Potential adjuvantic properties of innate immune stimuli

[Human Vaccines 5:6, 381-394 ; June 2009]; ©2009 Landes Bioscience

Review

Potential adjuvantic properties of innate immune stimuli Hemamali J. Warshakoon, Jennifer D. Hood, Matthew R. Kimbrell, Subbalakshmi Malladi, Wen Yan Wu, Nikunj M.Shukla, Geetanjali Agnihotri, Diptesh Sil and Sunil A. David* Department of Medicinal Chemistry; University of Kansas; Lawrence, KS USA

Key words: adjuvants, vaccines, immunostimulation, toll-like receptors, innate immunity, monophosphoryl lipid A, lipopeptides, imidazoquinolines, thiazoloquinolones, flagellin, CpG DNA, ssRNA

Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns and serve as primary sensors of the innate immune system. Ten members of the TLR family have so far been identified in the human genome. The ligands for these receptors are structurally highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-containing DNA (TLR9) and profilin present on uropathogenic bacteria (TLR 11). Complementing the TLRs are the nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs), which detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment, as well as the retinoic-acidinducible protein 1 (RIG-I-like receptors; RLRs) which sense single-stranded RNA of viral origin. The activation of PRRs by their cognate ligands leads to production of inflammatory cytokines, upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer cells, in addition to priming and amplifying antigen-specific T-, and B-cell effector functions. Thus, these stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. This review summarizes what is currently known about the immunopotentiatory and adjuvantic activities of innate immune stimuli.

Introduction—Adjuvants, Toll-Like Receptors (TLRs) and other Microbial Sensors “As interesting as this method is from a practical point of view, it may be just as interesting from a theoretical point of view, because of the research it could stimulate to understand the intimate mechanism of either the increase in antitoxin induced in this case, or the generation of antitoxins within the animal”—Gaston Ramon, 1926. *Correspondence to: Sunil A. David; Department of Medicinal Chemistry; University of Kansas; Multidisciplinary Research Building; Room 320D; 2030 Becker Drive; Lawrence, KS 66047 USA; Tel.: 785.864.1610; Fax: 785.864.1961; Email: [email protected] Submitted: 12/08/08; Revised: 02/08/09; Accepted: 02/16/09 Previously published online as a Human Vaccines E-publication: http://www.landesbioscience.com/journals/vaccines/article/ 8175 www.landesbioscience.com

Just as the prescient comment by Gaston Ramon was relegated to the last footnote of his 1926 paper,1 so has research on the mechanisms of action of adjuvants, until recently, languished as parenthetical annotations and addenda in the archives of immunology and vaccine development. Ramon defined immunological adjuvants as “substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone.” Interestingly enough, he was referring to his empirical findings that the addition of bread crumbs, tapioca, saponin and ‘starch oil’ to antigenic preparations greatly enhanced antibody responses to diphtheria or tetanus.2 A year later, the adjuvanticity of aluminum salts (primarily phosphate and hydroxide) was discovered by Glenny and coworkers.3 In the 83 years that have elapsed, the repertoire of investigational adjuvants has grown to encompass a very wide range of materials,4 but aluminum salt-based mineral salts (generically, and incorrectly, termed “alum”) have remained the only adjuvants currently approved by the FDA. Aluminum salts have enjoyed a good safety record, but they are weak adjuvants for antibody induction and induce a T helper-2 (TH2)-skewed, rather than a T helper-1 (TH1) response.5,6 Furthermore, not only are aluminum salts ineffective at inducing cytotoxic T lymphocyte (CTL) or mucosal IgA antibody responses, but also have a propensity to induce IgE responses, which have been associated with allergic reactions in some subjects.5,6 Very recent reports implicate the Nalp3 inflammasome, a component of the innate immune response, as the effector limb of alum-associated adjuvanticity.7-9 In 1962, Dresser observed that injection of purified soluble proteins not only failed to stimulate an immune response, but tolerized animals unless a bacterial extract was admixed with the protein immunogen.10 This led him to redefine adjuvanticity as “a property of a substance which can act as a physiological switch, directing at least some immunologically competent cells to respond by making antibody rather than by becoming immunologically paralyzed by the antigen,”11 confirming Johnson’s earlier observations that lipopolysaccharide (LPS) from Gram-negative bacteria exerted potent adjuvant properties,12 and perhaps paved the way for the subsequent discovery of the wide range of microorganism-derived adjuvants.13 TLRs are pattern recognition receptors present on diverse cell types that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).14,15 There are 10 TLRs in the human genome;

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Innate immune stimuli and adjuvanticity

Figure 1. Structures of enterobacterial lipopolysaccharide, diphosphoryl and monophosphoryl lipid A.

these are trans-membrane proteins with an extracellular domain having leucine-rich repeats (LRR) and a cytosolic domain called the Toll/IL-1 receptor (TIR) domain.15 The ligands for these receptors are highly conserved microbial molecules such as LPS (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double stranded RNA (TLR3), CpG motif-containing DNA (recognized by TLR9), and profilin present on uropathogenic bacteria (TLR 11).16,17 The activation of TLRs by their cognate ligands leads to production of inflammatory cytokines, and upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer (NK) cells (innate immune response), in addition to priming and amplifying T-, and B-cell effector functions18-21 (adaptive immune responses). Thus, TLR stimuli serve to link innate and adaptive immunity19 and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. TLR1, -2, -4, -5 and -6 respond to extracellular stimuli, while TLR3, -7, -8 and -9 respond to intracytoplasmic PAMPs, being associated with the endolysosomal compartment.15

TLR4 Agonists—Lipopolysaccharide, Monophosphoryl Lipid A and Synthetic Lipid A Mimics LPS is a structural constituent of the outer membrane of enterobacterial Gram-negative bacteria. LPS consists of a polysaccharide portion and a polyacylated diglucosamine lipid called lipid A. The polysaccharide portion consists of an O-antigen-specific polymer of repeating oligosaccharide units, the composition of which is highly varied among Gram-negative bacteria. A relatively well-conserved core hetero-oligosaccharide covalently bridges the O-antigen-specific chain with lipid A.22 Total synthesis of the structurally highly conserved lipid A has demonstrated unambiguously that this constitutes the core active moiety of LPS.23,24 As mentioned earlier, understanding the structural and mechanistic bases of adjuvanticity and toxicity is pivotal in rationally developing novel adjuvants. The progression from Johnson’s experiments in 1956,12 demonstrating the adjuvanticity of highly 382

toxic LPS, to Ribi’s ‘detoxified’ monophosphoryl lipid A in the 1980s,25-29 and culminating in extensive clinical trials and now awaiting imminent approval by the FDA and commercialization as MPLTM (Fig. 1) is instructive, and the lessons learned can be adduced to developing other TLR agonistic adjuvants. Kusumoto’s landmark total synthesis of lipid A, the toxic moiety of endotoxic LPS,23 allowed the formal verification that synthetic, 1-dephospholipid A, as well as monophosphoryl lipid A isolated by mild acid hydrolysis of Gram-negative bacteria-derived lipid A, were nontoxic, and yet immunostimulatory and adjuvantic.25,30,31 The mechanism of immunopotentiation of LPS and lipid A, however, remained unsolved until the discovery of TLR4-agonistic effects of LPS and lipid A derivatives in the late 1990s.21,32-37 It was not until 2007 that the basis of the dissociation between toxicity of diphosphoryl lipid A (DPLA) on the one hand, and the potent adjuvanticity (and nontoxicity) of monophosphoryl lipid A (MPLA), on the other, became apparent.38 Whereas DPLA, upon binding to TLR4, signals via both myeloid differentiation factor 88 (MyD88) as well as Toll-interleukin 1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways,14,39-45 MPLA appears to predominantly trigger the TRIF-dependent pathway.38 In the case of the LPS receptor TLR4, it is MyD88 that is associated with proinflammatory outcomes, while activation of TRIF-dependent pathways are correlated with immunostimulation.38 These results indicate that a TLR agonist that signals with a bias toward TRIF signaling is likely to be nontoxic and yet immunostimulatory. MPLTM, also known as AS04, has recently been approved for use in Europe and is defined as 3-O-desacyl-4'-monophosphoryl lipid A, obtained by mild hydrolytic treatment and purification of Salmonella minnesota R595 LPS, and absorbed on aluminum hydroxide or aluminum phosphate.46,47 The potent adjuvanticity and good safety profile of MPLA has spawned a large number of synthetic lipid A mimics including a new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs)48,49 such as Corixa’s (now GlaxoSmithKline) Ribi 529,50-52 and Eisai’s E6020 (Fig. 2).53,54 As mentioned earlier, aluminum salts

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Figure 2. Structural class of the Ribi and Eisai Lipid A-based synthetic TLR4 agonistic adjuvants now in preclinical testing.

predominantly induce antibody responses with negligible CTL expansion. Modern subunit vaccine targets, however, often require the induction of strong T helper (TH) and CTL responses, in addition to a robust humoral enhancement. The adjuvant activities of TLR agonists are usually a manifestation of multiple effects on the antigen presentation-effector apparatus.55 For instance, monophosphoryl lipid A induces dendritic cell maturation,56-60 upregulates MHC Class II molecules and CD80 and CD86,61-63 and also indirectly reduces the threshold for activation of TH1 cells.64-69

