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Adjuvants for the Future

Richard T. Kenney
Clinical R&D and Medical Affairs, Vaccines for Virus Diseases, North America
GlaxoSmithKline Biologicals, Columbia, Maryland, U.S.A.
Alan S. Cross
Center for Vaccine Development, University of Maryland School of Medicine,
Baltimore, Maryland, U.S.A.
INTRODUCTION
The past decade has seen dramatic progress in our understanding of immune mechanisms
and host defense. Along with the ability that vertebrates have to acquire immunity to
pathogenic antigens by expanding specific populations of T and B cells and making
cytokines and antibodies, scientists have discovered that like invertebrates, we have
multiple innate pathways to activate more generic host responses through a whole new
family of receptors. Over 80 years ago, Ramon demonstrated that it was possible to
artificially increase antigen-specific levels of diphtheria or tetanus antitoxin by the addition
of bread crumbs, agar, tapioca, starch oil, lecithin, or saponin to the vaccines (1). Since
then, aluminum salts have been the dominant substance used and are still the only
adjuvant currently used in licensed vaccines in the United States. The field has become
much more sophisticated recently with the introduction of numerous new adjuvants and
new concepts regarding the mechanisms of action. In this brief chapter, we review the
modern adjuvants used in a variety of current and experimental human vaccines. After a
more general discussion of adjuvants, including their definition, mechanisms of action, and
safety, we will discuss recent clinical trials of investigational adjuvants. For additional study
of this complex subject, including a historical perspective, the reader is referred to
published reviews of vaccine adjuvants (see Refs. 2–4).
DEFINITIONS
The term ‘‘adjuvant’’ (from the Latin adjuvare, meaning to help) was coined in 1926 by
Ramon for a substance used in combination with a specific antigen that produces a
stronger immune response than the antigen could if used alone (5). The enormous
diversity of compounds, which increase specific immune responses to an antigen and thus
function as vaccine adjuvants, makes any classification system somewhat arbitrary.
Adjuvants can be loosely categorized in terms of their source or their physical nature as (i)
mineral salts; (ii) mycobacterial, bacterial, and plant derivatives; (iii) surface-active agents
and microparticles; (iv) polymers, cytokines, vitamins, and hormones; and (v) synthetic
constructs. Those listed in Table 1 are examples of immunopotentiators used during the
past 25 years.
They are grouped according to origin rather than mechanism of action, because the latter
are incompletely understood for most adjuvants. All agents in Table 1 have
immunomodulating capabilities and are reported to augment the immune response to
specific antigens; nonspecific enhancers of the immune response that principally stimulate
innate immunity are largely excluded. A comprehensive list of adjuvants, beyond the scope
of this chapter, is available and updated by the NIAID (National Institutes of Health/National
Institute of Allergy and Infectious Diseases, U.S.A.) (6).
A ‘‘carrier’’ is an immunogenic protein to which a hapten or a weakly immunogenic antigen
is bound (7). It may also be a living organism (or vector) bearing genes for expression of
the foreign hapten or antigen on its surface. A naked DNA vaccine is a carrier in the sense
that it entails injection into the host of a plasmid-based DNA vector that encodes the
production of the protein antigen (8). Carriers increase the immune response by providing
T cell help to the hapten or antigen.
A ‘‘vehicle’’ provides the substrate for the adjuvant, the antigen, or the antigen-carrier
complex. Unlike the carriers listed in Table 1, vehicles are not themselves immunogenic.
Like carriers, most vehicles can enhance the immune response to antigens alone and are
sometimes considered to be another class of adjuvants. Scientists have also investigated
the result of combining adjuvants with different sources/mechanisms of action to increase
their immunostimulatory effect. These ‘‘adjuvant formulations’’ can, in some cases,
combine delivery improvement and immune modulation. Thus, in most cases, an adjuvant
formulation is composed of two or more adjuvants with complementary immunomodulating
effects, such as the adjuvant systems (ASs) being developed by GlaxoSmithKline (GSK).
Many examples of such adjuvant formulations have been tested in humans.
