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Pattern recognition receptors:
sentinels in innate immunity
and targets of new vaccine
adjuvants
Expert Rev. Vaccines 11(2), 237–256 (2012)
Colleen Olive
The Queensland Institute of Medical
Research, Locked Bag 2000, Royal
Brisbane Hospital, Herston, Brisbane,
Queensland 4006, Australia
Tel.: +61 7 3845 3703
Fax: +61 7 3845 3507
[email protected]
The innate immune system plays an essential role in the host’s first line of defense against
microbial invasion, and involves the recognition of distinct pathogen-associated molecular
patterns by pattern recognition receptors (PRRs). Activation of PRRs triggers cell signaling leading
to the production of proinflammatory cytokines, chemokines and Type 1 interferons, and the
induction of antimicrobial and inflammatory responses. These innate responses are also
responsible for instructing the development of an appropriate pathogen-specific adaptive
immune response. In this review, the focus is on different classes of PRRs that have been
identified, including Toll-like receptors, nucleotide-binding oligomerization domain-like receptors,
and the retinoic acid-inducible gene-I-like receptors, and their importance in host defense
against infection. The role of PRR cooperation in generating optimal immune responses required
for protective immunity and the potential of targeting PRRs in the development of a new
generation of vaccine adjuvants is also discussed.
Keywords : host defense • inflammasome • innate immunity • nucleotide-binding oligomerization domain-like
receptor • pattern recognition receptor • retinoic acid-inducible gene-I-like receptor • Toll-like receptor
• vaccine adjuvant
The mammalian immune system comprises
both innate and adaptive arms, which together
function to protect the host against an array of
invading microbial pathogens including bacteria, viruses, parasites and fungi. The innate
immune system comprises various immune cells
including DCs, macrophages and neutrophils
that sense and respond rapidly to aid in the elimination of microbial pathogens, thereby providing the first line of host defense against infection.
This early innate microbial sensing is achieved
via the recognition of distinct molecular motifs,
termed pathogen-associated molecular patterns
(PAMPs), of microbial components, such as proteins, lipids, nucleic acids and carbohydrates, by
evolutionarily conserved host germline encoded
pattern recognition receptors (PRRs) [1,2] . The
interactions between the various PRRs and their
cognate PAMPs trigger a complex cascade of
intracellular signaling pathways leading to the
production of cytokines, chemokines and Type 1
interferons (IFNs) that mediate the induction of
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10.1586/ERV.11.189
antimicrobial and inflammatory responses [1,2] .
The innate immune system is also responsible for
initiating an adaptive immune response specifically tailored to the invading microbe [3,4] . DCs
play a critical role in translating the appropriate signals from the innate to adaptive immune
system to mediate the regulation of adaptive
immunity (Figure 1) [5] . The adaptive immune system consists of B and T cells that express highly
diverse repertoires of B- and T-cell receptors,
respectively, and generates specificity in antibody
and cellular responses and long-term memory [6] .
There are several distinct families of PRRs
including those that are membrane-anchored
as well as those located in the cytosol. In addition to sensing microbial-derived molecules,
certain PRRs can be activated by endogenous
self-derived molecules referred to as ‘danger
signals’ or danger-associated molecular patterns
(DAMPs), which are released from dying host
cells as a result of tissue damage or stress [7] .
This review will focus on the following classes of
© 2012 Expert Reviews Ltd
ISSN 1476-0584
237
Review
Olive
PAMP
PAMP
PRR
PRR
PRR
PRRs: Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the retinoic
acid-inducible gene-I (RIG-I)-like receptors (RLRs).
Toll-like receptors
PRR
PAMP
PAMP
PRR
PRR
Dendritic
cell
IL-12p70
IL-10
TGF-β
IL-Iβ
IL-6
IL-23
TGF-β
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IL-10
Naive
Naive
Naive
T-bet
STAT4
Naive
GATA3
STAT5
Th1
Th17
Th2
IFN-γ
IL-4, IL-5
and IL-13
Foxp3
STAT5
RoRγt
STAT5
IL-17, IL-21
and IL-22
Treg
IL-10 and
TGF-β
TLR extracellular microbial sensors
TLR1, TLR2 & TLR6 ligands
TCR
CD40
OX40L
Peptide antigen
CD40L
CD28
MHCII
OX40
CD80, CD86
Expert Rev. Vaccines © Future Science Group (2012)
Figure 1. Shaping of the adaptive immune response by
dendritic cells. DCs are activated by microbial ligands that
stimulate various PRRs, such as Toll-like receptors, leading to the
production of cytokines that modulate the differentiation of naive
effector CD4 + Th cells into specific functional subsets by a process
called Th polarization. DCs that produce IL-12p70 and express the
costimulatory molecules CD40, CD80 and CD86 stimulate Th1
responses for combating intracellular bacteria and viruses,
whereas those that produce IL-1b, IL-6, IL-23 and TGF-b stimulate
Th17 responses, which are important in immunity against
extracellular bacteria and fungi. DCs that produce IL-10 and
express various costimulatory molecules (CD80, CD86, CD40 and
OX40L) stimulate Th2 cells for the control of extracellular
pathogens and helminths, whereas DCs that produce IL-10 and
TGF-b stimulate Treg responses in the absence of appropriate
costimulation. Tregs are important in the regulation of Th1, Th2
and Th17 responses. T cells themselves express certain master
lineage regulators, as indicated, which drive the relevant Th
responses.
PAMP: Pathogen-associated molecular pattern; PRR: Pattern
recognition receptor; TCR: T-cell receptor.
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The TLRs are expressed on various immune cells, including DCs,
and play an important role in bacterial, viral, fungal and protozoal
sensing. TLRs are Type 1 transmembrane proteins comprised of
extracellular leucine-rich repeats (LRRs) that mediate recognition of
PAMPs, transmembrane domains and cytoplasmic Toll������������
/�����������
IL-1 receptor (TIR) domains responsible for binding cytosolic TIR-containing
adapter molecules and initiating downstream cell signaling [8,9] . To
date, ten and 13 TLRs have been identified in humans and mice,
respectively [10] . TLRs 1–9 are conserved in both species [2] . TLR10
is nonfunctional in mice owing to a retrovirus insertion whereas
TLRs 11–13 are not present in the human genome [2] . Each type of
TLR recognizes a distinct PAMP [1,2,10] . The family of TLRs can
be divided into two subgroups based on their cellular localization:
one group contains TLRs that are expressed on the cell surface
(TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11) and the second
group contains TLRs that are expressed in intracellular compartments (TLR3, TLR7, TLR8 and TLR9), namely the endoplasmic
reticulum, endosomes, lysosomes and endolysosomes. The former
are important in sensing primarily bacterial cell wall components
whereas the latter recognize viral and bacterial nucleic acids. The
ligands recognized by individual TLRs will be described separately.
Table 1 presents a collation of these ligands together with those ligands
recognized by other PRRs, which will be discussed later.
TLR2 recognizes a wide array of structurally diverse PAMPs.
These include lipoproteins from Escherichia coli, Borrelia burgdorferi, mycoplasma, Mycobacterium tuberculosis and Treponema
pallidum [11–15] ; the diacylated mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) [16] and its stereoisomer
R-MALP [17] ; peptidoglycan (PDG) from Gram-positive bacteria such as Staphylococcus aureus, Streptococcus pneumoniae and
Streptococcus pyogenes [18–20] ; lipoteichoic acid from S. aureus and
S. pneumoniae [21] ; various fungi [22] ; zymosan from Saccharomyces
cerevisiae [23] ; lipoarabino­mannan from mycobacteria [24] ; glycosylphosphatidylinositol (GPI)-anchored mucin-like glycoproteins
from Trypanosoma cruzi [25] ; hemagglutinin protein from measles
virus [26] ; as well as whole pathogens (Chlamydia pneumoniae, HSV
and varicella-zoster virus) [27–29] . The extent of PAMP recognition
by TLR2 is thought to be primarily due to the ability of TLR2 to
form heterodimers with other molecules functioning as coreceptors
on the cell surface. For example, TLR2 forms heterodimers with
structurally related TLR1 [30] or TLR6 [16] , which can distinguish
between triacylated lipopeptides (bacterial as well as the synthetic
lipopeptide tripalmitoyl-S-glyceryl cysteine and Pam3Cys) and
diacylated mycoplasmal lipopeptide, respectively. The recognition
of zymosan has been shown to be mediated by the cooperation
of TLR2 with dectin-1 [23] , and in the TLR2-mediated recognition of some ligands, CD14 and lipopolysaccharide (LPS)-binding
Expert Rev. Vaccines 11(2), (2012)
Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
Review
Table 1. Distinct families of pattern recognition receptors recognize distinct pathogen-associated
molecular patterns from various microbes.
