World Journal of Vaccines, 2011, 1, 33-78
doi:10.4236/wjv.2011.12007 Published Online May 2011 (http://www.SciRP.org/journal/wjv)
1
Selection of Adjuvants for Enhanced Vaccine
Potency
Wei Wang1, Manmohan Singh2
1BioTherapeutics Pharmaceutical Sciences, Pfizer Inc, Chesterfield, USA; 2Novartis Vaccines and Diagnostics, Cambridge, USA.
Received February 16th, 2011; revised March 17th, 2011; accepted March 25th, 2011.
ABSTRACT
The advent o f mass vaccination has saved millio ns of human lives and revolu tionized the quality of life. Va ccination is
currently one of the most cost effective ways of managing healthcare costs in both emerging and developed countries.
Despite the long vaccine history and success, design and development of efficacious and safe vaccines has been tradi-
tionally semi-empirical. This is mainly due to our limited understanding of vaccination mechanism and its influencing
factors. The most important factor is arguably the type and concentration of vaccine adjuvants. Until recently, however,
only one type of adjuvant-aluminum salts, had been widely u sed within licen sed human va ccines in the US, even though
a variety of novel adjuvants have been evaluated in the past few decades. This review summarizes the key adjuvants that
have been evaluated in recent years with an intention to facilitate more efficient development of vaccine products to
combat human diseases.
Keywords: Adjuvant, Alum, Aluminum Salts, Immune Response
1. Introduction
The advent of mass vaccination significantly reduced the
morbidity or mortality of newborns and adults alike from
various infectious diseases, which are otherwise un-
avoidable as a vast majority of the global population
concentrate in cities with close contacts with one another.
It is estimated that universal influenza vaccination alone
saves 250,000 - 500,000 annual deaths worldwide [1].
The gradual decrease both bvin morbidity and mortality
has increased the life expectancy and quality of life as
well. With the global concerns for the ever-increasing
healthcare cost, vaccination remains one of the most cost
effective ways of managing healthcare costs in both
emerging and developed countries.
Human vaccines have now been used for over two
centuries since the first vaccination trial for cow-pox by
Edward Jenner. They have been proven to be very effec-
tive in preventing or controlling the occurrence and
spreading of numerous deadly diseases through im-
provement of the host’s innate and adaptive immune
systems. Despite the long vaccine history and success,
design and development of efficacious and safe vaccines
has been traditionally semi-empirical, even though re-
cently novel methods are being developed such as re-
verse vaccinology [2]. Various strategies have been in-
vestigated for the improvement of the vaccine efficacy.
Among these, use of a vaccine adjuvant has been a top
choice with many successes. However, until recent ap-
proval of AS04 adjuvant in a product licensed by GSK,
only one type of adjuvant-aluminum salts, had been
widely used within licensed human vaccines in the US,
even though a variety of novel adjuvants have been eva-
luated in the past few decades. Therefore, development
and identification of effective and safe vaccine adjuvants
is urgent and remains to be an area of extensive investi-
gation [3-5].
This review summarizes the key adjuvants that have
been evaluated in recent years that affect the vaccine
immunogenicity/efficacy with an intention to facilitate
more efficient development of vaccine products to com-
bat human diseases.
2. Major Players in an Immune Response
Many types of cells are involved in an immune response,
including dendritic cells, macrophages, mast cells, eosi-
nophils, neutrophils, B and T lymphocytes. A key player
in the immune response is the dendritic cells (DCs), the

Selection of Adjuvants for Enhanced Vaccine Potency
34
most effective antigen presenting cells (APCs) [6-8].
Activated DCs drain into the local lymph node, where
they present the antigen and direct the differentiation of
T helper (Th) cells into different effector cells, leading to
recognition of B and T cell epitopes.
A few types of receptors play important role in this
process. Pattern-recognition receptors (PRRs) recognize
conserved microbial structures-pathogen-associated mo-
lecular patterns (PAMPs) [9]. A major category of such
receptors are toll-like receptors (TLR1-9) for pathogen
recognition, DC activation, and induction of immune
response. They are widely expressed in cells of the im-
mune system, epithelial cells, endothelium, cardiomyo-
cytes and adipocytes for recognition of pathogens [9] and
could be utilized potentially for targeting activation
pathways by the same or different adjuvants [5,10]. Sti-
mulation of TLRs may lead to several events - stimulated
synthesis of anti-microbial substances and pro- inflam-
matory cytokines (TNF-, IL-1, IL-6, IL-8, IL-12, etc);
activation of dendritic cell maturation (increased expres-
sion of co-stimulatory molecules such as CD40, CD80,
CD86; and MHC antigens); and more effective antigen
presentation.
Antigen peptides are presented by DCs as a peptide/
MHC Class II protein complex, which binds to the TcRs
on T helper cells for activation of T helper cells - activa-
tion of the major biochemical pathways in the cytosol of
the T helper cells (signal 1). Interaction between CD28
on T helper cells and CD80 or CD86 on DCs activates
the second independent biochemical pathway (signal 2).
Activation of the two pathways within T helper cells
leads to the self proliferation by releasing IL-2 (T cell
growth factor). After many generations, the T helper
cells differentiate into effector T helper cells, memory T
helper cells and suppressor T helper cells.
The T helper cells differentiate into two major sub-
types: Th1 and Th2 cells. DCs are critical in controlling
the direction of T helper cells’ differentiation through
secretion of certain cytokines [11]. Secretion of IFN-,
IL-2, and IL12 promote Th1 differentiation, leading to
secretion of IFN-, IL-2, and IL12, proliferation of cyto-
toxic CD8 + T cells, macrophage activation, and produc-
tion of TNF-, TNF-β, and IgG2a. Secretion of IL-1β and
IL-18 promote Th2 differentiation, leading to secretion
of IL-4, IL-5, IL-6, IL-10, IL-13, and stimulation of
B-cell proliferation, and subsequent Ab production, with
a typical initial phase of IgM production, followed by
more specific IgG conversion with a few days to weeks
[12]. Enhancement of antigen-specific antibody produc-
tion is a critical post-vaccination event, as the natural
antibodies of the innate immune system is often ineffec-
tive due to their low-affinity and non-anamnesia in an
immune response [13]. Secretion of host IFN- is not
required to initiate a Th1 immune response [14].
Vaccine-induced immune responses can be generally
divided into these two different biased effects: Th1 vs
Th2 types. Many factors can contribute to the type of
specific immune response of a vaccine, including the
type and reletive amounts of antigens and adjuvants.
Even the intensity of an immune response can make a
difference. A weak immune response is generally Th2-
biased and therefore, increasing the intensity of response
may lead to a Th1 response [15]. The Th1 response to an
intramuscular flu vaccine (Fluarix) increased when the
dose was increased from 1/10th of a full dose to a full
dose, with a relatively similar Th2 response [16].
It is generally believed that activation of Th1 and Th2
effector cells are effective against intracellular and ex-
tracellular pathogens, respectively6. However, the actual
protection offered by these two pathways is still not so
clear-cut. For example, it is expected that Th2 immune
responses are required for protection against extracellular
bacteria, such as H. pylori but only a Th1-promoting
vaccine (H. pylori sonicate + CpG) showed protection in
mice [17]. Similar results were obtained where a strong
Th2-inducing protein vaccine was not as effective in the
protection of mice from virus challenge [18].
3. Most Commonly Used Adjuvant
Aluminum Salts
Aluminum salts have been used extensively both in vet-
erinary and human vaccines [19,20]. These salts include
aluminum hydroxide (more accurately as aluminum
oxyhydroxide AlO(OH)), aluminum phosphate (more
accurately as aluminum hydroxyphosphate, Al(OH)x
(PO4) y), and alum (KAl(SO4)2). Alum has been used
often exchangeably with the first two aluminum salts in
the literature [21]. The major characteristics of the two
major salt types are listed Table 1.
The regulatory limit for aluminum in biological prod-
ucts (including vaccines) is 0.85 mg/dose in the US and
0.125 mg/dose in Europe. This limit is high enough to
accommodate the needs of most vaccines. Nevertheless,
a minimal amount of such salts is recommended for use
as vaccine adjuvants to minimize possible side effect. In
addition, it is still being investigated whether the cause of
Gulf War Syndrome has anything to do with the subcu-
taneous administration of extra dose of aluminum hy-
droxide [22].
3.1. Adjuvantation Mechanisms
Aluminum salts can enhance the immunogenicity of
many types of vaccine antigens, including proteins [23],
virus [24], etc. Their mechanism of action for the stimu-
lation of the immune system remains a subject of con-

Selection of Adjuvants for Enhanced Vaccine Potency
35
tinuous investigation [20,21,25]. Several mechanisms
have been demonstrated, including recruitment of den-
dritic cells, retention of antigens, more efficient uptake
and presentation of antigens by dendritic cells, more ef-
fective activation of antigen-specific T cells, complement
activation and induction of necrosis at the injection site
[26-31]. More recently, several studies showed simulta-
neously that aluminum stimulates immune response to an
antigen through the nucleotide-binding oligomerization
domain- like (NOD-like) receptors (NLRs; cytosolic),
specifically NALP3 (Nacht Domain-, Leucine-Rich Re-
peat and PYD -Containing Protein 3). Aluminum adju-
vants activate an intracellular innate immune response
system, called the NALP3 inflammasome (also known as
cryopyrin, CIAS1 or NLRP3) [32] and via NALP3, in-
duce secretion of mature IL-1β, IL-18, and IL-33 in hu-
man and mouse macrophages [33]. Other particulate ad-
juvants, such as QuilA and chitosan, induce similar in-
flammasome activation, suggesting that activation of the
NALP3-inflammasome may be a common mechanism of
action for particulate adjuvants [33]. Therefore, any ac-
tivators of NLRP3 may be potentially effective adju-
vants.
Aluminum salts generally induce a Th2-type immune
response [29,34]. Th2 differentiation in mouse dendritic
cells (DCs) was apparently directed by the specific secre-
tion of IL-1β and IL-18 and lack of IL-12 secretion
(IL-12 directs differentiation of CD4 T cells to Th1 cells)
[29]. The Th2-response of alum is not purely a particu-
late-related consequence, as incubation of conjugated
ovulbumin to polystyrene beads (48 nm) induced sub-
stantial Th1 responses with moderate Th2 responses in
sheeps [35]. Generally, the Th2 effect is dependent on
the amount and type of aluminum salts used in the for-
mulation [29]. Differential effects have been seen be-
tween 500 and 158 µg of aluminum (Alhydrogel) with 25
µg of Bacillus anthracis recombinant protective antigen
(rPA) in inducing neutralizing antibodies [36].
On the other hand, aluminum salts may not always
enhance immunogenicity of an antigen. Aluminum hy-
droxide at 0.6 mg in 0.5 mL did not show any significant
effect in inducing serum anti-HA antibody formation
after immunization of healthy elderly adults with 7.5 ug,
15 µg, or 45 µg HA by IM injection [37]. Aluminum
hydroxide could even suppress the immune response of
vaccines in some cases [26,38].
3.2. Antigen Adsorption to Aluminum Salts
Aluminum salts have large surface areas for antigen adsorp-
tion (Table 1). Two major surface adsorption mechanisms
of antigens are known-electrostatic attraction and ligand
exchange [39,40]. Both can take place at the same time, but
when the antigen and aluminum salts carry the same type of
charge (repulsive to each other), ligand exchange plays a
key role [41,42]. Other mechanism of adsorption was also
proposed, including hydrophobic interactions [41], and oth-
er non-charge-associated surface interactions such as mo-
nosaccharides on the surface of aluminum oxide at PZC (pH
9.0) [43]. All of these mechanisms of adsorption may
co-exist for a single antigen [28].
Protein adsorption to aluminum salts is generally very
fast. The adsorption of several antigens - -casein, bo-
vine serum albumin (BSA), myoglobin and recombinant
protective antigen (rPA), to aluminum hydroxide took a
minute [44]. So is the adsorption of three recombinant
botulinum neurotoxin antigens to aluminum hydroxide39.
It may take a little longer if ligand exchange and/or
phosphorylation of aluminum hydroxide (in a phosphate
buffer) takes place [45]. Therefore, it is recommended
that the surface OH/PO4 ratio be determined in early
vaccine development stages [46].
Two parameters are often used to describe antigen ad-
sorption-dsorption capacity (or maximum adsorption)
and adsorption coefficient (the binding strength). The
adsorption capacity seems to be limited to a monolayer
coverage. This is certainly the case for ligand exchange-
ominant adsorption of a monoclonal antibody on alumi-
num hydroxide [40] and hepatitis B surface antigen
(HBsAg) on aluminum hydroxide [47]. For hepatitis B
surface antigen (HBsAg), the monolayer coverage is at
1.7 mg/mg Al [47].
Table 1. Major characteristics of aluminum salts.
Salts PZC Size Solubility Other propertiesPK properties References
Aluminum
hydroxide
AlO(OH)
11.4
Primary particles, fibers,
4.5 × 2.2 × 10 nm
Aggregates: 1 - 20 µm
in diameter
Well-shaped with pH
< 1 µg/mL at pH 5-9;
crystalline
Surface area of
primary parti-
cles: about 500
m2/g
17% absorption in 28 days
following IM injection in
rabbits; mainly distributed
in kidney and spleen
26
55
461
Aluminum
phosphate
Al(OH)x(PO4) y
9.6-4.0; 5.5
(prepared at
pH 3); 4.2
(prepared at
pH 7.5)
Primary particles, plates
around 50 nm;
Bell-shipped with pH;
Octahedrally/ tetrahe-
drally coordinated
aluminum
Well-shaped with pH
< 5 µg/mL at pH 5 -
6.5; amorphous
Density 2.05
g/mL at pH 3 to
2.15 g/mL at pH
7
70% absorption in 28 days
following IM injection in
rabbits; mainly distributed
in kidney and spleen
26;
54,462
55;
463
461