MECHANISMS OF ACTION
The effects of adjuvants can be strongly impacted by (i) the nature and dose of the
immunogen; (ii) the nature and dose of the adjuvant(s) or carrier in the formulation; (iii)
the stability of the formulation; (iv) the immunization schedule; (v) the route of
administration; (vi) the species of animal; and (vii) the genetic and other biologic variations
within species, including their immune status. The discovery of a class of receptors similar
to Toll, an essential receptor for innate defense against fungal infection in Drosophila,
changed the approach to understanding adjuvants over the past decade. Mammalian
homologues or tolllike receptors (TLRs) have been discovered that provide a means to
activate immune cells such as macrophages and dendritic cells with microbial ‘‘danger
signals’’ (9). Bacterial lipopolysaccharide (LPS), which has long been known to be
inflammatory if injected in very small quantities or if present as a trace contaminant in
vaccines, was found to be a ligand for the TLR4 receptor (10). Since then, lipopeptides,
flagellin, single- and double stranded RNA, and CpG DNA found principally in bacteria and
viruses have been linked to individual TLRs and provide a scientific basis for the
development of adjuvants (11–13). However, many existing adjuvants may not function via
the TLRs, and their mechanisms remain unknown (14).
It is known that adjuvants can select for or modulate humoral or cell-mediated immunity,
and they do this in several ways. First, antigen processing can be modified, leading to
vaccines that can elicit both helper T cells and cytotoxic lymphocytes (CTLs) (reviewed in
Ref. 15). Second, depending upon the adjuvant, the immune response can be stimulated in
favor of type 1 or type 2 immune responses (16). For example, complete Freund’s adjuvant
and the QS-21 adjuvant can elicit DTH and MHC class I CTL responses when mixed with
protein antigens, peptides, or inactivated viruses (17). Many other adjuvants, such as
aluminum salts (16) and nonionic block polymers (18) elicit principally antibody responses
when combined with protein antigens or inactivated organisms, perhaps by activating APCs
by an IL-4-dependent mechanism (19). Third, adjuvants can augment the immune
response by preferentially stimulating Th1 or Th2 CD4 T-helper cells (20). The Th1
response is accompanied by secretion of interleukin-2 (IL-2), interferon-g (IFN-g), and TNF-a
leading to a CMI response, including activation of macrophages and CTL and high levels of
IgG2a antibodies in mice. The Th2 response is increased by secretion of IL-4, IL-5, IL-6, and
IL-10, which provide better help for B cell responses, including those of IgG1, IgE, and IgA
isotypes in mice. Aluminum salts and MF59 principally stimulate the Th2 response (21,22),
while the Th1 response is stimulated by many adjuvants, such as muramyl dipeptide,
monophosphoryl lipid A (MPL), and QS-21 (3). Vaccine adjuvants can modulate antibody
avidity, specificity, quantity, isotype, and subclass against epitopes on complex
immunogens (23–26). For example, only certain adjuvants, vehicles, and adjuvant
formulations can induce the development of the protective IgG2a antibody isotype against
Plasmodium yoelii (27), a mouse model of malaria. While an adjuvant’s effect on
immunogenicity can be studied preclinically in animals, the models do not always
anticipate their level of efficacy or safety in humans (28,29). However, decades of basic
cellular research, preclinical experiments, and clinical safety and immunogenicity studies
have led to a significant expansion in the understanding of the mode of action of many
adjuvants. This is beginning to allow for rational design and combination of the optimal
adjuvants for a particular antigen in a specific population, which can lead to safer and more
effective vaccines.
SAFETY
During the past 75 years, many adjuvant compounds have been studied, but most were
never accepted for routine vaccination because of their immediate toxicity and fear of
delayed side effects. The current attitude regarding the risks and benefits of vaccination
favors safety over efficacy when a vaccine is given to a healthy population of children and
adults (30). In high-risk groups, including patients with cancer and AIDS, and for
therapeutic vaccines, an additional level of toxicity may be acceptable when the benefit of
the vaccine is substantial. However, the absolute safety of any vaccine cannot be
guaranteed, so the risks must be minimized. Undesirable reactions can be grouped as
either local or systemic.
The most frequent local adverse effects of vaccination are tenderness and swelling, with
the most severe ones involving the formation of painful induration and nodules at the
inoculum site. The mechanisms for such severe local reactions include formation of
inflammatory immune complexes at the inoculation site by combination of the adjuvanted
vaccine with preexisting antibodies resulting in an arthus-type reaction. In some cases,
poor biodegradability of the adjuvanted vaccine may result in prolonged persistence in the
tissues and reactive granuloma formation. Such local reactions are of concern for depot-
type adjuvants and living vectors such as BCG. Those adverse effects are rare events with
today’s vaccines. Clinical studies typically reveal this type of problem before a vaccine is
licensed and development is halted. Severe local reactions in humans followed
subcutaneous injections of incomplete Freund’s adjuvant (IFA), a mineral oil emulsion,
using early formulations made with a mannide monooleate stabilizer that contained free
fatty acid impurities. However, these lesions did not occur with IFA injected
intramuscularly, and that contained the stabilizer without impurities (reviewed in Refs. 28
and 31). IFA has been administered to more than a million people worldwide (31–34).