PRRs
PAMPs
Species (microbes)
TLR2/1
Triacyl lipopeptides
Bacteria and mycobacteria
TLR2/6
Diacyl lipopeptides
LTA
Zymosan
Phospholipomannan
Glucuronoxylomannan
tGPI-mucin
PDG
Porins
Lipoarabinomannan
Hemagglutinin protein
ND
dsRNA
poly(I:C)
LPS
MPL
Flagellin
Mycoplasma
Gram-positive bacteria
Fungi (Saccharomyces cerevisiae)
Fungi (Candida albicans)
Fungi (Cryptococcus neoformans)
Parasites (Trypanosoma cruzi)
Gram-positive bacteria
Bacteria (Neisseria)
Mycobacteria
Viruses (measles virus)
Viruses (HSV-1, HCMV)
Viruses (reovirus, RSV, West Nile virus)
Synthetic
Gram-negative bacteria
Synthetic
Flagellated bacteria
ssRNA
R-848 (imidazoquinolines)
CpG-DNA
CpG ODN
DNA
Hemozoin
Profilin
ND
Viruses (HIV, influenza virus)
Synthetic
Bacteria
Synthetic
Bacteria and viruses (HSV-1, HSV-2, MCMV)
Malaria parasite
Parasites (Toxoplasma gondii)
Uropathogenic bacteria
NOD1
Meso-diaminopimelic acid
NOD2
MDP
NLRP3
Whole pathogens
NLRP1
NALP1b
NLRC4
Toxins, LPS, MDP and RNA
Particulates and ATP
MDP
Microbial toxin
Flagellin
AIM2
dsDNA
Bacteria (Helicobacter pylori, Chlamydia pneumoniae, enteropathogenic
Escherichia coli, Campylobacter jejuni, Bacillus spp., Pseudomonas aeruginosa,
Listeria monocytogenes, Shigella flexneri)
Bacteria (Streptococcus pneumoniae, Staphylococcus aureus, Mycobacterium
tuberculosis, Salmonella typhimurum, Listeria monocytogenes, S. flexneri)
Bacteria (L. monocytogenes, S. aureus), viruses (influenza virus, adenovirus,
sendai virus, vesicular stomatitis virus, encephalomyocarditis virus, measles virus,
vaccinia virus) and fungi (C. albicans)
Bacteria
Host DAMPs
Bacteria
Bacteria (Bacillus anthracis)
Bacteria (Legionella pneumophila, Salmonella enterica serotype
Typhimurium, P. aeruginosa, S. flexneri)
Viruses (vaccinia virus) and bacteria (Francisella tularensis)
RNA (ssRNA bearing 5’ triphosphate, short
dsRNA, short poly(I:C))
RNA (long dsRNA, long poly(I:C))
RNA
Viruses (paramyxoviruses, orthomyxoviruses, flaviviruses, reoviruses, West Nile
virus, dengue virus)
Viruses (picornaviruses, reoviruses, West Nile virus, dengue virus)
Viruses
TLR
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TLR2
TLR3
TLR4
TLR5
TLR7/8
TLR9
TLR11
NLR
RLR
RIG-I
MDA5
LPG2
DAMP: Danger-associated molecular pattern; GPI: Glycosylphosphatidylinositol; HCMV: Human cytomegalovirus; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid;
MCMV: Murine cytomegalovirus; MPL: Monophosphoryl lipid A; MDP: Muramyl dipeptide; ND: Not determined; ODN: Oligodeoxynucleotide; PAMP: Pathogenassociated molecular pattern; PDG: Peptidoglycan; Poly(I:C): Polyinosinic-polycytidylic acid; PRR: Pattern recognition receptor; RSV: Respiratory syncytial virus;
TLR: Toll-like receptor.
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protein (LBP) were involved [21] . CD36 can also act together with
the TLR2/TLR6 heterodimer to mediate the sensing of some
TLR2 agonists [31] . TLR2 agonists induce mainly the production of
inflammatory cytokines; however, it has been reported that TLR2
activation in inflammatory monocytes in response to vaccinia virus
triggered the production of Type 1 IFN [32] .
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TLR4 ligands
TLR4 recognizes the major cell wall component LPS of Gramnegative bacteria [33,34] . Recognition of LPS by TLR4 requires
the accessory molecule myeloid differentiation protein-2 (MD-2)
[35,36] . Additional proteins are also involved in LPS binding. These
are LBP and the GPI-anchored CD14, which binds LBP to deliver
LPS to the TLR4–MD-2 complex [37] . TLR4 also recognizes
glycoinositolphospholipids from trypanosoma [38] , fusion protein
from respiratory syncytial virus [39] , envelope protein from mouse
mammary tumor virus [40] and pneumolysin from S. pneumoniae
[41] . TLR4 recognizes the plant-derived TLR4 mimetic Taxol [42]
and the LPS derivative monophosphoryl lipid A (MPL) [43] .
TLR5 & TLR11 ligands
TLR5 recognizes bacterial flagellin [44] . Specifically, it has been
demonstrated that TLR5 recognizes a conserved sequence of 13
amino acid residues on flagellin that is buried within the flagellar
filament and only accessible in monomeric flagellin [45] . TLR11
recognizes the profilin-like molecule from Toxoplasma gondii and
is the first defined ligand for this TLR [46,47] . TLR11 also recognizes uropathogenic bacteria but the specific ligand involved is
unknown [48] .
TLR intracellular microbial sensors
TLR3, TLR7 & TLR8 ligands
TLR3 recognizes dsRNA [49], which is a PAMP for most viruses that
originates from dsRNA viruses such as reoviruses [50] or is produced
as a replication intermediate for ssRNA viruses such as respiratory
syncytial virus, West Nile virus and encephalomyocarditis virus [51–
53] . TLR3 also recognizes the synthetic TLR3 agonist polyinosinicpolycytidylic acid (poly(I:C)) [50] . TLR7 and TLR8 are also viral
sensors but instead recognize ssRNA derived from RNA viruses such
as influenza virus and HIV [54,55] . In addition, they recognize the
imidazoquinoline derivatives imiquimod and resiquimod (R-848)
[56] and the guanine nucleotide analog loxoribine [57] .
TLR9 ligands
TLR9 recognizes unmethylated 2´-deoxyribose CpG motifs of
bacterial DNA [58] , as well as synthetic CpG oligodeoxynucleotides (ODNs) [59] . TLR9 is also a sensor of DNA viruses including
HSV-1, HSV-2 and murine cytomegalovirus [60–62] , and recognizes the pigment hemozoin from Plasmodium falciparum [63] .