Selection of Adjuvants for Enhanced Vaccine Potency
36
Similar levels of protein antigen adsorption were also
reported for several other proteins. The adsorptive capac-
ity of lysozyme, ovalbumin and bovine serum albumin at
pH 7.4 is 1.4 (Adju-Phos), 1.6 (Alhydrogel), and 2.2
(Alhydrogel) mg/mg Al, respectively [48]. On the other
hand, the adsorptive capacity of proteins can vary sig-
nificantly (> 10 times), depending on the source, prepa-
ration method, and age of the aluminum salts [44,45,
49,50]. If the protein and aluminum salts carry the same
charge, a significantly lower amount of antigen adsorp-
tion is expected. The maximum adsorption of endotoxin
(pI about 2) at pH 7.4 and 25 C is only 3 µg/mg Al on
aluminum phosphate [49]. The amount adsorbed under
such conditions would heavily depend on the number of
phosphorylation sites on the antigen [28,51]. Another
parameter similar to adsorptive capacity could also be
used and has been shown to be better for characterizing
time-induced changes in physical properties of these salts
[52].
The binding strength of antigen can be evaluated based
on its resistance to desorption in an elution buffer or in
an interstitial fluid [47]. It often correlates with the ad-
sorption capacities [45,49] and can be significantly dif-
ferent depending on study conditions [53]. Generally,
ligand exchange-based adsorption offers more binding
strength than that from electrostatic attraction. The ad-
sorption of hepatitis B surface antigen (HBsAg) was
strongly adsorbed on aluminum hydroxide with an ad-
sorptive coefficient of 6.0 ml/µg due to the presence of
phospholipids for ligand exchange [47]. The elution of
ovalbumin from aluminum hydroxide adjuvant upon ex-
posure to interstitial fluid was inversely related to the
degree of phosphorylation of the ovalbumin [28].
Many factors can influence the rate of antigen adsorp-
tion and adsorption capacity on aluminum salts. The
most critical one is arguably the solution pH, as this will
determine charged states of antigen and aluminum salts.
The influence of pH on antigen adsorption can be com-
plex because of the presence of multiple adsorption me-
chanisms, and differences in pH-dependent stability of
antigens, and solubility of both antigens and aluminum
salts [40]. Parabolic pH relationships were reported on
lysozyme adsorption on aluminum phosphate (minimum
at pH 4) [54] and ovalbumin (pI = 4.7) adsorption on
aluminum hydroxide (maximum at pH 4.3-6.2) [55]. In
rare cases, maximum adsorption can be found in a pH
range, where antigens and aluminum salts carry the same
charges [40].
Other solution factors can also play a significant role.
Presence of phosphate ions in a solution can significantly
reduce both the adsorption capacity and coefficient of
antigens on aluminum hydroxide due to a variable degree
of phosphorylation [42,45]. The adsorption capacity of a
monoclonal antibody at pH 7.4 decreased from 1.5 mg/
mg Al in water to 0.14 mg/mg Al in the presence of 100
mM phosphate [40]. The ionic strength of the solution
may change the adsorption capacity of an antigen based
on electrostatic interactions [40] but should not have a
significant effect on ligand exchange-based antigen ad-
sorption [47]. Presence of other proteins, excipients,
and/or multivalent anions (such as -hydroxycarboxylic
acid, citric acid, lactic acid and malic acid) would also
influence antigen adsorption. The adsorption capacity of
a monoclonal antibody at pH 7.4 decreased from 1.5
mg/mg Al in water to 1.1, 0.88, and 0.83 mg/mg Al, re-
spectively at 5 C, room temperature, and 37C in simu-
lated interstitial fluid (25 mg/mL BSA; 2.7 mq/L citrate,
5 mM phosphate, 154 mM·NaCl) [40]. Several excipients
have been shown to have negative effect on the antigen
adsorption, including EDTA on the adsorptive capacity
of recombinant protective antigen (pI of 5.6) on alumi-
num hydroxide [45], trehalose or combination of treha-
lose and Tween 20 on the total adsorption capacity of
trivalent protein antigens on Alhydrogel [51], sucrose on
the adsorption capacity of all three recombinant botuli-
num neurotoxin antigens [39]. The negative effect of
neutral molecules could be due to their stabilizing effect
on antigens, preventing antigens from effective hydro-
phobic interactions with the aluminum surface.
3.3. Degree and Strength of Adsorption vs
Immunogenicity
It has been a general belief that antigens need to be ad-
sorbed on aluminum salts for optimal immunogenicity
effect. However, the exact relationship between the de-
gree of antigen adsorption and in-vivo immunogenicity
has not been consistent. Subcutaneous administration of
lysozyme/aluminum hydroxide mixtures with different
degrees of adsorption of 3, 35 or 85% led to generation
of the same level of anti-lysozyme antibody titers in rab-
bits [23]. One study shows that administration of non-
adsorbed protein antigens (dephosphorylated -casein,
dephosphorylated ovalbumin, or lysozyme) with alumi-
num phosphate induced similar levels of antibody titers
to that for antigens adsorbed on aluminum phosphate in
mice [56]. Using recombinant N terminus of Als3p
(rAls3p-N) as a model antigen, Lin et al. [57] found that
more rAls3p-N was bound on aluminum hydroxide in
saline than in phosphate-buffered saline (PBS) but the
immunogenicity and efficacy were superior with antigens
in PBS.
Indeed, several studies showed that a tight binding
between antigens and aluminum salts would inhibit im-
munogenicity. Using -casein and dephosphorylated -
casein as model antigens and non-treated/phosphate-
treated aluminum hydroxide as adjuvants, it was shown

Selection of Adjuvants for Enhanced Vaccine Potency
37
that the geometric mean antibody titer in mice was in-
versely related to the adsorptive coefficient of antigens
[58]. Similar results were obtained with HBsAg as a
model antigen with aluminum hydroxide [53] or with
aluminum hydroxyphosphate sulfate in mice [59]. These
results clearly suggest that the immune response could
negatively correlate with the degree of adsorption of
proteins on aluminum adjuvants [40].
Would binding strength of an antigen in interstitial
fluid predict immunogenicity better, as this fluid mimics
the in-vivo tissue environment? Again, results are not
completely consistent, either. While the degree of ly-
sozyme adsorption on aluminum hydroxide in sheep in-
terstitial fluid correlated with the immune response (for-
mation of anti-lysozyme antibodies) after subcutaneous
administration [23], tight binding of antigens (non-re-
lease in interstitial fluid) has generated conflicting re-
sults-negative for endotoxin vaccine with aluminum hy-
droxide in rats [49] but positive for -casein with alumi-
num hydroxide in mice [27]. In certain cases, enhance-
ment of immunogenicity is achieved even when the anti-
gens are not adsorbed on aluminum salts [56]. Therefore,
the binding strength of antigen in interstitial fluid does
not predict immunogenicity well.
3.4. Storage and Process Stability of Aluminum
Salts
The storage stability of aluminum salts has not been re-
ported extensively, partly because of its long history of
use, and lack of accurate methods for monitoring its sta-
bility, such as particle size. These salts appear to be sta-
ble under normal storage conditions, but subtle changes
may occur. Aging of aluminum phosphate prepared un-
der uncontrolled pH conditions resulted in a drop in pH
(as much as 0.9 unit at P/Al ratio of 0.25) in 3 months at
room temperature [54].
Thermal treatment of aluminum hydroxide at 80C for
24 hours did not affect the adsorption capacity of oval-
bumin (pI 4.7) [50]. Higher temperature, however, does
cause certain changes. Amorphous aluminum phosphate
underwent deprotonation and dehydration when auto-
claved for 30 or 60 min at 121C, reducing the lysozyme
adsorption capacity, rate of acid neutralization at pH 2.5,
and the point of zero charge [60]. Autoclaving aluminum
hydroxide adjuvant increased the degree of crystallinity
in addition to deproto nation/dehydration, reducing the
protein adsorption capacity and viscosity [50,60].
The aluminum salts do not tolerate the freeze-thaw
process well, as freezing causes irreversible coagulation
[26]. Freeze-thawing (to -40C) of 0.2% Alhydrogel at pH
4.0 caused particle aggregation, which is inversely re-
lated to the cooling or thawing rate [61]. Freeze-thawing
of aluminum hydroxycarbonate gel caused coagulation,
leading to formation of visible aggregates without
changing the point of zero charge [62]. Single or re-
peated freezing of alum-containing hepatitis B vaccine
(to -10 C or lower) resulted in aggregation of the adju-
vant-antigen particles, which exacerbated with duration
of freezing, lower temperature, and the number of freez-
ing cycles [63]. The rate of freezing was inversely related
to the aggregate size (aluminum hydroxycarbonate) [62].
Certain processes could reverse freezing-induced ag-
gregation of aluminum salts, such as ultrasonic treatment
or homogenization [62]. Use of proper formulation ex-
cipients could potentially inhibit the freezing-induced
particle aggregation. The freezing-induced particle ag-
gregation in a hepatitis B vaccine could be prevented by
including PEG 300, propylene glycol, or glycerol [64].
At least 10% propylene glycol appeared to be needed for
complete protection from freezing-induced particle ag-
gregation [63]. The protective effect seems attributable to
their general lyoprotective effect rather than reduction in
freezing temperature. Other excipients are also found to
be effective in inhibiting freezing-reduced aggregation,
such as adsorbable polymers or surface-active agents
[62], and trehalose [61].
The additional drying step after freezing also leads to
aggregation of aluminum salts. It has been shown that
freeze-drying of 0.2% Alhydrogel caused particle aggre-
gation with bigger median diameter than that caused by
the freeze-thawing process alone [61]. Lyophilization of
a model antigen, bovine intestinal alkaline phosphatase
adsorbed on aluminum hydroxide, induced adjuvant ag-
gregation and reduced the antigen’s enzymatic activity
(up to 50% drop in activity) [65]. Such a process could
potentially reduce the in-vivo immunogenicity [66]. In
contrast, spray drying does not seem to cause particle
aggregation of aluminum hydroxide in the absence of
polymers [67].
The effect of process-induced aggregation of alumi-
num salts on immunogenicity may depend on the degree
of aggregation and type/level of antigen. While lyophili-
zation-induced particle aggregation (2-16 µ mean particle
diameter) did not translate into any change in immuno-
genicity of alkaline phosphatase in terms of anti-antigen
titers (IgG1) in mice [65], freezing-induced aggregation
of aluminum salt reduced significantly the immunogenic-
ity of hepatitis B vaccine in mice [63,64].
3.5. Stability of Antigen with Aluminum Salts
The stability of antigens adsorbed on aluminum salts has
not been studied extensively, partly due to the interfer-
ence of aluminum salts with different assays. Certainly,
the tertiary structures of antigens can change to a variable
degree upon adsorption to aluminum salts. The Tm’s of
three protein antigens, lysozyme, ovalbumin and bovine

Selection of Adjuvants for Enhanced Vaccine Potency
38
serum albumin, were all reduced Tm’s upon adsorption
onto aluminum salts [48]. Of course, change in antigen
structure or aggregation state may or may not negatively
impact the immunogenicity. In fact, the perturbed tertiary
structure could possibly facilitate the presentation of an-
tigens and thus contribute to the adjuvant activity of the
aluminum salts [48].
An option to bypass the interference of aluminum salts
is to analyze antigens after dissociation and separation
from the salts. Dissociation could be achieved through
pH adjustment with or without certain amount of cha-
otropic agents [68]. However, the dissociation process
may reverse or further alter the structural changes caused
by adsorption. The dissociated BSA and multiple anti-
gens (for Group A Streptococcus; GrAS) from aluminum
hydroxide were found to be structurally and functionally
similar to the untreated antigen controls by several me-
thods [68]. In such cases, the adsorption-induced struc-
tural changes in antigens can only be studied in the ad-
sorbed state.
Aluminum salts could potentially reduce the antigen
stability during storage. It was found that BSA formu-
lated with aluminum hydroxide showed more dimer for-
mation (31%) relative to the control (9% dimer) upon
storage [68]. The oxidation and deamidation of alumi-
num-adjuvanted rBoNTE(Hc) was faster than that with-
out adjuvant both at 4 and 30C, and could not be im-
proved by addition of 7.5% sucrose or combination of
sucrose and 0.01% Tween 20 [51]. One possible cause
for the protein instability is the different microenviron-
ment pH on the aluminum particle surface. It has been
shown that the surface of aluminum hydroxide is about 2
pH units higher than the bulk pH due to accumulation of
hydroxyls on the particle surface [69]. Therefore, for a
pH-sensitive antigen, the bulk pH needs to be adjusted
lower than its optimal value to maximize its stability on
the adjuvant surface. Addition of formulation excipients
could be effective, such as 20% propylene glycol in an
aluminum hydroxide-adjuvanted hepatitis B vaccine
(HBsAg) [70].
4. 2nd-Generation Adjuvants
The aluminum salts clearly have a limited and in some
cases, no adjuvantation effect clinically [71]. The limita-
tion prompted extensive search and development of non-
aluminum (2nd-generation) adjuvants in the past 20 years
[7,72,73]. Novel adjuvants are highly desired for more
potency, more balanced immune response and less side
effect/reactogenicity [72,73]. Significant progress has
been made as exemplified in the successful use of several
novel adjuvants on the European market (Table 2). In
general, such adjuvants works through several mecha-
nisms-antigen presentation, adjuvant-antigen complexa-
Table 2. Non-aluminum vaccine adjuvants currently used in
licensed products.
Adjuvants Adjuvant
composition
Representative
Products Indications
AS03
Squalene-based
oil-in-water
emulsion
Pandemrix Pandemic
flu
AS04 MPL + alum Fendrix
Cervarix
HBV
HPV
MF59
Squalene-based
oil-in-water
emulsion
Fluad Seasonal
flu
Virosomes
(150 nm)
Phosphatidylcholine
bilayer liposomes Inflexal V Seasonal Flu
tion/depot, enhanced antigen delivery to/into DCs, re-
cruitment of immune cells, and immunomodulation, etc.
4.1. Oligonucleotides
Oligonuceotides (ODNs) are extensively studied as vac-
cine adjuvants. A major sub-class of ODNs is the un-
methylated CpG ODNs resembling bacterial DNA struc-
ture. A series of review articles were recently published
addressing CpGs as stand-alone or secondary immuno-
therapeutic agent [74], approaches for enhancement of
immunostimulating effect of CpGs [75], microparticle-
mediated enhancement of immunostimulating effect of
CpGs [76,77]; dichotomous effects of CpG as an cancer
vaccine adjuvant [78], use of various methods [79] or
lipids [80] for improvement of CpG stability and delivery,
and use of CpG -antigen conjugates for improvement of
vaccine delivery and immunogenicity [81]. Non-CpG
ODNs as TLR9 agonist include 5'-TC dinucleotide
structure with a thymidine-rich sequence [82,83].
Immunomodulation of oligonuceotides (ODNs) is
through activation of toll-like receptor 9 (TLR9) [74].
TLR9 is localized both intracellularly (endosomes of
myeloid cells) and on the surface of epithelial cells [84].
TLR9 agonists directly induce the activation and matura-
tion of dendritic cells and enhance differentiation of B
cells into antibody-secreting plasma cells [74]. Since
TLR9 signaling is not absolutely required in mice [85],
other mechanisms of action could also be responsible for
their immune enhancement, such as up-regulation of
gene expression in mice [86], and formation of anti-
gen-adjuvant complexes [87,88]. Combined use of vac-
cines and such immunostimulants is emerging as one of
the innovative approaches in adjuvant development [89].
The CpG ODNs can be further classified into several
categories (A-, B-, and C-class) based on their relative
activity on B cell and NK cell activation and cytokine
production [90,91]. All classes can induce potent Th1
effects for a variety of antigens [90-96]. In reality, use of
CpGs often generates a balanced and more effective im-

