Arch. Pharm. Res.

Online ISSN 1976-3786

DOI 10.1007/s12272-017-0969-z Print ISSN 0253-6269

REVIEW

Vaccine adjuvants: smart components to boost the immune system

Rakesh Bastola1 • Gyubin Noh1 • Taekwang Keum1 • Santosh Bashyal1 •
Jo-Eun Seo1 • Jaewoong Choi1 • Yeonsu Oh2 • YoungSik Cho1 • Sangkil Lee1

Received: 28 February 2017 / Accepted: 26 September 2017

Ó The Pharmaceutical Society of Korea 2017

Abstract Vaccination is an effective approach to prevent Introduction

the consequences of infectious diseases. Vaccines
strengthen immunity and make individuals resistant to Vaccination is one of the most effective and cost-efficient
infections with pathogens. Although conventional vaccines ways to prevent many types of infectious diseases. It aims
are highly immunogenic, they are associated with some to induce a pathogen-specific immune response and pro-
safety issues. Subunit vaccines are safe, but they require vide long-lasting protection against infection (Lee and
adjuvants to stimulate the immune system because of their Nguyen 2015). Long-lasting and effective stimulation of
weaker immunogenicity. Adjuvants are entities incorpo- the immune system can be achieved by conventional vac-
rated into vaccines to increase the immunogenic responses cines composed of live attenuated or inactive pathogens,
of antigens. They play a crucial role in increasing the but these vaccines are associated with several safety issues
potency and efficacy of vaccines. Different adjuvants have including possible mutations that restore pathogenicity or
different modes of action; therefore, a better understanding incomplete inactivation of antigens. Meanwhile, subunit
of their immunology could provide guidance for the vaccines are safer but are less immunogenic. Thus, subunit
development of novel adjuvants. Numerous studies have vaccines need adjuvants to increase their immunogenicity
been conducted using different types of adjuvants to (Park et al. 2016). Adjuvants are commonly defined as
characterize their potency and safety; however, in practice, entities that are added to formulations of vaccines to
only few are used in human or animal vaccines. This increase the immunogenicity of antigens (Lee and Nguyen
review aims to introduce the different modes of action of 2015). They facilitate the use of smaller doses of antigen
adjuvants and give insight into the types of adjuvants that and permit immunization protocols using fewer doses of
possess the greatest potential for adjuvanticity. vaccine (Coffman et al. 2010). Furthermore, adjuvants help
to increase the stability of vaccines by making them less
Keywords Vaccines
Adjuvants
Modes of action
Types susceptible to degradation that can occur during storage or
of adjuvants upon injection. They also influence the onset, strength and
duration of the immune responses (Schijns and Lavelle
2011).
This review gives a brief overview of the role of adju-
vants in innate and adaptive immune responses, the modes
& Sangkil Lee of action and types of adjuvants, the current uses of
skdavid@kmu.ac.kr licensed adjuvants, and obstacles in development of new
1
adjuvants.
College of Pharmacy, Keimyung University, 1095
Dalgubeol-daero, Dalseo-Gu, Daegu 42601,
Republic of Korea
2
Department of Veterinary Pathology, College of Veterinary
Medicine and Institute of Veterinary Science, Kangwon
National University, Chuncheon 24341, Republic of Korea

