The perfect mix: recent progress in adjuvant research.

Abstract

Developing efficient and safe adjuvants for use in human vaccines remains both a challenge and a necessity. Past approaches have been largely empirical and generally used a single type of adjuvant, such as aluminium salts or emulsions. However, new vaccine targets often require the induction of well-defined cell-mediated responses in addition to antibodies, and thus new immunostimulants are required. Recent advances in basic immunology have elucidated how early innate immune signals can shape subsequent adaptive responses and this, coupled with improvements in biochemical techniques, has led to the design and development of more specific and focused adjuvants. In this Review, I discuss the research that has made it possible for vaccinologists to now be able to choose between a large panel of adjuvants, which potentially can act synergistically, and combine them in formulations that are specifically adapted to each target and to the relevant correlate(s) of protection.

Full text

Page 1

“In design sometimes one plus one equals three” J. Albers
“Come together” J. Lennon and P. McCartney
Adjuvants are usually defined as compounds that can
increase and/or modulate the intrinsic immunogenicity
of an antigen, and the term adjuvant is itself derived
from the latin adjuvare, meaning to help. Why do vac-
cines need help, and why do new vaccines need more
help than vaccines that are well established? To reduce
reactogenicity, new vaccines have a more defined com-
position that is often linked to lower immunogenicity
compared with previous whole-cell or virus-based
vaccines. Adjuvants are therefore required to assist
new vaccines to induce potent and persistent immune
responses, with the additional benefits that less antigen
and fewer injections are needed. Moreover, new vaccine
targets often require the induction of strong cellular
responses, including T helper (TH) cells and sometimes
cytotoxic T lymphocytes (CTLs), in addition to anti-
bodies. As aluminium salts, the adjuvants that have
been most widely used in humans so far, predominantly
induce antibody responses, discovering new adjuvants
is crucial for the development of vaccines that require
a cell-mediated response (for reviews on vaccines and
adjuvants see REFS 1–3).
Although much of the adjuvant research that was
carried out in the past can be seen as empirical, this
research did sometimes give rise to potent and useful
products. Nevertheless, there is a need to develop a new
generation of adjuvants that are rationally designed on
the basis of the recent progress that has been made in our
understanding of the immune response, particularly the
innate immune response. The first part of this Review
will present an overview of this progress, which has had
a dramatic impact on adjuvant research. The second part
will present the practical applications of this knowledge
and the new perspectives that have been opened in vac-
cinology as a result. As live vaccines and vectors usually do
not need adjuvants this Review will focus on inactivated
and inert vaccines.
It is important to stress that adjuvants are a heteroge-
neous group of compounds. The term adjuvant is often
used as a synonym for immunostimulant, but whereas
immunostimulants are generally single compounds with
intrinsic immunostimulant and/or immunomodula-
tory properties, adjuvants can be composed of differ-
ent constituents with different functions and activities,
including carrier/depot or targeting functions and
immunostimulant and/or immunomodulatory activity.
The current challenge facing adjuvant research is to find
the ‘perfect mix’; an optimal, safe formulation, the dif-
ferent components of which will be not only additive but
synergistic, and which will eventually drive the desired
immune response. In this Review I will attempt to show
that in adjuvant research, as in design, one plus one often
equals more than two, and that combining different vac-
cine constituents in appropriate formulations can further
enhance and target such synergy.
Research Department, sanofi
pasteur, Campus Merieux,
69280 Marcy l’Etoile, France.
e-mail: bruno.guy@
sanofipasteur.com
doi:10.1038/nrmicro1681
Published online 11 June 2007
T helper cell
(TH cell). A T cell that has cell-
surface antigen receptors that
bind fragments of antigens
displayed by MHC class II
molecules, which are
expressed at the surface of
antigen-presenting cells.
Activated TH cells express
cytokines and membrane-
associated co-stimulatory
molecules that help other
immune cells carry out their
specific functions. TH cells can
be divided into subsets
according to their cytokine-
secretion profiles.