Selection of Adjuvants for Enhanced Vaccine Potency
39
mune response. For example, use of CpG 2007 (22-mer)
not only enhanced antigen (hen egg lysozyme)-specific
humoral responses, but also induced long-lasting cell-
mediated immune response against the model antigen
(HEL) in calves after SC administration [87]. Similar
examples include CpG 1826 (20-mer) for OVA [88],
CpG 7909 (a 24-mer, B-class) for HBsAg [97] and for a
pneumococcal vaccine [98], three classes of CpGs for
hepatitis C virus [91], and CpG ODN 2006 for inacti-
vated gp120-depleted HIV-1 immunogen [99]. A bal-
anced effect can make a vaccine more effective against
challenging disease such as tuberculosis [100]. A bal-
anced immunogenicity effect can be also obtained with a
DNA vaccine administered with CpG-enriched plasmids
(5-20 CpG copies) [101].
CpG ODNs are quite effective in comparison with
other adjuvants. They were demonstrated to be more ef-
fective than alum for Trypanosoma cruzi (parasite) anti-
gens [93] and rabies virus vaccine [24] and even more
effective than modified complete Freund's adjuvant
(CFA with Mycobacterium butyricum instead of Myco-
bacterium tuberculosis) [66]. Because the effect of CpG
is clearly dose-dependent in several studies [93,102],
reducing the dose of CpG to 20 µg or less made it less
effective than a higher amount of aluminum hydroxide in
mice [96,103].
CpG ODNs are often added to aluminum-based vac-
cines for further improvement of the immune response.
Such examples include BioThrax (a licensed anthrax
vaccine) in mice (ip or sc) and guinea pigs [104], hepati-
tis B virus vaccine (Engerix-B) in chimpanzees [105],
poliovirus vaccine in mice [96], and Plasmodium falci-
parum Apical Membrane Antigen 1 vaccine in humans
[106]. This also seems to be the case for non-CpG ODN,
such as IMT504 (24-mer) with recombinant Hepatitis B
surface antigen in monkeys [107]. When CpGs are used
with aluminum-based vaccines, the immunostimulatory
effect of CpG may depend on the relative association of
CpG and antigen to the aluminum adjuvant [108]. Indeed,
the highest antibody response to a AMA1-C1/CPG
7909/Alhydrogel mixture in mice corresponds to a CPG
7909 concentration of saturated binding to Alhydrogel,
while unbound CpG 7909 was ineffective in enhancing
antibody response [109]. In contrast, changing the per-
centage of bound CpG 7909 on Alhydrogel in different
buffer systems did not change the peak level of antibody
formation in a phase I trial of a malaria vaccine [110].
Therefore, the relative importance of CpG binding to
aluminum adjuvants needs further verifications.
CpG ODNs could also improve the immune response
of mucosal vaccines, such as those applied in vagina and
GI tract [111]. CpG was shown to promote a strong anti-
gen- specific Th1-like immune response in the mucosa
and local lymph nodes after mucosal application with
glycoprotein D of herpes simplex virus type 2 (HSV-2)
and protection against mucosal viral challenge in mice
[112]. Intranasal administration seems to be especially
effective in inducing both systemic and mucosal reponses
[113]. Because of this, Intranasal administration of CpG
ODN in both murine leishmaniasis and toxoplasmosis
model in mice resulted in comparable results against
challenge as that after subcutaneous administration [114].
In comparison, oral delivery of CpG ODN has not gener-
ated consistent results. While oral delivery of CpG ODN
(20-mer) with purified hepatitis B surface antigen (HBsAg)
or tetanus toxoid (TT) in mice augmented both mucosal
and systemic immune responses [115], oral uptake of
uncoupled CpG ODN resulted in a complete failure of
treatment against murine leishmaniasis and toxoplasmo-
sis infection in mice presumably due to CpG degradation
[114]. To overcome the stability problem, Wang et al.
[116] designed “second-generation” immunomodulatory
oligonucleotides including: CpR, YpG, or R'pG (R =
2'-deoxy-7-deazaguanosine, Y = 2'-deoxy-5-hydroxy-
cytidine, and R' = 1-[2'-deoxy-beta-d-ribofuranosyl]-2-
oxo-7-deaza-8-methyl-purine). Indeed, these were sig-
nificantly more stable than CpG DNA following oral
administration and induced stronger local (IgA) and sys-
temic (serum IgG2a) immune responses to ovalbumin
than CpG DNA in mice.
On the other hand, use of CpGs is not always benefi-
cial for immunogenicity. The humoral responses to in-
tramuscular immunization with Fluarix in healthy volun-
teers were not significantly enhanced by inclusion of 1
mg CPG 7909 in healthy volunteers, although a positive
effect was seen at 1/10th dose of Fluarix16. A CpG ODN
(20-mer) was ineffective in enhancing the antibody titer
(IgG2a) of urea-solubilized p55 antigen (from HIV-1) in
mice, although positive adjuvant effect was seen when it
was formulated in an emulsion or when p55 was bound
to polylactide-co-glycolide microparticles [117]. Addi-
tion of CpG 1826 did not lead to additional immune en-
hancement for a peptide vaccine (complexed with an
immune-enhancement dsRNA adjuvant - pI:C/E749-57)
in mice [118] and for a montanide ISA720-adjuvanted
opossum vaccine in rats [119]. While co-administration
of CpG 1826 with a respiratory syncytial virus vaccine
increased the efficacy of the vaccine, co-administration
during primary infection actually enhanced the severity
of the disease in mice [120].
The neutral or negative effect of CpGs could be poten-
tially due to its inherent property. All classes of CpGs
were able to induce formation of IL-10 in healthy and
HCV PBMC, which is proposed to promote formation of
regulatory T cells (Treg), leading to inhibition of Th1-
type t cell responses [91]. Their effect on antigen integ-

Selection of Adjuvants for Enhanced Vaccine Potency
40
rity and the effect of other formulation excipients can
also be partially responsible. CpG caused dissociation of
antigen from Alhydrogel in the presence of phosphate
and formulation excipients strongly affected CpG 7909
adsorption on Alhydrogel [108]. Recent studies showed
that effect of CpG 7909 is also related to physical train-
ing in rats [121] and the immune status of the subjects
[122]. Therefore, use of CpGs needs to be carefully eva-
luated. Clinically, CpG seems to be well tolerated in
general but more frequent injection site pains and head-
aches were observed at a high dose in healthy volunteers
[16,102].
4.2. Emulsions
Traditionally, two types of emulsions are used in phar-
maceutical applications-water-in-oil (w/o) or oil-in- wa-
ter (o/w). Both types have been tried as vaccine adju-
vants. Complete Freund's adjuvant (CFA) is a histori-
cally- tested water-in-oil emulsion containing killed bac-
teria [123]. It has been proven to be a very effective ad-
juvant and generate balanced immune response [94]. The
humoral immunogenicity enhancement of CFA is more
effective than aluminum salts for a 42-amino acid amy-
loid-beta peptide antigen [124] and for cystein proteinase
antigen in mice [94]. However, severe toxicities have
been observed even at a reduced dose, such as weight
loss, leukocytosis, abdominal adhesions, granulomatous
peritonitis, and disrupted hyalimized myofibers in mice
[94,124]. Other animal toxicities include skin lesions in
rats and arthritis in dogs [66].
The toxicities of CFA led to the development of in-
complete Freund’s adjuvant (IFA). With less toxicities,
this adjuvant is less potent in mice [124]. In addition, the
water in oil emulsions were highly viscous and not stable
[123]. Further modified IFA systems (water-in-oil emul-
sions) were then developed, such as Montanide ISA 51,
which contains mineral oil and mannide monooleate as a
surfactant. This adjuvant is being tested clinically [125].
It appears to generate similar quality and intensity of
immunogenicity to aluminum hydroxide but side reac-
tions are not desirable, including granuloma, local pain,
tenderness and erythema [123]. Montanide ISA 720 is
another one (containing squalene, a metabolisable oil),
which was shown to increase the humoral response to a
malaria vaccine candidate in rhesus macaques and more
potent than Alhydrogel [126]. A dose escalating phase 1
trial of a vaccine containing recombinant Plasmodium
falciparum apical membrane antigen 1 (AMA1) formu-
lated in Montanide ISA 720 did not show any vac-
cine-related serious adverse events [127].
In comparison, oil-in-water (o/w) emulsions seem to
be safer than water-in-oil emulsions. A representative
o/w system is MF59 from Novartis, consisting of 4.3%
metabolizable oil squalene from shark liver, 0.5% poly-
sorbate 80, 0.5% sorbitan triolate, and 10 mM sodium
citrate with a size of 160 nm [30,34,128]. The proposed
mechanisms for immune enhancement by MF59 include
recruitment of APCs to the injection site, enhancement of
antigen uptake into APCs, and activation of innate im-
munity without activating TLR pathways [25,128]. Al-
though MF59 may be cleared independently from soluble
antigens after intramuscular injection [129], its efficacy
as adjuvant is likely attributable partly to its depot effect
and cellular infiltration [123]. The individual components
of the emulsion do not seem to be special, as replacement
of the oil and surfactant maintains satisfactory immu-
nological properties (antibody response to an antigen) in
mice [130].
MF59 has been used in licensed influenza vaccine
(Fluad) with good safety in more than 20 countries since
1997 [131,132]. It has been shown to be effective for a
HCV vaccine[133]. It can initiate greater, longer-lasting,
and broader immune responses than a nonadjuvanted
split flu vaccine in healthy young children [134,135] and
in adults [136]. As an adjuvant for flu vaccine, it is more
potent than aluminum-based adjuvants in terms of both
antibody and T-cell responses [131]. Therefore, MF59-
adjuvanted vaccines were found to be more effective
than a commercial product, such as hepatitis B virus
(HBV) vaccine (containing recombinant PreS2 and S
antigens) in healthy adult subjects [137]. However, the
species- and antigen-dependent variation in immuno-
genicity enhancement by MF59 appears to be a defi-
ciency of this adjuvant system [15,138]. MF59 does not
seem to induce significant side effects compared with
vaccines without MF59 in healthy adults [137,139]. Re-
cent studies show that MF59 is associated with more
reports of injection site pain and tenderness (local reac-
togenicity) relative to a non-adjuvanted flu vaccine in
children and young adults [140,141].
Other MF59-like oil-in-water (O/W) emulsions have
also been developed. One such system (10% squalene,
1.8% glycerol, 1.9% phosphatidylcholine, 0.09% Plu-
ronic F68) was shown to increase the immunogenicity of
an inactivated trivalent poliovirus vaccine [142]. Another
one (5% squalene, 4% Poloxamer 105 and 2% Abil-Care
as emulsifier) increased the immunogenicity of non- po-
tentiated rabies vaccine, more effective than aluminum
hydroxide [143]. CoVaccineHTTM, a submicron emul-
sion of squalene-in-water emulsion containing sucrose
fatty acid sulphate esters, has been shown to increase
the antibody responses for a whole inactivated influenza
A/ H5N1 virus vaccine through TLR4 signaling in mice
[144]. AF03, another oil-in-water emulsion adjuvant, was
found to induce stronger antibody responses to a pan-
demic influenza vaccine (at 0.3 µg HA) than non-adju-

