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An Overview of Novel
Adjuvants Designed for
Improving Vaccine Efficacy
Srinivasa Reddy Bonam,1,2 Charalambos D. Partidos,1,y
Sampath Kumar M. Halmuthur,2,* and Sylviane Muller1,3,*
Adjuvants incorporated in prophylactic and/or therapeutic vaccine formula-
Trends
tions impact vaccine efficacy by enhancing, modulating, and/or prolonging the
New-generation adjuvants are crucially
immune response. In addition, they reduce antigen concentration and the awaited to create new vaccines or
number of immunizations required for protective efficacy, therefore contribut- improve the efficacy of existing ones.
ing to making vaccines more cost effective. Our better understanding of the Pattern recognition receptors are cen-
molecular mechanisms of immune recognition and protection has led research tral to the innate immune response and
efforts to develop new adjuvants that are currently at various stages of devel- are considered as key targets for pro-
ducing effective vaccine generation.
opment or clinical evaluation. In this review, we focus mainly on several of these
promising adjuvants, and summarize recent work conducted in various labo- Several smart novel adjuvants have
emerged recently that act as immuno-
ratories to develop novel lipid-containing adjuvants
modulators or potent delivery systems.
Trends in Pharmacological Sciences, September 2017, Vol. 38, No. 9 http://dx.doi.org/10.1016/j.tips.2017.06.002 771
© 2017 Elsevier Ltd. All rights reserved.
adverse effects. Their discovery often emerges from studies dissecting the immunostimulatory Glossary
properties of bacterial components [5]. Vaccine adjuvants in current clinical use are listed in C-type lectin receptors (CLRs):
Table 1 and their advantages are depicted in Figure 1. Adjuvants stimulate the innate immune include DEC 205, macrophage
system directly by acting on dendritic cells (DCs), macrophages, and neutrophils, leading to the mannose receptor (MMR), dectin-1,2
macrophage-inducible C-type lectin
activation of the adaptive immune system (Figure 2). (Mincle), dendritic cell natural killer
lectin group receptor-1 (DNGR-1),
The innate immune system (Figure 2) has a critical role as a first line of defense that allows and dendritic cell-specific intracellular
adhesion molecule 3-grabbing
primary responses against microbes to occur. It confers an immediate nonspecific mechanism
nonintegrin (DC-SIGN). CLRs are
conserved carbohydrate recognition
receptors with or without calcium
Table 1. Examples of Vaccine Delivery Formulationsa,b dependency and form part of the
innate immune system with an
Adjuvant Composition Proposed mechanism of action Refs essential role in antifungal immunity.
+ Co-stimulation: in addition to the
MF59 Squalene, Span 85, Tween 80, and Upon injection, recruited CD11b [135,136]
citrate buffer cells and MHC-II+ cells and also first antigen-specific signal involving
promoted systemic expression of IL- the major histocompatibility complex
12 (p40) and IL5; further assisted the (MHC) peptide–T cell receptor (TCR)
sustained release of Ag at the interaction, a co-stimulatory signal (or
injection site and activated muscle ‘second signal’) is crucial for T cell
fibers followed by tissue-resident DCs activation. Co-stimulatory molecules
expressed on the membrane of
AS01 Liposomes containing MPLS and Activated Ag-loaded monocytes and [137] antigen-presenting cells (APCs) and
QS21 MHC-IIhigh DCs T cells act in a tandem ligand–
AS02 O/W emulsion containing MPLA and Enhanced both cellular and humoral- [138] receptor system that, upon
QS21 mediated immune responses completion of the full signal, initiates
T cell maturation, differentiation, and
AS03 a-tocopherol, squalene, polysorbate Promoted recruitment of monocytes [139]
proliferation. Co-stimulatory
80, and PBS and granulocytes; also increased
molecules include CD28-CD80/
secretion of cytokines (CCL2, CCL3,
CD86, or ICOS-ICOL complexes. B
IL-6, CSF3, and CXCL1) from APCs,
cell co-stimulatory molecules include
thereby enhancing Ag-specific
CD40-CD40L and complement-
adaptive immune responses
complement receptors.
AS04 Contains MPLA adsorbed onto a Directly activated APCs by inducing [121] Damage-associated molecular
particulate form of aluminum salt NF-kB, cytokine production, and patterns (DAMPs): host cellular
antigen-specific T cell activation components released during tissue
injury or death. DAMPs activate the
Virosomes Contain inactivated virus Virosomal-adjuvanted influenza [140]
immune system through PRRs;
vaccine Inflexal1 V increased the
nonspecific activation of APCs
geometric mean titers of antibodies
through DAMPs causes several
along with cell-mediated immunity
autoimmune diseases,
CAF01 Cationic liposomal vehicle containing Induced cell-mediated immunity [98] atherosclerosis, and myocardial
DDA with a glycolipid through release of IFN-g and IL-17 infarction.
immunostimulator (TDB) from activated T cells Danger signal: the immune system
is well organized to recognize danger
CAF04 Cationic liposomal vehicle containing Induced higher IFN- g responses than [103]
elements by discriminating self and
DDA with monomycoloyl glycerol CAF01
non-self. Exogenous dangers, such
analog (MMG)
as PAMPs, DAMPs, or stress
CAF05 Cationic liposomal vehicle containing Induced higher IFN-g response than [103] molecules detected by the APCs,
DDA with the immunostimulators TDB CAF01 and CAF04 instantaneously release signals, such
and poly(I:C) as heat shock proteins, uric acid,
reactive oxygen species,
Montanide ISATM 720 W/O emulsion containing non-mineral Formed a depot and induced cell- [127,141]
neuromediators, matrix proteins, and
oil with mannide mono-oleate family mediated immunity
cytokines, including IFN-g, IL-1b,
emulsifier
and IL-6. Dangers signals are a
Montanide ISATM 51 W/O emulsion containing mineral oil Formed a depot and induced strong [127] significant focus for therapeutic
with mannide mono-oleate family cytotoxic T cell response applications and diagnoses.