The perfect mix: recent
progress in adjuvant research
Bruno Guy
Abstract | Developing efficient and safe adjuvants for use in human vaccines remains both
a challenge and a necessity. Past approaches have been largely empirical and generally
used a single type of adjuvant, such as aluminium salts or emulsions. However, new vaccine
targets often require the induction of well-defined cell-mediated responses in addition
to antibodies, and thus new immunostimulants are required. Recent advances in basic
immunology have elucidated how early innate immune signals can shape subsequent
adaptive responses and this, coupled with improvements in biochemical techniques, has
led to the design and development of more specific and focused adjuvants. In this Review,
I discuss the research that has made it possible for vaccinologists to now be able to choose
between a large panel of adjuvants, which potentially can act synergistically, and combine
them in formulations that are specifically adapted to each target and to the relevant
correlate(s) of protection.
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Dendritic cell
(DC). ‘Professional’ antigen-
presenting cells that are found
in the T-cell areas of lymphoid
tissues and as minor cellular
components in most tissues.
They have a branched or
dendritic morphology and are
the most potent stimulators of
naive T-cell responses.
CD4+ T cell
A subpopulation of T cells that
express the CD4 receptor.
These cells aid in immune
responses and are therefore
referred to as T helper cells.
CD8+ T cell
A subpopulation of T cells that
express the CD8 receptor.
CD8+ T cells recognize antigens
that are presented on the
surface of host cells by MHC
class I molecules, leading to
their destruction, and are
therefore also known as
cytotoxic T lymphocytes.
Regulatory T (TReg) cell
A population of CD4+ T cells
that naturally express high
levels of CD25 (the interleukin-
2 receptor α-chain) and the
transcription factor forkhead
box P3 (Foxp3), and that have
suppressive regulatory activity
towards effector T cells and
other immune cells.
TH1 cell
(T helper 1 cell). A type of
activated TH cell that promotes
responses associated with the
production of a particular set of
cytokines, including interleukin
(IL)-2, interferon (IFN)-γ and
tumour-necrosis factor (TNF),
the main function of which is to
stimulate phagocytosis-
mediated defences against
intracellular pathogens.
TH2 cell
(T helper 2 cell). A type of
activated TH cell that
participates in phagocytosis-
independent responses
and downregulates
pro-inflammatory responses
that are induced by TH1 cells.
TH2 cells secrete interleukin
(IL)-4, IL-5 and IL-6.
A fresh look at innate immunity
Until recently, innate immunity was largely seen as less
‘noble’ than adaptive immunity, except for the few com-
ponents, such as complement and phagocytes, that had
the privilege of cooperating with antibodies to eliminate
pathogens. Innate immunity was thought of as merely the
first line of defence, providing non-specific microbicidal
activity and early inflammatory signals, and thus giving
more time for adaptive immunity to develop. However,
it is now clear that the adaptive immune response mainly
depends on the level and specificity of the initial ‘dan-
ger’ signals4 perceived by innate immune cells following
infection (and vaccination). Some innate and adaptive
cells have long been known to be mandatory for initiat-
ing immune responses by presenting antigen to T cells.
Among these antigen-presenting cells (APCs), dendritic
cells (DCs) are the key sentinels for priming naive
T cells5.
In parallel with the renaissance in innate immunity,
researchers have continued to characterize the adap-
tive response and clarify the respective roles of B cells,
and CD4+ T cells and CD8+ T cells. In particular, vari-
ous CD4+ TH cell subsets have been defined through
their cytokine profiles and ability to modulate B cell
and CD8+ CTL responses6–8 (BOX 1). In the continuous
football match involving the human immune system
and pathogens as the opposing teams, TH cells are the
key midfielders who conduct and organize the game
between the B-cell defenders and CTL strikers. It is
important to have referees keeping each game under
control and eventually signalling full time, and this
role is filled by regulatory T (TReg) cells, which are capable
of modulating both TH1 and TH2 responses9. Finally,
T- and B-cell memory responses have also been
dissected, allowing the identification of various means
of inducing different subsets of memory cells with
distinct functions10,11.
The overall consequence of these developments was
to strengthen the link between the initial innate and
subsequent adaptive response. The crucial steps
and signals required to induce T- and B-cell responses
have been defined, demonstrating that innate immune
signals modulate not only the magnitude of the adap-
tive response but also the quality of this response. For
instance, secretion of pro-inflammatory cytokines such
as interleukin (IL)-12 will drive TH1-cell polarization
and subsequent interferon (IFN)-γ secretion by T cells.