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The role of adjuvants in innate and adaptive TLR ligands are potent immunomodulators for a variety of
immune responses immune responses. Different components of bacteria and
viruses can be recognized by TLRs. Lipid, protein, and
If an antigen enters the body, the immune system responds nucleic acid components are different classes of TLR
to remove it (Kleinstein and Seiden 2000). The immune ligands (Akira 2011). When different TLR ligands stimu-
system can respond either quickly or slowly. The fast late cells, distinct patterns of gene expression are induced,
responding innate immune system acts as the first line of demonstrating differences in signaling pathways among
defense in the body, while the adaptive immune system TLRs that occur through selective usage of adaptor mole-
acts slowly and provides long-lasting immune responses cules like MyD88 (myeloid differentiation primary
(Pashine et al. 2005). The complement system and response gene 88) and TRIF (toll/interleukin-1 receptor
phagocytic cells are involved in innate immunity. Adaptive domain containing adaptor protein inducing interferon-b).
immune responses are initiated through antigen-mediated MyD88 activates nuclear factor-jB (NF-jB) leading to
stimulation of T cells and B lymphocytes that have antigen- production of inflammatory cytokines, whereas TRIF
specific surface receptors. CD4? T-helper (Th) cells and activates the transcription factor interferon regulatory fac-
CD8? cytotoxic T lymphocytes (CTLs) are two different tor 3 (IRF3) leading to production of type-I interferons
types of T cells, and Th cells are further divided into dif- (IFN) (Akira 2011). A well-known TLR ligand is
ferent subpopulations with Th1 and Th2 cells being lipopolysaccharide (LPS), which can activate TLR4 (Akira
important (Schwendener 2014). Innate immune cells 2011). Similarly, poly-I:C (polyinosinic:polycytidylic acid)
express pattern-recognition receptors (PRRs), which help activates TLR3, flagellin is recognized by TLR5, imida-
to detect infectious agents. C-type lectin-like receptors zoquinolines are recognized by TLR7/8, and CpG
(CLRs), nucleotide oligomerization domain (NOD) like oligodeoxynucleotides are recognized by TLR9 (Apostó-
receptors (NLRs), toll like receptors (TLRs) and retinoic lico et al. 2016). Therefore, adjuvants that have structures
acid-inducible gene-1 (RIG-1) like receptors (RLRs) are similar to the various ligands of PRRs can activate their
different families of PRRs. TLRs and CLRs are present on respective receptors, resulting in stimulation of innate
the surface of antigen presenting cells (APCs), while NLRs immunity.
and RLRs are located intracellularly (Awate et al. 2013). On the other hand, adjuvants can induce adaptive
Pathogen-associated molecular patterns (PAMPs) are immune responses either by enhancing T cell responses, by
molecular patterns present in pathogenic microorganisms stimulating humoral immunity, or both. Immune-stimulat-
like fungi, viruses, bacteria and parasites (Haghparast et al. ing complexes (ISCOMs) act by inducing antibody pro-
2016). APCs can recognize PAMPs via PRRs before or duction and balanced Th1 and Th2 immune responses
during the endocytosis of an antigen. After recognition, (Lövgren Bengtsson et al. 2011), whereas monophosphoryl
antigens undergo processing in APCs, and antigen peptides lipid A (MPL) elicits a Th1 response (Apostólico et al.
are loaded onto major histocompatibility complex (MHC) 2016; Cox and Coulter 1997), and cholera toxin (CT)
molecules. Antigen peptides complexed with MHC class II stimulates a Th2 response (Lee et al. 2009). The Th1
molecules can activate Th cells, which can result in stim- response involves cell-mediated immunity, while Th2 cells
ulation of cellular immunity (CTL responses) and/or activate humoral responses to neutralize extracellular
humoral immunity (opsonic and/or neutralizing antibodies antigens. There are challenges to the development of novel
production by B cells). Antigen peptides complexed with adjuvants that can generate protective CD8? T cell
MHC class I molecules can directly stimulate CD8? cells responses. In order to enhance differentiation of functional
leading to cellular responses (Skwarczynski and Toth CD8? T cells, a promising adjuvant should be mixed with
2016). an antigen in such a way that facilitates entry of the antigen
In general, adjuvants activate the innate immune system into the MHC class I processing pathway, triggering den-
through PRRs present in immune cells. The majority of dritic cell (DC) activation and inducing type-I IFN pro-
immunostimulatory adjuvants act as ligands for PRRs and duction (Coffman et al. 2010). A study by Bungener et al.
lead to secretion of cytokines by directly enhancing an demonstrated that ovalbumin delivered by fusion-active
activation pathway (Coffman et al. 2010). Receptor-ligand virosomes induced a class I MHC-restricted CTL response.
interactions lead to expression of genes that encode the They also highlighted the importance of virosomes as an
cytokines, chemokines, and costimulatory molecules ideal antigen delivery system that can induce cellular
responsible for priming, expansion, and polarization of immunity against encapsulated protein antigens (Bungener
immune responses. As a result, components of damaged or et al. 2005).
dying host cells also contribute to the function of adjuvants
due to inflammasomes (Coffman et al. 2010). In particular,