emulsifier Immunomodulators: any substance
that activates, suppresses (weakens)
a
Selection based on hits in preclinical and clinical studies. or changes (deviates) immune
b
Abbreviations: Ag, antigen; APC, antigen-presenting cells; AS, adjuvant system; CAF, cationic adjuvant formulations; CC/ responses. Immunomodulators can
CX, chemokine; CSF, colony-stimulating factor; DC, dendritic cell; DDA, dimethyl dioctadecyl-ammonium; IFN, inter-
modulate cytokines and chemokines
feron; IL, interleukin; MHC, major histocompatibility complex; MPLA, 3-O-desacyl-40 -monophosphoryl lipid A; NF,
nuclear factor; O/W, oil in water; PBS, phosphate-buffered saline; poly(I:C), polyinosinic-polycytidylic acid; QS, Quillaja secretion, thereby altering immune
saponaria; TDB, trehalose 6,6-dibehenate; W/O, water in oil.
acid content).
Lipopeptides
Macrophage-activating lipopeptide-2 (MALP-2), derived from the N-terminal moiety of Myco-
plasma and Escherichia coli, shows immunostimulatory activity that results from the induction
of B cell proliferation and increased secretion of TNF-a from macrophages [18]. Modification
studies on MALP-2 resulted in two analogs, Pam2CSK4 and Pam3CSK4 (Figure 4A,B), that
have proinflammatory properties. The N-terminal cysteine was shown to be a key residue in
these compounds [19]. Similar to MALP-2, both analogs were shown to interact with specific
receptors, namely TLR2 that heterodimerizes with TLR6 for the former, and TLR2 that
heterodimerizes with TLR1 for the latter (Figure 4C) [20,21]. This heterodimerization occurs
via an amide bond with distinct extra lipid chains [22]. Upon activation with Pam3CSK4, the
TLR2 receptor interacts with TLR1 and adopts a horse shoe-shape structure. The lipid portion
of Pam3CSK4 was found to fit into a narrow gap in the newly formed complex, while the
cysteine residue also inserted into the same gap. In the case of Pam2CSK4, in which an extra
amide bond is absent compared with Pam3CSK4, the lipid chain interacts with TLR2 and the
B cell
TH cell
Eosinophil APC
Complement Lysed target cell
NK cell T cell CTL
(apoptosis)
Innate immunity Adapve immunity
CD40 CD40L
CD80/86 CD28 TLRs
NLRs
MHC-II TCR
CLRs
ICOSL ICOS
Co-smulaon
Figure 2. Innate and Adaptive Immune Systems. The innate immune system constitutes a front line of defense and provides a nonspecific response against
invading pathogens. This response is mediated by various cells (granulocytes, monocytes, macrophages, dendritic cells, neutrophils, basophils, and natural killer cells,
and active molecules as proteins of the complement cascade) through recognition of PAMPs or DAMPs by PRRs. The innate immune response shapes adaptive
immunity resulting in the production of antigen-specific T and B lymphocytes. Abbreviations: APCs, antigen-presenting cells; CD, cluster of differentiation; CLRs, C-type
lectin receptors; CTL, cytotoxic lymphocytes; DAMPs, damage-associated molecular patterns; ICOS, inducible T cell costimulatory; MHC, major histocompatibility
complex; NKT, natural killer T cells; NLRs, nucleotide-binding, oligomerization domain (NOD)-like receptors; PAMPs, pathogen-associated molecular patterns; PRRs,
pathogen recognition receptors; TCR, T cell receptor; TLRs, Toll-like receptors.
peptide moiety interacts with TLR6, a molecular hot core of links that resembles the CD1d–
invariant NKT (iNKT) interaction (see below for more detail) [23]. TLR2 agonists represent an
effective family of adjuvants tested in vaccine formulations against different infectious agents,
including HIV [24], HBV, HPV [25], Leishmania [26], and Chlamydia [27], and in certain cancers,
such as melanoma [28].
Research has led to the development of novel Pam3CSK4 analogs [14]. In particular, carbo-
hydrate di- and tri-lipidated cysteine-based 1,2,3-triazoles have been developed that exhibit
adjuvant properties in vaccine formulations against bacterial, viral, and protozoan infections,
and in cancer [29]. A construct was built in which the polycationic peptide entity was swapped
DC
(ISCOMs)]
Innate immunity
Adjuvant T and B cells
Adapve
immunity
Memory cells
Immunosmulators
and nonreceptor-specific immune
acvators
Figure 3. Interactions of Adjuvant with the Bioactive Actors of Innate and Adaptive Immunity. Novel delivery
systems (e.g., emulsions, liposomes, polymers, and nanocarriers) and immunostimulants activate the innate immune
system. Antigen fragments activate adaptive immunity in inducing (or not) immunological memory. Adjuvants and
immunostimulatory compounds enhance vaccine efficacy primarily by stimulating innate immunity. They also boost
adaptive immunity, thus providing a long-term memory response against pathogens and foreign antigens. Abbreviation:
DCs, dendritic cells (acting as antigen-presenting cells, APCs).
with a hydrophilic carbohydrate entity tethered to a triazolyl moiety. The aim of this new design
was to examine the possibility of generating a functional mimic of Pam3CSK4. The carbohy-
drate entity imparts hydrophilicity, whereas the triazolyl moiety mimics the cationic nature of the
peptide portion.