These findings are summarized in BOX 1 and FIG. 1,
which present the different signals required to initiate a
potent immune response as signal 0 (antigen recognition
and APC activation), signal 1 (antigen presentation) and
signal 2 (co-stimulation)12. Adjuvants can act on each
of these three signals. Although targeting co-stimula-
tory molecules (signal 2) directly through antibodies,
cytokines or chemokines is an interesting possibility13,
caution is advised as this might result in non-focused
over-stimulation (discussed in more detail in the later
section on safety). This approach might be more usefully
applied in therapeutic settings rather than preventative
settings, and will not be discussed any further here.
TLRs and innate immunity
Innate immunity has regained a primary role, not only
chronologically but also functionally through its impor-
tance in shaping the adaptive response. Applied research
has directly benefited from these basic findings, thanks
in particular to the discovery of the Toll-like receptors
(TLRs) and other pattern-recognition receptors (PRRs).
Box 1 | Innate and adaptive immunity: from signals 0, 1 and 2 to TH0, 1 and 2 and beyond
The initiation of T helper (TH)-cell responses requires different signals from antigen-presenting cells (APCs). Signal 1 is
triggered by specific peptide presentation by class II major histocompatibility (MHC) molecules to the T-cell receptor (TCR).
However, unless another signal is given, anergy and abortive responses are induced. To avoid anergy, the co-stimulatory
signal, signal 2, is needed, through receptor–ligand interaction between APCs and T-cell antigens, such as CD40–CD40L
(CD154), OX40 (CD134)–OX40L (CD134L), CD80 (B7.1) or CD86 (B7.2)–CD28. Alternatively, interactions between CD80 or
CD86 and CD152 (CTLA4) or between PD1L (B7H1) or PDL2 (B7DC) and PD1 downregulate T-cell activation.
It was subsequently discovered that an additional, and earlier, signal called signal 0, was mandatory to activate APCs and
orientate the subsequent TH response. Signal 0 is mostly induced through the recognition of pathogen-associated
molecular patterns (PAMPs) by pathogen-recognition receptors (PRRs), including Toll-like receptors (TLRs)4,5. These signals
might also apply to class I MHC presentation by APCs for CD8+ T cell stimulation.
The TH1/TH2 cell dichotomy was first described 20 years ago6,7. The TH1-type response is accompanied by secretion of
interleukin-2 (IL-2) and interferon-γ (IFN-γ), favouring cell-mediated immune responses, activation of CD8+ cytotoxic T cells
(CTLs), and the generation in mice of the complement-fixing antibody IgG2a (IgG2c in strains lacking this IgG subclass),
and IgG3. The TH2-type response is characterized by secretion of IL-4, IL-5 and IL-6, providing B-cell help, and the
preferential induction of IgG1, IgE and IgA in mice, and IgE and IgG4 in humans. It is important to note that although TH1-
cell adjuvants promote the activation of CD4+ IFN-γ-secreting cells, this is not sufficient to trigger the activation of CTLs,
which also require class I MHC antigen presentation. More recently, IL-17-expressing TH17 cells have been linked to early
inflammation, defence against extracellular bacteria and parasites, and autoimmunity7.
In addition, regulatory T (TReg) cells, initially called suppressor cells, are undergoing a re-discovery and several types have
been identified, including CD4+ CD25+ Foxp3+ TReg cells, and Tr1 and TH3 TReg cells9. These cells regulate the balance
between tolerance and immune responses involving both T and B cells, in particular through the action of transforming
growth factor β and/or IL-10 and the membrane-bound molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)
and glucocorticoid-induced tumour necrosis factor receptor (GITR).
Finally, important progress has been made in elucidating how T- and B-cell memory is induced, identifying T effector and
central memory subsets, and still involving innate immune signals, in particular TLRs10,11,34 (see also BOX 2).
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TLR
Adjuvant B
Adjuvant C
Signal 0
Signal 1
Signal 2
Indirect effect
Indirect effect
TH cell
TH response
Internal
signal 0
Adjuvant A
APC
Pattern-recognition
receptor
(PRR). A host receptor (such as
Toll-like receptors (TLRs) or
NOD-like receptors (NLRs))
that can sense pathogen-
associated molecular patterns
and initiate signalling cascades
that lead to an innate immune
response. These can be
membrane bound (such as
TLRs) or soluble cytoplasmic
receptors (such as NLRs).
Pathogen-associated
molecular pattern
(PAMP). A molecular pattern
that is found in microorganisms
but not mammalian cells.