Despite the apparent long-term safety of this adjuvant (35), the risk/benefit ratio is felt to
be too high for commercial use.
To date, vaccine adjuvants have caused few severe acute systemic adverse effects. More
theoretical risks include the induction of autoimmunity or cancer. Fortunately, in 10-, 18-,
and 35-year follow-up studies, the incidence of cancer, autoimmune and collagen disorders
in 18,000 persons who received the IFA adjuvanted influenza vaccine in the early 1950s
was not different from that in persons given aqueous vaccines (32,35– 37). Autoimmunity
can be triggered by an infection through either specific or nonspecific mechanisms,
although this has been associated with vaccination only in rare circumstances, such as
when a form of Guillain-Barre´ syndrome was linked to the 1976 to 1977 vaccination
campaign against swine influenza (38). Extensive epidemiological studies have failed to
show an association of autoimmune disease with vaccination in nearly all instances (39).
Studies in animals can provide signals that would lead to further study. Anterior chamber
uveitis has been reported with MDP and several MDP analogues in rabbits (40) and
monkeys (41), and has been systematically sought in at least one adjuvant vaccine study
involving 110 volunteers, but was not detected (42). Adjuvant-associated arthritis (43,44)
has not been reported in humans, even after long-term follow-up (33,35,45). Anaphylactic
reactions, angioedema, urticaria, and vasculitis have been described following the
administration of the majority of vaccines, although severe events are rare (29).
Finally, a syndrome known as macrophagic myofasciitis (MMF), characterized by diffuse
arthromyalgias and fatigue in connection with muscle infiltration by macrophages and
lymphocytes, has been described in France (46), although a causal association with
vaccination has not been established.
REGULATORY ISSUES
In concert with the progress of the International Conference on Harmonisation (ICH) of
technical requirements for registration of pharmaceuticals for human use, worldwide
regulatory guidance on the development and testing of vaccines has expanded
significantly in the past few years. Documents covering nearly every aspect of drug and
biologic development are being created and revised in an effort to enhance and
standardize the quality, safety, and efficacy of pharmaceutical products
(http://www.ich.org, http://www.fda.gov/cber/guidelines.
htm, and http://www.emea.eu.int). There is little advice directed specifically at the
development of adjuvants in the United States, apart from their use in combination
vaccines (47).
The European Medicines Agency (EMEA) recently published a guidance that emphasizes a
number of additional points:
quality of manufacture of the adjuvant alone and in combination with vaccine antigens,
nonclinical proof of concept and toxicity studies, and clinical development that assesses
the adjuvant effect and dose required (48). It is important to note that as a rule, adjuvants
are not licensed on their own. Since each combination of one or more antigens with an
adjuvant has its own unique safety and efficacy profile, they are licensed and regulated as
individual vaccine products in combination.
ADJUVANTS USED IN LICENSED VACCINES FOR HUMANS
Several adjuvants are licensed with their vaccines for human use in various parts of the
world, including aluminum compounds, MF-59, virosomes, exotoxins, and AS04.
Aluminum Compounds
Aluminum salts, particularly aluminum hydroxide or phosphate, have been used for over 80
years (49) and have become the most widely used adjuvants in human vaccines. Vaccine
antigens can be adsorbed to the amorphous crystalline gel by electrostatic interactions
between proteins and the positively charged aluminum hydroxide. Alternatively, negatively
charged aluminum phosphate gels can bind proteins through a ‘‘ligand exchange’’
between hydroxyl and phosphate groups (21). Calcium phosphate has also been used to
adsorb DPT, inactivated polio vaccines, and allergens (50). The following licensed,
parenterally administered human vaccines are combined with aluminum: diphtheria,
pertussis, and tetanus alone or in various combinations with Haemophilus influenzae type b
(Hib), inactivated polio, hepatitis B, hepatitis A, a rabies vaccine, an anthrax vaccine, and
GardasilTM a human papillomavirus vaccine recently licensed by Merck.
The major advantage of using aluminum adjuvants is their safety record after billions of
doses, and the development of earlier, higher, and longer-lasting antibody after primary
immunization compared to primary immunization with soluble vaccines, particularly of
soluble toxoids, although the aluminum-adsorbed vaccines do not show any advantage
over soluble preparations for booster responses (51). While aluminum adjuvants can
stimulate Th2 type responses in mice and the production of cytokines such as IL-4 and IL-5,
as well as B cell production of IgG1 and IgE, they fail to stimulate Th1 responses such as
IFN-g production and B cell IgG2a secretion.
