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 bac-teria, 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 elim-ination of microbial pathogens, thereby provid-ing 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 pro-teins, 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
antimicrobial and inflammatory responses [1,2]. The innate immune system is also responsible for initiating an adaptive immune response specifi-cally tailored to the invading microbe [3,4]. DCs play a critical role in translating the appropri-ate signals from the innate to adaptive immune system to mediate the regulation of adaptive immunity (Figure 1) [5]. The adaptive immune sys-tem 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 addi-tion 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
Colleen OliveThe 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.
PRRs: Toll-like receptors (TLRs), nucleotide-binding oligomer-ization domain (NOD)-like receptors (NLRs) and the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs).
Toll-like receptorsThe 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 recep-/IL-1 recep-IL-1 recep-tor (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 compart-ments (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.
TLR extracellular microbial sensorsTLR1, TLR2 & TLR6 ligandsTLR2 recognizes a wide array of structurally diverse PAMPs. These include lipoproteins from Escherichia coli, Borrelia burg-dorferi, mycoplasma, Mycobacterium tuberculosis and Treponema pallidum [11–15]; the diacylated mycoplasmal lipopeptide macro-phage-activating lipopeptide-2 (MALP-2) [16] and its stereoisomer R-MALP [17]; peptidoglycan (PDG) from Gram-positive bacte-ria 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]; lipoarabino mannan from mycobacteria [24]; glyco-sylphosphatidylinositol (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 recogni-tion of some ligands, CD14 and lipopolysaccharide (LPS)-binding
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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Table 1. Distinct families of pattern recognition receptors recognize distinct pathogen-associated molecular patterns from various microbes.
PRRs PAMPs Species (microbes)
TLR
TLR2/1 Triacyl lipopeptides Bacteria and mycobacteria
Typhimurium, P. aeruginosa, S. flexneri)AIM2 dsDNA Viruses (vaccinia virus) and bacteria (Francisella tularensis)
RLR
RIG-I
MDA5LPG2
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
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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].
TLR4 ligandsTLR4 recognizes the major cell wall component LPS of Gram-negative 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 ligandsTLR5 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 recog-nizes uropathogenic bacteria but the specific ligand involved is unknown [48].
TLR intracellular microbial sensorsTLR3, TLR7 & TLR8 ligandsTLR3 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 polyinosinic-polycytidylic 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 ligandsTLR9 recognizes unmethylated 2 -́deoxyribose CpG motifs of bacterial DNA [58], as well as synthetic CpG oligodeoxynucleo-tides (ODNs) [59]. TLR9 is also a sensor of DNA viruses including HSV-1, HSV-2 and murine cytomegalovirus [60–62], and recog-nizes the pigment hemozoin from Plasmodium falciparum [63].
TLR signalingTLR 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 fac-tors (IRFs), culminating in the production of proinflammatory
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 differentia-tion factor primary-response gene 88 (MyD88), TIR-domain-containing 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 pathwayTLRs 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 sig-naling and once activated, IRAK-4 subsequently activates IRAK-1 and/or IRAK-2 by phosphorylation, which is followed by recruit-ment 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-bind-ing 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 impor-tant 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 MyD88-dependent 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 TRAF6-dependent ubiquitination, are required for activation of IRF7 [1,85–87]. IRAK-1 and IKKa are IRF7 kinases and phosphory-late IRF7. It has also been reported that TRAF3 is required for TLR7- and TLR9- dependent IFN-a production and interacts
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with IRAK-1 to phosphorylate IRF7 [88]. Phosphorylated IRF7 subsequently translocates to the nucleus and activates the tran-scription of Type 1 IFNs.
TRIF-dependent signaling pathwayTLR3 signals via the TRIF-dependent pathway involving the recruitment of TRAF3 and activation of the noncanonical IKK-related 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 expres-sion 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
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 receptorsNLRs 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
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 TAK1-mediated 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.