TLR1/TLR2/TLR6 Agonistic Bacterial Lipoteichoic Acid and Lipopeptides The exoskeleton of the Gram-positive organism, similar to that of Gram-negative bacteria, is comprised of underlying peptidoglycan www.landesbioscience.com

(PGN), a super-sized polymer of β-1→4-linked N-acetylglucosamine-N-acetylmuramic acid glycan strands that are cross-linked by short peptides.70 Unlike in Gram-negative bacteria which bear LPS on the outer leaflet of the outer membrane, the external surface of the peptidoglycan layer is decorated with lipoteichoic acids (LTA; Fig. 3),71 which are anchored in the peptidoglycan substratum via a diacylglycerol moiety and bear a surface-exposed, polyanionic, 1-3-linked polyglycerophosphate appendage which varies in its subunit composition in LTAs from various Gram-positive bacteria; in S. aureus, the repeating subunit contains D-alanine and α-D-Nacetylglucosamine.72 Lipoproteins are found in the bacterial cytoplasmic membrane and are also common constituents of the cell wall of both Gramnegative and Gram-positive bacteria.73 The free amine of the N-terminus of lipoproteins are acylated with a S-(2,3-diacyloxypropyl) cysteinyl residue which constitutes the immunostimulatory moiety74 as shown by studies on synthetic peptides containing the diacylthioglycerol unit (see below).72 In contradistinction to enterobacterial LPS which is recognized by TLR4, PGN,75 LTA,76 lipopeptides,77 as well as some non-enterobacterial LPS78 signal via TLR2. One of the earliest reports on bacterial components other than LPS was the study by Melchers et al.79 showing that purified lipoprotein from the outer membrane of E. coli was a B-lymphocyte mitogen, expressing activity in LPS-nonresponder C3H/HeJ mice. Lipoprotein fractions isolated from Gram-positive organisms as well as mycoplasmal cultures indicated potent macrophage-activating and CTL-inducing properties.80-83 A lipopeptide fraction termed mycoplasma associated lipopeptide-2 kDa (MALP-2; Fig. 4) was subsequently isolated and structurally characterized,74 leading to the identification of S-(2,3-dihydroxypropyl)cysteine as the structural core of MALP-2.84 Recent structure-activity relationships have sought to examine the stereochemistry, acylation pattern, and the contributions of the peptide unit.85-88 In contradistinction to Gram-negative LPS which is recognized by TLR4,21,32-37 the lipopeptides signal via TLR2.77,85,87,89 Although diacyl lipopeptides have been reported to be agonists of heterodimers of TLR2/ TLR6,86,90,91 and triacylated species were thought to signal via TLR2/TLR1,77,87,89 the recognition of the lipopeptides by TLR2 has been recently shown to be relatively independent of TLR1 and TLR6, and are mediated predominantly via TLR2.92,93 Structural basis for the interactions of PAM2CSK4 with TLR2 and PAM3CSK4 with TLR2/TLR1. The crystal structures of both PAM2CSK4 and PAM3CSK4 with TLR1-TLR2 heterodimers have recently been determined (Fig. 5).94 This not only provides a structural basis for differential recruitment of TLR6 and TLR1 to heterodimerize with TLR2 by diacyl and triacyl lipopeptides, but also allows, for the first time, a rational, structure-based template for the ab initio design of novel analogues of TLR2. The N-acyl group of the triacyl PAM3CSK4 interacts with a hydrophobic groove in TLR1, driving the formation of an ‘m’ shaped TLR2-TLR1 dimer (Fig. 5), whereas both ester-linked acyl groups of the diacyl PAM2CSK4 are inserted into a hydrophobic pocket in TLR2, rendering the diacyl lipopeptide a pure TLR2 ligand.94 The correct stereoisomer is necessary for the appropriate orientation of the ester-linked acyl groups in the hydrophobic pocket of TLR2. The external tetralysine peptide unit is almost fully solvent-exposed and does not participate in significant interactions with TLR2, which is consistent with

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Figure 3. Structure of lipoteichoic acid (LTA), a constituent of the cell envelope of Gram-positive organisms.

our observation that the PAM2CS unit is sufficient to confer full TLR-agonistic activity. Effects of the TLR2 agonistic lipopeptides on professional antigen presenting cells and effector lymphocytes. The exposure of bone marrow-derived dendritic cells of C57/BL6 mice to the lipopeptide FSL-1 has been shown to result in a TLR2-dependent upregulation of MHC Class II and CD80/CD86 co-stimulatory molecules, with enhanced expression of CD11b and CD11c, associated with the production of TNFα and IL-12.95 It is noteworthy that, in this study, whereas LPS also induced IL-10 production, FSL-1 only induced a pro-TH1-type cytokine response.95 These results have been independently confirmed in BALB/c mice using MALP-2, and extended to show that the lipopeptide also upregulates immunoproteasome (LMP2, LMP7 and MECL1) activity in a dose-dependent manner, suggesting that lipopeptides may indirectly enhance MHC Class I-restricted responses by accelerated antigen processing and peptide presentation.96 Importantly, the effects of lipopeptides on APC maturation and antigen presentation have also been demonstrated in human DCs.97 MALP-2 directly stimulates murine B lymphocytes without accessory APC help in a TLR2- and dose-dependent fashion. The expansion in the resting CD19+ population was accompanied by upregulation of both MHC Class I and II molecules, as well as CD25 (IL2Rα), CD40, CD80 and CD86.98 Triacyl PAM3CSK4 has been shown to induce IL-6 production in human tonsillar B cells.99 In addition, PAM2CSK4 and MALP-2 induce isotypic switching and differentiation of naïve human B lymphocytes to IgG-secreting plasma cells,100 indicating a functional association between BCR stimulation and TLR activation.100 Triacyl PAM3CSK4 has been shown to dramatically enhance MHC Class I-restricted CTL proliferation to an immunodominant influenza peptide Mx58-66 in a human autologous DC/CD8+ T cell co-culture model.101 The peptide-specific CTLs (assessed by tetramer staining) were also found to be strong producers of IFNγ. Lipopeptide-primed DCs also stimulated the proliferation of allogeneic naïve CD8+ CTLs.101 Intranasal immunization of mice with influenza-specific peptide resulted in greatly enhanced numbers of IFNγ-secreting CD8+ CTLs when lipopeptides were used as adjuvants.102 Similarly, MALP-2 induces robust, long-lived CTL responses against HIV-1 Tat protein.103,104 These results collectively indicate that the adjuvanticity of the lipopeptides encompasses a strong CTL response. Reports as early 384

Figure 4. The structure of MALP-2, the first TLR2 agonistic, immunostimulatory lipopeptide identified. The lipopeptide comprises of a diacylthioglycerol unit (blue) in thioether linkage (purple) with cysteine (red). The α-amino group of cysteine is acylated (R = palmitoyl) in triacyl lipopetides. The peptide unit (green) can be variable, and does not appear to be a determinant of immunostimulatory activity.

Figure 5. Crystal structure of PAM3CSK4 (shown in spacefill) bound to a heterodimer of TLR1 and TLR2 (PDB: 2Z7X). The N-acyl group of the triacyl PAM3CSK4 interacts with a hydrophobic groove in TLR1 (cyan) whereas both ester-linked palmitoyl chains are inserted into a hydrophobic pocket in TLR2 (gray), driving the formation of an ‘m’ shaped TLR2-TLR1 heterodimer. The correct stereoisomer is necessary for the appropriate orientation of the ester-linked acyl groups in the hydrophobic pocket of TLR2. The external tetralysine peptide unit is almost fully solvent-exposed and does not participate in significant interactions with TLRs.

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TLR5 Agonistic Flagellin

Figure 6. Crystal structure of a truncated flagellin homologue from Sphingomonas (PDB:2ZBI), consisting of two domains: an alpha-domain rich in α-helices that forms the N- and C-terminal regions and a beta-domain rich in β-strands that constitutes the central region. The alpha-domain is structurally similar to the D1 domain of Salmonella typhimurium flagellin.

as 1997 indicated that PAM3CSK4 markedly enhanced the humoral response to haptens conjugated to poly-L-lysine; adjuvanticity was also observed by these authors for the lipopeptide-TH1 cell epitope conjugates in in vitro immunization protocols.105 It was subsequently shown using human lymphocytes ex vivo, that PAM3CSK4-dependent CD4+ T cell differentiation and proliferation required professional APC, and that naïve CD4+ T cell differentiation progressed with faster kinetics in the presence of the adjuvant. The lipopeptide enhanced the frequency of in vitro expanded TH cells specific for tetanus toxoid and hepatitis B surface antigen peptides, with the majority of the expanded CD3+/CD4+ cells being positive for IFNγ.106 A recent study indicates that the lipopeptides lower the threshold for TH1 responses by TLR2-dependent secretion of IL-12p70 by DC.107 www.landesbioscience.com