The mechanism of adjuvanticity is still a subject of debate and includes formation of a
depot at the injection site allowing slow release of antigen, stimulation of immunoreactive
cells via activation of complement, activation of macrophages, and efficient uptake of
aluminum-adsorbed antigen particles by antigenpresenting cells because of their
particulate nature and optimum particle size (<10 mg) (16,51).
The limitations of aluminum adjuvants include (i) the potential for induction of occasional
painful nodules or swelling and erythema at the inoculation site, and the induction of
antigen-specific IgE antibody that correlates with such local reactions (51,52), although the
incidence of systemic immediate hypersensitivity is probably less than one in a million
(53).
(ii) Aluminum has been detected at the site of subcutaneous injections for up to one year in
animals (51), so it is not readily ‘‘biodegradable.’’ In addition, the aluminum compounds
have several immunological drawbacks including (iii) their inability to enhance humoral
immunity against certain vaccines in humans such as typhoid (54), influenza hemagglutinin
antigen (55), and Hib capsular polysaccharide-tetanus toxoid conjugate (56), and (iv) their
near total inability to elicit cell-mediated immune responses, particularly cytotoxic T-cell
responses to intracellular organisms (16). Finally, (v) careful formulation of aluminum
adjuvant preparations is required for reproducibility, they cannot be sterilized by filtration,
and they cannot be frozen or readily lyophilized (51).
Microfluidized Oil/Water Emulsion (MF59)
A series of squalene emulsions were prepared using a microfluidizer to generate small
particle (200–300 nm), oil-in-water (O/W) emulsions that had low-viscosity and were
biodegradable (57). The most stable emulsion, termed MF59, consists of 4.3% (vol/vol)
squalene and 0.5% (vol/vol) each of the surfactants Tween 80 (polyoxyethylene sorbitan
monooleate) and Span 85 (sorbitan trioleate). Overall, MF59 generates antibody titers
consistently higher than those obtained with aluminum hydroxide, equal to or higher than
IFA, and equal to or lower than CFA, although it does not stimulate antibody responses
against squalene (58). Results of timed injection studies suggest that MF59 microdroplets
activate the immune system in the absence of antigen. It is postulated that macrophage
uptake of the emulsion droplets results in cytokine production, which leads to an enhanced
immune response in the presence of the antigen (57). MF59 has been tested in a variety of
animal species, showing a good safety profile and a significant increase of the immune
response to several subunit antigens including CMV, HSV, HIV, HCV, HBV, and influenza
antigens.
Novartis Vaccines (formerly Chiron Biocine, Siena, Italy) registered an influenza vaccine
adjuvanted with MF59 as FLUADTM in much of Europe, which has been given to more than a
million people (59). The MF59 formulation has also been tested in combination with
pandemic influenza antigens, recombinant HSV glycoproteins, hepatitis B virus PreS2/S
antigens, and HIV envelope proteins with various degrees of success (22).
Study populations have included healthy adults (HSV, HBV, HIV, influenza) (60), elderly
populations (influenza) (61), and infants and children (HIV) (57). Overall, MF59 has had
acceptable reactogenicity profiles, although in the NIH comparison trial of multiadjuvanted
HIV gp 120 vaccine described above, MF59 MTP-PE (in addition to SAF threonyl-MDP)
induced significantly more moderate to severe local reactions than did other adjuvants
(62).
Virosomes
Immunopotentiating reconstituted influenza virosomes (IRIV) are 150 nm unilamellar
vesicular proteoliposomes composed of influenza H1N1 surface glycoproteins intercalated
in a mixture of natural and synthetic phospholipids (63). The influenza HA antigen binds to
sialic acid on the surface of antigen-presenting cells that take up the particles by receptor-
mediated endocytosis and, subsequently, by pH induced membrane fusion with the
phagolysosomal membrane. IRIV can act as antigen carriers to deliver many types of
antigens bound or conjugated to the surface or internalized. Given the unique properties of
the system, after proteolytic degradation, the antigenic peptides can become complexed
with both MHC class I and class II molecules to be expressed on the surface of the APC.