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Modes of action of adjuvants which is apoptosis-associated speck-like protein containing

a caspase recruitment domain (CARD). Stimulation of the
Depot formation (Fig. 1), recruitment of immune cells, NLRP3 inflammasome results in caspase-1 activation and
activation of the inflammasome, enhancement of antigen this leads to cleavage of proforms of interleukin (IL)-1b,
presentation by MHC molecules, and immunomodulation IL-18, and IL-33 into their bioactive forms (Awate et al.
are different modes of action of adjuvants discussed in this 2013). Alum functions as an adjuvant by activating the
review and these are summarized in Fig. 2. NLRP3 inflammasome (Awate et al. 2013; Tritto et al.
2009).
Depot formation
Enhancement of antigen presentation by MHC
Adjuvants entrap, adsorb, or aggregate antigens and release molecules
them slowly over a long period of time (Bowersock and
Martin 1999). This depot effect at the site of injection also Adjuvants like alum or oil-based emulsions are believed to
prevents the loss of antigens by liver clearance (Cox and exhibit their adjuvanticity by targeting antigens to APCs,
Coulter 1997). Liposomes act by the depot effect (Gupta leading to enhancement of antigen presentation by MHC
and Siber 1995). molecules on APCs, and stimulation of adaptive immune
responses. However, still the role of adjuvants in the
Recruitment of immune cells enhancement of antigen presentation by MHC molecules
for the development of adaptive immune response has not
Adjuvants create localized pro-inflammatory immune been clearly evaluated (Awate et al. 2013).
responses at the site of injection leading to recruitment and
activation of immune cells. Local inflammation may also Immunomodulation
precipitate redness, swelling and local pain (Gerdts 2015).
MF59 acts by recruiting immune cells at the injection site Immunomodulatory adjuvants trigger the immune system
(Seubert et al. 2008). by modifying the cytokine network. Primarily, adjuvants
affect the type of immunity by increasing the concentra-
Activation of the inflammasome tions of some cytokines while decreasing concentrations of
others. Interferon gamma (IFN-c), IL-2 and IL-12 are
PRRs are recognized and/or are activated by adjuvants. linked with Th1 responses and cell-mediated immunity,
Inflammasome belongs to the NLR family. NOD like whereas IL-4, IL-5, IL-6, IL-13, and possibly IL-10 are
receptor protein 3 (NLRP3) is a highly studied inflamma- linked with Th2 responses and humoral immunity (Spickler
some receptor for adjuvanticity. The NLRP3 inflamma- and Roth 2003). MPL acts by stimulating Th1 responses
some is intra-cytoplasmic multiprotein complex that (Apostólico et al. 2016; Cox and Coulter 1997).
contains a NLRP3 receptor, procaspase-1, and an ASC,

Fig. 1 Depot formation

(redrawn and modified from
Smith et al. 2013). Adjuvants
entrap, adsorb or aggregate
antigens at the site of injection
and release them in a sustained
manner

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Fig. 2 Modes of action of adjuvants (redrawn and modified from InvivoGen 2011). Adjuvants form depot of antigens and recruit immune cells
at the site of injection. They can active PRRs of recruited APCs before or during endocytosis of antigens, after which antigens are processed and
presented to T cells resulting in cellular and/or humoral responses

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Vaccine adjuvants: smart components to boost the immune system

Types of adjuvants vaccine without adjuvant. In addition, inactivated

poliomyelitis vaccine was found to be more immunogenic
The modes of action of some common adjuvants are listed when adsorbed onto aluminum phosphate and the human
in Table 1. diploid cell strain rabies vaccine showed higher immuno-
genicity when adsorbed onto aluminum hydroxide (Gupta
Mineral salts et al. 1993). In a study conducted by Fazeli et al., using
aluminum phosphate as an adjuvant, a hepatitis B vaccine
Mineral salts like calcium phosphate and aluminum salts formulation exhibited higher adjuvanticity than a prepara-
such as aluminum phosphate and aluminum hydroxide, are tion with aluminum hydroxide (Fazeli et al. 2008). Thus,
commonly used as adjuvants in vaccines. Aluminum the potency of aluminum salts might depend on the type of
phosphate and aluminum hydroxide have different physical antigen used in the vaccine formulation.
and adjuvant properties, but both are imperfectly known as In the past, aluminum salts were believed to act by
alum. Chemically, alum is potassium aluminum sulfate, forming a depot at the site of injection and by enhancing
and it was initially used for purifying protein antigens like the recruitment of APCs and uptake by them. Recently, it
tetanus toxoid and diphtheria toxoid (Sivakumar et al. has been recommended that antigens adsorbed on the sur-
2011). Aluminum hydroxide-adsorbed cholera vaccine face of aluminum salts should be delivered in particulate
exhibited higher protection in comparison to cholera multivalent form, which are easily processed by APCs
(Leroux-Roels 2010). It also has been shown that