Flagellin, a 50 kDa protein constituent of bacterial flagellae was first recognized by Aderem’s group in 2001 to be specifically recognized by cell-surface associated TLR5 in a MyD88-dependent manner.108 The crystal structure reveals an α-domain rich in α-helices that forms the N- and C-terminal regions and a β-domain rich in β-strands that constitutes the central region (Fig. 6).109 Random and site-directed mutagenesis of flagellin from enteroaggregative E. coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation in Caco-2 cells.110 The transposon linker insertions which eliminate inflammatory activity (2H3, E8, F11, C5) are located near the midpoint of the α-helix formed by the C-terminal conserved domain of the protein (Fig. 6).110 Further exploration of the correlates of the primary sequence of flagellin and TLR5 agonism have now defined two regions: a 14-residue N-motif peptide (AA 95–108) and a 9-residue C-motif peptide (AA 441–449), deletions or mutagenesis of which result in abrogation of TLR5 signaling.111 Flagellin has been demonstrated to induce maturation and chemokine production in human dendritic cells,112 resulting in stimulation of TH1 responses via IL-12p70 production, which depends on the phosphorylation of p38 and c-Jun N-terminal kinase 1/2 (JNK).113 Flagellin was also found to stimulate NK cells with resultant extrusion of α-defensins.114 In an in vivo murine model, flagellin increased NK cell number and activation status in draining lymph nodes after footpad injection; this was evident also in an ex vivo coculture model using carboxy-fluorescein diacetate succinimidyl ester-labeled NK cells.115 It has also been reported that flagellin synergizes with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent (anti-CD2 mAbs or IL-2) stimuli to upregulate proliferation and IFNγ, IL-8 and IL-10 but not IL-4 production by human CD4+ T cells.116 As described above, as a consequence of the potent effects of flagellin on both the innate and adaptive limbs of the immune system, the protein has indeed been demonstrated to have potent adjuvantic effects in experimental vaccine constructs.117-122 However, the safety profile of flagellin is yet to be unequivocally established. Of particular note, it has been observed that the administration of flagellin at doses comparable to or lower than that of bacterial LPS induces prominent local and systemic immune/inflammatory responses in vivo.123 It is possible that, analogous to examination of part-structures of LPS and lipid A which led to the derivation and use of MPLA, peptidic fragments of flagellin (see above) may also be immunostimulatory, but with greatly attenuated local and systemic proinflammatory effects.

Intracellular TLR3 Activation: dsRNA and Poly (I:C) The elaboration of type I interferon (IFNα/β) by virusinfected cells is a pivotal event in their antiviral immune responses. Type I IFN gene transcription is induced through distinct signaling pathways by viral infection or by treatment with double-stranded (ds) RNA, which is an intermediate of virus replication. TLR3 was found to recognize double-stranded RNA (dsRNA) and signal downstream to activate NFκB and the IFNβ promoter.124-129 The crystal structure of the ectodomain (ligandbinding domain) of murine TLR3 has recently become available (Fig. 7).130 A synthetic analog of naturally occurring dsRNA,

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polyriboinosinic:polyribocytidylic acid (poly(I:C)), is often used experimentally to probe TLR3 responses.131 The IFN-inducing properties of poly (I:C) has long been recognized, albeit empirically, predating the discovery of the involvement of TLR3 in its specific recognition by several decades. The safety profile of Ampligen®, also known as polyI:polyC12U [5'-Inosinic acid, homopolymer, complexed with 5'-cytidylic acid polymer with 5'-uridylic acid (1:1)] has been established in the context of HIV therapy, and was found to be generally well-tolerated.132 Poly[I:C(12)U] was found to be effective in inducing optimal phenotypic (elevated levels of MHC-Class I/Class II, CD83, CCR7, CD86 and CD40 molecules) and functional maturation of human DC in vitro, and capable of promoting the production of the TH1-type cytokine IL-12.133 The production of Type I IFN is thought to be crucial in enhancing the primary antibody response to soluble proteins, and in the stimulation of the production of all subclasses of IgG, and consequent induction of long-lived antibody production and immunological memory.134 Poly(I:C) also induced functional CD8+ T-cell responses against OVA peptides in murine models; antigen-specific CD8+ responses were found not onto be strongly Type I IFN-dependent,135 but also on the cytokine milieu provided by activated NK cells.136 Although the strong adjuvantic properties of poly I:C have been validated in multiple immunization models including the induction of mucosal (IgA-mediated) immunity,137-142 it remains to be seen whether the in vivo stability of poly(I:C) formulations would be sufficient to permit a detailed evaluation of its potential as an adjuvant in human trials.

Intracellular TLR7 Activation: Imidazoquinoline Ligands Single-stranded RNA (ssRNA) molecules of viral as well as nonviral origin (poly dT; thymidine homopolymer phosphorothioate) induce the production of inflammatory cytokines, mediated by the recognition in the endosomal compartment by TLR7.143145 Reviews on oligoribonucleotide TLR7 agonists have been published recently.146-148 The recognition of ssRNA by TLR7 is both sequence- and motif-dependent,149-152 and methylation of the 2' hydroxyl group in uridine leads not only to abrogation of TLR7 activation,153,154 but also appears to antagonize the receptor.155,156 These effects, understandably, have important consequences on the development of siRNA as viable therapeutic modalities,157-159 and it is to be expected that future advances in controlling and modulating adventitious innate immune stimulation by siRNA will also add significantly to our understanding of the biology of TLR7 and, hopefully will lead to a rational evaluation of ssRNA as potential immune adjuvants. There is considerable interest in small molecule, non-polymeric (non ssRNA-derived), synthetic TLR7 agonists which include the imidazoquinolines [Imiquimod, Resiquimod (R-848) and Gardiquimod],160-162 as well as guanosine analogues such as loxoribine163-165 (Fig. 8). Long before the fact that these synthetic analogues specifically engaged and activated TLR7 was known, it was recognized that these compounds were potently adjuvantic163-169 in addition to displaying anti-viral effects via the induction of IFNα/β.143,162,170,171 It became apparent as early as 1991 that loxoribine (7-allyl 8-oxoguanosine) potentiated anti-tetanus-specific IgG antibody anamnestic responses in a dose-dependent manner in human peripheral blood 386

Figure 7. Crystal structure of murine TLR3 ectodomain (ECD) complexed with double-stranded RNA (dsRNA). Each TLR3-ECD (shown in cyan and gray mesh of the van der Waals surface) binds dsRNA (shown in solid blue and red van der Waals surface) at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains stabilizes the dimer.

mononuclear cells,165 as well as strongly activating NK cells in an IL-12-dependent manner.163,166,167 In human blood stimulated ex vivo with TLR7 agonists, upregulation of the surface expression of co-stimulatory molecules such as CD40, CD80 and CD86 occurred in both CD11c- plasmacytoid DCs and CD11c+ myeloid DCs; furthermore, the TH1 stimulatory ability of both DC subsets was enhanced in response to TLR7 ligands.172 Because TLR7 agonists transmit a T-helper-like signal to antibody-producing B cells, it is a highly effective adjuvant even for synthetic peptides that lack T-cell epitopes, possibly even replacing the function of T-helper cells, and providing a potential T-cell-independent vaccination strategy.168 One potential drawback is that small molecules such as the imidazoquinolines would quickly diffuse out of the vaccination site, thereby limiting their utility as an adjuvant. This has been addressed by conjugating the TLR7 ligand to the immunogen(s) of interest. For instance, non-human primates immunized with HIV Gag protein-TLR7 agonist conjugate showed dramatic enhancement of the magnitude and the quality of TH1 responses, as well as eliciting