The initial application of this system was with a virosomal hepatitis A vaccine. Berna
Biologics, Ltd. (now owned by Crucell, Leiden, The Netherlands) registered EpaxalTM in
several European, Asian, and South American countries after clinical testing, which showed
an acceptable immune response and a significant reduction in local reactions compared to
the conventional alumadsorbed vaccine (64,65). A second example, Inflexal VTM (Solvay
Pharmaceuticals, Brussels, Belgium), is a trivalent influenza vaccine that is made by mixing
three monovalent virosomes, each one containing the seasonal HA and NA glycoproteins
recommended annually by WHO (63). This technology was licensed by Solvay, and their
virosomal influenza vaccine has been marketed as InvivacTM since 2004, showing similar
immunogenicity to FLUAD and decreased reactogenicity (66). The virosome system is
being further developed for use with a DPT vaccine, as well as other antigens.
Exotoxins
The bacterial ADP-ribosylating exotoxins (bAREs) represent a potent group of proteins that
have been studied as enteric, nasal, and topical adjuvants for decades, and this category
includes both licensed (albeit since withdrawn) and experimental vaccines. The only
licensed vaccine that included a bARE as adjuvant was the intranasal virosome-based
influenza vaccine that included a low dose of the E. coli heat-labile toxin (LT) for mucosal
immunization (61,67). In pre-licensure trials, the vaccine was well-tolerated and elicited
secretory IgA mucosal responses to influenza hemagglutinin, as well as serum antibody
responses (68,69). However, post-licensure surveillance indicated that the vaccine was
associated with an increased occurrence of Bell’s palsy, and it was concluded that the
intranasal administration of wild-type LT was likely to be an important contributing factor
(70,71). Interestingly, extensive preclinical toxicology studies did not predict such adverse
reactions (72).
AS04
GSK Biologicals has been developing novel AS for more than a decade. These are unique
combinations of different compounds with immunomodulating abilities that can tailor an
immune response to a specific disease and target population.
One of these, AS04, is a combination of aluminum salt and 3-O-desacyl-40-MPL, a purified,
detoxified derivative of bacterial lipopolysaccharide. MPL acts through binding to TLR4, a
toll-like receptor found on macrophages and dendritic cells, to enhance the secretion of
inflammatory cytokines and chemokines.
AS04 was combined with hepatitis B antigen to create FENDrixTM, which is approved in by
the EMEA for the prevention of hepatitis B in high-risk pre-hemodialysis and hemodialysis
patients over 15 years. Clinical trials demonstrated
that the hepatitis B/AS04 vaccine resulted in higher antibody levels, enhanced cell-
mediated immunity and increased rates of seroprotection compared to a classical hepatitis
B vaccine adjuvanted with aluminum salt alone (73). In addition, a cervical cancer vaccine
called CervarixTM, which is formulated with human papillomavirus antigens as virus-like
particles (VLPs) of type 16 and 18 and adjuvanted with AS04, was recently approved in
Australia and has been submitted for licensure in several other areas of the world (74–76).
EXPERIMENTAL ADJUVANTS IN HUMANS
The number of commercially feasible adjuvants tested in animals and humans (Table 1) is
too large to review in this short chapter. Instead, a smaller number of modern adjuvants or
adjuvant formulations used to enhance a variety of experimental vaccines in humans
(Table 2) will be considered. The development of experimental adjuvants has been driven
principally by the failure of aluminum compounds to (i) enhance many vaccines in man
(31), (ii) enhance subunit vaccine antigens in animals (28,144,145), and (iii) to stimulate
cytotoxic T-cell responses (146). In many instances, several adjuvants have been combined
in one adjuvant formulation, hoping to obtain a synergistic or additive effect.
Emulsion-Based Formulations
Two basic concepts have emerged in the manufacture of aqueous and oil combinations
that describe the dispersion of one liquid as particles within a second liquid that is
continuous (147). Surfactants, which are compounds that contain both polar and nonpolar
groups, are added to stabilize the emulsions.
Their hydrophilic/lipophilic balance determines the state of the emulsion that forms. Water-
in-oil (W/O) emulsions that Freund used were initially very unstable and viscous and caused
strong local reactions, yet they are very efficient at inducing an immune response to weak
antigens. Newer oils and surfactants are now used, which allow the development of stable
fluid emulsions that are safer (147).
Mineral oils in W/O emulsions, such as IFA, stay at the injection site, and are slowly
eliminated by macrophages or metabolized to fatty acids, triglycerides, phospholipids, or
sterols (148). Protein antigens are released very slowly from this matrix. Proprietary, highly
refined emulsifiers from the mannide monooleate family in a natural metabolizable oil
solution were developed by SEPPIC (Paris, France), named MontanideTM ISA 51 and ISA 720.
Both Montanide adjuvants induce a strong immune response, but severe local reactions
may limit their use.
