th2 vs. Th1 cellular immunity). The outputs can be integrated
into a single score for ranking various candidates using an aggregate tool such as the
desirability index approach [88]. A number of groups have demonstrated desirability index
or DoE-based optimization in the development of preclinical and even clinical vaccine
products, including the optimization of the ratio of 3M-052 and GLA in a preclinical Enta-
moeba histolytica vaccine candidate or the optimization of the excipient content and stability
of a lyophilized subunit tuberculosis antigen with GLA-SE adjuvant that has now been
validated in a Phase 1 clinical trial [46,88,126–130]. Applying the high-dimensional data
that can be generated using systems vaccinology approaches together with the efficient
multi-parameter optimization of DoE techniques enables extracting as much information
as possible while minimizing the size of groups required for testing.
8. Conclusions
Considering that many vaccines are well served by existing adjuvants, what benefit
could new vaccine adjuvants address that is not currently met by existing products?
One important practical point is access to the adjuvant. In many cases, it may not be
possible to access existing adjuvants due to licensing restrictions, limited supply, or related
business decisions. Additionally, new, rationally designed adjuvants might lead to more
desirable immune response qualities or better meet the needs of specific populations or
geographies. In any case, it is essential that the added value of any new adjuvant system
be clearly benchmarked to well-accepted, widely available adjuvants, such as Alum or
squalene emulsion [131]. Another practical factor that should be considered early in
the development of new adjuvant systems is the balance of simplicity and innovation.
In general, most pharmaceutical systems benefit from being as simplified as possible,
from formulation and composition to manufacturing and distribution. A simpler product
that contains fewer ingredients, and is manufactured in fewer steps, has fewer potential
points of failure, fewer risks of supply chain disruption, reduced regulatory burden, and a
lower production cost. However, there are obvious benefits to mindfully accepting some
additional risk or complication in order to generate a more effective product. For example,
over the last 40 years, numerous HIV vaccine candidates based on standard approaches
have failed [132], so there is a clear need to innovate to develop new vaccine and adjuvant
technologies to address these challenges. However, such innovations must still be globally
accessible in terms of material availability, vaccine stability, dosing strategy, and scale of
manufacturing. These factors need to be considered early on in development to ensure that
clinical translation is feasible (see Box 1).
Although vaccines containing novel adjuvants have historically been one of the most
challenging products to bring to the clinic, multiple adjuvant-containing vaccines have
been approved in the last ~15 years, with SARS-CoV-2 substantially accelerating this trend.
Furthermore, initial clinical progress has been achieved in the development of thermostable
vaccine adjuvant platforms as well as the implementation of systems immunology ap-
proaches to analyze clinical samples. In parallel to these clinical advancements, significant
preclinical progress has been made regarding sustainable sourcing of adjuvant raw materi-
als, alternative routes of adjuvant delivery, and control of vaccine and adjuvant delivery
kinetics, providing new opportunities to optimize vaccine adjuvant design, development,
and manufacturing. In general, vaccine adjuvant development is a highly contextual en-
deavor, and there is no one-size-fits-all solution. However, there is hope that progress in
vaccine adjuvant development and accessibility can be accelerated further by taking into
account the practical aspects of adjuvant development discussed here.
Box 1. Summary of recommendations for new adjuvant development.
1. Derisk the environmental and supply chain sustainability of materials. Consider alternate
compounds or production methods to reduce dependency on animal or unsustainable sources.
2. Incorporate thermostability approaches early in development.
3. Define the need that a new adjuvant would address (e.g., mucosal immunity and durability of
immunity) and tailor subsequent activities to this need.
4. Keep compositions as simple as possible but consider the added value of combination ap-
proaches when necessary.
5. Employ experimental design methods and systems immunology approaches to comprehen-
sively characterize the immune profile of the new adjuvant.
Author Contributions: Conceptualization, C.B.F. and W.R.L.; writing—original draft preparation,

C.B.F. and W.R.L.; writing—review and editing, C.B.F. and W.R.L.; visualization, W.R.L.; funding
acquisition, C.B.F. All authors have read and agreed to the published version of the manuscript.
Funding: Research reported in this publication was supported by the National Institute of Allergy
and Infectious Diseases of the National Institutes of Health under award number R01AI135673
($4.1 million). Moreover, 80% of the total project costs were financed with federal money. The content
is solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: C.B.F. is an inventor on numerous patents and patent applications claiming
adjuvant and formulation compositions and methods. The funding agency had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
and in the decision to publish the results.
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Pharmaceutics 2023, 15, 1850. https://doi.org/10.3390/pharmaceutics15071850 https://www.mdpi.com/journal/pharmaceutics

4. Alternative Routes of Delivery

5. Control of Exposure Duration and Kinetics

development of a broadly neutralizing antibody response [68,69]. This is especially relevant

6. Combining Multiple Adjuvants and Vaccine Platforms

Pharmaceutics 2023, 15, 1850

7. Optimization via Aggregate Metrics

Box 1. Summary of recommendations for new adjuvant development.

Author Contributions: Conceptualization, C.B.F. and W.R.L.; writing—original draft preparation,

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