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have mixed TLR7/8 agonistic activity, but a thiazoloquinolone compound (3M-002) is characterized as a pure TLR8 agonist (Fig. 9).188 The prominent NFκB- and JNK-mediated stimulatory effects of TLR8agonists on antigen-presenting cells188,189 as well as the activation of NK cells,190 leads to robust IL-12-driven TH1-type responses.191 Studies of human neonatal APCs cultured in vitro have demonstrated that TLR7/8 agonists possess unique efficacy to Figure 8. Representative synthetic TLR7 agonists of the imidazoquinoline (imiquimod, gardiquimod) and induce the TH1-polarizing cytokine TNFα the substituted purine (loxoribine) classes. at both the transcriptional and translational levels.191 TLR8 agonists, including the imidazoquinoline congeners R848 (TLR7/8) and 3M-003 (TLR7/8), the amidinothiazoline 3M-002 (TLR8), as well as ssRNAs (TLR8) induce a marked production of the TH1-polarizing cytokines TNFα and IL-12 from neonatal APCs that substantially exceeds responses induced by TLR2, -4 or -7 agonists.191 TLR7/8 agonists are thus uniquely efficacious in activating co-stimulatory responses in neonatal APCs, suggesting that these agents are promising candidate adjuvants for enhancing immune Figure 9. Representative structures of synthetic, mixed TLR7/8 imidazoquinoline agonists (R488, CL097) responses in newborns. and a pure thiazoloquinolone TLR8 agonist (3M-002/CL-075). Reversal of Treg suppressive effects by TLR8 agonists. Human T-regulatory cells Gag-specific CD8+ CTL responses compared with animals immu- (Tregs), classified immunophenotypically as naturally occurring nized with HIV Gag protein and the TLR7 agonist as a noncovalent (CD4+CD25+Foxp3+) or induced (CD4+CD25high), downregulate mixture.173 It must be noted, however, the method of conjugation and suppress a broad array of immune responses, including the in the above-referenced study was by UV irradiation of antigen- non-specific suppression of both CD4+ and CD8+ T-cells via cellimidazoquinoline mixtures, followed by exhaustive dialysis.173 There cell contact and via production of immunosuppressive cytokines 192-198 Tregs express abundant TLR8 are no photoactivable groups on the molecule, and it is not clear such as IL-10 and TGFβ. mRNA, and TLR8 agonists have been shown to reverse Treg funcwhether proteins can be truly covalently modified with UV irradiation via a TLR8-MyD88-IRAK4 (IL-1-receptor-associated kinase 4) tion alone, and it would be preferable to introduce biocompatible 199 Engagement and activation of TLR8, therefore, signaling pathway. 174-176 electrophiles such as the isothiocyanate group or succinimidyl esters,177-179 with a linker, so that formal, covalent adduction with strongly induces innate immunity and enhances adaptive immunity. above, the producprotein antigens can be performed under aqueous conditions. In addition to the direct effects on Tregs described tion of IL-6 in the milieu renders CD4+ cells refractory to Treg A number of preclinical studies with TLR7 agonists as adjuvants are suppression.200 currently in progress.180-185 Potential problems with pure, high-potency TLR8 agonists, and a case for a mixed TLR7/TLR8 activator. Pure TLR8 agonists Intracellular TLR8 Activation: Imidazoquinoline are perhaps unique in their ability to very strongly induce the producand Thiazoloquinolone Ligands tion of proinflammatory cytokines such as TNFα. This property is TLR7 and TLR8 are phylogenetically and structurally related thought to be related to the ability of the TLR8 pathway to strongly and, like TLR7, ssRNA molecules are recognized in the endosomal induce TLR-mediated intracellular signaling via the p38 MAPK and compartment also by TLR8, but significant species specificities exist: NFκB pathways.191 Local and/or systemic toxicity is to be anticipated murine TLR7 and human TLR8 mediate the recognition of GU-rich and is therefore a potential concern with potent TNFα-inducing ssRNA.186 In human cells, TLR8 agonists activate myeloid dendritic molecules; indeed such an effect may have been contributory to the cells, monocytes and monocyte-derived dendritic cells, with the GI toxicity that has been observed with clinical trials on Isatoribine 201-203 production of GM-CSF/IL-4/TGFβ. TLR7-selective agonists were given orally. found to be more effective than TLR8-selective agonists at inducing Intracellular TLR9 Activation: CpG ds-DNA IFNα and IFNγ-regulated chemokines, whereas TLR8 agonists induce a dominant proinflammatory cytokine profile including TNFα, Unmethylated “CpG motifs” which are present at high frequency IL-12 and MIP-1α.187 Not surprisingly, several imidazoquinolines in bacterial but not mammalian DNA due to a combination of www.landesbioscience.com

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CpG suppression and CpG methylation are ligands for endosomal TLR9, which is expressed primarily in B lymphocytes and plasmacytoid dendritic cells (pDCs).204-207 Synthetic oligodeoxynucleotides (ODNs) expressing CpG motifs mimic the activity of bacterial DNA, and activate multiple cell types including lymphocytes, NK cells and monocytes. The recognition of CpG motifs by TLR9 is species-specific, and a single base difference impacts the magnitude of the resulting immune response. Structurally distinct classes of CpG DNA molecules are recognized by TLR9, and responses are determined by nucleotide sequence, backbone modifications and sequence context (motif).208-211 CpG DNA was originally described as two functionally dimorphic classes by Dennis Klinman;212 “D”-type ODNs were found to trigger a TH1-dominated immune response driven by NK cell-mediated IFNγ production and upregulation of CD83 and CD86 co-stimulatory molecules, while “K”-type ODNs activated human monocytes and were also mitogenic to B cells.213-215 The D/K classification has largely been supplanted by an expanded A/B/C/S classification which denote different sequence motifs, base-pair composition, and the presence or absence of palindromic sequences.210,216,217 The pleiotropic effects on APCs (pDCs), as well as effector B, NK- and γδ-T cells confer potent adjuvantic properties to CpG ODN. Comparisons of different adjuvants in murine models have demonstrated CpG ODN to be outstanding at inducing TH1-type responses, with the TH1 bias maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund’s adjuvant (IFA) that normally promote a TH2 bias.218-220 In a human trial comparing alum-adsorbed hepatitis B surface antigen (HBsAg alone) or with a CpG adjuvant (CpG 7909; VaximmuneTM), HBsAg-specific antibody responses appeared sooner, with higher antibody titers at all time points from 2 weeks up to 48 weeks in CPG 7909 recipients compared with those individuals who received vaccine alone. The majority of individuals receiving CPG 7909 also developed protective levels of anti-HBs IgG within just 2 weeks of the priming vaccine dose, compared with none of the subjects receiving the commercial vaccine alone.221 The use of CpG ODN also increased the proportion of antigen-specific high-avidity antibodies, indicating augmentation of the late-affinity maturation in the activated plasmocytes.222 It is pertinent to note in this context that CpG oligodeoxynucleotides are distinguished in their ability to expand the long-lived memory B cell populations.223 The safety profile of several TLR9 agonists in humans has been documented in multiple clinical trials. A maximal tolerated dose in humans has not been reported to date, even though doses of up to 0.81 mg per kg have been used. The primary adverse events appear to be limited to dose-dependent local injection reactions (such as erythema, pain, swelling, induration, pruritis or warmth at the site of injection) or systemic flu-like reactions (such as headache, rigors, myalgia, pyrexia, nausea and vomiting).224 CpG nucleotides constitute the adjuvant in the only licensed anthrax vaccine, AVA.225,226

Peptidoglycan Fragment Sensing by Intracellular Nod-Like Receptors The nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs) constitute a family of about 20 cytosolic pattern recognition molecules in mammals which function as microbial sensors of intracellular infections.227-230 388

The NLRs complement the TLRs (described in preceding sections) and the RIG-I-like receptors (RLRs; discussed below). Nod1 and Nod2 are the members to which specific ligands have presently been assigned; both detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment.230-234 PGN is the major constituent of bacterial cells walls, providing shape and mechanical rigidity, and is composed of oligosaccharide subunits that contain alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked to each other by short peptides. A structural distinction between Gram-positive and Gram-negative PGN resides in the amino acid composition of the intervening peptides; in Gram-positive bacteria, the amino acid is commonly a lysine, whereas a diaminopimelic acid (DAP) is found in most Gram-negative bacteria.235-237 PGN is constantly remodeled by specific hydrolases during either in the course of bacterial fission, or as a consequence of phagocytosis. Degradation of PGN leads to the formation of muropetides which have strong immunomodulatory properties. In antigen-presenting cells, activation of Nod1 and Nod2 leads to the production of proinflammatory cytokines (IL-1β, IL-6, TNFα, IL-18), and chemokines (IL-8); it has also been shown that Nod1 and Nod2 agonists act in synergistic fashion with TLR agonists in inducing cytokine production and dendritic cell maturation.238-242 Nod1 and Nod2 activation also drive adaptive immune responses. Kobayashi et al. have shown that Nod2 mediates adjuvant activity through the production of IgG1-type antibodies to T-cell-dependent antigens.243 It is of note that Nod1 activation in isolation appears to elicit predominantly an antigen-specific TH2 immunity. However, in synergy with TLRs, Nod1 triggering is crucial for priming of TH1, TH2, as well as TH17 immune responses in a murine model.244 Given the exquisite species specificity of PAMPs in general,245-252 and of the NLRs themselves,253,254 a definitive evaluation will have to await careful evaluation in human ex vivo models.

Intracellular ssRNA Sensing by RIG-I-Like Receptors (RLRs) RLRs constitute a family of cytoplasmic RNA helicases that are critical for host antiviral responses. RIG-I (retinoic-acid-inducible protein 1) and MDA-5 (melanoma-differentiation-associated gene 5) sense viral RNA, leading to production of Type I IFNs in infected cells.255-257 It is now known that RIG-I recognizes a specific set of RNA viruses (Flaviviridae, Paramyxoviridae, Orthomyxoviridae and Rhabdoviridae), whereas MDA-5 is responsible for the antiviral defense against a reciprocal set of RNA viruses (Picornaviridae).258-260 It was originally thought that dsRNA was detected by the RLRs (as does TLR3, discussed above), but it was only in 2006 that it became clear that the notion was incorrect. It was demonstrated that in influenza A virus infection (which does not generate dsRNA), RIG-I is activated by viral genomic ssRNA bearing uncapped 5'-phosphates, a feature that is common in viral RNA but absent in normally processed and capped cellular RNA. This clearly identifies the RLRs as ssRNA sensors and further suggests that their ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.261263 The TLR and RLR signaling pathways thus complement each other and may operate in tandem, recognizing foreign dsRNA and ssRNA in various cells; the exception is that of plasmacytoid

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dendritic cells, which appear to solely utilize TLR3 for RNA recognition.255 RIG-I is an ATPase, and the structural basis of the interactions of RNA with the C-terminal regulatory domain (CRD) has just become available.264 RIG-I binds viral RNA in a 5'-triphosphate-dependent manner and activates the RIG-I ATPase by RNA-dependent dimerization. The crystal structure reveals a zinc-binding domain that is structurally related to GDP/GTP exchange factors of Rab-like GTPases.264 A detailed characterization of the immunostimulatory and adjuvantic effects of RLR activation is yet to be established.