TLR2
T TLR2 TLR6
TLR1
(A) (B)
MyD88
TIRAP
IRAK
TRAF6
Nucleus
IKKγ
NF-κB IκB
NF-κB IKKα IKKβ
Proinflammatory cytokines
Pam3CSK4 Pam2CSK4
(D) (E)
6e 6h
250 <0.001
<0.0001
Anbody ter (x 103 )
200
150
100
50
0
e
e
um
6h
4
on
6
SK
A+
A+
Al
al
3C
OV
A+
OV
A
m
OV
OV
Pa
A+
OV
There is abundant literature highlighting the adjuvanticity of CpG ODNs in preclinical and
clinical studies (Table 2) [32,33]. CpG ODNs conjugated with HBV surface antigen resulted in
a 1000-fold increase in the antibody response compared with antigen alone [34]. In contrast
to other TLR ligands, CpG ODNs have been shown to elicit superior CD8+ cell-mediated
immunity [35], due to the activation of Th1 cytokine production (Figure 5B) [36]. It has also
been shown that CpG ODN 1668 associated with carbon nanotubes and a Tat peptide of HIV
potentiated the immune response [37,38]. Vaccine formulations including CpG ODNs have
been shown to elicit strong humoral immune response against a variety of pathogens,
including anthrax, Leishmania, HBV, influenza, measles, orthopox, and tetanus toxoid
[39–43]. In a Phase I trial, a malaria vaccine comprising CpG ODNs and the apical membrane
antigen resulted in fivefold higher antibody titers compared with antigen alone [44]. Moreover,
CpG ODNs showed significant adjuvant activity against Engerix B (a HBV vaccine for adults)
by increasing antibody titers and enhancing T cell proliferative responses in HIV-infected
immunocompromised subjects [45]. Improved antibody and CD8+ T cell responses were also
described when CpG ODNs were combined with Melanoma A, New York-oesophageal
cancer 1, and anthrax vaccines [46–48]. Finally, the NuThraxTM vaccine candidate from
Bioemergent is based on BioThrax1 (Anthrax Vaccine Adsorbed) combined with the CpG
ODN 7909 as an adjuvant. A Phase III study is planned (first subject enrollment targeted for
2018). Millions of doses of NuThrax are being stockpiled and the US FDA is close to licensing
this product (making it the first CpG-containing vaccine to achieve licensure). The FDA could
authorize NuThrax for emergency as early as 2018 [49]. CpG ODNs have also been combined
with other adjuvants or chemically linked to different nanoparticles or antigens to improve
vaccine immunogenicity [50–52].
Figure 4. Acylated Peptide-Induced Signaling Pathway. Pam2CSK4 (A) and Pam3CSK4 (B) acyl lipopeptides bind to the TLR2 present on the cytoplasmic
membrane (C), resulting in TLR2 heterodimerization with TLR6 and TLR1, respectively. As a consequence, these heterodimers interact with MyD88, leading to activation
of the IRAK-TRAF6 cascade, followed by activation of various kinases, which finally leads to the binding of NF-kB to its promoter region. This final step is responsible for
the production of proinflammatory cytokines. (D,E) Newly developed Pam3CSK4 derivatives (the structure of the hit molecules is shown). (F) OVA-specific IgG response
in BALB/c mice that were immunized subcutaneously with OVA alone, OVA + alum (Alhydrogel1 adjuvant 2%), OVA + Pam3CSK4, OVA + 6e molecule, or OVA + 6h
molecule (100 mg/100 ml/mouse in each group; five mice per group). Mice received two injections at days 0 and 14 and were bled at day 28. Anti-OVA antibodies were
measured by indirect ELISA. Briefly, the plates were coated with OVA (1 mg/well) in carbonate buffer, pH 9.5, and then incubated at 4 C overnight. After three
consecutive washings in PBS containing 0.05% v/v Tween-20 (PBS-T), and blocking nonspecific binding sites remaining on the plastic plates by adding 200 ml 1% (v/v)
bovine serum albumin at room temperature for 1 h followed by three other washings, standards and samples diluted in PBS-T were added to the plates and incubated at
37 C for 2 h at room temperature. Plates were again washed three times with PBS-T, followed by addition of antimouse IgG-horseradish peroxidase (IgG-HRP; Jackson
ImmunoResearch diluted in PBS-T). After 30 min at room temperature, plates were again washed three times with PBS-T and the HRP substrate (H2O2) and 3,3',5,5;-
tetramethylbenzidine (TMB) chromogen were added. Plates were incubated at room temperature (4–30 min) for color development. Color reaction was stopped by
adding 50 ml HCl. Absorbance was measured at 450 nm [142]. The values are presented as mean titers SD. **P <0.001, ***P <0.0001 (Mann–Whitney U test).
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IRAK, IL-1 receptor-activated kinase; MyD88, adaptor protein myeloid differentiation gene 88; NF-kB,
nuclear factor-kB; OVA, ovalbumin; TIRAP, Toll-interleukin 1 receptor domain containing adaptor protein; TLR, Toll-like receptors; TRAF, tumor-necrosis factor
receptor-associated factor.