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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 con-tains 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 nucleo-tide-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 activa-tion of caspase-1-activating platforms called inflammasomes [99]. This review will discuss those NLRs where the particular microbial motifs recognized have been characterized.
NOD1 & NOD2NOD1 and NOD2 are members of the NLRC family and contain either one or two CARDs in their N-terminal domains, respec-tively. Both NOD1 and NOD2 respond to intracellular bacterial cell wall PDG components, released during bacterial growth or degradation of PDG. Specifically, NOD1 recognizes meso-diami-nopimelic acid from Gram-negative bacteria and certain Gram-positive bacteria [100], whereas NOD2 recognizes muramyl dipep-tide (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. tuber-culosis [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 reg-ulatory subunit of IKK, and promotes its ubiquitination and activation of the catalytic subunits IKKa and IKKb. IKK phos-phorylates 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
syncytial virus, leading to IRF3 activation and the production of Type 1 IFN [116], and to promote responses to the parasite T. gon-dii [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].
InflammasomesNLRs play a critical role in the assembly and activation of inflam-masomes, 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 rec-ognize specific substances whereas the NLRP3 inflammasome responds to an array of structurally and chemically diverse trig-gers. Inflammasomes control activation of proinflammatory cas-pase-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 essen-tial 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 inflammasomesThe 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 con-tains 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-kB-activating 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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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 inter-action. 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 mecha-nisms of NLRP3 inflammasome activation have been proposed including low intracellular potassium concentration [152], which results from stimulation of the ATP-gated P2X
7 receptor that
promotes potassium efflux and opening of the hemichannel pan-nexin-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 pro-tection 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 inflammasomesNLRC4 (also called IPAF) is structurally similar to NOD1 and contains a CARD–NACHT–LRR complex, which allows direct interaction with CARD-containing procaspase-1 follow-ing NLRC4 oligomerization (Figure 3b) [160]. Activation of cas-pase-1 through the NLRC4 inflammasome results in the secre-tion 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 aerugi-nosa and Shigella flexneri [161–164]. NLRC4 inflammasomes rec-ognize 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
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 inflammasomesThe NLRP1 (NALP1) inflammasome contains NLRP1, cas-pase-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 exten-sion 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 cas-pase-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 inflammasomesThe PYHIN (pyrin and HIN200 domain-containing protein) family member absence in melanoma 2 (AIM2) has been iden-tified as a cytosolic dsDNA sensor that activates caspase-1-me-diated 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 proinflam-matory response to the Gram-negative bacterium Francisella tularensis [177,178]. In addition to viral DNA, AIM2 was identi-fied 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 acid-binding 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 receptorsThe 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
Pattern recognition receptors: sentinels in innate immunity & targets of new vaccine adjuvants
expression of Type 1 IFN-inducible genes. The RLR family of intracellular receptors consists of three members, namely, RIG-I, 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, includ-ing Newcastle disease virus, vesicular stomatitis virus and Sendai virus, and orthomyxoviruses such as influenza virus. Japanese encephalitis virus and hepatitis C are also recognized (flavivi-ruses). 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 rec-ognition [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 sig-naling [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 trans-location of IRF3/IRF7 and NF-kB mediates transcriptional acti-vation 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 5́ -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
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 P2X7 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: Danger-associated 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.
shortened length poly(I:C), whereas MDA5 senses long dsRNA such as the dsRNA analog poly(I:C) [191].
Cooperation between PRRs: synergy & antagonismOwing 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 effec-tor function in mice in vivo. Other studies have shown the syn-ergistic 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 meningo-coccal 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 dem-onstrated 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 vac-cine-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 signal-ing 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 com-bined 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 ago-nists, 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 designVaccine 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 sub-unit 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 accept-able safety profiles, thus lacking major adverse side effects, are in particularly high demand. In addition to facilitating increased
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 cur-rent 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-in-water emulsions MF59 and AS03, and reconstituted influenza virosomes are currently on the market) [223].