Selection of Adjuvants for Enhanced Vaccine Potency
41
vanted vaccine in both naive and seasonal flu-primed
mice [145].
A frequently studied series of oil-in-water emulsion
adjuvant system is the AS series. A representative of this
series is AS03, which is a 10% oil-in-water emulsion-
based adjuvant system comprising squalene, -toco-
pherol and emulsifying agent Tween 80 [146-148]. AS03
has been used in the product Prepandrix (H5N1 vaccine
from GSK) in EU for a few years. This adjuvant has been
shown to enhance the initial priming effect of pandemic
influenza vaccination and promote a rapid humoral re-
sponse to a single boosting dose with a heterologous
strain, not only reducing the dose of the antigen (up to 24
times) but also offering a better cross-protection against
drifted strains in young, elderly, or Asian subjects
[149,150]. In different studies, administration of AS03-
adjuvanted H1N1 pandemic vaccine generated same
level of immunogenicity as unadjuvanted vaccine at a
four-fold higher dose in adults [151], and long-term pro-
tection in health care workers [152]. On the other hand,
this adjuvant system showed limited effect for a H1N1
-like virus vaccine after administration of two doses in
hematopoietic stem cell transplantation recipients [153].
Although the safety profiles of AS03 is acceptable, reac-
tions such as fever tend to increase after a second dose
for a H1N1 vaccine in children of 6-35 months [154],
and delayed focal lipoatrophy was reported recently after
its use in a H1N1 flu vaccine [155]. These vaccine sys-
tems are certainly promising but the added complexity of
emulsion preparation and difficulty in vaccine charac-
terization are clear disadvantages.
4.3. Iscomatrix
The immunostimulating complex (ISCOM) is an anti-
gen-containing particulate system while ISCOMATRIX
is the antigen-free, and structurally-similar system [156-
158]. It was first described more than 2 decades ago as a
novel structure for antigenic presentation of membrane
proteins with potent immunomodulatory capability. IS-
COMATRIX system consists of a Quil A-based saponin
mixture (see QS21 below) combined with cholesterol
[157]. This system enhances immunogenicity through
several mechanisms, including recruitment and activation
of APCs, extension of antigen presentation in the drain-
ing lymph node, enhancement of CD8 cross-presentation,
induction of IFN- and IL-6, etc [30,159]. Association of
antigen with ISCOMATRIX seems necessary for the
optimal induction of cytotoxic T lymphocyte (CTL) re-
sponses [160].
ISCOMATRIX as an adjuvant promotes both humoral
and cellular immune responses due to the powerful im-
munomodulatory capability of saponin both preclinically
and clinically [157,158]. Subcutaneous injection of IS-
COMATRIX®-adjuvanted 4 dengue virus envelope pro-
teins (10 µg) resulted in adequate protection in both
mouse and monkey challenge models [161]. Such an
immune enhancement effect of ISCOMATRIX (50 µg)
on recombinant HIV gp120 vaccine can be significantly
greater than that aluminum hydroxide [162]. Similarly,
immunization of patients with a mixture of HPV16 E6E7
fusion protein and ISCOMATRIX adjuvant induced an-
tigen specific cell mediated immunity in terms of anti-
body formation, delayed type hypersensitivity, in vitro
cytokine release, and CD8 T cell responses [163]. To
mitigate the potential safety issues related to ISCOMA-
TRIX, Matrix M, the particles made of two selected and
purified fractions of saponin, was developed and found to
be effective to initiate strong immediate and long-term
humoral immune response for influenza H5N1 vaccine
with a balanced Th1/Th2 cytokine profile and high cross-
reactivity against drifted H5N1 viruses in mice [164].
The lipophilic nature of the ISCOMATRIX also makes
it an effective mucosal adjuvants. It was shown to induce
more effective pulmonary protection (10-100 fold dose
sparing) against viral challenge when it is used intrana-
sally with split influenza vaccines in mice [165]. Deep
pulmonary delivery of several ISCOMATRIX-based
vaccines has been shown to induce antigen-specific mu-
cosal and systemic immunity [166,167]. It was also
shown to induce local and systemic immune responses
against orally delivered protein antigens, partly due to the
enhancement of antigen absorption in mice [159].
The ISCOMATRIXes are promising vaccine adjuvants.
Due to the side effects of component saponins (see be-
low), their uses as vaccine adjuvants would be limited for
serious indications. Further modification of this system is
expected to reduce its side effect while maintain or im-
prove its adjuvatation effect.
4.4. Liposomes/Proteoliposome/Virosomes
Liposomes have been shown to up-regulate several che-
mokine genes, including CCL2, CCL3 and CCL4, in
dendritic cells [168]. Liposomes can facilitate in vivo
migration of antigens [169] and deliver encapsulated
antigen into cytosol of the antigen presenting cells for
both cell mediated as well as humoral immune responses
[170,171]. It is believed that uptake of liposomes is gen-
erally through a passive phagocytic or endocytic process,
not by fusing with cellular membranes [172]. Charged
liposomes can bind to antigen readily and enhance the
uptake of antigen and the efficiency of antigen presenta-
tion [173]. Liposome- antigen complexes could induce
significantly higher cellular immune responses that anti-
gen carried by aluminum hydroxide after subcutaneous
administration in mice [174]. Other additives, such as
vitamine E, can be included to improve the adjuvant ef-

Selection of Adjuvants for Enhanced Vaccine Potency
42
fect (novasomes) [175].
Liposomes can induce different types and levels of
immune response for a variety of vaccines/antigens, such
as cytosolic proteins (sAg) of Plasmodium yoelii ni-
geriensis [170], tuberculosis vaccine candidate Ag85B-
ESAT-6 [176], flu vaccine [169,177], leishmanial anti-
gen vaccine [178], and DNA vaccine [179]. Liposomes
can generate more efficient immune response [180] and
less side effect (limited IgE levels) than aluminum-ad-
juvanted vaccines [171]. Greater effectiveness was ob-
served in mice for a leishmaniasis vaccine adjuvanted
with cationic liposomes than those adjuvanted with BCG
or MPL plus trehalose dicorynomycolate [178]. Vax-
fectin, a cationic lipid-based adjuvant, was shown to in-
crease the immune response for a seasonal influenza
vaccine with a  10-fold dose sparing effect in mice and
the type of effect (Th1 or Th2) could be directed simply
by varying the ratio of adjuvant to antigen [181,182].
Similar effect could also be obtained by adding/varying
other liposome components such as lipid A [183] or an
immunomodulator ,'-trehalose 6,6'-dibehenate (TDB)
[184].
The type and degree of immunogenicity enhancement
by liposomes depends on liposome’s composition, size,
the type of antigens [185], and interestingly, the chirality
of lipids [186] Liposomes made of S. cerevisiae mem-
brane lipids are more effective than egg PC liposomes in
inducing IgG2a, IFN-, and IL-4 cytokine [170]. Cationic
lipid vesicles (comprising a cationic cholesterol deriva-
tive, DC-Chol) bind strongly split influenza vaccine an-
tigens and induced robust anti-influenza immune re-
sponses while neutral cholesterol/DOPC liposomes dis-
played virtually no stable antigen binding and no adju-
vant effect in mice [187]. Replacing the polyalkylamine
head group spermine with spermidine (with one less
secondary amine) in a polycationic liposome reduced the
enhancement of the immune response to a flu vaccine by
~50% in mice [177]. Saturated phospholipids are more
effective than unsaturated phospholipids in the enhance-
ment of allergen-specific IgG response upon immuniza-
tion in mice [171]. Increasing the amount of fusogenic
lipids (DOPE) could further enhance the Th1 response to
model OVA antigen in mice [188]. Bile salt-incorporated
lipid vesicles (bilosomes) of 980 nm in size containing
influenza A antigen generated significantly more Th1
biased response than vesicles of 250 nm in mice [189].
More complexed lipid-based vesicles were also de-
veloped as potential adjuvants, including proteolipo-
somes (PL), cochleate structures (CS), and virosomes.
Proteoliposomes in different sizes contain bacterial
membrane components (e.g. the outer membrane of N.
meningitidis B), including LPS, phospholipids, and trac-
es of bacterial DNA to improve immunogenicity
[190,191]. Cochleate structures (AFCo1) are made
through interaction of divalent cations with anionic lipids
in proteoliposomes. Virosomes mimics the structure of a
virus. Proteoliposomes can up-regulate MHC-II, CD40,
CD80, and CD86 expression and production of TNF
and IL12(p70) in dendritic cells [192]. They were found
to be effective in inducing a Th1-type immune response
(production of IgG2a and IFN-) to allergens even in the
presence of alum in mice [193]. Proteoliposomes can
also be used as effective mucosal adjuvants. Intranasal
administration of proteoliposomes containing LPS into
mice led to high anti-LPS IgG titers [194]. Similarly,
intranasal administration of proteoliposomes (AFPL1; 70
nm in size) containing glycoprotein D (gD) of herpes
simplex virus type 2 (HSV-2) induced gD-specific IgG
antibody formation, leading to partial protection against
genital herpes infection in mice [190]. In comparison,
intranasal administration of AFPL1-derived cochleate
structures (AFCo1) containing the same protein elicited a
complete protection in the study [190]. Indeed, the coch-
leate structures can be more potent than aluminum salts
in inducing high levels of both IgG1 and IgG2a for a
variety of pathogen-derived antigens in mice [195]. Vi-
rosomes have been successfully used in three commercial
products [196]. The representative product is Inflexal V,
a virosomal adjuvanted influenza vaccine, which is made
of phosphatidylcholine bilayer liposomes, containing
neuraminidase and hemagglutinin. By mimicking natural
infection, the vaccine has shown to have good efficacy
for people of all age groups [197]. The entire virosome
has a diameter of 150 nm and have been proven safe with
mostly mild to moderate symptoms resolvable within a
few days [198].
The effect of liposomes on DNA vaccines has not been
consistent. Intramuscular injection of Vaxfectin®- adju-
vanted vaccines containing five P. falciparum protein-
encoding plasmids enhanced both antibody and cellular
immune responses to each component of the mul-
ti-antigen vaccine with no apparent antigenic competition
in mice [199]. In human trials, intramuscular injections
of Vaxfectin®-adjuvanted H5 hemagglutinin- encoding
DNA vaccine led to 4-fold rises in hemagglutination in-
hibition (HI) titers in 47% - 67% of subjects [200].
However,  20% of subjects showed such a response
after immunization with a trivalent Vaxfectin®- adju-
vanted DNA vaccine [200]. While liposomes, made of
phosphatidyl choline (PC), dioleoyl phosphatidyl etha-
nolamine (DOPE), and dioleoyloxy trimethyl ammonium
propane (DOTAP) in a molar ratio of 4:2:1, is a good
adjuvant for DNA vaccination against hepatitis infection
in Rhesus monkeys [201], subcutaneous administration
of hepatitis E virus neutralizing epitope-encoding or he-
patitis B virus surface antigen-encoding DNAs entrapped

Selection of Adjuvants for Enhanced Vaccine Potency
43
in liposomes consisting of the same types of lipids does
not elicit antibody response in mice [202]. In fact, li-
pid-DNA complexes actually appear to inhibit the ex-
pression of the complexed DNA comparing with naked
DNA after IM injection [172].
The above results show that lipid-based systems are
effective and promising vaccine adjuvants with excep-
tions, especially for DNA vaccines. The versatility of
these adjuvant systems resides in the availability of a
variety of lipids and other additives to accommodate dif-
ferent types of antigens [4,172,184,185].
4.5. Polymeric Solutions or Particulates
Polymeric solutions, micro-or nano-particulates can be
used as effective adjuvants either alone [203] or in a
complexed form with antigens [204] and/or other adju-
vants [205]. Various polymers have been demonstrated
to have such properties, such as polylactic acid (PLA),
poly (lactide- co-glycolide) acid (PLGA) [206,207], chi-
tosan [208-211] or modified chitosan [207], poly(-glu-
tamic acid) (-PGA) [212,213], polypeptides [204],
starch [214,215], polystyrene [35,205], polyphosphazene
[216], poly (prolylene sulfide) [217], polyethylene glycol
[218], alginate [219], and their copolymers [220,221].
They enhance immunogenicity through several mecha-
nisms, including enhancement of antigen uptake by den-
dritic cells (in vitro) [212], maturation of dendritic cells
[212], promotion of proliferation of antigen-specific T
cells [212], stimulation of both B and T lymphocytes
[208], facilitation of activation of dendritic cells [222],
viscosity-induced retention of antigens in the vaccination
site [210] and controlled release of antigen by particles
[223,224]. Uptake of such nanoparticulates could take
place by DCs through both clathrin and pinocytic path-
wasys [217]. The event leads to activation of the NALP3
inflammasome, promoting innate and antigen-specific
cellular immunity [77]. Such particulate adjuvants could
be as effective as aluminum hydroxide [221] or even the
potent complete Freund's adjuvant (CFA) [212].
A widely studied polymer is polylactic acid (PLA) or
poly (lactide-co-glycolide) acid (PLGA). Antigens can
either be adsorbed on the surface or encapsulated inside
the polymeric particles. Intramuscular administration of
adsorbed antigen - Neisseria meningitidis serotype B
(Men B) (99% adsorption efficiency) on PLGA particu-
lates with CpG significantly increased the anti-Men B
serum antibody titers and serum bactericidal titer in mice
[206]. The immunogenicity enhancement can be compa-
rable to that by traditional aluminum hydroxide-adju-
vanted vaccine [225]; or even better than that by more
advanced adjuvant such as Montanide ISA 720 [226].
Using poly (lactide-co-glycolide) as a model polymer
and Neisseria meningitidis serotype B (Men B) as a
model antigen, encapsulating CpG within PLG micropar-
ticles induced statistically significant higher antibody,
bactericidal activity and T cell responses when compared
to the soluble form of CpG [206].
Another widely studied polymer is chitosan. Addition
of 0.5% of a chitosan derivative to an inactivated influ-
enza vaccine resulted in a four or six to tenfold increase
in antibody titers after intramuscular injection of a single
or two doses in mice, respectively [227]. Their effect of
immune enhancement can be better than the traditional
aluminum adjuvants. Intramuscular administration of
chitosan nanoparticle (160 - 200 nm)-encapsulated re-
combinant hepatitis B surface antigen (rHBsAg; 10 µg)
induced a 9-fold higher anti-HBsAg IgG levels compared
to the conventional alum-adsorbed vaccine in mice [223].
It was also shown to be more effective than PLGA or
PLA for HBsAg vaccine [224]. Chitosan as a mucosal
adjuvant enabled a M2 flu vaccine against heterologous
virus challenge after intranasal administration in mice
[209]. Mannosylation of chitosan can further enhance its
activity as a mucosal adjuvant [228].
The degree and types of immunogenicity enhancement
may be dependent on the types of polymers and prepara-
tion methods. While immunization with zinc-chitosan
particles bound to histidine-tagged recombinant protein
antigen promoted an IgG1 response in mice, subcutane-
ous immunization with a simple mixture of an influenza
hemagglutinin (HA) vaccine and amphiphilic poly (-
glutamic acid)-graft-l-phenylalanine copolymers (-PGA
-NPs) led to a more balanced immune response than
aluminum-adjuvanted vaccines in mice [229]. Some po-
lymer particulates may also exert their immune en-
hancement activity through conjugation with antigens,
such as starch microparticles for protein antigens in mice
[214,215], and polystyrene beads for protein antigens in
sheeps [35]. A more recent study suggests that a reduci-
ble linker (-S-S-) could be effective than non-reducible
linkers in immunogenicity enhancement [217].
Other physical properties of particulates such as size
and charge can have a significant effect on the immuno-
genicity. While no significant difference was found in the
immune response after intramuscular administration of
MenB antigen adsorbed on PLGA nanoparticles (110 nm)
or microparticles (800-900 nm) in mice [225], PLGA-
CTAB particles of 0.3 µ were more effective than larger
particles (1 and 30 µ) in the elicitation of transgene- spe-
cific serum IgG responses [230]. The greater effect with
smaller particles could be related to two factors-higher
adsorption capacity of smaller particles and greater effi-
ciency of particle uptake by dendritic cells. It has been
shown that the antigens adsorbed on aluminum particles
of 3 µ are internalized more effectively than those of 17
µ [28]. Charged nanoparticles could present antigens