A type
Acvate maturaon of Acvate pDCs to secrete Acvate pDCs to produce
pDCs and their secreon TNF-α, and B cells to IFN-α, and
of IFN-α; no effect on B proliferate and secrete B cells to secrete IL-6 T
G T
cells IgM
C C
G T
A C
Mainly remain in early Quickly transported Have intermediate G
endosomes and interact through early properes of both A- and C
with adaptor proteins endosomes into late B-type ODNs T
(e.g., MYD88) to trigger endosomes for signal
the IFN-α signaling transducon
cascade
B type
CpGODN
(B)
Nucleus
Endosome
TL
R9
AP–1
MYD88
IRAK
NF-κB AP–1
TRAF6
CpG ODN
JNK
NF-κB IκB
DC B cell
•Maturaon
•Differenaon
•Proliferaon
Eosinophil Monocyte Macrophage TH1 cell CTL
NK cell
CD80
CD40
CD40
Chemokines (IP10, Mig, and I-TAC) Memory cells
Figure 5. Structure and Mode of Action of CpG Oligodeoxynucleotides (CpG ODNs). (A) The three different types of CpG ODN (A, B, and C), their structures,
and properties. (B) CpG ODNs modulate innate and adaptive immune responses in several ways. (A) The CpG ODN–TLR9 signaling pathway. TLR9 receptors are
present on the endosomal membrane. After internalization, CpG ODN activates elements of the MyD88/IRAK/TRAF6 pathway, leading to the simultaneously activation
of two kinase pathways (MAPK/c-JUN and NF-kB) and the AP-1 and NF-kB promoter genes. (B) CpG ODN activates directly DCs and B cells acting as APCs. Activated
The mechanism of action of QS-21 at the molecular level remains unclear. According to current
understanding, the free aldehyde side chains of QS-21 are the primary interacting components
that bind amino acid residues of proteins present on the surface of immune cells. This leads to
the formation of a Schiff base or imine [59]. This interaction, on T cells and antigen-presenting
cells (APCs), appears to contribute to the initial triggering signal for downstream signaling
(Figure 6B) [5,68].
DCs and B cells also activate other immune cells, such as neutrophils, monocytes, macrophages, Th1-type CD4+ T cells, CTL, and NK cells, which then mature,
differentiate, and proliferate. APCs construct a base by forming co-stimulatory signals and provide strong memory responses. Abbreviations: APCs, antigen-presenting
cells; Fc, fragment crystallizable region; IFN, interferon; IP10, IFN-g-inducible protein 10 (or CXCL1); I-TAC, interferon-inducible T cell alpha chemoattractant (or
CXCL11); JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MHC-II, class II major histocompatibility complex; Mig, monokine-induced by IFN-g
(or CXCL9); pDC, plasmacytoid dendritic cells. For definitions of other abbreviations, please see the main text and the legend to Figure 2.
US 5182109A National Institute of Health; Vaccine preparation comprising a 1993 Vaccine comprises different toxin
The Kitasato Institute, both of Tokyo bacterial toxin adjuvant antigens
(JP)
EP 0781 559 A2 Juridical Foundation, The Chemo- Oil adjuvant vaccine and method for 1996 W/O emulsion with different surfactant
Sero-Therapeutic Research Institute preparing same and polymer ratios
Kumamoto-ken (JP)
PCT/KR96/00053 LG Chemical Ltd., Seoul, (KR) Quillaja saponin adjuvant and vaccine 1998 Comprises saponin component from
formulation containing same the bark of Quillaja saponaria Molina as
an immune adjuvant
US 6,406,705 B1 University of Iowa Research Use of nucleic acids containing 2002 Unmethylated CpG ODN and non-
Foundation, Iowa City (USA); Coley unmethylated CpG dinucleotide as an nucleic acid adjuvant
Pharmaceutical GmbH, Langenfeld adjuvant
(DE); Ottawa Health Research Institute,
Ottawa (CA)
PCT/US98/08652 Protection Unlimited, Inc., Wilmington, Nerve growth factor as a vaccine 2003 Nerve growth factor acts as an adjuvant
DE (USA) adjuvant to enhance effectiveness of vaccine
US 2004/0101534 A1 Rothwell’ Figg’ Ernst & Manbeck, P.C. Adjuvant-free peptide vaccine 2004 Vaccine comprises fusion peptide
Washington (USA) synthetically derived from
cytomegalovirus with CpG ODN as an
adjuvant
US 6,881,561 B1 Cheil Jedang Corporation, Seoul (KR) Endonuclease of immune cell, process 2005 Immune adjuvant comprising
for producing the same, and immune approximately
adjuvant using the same 10-bp single-stranded oligonucleotide
with a CpG motif; produced by
treatment of bacterial DNA with
endonuclease
PCT/EP00/01046 Eurocine AB, Stockholm, Solna (SE) Vaccine composition 2005 TB vaccine that comprises
mycobacterial
US 2005/0158330 A1 NOF Corporation, Tokyo (JP); Juridical Oil adjuvant vaccine 2005 Vaccine comprises W/O/W-type oil
Foundation, Kurnarnoto-shi (JP) adjuvant
PCT/NL2004/000437 Bestewil Holding B.V., Amsterdam (NL) Functionally reconstituted viral 2009 Membrane protein from pathogens or
membranes containing adjuvant tumor cells with amphiphilic adjuvant
PCT/EP05/05786 SmithKlineBeecham Corporation Vaccine compositions comprising 2009 Vaccine comprises influenza viral
Corporate Intellectual Property, virosomes and a saponin adjuvant antigen, QS21, and sterol
Pennsylvania (USA)
PCT/KR2007/006889 SNU R&DB Foundation, Seoul (KR) a-GalCer derivatives, pharmaceutically 2010 a-GalCer-containing triazole moiety at
acceptable salts thereof, preparation the amide position
method, and pharmaceutical
composition for the immune adjuvant
containing the same as an active
ingredient
PCT/EP08/57998 GlaxoSmithKline Corporate Intellectual Vaccine comprising Streptococcus 2010 Vaccine comprises different