Table 1 Modes of action of some common adjuvants

Adjuvant Mode of action References

Aluminium Activation of innate immune responses Leroux-Roels (2010)

salts
Emulsion Innate inflammatory responses, activation and recruitment of APCs, enhancement of Leroux-Roels (2010)
(o/w) antigen persistence at site of injection and delivery to immune-competent cells
Emulsion Induction of local inflammation, activation and recruitment of APCs Leroux-Roels (2010)
(w/o)
Liposomes Depot formation, presentation of antigens to APCs Gupta and Siber (1995)
Virosomes Delivery of antigens to APCs Copland et al. (2005)
QS21 Presentation of antigens to APCs, induction of CTL production, stimulating both Th1 and Leroux-Roels (2010)
Th2 cytokine secretion
ISCOMs Induction of balanced Th1 and Th2 responses Lövgren Bengtsson et al. (2011)
?
PLGA Cross-presentation of antigens to CD8 T cells Apostólico et al. (2016)
Chitosan Translocation of tight junctions of cells Apostólico et al. (2016)
MPI Stimulation of both Th1 and Th2 responses Aguilar and Rodriguez (2007)
IFN-c Up-regulation of Th1 responses Cox and Coulter (1997)
IL-1 Maturation of T and B cells Cox and Coulter (1997)
IL-2 Up-regulation of Th1 responses Cox and Coulter (1997)
IL-4 Up-regulation of Th2 responses Cox and Coulter (1997)
IL-12 Induction of strong Th1 shift Cox and Coulter (1997)
GM-CSF Activation and recruitment of APCs Aguilar and Rodriguez (2007)
VLPs Direct activation of B cells, stimulation of DCs, induction of cross-presentation to CD8? T Apostólico et al. (2016)
cells
CT Stimulation of Th2 responses Lee et al. (2009)
CpG motifs Stimulation of Th1 responses Aguilar and Rodriguez (2007)
MPL Stimulation of Th1 responses Apostólico et al. (2016) and Cox
and Coulter (1997)
MDP- Stimulation of Th1 responses Cox and Coulter (1997)
lipophilic
MDP- Stimulation of Th2 responses Cox and Coulter (1997)
hydrophilic