O/W preparations containing small particles of oil dispersed in an aqueous continuous
phase are more easily cleared from the injection site. The droplets with antigen can be
endocytosed by APCs or readily pass from the injection site to lymphatics (149). A major
part of the effort to develop immunostimulators as vaccine adjuvants has been devoted to
the characterization of mycobacterial cell wall components and their analogues as
additions to these preparations (31,150,151). The most studied component of the cell wall
has been the muramyl dipeptide, N-acetylmuramyl-L-alanyl-Disoglutamine (MDP). Three
promising derivatives of MDP were developed because of its residual toxicity and
pyrogenicity; they include a butyl-ether derivative (MurabutideTM) (152,153), threonyl-
MDP(57,154), and muramyl tripeptide dipalmitoyl phosphatidylethanolamine (MTP-PE)
(57,145).
Because MDP in water provides only a modest adjuvant effect in mice (155,156) and
humans (152), threonyl-MDP and MTPPE have been administered in oil emulsion vehicles in
attempts to improve potency. The Syntex Adjuvant Formulation (SAF) preparation (Syntex
Research, acquired by Roche in 1995) is an O/W emulsion vehicle. The vehicle contains 5%
squalane, 2.5% PluronicTM L121, and 0.2% polysorbate 80 (TweenTM 80) in phosphate-
buffered saline, pH 7.4 (149,157). Squalane, used in several modern adjuvant emulsions, is
metabolizable oil used in many over-the-counter drugs and cosmetics. Pluronic 121 is a
nonionic block copolymer discussed below. SAF elicits both cell-mediated (lymphocyte
blastogenic) and humoral responses, but it is highly reactogenic so it is no longer studied
as a vaccine adjuvant. GSK Biologicals is also developing a proprietary O/W emulsion-based
AS for use in a prepandemic H5N1 influenza vaccine to provide heterologous immunity
(115). Finally, MF-59 has been studied by Chiron in various experimental vaccines for HIV,
herpes simplex, and HPV (57,90,94,158).
Monophosphoryl Lipid A
The adjuvant effect of LPS was described in 1956 (159). Most of the adjuvanticity and
toxicity of LPS are associated with the lipid A region of the molecule (160). The LPS of
Salmonella minnesota R595 has been detoxified without destroying its adjuvant activity by
exposing the LPS to mild hydrolytic treatment (161). The resultant monophosphoryl
derivative of lipid A, called MPL, is a highly adaptable molecule that can be used effectively
in many adjuvant formulations (162). The immunopotentiating nature of MPL may be
associated with its capacity to induce cytokines such as IL-12 (163), IFN-g, IL-1, and IL-2 in
mouse and human macrophages (164–166). MPL promotes antigen-specific DTH and a
predominant murine IgG2a immunoglobulin response characteristic of TH1 help (167).
Numerous animal and human studies testify to the utility of MPL as an adjuvant, used alone
or combined effectively with other adjuvants and vehicles for capsular polysaccharide,
protein, and peptide antigens (74,123,162). In the past decade, many clinical studies have
utilized MPL or DETOXTM (MPL plus cell wall skeleton of Mycobacterium phlei in a squalane-
inwater emulsion vehicle) as vaccine adjuvants in volunteers (75,76,79–
82,95,119,123,130,131,143,168–170) (Table 2). More recently, synthetic lipid A mimetics
(aminoalkyl glucosaminide 4-phosphates) that share most of the properties of MPL, have
been developed by Corixa, and are now being developed by GSK (83,171,172).
Several AS under development by GSK Biologicals contain MPL combined with O/W
emulsions. AS02 (formerly known as SBAS2) is a proprietary O/W emulsion containing MPL
and QS-21 that causes strong antibody responses as well as Th1 and CTL cellular
responses. Phase 1/2 studies have been conducted in hepatitis (82), HIV (95), and with
multiple HIV vaccine formulations (96). AS02 has been broadly studied in malaria, most
recently with RTS,S, a circumsporozoite (CS) subunit antigen fused to the hepatitis S
antigen (119,130,132–134), or with FMP1, a 42-kDa fragment of the merozoite surface
protein-1 (131,143). RTS,S with AS02 demonstrated efficacy in Phase 2b field trials in The
Gambia (168) and Mozambique (135–137).
AS04 (described above) is comprised of aluminum salts and MPL for use in licensed
vaccines. This AS has also been studied in HSV (173).
Exotoxins
Recombinant LT, which is one of the most potent mucosal adjuvants (174), has been shown
to be safe and immunogenic by transcutaneous immunization (TCI) in humans (175).