TLR signaling
TLR activation triggers several intracellular signaling pathways,
which lead to activation of the transcription factors NF-kB,
activator protein-1 (AP-1) and various interferon regulatory factors (IRFs), culminating in the production of proinflammatory
240
cytokines and Type 1 IFNs, and expression of IFN-inducible
genes (Figure 2) . Following recognition of its cognate PAMP, the
TLR dimerizes and recruits one or more adapter molecules to
the receptor complex through a TIR–TIR homotypic protein
interaction. There are five known adapters; myeloid differentiation factor primary-response gene 88 (MyD88), TIR-domaincontaining adapter inducing IFN-b (TRIF; also known as
TICAM-1), MyD88-adapter-like (MAL) protein (also known as
TIRAP), TRIF-related adapter molecule (TRAM) and sterile-a
and Armadillo motif containing protein (SARM) [9] , although
TLR signaling can be divided into two major branches – MyD88
and TRIF – corresponding to the central signaling adapter used.
Both pathways will be discussed; however, the reader is directed
to other more comprehensive reviews on TLR signaling [64–70] .
MyD88-dependent signaling pathway
TLRs apart from TLR3 signal through MyD88, leading to the rapid
activation of NF-kB, MAPKs/AP-1 and IRF5, and induction of
proinflammatory cytokine production (Figure 2) . Signaling involves
the recruitment of IL-1R-associated kinase (IRAK) family members
to MyD88 through homotypic interactions between their death
domains. IRAK-4 is essential for activation of TLR-mediated signaling and once activated, IRAK-4 subsequently activates IRAK-1
and/or IRAK-2 by phosphorylation, which is followed by recruitment of TNF receptor-associated factor 6 (TRAF6) [71–74] . TRAF6
is a ubiquitin E3 ligase and in conjunction with the E2 enzyme
UBC13/UEV1A catalyzes the lysine 63-linked autoubiquitination
of TRAF6 [75] . In addition, lysine-63-linked polyubiquitin chains
are synthesized [76] , and act as a scaffold to recruit transforming
growth factor-b-activated kinase 1 (TAK1) via its ubiquitin-binding subunit TAB2/3. This leads to TAK1 polyubiquitination, and
recruitment of the canonical inhibitor of kB kinase (IKK) complex
via its ubiquitin-binding and regulatory subunit NF-kB essential
modulator (NEMO; also known as inhibitor of NF-kB kinase g
[IKKg]) [77,78] . The catalytic subunit of the IKK complex IKKb is
phosphorylated by TAK1, and in turn phosphorylates IkB leading to
its degradation, and concomitant nuclear migration and activation
of NF-kB [79] . TAK1 also triggers MAPK activation [79] leading to
the activation of AP-1. Interaction of the transcription factor IRF5
with MyD88, IRAK-1, IRAK-4 and TRAF6 results in the nuclear
translocation of IRF5, which activates cytokine gene expression [80] .
TRAF6-mediated lysine 63-linked ubiquitination of IRF5 is important for its entry into the nucleus to initiate target gene transcription
[81] . For TLR2- and TLR4-mediated MyD88-dependent signaling,
the adapter MAL is required [82–84] .
In addition to activation of NF-kB, AP-1 and IRF5 via MyD88dependent signaling pathways, stimulation of TLRs 7, 8 and 9
leads to the MyD88-dependent activation of IRF7, and induction
of IFN-a [85] , especially in a subset of DCs called plasmacytoid
DCs [86] . The formation of a complex consisting of MyD88,
TRAF6, IRAK-4, IRAK-1, IKKa and IRF7, as well as TRAF6dependent ubiquitination, are required for activation of IRF7
[1,85–87] . IRAK-1 and IKKa are IRF7 kinases and phosphorylate IRF7. It has also been reported that TRAF3 is required for
TLR7- and TLR9- dependent IFN-a production and interacts
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Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
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TLR4
TLR5/TLR11
TLR2/TLR1/TLR6
TRAM
MAL TRIF
MyD88
MAL
MyD88
Endosome
MyD88
TLR3
TLR9
TLR7/
TLR8
IRAK4/1/2
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DNA
dsRNA ssRNA
TIR
MyD88
UBC13
TRAF6
UEV1A
IKKα/β/NEMO
IRF5
TAB2/3 TAB1
TAK1
TRIF
TRADD
Pellino-1
RIP1
IRAK4/1
TRAF6
IKKα
TRAF3
TRAF3
MAPK
IKKε/TBK1
NF-κB
AP-1
IRF3
Proinflammatory
cytokines
IRF7
NF-κB/AP-1
Type 1 IFNs
Nucleus
IRF5
Proinflammatory
cytokines
Expert Rev. Vaccines © Future Science Group (2012)
Figure 2. Toll-like receptor signaling. Upon binding of the respective ligands to TLRs, a complex series of intracellular signaling
pathways is initiated, which involves adapter molecules and various downstream effector molecules, that culminate in the production of
proinflammatory cytokines, chemokines and Type 1 IFNs and expression of IFN-inducible genes. Activation of cell surface TLRs stimulates
MyD88-dependent signaling, leading to activation of the canonical IKK complex, translocation of NF-kB to the nucleus and TAK1mediated activation of MAPK and AP-1. Activation of cytosolic TLRs 7, 8 and 9 also activates MyD88-dependent activation of IRF7.
Activation of cytosolic TLR3 activates TRIF-dependent signaling, leading to activation of the noncanonical IKK complex, and subsequent
activation of IRF3 and IRF7. TRIF-dependent signaling also links, via TRAF6 and TAK1, to activation of NF-kB and AP-1. Activation of TLR4
can engage either MyD88- or TRIF-dependent signaling pathways.
IFN: Interferon; TLR: Toll-like receptor.
with IRAK-1 to phosphorylate IRF7 [88] . Phosphorylated IRF7
subsequently translocates to the nucleus and activates the transcription of Type 1 IFNs.
TRIF-dependent signaling pathway
TLR3 signals via the TRIF-dependent pathway involving the
recruitment of TRAF3 and activation of the noncanonical IKKrelated kinases TANK-binding kinase 1 (TBK1) and IKK-e (also
known as IKK-i), which phosphorylate IRF3 and IRF7 [89–91]
and induce their nuclear translocation with concomitant expression of Type 1 IFN genes, particularly IRF3-mediated IFN-b
production [92] .
In addition to Type 1 IFN production, activation of the TRIF
pathway leads to NF-kB and AP-1 activation, and the production
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of proinflammatory cytokines involving the TRAF6–IKK–TAK1
axis [93] . TRIF also recruits receptor-interacting protein (RIP)1
through the distinct RIP homotypic interaction motif. RIP1 then
undergoes lysine 63-linked polyubiquitination. TRADD and
Pellino-1 appear to be involved in ubiquitination and activation
of RIP1 [94,95] , which together form a complex with TRIF and
TRAF6 for the activation of TAK1, in turn activating the NF-kB
and MAPK pathways. TLR4 can signal via the TRIF pathway
utilizing the bridging adapter TRAM [96,97] .
NOD-like receptors
NLRs are a class of cytosolic PRRs that sense a wide array of
ligands and are involved in the regulation of innate immune
responses and cell death pathways. The NLRs are involved
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primarily in recognition of intracellular bacterial-derived PAMPs,
and as sensors of DAMPs. In humans, 23 NLRs have been defined
whereas >34 NLR genes are present in the mouse genome [98] .
NLRs comprise three domains: the N-terminal protein-binding
effector domain, which mediates downstream signaling and contains a caspase activating and recruitment domain (CARD), pyrin
domain (PYD), baculovirus inhibitory of apoptosis repeat (BIR)
domain, or an acidic domain. There is a centrally located nucleotide-binding and oligomerization (NACHT) domain important
for ligand-induced, ATP-dependent self-oligomerization and a
C-terminal LRR domain that binds microbial PAMPs or host
DAMPs [98] . The family of NLRs can be distinguished into
four subfamilies – NLRA (acidic transactivation-containing),
NLRB (BIR-containing neuronal apoptosis inhibitory proteins),
NLRC (CARD-containing NODs) and NLRP (PYD-containing
NALPs) – based on the domain type of the variable N-terminal
region as indicated [98] . NLR-mediated recognition of PAMPs
either drives the activation of MAPKs and NF-kB to induce the
production of proinflammatory cytokines, or induces the activation of caspase-1-activating platforms called inflammasomes [99] .