Examples include bacterial
lipopolysaccharides,
hypomethylated DNA, flagellin
and double-stranded RNA.
Following host invasion, microorganisms express-
ing pathogen-associated molecular patterns (PAMPs) are
recognized by innate immune cells through specific
and non-polymorphic PRRs. Among the PRRs, TLRs
have been shown to have a crucial role in the early steps
of the immune response to infection. These receptors
were discovered in human cells14 and named after the
Drosophila melanogaster Toll gene, the protein product
of which is involved in innate immunity and develop-
ment15. The importance of Toll signalling in mammals
was confirmed by the observation that TLR4 is involved
in lipopolysaccharide (LPS) recognition16. This seminal
finding opened the way for an ever-increasing number of
investigations focusing on the different roles of TLRs in
APC activation and T- and B-cell responses (for reviews
see REFS 17,18).
BOX 2 and FIG. 2 present some key features of TLR dis-
tribution and localization, TLR agonists and downstream
signalling pathways and their role in the modulation of
the adaptive response. TLRs are expressed on the surface
or in the endosomes of different APC subtypes19,20 and
they respond to, among other stimuli, specific bacterial,
viral, fungal or protozoan signals. This activates APCs
and modulates and shapes the adaptive response, for
instance, by shifting the balance towards TH1 or TH2 cells
(REFS 21,22) and activating or inhibiting TReg cells.
Importantly, TLRs not only trigger APC activation
(signals 0 and 2), they also have a role in antigen pre-
sentation (signal 1). The presence of both antigen and
TLR is required for optimal antigen presentation and
activation: TLRs control the generation of T-cell receptor
(TCR) ligands from the phagosome, which ensures that
the contents derived from microbial pathogens are prefer-
entially presented to T cells by the activated APC. This
was first demonstrated in vitro23, and the importance of
such selection has also been demonstrated in vivo with
the generation of an immunodominant response against
Toxoplasma profilin, a TLR11 agonist24. This highlights
the need for antigen and TLR-agonists to be co-delivered,
in order to be present in the same phagosome cargo and
induce optimal antigen presentation and stimulation of
the subsequent T-cell response. Moreover, direct TLR
stimulation is also needed to fully activate B cells25,26.
Figure 1 | Where do adjuvants act? The initiation of T helper (TH)-cell responses
requires three signals, referred to as signal 0, signal 1 and signal 2 (see BOX 1).
In theory adjuvants can act — alone or in combination — on each of these three signals,
and an adjuvant acting on each signal is shown. Most of the recently developed
specific adjuvants, such as Toll-like receptor (TLR) agonists, can be considered as
type A adjuvants: they act on signal 0, and indirectly on signal 2, by activating antigen-
presenting cells (APCs) and triggering the secretion of cytokines such as interleukin
(IL)-12. In addition, TLR agonists can act on signal 1 by favouring efficient presentation of
the co-administered antigen (Ag). Some TLR agonists also directly trigger signal 0 on
regulatory T cells and B cells expressing some of the corresponding receptors. Adjuvants
and formulations targeting APCs or favouring Ag capture can be viewed as type B
adjuvants acting on signal 1, as their effect is eventually mediated by enhanced Ag
presentation to T cells. Liposomes, microspheres and some emulsions are in this category.
As stressed in this Review, targeting signal 1 is not sufficient and an immunostimulatory
signal should be co-delivered for an optimal response. In this regard, some specific
ligands of co-stimulatory molecules can directly enhance signal 2, acting as type C
adjuvants; such compounds must however be used with caution.
Box 2 | Toll-like receptors: expression, ligands and signalling
Toll-like receptors (TLRs) are pathogen-recognition receptors (PRRs). They are membrane-anchored proteins exposing
leucine-rich-repeat (LRR) domains in extracellular or luminal (endosomal) compartments, and can therefore interact
with pathogen-associated molecular patterns (PAMPs). The cytoplasmic tail of TLRs contains Toll–interleukin 1
receptor (TIR) domains, that can interact with downstream signalling pathways. TLRs mostly recognize foreign
antigens, although it has been shown that endogenous proteins such as heat-shock proteins can stimulate some TLRs.
Evidence of direct binding of agonists to the corresponding TLRs is still lacking in most cases; for instance, TLR4 does
not bind lipopolysaccharide (LPS) directly123.