Selection of Adjuvants for Enhanced Vaccine Potency
44
effectively thorough electrostatic interactions with anti-
gens. Immunization of HIV-1 Tat (1-72) protein (1 µg
antigen dose) coated on anionic nanoparticles generated
higher antibody titers than alum-adjuvanted vaccine
[231]. Polyphosphazene (ionic, cross-linkable water so-
luble polymers) has been shown to have synergistic ef-
fect on enhancement of the secretion of cytokines in vitro
when used with CpG and indolicidin, likely due to for-
mation of antigen-adjuvant complexes [87]. Cationic
block co-polymers and DNA can easily form complex
structures via electrostatic interaction and were shown to
elicit broad and long-lasting antigen-specific humoral
and cellular responses after intramuscular administration
in mice [232].
Polymeric particles are also effective mucosal adju-
vants. Examples include oral administration of PLGA
microsphere-encapsulated concentrated rabies virus (CRV)
in mice [233], or polyacryl starch microparticle- conju-
gated diphtheria toxin in mice [234]. The oral uptake of
particulate antigens is presumably through the Peyer's
patches in the GI tract [215]. Nasal administration has
not resulted in consistent results. No immune response
was seen after intranasal immunization with a mixture of
-PGA-NPs and influenza virus hemagglutinin (HA) in
mice [235]. While PLGA-encapsulated OVA did not
initiate induce detectable antibody titers after nasal ad-
ministration in mice, high titers were observed when po-
sitively charged N- trimethyl chitosan was used [207].
This could be due to a difference in the binding strength
between the antigen and polymeric particles.
The above examples demonstrated effectiveness of
polymers as vaccine adjuvants. Similar to lipid-based
adjuvant systems, polymeric systems appear also versa-
tile, due to the availability of a variety of polymers and
their processability for surface modification and direct
conjugation with antigens. It is expected that such adju-
vant systems would be further explored and optimized
for enhancement of vaccine efficacy.
4.6. Saponins and QS21
Saponins are natural glycosides of steroids or triterpenes.
They possess certain adjuvant activities, due to their
structural features of sugar chain(s), its length, and hy-
drophilic and lipophilic properties [236]. Therefore, their
adjuvant activity can be further modified by structural
modifications such as GPI-0100 [237]. The widely used
saponin-based adjuvants are Quil A, isolated from the
bark of Quillaja saponaria (QS) Molina, and QS21, a
more purified form, is the 21st of 22 fractions in RP-
HPLC trace of semipurified QS bark extracts [238].
These adjuvants have been evaluated in numerous pre-
clinical and clinical trials [239]. They have been proven
to be very effective in enhancing the immunogenicity of
various vaccines, including Aβ(1-15) in mice [240];
foot-and -mouth disease virus (FMDV) antigen in mice
[241]; L1 and A33 proteins in mice [242]; vaccinia virus
proteins in monkeys [242]; parasitic antigens in sheeps
[243]; keyhole limpet hemocyanin (KLH) conjugate an-
tigens [238,244,245]. The effect is often toward a Th1-
type response in comparison with aluminum-based vac-
cine such as Aβ(1-15), PADRE-Aβ(1-15)-MAP) [240],
L1 and A33 proteins in mice[242], parasitic antigen
(Fasciola hepatica) in sheeps [243].
Quil A or QS21 is often much more effective than
aluminum salts in immunogenicity enhancement for
many antigens, such as Aβ (1-15) in mice [240], and
SPf66 (a synthetic malaria peptide vaccine) [246]. Ex-
ceptions do exist. QS21 did not exert any significant ef-
fect on either binding or neutralizing antibody titers after
IM immunization of gp120 HIV-1(MN) protein (rsgp120)
at doses of 100, 300, and 600 µg, even though it is effec-
tive at a lower antigen dose [247]. Similarly, QS21 adju-
vant was not effective in enhancing the immunogenicity
of an inactivated influenza vaccine in healthy young
adults, in terms of serum titers, T cell cytotoxicity, and
IFN- levels [248].
A key issue of saponins is their induction of red blood
cell hemolysis, and they are painful to inject with high
local reactogenicity [30,239]. The more purified form
QS21 is much better tolerated but the side effects still
prevent its use as an effective adjuvant. These side ef-
fects include moderate to severe pain for a recombinant
HIV protein vaccine [247], site pain and postvaccination
myalgias for inactivated influenza vaccine in healthy
young adults [248], malaise, headache, fever, and nausea
[247]. Two out of 89 subjects developed severe vaccine
allergy following the third dose of 1/3 QS21/SPf66 for-
mulations (a synthetic malaria peptide vaccine) [246].
Because of these potential toxicities, alternative
sources or structural modifications of saponins were then
sought. Platycodin D (PD), a saponin from the root of
Platycodon grandiflorum without the acyl domain, is less
hemolytic, very stable in aqueous solution, and enhanced
the immunogenicity of hepatitis B surface antigen
(HBsAg) in mice [249]. This led to deacylation of QS21
but the deacylated QS21 was less effective as an adjuvant
for ovulbumin in inducing IgG1 responses and inactive
in inducing IgG2a or CTL responses at any doses in mice
[250]. A semi-synthetic saponin (GPI-0100) was found
to enhance the immunogenicity of a single tandem fusion
protein in mice [251] and enhance antibody titers against
the glycolipid Globo H and the glycosylated mucin
MUC2 with only occasional grade II local toxicity at a
dose of 5000 µg in cancer patients [252]. Less haemo-
lytic saponins were also found in Chinese herbs [253].
Some are effective, such as Achyranthes bidentata (herb)

Selection of Adjuvants for Enhanced Vaccine Potency
45
saponins [254], Glycyrrhiza uralensis (herb) saponins
(GLS) [255], Bupleurum chinense (herb) saponins [256],
and Panax ginseng (root) saponins [257].
The side effects of QS21 is a clear disadvantage for
this adjuvant. Additional structural modifications could
potentially overcome this deficiency but may lose adju-
vantation effect at the same time. It is expected that fu-
ture use of this adjuvant would be limited to serious in-
dications.
4.7. Virus-Like Particles (VLPs)
Virus-like particles (VLPs) has been widely used for
vaccination against the corresponding virus such as in-
fluenza virus [175,258]. VLPs possess key immunologic
features of viruses-repetitive surfaces, particulate struc-
tures and induction of innate immunity through activa-
tion of pathogen-associated molecular-pattern recogni-
tion receptors. Therefore, they can facilitate presentation
of a foreign antigen or hapten to the immune system
through chemical conjugation or genetic fusion.
Most VLP-conjugate vaccines seem to be effective.
Examples include CCR5 peptide-VLP conjugates for
HIV [259], M2 peptide-VLP conjugate for influenza
[260], nicotine-VLP conjugates via succinimate linkers
for nicotine addiction [261]. It should be noted that VLPs
may not be very stable in an aqueous solution and could
be responsible for lower-than-expected immunogenicity
[262]. Conjugation of VLPs with haptens may improve
the stability of VPLs [260]. DNA vaccines containing
proper antigens can be designed to form VLPs in vivo,
inducing strong cellular immune responses [263]. Multi-
ple tandem copies of antigens can be incorporated in
such a vector for enhanced immunogenicity [264].
Virus replicon particles (VRP) can also be used as an
effective systemic, cellular or mucosal adjuvant. Intra-
muscular delivery of equine encephalitis VRP-conju-
gated OVA induced dose-dependent immune response in
mice [265]. Alphavirus replicon particles have been
shown to enhance the immunogenicity and effectiveness
of Fluzone® vaccine in rhesus macaques [266] and to
induce high antibody titers to the influenza hemaggluti-
nin (HA) protein in pigs [267].
VLPs hold great promise as effective vaccine adju-
vants, and particularly as effective carriers for non-im-
munogenic haptens. A clear disadvantage is the com-
plexity of expression, production, and purification of
VLPs, adding significant manufacturing cost to the final
vaccine product. Additional chemical conjugation steps
would further reduce the final yield and incur additional
production cost.
4.8. Carrier Proteins
Small-molecule drugs or even protein antigens may not
initiate any or adequate immune response. They can be
conjugated or fused to an effective protein antigen to
generate adequate immune response. Such carrier pro-
teins have been recently reviewed, such as keyhole lim-
pet hemocyanin (KLH), and bacterial proteins [268].
KLH belongs to the largest oxygen-transporting proteins
in nature and the glycoprotein moiety is critical for the
antigenicity of the molecule. Yet, use of KLH conjugate
alone may or may not initiate adequate immune response
and another adjuvant often has to be used. Such combi-
nations include KLH conjugates with QS21 for cancer
antigen (GD3; ganglioside) [238], with Freund’s adju-
vant for influenza virus A M2 peptide [269], and with
other adjuvants for cancer antigens (MUC1 and GD3)
[237,244]. The structure of linkers between hapten/anti-
gen and KLH carrier protein may have a significant ef-
fect on the immunogenicity and selectivity of the conju-
gated vaccine [270].
Conjugation or fusion of a hapten or antigen with a
bacterial or parasitic protein is frequently used. A class
of such proteins is the bacterial heat shock proteins
(HSPs). Success examples include HSP70 conjugates
with viral MHC Class I-restricted epitope (for Herpes
Simplex Virus Type-1) [271], and HSP65 conjugates
with a protein containing linear repeats of the gonadotro-
pinreleasing hormone (GnRH3), the hinge region of hu-
man IgG1 (hinge),or a T-helper epitope from the measles
virus protein (MVP) [272]. The immunogenicity en-
hancement can be comparable to that with Freund's ad-
juvant. Because HSPs can effectively induce partial ma-
turation of DCs in vitro [273], such proteins, as free
forms, can also enhance immune responses of antigens
but the effect may not be as strong as other adjuvants
such as CFA or LPS in mice [273]. Plasmid expressing
heat shock proteins (HSPs) could achieve the same effect
[274].
Other bacterial proteins can also be effective. Fusion
of the Brucella spp. lumazine synthase (BLS), a highly
immunogenic (decameric) protein, with a model virus
protein domain (C486 bovine rotavirus VP8 core protein
(VP8d)) led to almost 100% protection against homolo-
gous challenge with C486 bovine rotavirus [275]. Con-
jugation of synthetic peptides of influenza virus A M2
extracellular domain with Neisseria meningitidis outer
membrane protein complex (OMPC) was able to confer
protection against lethal challenge of either H1N1 or
H3N1 virus in the presence of Freund’s adjuvants in
mice [269].
In general, carrier proteins as vaccine adjuvants seem
to have limited adjuvantation effects, as the protein itself
may have limited immunogenicity. Therefore, additional
adjuvants may have to be used to initiate adequate im-
mune response.