This review will discuss those NLRs where the particular microbial
motifs recognized have been characterized.
NOD1 & NOD2
NOD1 and NOD2 are members of the NLRC family and contain
either one or two CARDs in their N-terminal domains, respectively. Both NOD1 and NOD2 respond to intracellular bacterial
cell wall PDG components, released during bacterial growth or
degradation of PDG. Specifically, NOD1 recognizes meso-diaminopimelic acid from Gram-negative bacteria and certain Grampositive bacteria [100] , whereas NOD2 recognizes muramyl dipeptide (MDP) from Gram-positive and Gram-negative bacteria [101] .
These PRRs also recognize various types of pathogenic bacteria;
for example, Helicobacter pylori [102] , Campylobacter jejuni [103] ,
C. pneumoniae [104] and Enteropathogenic E. coli [105] are sensed
by NOD1, whereas NOD2 senses S. pneumoniae [106] , M. tuberculosis [107] and S. aureus [108] (Table 1) . Activation of NOD1 and
NOD2 via PAMP recognition initiates oligomerization of these
sensors, leading to recruitment of a CARD-containing adapter
protein known as RIP2 (also called RIP-like interacting CLARP
kinase [RICK]) by homotypic CARD–CARD interactions. RIP2
binds directly to NF-kB essential modulator (NEMO), the regulatory subunit of IKK, and promotes its ubiquitination and
activation of the catalytic subunits IKKa and IKKb. IKK phosphorylates the inhibitor, IkB, leading to its degradation and the
release of NF-kB, which subsequently translocates to the nucleus
to induce the transcriptional upregulation of proinflammatory
genes. Polyubiquitination of RIP2 mediates the recruitment of
TAK1, also leading to activation of the IKK complex [109–111] .
Both RIP2 and TAK1 are required for MAPK activation [112,113] .
Recently, a role for NOD1 in host defense against the parasite
T. cruzi has been reported [114] . NOD1 has also been shown to
induce the production of Type 1 IFN, which contributes to host
defense against H. pylori involving activation of IRF7 [115] . NOD2
has been shown to also respond to viral ssRNA, such as respiratory
242
syncytial virus, leading to IRF3 activation and the production of
Type 1 IFN [116] , and to promote responses to the parasite T. gondii [117] . In another study, NOD2 signaling played a critical role
in the production of Type 1 IFNs in response to M. tuberculosis
in an IRF5-dependent manner [118] . Recent studies have shown
that NOD2 plays a role in the intestinal clearance of the enteric
bacterium Citrobacter rodentium involving the chemokine CCL2
and CCL2-dependent recruitment of inflammatory monocytes
[119] . In addition, NOD2 played an important role in sensing of
S. pneumoniae, resulting in stimulation of CCL2 by macrophages
and bacterial clearance [120,121] .
Inflammasomes
NLRs play a critical role in the assembly and activation of inflammasomes, which are large cytosolic multi-protein innate immune
signaling complexes that activate caspase-1 [122–125] . To date, the
NLR protein family members NLRP1, NLRP3 and NLRC4
(IPAF), and the non-NLR protein AIM2 have been identified as
capable of forming inflammasomes. Inflammasomes are activated
in response to various microbial signals as well as endogenous
DAMPs. The NLRP1, NLRC4 and AIM2 inflammasomes recognize specific substances whereas the NLRP3 inflammasome
responds to an array of structurally and chemically diverse triggers. Inflammasomes control activation of proinflammatory caspase-1, which is constitutively expressed as an inactive pro-form
in the cytosol. Procaspase-1 contains a CARD that can either
interact directly with the CARD of NLRC4 or NLRP1 or with
the CARD of the adapter protein apoptosis-associated speck-like
protein containing a CARD (ASC). The PYD domain of ASC
also associates with the PYD domains of NLRPs and AIM2. The
CARD–CARD interaction leads to the autocatalytic processing
of procaspase-1 to the active form caspase-1. Caspase-1 is essential for the maturation and release of bioactive proinflammatory
cytokines IL-1b and IL-18 by proteolytic cleavage of their inactive
pro-forms. These cytokines play crucial roles in directing host
responses to infection and injury.
NLRP3 inflammasomes
The NLRP3 inflammasome (also known as NALP3 or cryopyrin)
is composed of NLRP3, ASC and procaspase-1, and is the most
widely characterized inflammasome. Structurally, NLRP3 contains an N-terminal PYD, central NACHT and C-terminal LRRs
(Figure 3A) . The NLRP3 inflammasome is activated by diverse
stimuli including both microbial components (from all classes
of pathogens) and a plethora of DAMPs. Activation of NLRP3
inflammasomes requires two signals: a priming signal that acts
to induce the expression of pro-IL-1b (signal 1) and an additional
stimulus that activates the inflammasomes (signal 2) and triggers
caspase-1 activation [122] . The first signal is provided by NF-kBactivating stimuli, including a wide variety of PAMPs such as LPS
and cytokines [122] . The second signal is triggered by exposure to
various NLRP3 activators (Table 1) including extracellular ATP
[126,127] , microbial ligands such as bacterial MDP [128] , malarial
hemozoin [129] , bacterial RNA [130] , viral DNA [131] , viral RNA
[132] and the dsRNA analogue poly(I:C) [132,133] , viruses including
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Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
sendai virus, adenovirus, influenza virus, vesicular stomatitis virus,
encephalomyocarditis virus, measles virus and vaccinia virus
[134–138] , and various toxins including the potassium ionophore
nigericin [127] and the marine toxin maitotoxin [127] , as well as
bacterial pore-forming toxins such as listeriolysin O from Listeria
monocytogenes, aerolysin from Aeromonas hydrophila and hemolysin
from S. aureus [139–141] . In addition to sensing viral nucleic acids
[131,132] , the mechanisms by which viral infection activates the
NLRP3 inflammasome may involve changes in intracellular ionic
concentrations [142] or disruption of lysosomal membranes [134] .
Stimulation with host-derived particulates such as uric acid [143]
and cholesterol crystals [144,145] , as well as other crystal structures
(silica, asbestos, aluminum hydroxide and fibrillar amyloid-b)
[146–151] , also activate caspase-1 in an NLRP3-dependent manner.
Recognition of PAMPs induces oligomerization of NLRP3, which
promotes clustering of ASC with NLRP3 via a PYD–PYD interaction. ASC and procaspase-1 then interact via their CARDs to
yield caspase-1. The molecular mechanisms that mediate NLRP3
inflammasome activation are not fully understood. Several mechanisms of NLRP3 inflammasome activation have been proposed
including low intracellular potassium concentration [152] , which
results from stimulation of the ATP-gated P2X7 receptor that
promotes potassium efflux and opening of the hemichannel pannexin-1 [153,154] , generation of reactive oxygen species [155] and
lysosomal destabilization after uptake of crystalline or particulate
NLRP3 activators, resulting in the release of cathepsin B (Figure 3A)
[156] . Activation of the NLRP3 inflammasome is required for protection against bacterial infections such as L. monocytogenes and
S. aureus [127] . NLRP3 inflammasomes are also critical sensors
in innate antifungal immunity and can be activated by Candida
albicans, S. cerevisiae, the fungal b-glucan curdlan and purified
zymosan [157,158] . Signaling via the tyrosine kinase SYK has been
shown to control both pro-IL-1b synthesis and inflammasome
activation, which involved reactive oxygen species production and
potassium efflux, in response to C. albicans [159] .