As shown in FIG. 2, TLRs 1, 2, 4, 5, 6 and 11 are surface-exposed, whereas TLRs 3, 7, 8 and 9 are located within
endosomes. In addition to differences in cellular sub-compartmentalization, there are also differences in their cellular
distribution: in humans, myeloid dendritic cells (DCs) express all TLRs except TLR7 and 9, which are selectively
expressed by plasmacytoid DCs20. Epidermal Langerhans cells express mRNA encoding TLRs 1, 2, 3, 5, 6 and 10, but do
not directly sense TLR4 or TLR-7/8 agonists19.
TLR sensing of their natural or synthetic agonists can orientate the immune response towards a TH1- or TH2-cell
response21,22 (FIG. 2) and promote regulatory T (TReg) cell activity29–31. In particular, LPS agonists, imidazoquinolines and
unmethylated CpG oligonucleotides induce TH1 responses after sensing by TLR4, 7/8 and 9, respectively.
In this regard, all TLRs were initially considered to transduce similar transactivation and phosphorylation pathways
through their common adaptor protein MyD88, leading to the activation of the transcription factor nuclear factor (NF)-κB
and the JNK and p38 kinases. This pathway is crucial for inducing antigen-specific TH1 responses, as observed in MyD88-
deficient mice. However, although most TLRs trigger MyD88-dependent pathways, TLR3 stimulates MyD88-independent
pathways and TLR4 can signal through both pathways; for both TLR3 and TLR4 this depends on their coupling with the TRIF
(TIR-domain-containing adaptor protein inducing IFN-β). In addition, TLR negative regulators have progressively been
identified (interleukin-1 receptor-associated kinase M (IRAK-M) and Tollip), which help to keep inflammation under control.
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Scavenger receptors
Gram positive and Gram negative
polyanionic ligands, LDL, apoptotic cells
CLRs
Polysaccharides
Cross–talk
RLHs (viruses)
5′-triphosphate RNA, ssRNA
(VSV, paramyxoviruses,
influenza virus, JEV)
Poly (I:C), picornavirus
RIG-1
MDA5
Peptidoglycan, muropeptides, DAP
NLRs (bacteria)
Peptidoglycan, muropeptides, MDP
Bacterial RNA, uric acid crystals, toxins, MDP
NOD1
NOD2
NALP3
dsRNA, Poly (I:C)
ssRNA, Imidazoquinolines
(Imiquimod, resiquimod (R848))
ssRNA, Imidazoquinolines
(resiquimod (R848))
Unmethylated CpG DNA,
synthetic oligonucleotides
Endosome TLRs and agonists
TREMs
Ligands?
Triacyl lipopeptides, Pam3Cys
Diacyl lipopeptides,
Lipoteichoic acids, Zymosan
LPS, MPL, LPS analogues, Taxol
Flagellin
Uropathogenic bacteria,
Toxoplasma profilin
Surface TLRs and agonists
TH1
TH1
TH1
TH0, TH2
TH0, TH2
TH1
TH1
TH1
TH1
7
7
8
8
9
9
2
1
11
3
5
4
6
2
However, some researchers have questioned the need
for TLRs in inducing antibody responses27,28, and this
will be discussed in more detail later.
TLR stimulation also regulates TReg activity, directly
through TLR recognition on TReg cells, or indirectly
through APC–TReg interactions29. The consequence of
direct stimulation is to activate TReg cells, as has been shown,
for example, for TLR2 and TLR4 (REFS 30,31), or to reverse
their inhibitory effect, as has been shown for TLR8 (REF. 32).
Indirect stimulation through TLR-linked APC stimulation
inhibits TReg cell differentiation33. This shows that TLR
recognition not only induces inflammatory responses,
but can also regulate these responses by triggering
the expansion of TReg cells, which will limit potentially
harmful responses through a feedback mechanism once
the infection is resolved. Finally, it has been shown that
TLR-induced signals are important for the generation of
memory CD4+ T cells, but not for their activation34.
It was then logical to translate these basic discover-
ies into practical applications in adjuvant research. TLR
agonists can act as a coach for the immune system team:
they select and stimulate the most appropriate players
with respect to the optimal strategy to oppose each par-
ticular team of pathogens. Vaccines could benefit from
having such coaches, and this will be developed in the
following sections.