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aluminum salts exhibit their adjuvanticity by activating the in experimental vaccines for rats, mice, dogs, and cats
NLRP3 inflammasome (Awate et al. 2013; Tritto et al. using different kinds of synthetic, recombinant, and natural
2009). The formation of granulomas, allergenicity, neuro- antigens (Petrovsky and Aguilar 2004).
toxicity and stimulation of IgE production are potential Several studies have showed synergistic actions of
side effects of aluminum salts (Petrovsky and Aguilar emulsions in combination with other compounds. A study
2004). Meanwhile, calcium phosphate stimulates IgG by Bayry et al. suggested that the combination of Mon-
production, but does not increase the production of IgE tanide ISA 206 and recombinant protein ISCOMs could be
antibodies. It has similar properties to aluminum salts and a good choice for achieving early protective titers and long-
is a well-tolerated compound in humans (Petrovsky and lasting immunity (Bayry et al. 1999). In another study done
Aguilar 2004). In France, calcium phosphate had been used by Song et al., ginseng stem-leaf saponins (GSLS) and
regularly in childhood and adult vaccines for many years. mineral oil adjuvant acted synergistically and promoted
However, later, the use of calcium phosphate in vaccines both Th1 and Th2 immune responses (Song et al. 2009).
was prohibited because vaccine formulations prepared with
calcium phosphate had problems in passing the test for Liposomes, virosomes and niosomes
potency of tetanus and diphtheria components in accor-
dance with the regulations of the World Health Organiza- Liposomes are spherical-shaped vesicles that can be
tion and European Pharmacopeia (Gupta 1998). formed from cholesterol and non-toxic natural phospho-
lipids. They may contain one or more lipid bilayers
Oil-based emulsions enclosing an aqueous unit. Lipid composition, size, surface
charge and preparation methods directly affect the prop-
An emulsion is dispersion of two immiscible phases e.g., erties of liposomes. Liposomes are biodegradable, bio-
water and oil. Surfactants are added to stabilize emulsion compatible and less toxic (Akbarzadeh et al. 2013). They
systems (Aucouturier et al. 2001). Oil-in-water, water-in- can induce both humoral and cell-mediated immune
oil, and multiple emulsions including water-in-oil-in-water responses. Their adjuvanticity results from the formation of
and oil-in-water-in-oil are different classes of emulsions a depot at the injection site and the presentation of antigens
(Shah et al. 2015). The adjuvanticity of oil-in-water (o/w) to APCs (Gupta and Siber 1995). Liposomes can easily
emulsions involve innate inflammatory responses and adsorb to mammalian cells and release antigens. In addi-
recruitment and activation of APCs. They also facilitate the tion, they can easily be phagocytozed by APCs and then
persistence of antigens at the site of injection and enhance can easily release entrapped antigens (Bowersock and
the delivery of antigens to immune-competent cells (Ler- Martin 1999). The surface charge of the vesicles, rigidity of
oux-Roels 2010). Meanwhile, water-in-oil (w/o) emulsions the bilayer, epitope density, and the association of antigen
are thought to act by inducing local inflammation and by with the liposome structure are different factors that
enhancing recruitment and activation of APCs (Leroux- influence the immunogenic responses of liposomes (Cop-
Roels 2010). Inflammatory reactions, granuloma forma- land et al. 2005). The immunoadjuvant action of liposomes
tion, and ulcers at the site of injection are some common can be enhanced by different methods including receptor-
side effects associated with emulsions (Aguilar and mediated targeting to macrophages, the use of co-adju-
Rodriguez 2007). vants, modification of structural characteristics of vesicles
Complete Freund’s Adjuvant (CFA) and Incomplete and the use of cytokines (Gregoriadis et al. 1996). In a
Freund’s Adjuvant (IFA) are w/o emulsions. CFA contains study conducted by Nakanishi et al., positively charged
heat-killed mycobacteria, which IFA lacks. CFA induces liposomes containing soluble antigens were more potent
long-lasting local inflammation, which can cause pain and inducer of antigen-specific cytotoxic T lymphocyte
lead to ulceration at the injection site in animals. Therefore, responses and delayed-type hypersensitivity responses
regulatory guidelines are followed while performing compared to neutral and negatively charged liposomes with
experiments with CFA in experimental animals (Apostó- the same concentrations of antigens (Nakanishi et al.
lico et al. 2016). MF59 is o/w emulsion, which is safe and 1999). The high cost, stability and pain at the site of
highly potent adjuvant allowing for lower concentrations of injection are drawbacks associated with the use of lipo-
antigens in vaccines (Schultze et al. 2008). AS03 is o/w somes as adjuvant (Sivakumar et al. 2011).
emulsion that contains squalene and a-tocopherol, which is Virosomes are reconstituted viral envelopes composed
the major bioavailable form of vitamin E. Vitamin E has of membrane lipids and viral glycoproteins. They lack
anti-oxidant and immunostimulatory activities. Thus, a- genetic material so they cannot replicate (Apostólico et al.
tocopherol must be included in AS03 to obtain high anti- 2016). Immunopotentiating reconstituted influenza viro-
body response (Morel et al. 2011). Another example of an somes (IRIV) are a widely used virosomal system. These
oil-based emulsion is Montanide, which is commonly used virosomes contain hemagglutinin (HA) and neuraminidase

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Vaccine adjuvants: smart components to boost the immune system