NLRC4 inflammasomes
NLRC4 (also called IPAF) is structurally similar to NOD1
and contains a CARD–NACHT–LRR complex, which allows
direct interaction with CARD-containing procaspase-1 following NLRC4 oligomerization (Figure 3B) [160] . Activation of caspase-1 through the NLRC4 inflammasome results in the secretion of IL-1b and IL-18 followed by caspase-1-dependent cell
death (pyroptosis) [160] . Several studies have suggested that ASC
is crucial for maximal NLRC4-mediated caspase-1 activation
and IL-b secretion but is not required for NLRC4-mediated cell
death [161–163] . NLRC4 inflammasomes are activated in response
to Gram-negative bacteria possessing functional Type 3 or Type 4
secretion systems (T3SS or T4SS) such as Legionella pneumophila,
Salmonella enterica serotype Typhimurium, Pseudomonas aeruginosa and Shigella flexneri [161–164] . NLRC4 inflammasomes recognize the T3SS or T4SS either indirectly by detecting bacterial
protein flagellin, or directly by detecting the rod protein of the
bacterial T3SS [165] . Flagellin and rod protein are delivered to the
cytosol by the bacterium through the T3SS [165] . L. monocytogenes
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is also detected in the cytosol by NLRC4 inflammasomes [166] . In
the case of L. pneumophila, flagellin is required for activation of
the NLRC4 inflammasome via interaction with the NLR member
NAIP5 [167] . Flagellin, however, is not essential for activation of
NLRC4 as the non-flagellated bacterium S. flexneri and a mutant
strain of P. aeruginosa, which is deficient in flagellin, activated
caspase-1 independently of flagellin [162,163] .
NLRP1 inflammasomes
The NLRP1 (NALP1) inflammasome contains NLRP1, caspase-1, caspase-5 and ASC [168] . NALP1 contains an N-terminal
PYD, central NACHT followed by C-terminal LRRs (Figure 3C) .
Unlike other NALPs, NALP1 also contains a C-terminal extension composed of a FIIND domain followed by a CARD domain
[169] . It has been proposed that both caspase-1 and caspase-5 are
activated upon assembly with NALP1 and ASC to form the
inflammasome, in which the N-terminal PYD of NALP1 binds
ASC to recruit and activate procaspase-1, whereas the C-terminal
CARD activates caspase-5 [169] . However, a recent report showed
that ASC was not essential for NLRP1 inflammasome and caspase-1 activation [170] , indicating that the C-terminal CARD can
directly activate procaspase-1. Human NLRP1 recognizes the
bacterial cell wall component MDP [170] , whereas the murine
variant NLRP1b, which lacks the N-terminal PYD, senses Bacillus
anthracis lethal toxin and activates procaspase-1 [171] .
AIM2 inflammasomes
The PYHIN (pyrin and HIN200 domain-containing protein)
family member absence in melanoma 2 (AIM2) has been identified as a cytosolic dsDNA sensor that activates caspase-1-mediated secretion of IL-1b [172–175]. The AIM2 inflammasome is
composed of AIM2, ASC and procaspase-1, and is proposed to
function in the cytosolic surveillance of DNA viruses. As with
NLRP3, AIM2 contains a PYD domain that interacts with ASC
via homotypic PYD–PYD interactions, allowing the ASC CARD
to recruit procaspase-1 to the complex (Figure 3D) . The C-terminal
HIN200 domain is responsible for binding cytoplasmic DNA by
means of its oligonucleotide/oligosaccharide-binding domain.
AIM2 was shown to be essential for inflammasome activation
in response to vaccinia virus and mouse cytomegalovirus [176] . It
has been reported that AIM2 is critical for the host proinflammatory response to the Gram-negative bacterium Francisella
tularensis [177,178] . In addition to viral DNA, AIM2 was identified as a detector of L. monocytogenes DNA [179] . The endoplasmic
reticulum-associated molecule stimulator of IFN gene (STING)
has been shown to be critical for regulating the production of IFN
in response to cytoplasmic DNA [180] . The cytosolic nucleic acidbinding protein LRRFIP1 has recently been identified as another
DNA sensor and contributed to the IRF3-mediated production of
IFN-b induced by L. monocytogenes [181] .
Retinoic acid-inducible gene-I-like receptors
The RLRs are cytosolic RNA helicases that sense viral RNAs
leading to activation of MAPK, NF-kB and IRF3/IRF7, and
the production of inflammatory cytokines, Type 1 IFNs and
243
244
PYD
PYD
PYD
IL-1β, IL-18
CARD
Caspase CARD
PYD ASC
PYD
LRR
LRR
NACHT
NACHT
LRR
LRR
Bacillus anthracis toxin
CARD PYD ASC
Caspase CARD
MDP
NACHT
IL-1β, IL-18
ASC
PYD ASC
NACHT
Reactive
oxygen
species
PAMP/DAMP
CARD
NLRP1
CARD Caspase
CARD Caspase
CARD NLRP1
NLRP3
NLRP3
Pannexin-1
Figure 3. Pathogen sensing by different inflammasomes.
pro-IL-1β
pro-IL-18
pro-IL-1β
pro-IL-18
ATP
P2X7
K+ efflux
PYD
Caspase-1
(Active)
CARD
Procaspase-1
Caspase CARD
(Inactive)
Caspase CARD
CARD
Cathepsin B
Uric acid
crystals
Asbestos, silica,
alum, amyloid-β
SIGNAL 1 (priming)
PRR, cytokine
PAMP/DAMP
pro-IL-1β
pro-IL-18
pro-IL-1β
pro-IL-18
Cell
death
CARD
IL-1β, IL-18
CARD
Caspase CARD
PYD
PYD
PYD
PYD
dsDNA
NACHT
HIN200
HIN200
LRR
LRR
AIM2
AIM2
NLRC4
NLRC4
?
T3SS
Shigella
Expert Rev. Vaccines © Future Science Group (2012)
ASC
ASC
DNA virus
IL-1β, IL-18
CARD
CARD ASC
Caspase CARD
PYD
NACHT
Flagellin
T3SS or T4SS
Salmonella, Legionella
Pseudomonas
CARD ASC
CARD
?
Caspase CARD
PYD
Caspase CARD
NAIP5
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Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
Review
Figure 3. Pathogen sensing by different inflammasomes (see page on the left). The NLRP3 inflammasome (A) is composed of
NLRP3, ASC and procaspase-1 and is activated by various stimuli after an initial priming signal to induce the expression of pro-IL-1b.
NLRP3 contains an N-terminal PYD, central NACHT and C-terminal LRRs. PYD recruitment of ASC followed by CARD–CARD interaction
with procaspase-1 leads to activation of caspase-1. Inflammasome activation may involve stimulation of the ATP-gated P2X 7 receptor that
promotes potassium efflux and opening of the hemichannel pannexin-1, generation of reactive oxygen species and lysosomal
destabilization/release of cathepsin B after uptake of crystalline or particulate NLRP3 activators. NLRC4 contains a CARD–NACHT–LRR
(B), which along with ASC optimally activates CARD-containing procaspase-1, leading to secretion of IL-1b and IL-18 followed by cell
death (pyroptosis). NLRC4 inflammasomes are activated by Gram-negative bacteria possessing functional T3SS or T4SS by detecting
flagellin, or the rod protein of T3SS. For Legionella pneumophila, activation of the NLRC4 inflammasome requires NAIP5. The NLRP1
inflammasome (C) contains NLRP1, which consists of an N-terminal PYD, central NACHT followed by C-terminal LRRs terminating in a
CARD domain, caspase-1, caspase-5 and ASC. PYD binds ASC to recruit and activate procaspase-1, whereas the C-terminal CARD
activates caspase-5 and may also activate caspase-1. Human NLRP1 recognizes MDP, whereas the murine variant NLRP1b lacking
N-terminal PYD senses the lethal toxin of Bacillus anthracis. The AIM2 inflammasome (D) is composed of AIM2, ASC and procaspase-1,
and is proposed to function in the surveillance of DNA viruses by sensing cytosolic dsDNA and activating caspase-1. AIM2 contains a PYD
domain that interacts with ASC via homotypic PYD–PYD interactions, allowing the CARD domain of ASC to recruit procaspase-1.