Property, North Carolina (USA) pneumoniae capsular polysaccharide Streptococcus pneumoniae capsular
conjugates saccharide conjugates
PCT/FR08/51807 Air Liquide USA LLC, Intellectual Method for preparing a vaccine 2010 Composition comprises an adjuvant
Property, Houston, Texas (USA) composition comprising at least one such as inverse latex or polymer mixed
antigen and at least one adjuvant into an antigenic medium
US 7,749,979 B2 National Pingtung University of Science CpG DNA adjuvant in avian vaccines 2010 CpG DNA
and Technology, Pingtung County (TW)
US 7,771,726 B2 New York University, New York (USA); Use of synthetic glycolipids as universal 2010 Synthetic a-C-GalCer
The Research Foundation of the City adjuvants for vaccines against cancer
University of New York, New York and infectious diseases
PCT/EP10/51882 GlaxoSmithKline Biologicals S.A. Inactivated dengue virus vaccine with 2011 Inactivated dengue virus antigen in
Rixensart (BE) aluminium-free adjuvant combination with aluminum-free
adjuvant
PCT/EP2006/069979 GlaxoSmithKline Biologicals S.A., Vaccine comprising an O/W emulsion 2012 O/W emulsion comprises
Rixensart (BE) adjuvant metabolizable oil, tocol, emulsifying
agent with various antigens and
methods of preparation
EP 2 589 392 A2 Sanofi Pasteur, Lyon (FR) Process for stabilizing an adjuvant 2013 Process for stabilizing a vaccine
containing a vaccine composition composition with a stabilizer
PCT/US2004/005152 HasumiLlc (Dba Shukokai International) Human lymphocyte vaccine adjuvant 2013 Supernatant collected from stimulated
New York (USA) cultured human lymphocytes
US 8,540,955 B2 Wyeth LLC, Madison (USA) Process for producing aluminium 2013 Provides improved methods for
phosphate producing the aluminium adjuvant
AlPO4
PCT/U52008/057355 The Regents of the University of Method of preparing an 2013 Preparation of freeze-dried vaccine
Colorado, A Body Corporate, Denver immunologically active adjuvant-bound comprising an aluminium salt adjuvant,
(USA) dried vaccine composition a recombinant Clostridium botulinum
neurotoxin protein, and a glass-forming
agent
US 8,624,004 B2 GlaxoSmithKline Biologicals S.A., Purification of HBV antigens for use in 2014 HBV antigen that includes cysteine
Rixensart (BE) vaccines
PCT/FR2011/050069 Société d’Exploitation de Produits pour Adjuvant for preparation of vaccine 2014 Short and long-chain hydrocarbons
les Industries Chimiques, Paris (FR) compositions intended for prevention with Coccidia
of coccidiosis
US 8,795,678 B2 Academia Sinica, Taipei (TW) TLR2 agonists and methods of use 2014 FMDV capsid proteins VP1 and VP3
thereof activate TLR2
US 8,889,616 B2 Oncothyreon Inc., Seattle (USA) MUC1-based glycolipopeptide vaccine 2014 Liposomal vaccine formulations
with adjuvant comprising an adjuvant and an
immunogen for immunotherapy
PCT/US2013/034372 Kansas State University Research Vaccine adjuvant 2015 Oil-based adjuvants comprise a plant-
Foundation, University of Tennessee derived surfactant, such as gum arabic,
Research Foundation, Manhattan an aqueous component, and an oil
(USA)
US2016/0200758A1 Council of Scientific and Industrial 1,2,3-Tiazole-tethered carbohydrate 2016 Lipidated cysteine-based triazoles as
Research, Delhi (India) di- and tri-lapidated cysteine vaccine adjuvants covered both
conjugates useful as vaccine therapeutic and prophylactic vaccines
adjuvants, and process for preparation against bacterial, viral, and protozoan
thereof infections, and cancer
a
The mentioned patents are all in the field of vaccines and vaccine adjuvants.
b
Abbreviations: bp, base pairs; CpG ODN, CpG dinucleotide; FMDV, Foot-and-mouth disease virus; TLR, Toll-like receptor; VP, viral protein; W/O/W, water/oil/water.
a-GalCer
The synthesis of a-GalCer, popularly known as KRN7000 (Figure 7A), was based on structure–
activity relationship (SAR) studies pertaining to Agelasphine-9b. The latter was first isolated
from the Okinawan sea (Japan) sponge Agelas mauritianus. The curiosity-driven discovery of
agelasphine-9b was based on the bioactive cerebrosides that it contains. Glycosphingolipids
are widely distributed in living organisms [71] and a-GalCer is mostly known as an activator of
type 1-murine and human iNKT cells. As NK cells, NKT cells express an invariant ab T cell
Parally
Oligosaccharide Oligosaccharide hydro- Pentose
philic lipid
(B)
Saponin
Ag CHO CH
NH2 Imine
N
Ca2+
Na+
K+ ERK2
Lipid body
MAPK
ER MHC-I TCR
Transcripo n
Pepde
Cell survival
Proliferaon
APC Migraon
CD8+ T cells
(C) (D)
OVA-specific IgG
<0.00 01
40
Anbody ter (x 103)
<0.000 1
30
20
10
15g
0
A g
OV um 15
+Al A+
VA OV
O
iNKT
IFN -γ
α-GalCer TCR
(C) Lipid angen
CD1d
Monocytes
NK cells
APC
IL-12
α-C-GalCer
(D)
Macrophages
CD8+ T cells
(E)
CD4+ T cells
Figure 7. CD1d–a-GalCer–iNKT Ternary Complex Activation of Bystander Cells. (A) Pictorial representation
summarizing the formation of the binary complex of CD1d–a-GalCer that releases IL-12 from APCs and the formation of
the ternary complex CD1d–a-GalCer-iNKT that releases cytokines IL-4 and IFN-g from iNKT cells. The release of large
amounts of cytokines activates bystander cells to migrate, proliferate, and differentiate. (B) a-GalCer; (C) a-C-GalCer
analog; (D,E) novel benzyloxyalkyl-substituted 1,2,3-triazolyl a-GalCer analogs (T1204B and T1206B). Abbreviations:
iNKT, invariant natural killer T cells. For definitions of other abbreviations, please see the main text.