Selection of Adjuvants for Enhanced Vaccine Potency
46
4.9. Toxins
Toxins represent one of the most potent adjuvants, espe-
cially for mucosal vaccines. Bacterial toxins are consid-
ered the most potent mucosal adjuvants such as heat-
abile enterotoxin and cholera toxin [34]. Intranasal de-
livery of ovalbumin with a recombinant bacterial entero-
toxin Zonula occludens toxin (Zot; 45kD single polypep-
tide chain) induced high Ag-specific serum IgG titers
over a year and is effective also through other mucosal
routes in mice [276]. It can increase reversibly the intes-
tinal mucosa permeability by affecting the structure of
tight junctions. Several mutant heat-labile enterotoxins
were found to be effective as adjuvants for nasal admini-
stration of pneumococcal vaccine [277], deglycosylated
chain A ricin (DGCA) [278], or influenza vaccine [279],
and for vaginal administration of inactivated caprine
herpes virus 1 vaccine [280].
Different toxins may offer different degrees of effect.
Zonula occludens toxin (a bacterial enterotoxin; 45 kD
single polypeptide chain) is highly efficacious when
compared to the mucosal adjuvant Escherichia coli
heat-labile enterotoxin (LT) [276]. However, two major
issues prevent them from use as vaccine adjuvants-their
toxicities and the residual bacterial endotoxins. Options
to bypass their toxicities include use of subunits of
toxins, toxoids, or structurally modified versions [281].
The toxin subunits could be still effective [282,283]
but a less adjuvantation effect would be expected rela-
tive to the complete toxin [284]. Several non-toxic LT
mutants have been recently reviewed [285]. A suc-
cessful one is the nontoxic mutant of diphtheria toxin
CRM197 [286].
Conjugation of these less effective toxin subunits or
toxoids to antigens could potentially restore the original
efficiency in immunogenicity enhancement. Successful
examples include fusion of subunit B of LT to a viral
protein [287], conjugation of toxoids to capsular poly-
saccharides of many invasive bacteria such as Strepto-
coccus pneumoniae, H. influenzae type b, meningococci,
pneumococci [2,288,289], and conjugation of LT sub-
units with CpG [111,290]. It should be noted that the
bacterial toxins can initiate a strong humoral immune
response, which may overshadow the response to the
conjugate antigen [7]. Adjuvants could also potentially
enhance antibody formation against both the conjugate
antigen and the toxins [244].
The above examples demonstrated that toxins or their
derivatives are effective antigen carriers for immuno-
genicity enhancement, especially for mucosal vaccina-
tions. The potential immune response against both the
original antigens and toxins need to be evaluated for pos-
sible mutual interferences.
4.10. Lipopolysaccharides/Polysaccharides
Lipopolysaccharides (LPS) are proven to be effective
adjuvants in many studies. Mechanistically, LPS acti-
vates TLR4 and directs DCs for a Th1 response [11,29].
LPS can facilitate internalization of particles by dendritic
cells [291], stimulate significantly expression of CD40,
CD80, CD86 and CD275 in dendritic cells, and also sti-
mulate release of IL-6, IL-12p40, and IL-12p70 [29].
Generation of a Th1 response can also be induced by
LPS fused with calreticulin/peptide complex in mice
[292]. If bound to proteins, bacterial polysaccharides
could induce both T and B cell arms of an immune re-
sponse [268].
The potential toxicities of these compounds, especially
those from Gram-negative bacteria are the barrier for
human use [293]. The purified Neisseria meningitidis
serogroup B lipopolysaccharide (LPS) has high en-
dotoxic activity but can be detoxified through structural
modifications. LPS analog LpxL1 from Neisseria menin-
gitidis is non-toxic and incorporation of this analog
LpxL1 in influenza H5N1 virosomes induced signifi-
cantly enhanced H5N1-specific total IgG titers as com-
pared to non-adjuvanted virosomes in mice [294]. Con-
jugation with tetanus toxoid (TT) reduced the endotoxic
activity of LPS by 2400 times and the conjugated
LPS-TT elicited higher anti-TT IgG2a and IgG1 levels
than unconjugated TT in mice [295]. In addition, high
levels of anti-LPS IgG and IgG subclasses were also de-
tected in sera.
Many polysaccharides have been shown to potentiate
immune response, such as -inulin, a human polysaccha-
ride for HBsAg [293], Advax, a inulin-based polysaccha-
ride for Japanese encephalitis vaccine in mice [296], PAP,
a water-soluble polysaccharide from the mycelium of
Polyporus albicans (edible fungus) for subcutaneous
immunization of mice with ovalbumin [297], BOS 2000
(polysaccharides from Boswellia serrata) for hepatitis B
in mice [298], lentinan (a (1-3)-beta-D-glucan from the
mushroom Lentinus edodes) or its sulfated form for
Newcastle disease (ND) vaccine [299]. Their effects can
be more effective than alum-based vaccine [296,298].
Intradermal administration of bovine serum albumin
conjugated to microparticulate β-glucan (MG) from yeast
Saccharomyces cerevisiae enhanced the primary IgG
antibody response to BSA in a manner comparable to the
prototypic complete Freund's adjuvant in mice [300].
It seems that the endotoxic activity of LPS needs to be
reduced before it can be used safely as a vaccine adju-
vant. Detoxification could be done through purification,
structural modification, or conjugation to another toxoid.
In comparison, polysaccharides seem to be much safer
but their adjuvantation effect may not be adequate. As

Selection of Adjuvants for Enhanced Vaccine Potency
47
for toxins, the simultaneous immune response to both the
original antigen and LPS needs to be evaluated for mu-
tual interferences.
4.11. Monophosphoryl Lpid A/Lipids
Monophosphoryl lipid A (MPL or MPLA) is a detoxified
(chemically modified) form of the endotoxin lipopoly-
saccharide (from Salmonella minnosota). Its properties
and applications have been reviewed recently [301].
Similar to LPS, it exerts its action through toll-like re-
ceptor 4. OM-174, a lipid A analog when injected intra-
venously or subcutaneously in mice, induces the migra-
tion of DCs from the periphery to the T cell areas of
lymphoid organs, and their maturation into cells ex-
pressing high levels of MHC class II and co-stimulatory
molecules, with its potency close to that of Escherichia
coli lipopolysaccharide (LPS) [302].
Both Th1- or Th2-biased response was observed with
MPL-adjuvanted vaccines. Administration of the full
length recombinant Toxoplasma GRA2 or GRA6 protein
antigen in combination with MPL adjuvant led to a Th1
response in mice [303]. Similar Th1 effects were also
observed with vaccine Fluzone [304] or recombinant
antigens [305], both adjuvanted with synthesized hesaa-
cylated lipid A derivatives in mice and Leishmania sterol
24-c-methyltransferase antigen with MPL-SE [306]. In
contract, subcutaneous immunization of MPL-adjuvanted
ovalbumin or glutaraldehyde-modified ragweed pollen
extract enhanced IgG1 titer (barely increased IgG2a) in
mice [307].
Many other lipids have been shown to have immuno-
modulating effect, certainly for those from microorgan-
isms [308,309]. Lysophosphatidylcholine (LPC) mixed
with various antigens induced cytotoxic T cell responses
and production of antigen-specific antibodies with an
efficiency similar to Alum in mice [310]. Synthetic al-
kylglycerol analogues in a mixture with ovulbumin have
been shown to increase anti-Ova IgG antibody produc-
tion in sera of immunized mice and the relative levels of
IgG1 and IgG2a depended on the carbon chain length
[311]. Immunization of mice with an HBV vaccine with
various doses of β-glucosylceramide (β-GC), β-lacto-
sylceramide (β-LC), or a combination of both augmented
both the anti-HBV titers and the percentage of mice ex-
hibiting high titers [312]. A similar lipid, -galactosyl-
ceramide (-GalCer) increased the immune response
after repeated intranasal or oral delivery of HIV peptide
antigens [313]. This compound was able to stimulate
repeatedly NKT cells without inducing anergy of NKT
cells [314]. More recently, step-wise screening of 25
-GalCer analogues led to the identification of a glycol-
ipid 7DW8-5, which is a more potent adjuvant than -
GalCer on HIV and malaria vaccines in mice [315,316].
Mucosal applications of lipids can also be effective.
Several studies have shown that intranasal delivery of a
HIV-1 genetic vaccine (plasmids) in the presence of a
cationic lipid adjuvant, the Eurocine N3, resulted in va-
ginal and rectal IgA responses as well as systemic hu-
moral and cellular responses in mice [317,318]. Initial
immunization can be boosted intranasally with a gp41
peptide in an anionic L3 adjuvant for a lower dose of
DNA [317].
Although MPL is currently a component of a licensed
cancer vaccine (Melacine), its potential toxicity led to
synthesis of MPL analogues. Three such analogues, syn-
thesized by different substitutions at 3-O-position of the
reducing sugar, were all found as effective as the natural
compound in inducing antigen specific T-cell prolifera-
tion and interferon- production with a liposome vaccine
[319]. A hexaacylated lipid A derivative was recently
found to be more potent than MPL on DC maturation and
expression of cytokines/chemokines [305]. The explora-
tion of MPL derivatives or other glycolipids would con-
tinue in the development of future vaccines.
4.12. Cytokines
Cytokines have been tried as valuable adjuvants. They
can be used either systemically and mucosally, such as
mutant TNF-α [320,321]. The majority of tested cyto-
kines are briefly summarized below.
4.12.1. Interferons
Type I interferons (IFN-α, IFN-β and IFN-ω) are cyto-
kines with multiple biological activities in innate immu-
nity, dendritic cell maturation/differentiation, etc. IM
vaccination of mice with a flu vaccine with IFN-α gener-
ated clear-cut IgG production (IgG2a rather than IgG1
and also IgA) and complete survival to infection with flu
virus [322]. In a similar study, intramuscular injection of
flu vaccine Vaxigrip ad-mixed with IFN-α markedly in-
creased the serum levels of all four classes of flu-specific
IgGs (IgG, IgG1, IgG2a, and IgA) in a dose-dependent
manner in mice [323]. Coadministration of inter-
feron-2b (Intron A) with hepatis B vaccine (Egerix B)
by IM initiated earlier and higher seroprotection with
improved Th1 response in haemodialysis patients [324].
Plasmid encoding IFN- can also be an effective adju-
vant for protein antigens [325].
IFNs can be effective in mucosal vaccinations. A sin-
gle intranasal administration of IFNβ-adjuvanted vac-
cine resulted in a full protection of 100% of mice against
virus challenge while vaccine alone was only partially
effective (40%) [326]. However, the efficacy seen in
animals has not translated into efficacy in humans. Addi-
tion of IFN-α to a trivalent flu vaccine did not change
significantly the serum neutralizing antibody response

Selection of Adjuvants for Enhanced Vaccine Potency
48
nor the IgA antibody response in respiratory secretions of
humans after nasal administration [327].
4.12.2. Granulocyte-Macrophage Colony Stimulating
Factor
Granulocyte-macrophage colony stimulating factor (GM-
CSF) can attract and stimulate macrophages, leading to
increased antigen presentation [328]. It increased the rate
of immune response (earlier seroconversion) and anti-
HBs titers in human subjects relative to hepatitis B virus
vaccination alone [329]. Administration of plasmids en-
coding GM-CSF can also be effective for DNA vaccines,
such as HIV-1 Gag DNA vaccine [330-332] and murine
vaccinia env pDNA vaccine [331,332].
On the other hand, results from human trials have not
been consistent. In some studies, GM-CSF appeared to
have minimal effect or even a suppressive effect, appar-
ently related to the dose level and frequency [328]. In a
recent clinical trial, use of GM-CSF as adjuvant in a
novel multiepitope peptide vaccine was minimally im-
munogenic (6 of 80 volunteers) in developing transient
HIV-specific responses [333]. In addition, the vaccine
induced formation of anti-GM-CSF antibody in the ma-
jority of GM-CSF recipients333. In another example,
subcutaneous administration of GM-CSF as an adjuvant
for a tetanus toxoid-adjuvanted influenza or hepatitis A
vaccine did not augment the antibody responses in nor-
mal volunteers relative to vaccination alone [334]. In fact,
subjects who received GM-CSF had statistically signifi-
cant lower increases in anti-tetanus antibodies.
4.12.3. Interleukins
Several interleukins have been tried as vaccine adjuvants
and proven to be effective. IL-12 has been extensively
tested for its adjuvant activity not only systemically but
mucosally [335]. IL-12 promotes differentiation of CD4+
cells towards a Th1 immune response. Its effect seems
due at least partially to its inflammatory properties. S. C.
injection of recombinant human IL-12 (rhIL-12) in pa-
tients with renal cell cancer induced dose-dependent sys-
temic activation of multiple inflammatory mediator sys-
tems [336]. Increasing the dose of rhIL-12 in a human
cytomegalovirus (CMV) vaccine gradually increased the
peak anti-CMV gB IgG titers and CMV viral lysate- spe-
cific CD4+T cell proliferation [337]. DNA expressing
IL-12 was also shown to be an effective adjuvant for
SIVmac239 gag pDNA vaccine in rhesus macaques
[338], VSG antigens expressed by Trypanosoma brucei,
and pneumonic plague after both systemic or nasal ad-
ministration in mice [339,340].
However, use of IL-12 as an adjuvant should be lim-
ited to severe diseases as it increases incidence of local
and systemic side effects. Such side effects include injec-
tion site pain, fever, headache, myalgia, general pain,
chills, and increased cough side effects observed when
used concurrently with a pneumococcal polysaccharide
vaccine in healthy volunteers [341]. Therefore, the
amount of IL-12 as an adjuvant was suggested not to
exceed a dose of 0.1 µg/kg, in order to avoid severe sys-
temic inflammatory responses [336].
IL-15 is a powerful immune stimulatory cytokine with
a wide range of biological activities. It plays critical roles
in the activation, proliferation and differentiation of CD8
+ T-cells and NK-cells. SC vaccination of mice with a
multi-valent TB vaccine with IL-15 induced comparable
CD4 + T cell and greater CD8+T cell and antibody re-
sponses against Mycobacterium tuberculosis compared
with the standard BCG vaccine [342]. Insertion of IL-15
in a recombinant TB virus make it an effective vaccine
for generation of consistent anti-TB responses in mice
and equal protection as BCG vaccine against M. tuber-
culosis challenge after heterologous prime/boost regimen
(priming with a fusion TB antigen adjuvanted with di-
methyldiotacylammonium bromide/monophosphoryl lipid
A and boosting with the recombinant TB virus) [343].
Combining IL-15 with CpG generated synergistic effect
on the immunogenicity of a SIV mucosal vaccine [344].
Similarly, plasmids encoding IL-15 could also be effec-
tive for a variety of vaccines tested in mice, such as a
tumor antigen-derived peptide vaccine [345], HIV-1
DermaVir vaccine formulated with HIV-1 Gag plasmid
(more potent than IL-7) [346] and flaviviruse DNA vac-
cine [347].
Recombinant IL-2 has been shown to increase the
immune response (Th1) of 78kDa antigen of Leishmania
donovani in mice and provided better protection against
challenge than other adjuvants, including monophos-
phoryl lipid A, liposomal encapsulation, and even
Freund's adjuvant [348]. Metronomic low dose of IL-2 as
a biological adjuvant has been applied in combination
with a recombinant vaccinia virus vaccine encoding
prostate-specific antigen (PSA) can induce prostate-spe-
cific immune responses with markedly reduced toxicities
[349].
Other interleukins that have been tested for adjuvanta-
tion effect, include IL-1α and IL1β. Intranasal immuniza-
tion with pneumococcal surface protein A or tetanus tox-
oid with IL-1β induced immune protection equivalent to
that induced by parenteral immunization with alum-
based vaccines in mice [350].
The above results show that cytokines can be used as
potential adjuvants with a variety of vaccines. Combina-
tion of cytokines can be more effective than a single one,
either as a co-expressing plasmid or a mixture of proteins
[351,352]. Nonetheless, their positive effects as adju-
vants have not been consistent in many cases. Further
studies are needed to understand their different behav-