ASC: Apoptosis-associated speck-like protein containing a CARD; CARD: Caspase activating and recruitment domain; DAMP: Dangerassociated molecular pattern; MDP: Muramyl dipeptide; LRR: Leucine-rich repeat; NACHT: Nucleotide-binding and oligomerization;
PAMP: Pathogen-associated molecular pattern; PRR: Pattern recognition receptor; PYD: Pyrin domain; T3SS: Type 3 secretion system;
T4SS: Type 4 secretion system.
expression of Type 1 IFN-inducible genes. The RLR family of
intracellular receptors consists of three members, namely, RIGI, melanoma differentiation-associated gene 5 (MDA5) and
Laboratory of Genetics and Physiology gene 2 (LGP2) [182,183] .
These sensors recognize different RNA viruses (Table 1) [184,185] . For
example, RIG-I recognizes the ssRNA paramyxoviruses, including Newcastle disease virus, vesicular stomatitis virus and Sendai
virus, and orthomyxoviruses such as influenza virus. Japanese
encephalitis virus and hepatitis C are also recognized (flaviviruses). MDA5 is involved in the recognition of picornaviruses
such as encephalomyocarditis virus, Mengo virus and Theuiler’s
virus. Both sensors have been reported to recognize reoviruses,
West Nile virus and dengue virus. All RLRs are members of the
DExD/H family of RNA helicases and contain a central H-box
RNA helicase/ATPase domain, which is required for ligand recognition [185] . RIG-I and MDA5 also have a C-terminal regulatory
domain, which prevents constitutive activation of the protein,
thereby repressing downstream signaling [174] . The N termini of
RIG-I and MDA5 contain two tandem CARDs [185] . Following
activation of RLRs, these CARDs bind to a CARD-containing
adaptor protein called mitochondrial antiviral signaling (MAVS)/
IFN-b promoter stimulator (IPS)-1 to initiate downstream signaling [186] . IPS-1 then recruits TRADD, which in turn recruits
the E3 ubiquitin ligase TRAF3 and the adapter protein TANK,
and forms a complex with Fas-associated death domain protein
(FADD) and RIP1. This leads to activation of MAPK and NF-kB
through the canonical IKK complex, and activation of IRF3 and
IRF7 via the IKK-related kinase complex [187] . The nuclear translocation of IRF3/IRF7 and NF-kB mediates transcriptional activation of IFN and inflammatory cytokine genes leading to Type 1
IFN and cytokine production. Signaling via RIG-I also requires
the membrane-bound protein called STING [188] . LGP2, however,
lacks CARD domains and has been shown to act as a positive
regulator of RIG-I and MDA5-mediated viral responses [189] . It
has been reported that RIG-I via its C-terminal domain recognizes
ssRNA bearing 5´-triphosphate, which is a potential mechanism
allowing RIG-I to discriminate between self and nonself RNA
[190] . In addition, RIG-I recognizes short dsRNA including a
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shortened length poly(I:C), whereas MDA5 senses long dsRNA
such as the dsRNA analog poly(I:C) [191] .
Cooperation between PRRs: synergy & antagonism
Owing to their complex nature, pathogens present a wide variety
of ligands that may be recognized by the innate immune system
and this most likely involves multiple PRRs. The cooperation and
crosstalk between different PRR signals mediates activation of an
effective immune response and host defense against infection.
Collaboration between TLRs in particular has been demonstrated
in host resistance to M. tuberculosis, HSV and T. cruzi infections
[192–194] . Several studies have demonstrated the cooperation of
different TLRs in DC activation by stimulating multiple TLRs
with TLR agonists, which resulted in a synergistic upregulation of
cytokines, especially the Th1-polarizing cytokine IL-12p70 [195–
201] . For example, in human and mouse DCs, TLR3 and TLR9
acted in synergy with TLR7/8 and TLR9, leading to DCs with
enhanced and sustained Th1-polarizing capacity [196] . Warger
et al. [201] showed that peptide-loaded DCs activated by TLR
synergy led to a marked increase in cytotoxic T-lymphocyte effector function in mice in vivo. Other studies have shown the synergistic enhancement of IL-6 [197,199,201–203] , TNF-a [197,199,203] ,
IL-12p40 [202,203] , IL-10 [199,204] , IL-23 [196] and IL-1b [196] in DCs
in response to certain TLR ligands. The potential of using TLR
synergy to improve immune responses and immunotherapy has
recently been demonstrated using a Leishmania vaccine candidate
together with TLR4 and TLR9 agonists, which protected against
cutaneous leishmaniasis in a mouse model [205] . A second study
has shown that a triple TLR agonist combination increased the
protective efficacy of an HIV envelope peptide vaccine in mice
by augmenting the quality of T-cell responses needed for viral
clearance [206] . Another study by Chen et al. [207] showed the
involvement of TLR2, TLR4 and TLR9 in the generation of
optimal cytokine and antibody responses to a group B meningococcal outer membrane protein complex vaccine and protection
of animals from lethal sepsis. It is important to mention here
that combining TLR agonists can lead to antagonism, as demonstrated using an adeno-associated virus vector cancer vaccine
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administered to mice with TLR7 and TLR9 agonists, in which
Th1 responses and DC activation were reduced [208] . Ghosh et al.
[209] have also demonstrated that TLR–TLR crosstalk may result
in synergistic or antagonistic regulation of cytokine responses,
and identified certain combinations of TLR agonists that may
or may not be advantageous over single agonists for generating
optimal cytokine responses. Understanding how best to balance
the different response outcomes as a result of simultaneous TLR
stimulation is therefore crucial in generating an effective vaccine-induced immune response for protection against a particular
pathogen.
The molecular mechanisms by which TLR agonists act in
synergy is unclear but may partly involve activation of signaling through both the MyD88- and TRIF-dependent pathways
[201,210,211] . Stimulation of both pathways, however, does not always
lead to TLR synergy. For example, Zhu et al. [211] showed a lack of
synergy for IL-12 by DCs stimulated with macrophage-activating
lipoprotein (MALP)-2 (TLR2–TLR6 ligand) and CpG-ODN,
and Makela et al. [199] showed a lack of synergy between TLR2,
TLR5 and TLR7/8, and TLR5 or TLR7/8. Factors that may
influence synergy between different TLRs include the cellular
localization of individual TLRs, the association of TLRs with
other receptors or accessory molecules that link TLR to distinct
signaling pathways, the effect of the interaction of different TLR
signaling pathways on cytokine regulation, and the effect of combined triggering of different TLRs on the negative regulatory
mechanisms that modulate TLR responses [212–215] .
TLRs have been shown to act synergistically with other PRRs
expressed on DCs. Evidence for this has been demonstrated by the
stimulation of DCs with TLR4 and either NOD1 or NOD2 agonists, which acted synergistically to induce DC maturation and
the production of proinflammatory cytokines IL-6 and IL-12p40
[216] . Tada et al. [217] also demonstrated synergistic effects on DCs
when combining NOD1 and NOD2 agonists with various TLR
agonists in the generation of IL-12p70 and Th1 cells. Recently,
it has been demonstrated that stimulation of DCs with a NOD2
ligand and TLR2 agonist potentiated the production of IL-23
[218] . Other examples of cooperation between different PRRs
include the cooperation of dectin-1 with TLRs [219] , mannose
receptor targeting with CpG ODNs [220] , combined chemical
insult with TLR ligands [221] , and commensal-related bacteria
with the TLR3 agonist poly(I:C) [222] .