(NA) proteins, which are intercalated within the lipid hydrophobic regions by chemical linkage of palmitic acid
membrane (Apostólico et al. 2016). The structure of viro- tails are two different approaches to overcome the prob-
somes is similar to unilamellar liposomes (Copland et al. lems of incorporating hydrophilic protein antigens in
2005). Virosomes can deliver antigens into the cytoplasm ISCOMs (Smith et al. 1998). An ISCOM-matrix is similar
of APCs either by fusing with the endosomal membrane or to an ISCOM, but it does not contain antigen (Sjölander
by being endocytosed (Copland et al. 2005). In a study et al. 2001). ISCOMs are effective at low antigen con-
conducted on influenza virosomes, Cusi et al. showed that centrations and adjuvant doses, and lead to the induction of
immunization of respiratory syncytial virus-F protein with long-lasting humoral and cellular immune responses. In
influenza virosomes in combination with the mucosal comparison to free form of saponins, incorporation of
adjuvant Escheriagen (Escherichia coli heat-labile toxin) in saponins into ISCOMs results in decreased hemolytic
BALB/c mice resulted in a mucosal IgA response, high activity (Sjölander et al. 2001). ISCOMs act by inducing
serum IgG levels and a balanced Th1/Th2 cytokine profile the production of high concentrations of long-lasting anti-
(Cusi et al. 2002). bodies and by inducing a balanced Th1 and Th2 immune
Niosomes structurally resemble liposomes and have response with multifunctional T cells and CTLs (Lövgren
similar physical properties. They are formulated using non- Bengtsson et al. 2011). Rimmelzwaan et al. demonstrated
ionic surfactants, which are cheaper and chemically that cynomolgus macaque monkeys immunized with an
stable (Khan and Irchhaiya 2016). Niosomes also act as ISCOM-based vaccine produced high titers of specific
adjuvant. For example, if bovine serum albumin (BSA) is IgM, IgA and IgG serum antibodies as well as high titers of
incorporated within niosomes, it results in the formulation haemagglutination-inhibiting and virus-neutralizing serum
having an effectiveness equivalent to that achieved by antibodies in comparison to monkeys immunized with a
using CFA. Niosomes are safe when administered par- classical subunit vaccine (Rimmelzwaan et al. 1997).
enterally (Kersten and Crommelin 1995).
Polymeric particles
Saponins and immune-stimulating complexes
(ISCOMs) Synthetic polymers like poly(D,L-lactide-co-glycolide)
(PLG), poly(D,L-lactic-coglycolic acid) (PLGA), poly(g-
Saponins are extracted from the bark of a tree, Quillaja glutamic acid), poly(ethylene glycol) and polystyrene are
saponaria. The use of crude extracts of plant matter con- used in vaccines as adjuvant (Zhao et al. 2014). PLG and
taining saponins is associated with different adverse effects PLGA nanoparticles are biocompatible and biodegradable
when used in vaccine (Gupta and Siber 1995). The active (Zhao et al. 2014). PLGA has the ability to cross-present
adjuvant component, known as Quil A, can be partially antigens to CD8? T cells as it can easily reach MHC class I
purified from saponins using methods like dialysis, ion molecules (Apostólico et al. 2016). PLG have been used as
exchange and gel filtration chromatography. Quil A exhi- suture material in humans and animals for many years.
bits higher potency and reduced local reactions compared They also have been used for the controlled release of
to saponins (Gupta and Siber 1995). QS21 can be obtained drugs in drug delivery systems (Singh and O’Hagan 2003).
by purification of Quil A and acts by enhancing presenta- A study conducted by Eldridge et al. showed that the
tion of antigens to APCs and by inducing CTL production, delivery of staphylococcal enterotoxin B toxoid via
stimulating both Th1 and Th2 cytokine secretion (Leroux- poly(D,L-lactide-co-glycolide) microspheres with sizes
Roels 2010). Quil A has been previously used in veterinary from 1 to 10 lm in diameter induced an immune response
vaccines (Kersten and Crommelin 2003). The Quil A which was approximately 500 times that shown by
molecule has three distinct polar areas: hydrophilic-hy- nonencapsulated toxoid (Eldridge et al. 1991).
drophobic-hydrophilic. The hydrophilic areas are com- Polysaccharide-based natural polymers like chitosan,
posed up of carbohydrates, whereas quillaic acid forms the inulin, pullulan and alginate are also used as adjuvant
hydrophobic region (Kersten and Crommelin 2003). (Zhao et al. 2014). Chitosan-based nanoparticles are
Saponin mixture Quil A, cholesterol and phospholipids biodegradable, nontoxic, biocompatible, and their shapes
are components of ISCOMs. ISCOMs are spherical in and sizes can be easily modified as desired (Zhao et al.
shape and have a size about 40 nm (Kersten and Crom- 2014). Chitosan is a mucosal adjuvant. It acts in vitro by
melin 2003). Hydrophobic molecules like glycoproteins translocating the tight junctions of cells which enhances the
from the membranes of viruses and bacteria can be incor- transepithelial transport of antigens and reduces the
porated in ISCOMs during their formation, but it is difficult mucociliary clearance rate, stimulating phagocytosis of
to incorporate purified hydrophilic protein antigens (Smith antigens (Apostólico et al. 2016). Inulin is a carbohydrate
et al. 1998). Chemical modifications of proteins by acidi- obtained from the roots of plants that belong to the Com-
fication to expose hydrophobic groups and addition of positae family. Micro-particulate inulin (MPI) stimulates