receptor (TCR) and also markers, such as NK1.1, DX5, and CD161 [72]. Unlike conventional
CD4+ and CD8+ T cells, NKT cells recognize exogenous or endogenous glycolipids presented
by the MHC-I-like molecule CD1d. Although there are minor variations among human and
mouse NKT cells, the latter are considered to be 100% CD1d restricted. Interactions between
NKT and a-GalCer or between analogs and CD1d and their immunomodulatory properties
have been extensively studied over the past two decades. Many exogenous (natural and
synthetic) and endogenous CD1d-restricted iNKT stimulators have been reported [73–76].
X-ray crystallography studies revealed that a-GalCer is primarily recognized by the nonclassical
MHC-I or CD1d molecules present on APCs. CD1d forms two pockets, A’ and F’, for binding
two lipid chains in a-GalCer (the acyl and sphingosine chains) and forms an a-GalCer/CD1d
binary complex [77,78]. Furthermore, CD1d presents a-GalCer to the TCR of iNKT cells and
forms a NKT/a-GalCer/CD1d ternary complex. This ternary complex is one of the largest ever
recorded for any natural TCR ligands [79,80].
Figure 6. Mechanisms Involved in the Adjuvant Activity of Saponins. (A) Structural elements of QS-21. (B) Aldehyde-containing saponins (e.g., QS-21) act via
two different mechanisms. One is via the formation of a lipid body that participates in the presentation of MHC antigens by APCs. The second one is direct activation of T
cells by interacting with amino acid residues present in the TCRs. These interaction steps are followed by the activation of ERK2, MAPK, and transcription factors,
leading to cytokine release, and cell proliferation, migration, and differentiation. (C) Compound 15g, a novel 1,2,3-triazolyl conjugate with a carbohydrate-steroid entity.
(D) OVA-specific IgG response in BALB/c mice that were immunized subcutaneously with OVA alone, OVA + alum, and OVA + 15g (100 mg/mouse in each group; five
mice per group). Mice received two injections at days 0 and 14 and were bled at day 28. Anti-OVA antibodies were measured by indirect ELISA (please see the legend to
Figure 4 for technical details). The values are presented as mean SD. ***P <0.0001 (Mann–Whitney U test). Abbreviations: Ag, antigen; ER, endoplasmic reticulum;
ERK2, extracellular signal-regulated kinases. For definitions of other abbreviations, please see the main text and the legends to Figures 2 and 3.
Liposomes
Liposomes are lipid vesicles with phospholipid bilayers. They have the capacity to act both as
delivery systems for antigens and as immunomodulators [96–98]. They protect antigens from
degradation, deliver them to APCs, and can be used for mucosal delivery. The lipid membrane
comprises phosphatidylcholine, which is nonimmunomodulatory but shows fusogenic activity,
thus facilitating better uptake by APCs. Frequently, other compounds, such as TLR ligands,
need to be incorporated for optimal adjuvanticity [99]. It has taken many years of research to
design liposome-containing formulations that are clinically usable as safe vaccine delivery
systems. Liposomes and virosomes are present in vaccine suspensions against influenza virus
(Inflexal V), HAV (Epaxal), and Plasmodium falciparum for malaria (RTS,S/AS01/MosquirixTM,
approved for use by European regulators in July 2015) [100]. In addition, some oral liposome
formulations with recombinant heat shock protein 60 were shown to be promising against
Helicobacter pylori infection in preclinical studies [101].
Cholesterol-containing liposomes have also been used to attenuate adjuvant toxicity. For
example, a liposome formulation was shown to reduce the toxic effects of QS-21 (hemolytic
activity) and MPLA (endotoxic activity) [102]. Moreover, liposomes containing cationic charges
have demonstrated superior delivery properties and elicited significantly higher protective
response against Mycobacterium spp. (Table 1) [103].
Immune-Stimulating Complexes
Immune-stimulating complexes (ISCOMs) are spherical open cage-like structures (typically
40 nm in diameter) that spontaneously form when cholesterol, phospholipids, and specific
saponins (mostly Quil A) are mixed at a specific ratio. The ISCOM matrix can directly associate
with antigens or be formed first and then added to the formulation at a later point. Given their
structure, ISCOMs can achieve efficient antigen delivery into DCs that results in the induction of
antigen-specific T cell responses, longlasting antibody responses, and balanced Th1/Th2
immunity [109–111]. To improve their efficacy, ISCOMs have also been used in combination
with immune-stimulating molecules, such as TLR ligands or cholera toxin A (CTA) [112,113].