Figure 2 | Potential targets for adjuvants and formulations. Different types of receptors on antigen-presenting cells
(APCs) can be targeted by adjuvants. Each particular type of APC can express a restricted array of these receptors, which
can regulate each other’s signalling through some cross-talk within the cell. First, some synthetic agonists of Toll-like
receptors (TLRs) (the names of synthetic agonists are highlighted in red) are among the most promising and efficient
adjuvant candidates, able to sense surface- and endosome-located TLRs, the activation of which will orientate T helper
(TH) 1 and/or TH2 responses. The cytosolic NOD-like receptors (NLRs) and RIG-like helicases (RLHs) can sense signals
from intracellular bacteria and viruses, respectively. Although, so far, less development has been carried out in humans
with synthetic agonists of these receptors, compounds such as muramyl dipeptide (MDP) have been used with some
success in the veterinary field. Other receptors such as C-type lectin receptors (CLRs) and scavenger receptors can
constitute targets for formulations aiming at enhancing antigen capture and presentation should a stimulatory signal be
co-delivered to avoid tolerance induction. Finally, TREM receptors (triggering receptors expressed on myeloid cells) are
also involved in early inflammation, although their ligands are still unknown. DAP, diaminopimelic acid; JEV, Japanese
encephalitis virus; LDL, low density lipoprotein; LPS, lipopolysaccharide; MPL, monophosphoryl lipid A; NOD,
nucleotide-binding oligomerization domain; Pam3Cys, tripalmitoyl-S-glyceryl cysteine; VSV, vesicular stomatitis virus.
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Tolerance
Immunological tolerance
(central or peripheral) results
from different mechanisms
preventing the immune system
mounting responses against
(self) antigens. Peripheral
tolerance occurs when mature
lymphocytes encounter
antigens and undergo anergy,
deletion or suppression. In
particular, T-cell anergy is
defined by defective
proliferation by previously
primed T cells following
restimulation, reflecting a
selective defect in the
activation of some TCR-
induced signalling pathways.
Natural killer T cells
(NKT cells). A subpopulation of
T cells that expresses both
NK-cell and T-cell markers.
γδ T cells
A minor population of T cells
that express the γδ T-cell
receptor (TCR), and that are
more abundant in epithelial-
rich tissues such as the skin,
gut and reproductive tract.
Like NKT cells, γδ T cells can
be cytolytic and produce high
levels of cytokines and
chemokines.
Non-TLR innate receptors
TLRs sense extracellular signals, and it therefore fol-
lows that intracellular cytosolic counterparts should
sense intracellular signals. Such cytosolic sensors
include NODs (nucleotide-binding oligomerization
domain proteins), NOD-like receptors (NLRs) and
RIG-like helicases (RLHs), which sense signals from
intracellular bacteria and viruses, respectively35 (FIG. 2).
As for TLRs, agonists of such receptors could in theory
be used as adjuvants, although their effects would be
less focused as NLRs and RLHs are more ubiquitous
than TLRs.
NODs and NLRs. NOD proteins and NLRs have in com-
mon a C-terminal leucine-rich-repeat (LRR) domain,
a central nucleotide-binding domain and N-terminal
protein–protein interaction CARD (caspase activation
and recruitment domain) and pyrin or BIR (baculovirus
inhibitor-of-apoptosis repeat) domains36. NOD1 and
NOD2 detect distinct substructures from bacterial pep-
tidoglycan: NOD2 detects muramyl dipeptide (MDP)
from Gram-negative and Gram-positive bacteria and
NOD1 senses meso-diaminopimelic acid (meso-DAP),
which is found in Gram-negative bacteria and in Gram-
positive rods37. NLRs sense danger-associated host
components such as uric acid crystals35. MDP has been
successfully used as an adjuvant in veterinary vaccines,
such as in leishmaniasis, inducing TH1 responses and
protection38. A synthetic lipophilic glycopeptide, mur-
amyl-tripeptide-phosphatidylethanolamine (MTP-PE),
was also shown to have adjuvant and immunostimulatory
properties in humans.