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both Th1 and Th2 responses but does not induce IgE. alternatives to live, attenuated and inactivated vaccination
Additionally, it does not cause local or systemic toxicity. approaches (Trovato and De Berardinis 2015). Because of
Inulin gets metabolized into simple sugars, so it does not their polyvalent structure, they can accommodate multiple
have the safety issues that are generally associated with copies of antigens. They also can ensure tissues-specific
alum-based adjuvants (Aguilar and Rodriguez 2007). targeting, which depends on the origin of virus (Chro-
boczek et al. 2014). In a recent study, Fontana et al.
Cytokines demonstrated the ability of rabies virus-like particles (RV-
VLPs) to stimulate the production of rabies virus neutral-
Cytokines are proteins released in response to infection. izing antibodies and suggested that RV-VLPs are good
Signals provided by cytokines help to direct immune rabies vaccine candidate (Fontana et al. 2015).
response towards either cell-mediated or antibody-medi-
ated response in mammals. Thus, they play an important Bacterial derivatives
role to control the immune system (Asif et al. 2004). Dif-
ferent cytokines exert their adjuvant activity through dif- Bacterial toxins like CT and E. coli heat-labile enterotoxin
ferent pathways. IFN-c up-regulates Th1 responses and (LT) are good mucosal adjuvants in experimental animals
enhances MHC expression, IL-2 up-regulates Th1 respon- (Simmons et al. 1999). CT is a major enterotoxin produced
ses, IL-4 up-regulates Th2 responses, IL-1 stimulates by Vibrio cholera. It induces a strong Th2 response and
maturation of T and B cells, and IL-12 induces a strong suppresses Th1 responses (Lee et al. 2009). Adjuvant
Th1 shift (Cox and Coulter 1997). Granulocyte–macro- properties of CT and LT include the capacity to promote
phage colony stimulating factor (GM-CSF) activates and antigen uptake through greater gut permeability, antigen
recruits APCs leading to enhancement of primary immune presentation by APCs, and induction of cytokines that lead
response. The requirement for multiple doses and its tox- to immunoglobulin class switching. LT is considered to be
icity are limitations for practical application of GM-CSF as safer adjuvant for mucosal vaccination as it is 10-fold less
an adjuvant (Aguilar and Rodriguez 2007). If a large toxic than CT in animals (Katz et al. 1997). To reduce the
amount of cytokines enters systemic circulation, adverse enterotoxicity of native LT, double mutant LT (dmLT) has
effects including severe shock or even death, may occur. been developed (Leach et al. 2012) and is a powerful and
Cytokines also stimulate autoimmunity. For example, safe detoxified enterotoxin that can act as mucosal adjuvant
overdose of IL-2 is associated with autoimmune diseases. for coadministered antigens (Norton et al. 2011). In dif-
Use of cytokine inducers like avridine, gliding bacterial ferent mouse models, dmLT increased immune responses
adjuvant (GBA) and MPL are an alternative approaches to to whole-cell vaccines for enterotoxigenic E. coli, Heli-
reduce the toxicity associated with cytokines (Spickler and cobacter pylori, and Streptococcus pneumoniae (Leach
Roth 2003). et al. 2012). Ottsjö et al. suggested that dmLT, as an
adjuvant, could be included in the mucosal vaccine for H.
Virus-like particles (VLPs) pylori infection (Ottsjö et al. 2013). In another study con-
ducted by Norton et al., incorporation of dmLT in inacti-
VLPs are viral proteins that spontaneously form particles vated poliovirus vaccine (IPV) immunizations increased
resembling virions. They do not contain viral genes, but mucosal immunity and the longevity of anti-poliovirus
retain the authentic conformation of the virus capsid pro- responses (Norton et al. 2015).
teins as seen in attenuated and inactivated viral vaccines Bacterial DNA-based molecules also exhibit immunos-
(Young et al. 2006). Since they mimic the structure of the timulatory properties. CpG motifs are six base-long DNA
virus, lower doses of antigen, compared to subunit vacci- sequences, which contain a central CpG dinucleotide. They
nes, are enough to stimulate a similar protective response occur twenty times more within bacterial DNA than within
(Noad and Roy 2003). Depending on the parental virus, the mammalian DNA. In mammalian cells, they are recognized
structures of VLPs can be either enveloped or non-en- by TLR9 which leads to secretion of type I IFNs (a and b)
veloped. Enveloped VLPs contain a host cell membrane and IL-12 by cells of the innate immune system, stimu-
(an envelope) and the antigen of interest, whereas non- lating a Th1 response (Aguilar and Rodriguez 2007).
enveloped VLPs contain only pathogen components with LPS is also a good adjuvant. Chemically, it has three
self-assembling capacity (Apostólico et al. 2016). VLPs principal regions. Among them, the Lipid A region func-
can directly activate B cells. Additionally, they can stim- tions as both as a mitogen and adjuvant (Skidmore et al.
ulate and be taken up by dendritic cells (DCs) resulting in a 1975). MPL is a detoxified derivative of LPS obtained from
T cell response. Furthermore, they can induce cross-pre- Gram-negative bacteria (Salmonella minnesota R595)
sentation to CD8? T cells (Apostólico et al. 2016). VLPs (Apostólico et al. 2016). MPL induces IL-12 and IFN-c
are unable to replicate so they are considered safe. They are