Concluding Remarks While it is indisputable that progress in developing adjuvants had been stymied due to the lack of a detailed understanding of the mechanistic bases underlying their immunopotentiatory properties as well as the pronounced systemic toxicity of bacterial products, it is astonishing that purely empirical observations [which predate by many decades the discovery of what is taken for granted today, such as the professional antigen presenting cell (APC), or the immunological synapse] had fueled incremental advances, driving the field slowly to a point where it is now poised to reach ‘critical mass’ due to the convergence of the decade-old science of innate immunity, and the nascent field of TLRs, and other Pathogen-Associated Molecular Pattern (PAMP)-recognizing elements such as the nucleotidebinding oligomerization domain (NOD)-like and retinoic acid inducible gene (RIG)-like receptors. Safety and the lack of toxicity, alongside efficacy are, obviously, the central issues in the choice of a vaccine adjuvant. The search for immunopotentiators with a wide margin of safety continues, but the pace of discovery, until very recently, had been hampered by the lack of understanding of the structure-function correlates underlying immunostimulatory versus inflammatory activities. Although the development of specific TLR knockout mouse strains35 made an enormous impact on the field of innate immunity, significant differences between murine and non-rodent species exist not only in receptor specificity to TLR ligands, but also in the cellular responses to them. As has been observed with TLR4 ligands such as taxol,265 lipid IVa, and E5531, a synthetic lipid A analogue,266 recent evidence suggests that significant interspecies differences exist for TLR2 also, as exemplified by variations in specificities for lipopeptide recognition in chimeric TLR constructs.267 Furthermore, the coupling of these pattern recognition receptors to downstream adaptor molecules also appear to be distinct as shown by disparities in clinical outcomes in humans with IRAK-4 deficiency versus the susceptibility to pathogens in knockout mice.268 It is also important to note that significant polymorphisms in TLRs exist which have been shown to be associated with altered innate immune responses269-271 and, by extension, would therefore also be expected to have pronounced effects on the shaping of adaptive immune responses. In practical terms, these considerations emphasize the necessity of examining human innate immune responses (preferably using primary cells ex vivo, with a sufficiently large sample size to account for heterogeneities in responses), even in preliminary studies aimed at screening for immunopotentiatory activities. An interdisciplinary approach encompassing structure-activity elucidation of TLR-active substances and in vitro evaluation of indices of immunopotentiation, coupled with focused animal www.landesbioscience.com

model studies that take into consideration not only adjuvanticity, but also issues of species-specificities of TLR-specific responses and of local and systemic toxicities will likely accelerate the discovery and development of clinically useful adjuvants in the years to come. References 1. Ramon G. Procedes pour accroïtre la production des antitoxins. Ann Inst Pasteur 1926; 40:1-10. 2. Ramon G. Sur l’augmentation anormale de l’antitoxine chez les chevaux producteurs de serum antidiphtherique. Bull Soc Centr Med Vet 1925; 101:227-34. 3. Glenny AT, Pope CG, Waddington H, Wallace V. The antigenic value of toxoid precipitated by potassium-alum. J Path Bact 1926; 29:38-45. 4. Vogel FR, Powell MF. A compendium of vaccine adjuvants and excipients. Pharm Biotechnol 1995; 6:141-228. 5. Relyveld EH, Bizzini B, Gupta RK. Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoids. Vaccine 1998; 16:1016-23. 6. Gupta RK. Aluminum compounds as vaccine adjuvants. Adv Drug Deliv Rev 1998; 32:155-72. 7. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008. 8. Seubert A, Monaci E, Pizza M, O’Hagan DT, Wack A. The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J Immunol 2008; 180:5402-12. 9. Kool M, Soullie T, van Nimwegen M, Willart MA, Muskens F, Jung S, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 2008; 205:869-82. 10. Dresser DW. Specific inhibition of antibody production II. Paralysis induced in adult mice by small quantities of protein antigen. Immunology 1962; 5:378-88. 11. Dresser DW. An assay for adjuvanticity. Clin Exp Immunol 1968; 3:877-88. 12. Johnson AJ, Gaines S, Landy M. Studies on the O antigen of Salmonella typhosa V. Enhancement of the antibody response to protein antigens by the purified lipopolysaccharide. J Exp Med 1956; 103:225-33. 13. Ishii KJ, Akira S. Toll or toll-free adjuvant path toward the optimal vaccine development. J Clin Immunol 2007; 27:363-71. 14. Kawai T, Akira S. TLR signaling. Semin Immunol 2007. 15. Kumagai Y, Takeuchi O, Akira S. Pathogen recognition by innate receptors. J Infect Chemother 2008; 14:86-92. 16. Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS et al. TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 2005;308:1626-9. 17. Takeda K, Akira S. Toll-like receptors. Curr Protoc Immunol 2007; 14. 18. Akira S. Toll-like receptors and innate immunity. Adv Immunol 1902; 78:1-56. 19. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunol 2001; 2:675-80. 20. Cottalorda A, Verschelde C, Marcais A, Tomkowiak M, Musette P, Uematsu S, et al. TLR2 engagement on CD8 T cells lowers the threshold for optimal antigen-induced T cell activation. Eur J Immunol 2006; 36:1684-93. 21. Kaisho T, Akira S. Toll-like receptors as adjuvant receptors. Biochim Biophys Acta 2002; 1589:1-13. 22. Galanos C, Lüderitz O. Lipopolysaccharide: Properties of an amphipathic molecule. In Handbook of endotoxin Proctor, R.A. Ed. Rietschel ET, Ed.; Elsevier Science Publisher: Amsterdam 1984; 46. 23. Galanos C, Lüderitz O, Rietschel ET, Westphal O, Brade H, Brade L, et al. Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities. Eur J Biochem 1985; 148:1-5. 24. Rietschel ET, Brade H, Brade L, Brandenburg K, Schade UF, Seydel U, et al. Lipid A, the endotoxic center of bacterial lipopolysaccharides: relation of chemical structure to biological activity. Prog Clin Biol Res 1987; 231:25-53. 25. Baker PJ, Hiernaux JR, Fauntleroy MB, Prescott B, Cantrell JL, Rudbach JA. Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A. Infect Immun 1988; 56:1076-83. 26. Baker PJ, Hiernaux JR, Fauntleroy MB, Stashak PW, Prescott B, Cantrell JL, et al. Ability of monophosphoryl lipid A to augment the antibody response of young mice. Infect Immun 1988; 56:3064-6. 27. Johnson AG, Tomai M, Solem L, Beck L, Ribi E. Characterization of a nontoxic monophosphoryl lipid A. Rev Infect Dis 1987; 9:512-6. 28. Qureshi N, Mascagni P, Ribi E, Takayama K. Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination. J Biol Chem 1985; 260:5271-8. 29. Qureshi N, Takayama K, Ribi E. Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium. J Biol Chem 1982; 257:11808-15.