RIG/MDA. Retinoic acid-inducible gene I (RIG-I) and
melanoma-differentiation-associated gene 5 (MDA5) are
RLH proteins that share two N-terminal CARD domains
followed by an RNA helicase domain. These sensors are
activated by viral infection, and trigger cooperative
activation of nuclear factor (NF)-κB and IFN regula-
tory factors 3 and 7 (IRF3/7) to induce antiviral type I
IFNs (IFNα and IFNβ). RIG-I responds to RNA viruses,
including paramyxoviruses, influenza virus and Japanese
encephalitis virus, whereas MDA5 detects picorna-
viruses39. The ligand for RIG-I was recently shown to be
triphosphate RNA (3pRNA)40,41.
Other receptors involved in antigen capture and rec-
ognition. APCs express other receptors involved in
antigen capture and recognition, such as scavenger
receptors, C-type lectin receptors (CLRs) and trig-
gering receptors expressed on myeloid cells (TREMs)
(FIG. 2). Scavenger receptors bind polyanionic ligands
and can internalize not only pathogens but also host
components such as apoptotic cells and modified
low-density lipoproteins42. CLRs, including DC-SIGN
and the mannose receptor, can bind a wide range
of viruses, bacteria and fungi through recognition of
sugar moieties, such as N-acetyl-glucosamine, man-
nose, N-acetyl-mannosamine, fucose and glucose43.
However, it appears that the first role of scavengers and
CLRs is not in pathogen recognition, and pathogens
seem to be able to use these host–host recognition
systems for their own benefit. In contrast to TLRs,
the primary function of which is to discriminate
between self and non-self, targeting scavengers
and CLRs require a co-stimulatory signal to induce
immunity rather than tolerance. This has been shown,
for instance, for the DEC-205 receptor, a decalec-
tin involved in uptake and presentation by DCs.
Targeting this receptor in the absence of a maturation
stimulus leads to CD8+ T-cell tolerance44. A ‘yin yang’
regulation has been proposed to describe the crosstalk
between TLRs and CLRs45. These receptors neverthe-
less have a practical interest for vaccine researchers,
as their targeting could focus antigen and adjuvant
to the target cells, increasing both immunogenicity
and safety. Finally, TREMs have been characterized
as amplifiers of immune responses but so far their
specific ligands have not been identified46.
In addition to these receptors, two additional players
acting at the border between innate and adaptive immu-
nity are worth mentioning, natural killer T (NKT) cells and
γδ T cells. NKT cells can be activated in particular by a
synthetic glycolipid, α-galactosylceramide (α-GalCer).
This compound and its derivatives have demonstrated
immunomodulatory properties in various settings,
including experimental models of cancer47. The γδ
T cells are specifically activated by small antigens, such
as organic phosphoesters, which are also potential adju-
vants. Moreover, some γδ T cells can themselves act as
APCs (for a review see REF. 48).
Practical applications to the adjuvant field
Synthetic TLR agonists. The identification of natural
TLR agonists has led to the design of synthetic ligands
that can target TLRs more precisely and safely than
pathogen-derived ligands, and which were selected by
their ability to bind receptors and activate downstream
signalling pathways49. Interestingly, some adjuvants and
immunostimulants that had been identified previously
were later shown to be TLR agonists: unmethylated viral
or bacterial CpG DNA and oligonucleotides are agonists
for TLR9; poly (I:C) for TLR3; lipopeptides and tripal-
mitoyl-S-glyceryl cysteine (Pam3Cys) for TLR2; LPS
and its derivatives for TLR4; and imidazoquinolines for
TLR7/TLR8 (REF. 50).
Additional TLR agonists have since been synthe-
sized and tested in different experimental models, such
as second-generation oligonucleotide TLR9 agonists,
with sequences that have been optimized with respect
to the species (for instance mouse or human), type of
immune response51,52 and stability. The identification of
TLRs also allowed the generation of new tools such as
transformed cell lines expressing a given array of TLRs,
in which activation of downstream signalling pathways
can be followed using, for example, an NF-κB–luciferase
reporter gene. Such cell lines have been used to investi-
gate the activity of new TLR4 agonists with a modified
backbone53. Other second-generation TLR4 agonists
have been obtained, such as the RC529 adjuvant54. New
synthetic TLR7/8 agonists such as 3M-019 have also
been synthesized55.
REVIEWS
NATURE REVIEWS | MICROBIOLOGY
VOLUME 5 | JULY 2007 | 509
FOCUS ON VACCINES — PROGRESS & PITFALLS
© 2007 Nature Publishing Group