Exploiting the innate immune system in vaccine
adjuvant design
Vaccine development is experiencing a shift from traditional
whole cell vaccines to the development of more defined and
safer subunit vaccines. This has concomitantly created a major
and growing demand for the use of immunopotentiators in subunit vaccines, which are intrinsically poorly immunogenic, and
development of a new generation of vaccine adjuvants. The most
common barrier to the development of new adjuvants is safety
issues; therefore, those that can be demonstrated to have acceptable safety profiles, thus lacking major adverse side effects, are in
particularly high demand. In addition to facilitating increased
246
uptake of antigen by APCs, new vaccine adjuvants are designed
to facilitiate the recruitment and activation of DCs by stimulating
PRRs, thereby enabling the transition from the innate to adaptive
immune system for priming of B- and T-cell responses. The current vaccine adjuvants licensed for use in human vaccines (Table 2)
are limited (alum, the alum–TLR4 agonist MPL combination
Adjuvant System [AS]04 [GlaxoSmithKline Biologicals], oil-inwater emulsions MF59 and AS03, and reconstituted influenza
virosomes are currently on the market) [223] .
Licensed vaccine adjuvants & mechanisms of action
Aluminum-based adjuvants such as salts (aluminum phosphate or
aluminum hydroxide), which are referred to as alum, are widely
used in human vaccination and although these have been highly
successful, the mechanisms of action of alum remain unclear. In
several studies, activation of the NLRP3 inflammasome by alum
was shown to be a crucial mediator for adjuvanticity [148–150] .
However, other studies indicated that NLRP3 inflammasome
activation is not essential for alum’s adjuvant activity [224,225]
but is critical for mediating IL-1b secretion [225] . More recent
studies have suggested potentially new mechanisms mediating
the adjuvanticity of alum. These are by alteration of membrane
lipid structures [226] and by host cell DNA released as a result of
cell death [227] . MF59 is well tolerated in humans and has been
licensed for use in an influenza vaccine for the elderly (Fluad®,
Novartis). MF59 is a squalene-based emulsion and promotes
influenza antigen-specific CD4 + T-cell responses and strong and
long-lasting memory T- and B-cell responses [228] . The adjuvanticity of MF59 has, as with alum, been recently reported to be
independent of the NLRP3 inflammasome [224] . Another oil-inwater emulsion, AS03, containing squalene and tocopherol, is in
use in a pandemic influenza vaccine [229] , and a virosomal adjuvanted vaccine called Inflexal® (Crucell [former Berna Biotech
Ltd]), which has been on the market for >10 years, is in use in
a seasonal influenza vaccine for all age groups [230] . However,
the mechanisms of action of these vaccine adjuvants are less
understood.
It has recently been demonstrated that a combination of aluminum salts and MPL primed antigen-specific memory CD8 +
T cells, which significantly protected mice from influenza A
challenge [231] . These results suggested that these adjuvants could
be used in human vaccines to prime protective memory CD8 +
T cells. AS04 is a combination of aluminum salts and MPL,
and two AS04-adjuvanted vaccines are licensed; FENDrix® and
Cervarix® vaccines (both GlaxoSmithKline Biologicals), which
confer protective immunity against HBV and HPV, respectively
[232] . AS04 induces a transient localized innate immune response
and activation of DCs, leading to activation of antigen-specific
T cells and enhanced adaptive immunity [233] . Unlike alum,
which promotes Th2 responses [234] , MPL modulates the quality
of the immune response towards a balanced Th1/Th2 response
[232,235] . MPL is a detoxified derivative of LPS from Salmonella
minnesota, and is the first of a new generation of defined vaccine adjuvants to achieve widespread use in human populations
since the approval of alum [223] . While LPS is highly toxic,
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Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
causing strong inflammatory responses, the LPS derivative MPL
enhances adaptive immunity without causing excessive inflammation. The mechanism by which MPL enables potent but safe
adjuvanticity appears to be a result of biased TRIF signaling and
selective activation of p38 MAPK [43,236] . It has also recently
been reported that MPL fails to activate caspase-1, leading to
defective production of the proinflammatory cytokine IL-1b,
and that this was attributed to TRIF-biased TLR4 activation
by MPL and the impairment of NLRP3 inflammasome activation, which requires MyD88 [237] . These findings highlight the
potential of altering signaling pathways for improving the safety
of vaccine adjuvants.
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Vaccine adjuvants in clinical development
Single PRR-based adjuvants
Other TLR agonists in clinical stages of development are emerging as potential vaccine adjuvant candidates (Table 2; reviewed in
[238]). For example, TLR7/8 agonists are potent vaccine adjuvants
for inducing T-cell-mediated immunity [239] , and the TLR9 agonist CpG 7909 combined with alhydrogel has been used in clinical trials of a P. falciparum blood-stage malaria vaccine [240,241] .
The TLR3 agonist polyI:polyC12U (Ampligen® ; Hemispherx
Biopharma) is also a promising mucosal adjuvant for intranasal
H5N1 influenza vaccination [242] . The TLR5 agonist flagellin has
been used in several experimental studies of flagellin-based vaccines, mostly as recombinant flagellin–antigen fusion proteins,
and flagellin-based vaccines for infectious diseases have entered
clinical trials (reviewed in [243]).
Review
Vaccine adjuvants targeting combination of PRRs
Evidence is now emerging that many empiric vaccines and adjuvants inherently stimulate PRRs. For example, the yellow fever
vaccine 17D, one of the most effective vaccines available, has been
shown to activate multiple DC subsets through stimulation of various TLRs [244] , supporting the strategy of targeting multiple PRRs,
especially TLRs, to optimize vaccine-induced immune responses.
In addition to AS04, discussed above, a combination of MPL
with the saponin QS-21 is contained within AS01 and AS02,
which are liposome and emulsion-based formulations, respectively.
As with AS04, clinical studies of AS01- and AS02-adjuvanted vaccines have demonstrated acceptable safety profiles [232] . Various
candidate AS-adjuvanted vaccines have been evaluated in clinical trials using AS01 and AS02, including malaria, HIV and
TB (reviewed in [232]). There are several other combination-type
adjuvants in clinical development, incuding IC31, which consists of a synthetic antimicrobial cationic peptide and synthetic
ODN, ODN1a [245–247] . Cationic liposomes (CAF01) composed
of dimethyl-dioctadecyl-ammonium and the immune modulator trehalose 6,6’-dibehenate are promising adjuvants and have
entered clinical trials for use in a TB vaccine [248,249] . Iscomatrix
adjuvant has been shown to induce antibody and cell-mediated
immune responses including CD8 + T-cell responses [250] . These
findings suggest that certain combinations of PRR agonists, such
as multiple TLR agonists or individual TLR agonists in combination with non-TLR agonists, could have advantages over the
use of a single agonist in amplifying or modulating the immune
response. The challenge will be in identifying which combinations
Table 2. Vaccine adjuvants and modulation of immune response outcomes.
Adjuvant
Components
Immune response outcomes
Alum
Aluminum salts
Ab, Th2
MF59
Oil-in-water emulsion
Ab, Th1/Th2, long-lived memory
AS03
Oil-in-water emulsion
Ab, B-cell memory, Th
AS04
Alum-absorbed MPL
Ab, Th1
Virosomes
Liposome, neuraminidase and hemagglutinin
Ab, Th1
Licensed vaccine adjuvants
Vaccine adjuvants in clinical development
Poly(I:C)
Synthetic dsRNA, TLR3 agonist
Ab, Th1, CD8 + T cells
Flagellin
FL-antigen fusion proteins, TLR5 agonist
Ab Th1/Th2, CD8 + T cells
Imidazoquinolines
Small molecules, TLR7 and TLR8 agonists
Ab, Th1, CD8 + T cells
CpG 7909, CpG 1018
CpG ODNs alone or combined with alum/emulsion,
TLR9 agonists
Ab, Th1
AS01
Liposome, MPL and saponin (QS-21)
Ab, Th1
AS02
Oil-in-water emulsion, MPL and saponin
Ab, Th1
IC31
Synthetic peptide KLK and ODN
Ab, Th1, long-lived memory
CAFO1
Cationic liposomes (DDA and TDB)
Ab, Th1
ISCOMS/ISCOMATRIX
Saponin-based particulate
Ab, broad Th, CD8 + T cells
Ab: Antibody; AS: Adjuvant system; CAF01: Cationic adjuvant formulation 01; DDA: Dimethyl-dioctadecyl-ammonium; ISCOM: Immune-stimulating complex;
MPL: Monophosphoryl lipid A; ODN: Oligodeoxynucleotides; Poly(I:C): Polyinosinic-polycytidylic acid; TDB: Trehalose 6,6’-dibehenate; TLR: Toll-like receptor.