Human Vaccines

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30. Baker PJ, Hraba T, Taylor CE, Stashak PW, Fauntleroy MB, Zahringer U, et al. Molecular structures that influence the immunomodulatory properties of the lipid A and inner core region oligosaccharides of bacterial lipopolysaccharides. Infect Immun 1994; 62:2257-69. 31. Baker PJ, Hraba T, Taylor CE, Myers KR, Takayama K, Qureshi N, et al. Structural features that influence the ability of lipid A and its analogs to abolish expression of suppressor T cell activity. Infect Immun 1992; 60:2694-701. 32. Beutler B, Poltorak A. The sole gateway to endotoxin response: how LPS was identified as TLR4, and its role in innate immunity. Drug Metab Dispos 2001; 29:474-8. 33. Beutler B. Tlr4: central component of the sole mammalian LPS sensor. Curr Opin Immunol 2000; 12:20-6. 34. Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998; 282:2085-8. 35. Akira S, Takeda K. Functions of toll-like receptors: lessons from KO mice. C R Biol 2004; 327:581-9. 36. Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004; 16:3-9. 37. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999; 11:443-51. 38. Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 2007; 316:1628-32. 39. Bowie A, O’Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol 2000; 67:508-14. 40. McGettrick AF, Brint EK, Palsson-McDermott EM, Rowe DC, Golenbock DT, Gay NJ, et al. Trif-related adapter molecule is phosphorylated by PKC{epsilon} during Toll-like receptor 4 signaling. Proc Natl Acad Sci USA 2006; 103:9196-201. 41. Miggin SM, O’Neill LA. New insights into the regulation of TLR signaling. J Leukoc Biol 2006; 80:220-6. 42. O’Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 2007; 7:353-64. 43. Watters TM, Kenny EF, O’Neill LA. Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. Immunol Cell Biol 2007. 44. Hirotani T, Yamamoto M, Kumagai Y, Uematsu S, Kawase I, Takeuchi O, et al. Regulation of lipopolysaccharide-inducible genes by MyD88 and Toll/IL-1 domain containing adaptor inducing IFNb. Biochem Biophys Res Commun 2005; 328:383-92. 45. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 2003; 301:640-3. 46. Garcon N, Chomez P, Van Mechelen M. GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives. Expert Rev Vaccines 2007; 6:723-39. 47. Tagliabue A, Rappuoli R. Vaccine adjuvants: the dream becomes real. Hum Vaccin 2008; 4:347-9. 48. Johnson DA, Sowell CG, Johnson CL, Livesay MT, Keegan DS, Rhodes MJ, et al. Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs). Bioorg Med Chem Lett 1999; 9:2273-8. 49. Johnson DA, Keegan DS, Sowell CG, Livesay MT, Johnson CL, Taubner LM, et al. 3-O-Desacyl monophosphoryl lipid A derivatives: synthesis and immunostimulant activities. J Med Chem 1999; 42:4640-9. 50. Johnson DA. Synthetic TLR4-active glycolipids as vaccine adjuvants and stand-alone immunotherapeutics. Curr Top Med Chem 2008; 8:64-79. 51. Baldridge JR, McGowan P, Evans JT, Cluff C, Mossman S, Johnson D, et al. Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Ther 2004; 4:1129-38. 52. Persing DH, Coler RN, Lacy MJ, Johnson DA, Baldridge JR, Hershberg RM, et al. Taking toll: lipid A mimetics as adjuvants and immunomodulators. Trends Microbiol 2002; 10:32-7. 53. Hawkins LD, Ishizaka ST, McGuinness P, Zhang H, Gavin W, DeCosta B, et al. A novel class of endotoxin receptor agonists with simplified structure, toll-like receptor 4-dependent immunostimulatory action, and adjuvant activity. J Pharmacol Exp Ther 2002; 300:655-61. 54. Ishizaka ST, Hawkins LD. E6020: a synthetic Toll-like receptor 4 agonist as a vaccine adjuvant. Expert Rev Vaccines 2007; 6:773-84. 55. Guy B. The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 2007; 5:505-17. 56. De Becker G, Moulin V, Pajak B, Bruck C, Francotte M, Thiriart C, et al. The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells. Int Immunol 2000; 12:807-15. 57. Kamath AT, Pooley J, O’Keeffe MA, Vremec D, Zhan Y, Lew AM, et al. The development, maturation and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000; 165:6762-70. 58. Ismaili J, Rennesson J, Aksoy E, Vekemans J, Vincart B, Amraoui Z, et al. Monophosphoryl lipid A activates both human dendritic cells and T cells. J Immunol 2002; 168:926-32. 59. Ten Brinke A, Karsten ML, Dieker MC, Zwaginga JJ, van Ham SM. The clinical grade maturation cocktail monophosphoryl lipid A plus IFNgamma generates monocyte-derived dendritic cells with the capacity to migrate and induce Th1 polarization. Vaccine 2007; 25:7145-52. 60. Lapteva N, Seethammagari MR, Hanks BA, Jiang J, Levitt JM, Slawin KM, et al. Enhanced activation of human dendritic cells by inducible CD40 and Toll-like receptor-4 ligation. Cancer Res 2007; 67:10528-37.

390

61. Yang QB, Martin M, Michalek SM, Katz J. Mechanisms of monophosphoryl lipid A augmentation of host responses to recombinant HagB from Porphyromonas gingivalis. Infect Immun 2002; 70:3557-65. 62. Martin M, Michalek SM, Katz J. Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A. Infect Immun 2003; 71:2498-507. 63. Zhang P, Lewis JP, Michalek SM, Katz J. Role of CD80 and CD86 in host immune responses to the recombinant hemagglutinin domain of Porphyromonas gingivalis gingipain and in the adjuvanticity of cholera toxin B and monophosphoryl lipid A. Vaccine 2007; 25:6201-10. 64. Johnson AG, Tomai MA. A study of the cellular and molecular mediators of the adjuvant action of a nontoxic monophosphoryl lipid A. Adv Exp Med Biol 1990; 256:567-79. 65. Baker PJ, Hiernaux JR, Fauntleroy MB, Prescott B, Cantrell JL, Rudbach JA. Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A. Infect Immun 1988; 56:1076-83. 66. Sasaki S, Tsuji T, Hamajima K, Fukushima J, Ishii N, Kaneko T, et al. Monophosphoryl lipid A enhances both humoral and cell-mediated immune responses to DNA vaccination against human immunodeficiency virus type 1. Infect Immun 1997; 65:3520-8. 67. Vernacchio L, Bernstein H, Pelton S, Allen C, MacDonald K, Dunn J, et al. Effect of monophosphoryl lipid A (MPL) on T-helper cells when administered as an adjuvant with pneumocococcal-CRM197 conjugate vaccine in healthy toddlers. Vaccine 2002; 20:3658-67. 68. Thompson BS, Chilton PM, Ward JR, Evans JT, Mitchell TC. The low-toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells. J Leukoc Biol 2005; 78:1273-80. 69. von BV, Hermes A, von Baehr R, Scherf HP, Volk HD, Fischer von WeikersthalDrachenberg KJ, et al. Allergoid-specific T-cell reaction as a measure of the immunological response to specific immunotherapy (SIT) with a Th1-adjuvanted allergy vaccine. J Investig Allergol Clin Immunol 2005; 15:234-41. 70. Vollmer W, Holtje JV. The architecture of the murein (peptidoglycan) in gram-negative bacteria: vertical scaffold or horizontal layer(s)? J Bacteriol 2004; 186:5978-87. 71. Baddiley J. Bacterial cell walls and membranes: discovery of the teichoic acids. Bioessays 1989; 10:207-10. 72. Fischer W. Lipoteichoic acid and lipids in the membrane of Staphylococcus aureus. Med Microbiol Immunol 1994; 183:61-76. 73. Henderson B, Poole S, Wilson M. Bacterial modulins: a novel class of virulence factors which cause host tissue pathology by inducing cytokine synthesis. Microbiol Rev 1996; 60:316-41. 74. Muhlradt PF, Kiess M, Meyer H, Sussmuth R, Jung G. Isolation, structure elucidation and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J Exp Med 1997; 185:1951-8. 75. Dziarski R, Gupta D. The peptidoglycan recognition proteins (PGRPs). Genome Biol 2006; 7:232. 76. Schroder NW, Morath S, Alexander C, Hamann L, Hartung T, Zahringer U, et al. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 2003; 278:15587-94. 77. Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, Ulmer AJ. Lipopeptide structure determines TLR2 dependent cell activation level. FEBS J 2005; 272:6354-64. 78. Nahori MA, Fournie-Amazouz E, Que-Gewirth NS, Balloy V, Chignard M, Raetz CR, et al. Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J Immunol 2005; 175:6022-31. 79. Melchers F, Braun V, Galanos C. The lipoprotein of the outer membrane of Escherichia coli: a B-lymphocyte mitogen. J Exp Med 1975; 142:473-82. 80. Muhlradt PF, Quentmeier H, Schmitt E. Involvement of interleukin-1 (IL-1), IL-6, IL-2 and IL-4 in generation of cytolytic T cells from thymocytes stimulated by a Mycoplasma fermentans-derived product. Infect Immun 1991; 59:3962-8. 81. Quentmeier H, Schmitt E, Kirchhoff H, Grote W, Muhlradt PF. Mycoplasma fermentansderived high-molecular-weight material induces interleukin-6 release in cultures of murine macrophages and human monocytes. Infect Immun 1990; 58:1273-80. 82. Quentmeier H, Schumann-Kindel G, Muhlradt PF, Drexler HG. Induction of proto-oncogene and cytokine expression in human peripheral blood monocytes and the monocytic cell line THP-1 after stimulation with mycoplasma-derived material MDHM. Leuk Res 1994; 18:319-25. 83. Deres K, Schild H, Wiesmuller KH, Jung G, Rammensee HG. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 1989; 342:561-4. 84. Muhlradt PF, Meyer H, Jansen R. Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans. Biochemistry 1996; 35:7781-6. 85. Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K, Morr M, et al. Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J Immunol 2000; 164:554-7. 86. Morr M, Takeuchi O, Akira S, Simon MM, Muhlradt PF. Differential recognition of structural details of bacterial lipopeptides by toll-like receptors. Eur J Immunol 2002; 32:3337-47. 87. Okusawa T, Fujita M, Nakamura J, Into T, Yasuda M, Yoshimura A, et al. Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6. Infect Immun 2004; 72:1657-65. 88. Reutter F, Jung G, Baier W, Treyer B, Bessler WG, Wiesmuller KH. Immunostimulants and Toll-like receptor ligands obtained by screening combinatorial lipopeptide collections. J Pept Res 2005; 65:375-83.