Antigens can be formulated with LT and delivered using a topical patch for delivery to the
dense population of dendritic cells that are resident in the epidermis. LT-specific IgG and
IgA antibodies were present in both stool and urine, implying the induction of a strong
mucosal immune response. The potent activation of epidermal Langerhans cells allows LT
to adjuvant the response to a coadministered enterotoxigenic Escherichia coli(ETEC)
antigen as well (176). Serological and antibody-secreting cell (ASC) responses to the LT
and the E. coli surface antigen CS6 were comparable to those seen following a protective
oral challenge, suggesting that TCI can potentially elicit effective immunity similar to
natural infection with ETEC, although local rashes may limit its broad applicability (176). LT
is being studied as an immunostimulant to be used with TCI in conjunction with an injected
influenza vaccine (104,177). Other groups are using detoxified mutants of LT to explore the
potential for oral or intranasal vaccination (178–180), however, there is at least a
theoretical concern that the LT could traffic to the brain and could cause inflammation
there (72).
Saponins
Saponins are triterpene glycosides that can be isolated from the bark of the Quillaja
saponaria Molina tree, a species native to South America (181). A partially purified saponin,
Quil A, has been used widely as an adjuvant in veterinary vaccines (182).
Quil A is a heterogeneous mixture of glycosides. Analysis by high performance liquid
chromatography (HPLC) reveals at least 24 peaks that vary in their adjuvanticity and
toxicity in mice (183). Quil A has also been tested extensively as part of immune-
stimulating complexes known as ISCOMsTM, which are colloidal cage-like 40 nm particles
consisting of antigen, cholesterol, phospholipids, and Quil A (184). Despite their potent
adjuvanticity, ISCOM vaccines have only recently been administered to humans because of
the local and systemic toxicity of Quil A in mice (184,185). An influenza-ISCOM vaccine for
humans containing a less toxic saponin fraction is under development, which shows a
strong cellular immune response (103,186).
QS-21 (StimulonTM) is one of at least 24 structurally distinct triterpene glycosides isolated
from Quil A, and is being developed by the Antigenics, Inc. (Framingham, Massachusetts,
U.S. ). It demonstrated the proper balance of low mouse toxicity and maximum
adjuvanticity, and it eliminated the problem of lot-to-lot variation characteristic of Quil A
(183).
QS-21 is novel in that it can improve the immunogenicity of protein and polysaccharide
antigens (187) in a variety of small animals, dogs, or primates. It also uniquely stimulates
both humoral and cell mediated immunity, including potent class I-restricted cytotoxic T
lymphocyte responses to subunit antigens (188). The addition of QS-21 to malarial peptide
vaccines promoted CD4 and CD8 T cell responses (189). As noted above QS-21 is
synergistic with MPL in stimulating the response to several vaccines in AS02.
Nonionic Block Copolymers
The copolymer adjuvants are simple linear chains or blocks of polymers of hydrophobic
polyoxypropylene, flanked by two chains of hydrophilic polyoxyethylene. A large number of
copolymer adjuvants have been synthesized by varying the constituent chains (190).
Nonionic block copolymers are currently used commercially in over-the-counter products,
including shampoos, mouthwashes, and cosmetics. Copolymers are adhesive molecules
that bind antigens to hydrophobic surfaces, such as oil drops or cells (191). Evidence
suggests that proteins bound to copolymer are held firmly in a condensed fashion, and
retain much of their native B-cell epitope confirmation when presented to macrophages
and dendritic cells for immune processing (191).
The activation of complement by contact with the copolymer surface augments the
adjuvant effect. Several preparations of block polymers developed by Vaxcel, Inc.
(Norcross, Georgia, U.S.) are awaiting clinical trial (190,192).
Cytokines
The use of cytokines as vaccine adjuvants has been encouraged due to a better
understanding of cytokine mechanisms and the commercial availability of recombinant
interferon-g (IFN-g) and granulocyte-macrophage stimulating factor (GM-CSF). Many
cytokines (e.g., IL-3, IL-6, IL-11, GM-CSF) are capable of enhancing various immune
responses when administered repeatedly. But the cytokines with the greatest potential are
those administered in a single dose at or near the time of antigen injection; cytokines
administered in this practical way include IFN-a, IFN-g, IL-1, IL-2, IL-12, and GM-CSF. A
cytokine can enhance, inhibit, or have no effect, depending on the dose, timing, and
animal species, and which of these effects predominates is not always predictable
(193,194).