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of PRR agonists induce optimal protective responses depending
on the vaccine target.
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Immunomodulation by targeting PRR signaling
pathways
In addition to targeting PRRs in vaccine adjuvant design and
potentially manipulating signaling pathways for improving adjuvant safety, another possible and novel strategy for modulating
immune responses against specific pathogens is targeting specific
PRR-induced signaling pathways; for example, MAPK pathways,
which seem to be important in skewing T-helper cell polarization.
It has been shown that LPS stimulated the production of IL-12p70
by DCs influencing a Th1 bias dependent on phosphorylation
of p38 MAPK and JNK, whereas Pam3Cys preferentially activated ERK and induced little IL-12, indicating a role for ERK in
Th2 polarization [251] . DCs from ERK1 knockout mice showed
enhanced IL-12p70 and reduced IL-10 production in response
to TLR stimulation, again suggesting ERK biases the immune
response towards Th2 [252] . There is evidence that altered cytokine
production by TLR2- and TLR4-primed DCs upon TLR restimulation may be attributable to an altered balance of signaling
pathways [253] . Moreover, a recent report has demonstrated that
inhibition of p38 MAPK signaling in DCs enhanced protective
efficacy of a pertussis vaccine formulated with CpG by suppressing IL-10-secreting Treg cells [254] . These data clearly illustrate
that TLR agonists can be made more effective by manipulating
downstream signaling pathways.
PRR output & design/choice of adjuvant
Understanding host defense against infection and in particular knowledge of the distinct type of immune response
that is naturally induced for protection against a given pathogen will be important for the design of new adjuvants or in
directing the choice of existing adjuvants to induce similar
protection in response to a rationally designed vaccine. The
type of PRR or unique combination of PRRs stimulated by
a pathogen are responsible for controlling the outcome of the
adaptive response, as is the case for the various PRR agonists.
For example, most TLR agonists induce antibody and Th1
responses [238] , although some can induce Th2 [255] and possibly
Th17 responses [256] . Nod1 stimulation has also been shown to
induce immunity with predominately a Th2 polarization profile
[257] . Studies have evaluated cytokine profiles induced in DCs
after TLR stimulation with individual TLR agonists [256,258,259] ;
however, knowledge of the response outcomes including cytokine and chemokine signature profiles as well as the ratio of
T-cell subtypes generated upon activation of combination PRRs
is lacking, and would help in the design of vaccine formulations
employing appropriate combination adjuvants in the future that
can broaden the development of new vaccines against infectious diseases. Other issues for consideration when designing
or choosing an adjuvant formulation are the vaccine antigen
target and delivery system used as well as the route of vaccination. Clearly TLR agonists are the most clinically advanced
in adjuvant development, but it is important to mention here
248
some additional considerations regarding their use as vaccine
adjuvants, such as variation in TLR expression and influence of
age on TLR responsiveness [238] . Another important consideration is the potential for the induction of excessive inflammatory responses and risk of autoimmunity. In particular, innate
immune signaling triggered by endogenous ligands and activation of nucleic acid-sensing TLR3, TLR7/8 and TLR9 has
the potential to provide the development of autoimmune or
autoinflammatory diseases due to loss of tolerance resulting in
aberrant recognition of self-nucleic acids [260] . Therefore, the
balance between providing the appropriate host defense against
a pathogen for safe-guarding against infection and minimizing
possible adverse responses needs to be met to ensure safe adjuvanticity. The identification of detoxified derivatives of TLR
agonists, as has been described by the use of MPL as a derivative of LPS, is a possible option to help towards improving the
safety of vaccine adjuvants.
Expert commentary
The detection of PAMPs by TLRs, NLRs and RLRs activates
proinflammatory signaling pathways to mount an effective anti­
microbial response targeting the invading pathogen. While all
of the PRRs may be suitable vaccine adjuvant targets, TLRs
have been particularly well characterized and widely investigated as new vaccine adjuvant targets. TLR agonists are being
used to mimic natural ligands of pathogens and activate intracellular TLR signaling in an approach to enhance the immunogenicity of subunit vaccines and modulate the outcome of
immune responses. TLR-based activation can be built upon
to include other individual PRR-based as well as combined
PRR-based strategies in the innate–adaptive paradigm to generate immunity and tailor a new generation of vaccine adjuvants. Understanding how the innate immune system senses
individual pathogens and the crosstalk among PRRs is crucial
for understanding the complexities of innate immune regulation. Furthermore, knowledge of these processes, especially
the immunological outcomes of PRR signaling, will provide
new insights facilitating the design of novel vaccine adjuvants
that target PRRs and appropriately guide protective immunity. A better understanding of how individual cell signaling
pathways such as MAPKs influence regulation of the adaptive
immune system, thereby underlying immunity to infection,
will be important in enabling the generation of more effective
and safe vaccine adjuvants that not only target PRRs but also
manipulate downstream signaling pathways to influence the
outcome of the immune response.
Five-year view
Within the next 5 years, we would expect to see major advances
in the development of TLR-based vaccine adjuvants and an
expansion in the breadth of adjuvants licensed for human vaccines. Consequently, we will see a greater number of safe and
effective prophylactic vaccines for infectious diseases that contain formulations of TLR agonists. New vaccine formulations
may incorporate multiple TLR agonists and other PRR agonists
Expert Rev. Vaccines 11(2), (2012)
Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
in order to take advantage of their synergistic effects on cytokine production and in generating effective immune responses.
Targeting various PRRs together with manipulating cell signaling pathways may potentially be used for optimally directing
specific types of immune responses elicited by rationally designed
vaccines, and in generating safer vaccines. The success of defined
subunit vaccines in the next 5 years will correlate with progress
in understanding PRR adjuvanticity and response outcomes, cell
signaling mechanisms, advances in vaccine formulation development including delivery modalities, and in finding a balance
between effective immune stimulation and potentially excessive
systemic inflammatory responses.
Review
Acknowledgements
The author gives special thanks to Madeleine Flynn (QIMR) who has worked
with her on the illustrations presented in this review.
Financial & competing interests disclosure
This work was supported by the National Health and Medical Research
Council of Australia��������������������������������������
. The author has no other relevant affiliations
��������������
or finan�����
cial involvement with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
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Key issues
• New vaccine adjuvants or combinations therein are crucial to the success of the new subunit vaccine formulations.
• DCs are critical in bridging the innate and adaptive arms of the immune system and control the magnitude, quality and duration of the
adaptive immune response.
• Directly stimulating pattern recognition receptors (PRRs) on DCs is a viable strategy for the development of new-generation vaccine
adjuvants that enhance or modulate humoral and cellular immunity, and could be a beneficial approach for many vaccine formulations.
• The success of TLR4-containing vaccines demonstrates the potential to develop new or improved vaccine adjuvants based on defined
PRRs.
• Pathogen sensing involves activating complex sets of PRRs for the induction of an effective immune response and host defense against
infection.
• Understanding the mechanisms by which various pathogens are recognized by PRRs and the crosstalk between different PRRs involving
synergy/antagonism will be beneficial to the development of effective vaccine adjuvants.
• Multiple PRR-based vaccine adjuvants may be required to elicit optimal immune responses.
• Knowledge of PRR adjuvanticity and output, and signaling pathways that guide particular types of immune response will be crucial for
successful vaccine adjuvant design in promoting appropriate immune responses for protection against a given pathogen/disease.
• Safety is paramount and therefore vaccine adjuvants that induce local immune activation without inducing systemic inflammatory
responses that might lead to unwanted adverse side effects are important.
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