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89. Spohn R, Buwitt-Beckmann U, Brock R, Jung G, Ulmer AJ, Wiesmuller KH. Synthetic lipopeptide adjuvants and Toll-like receptor 2—structure-activity relationships. Vaccine 2004; 22:2494-9. 90. Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J, Schoen RT, et al. Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in. Nat Med 2002; 8:878-84. 91. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002; 169:10-4. 92. Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, Akira S, et al. TLR1- and TLR6-independent recognition of bacterial lipopeptides. J Biol Chem 2006; 281:9049-57. 93. Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, Akira S, et al. Toll-like receptor 6-independent signaling by diacylated lipopeptides. Eur J Immunol 2005; 35:282-9. 94. Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, et al. Crystal structure of the TLR1TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 2007; 130:1071-82. 95. Kiura K, Kataoka H, Nakata T, Into T, Yasuda M, Akira S, et al. The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2. FEMS Immunol Med Microbiol 2006; 46:78-84. 96. Link C, Gavioli R, Ebensen T, Canella A, Reinhard E, Guzman CA. The Toll-like receptor ligand MALP-2 stimulates dendritic cell maturation and modulates proteasome composition and activity. Eur J Immunol 2004; 34:899-907. 97. Espuelas S, Roth A, Thumann C, Frisch B, Schuber F. Effect of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells. Mol Immunol 2005; 42:721-9. 98. Borsutzky S, Kretschmer K, Becker PD, Muhlradt PF, Kirschning CJ, Weiss S, et al. The mucosal adjuvant macrophage-activating lipopeptide-2 directly stimulates B lymphocytes via the TLR2 without the need of accessory cells. J Immunol 2005; 174:6308-13. 99. Mansson A, Adner M, Hockerfelt U, Cardell LO. A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation. Immunology 2006; 118:539-48. 100. Ruprecht CR, Lanzavecchia A. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur J Immunol 2006; 36:810-6. 101. Reschner A, Moretta A, Landmann R, Heberer M, Spagnoli GC, Padovan E. The esterbonded palmitoyl side chains of Pam3CysSerLys4 lipopeptide account for its powerful adjuvanticity to HLA class I-restricted CD8+ T lymphocytes. Eur J Immunol 2003; 33:2044-52. 102. Lau YF, Deliyannis G, Zeng W, Mansell A, Jackson DC, Brown LE. Lipid-containing mimetics of natural triggers of innate immunity as CTL-inducing influenza vaccines. Int Immunol 2006; 18:1801-13. 103. Borsutzky S, Fiorelli V, Ebensen T, Tripiciano A, Rharbaoui F, Scoglio A, et al. Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant. Eur J Immunol 2003; 33:1548-56. 104. Borsutzky S, Ebensen T, Link C, Becker PD, Fiorelli V, Cafaro A, et al. Efficient systemic and mucosal responses against the HIV-1 Tat protein by prime/boost vaccination using the lipopeptide MALP-2 as adjuvant. Vaccine 2006; 24:2049-56. 105. Mittenbuhler K, Loleit M, Baier W, Fischer B, Sedelmeier E, Jung G, et al. Drug specific antibodies: T-cell epitope-lipopeptide conjugates are potent adjuvants for small antigens in vivo and in vitro. Int J Immunopharmacol 1997; 19:277-87. 106. Ghielmetti M, Zwicker M, Ghielmetti T, Simon MM, Villiger PM, Padovan E. Synthetic bacterial lipopeptide analogs facilitate naive CD4+ T cell differentiation and enhance antigen-specific HLA-II-restricted responses. Eur J Immunol 2005; 35:2434-42. 107. Bohnenkamp HR, Papazisis KT, Burchell JM, Taylor-Papadimitriou J. Synergism of Tolllike receptor-induced interleukin-12p70 secretion by monocyte-derived dendritic cells is mediated through p38 MAPK and lowers the threshold of T-helper cell type 1 responses. Cell Immunol 2007; 247:72-84. 108. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001; 410:1099-103. 109. Maruyama Y, Momma M, Mikami B, Hashimoto W, Murata K. Crystal structure of a novel bacterial cell-surface flagellin binding to a polysaccharide. Biochemistry 2008; 47:1393-402. 110. Donnelly MA, Steiner TS. Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5. J Biol Chem 2002; 277:40456-61. 111. Murthy KG, Deb A, Goonesekera S, Szabo C, Salzman AL. Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro. J Biol Chem 2004; 279:5667-75. 112. Means TK, Hayashi F, Smith KD, Aderem A, Luster AD. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol 2003; 170:5165-75. 113. Agrawal S, Agrawal A, Doughty B, Gerwitz A, Blenis J, Van Dyke T, et al. Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J Immunol 2003; 171:4984-9. 114. Chalifour A, Jeannin P, Gauchat JF, Blaecke A, Malissard M, N’Guyen T, et al. Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alphadefensin production. Blood 2004; 104:1778-83. 115. Tsujimoto H, Uchida T, Efron PA, Scumpia PO, Verma A, Matsumoto T, et al. Flagellin enhances NK cell proliferation and activation directly and through dendritic cell-NK cell interactions. J Leukoc Biol 2005; 78:888-97. www.landesbioscience.com

116. Caron G, Duluc D, Fremaux I, Jeannin P, David C, Gascan H, et al. Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 upregulate proliferation and IFNgamma production by memory CD4+ T cells. J Immunol 2005; 175:1551-7. 117. Honko AN, Sriranganathan N, Lees CJ, Mizel SB. Flagellin is an effective adjuvant for immunization against lethal respiratory challenge with Yersinia pestis. Infect Immun 2006; 74:1113-20. 118. Huleatt JW, Jacobs AR, Tang J, Desai P, Kopp EB, Huang Y, et al. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 2007; 25:763-75. 119. McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P, et al. A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity. J Infect Dis 2007; 195:1607-17. 120. Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs. PLoS ONE 2008; 3:2257. 121. Bargieri DY, Rosa DS, Braga CJ, Carvalho BO, Costa FT, Espindola NM, et al. New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin. Vaccine 2008. 122. Mizel SB, Graff AH, Sriranganathan N, Ervin S, Lees CJ, Lively MO, et al. A Fusion Protein, Flagellin/F1/V, is an Effective Plague Vaccine in Mice and Two Species of Nonhuman Primates. Clin Vaccine Immunol 2008. 123. Liaudet L, Deb A, Pacher P, Mabley JG, Murthy KG, Salzman AL, et al. The FlagellinTLR5 Axis: Therapeutic Opportunities. Drug News Perspect 2002; 15:397-409. 124. Matsumoto M, Funami K, Oshiumi H, Seya T. Toll-like receptor 3: a link between toll-like receptor, interferon and viruses. Microbiol Immunol 2004; 48:147-54. 125. Hoebe K, Beutler B. LPS, dsRNA and the interferon bridge to adaptive immune responses: Trif, Tram and other TIR adaptor proteins. J Endotoxin Res 2004; 10:130-6. 126. Sen GC, Sarkar SN. Transcriptional signaling by double-stranded RNA: role of TLR3. Cytokine Growth Factor Rev 2005; 16:1-14. 127. Sioud M. Innate sensing of self and non-self RNAs by Toll-like receptors. Trends Mol Med 2006; 12:167-76. 128. Kawai T, Akira S. Antiviral signaling through pattern recognition receptors. J Biochem 2007; 141:137-45. 129. Uematsu S, Akira S. Toll-like receptors and Type I interferons. J Biol Chem 2007; 282:15319-23. 130. Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, et al. Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 2008; 320:379-81. 131. Matsumoto M, Seya T. TLR3: interferon induction by double-stranded RNA including poly(I:C). Adv Drug Deliv Rev 2008; 60:805-12. 132. Thompson KA, Strayer DR, Salvato PD, Thompson CE, Klimas N, Molavi A, et al. Results of a double-blind placebo-controlled study of the double-stranded RNA drug polyI:polyC12U in the treatment of HIV infection. Eur J Clin Microbiol Infect Dis 1996; 15:580-7. 133. Navabi H, Jasani B, Reece A, Clayton A, Tabi Z, Donninger C, et al. A clinical grade poly I:C-analogue (Ampligen((R))) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro. Vaccine 2008. 134. Le Bon A, Schiavoni G, D’Agostino G, Gresser I, Belardelli F, Tough DF. Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 2001; 14:461-70. 135. Durand V, Wong SY, Tough DF, Le Bon A. Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFNalpha/beta. Immunol Cell Biol 2004; 82:596-602. 136. Salem ML, El Naggar SA, Kadima A, Gillanders WE, Cole DJ. The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu. Vaccine 2006; 24:5119-32. 137. Adams M, Navabi H, Jasani B, Man S, Fiander A, Evans AS, et al. Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R). Vaccine 2003; 21:787-90. 138. Ichinohe T, Watanabe I, Ito S, Fujii H, Moriyama M, Tamura S, et al. Synthetic doublestranded RNA poly(I:C) combined with mucosal vaccine protects against influenza virus infection. J Virol 2005; 79:2910-9. 139. Ma Y, Ross AC. The anti-tetanus immune response of neonatal mice is augmented by retinoic acid combined with polyriboinosinic:polyribocytidylic acid. Proc Natl Acad Sci USA 2005; 102:13556-61. 140. Cui Z, Qiu F. Synthetic double-stranded RNA poly(I:C) as a potent peptide vaccine adjuvant: therapeutic activity against human cervical cancer in a rodent model. Cancer Immunol Immunother 2006; 55:1267-79. 141. Ichinohe T, Kawaguchi A, Tamura S, Takahashi H, Sawa H, Ninomiya A, et al. Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge. Microbes Infect 2007; 9:1333-40. 142. Houston WE, Crabbs CL, Stephen EL, Levy HB. Modified polyriboinosinic-polyribocytidylic acid, an immunological adjuvant. Infect Immun 1976; 14:318-9. 143. Lee J, Chuang TH, Redecke V, She L, Pitha PM, Carson DA, et al. Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc Natl Acad Sci USA 2003; 100:6646-51.