CA2628206 A1 Animal (such as fish) or vegetable Tweens, Spans, Brijs, lecithin, triton X-100,
source copolymers of ethylene oxide, propylene oxide,
and/or butylene oxide; mixture of surfactants
US 5885590 A Vegetable oil, animal oil, mineral oil, Polyoxyethylene polyoxy propylene block
isopropyl myristate, and copolymer, poloxamer 331, poloxamer 461,
polyoxypropylene poloxamer 520.5, sorbitan monooleate, sorbitan
tristearate, Arlacel 186, poloxamer 403,
polyoxyethylene sorbitan monooleate, poloxamer
188, and mixtures thereof
a
Selection based on the patented product (oil, surfactant, and adjuvant).
% populaon
High stability
5
Easily metabolizable in biological systems
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Figure 8. Emulsion-Based Vaccine Delivery Systems. (A) Prerequisites for emulsions to be used as adjuvants. (B) Efficacy of an oleic acid nanoemulsion for
vaccine delivery. An O/W emulsion comprising OVA as a model antigen and MPLA as an adjuvant was administered intranasally to C57/Bl6 mice (N = 5). Antigen-
specific IgA from nasal washes and IgG from the serum of immunized mice were revealed by indirect ELISA (please see the legend to Figure 4 for technical details).
Immunophenotyping for co-stimulator markers CD19+ and CD86+ was performed according to the method of Colovai et al. [143] with slight modifications. Briefly,
3 105 splenocytes (3 105cells/tube) were centrifuged at 2000 rpm for 15 min. BD Fc Block (10; 1 ml) was then added for 15–20 min at 4 C in the dark to block
TM
nonspecific Fc receptor-mediated antibody binding. After two successive washings with 1% (v/v)-fetal calf serum-containing PBS, cells were centrifuged at 2000 rpm
for 5 min. They were then labeled using 7 ml (determined according to previous titrations) of anti-CD19-PE and anti-CD86-allophycocyanin fluorochrome-conjugated
antibodies and 2 ml of fluorochrome-conjugated isotype control (all from BD Biosciences). Cells were then incubated on ice for 30 min in the dark, centrifuged with 1%
(v/v) fetal calf serum-containing PBS and analyzed using a BD FACSVerse flow cytometer. Compensation was established using BD Biosciences compensation beads.
Postacquisition flow cytometry analyses were performed using the FACS Suite software. Abbreviations: E, emulsion; ELISA, enzyme-linked immunosorbent assay; Fc,
fragment crystallizable region; MPLA, monophosphoryl lipid A; OVA, ovalbumin; PE, phycoerythrin.
Oil-in-Water Emulsions
O/W emulsions are extremely safe compared with W/O formulations. The antigen is available in
the continuous phase and can easily diffuse into tissues. However, the addition of immuno-
modulators, such as MPLA, QS21, MDP, or imiquimod, was found to be necessary to achieve a
strong immune response. The oil phase in most emulsions comprises metabolizable oils, such
as vegetable (sunflower, soybean, corn, olive, and mustard) and animal based-oils, such as fish
oils (cod liver oil, shark liver oils, or whale oil) and squalene and squalane. These oils are
biocompatible and biodegradable [114,115]. O/W emulsions associated with immunomodu-
lators with established mechanisms of action, such as MF59, AS02, AS03, and AS04, showed
efficacy in preclinical and clinical studies against infectious diseases (Table 1). MF59 composi-
tion contains squalene with varying amounts of muramyl tripeptide phosphatidyl-ethanolamine
(MTP-PE). Fluad1, seasonal influenza vaccine with MF59 was shown to produce high antibody
titers against influenza in older patients [116,117]. An AS02 emulsion associated with 3-O-
desacyl-40 -MPLA (a TLR4 agonist) and QS-21 has been associated with many antigenic
vaccine preparations. For example, RTS,S/AS02 effectively elicited protective immune
responses against P. falciparum [118]. Others, such as TB vaccines, comprising Mtb72f
(Mycobacterium tuberculosis fusion protein), Mtb41, or Ag85B-ESAT-6 (secreted antigenic
proteins) with AS02; HBV vaccines, comprising HBV hyperimmunoglobulin (HBIG) or SL* (small
HBV envelope protein) with AS02; HIV vaccines, comprising gp120 (an envelope glycoprotein)
and AS02; and cancer vaccine, comprising melanoma-associated antigen 3 (MAGE-A3) and
AS02 have elicited potent immune responses in preclinical and clinical studies [119]. An AS03
emulsion composed of biodegradable oils (squalene and a-tocopherol) and a surfactant
(polysorbate 80) in phosphate-buffered saline (PBS) also showed some efficacy. Even though
this adjuvant system did not show encouraging results with malaria and HIV vaccines, it
demonstrated strong immunostimulatory activity and surrogate protection against H5N1
and H1N1 pandemic influenza in both children and adults [120].The AS04 emulsion contains
MPLA with a particulate form of aluminum salt. CervarixTM and FENDrixTM are two licensed
vaccines containing AS04 against HPV and HBV [121] and others, such as herpes simplex virus
(HSV), respiratory syncytial virus (RSV), and Epstein–Barr virus (EBV) are under clinical evalua-
tion [119]. This system is also exploited in other infectious diseases [122].