Selection of Adjuvants for Enhanced Vaccine Potency
49
iors.
4.13. Specific Peptides/Proteins
Generally, peptides are too small to change the immuno-
genicity of an antigen. However, peptides of special
structures do have such an effect. Among all peptides
studied, IC31 seems to be among the most widely re-
ported. Actually, it is a vehicle made of both cationic
antimicrobial peptide KLKL(5)KLK and ODN and
therefore, one component can affect the effect of the oth-
er [353]. It generally promotes efficient Th1 responses
[88,353]. This adjuvant was found to increase the im-
munogenicity of a mycobacterial vaccine antigen, and
offered a comparable protective efficacy to the standard
BCG vaccine in mice [354]. In a different study, IC31
was shown to increase the HI titers and induction of
IFN- producing CD4+T cells after single vaccination
with influenza vaccine in both young and aged mice
[355].
Several other small peptides were found to be effective
as adjuvants. Co-administration of WKYMVm (a syn-
thetic peptide) with HIV, HBV or Influenza DNA vac-
cines selectively enhanced the vaccine-induced CD8(+)
T cell responses in a dose-dependent manner in mice
[356]. Another one is complement component C5a -
YSFKPMPLaR (EP54). B and T cell epitopes attached to
EP54 (0.2 µM) were easily internalized by human DCs,
which induced activation of genes specific for the Th1
and Th2 cytokines [357]. Another complement compo-
nent C5a65-74 (EP67) was also shown to induce Th1
(inflammatory) cytokines from C5a receptor-bearing
antigen presenting cells and immunization of EP67-val-
bumin resulted in higher OVA-specific antibody titers
(IgG1, IgG2a and IgG2b) in mice [358]. Its adjuvantation
effect seems to be better alum or CpG, as shown in a
recent study [359].
Host defense peptides (HDPs) are small and positively
charged peptides. Indolicidin (a bovine host defense pep-
tide; cationic;13-mer), CpG1826 (20-mer) and poly-
phosphazene can form a complex, enhancing antibody
formation and cell-mediated response in mice88. Substi-
tution of the proline residues in indolicidin with arginine
increased the synergistic adjuvant effect of the peptide.
Similarly, a synthetic cationic defense peptide (HH2; 12
mer) forms a complex with CpG 10101 and significantly
increases the immunogenicity of pertussis toxoid in terms
of toxoid-specific antibody formation (both IgG and IgA)
relative to toxoid alone in mice [360]. A modified (Arg
and IsoLeu replaced) HDP – Bac2A (11 aa) was also
shown to increase the immune response (Th2) to subcu-
taneous OVA and enhance the adjuvant activity of CpG
to OVA in mice [361]. A hypothesis for the improved
adjuvant activity of the modified peptides is the en-
hanced stability.
Peptides derived from microorganisms can be used as
effective adjuvants, such as muramyl glycopeptides,
analogs of muramyl dipeptide (MDP), glucosaminyl-
muramyl dipeptide (GMDP), and desmuramyl peptides.
Changes in both the sugar and the peptide structures can
improve the immunostimulating and adjuvant activity
and suppress adverse side effects [362]. Coadministration
of GK1 (19-mer from Taenia crassiceps cysticerci) with
an influenza vaccine increased levels of anti-influenza
antibodies (higher IgG levels than that with aluminum
hydroxide), reduced the local inflammation that accom-
panied influenza vaccination itself, and favored virus
clearance after infection in mice [363].
CEL-1000 (18-mer) is an analogue of peptide G (a
peptide from human MHCII beta chain, aa 135-149),
which is known to enhance immune responses of other
immunogenic peptides. They can be conjugated to HIV
(HGP-30) and malaria peptides as potential vaccines. SC
administration of the conjugate CEL-1000-HGP-30 led
to a 4-10-fold higher titer in antibody response than seen
with several other peptide conjugates (such as KLH) in
mice [364]. Improved adjuvant activity of CEL-1000 for
the peptide conjugates was also demonstrated by a shift
in the antibody isotypes toward a Th1 response (IgG2a).
CEL-1000 did not induce detectable self-directed or
cross reactive antibodies [364].
These peptides can also be used as mucosal vaccine
adjuvants. Macrophage-activating lipopeptide (MALP;
2kD; a TLR2/6 agonist) has been proven to be an effec-
tive adjuvant in several studies. Intranasal vaccination
with HIV-1 matrix protein p17 and immunomodulator
MALP-2 (a synthetic derivative of the MALP) stimulated
strong humoral and cellular immune responses both at
systemic and mucosal levels [365]. Similar results were
reported in heterologous prime/boost vaccination- in-
tradermal priming with the HIV-1 Tat protein and intra-
nasal boosting with the Tat protein co-administered with
the mucosal adjuvant MALP-2 in mice [366]. The pegy-
lated form of MALP-2 was also effective in inducing
strong humoral and cellular immune responses after in-
tranasal vaccination in mice [367].
Lactoferrin is a natural immune modulator and has
been shown to decrease pro-inflammatory cytokines and
chemokines, increase regulatory cytokines, enhance the
ability of BCG-infected BMDCs to respond to IFN-
activation through up-regulation of DC maturation
markers, increase IFN- production, promote generation
of antigen-specific T cells, etc [368,369]. Indeed, it was
shown that co-administration of lactoferrin with a BCG
vaccine increased host protection against Mycobacterium
tuberculosis (MTB) infection in terms of organ bacterial
load, lung histopathology, and significant reduction in

Selection of Adjuvants for Enhanced Vaccine Potency
50
tissue CFUs compared with BCG alone in mice [370]. In
addition, it was shown that the sialylated form of lacto-
ferrin was more effective in increasing the immune re-
sponse in terms of IFN-, IL-6 and IL-12 production in
mice and in protecting the animals against infection in
challenge studies [371].
CD40 is a co-stimulatory receptor on B lymphocytes.
Agonistic antibodies against CD40 have great potential
as immunological adjuvants to induce strong antibody
responses against conjugated antigen [372,373]. It is be-
lieved that the adjuvant effect is mediated at least in part
through enhanced antigen presentation by specific B cells
[373]. Such immune enhancement activity of CD40 an-
tibody is similar to that of CD40 ligand, which has been
shown to enhance cellular immune activity in mice [374].
Conjugation of anti-CD-40 to antigen (HSV glycoprotein
D) enhanced the immunogenicity of the antigen [375].
Similarly, CCR5 agonists could also be used as potential
adjuvants [376].
Another similar concept is the use of immune complex
to enhance the immunogenicity of antigens. It has been
shown that interaction of anti-CD4-binding site (CD4bs)
Abs with HIV-1 gp120 induces conformation changes
that lead to enhanced antigenicity and immunogenicity of
neutralizing epitopes in the V3 loop and C1 regions of
gp120 of several subtypes and the immune complexes as
immunogens induced serum Abs to gp120 and V3 at sig-
nificantly higher titers than those induced by the respec-
tive uncomplexed gp120s [377]. Fusion of the c-terminal
domain of gp96 to transmembrane and extracellular do-
main of rat Her2/neu enhanced the immunogenicity of
Her2/neuIt DNA vaccine [378]. It is possible that such an
immunogenicity enhancement is due to increased rigidity
of the protein antigen. Such a rigidity concept may ex-
plain why administration of plasmids encoding aggrega-
tion-prone103Q-GFP generated significantly higher anti-
GFP antibody titer than plasmid encoding control or so-
luble 25Q-GFP [379].
The above results demonstrated availability of a vari-
ety of peptides and proteins as potential vaccine adju-
vants for different kinds of vaccines. The concept of us-
ing small but immunogenic peptides is plausible, as they
are easy to synthesize and can be further modified for
optimization of their adjuvantatioun effect. Development
of a peptide vaccine adjuvant could be one of the focus
areas in the future.
4.14. RNA-Like Compounds
Foreign RNAs and RNA-like compounds can initiate
immune stimulatory effects [380,381]. Such effects in-
clude activation of TLR3, recruitment of mature APCs,
and induction of proinflammatory cytokines [381]. A
synthetic and seemingly popular dsRNA is poly(I:C)
(polyinosine- polycytidylic acid). The immune stimula-
tory effect of poly (I:C) is likely through activation of
TLR3 [382,383], and possibly other receptors as well
[380]. Stimulation of bone marrow-derived murine den-
dritic cell populations with poly(I:C) results in Th1- po-
larized maturation of dendritic cells in mice [92]. Indeed,
poly (I:C) treatment in mice was associated with a rapid
induction of inflammatory cytokines in the serum, in-
cluding IL-6, IL-10, MCP-1, TNF-, IFN-, and IFN-,
and selective increases in the numbers of NK
(NK1.1(+)CD11b(+)) cells [382]. These effects make it
an effective adjuvant for pneumococcal surface protein A
vaccine against secondary pneumococcal pneumonia in
mice [384].
The immunostimulation of poly (I:C) can be more ef-
fective than other adjuvants. Poly (I:C) complex with a
peptide epitope - E749-57 (derived from the HPV 16 E7
protein) was shown to induce strong E749-57-specific
CTL responses, leading to significant regressions of
model human cervical cancer tumors - an effect much
better than E749-57 conjugated with CpG1826 in mice
[118]. Combination of poly (I:C) with other adjuvants
could further enhance the adjuvantation effect. Inclusion
of poly(I:C) (100 µg) in a Montanide-ISA51/GP33 (50
µg, peptide 33-41 of LCMV glycoprotein) vaccine sig-
nificantly enhanced the proliferation of antigen-specific
CD8+T cells, greater than CpG 1826 (50 µg)-adjuvanted
vaccine after SC administration in mice [383]. Another
example is the combination with zymosan, a cell wall
extract from Saccharomyces cervisiae for a flu vaccine
for intranasal administration [385].
Recently, another similar adjuvant, polyI:polyC12U
(Ampligen®), a toll-like receptor 3 agonist, has been
shown to be more effective as a mucosal adjuvant for
intranasal H5N1 influenza vaccination (formalin- inacti-
vated A/Vietnam/1194/2004 strain) than for subcutane-
ous administration, and nasal vaccination provided pro-
tection against both homologous and heterologous viral
challenges in mice [386] and against homologous viral
challenge in monkeys [387]. Poly-ICLC, a stabilized
poly (I:C) by polylysine and carboxymethylcellulose, is
also effective adjuvant for a malaria vaccine in primates
[388].
It seems that these RNA-like compounds can be effec-
tive as vaccine adjuvants. Their relative adjuvantation
and potential side effects will need to be compared with
other types of adjuvants. The future of these compounds
as vaccine adjuvants remains to be seen.
4.15. Surfactant-Like Compounds
Surfactants can be used as potential adjuvants, such as
fatty acid derivatives, dimethyl dioctadecyl ammonium
bromide (DDAB), and poloxamers. Mannide monoo-