Licensed vaccine adjuvants & mechanisms of actionAluminum-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 adjuvan-ticity of MF59 has, as with alum, been recently reported to be independent of the NLRP3 inflammasome [224]. Another oil-in-water emulsion, AS03, containing squalene and tocopherol, is in use in a pandemic influenza vaccine [229], and a virosomal adju-vanted 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 alu-minum 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 vac-cine adjuvants to achieve widespread use in human populations since the approval of alum [223]. While LPS is highly toxic,
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causing strong inflammatory responses, the LPS derivative MPL enhances adaptive immunity without causing excessive inflam-mation. 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 activa-tion, which requires MyD88 [237]. These findings highlight the potential of altering signaling pathways for improving the safety of vaccine adjuvants.
Vaccine adjuvants in clinical developmentSingle PRR-based adjuvantsOther TLR agonists in clinical stages of development are emerg-ing 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 ago-nist CpG 7909 combined with alhydrogel has been used in clini-cal 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 vac-cines, mostly as recombinant flagellin–antigen fusion proteins, and flagellin-based vaccines for infectious diseases have entered clinical trials (reviewed in [243]).
Vaccine adjuvants targeting combination of PRRsEvidence is now emerging that many empiric vaccines and adju-vants 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 vari-ous 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 vac-cines have demonstrated acceptable safety profiles [232]. Various candidate AS-adjuvanted vaccines have been evaluated in clini-cal 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 con-sists of a synthetic antimicrobial cationic peptide and synthetic ODN, ODN1a [245–247]. Cationic liposomes (CAF01) composed of dimethyl-dioctadecyl-ammonium and the immune modula-tor 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 combi-nation 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.
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of PRR agonists induce optimal protective responses depending on the vaccine target.
Immunomodulation by targeting PRR signaling pathwaysIn addition to targeting PRRs in vaccine adjuvant design and potentially manipulating signaling pathways for improving adju-vant 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 acti-vated 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 restim-ulation 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 suppress-ing 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 adjuvantUnderstanding host defense against infection and in par-ticular knowledge of the distinct type of immune response that is naturally induced for protection against a given patho-gen 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 cyto-kine 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 infec-tious 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 vaccina-tion. Clearly TLR agonists are the most clinically advanced in adjuvant development, but it is important to mention here
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 consider-ation is the potential for the induction of excessive inflamma-tory responses and risk of autoimmunity. In particular, innate immune signaling triggered by endogenous ligands and acti-vation 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 adju-vanticity. The identification of detoxified derivatives of TLR agonists, as has been described by the use of MPL as a deriva-tive of LPS, is a possible option to help towards improving the safety of vaccine adjuvants.
Expert commentaryThe 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 investi-gated as new vaccine adjuvant targets. TLR agonists are being used to mimic natural ligands of pathogens and activate intra-cellular TLR signaling in an approach to enhance the immu-nogenicity 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 gen-erate immunity and tailor a new generation of vaccine adju-vants. Understanding how the innate immune system senses individual pathogens and the crosstalk among PRRs is crucial for understanding the complexities of innate immune regu-lation. 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 immu-nity. 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 viewWithin 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 vac-cines. Consequently, we will see a greater number of safe and effective prophylactic vaccines for infectious diseases that con-tain formulations of TLR agonists. New vaccine formulations may incorporate multiple TLR agonists and other PRR agonists
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in order to take advantage of their synergistic effects on cyto-kine production and in generating effective immune responses. Targeting various PRRs together with manipulating cell signal-ing 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 devel-opment including delivery modalities, and in finding a balance between effective immune stimulation and potentially excessive systemic inflammatory responses.
AcknowledgementsThe author gives special thanks to Madeleine Flynn (QIMR) who has worked with her on the illustrations presented in this review.
Financial & competing interests disclosureThis work was supported by the National Health and Medical Research Council of Australia. The author has no other relevant af� liations or � nan-. The author has no other relevant af�liations or �nan-cial involvement with any organization or entity with a �nancial interest in or �nancial 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.
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.
ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest
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