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production (Apostólico et al. 2016) and stimulates Th1 Obstacles in adjuvant development
responses (Apostólico et al. 2016; Cox and Coulter 1997).
GBA is a polymer of amino sugars, which is obtained Adjuvanted vaccines show higher reactogenicity at the
from the bacterial genus Cytophaga. It is a safe and injection site in comparison to non-adjuvanted vaccines
effective adjuvant that can stimulate the release of (Leroux-Roels 2010). Thus, adjuvants not only increase the
cytokines in cats, mice and humans (Spickler and Roth immunogenic responses of antigens, but also can lead to
2003). adverse reactions. Local inflammation at the injection site,
Muramyl dipeptide (MDP) is an active component of and granuloma and sterile abscess formation are local
peptidoglycan obtained from mycobacteria (Spickler and adverse reactions associated with adjuvants. Systemic
Roth 2003). MDP and its different derivatives can induce reactions like malaise, fever, adjuvant arthritis and anterior
IL-1 production. Lipophilic derivatives of MDP stimulate uveitis are also seen in laboratory animals (Vogel 2000).
Th1 responses, whereas hydrophilic derivatives of MDP Although many adjuvant candidates are available, very
stimulate Th2 responses (Cox and Coulter 1997). few of them are licensed and used as successful adjuvants.
Safety is the major reason for failure of adjuvants. Gen-
erally, short-term safety issues impede the development of
Current uses of licensed adjuvants adjuvants. Intermediate and long-term safety issues are
more difficult to overcome (O’Hagan and De Gregorio
Alum is widely used and it has been approved in the United 2009). Thus, the field of adjuvant development should not
States (Mbow et al. 2010). Alum-based vaccines were only identify more inflammatory adjuvants, but also should
licensed more than 70 years ago (O’Hagan 2007). In 1997, focus on new methods to separate the potency of adjuvants
an influenza vaccine containing an alternative adjuvant from their reactogenicity (Petrovsky 2013).
known as MF59 was successfully launched in the European Moreover, because of the complex nature of the immune
market (O’Hagan 2007). In addition, AS04, a combination system, there is no in vitro assay that can completely
adjuvant containing MPL adsorbed to alum, has been simulate in vivo immune responses. Thus, most preclinical
approved in Europe and licensed in the United States studies are done in animal models. Such studies can pro-
(Mbow et al. 2010). Additionally, Montanide ISA51 is vide some information regarding the potency and safety of
used as an adjuvant in a non-small-cell lung cancer vaccines, but may not guarantee that similar effects will be
(NSCLC) vaccine, which has been registered in Cuba and observed in humans or veterinary target animals (Schijns
Chile (Garçon et al. 2011). Examples of licensed adjuvants and Lavelle 2011).
with their applications are summarized in Table 2.

Table 2 Licensed adjuvants

Adjuvant Vaccine References

Alum Hepatitis A, Hepatitis B, diphtheria/tetanus/pertussis (DTP), human papillomavirus, Apostólico et al.

Haemophilus influenza type B, Pneumococcus (2016)
Virosomes Influenza, Hepatitis A Leroux-Roels
(2010)
MF59 Influenza (H1N1, H5N1, seasonal) Garçon et al.
(2011)
AS04 Hepatitis B, human papillomavirus Apostólico et al.
(2016)
AS03 Influenza (H5N1, H1N1) Garçon et al.
(2011)
Thermo-reversible oil-in- Influenza (H1N1) Garçon et al.
water emulsion (2011)
Montanide ISA51a Cancer Garçon et al.
(2011)
a
Montanide ISA51 is used in cancer vaccine for immunotherapy

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 R. Bastola et al.

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