The adjuvant effects of these cytokines in animals or humans have been reviewed in detail
(195–197), although trial results to date have failed to document a strong adjuvant effect
for cytokines in humans (198). The cytokine most intensively studied for its adjuvant
activity is GM-CSF. It has been used with hepatitis B vaccine in patients with chronic renal
failure (199,200) as well as in patients with HIV infection (201). In each of these studies, it
enhanced the vaccine response, but was usually administered 24 hours before the vaccine.
In contrast, when given concurrently with hepatitis A, influenza, and tetanus-diphtheria
toxoid vaccines, a lower response was observed (202), which emphasizes that the effect of
the timing of GM-CSF administration requires further study. Nevertheless, two
metaanalyses concluded that the use of GM-CSF with hepatitis B vaccines both accelerated
and increased the response rate (203,204). GM-CSF has also been used with a leishmania
vaccine (128). Another cytokine, flt3 ligand, enhances dendritic cell numbers and function,
but this effect was not translated into an improved antibody response (89,205). The use of
rIL-12 with an experimental vaccine for CMV improved both humoral and cellular immune
responses in human subjects (77).
Immunostimulatory Oligonucleotides: CpGs
Just as bacterial DNA can activate immune cells, synthetic oligodeoxynucleotides (ODN)
containing unmethylated CpG dinucleotides in particular base contexts (CpG motifs)
stimulate the innate immune system to induce protection in mice and primates (206,207).
Either alone or in combination with a vaccine, they can activate human B cells, DC, and NK
cells (208) and trigger an immune cascade that includes the production of cytokines,
chemokines, and IgM to protect against infection. CpG ODN are extremely efficient
inducers of Th1 immunity and CTL, and can allow a 10- to 100-fold reduction in the dose of
antigen, presumably because of the increased efficiency of antigen presentation by DC
(209). The administration of CpG ODN is currently being tested as a stand-alone treatment
for cancer and asthma, and as a vaccine adjuvant (210–213). Acting through TLR9
receptors present on B cells and plasmacytoid dendritic cells, CpG has been shown in many
studies to both accelerate and enhance the response to hepatitis B vaccines in human
subjects (214,215) with protective and sustained levels of anti-hepatitis B surface antigen
achieved with fewer doses of vaccine. The adjuvant improved the anti-HBs antibody levels
in HIV patients (85), and enhanced the affinity of anti-HBs antibodies independently of the
titers achieved (86). Immunostimulatory sequences have been used with hepatitis B
vaccine proteins (87,214,216). While the addition of CpG ODN to hepatitis B vaccines has
improved the immune response to this vaccine, no improvement was observed when
added to an influenza vaccine (108). However, CpGs did enhance the response to that
same vaccine when given at 1/10, the standard dose.
SUMMARY AND CONCLUSION
Every adjuvant has a complex and often multifactorial immunological mechanism, usually
poorly understood in vivo, although the discovery of the TLRs and mechanisms of innate
immunity will help guide adjuvant development over the next decade. Adjuvant safety,
including the real and theoretical risks of administering vaccine adjuvants to humans, is a
critical component that can enhance or retard adjuvant development.
In addition to the problem of safety, several other issues impede the orderly development
of adjuvanted vaccines. These include inconsistent immunopotentiation by candidate
adjuvants, marked variation in response to the same adjuvant by different animal models,
and the inability to consistently predict protective efficacy by immunoassays. However,
decades of basic cellular research, preclinical experiments, and clinical safety and
immunogenicity studies have led to a significant expansion in understanding the role of
adjuvants in recent years. Hopefully, this will open new doors in vaccine research.
The most studied experimental adjuvants in man include aluminum compounds, oil-based
emulsions with or without muramyl dipeptide, monophosphoryl (detoxified) lipid A, MPL,
the triterpene glycoside QS-21, nonionic block copolymers, several cytokines, especially
GM-CSF, and CpG ODNs. In preclinical studies of adjuvants and vaccines, depending on the
antigen used, the same adjuvant can enhance, inhibit, or have no effect at all. The more
important determinants of immunogenicity include the nature and dose of the immunogen,
the schedule and route of administration, the population being immunized, the stability of
the adjuvant formulation, and the choice of adjuvants used alone or in combination. The
increasing understanding of these determinants is fundamental to the further development
of new vaccines. This is beginning to allow for rational design and combination of the
optimal adjuvants for a particular antigen in a specific population, which has the potential
to lead to safer and more effective vaccines. In addition to immunologic enhancement
without toxicity and successful protection against challenge, choice of adjuvant for a
clinical trial may depend on cost and commercial availability. Rational development of
classical and novel adjuvants will continue to be one of the most important challenges for
the vaccinologist to be able to address persistent unmet medical needs.

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