Selection of Adjuvants for Enhanced Vaccine Potency
51
leates induced both IgG1 and IgG2a antibody responses
in mice in a dispersed form without base oil [389]. On
the other hand, injection of certain surfactants can pro-
duce local irritation reactions [390]. Therefore, such sur-
factants are usually used in an emulsion. CoVaccine HT
is a microemulsion of squalene-in-water (squalene drop-
lets of 130 nm) containing the key sucrose derivatives
with seven lauric acid esters and one sulphate ester im-
mobilized on the oil droplets. This adjuvant system en-
hanced both humoral and cell-mediated responses against
a wide range of antigens, e.g. inactivated viruses, bacte-
rial subunits, recombinant proteins, virus-like particles
and peptide-protein conjugates in large non-rodent ani-
mal models and showed significantly lower reactogenic-
ity than simple mineral oil emulsions (W/O or O/W)
[391].
Other surfactant-like compounds may have similar ef-
fects. Addition of a nonionic surfactant-like block copoly-
mer CRL1005 (95:5=polyoxypropylene: polyoxyethyl-
ene) to an inactivated whole influenza virus vaccine sig-
nificantly enhanced virus-specific IgG and hemaggluti-
nation-inhibition (HI) antibody responses in mice fol-
lowing subcutaneous vaccination [392]. Addition of a
cationic surfactant-like preservative, benzalkonium chlo-
ride (BAK) in a plasmid DNA vaccine formulation (hu-
man immunodeficiency virus-1 gag) containing a non-
ionic triblock copolymer adjuvant (CRL1005) leads to an
enhancement in the gag-specific cellular immune re-
sponse [393]. Use of a lipopeptide (P3CSK4) in a respi-
ratory syncytial virus (RSV) vaccine led to a balanced
Th1/Th2 response and immunity to virus challenge in
mice and cotton rats [394].
Surfactants as adjuvants have also been used in muco-
sal vaccines. Nasal administration of antigens diphtheria
toxoid, tetanus toxoid and BBG2Na (recombinant frag-
ment of the G protein of respiratory syncytial virus) with
dimethyldioctadecylammonium bromide (DDA) induced
both mucosal and systemic immune responses [395].
DDA was found equally effective as Adju-Phos when
used with BBG2Na for protection against viral challenge
in mice and rats [395,396]. Intranasal administration of
influenza hemagglutinin (HA) vaccine with Surfacten, a
modified pulmonary surfactant, induced higher protec-
tive mucosal immunity in the airway without inducing a
systemic response in mice [283].
4.16. Bacteria and Other Related Components
Bacteria or their components represent natural foreign
antigens and may enhance immune response of other
antigens. Lactobacillus GG has been shown to have cer-
tain adjuvantation effect for live-attenuated flu vaccine in
healthy adults [397]. Gram-positive enhancer matrix
(GEM) particles derived from Lactococcus lactis was
effective for intranasal vaccination with subunit flu vac-
cine in mice [398]. Other examples include a mixture
from heat-killed Mycobacterium vaccae preparation [399]
or a particular component, such as Neisseria meningitidis
outer membrane [262] or mycobacterial binding protein 1
[400].
4.16.1. Peptidoglycans
Peptidoglycans are bacterial origin and have certain im-
munostimulant activity. These compounds activate cells
primarily via the cytosolic NLR family member NOD2
and therefore lead to enhancement of antibody produc-
tion [401]. Peptidoglycan monomer (PGM), a natural
compound of bacterial origin (Brevibacterium divarica-
tum), is a non-toxic, non-pyrogenic, water-soluble im-
munostimulator. Immunization of mice (twice) with
OVA + PGM induced significantly higher anti-OVA IgG
levels than OVA alone [402,403]. In comparison, the
individual PGM components - the pentapeptide or the
disaccharide, are not effective [402]. Addition of pepti-
doglycan monomer into OVA-containing liposomes
switched the response from Th1 to Th2 type after subcu-
taneous injection in mice [188].
Other peptidoglycan fragments, known as muramyl
peptides, are also effective. A series of di, tetrasaccharide
peptides and their stearoyl derivatives were found to ac-
tivate NF-kB pathway through NOD2 [404]. While mu-
ramyl peptides preferentially stimulate IgG1 production,
the tetrasaccharide containing muramyl peptide induces
additional production of IgG2b subclasses [404]. How-
ever, muramyl dipeptide (MDP), a component of the
peptidoglycan polymer, has minimal adjuvant properties
for antibody production compared to the TLR agonist
lipopolysaccharide under a variety of immunization con-
ditions [401].
4.16.2. cdiGMP
Cyclic dimeric guanosine monophosphate (cdiGMP) is a
bacterial intracellular signaling molecule capable of sti-
mulating protective innate immunity against various
bacterial infections [405]. Its application as a systemic or
mucosal vaccine adjuvant has been recently reviewed
[406]. Subcutaneous co-administration of β-galactosidase
(β-al) and bis-(3',5')-cyclic dimeric guanosine mono-
phosphate (cdiGMP) elicited strong cellular immune
responses, characterized by a balanced Th1/Th2 pattern,
and significantly higher antigen-specific serum IgG titers
than β- al alone in mice [407]. In a different study, it was
found that intraperitoneal coadministration of cdiGMP
with pneumolysin toxoid (PdB) or pneumococcal surface
protein A (PspA) resulted in significantly higher anti-
gen-specific antibody titers and increased survival of
mice, compared to alum adjuvant [405].

Selection of Adjuvants for Enhanced Vaccine Potency
52
4.16.3. Spores
The negatively charged and hydrophobic surface of bac-
terial spores can adsorb protein antigens for enhanced
immunogenicity [408]. It was found that mice immu-
nized intranasally or intragastrically can be protected
against challenge with different kinds of toxins [408].
Killed or inactivated spores appear equally effective as
live spores in these studies. Intranasal administration of
two model antigens, tetanus toxoid fragment C (TT) and
ovalbumin (OVA), with Bacillus subtilis spores (safe and
fully tolerated by ingestion in man) increased T cell re-
sponse and specific IgA both in the local respiratory and
distal vaginal mucosa, as well as increased antigen-spe-
cific IgG antibody in draining LN and blood in mice
[409].
4.16.4. Flagellin
IM administration of influenza virus epitope peptides
carried by recombinant flagellin (originated from non-
virulent salmonella bacteria) induced both humoral and
cellular responses and conferred some protection against
lethal challenge (H5N1) [410]. The intensive response to
flagellin is mediated by toll-like receptor 5, linking in-
nate and adaptive immunity11. Because of this, a pow-
erful vaccine can be created by fusion of flagellin with an
antigen [411].
In general, bacterial components can be effective ad-
juvants, as they are natural antigens. If these components
are not synthetically obtained, a key issue in using such
components is to control the level of potential bacterial
contaminants. Therefore, purification of such compo-
nents could be intensive during production, adding addi-
tional cost to the vaccine products.
4.17. Miscellaneous Compounds/Means as
Adjuvants
Many other compounds have been shown to provide
immunogenicity enhancement. Topical application of
Imiquimod (5% cream) over the site of subcutaneous
injection with ovalbumin enhanced anti-OVA antibody
responses 100-fold and markedly increased cellular re-
sponses in mice [412]. So is Resiquimod for the same
model antigen [413]. These are TLR 7/8 agonists, which
can be more effective adjuvants than alum for HBsAg
[414]. Naloxone, an opioid receptor antagonist, improved
the immunogenicity of heat-killed Listeria monocyto-
genes vaccine [415]. Broystatin 1, a protein kinase C
modulator, could stimulate chemokine release from den-
dritic cells and be used for cancer vaccines [416]. To-
matine, a glycoalkaloid (rod-like structures under micro-
scope), was shown to be capable of stimulating potent
antigen- specific humoral and cellular immune responses
leading to protection against malaria, Francisella tularen-
sis and regression of experimental tumors [417]. Other
plant isolates were found to promote both Th1 and Th2
effects in mice such as paclitaxel (a diterpenoid) for
ovalbumin [418], RLJ-NE-299A, a mixture of iridoid
glycosides for hepatitis B surface antigen (HBsAg) [419],
and acylated derivatives of iridoid glycosides for oval-
bumin [420]. Other compounds as potential adjuvants
include anti-viral drugs such as Ribavirin [421], the ac-
tive form of vitamin D3 (1,25(OH)2D3) [422,423], cal-
cium phosphate [293,424], and herbs such as Astragalus
membranaceus and Scutellaria baicalensis [425,426], or
herbal extract [427] and tumor cell components [428,
429].
A recent report shows that green laser (520 nm) can
mobilize APCs and facilitate antigen uptake at the vac-
cine injection site, leading to increased humoral and
cell-me- diated immune responses to OVA after im ad-
ministration in mice [430]. Such a novel concept needs to
be verified, regarding its relative adjuvantation and side
effects.
5. Adjuvant Combinations
The above adjutants may enhance immunogenicity
through different mechanisms. Therefore, combining two
or more adjuvants may further enhance the degree of
immune response due to simultaneous, multiple mecha-
nisms of actions [385]. In several studies, combination of
aluminum salts and deacylated MPL was shown to in-
duce a robust and persistent immune response with HPV-
16/18 protein VLP vaccine in women (Cervarix, GSK
Biologicals) [431] and more effective with a hepatitis B
vaccine than the commercial product Engerix-B in hu-
mans [432]. Another reason for combing adjuvants is to
change the type of immune response. For example, com-
bining emulsion and a synthetic lipid A in Fluzone vac-
cine significantly and synergistically increased Th1 type
response in mice [304]. Use of CpG often changes a
Th2-type immune response of aluminum-based adjuvants
to a Th1-type immune response [107,242,433].
A variety of adjuvant combinations have been tried.
Aluminum salts are often used with CpG ODNs to en-
hance and/or balance the immunogenicity of many dif-
ferent types of antigens [264,434,435]. Naloxone (an
opioid receptor antagonist) has been recently evaluated
with aluminum salts with use [436,437]. Liposomes can
easily accommodate another adjuvant for enhanced im-
munogenicity, such as CpG ODNs [119,438-440], TLR7
ligand-3M-019 [441], Quil-A [442], MPL [443], and
DNA [444]. Liposomes have been shown to enhance the
uptake of CpG ODN (16-mer) by immune cells in spleen
and lymph nodes [440] and DC maturation by these ad-
juvants [438]. Other combinations include MPA/treha-
lose dicorynomycolate for a protein vaccine [445], chi-

Selection of Adjuvants for Enhanced Vaccine Potency
53
tosan/LTK63 (a nontoxic E. coli enterotoxin mutant) for
DT-conjugated group C meningococcal polysaccharide
antigen [446], chitosan/muramyl di-peptide (MDP) for
Helicobacter pylori urease [230], PLG microparticles/
MF59 for recombinant protein antigens [447], PLGA
particles/LPS for West Nile encephalitis [291], pro-
teosome/LPS for influenza virus [448], QS-21 combina-
tions with a variety of adjuvants for KLH-conjugated
peptide vaccine [237], and FML (a glycoprotein complex)
combinations with a variety of adjuvants for visceral
leishmaniasis (VL) [449]. More complex adjuvant sys-
tems (3 adjuvant components) have also been tried.
These include, GM-CSF/IL-2/emulsion for HPV16 E7
peptide antigen [352], liposome/polycation/DNA (LPD)
particles for HPV 16 E7 protein antigen [450], CpG
(1826)/indolicidin/ olyphosphazene for OVA[88], QS21/
GM-CSF/MPL/emulsion system for KLH-conjugated
peptide antigens [244]. Although some of these combina-
tions have been compared, the results are difficult to in-
terpret as their doses and preparations are different
[237,449].
A widely investigated adjuvant combinations contain-
ing 2 adjuvant components are the AS series of adju-
vants, such as AS01 (liposomes containing deacylated
MPL and QS21) [451], AS02 (an oil-in-water emulsion
containing deacylated MPL and QS21) [452,453], and
AS05 (liposomes containing aluminum hydroxide, dea-
cylated MPL and QS21 in a weight ratio of 10:1:1) [454].
These systems seem to be effective in enhancing both
humoral and cellular immunogenicity of different types
of antigens in human subjects [452,455] and can be more
effective than commercial vaccines for certain patient
populations 453. In a comparative study on these systems,
AS01B offered the best enhancement in cellular immu-
nity for a malaria vaccine relative to AS02A, and AS05
both in Rhesus macaques [454] and human subjects
[456]. The superiority of AS01B was confirmed in a re-
cent study with recombinant hepatitis B surface antigen
in terms of cellular immune responses among several AS
series of adjuvants and all the AS adjuvants (MPL/QS21)
are more effective than CpG (dosed at 0.5 mg) in healthy
adults [457]. These studies clearly indicate that lipo-
somes are superior to emulsions, suggesting that more
rigid particulates might be more immunogenic.
It should be noted that even combinations of adjuvants
may have limited effect, such as use of AS systems in a
malaria vaccine [458]. Such combinations could further
enhance the side effects of individual adjuvants and po-
tentially lead to greater and perhaps different types of
effects from those generated by individual adjuvants. On
the other hand, infinite enhancement of vaccine immu-
nity may not the best choice for developing vaccines
against certain viruses. This is because vaccines can ac-
tually enhance the susceptibility of the host to virus in-
fection, such as certain flavi-, corona-, paramyxo-, im-
munodeficiency-, and lentivirus vaccines, likely due to
antibody-dependent enhancement of viral entry [459].
6. Summary
Development of the first-generation human vaccines
has been largely semi-empirical. Aluminum salts are the
only type of adjuvants used in these vaccines. Although
the efficacy and safety of aluminum salts have been well
established through the long history of use, a minimum
quantity is recommended for use in a vaccine, as we con-
tinue to gather new evidence for their potential side ef-
fect.
Due to the limited adjuvantation effect of aluminum
salts, constant mutation of existing microbes, and ever
identification of new disease-causing microbes, extensive
search of more effective adjuvants has been the focus of
many scientists for many years. Although many novel
adjuvants have been identified, few have been commer-
cialized in limited market regions. This is partly due to
the difficulty in elucidating the exact mechanisms of ad-
juvantation, especially for multi- component adjuvant
systems, and lack of long-term safety data. Another fact
is that none of these novel adjuvants seem to work well
with different kinds of vaccines. Therefore, further in-
vestigations would continue to address these issues.
Among all adjuvants examined, it seems difficult to
pinpoint the most effective one, as these adjuvants are
evaluated in different laboratories, with different animal
models, and for different indications. Even some adju-
vants were compared side-by-side by the same investi-
gators, the amount of adjuvant may still be different and
thus, conclusions from these studies are still debatable. It
is expected that future comparative studies would be de-
signed such that data could support selection of the right
adjuvant. Novel adjuvants will be continually searched
and evaluated, but their successes will be based on our
further understanding of the interactions between human
body and foreign microorganisms on a molecular level
[460-463]. Ideally, such adjuvants would offer earlier,
robust and durable immunity with less adverse events.
A clear trend in the development of effective adjuvant
is to use an adjuvant system consisting of two or more
adjuvants. Good examples are the AS systems, as dis-
cussed above. Most adjuvant systems seem to be more
effective than individual adjuvants due to additive or
frequently synergistic effect, which may result from
combining the same or different classes of adjuvants
[344,352]. One the other hand, use of such systems
should not be first choice, unless no individual adjuvant
would be able to provide adequate immunogenicity en-
hancement. If an adjuvant system is to be used, it should

Selection of Adjuvants for Enhanced Vaccine Potency
54
ideally comprise no more than two adjutants. Any adju-
vant system consisting of three or more adjuvant would
need much more studies to understand the interactions
among all the adjuvants, and between adjuvants and an-
tigens. It would be very difficult to decipher the contri-
bution of individual adjuvants, and their mechanisms of
actions. In addition, preparation of such a system with
antigen and subsequent characterization would be more
laborious and batch-to-atch consistency would not be
easy to control.
The human society is still facing many uncurable dis-
eases today. Given the high cost of drug product devel-
opment, the high pressure of reducing health care cost,
and the public desire for a better quality of life, the de-
velopment of both prophylactic and therapeutic vaccines
would remain a top focus of many pharmaceutical com-
panies and public health leaders.
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