Water-in-Oil Emulsion
In W/O emulsions, the antigen is present in aqueous droplets entrapped in the continuous phase of
oil and, therefore, has a tendency to remain at the site of injection. This accumulation can induce
granulomas at the injection site and also causes sharp pain [123–125]. However, this type of
emulsion generates high immunogenicity especially due to the sustained release of antigen from
the oily phase. Nevertheless, W/O emulsion are of limited use in humans. However, many
veterinary vaccines are based on W/O emulsions and some are in clinical trials for the treatment
of certain cancers, such as melanoma [126]. Freund's adjuvant, a classical W/O emulsion system
with nonmetabolizable oils, produced high antibody responses against many antigens with a Th1-
biased cytokine response, although it is not authorized for human use. Other W/O emulsions that
have gained interest as adjuvant delivery systems include Montanide ISATM 51 and Montanide
ISATM 720. These emulsions comprise mineral oil (Drakeol1 6VR) (Montanide ISATM 51) and
nonmineral oil (squalene) (Montanide ISATM 720) with a surfactant from the mannide monooleate
family (Table 1) [127]. Both formulations have been used in preclinical and clinical vaccine trials
against HIV infection, malaria, and breast cancer [117].
Basic research on adjuvants has come a long way in identifying and or synthesizing novel
molecules that exhibit potent immunostimulatory properties with minimal adverse effects.
These included autoimmune diseases (rabies vaccine), disseminated encephalomyelitis (small
pox vaccine), and paralysis of children (polio vaccine). New vaccine adjuvants currently in
clinical use, such as MF59 and AS03, minimize the number of injections, and exhibit fewer
adverse effects and improved vaccine efficacy.
Many preclinical and clinical studies have demonstrated the use of purified TLR agonists as
adjuvants. Ligands of TLR4 are currently used in human licensed vaccines for the prevention of
HPV and HBV infections [9,10]. With the increased understanding of TLR4 receptor–ligand
interactions, chemists have synthesized new compounds that exhibit immunostimulatory
properties with minimal adverse effects. Such work has led to the development of novel
Pam3CSK4 analogs [12], such as carbohydrate di- and tri-lipidated cysteine-based 1,2,3-
triazoles that exhibit adjuvant properties in vaccine formulations against bacterial, viral, and
protozoan infections, and against cancer [27].
Non-invasive vaccine delivery remains a primary focus of research that could impact mass
vaccination campaigns worldwide [131–133]. In this context, developing vaccine formulations
that are safe, effective, and available at low cost is a priority (see Outstanding Questions).
Stringent strategies should be followed for the development of vaccine formulations that
include adjuvants, that result in the desired immune responses, that have immunopotentiating
activity that result in long-term immunity and protection. Extensive research has already led to
the development of several novel adjuvants that are currently being evaluated in clinical trials or
have been licensed. Along this line, research efforts have focused on developing novel lipid-
containing adjuvants that are safe and improve the efficacy of vaccine, notably for non-invasive
delivery. However, detailed information regarding the interactions between cell surface recep-
tors after stimulation by adjuvant molecules and the compatibility of these novel adjuvants with
existing or novel formulations is required. As our understanding of the molecular mechanisms
involved in immune protection improves, and with the advent of new methods in synthetic
chemistry, considerable breakthroughs in vaccine development, along the lines of those
summarized here, are anticipated in the near future. Novel technologies, such as new glyco-
conjugation methods, reverse vaccinology, and next-generation sequencing techniques,
should lead to novel vaccine strategies including, but not limited to, HBV, pertussis toxin,
Lyme borreliosis, and HPV. Such vaccines could be developed with or without adjuvants [134].
Thus, the goal of the research community is to ensure a high level of broad protection by
building new concepts that are no longer based on structural vaccinology (given the variability of
target epitopes and the constant emergence of new pathogens and tumor antigens), but on
introducing new immunostimulants and novel delivery systems that combine efficacy (with
longlasting memory immune responsiveness) and safety.
Author Contributions
All the authors listed made substantial direct and intellectual contributions to this article and approved it for publication.
Acknowledgments
We thank Dennis Klinman for insightful comments. Some figures were made from the material provided by Servier Medical
Art (www.servier.fr) under the CC 3.0 FR license. Research in S.M.’s laboratory is funded by the French Centre National de
la Recherche Scientifique (CNRS), the Laboratory of Excellence Medalis (ANR-10-LABX-0034), and the EquipEx program
I2MC (ANR-11-EQPX-022), Initiative of Excellence (IdEx), Strasbourg University, France. Research in H.M.S.K.’s labora-
tory is funded by the Council of Scientific and Industrial Research (CSIR), Government of India for the founding of the 12th
five-year plan CSC0205 (Development of Novel Vaccine Adjuvant, DENOVA). S.R.B. thanks the Department of Science
and Technology (DST), Government of India, and the Centre Franco-Indien pour la Promotion de la Recherche Avancée
(CEFIPRA) for the award of a Raman-Charpak fellowship.
References
1. Kaushik, D. and Sehgal, D. (2008) Developing antibacterial 4. Chan, Y.K. and Gack, M.U. (2016) Viral evasion of intracellular
vaccines in genomics and proteomics era. Scand. J. Immunol. DNA and RNA sensing. Nat. Rev. Microbiol. 14, 360–373
67, 544–552 5. Marciani, D.J. (2003) Vaccine adjuvants: role and mechanisms
2. Leclerc, C. and Ronco, J. (1998) New approaches in vaccine of action in vaccine immunogenicity. Drug Discov. Today 8,
development. Immunol. Today 19, 300–302 934–943
3. Nascimento, I. and Leite, L. (2012) Recombinant vaccines and 6. Akira, S. et al. (2006) Pathogen recognition and innate immunity.
the development of new vaccine strategies. Braz. J. Med. Biol. Cell 124, 783–801
Res. 45, 1102–1111 7. Hoebe, K. et al. (2004) The interface between innate and adap-
tive immunity. Nat. Immunol. 5, 971–974