Human Vaccines

391


144. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004; 303:1529-31. 145. Crozat K, Beutler B. TLR7: A new sensor of viral infection. Proc Natl Acad Sci USA 2004; 101:6835-6. 146. Diebold SS. Recognition of viral single-stranded RNA by Toll-like receptors. Adv Drug Deliv Rev 2008; 60:813-23. 147. Schlee M, Barchet W, Hornung V, Hartmann G. Beyond double-stranded RNA-type I IFN induction by 3pRNA and other viral nucleic acids. Curr Top Microbiol Immunol 2007; 316:207-30. 148. Hornung V, Barchet W, Schlee M, Hartmann G. RNA recognition via TLR7 and TLR8. Handb Exp Pharmacol 2008; 71-86. 149. Sioud M. Does the understanding of immune activation by RNA predict the design of safe siRNAs? Front Biosci 2008; 13:4379-92. 150. Sioud M, Furset G. Molecular Basis for the Immunostimulatory Potency of Small Interfering RNAs. J Biomed Biotechnol 2006; 2006:23429. 151. Sioud M. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol 2005; 348:1079-90. 152. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 2005; 23:457-62. 153. Kariko K, Weissman D. Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development. Curr Opin Drug Discov Devel 2007; 10:523-32. 154. Ishii KJ and Akira S. TLR ignores methylated RNA? Immunity 2005; 23:111-3. 155. Wang D, Bhagat L, Yu D, Zhu FG, Tang JX, Kandimalla ER, et al. Oligodeoxyribonucleotidebased antagonists for Toll-like receptors 7 and 9. J Med Chem 2009; 52:551-8. 156. Robbins M, Judge A, Liang L, McClintock K, Yaworski E, MacLachlan I. 2'-O-methylmodified RNAs act as TLR7 antagonists. Mol Ther 2007; 15:1663-9. 157. Lopez-Fraga M, Wright N, Jimenez A. RNA interference-based therapeutics: new strategies to fight infectious disease. Infect Disord Drug Targets 2008; 8:262-73. 158. Behlke MA. Chemical modification of siRNAs for in vivo use. Oligonucleotides 2008; 18:305-19. 159. Alvarez-Salas LM. Nucleic acids as therapeutic agents. Curr Top Med Chem 2008; 8:1379-404. 160. Dockrell DH, Kinghorn GR. Imiquimod and resiquimod as novel immunomodulators. J Antimicrob Chemother 2001; 48:751-5. 161. Garland SM. Imiquimod. Curr Opin Infect Dis 2003; 16:85-9. 162. Miller RL, Meng TC, Tomai MA. The antiviral activity of Toll-like receptor 7 and 7/8 agonists. Drug News Perspect 2008; 21:69-87. 163. Pope BL, Chourmouzis E, Sigindere J, Capetola RJ, Lau CY. In vivo enhancement of murine natural killer cell activity by 7-allyl-8-oxoguanosine (loxoribine). Int J Immunopharmacol 1992; 14:1375-82. 164. Reitz AB, Goodman MG, Pope BL, Argentieri DC, Bell SC, Burr LE, et al. Small-molecule immunostimulants. Synthesis and activity of 7,8-disubstituted guanosines and structurally related compounds. J Med Chem 1994; 37:3561-78. 165. Gupta S, Vayuvegula B, Gollapudi S. Substituted guanine ribonucleosides as B cell activators. Clin Immunol Immunopathol 1991; 61:21-7. 166. Pope BL, Chourmouzis E, Sigindere J, MacIntyre JP, Capetola RJ, Lau CY. In vivo activation of natural killer cells and priming of IL-2 responsive cytolytic cells by loxoribine (7-allyl-8-oxoguanosine). Cell Immunol 1993; 147:302-12. 167. Pope BL, Chourmouzis E, Victorino L, MacIntyre JP, Capetola RJ, Lau CY. Loxoribine (7-allyl-8-oxoguanosine) activates natural killer cells and primes cytolytic precursor cells for activation by IL-2. J Immunol 1993; 151:3007-17. 168. Goodman MG. A new approach to vaccine adjuvants. Immunopotentiation by intracellular T-helper-like signals transmitted by loxoribine. Pharm Biotechnol 1995; 6:581-609. 169. Ambach A, Bonnekoh B, Nguyen M, Schon MP, Gollnick H. Imiquimod, a Toll-like receptor-7 agonist, induces perforin in cytotoxic T lymphocytes in vitro. Mol Immunol 2004; 40:1307-14. 170. Durand V, Wong SY, Tough DF, Le Bon A. Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFNalpha/beta. Immunol Cell Biol 2004; 82:596-602. 171. Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, Gellert T, et al. The Tolllike receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol 2003; 33:2987-97. 172. Ito T, Amakawa R, Kaisho T, Hemmi H, Tajima K, Uehira K, et al. Interferon-alpha and interleukin-12 are induced differentially by Toll-like receptor 7 ligands in human blood dendritic cell subsets. J Exp Med 2002; 195:1507-12. 173. Wille-Reece U, Flynn BJ, Lore K, Koup RA, Kedl RM, Mattapallil JJ, et al. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates. Proc Natl Acad Sci USA 2005; 102:15190-4. 174. Zhang Y, Li J, Tang L. Cancer-preventive isothiocyanates: dichotomous modulators of oxidative stress. Free Radic Biol Med 2005; 38:70-7. 175. Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat Res 2004; 555:173-90. 392

176. Keum YS, Jeong WS, Kong AN. Chemopreventive functions of isothiocyanates. Drug News Perspect 2005; 18:445-51. 177. Parish CR. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 1999; 77:499-508. 178. Lyons AB. Divided we stand: tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol Cell Biol 1999; 77:509-15. 179. Fazekas de St GB, Smith AL, Koh WP, Girgis L, Cook MC, Bertolino P. Carboxyfluorescein diacetate succinimidyl ester and the virgin lymphocyte: a marriage made in heaven. Immunol Cell Biol 1999; 77:530-8. 180. Adams S, O’Neill DW, Nonaka D, Hardin E, Chiriboga L, Siu K, et al. Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant. J Immunol 2008; 181:776-84. 181. Zhang WW, Matlashewski G. Immunization with a Toll-like receptor 7 and/or 8 agonist vaccine adjuvant increases protective immunity against Leishmania major in BALB/c mice. Infect Immun 2008; 76:3777-83. 182. Fransen F, Boog CJ, van Putten JP, van der LP. Agonists of Toll-like receptors 3, 4, 7 and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup B. Infect Immun 2007; 75:5939-46. 183. Ma R, Du JL, Huang J, Wu CY. Additive effects of CpG ODN and R-848 as adjuvants on augmenting immune responses to HBsAg vaccination. Biochem Biophys Res Commun 2007; 361:537-42. 184. Johnston D, Zaidi B, Bystryn JC. TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines. Cancer Immunol Immunother 2007; 56:1133-41. 185. Wille-Reece U, Flynn BJ, Lore K, Koup RA, Miles AP, Saul A, et al. Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. J Exp Med 2006; 203:1249-58. 186. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Speciesspecific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004; 303:1526-9. 187. Gorden KB, Gorski KS, Gibson SJ, Kedl RM, Kieper WC, Qiu X, et al. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol 2005; 174:1259-68. 188. Philbin VJ, Levy O. Immunostimulatory activity of Toll-like receptor 8 agonists towards human leucocytes: basic mechanisms and translational opportunities. Biochem Soc Trans 2007; 35:1485-91. 189. Qin J, Yao J, Cui G, Xiao H, Kim TW, Fraczek J, et al. TLR8-mediated NFkappaB and JNK activation are TAK1-independent and MEKK3-dependent. J Biol Chem 2006; 281:21013-21. 190. Hart OM, Athie-Morales V, O’Connor GM, Gardiner CM. TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFNgamma production. J Immunol 2005; 175:1636-42. 191. Levy O, Suter EE, Miller RL, Wessels MR. Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells. Blood 2006; 108:1284-90. 192. Smith TR and Kumar V. Revival of CD8+ Treg-mediated suppression. Trends Immunol 2008; 29:337-42. 193. Gupta S, Shang W, Sun Z. Mechanisms regulating the development and function of natural regulatory T cells. Arch Immunol Ther Exp (Warsz) 2008; 56:85-102. 194. Cools N, Ponsaerts P, Van Tendeloo VF, Berneman ZN. Regulatory T cells and human disease. Clin Dev Immunol 2007; 2007:89195. 195. Germain RN. Special regulatory T-cell review: a rose by any other name: from suppressor T cells to Tregs, approbation to unbridled enthusiasm. Immunology 2008; 123:20-7. 196. Wan YY, Flavell RA. Regulatory T cells, transforming growth factor-beta, and immune suppression. Proc Am Thorac Soc 2007; 4:271-6. 197. Kim CH. Trafficking of FoxP3+ regulatory T cells: myths and facts. Arch Immunol Ther Exp (Warsz) 2007; 55:151-9. 198. Long ET and Wood KJ. Regulatory T cells—a journey from rodents to the clinic. Front Biosci 2007; 12:4042-9. 199. Sutmuller RP, Morgan ME, Netea MG, Grauer O, Adema GJ. Toll-like receptors on regulatory T cells: expanding immune regulation. Trends Immunol 2006; 27:387-93. 200. Pasare C and Medzhitov R. Toll pathway-dependent blockade of CD4+CD25+ T cellmediated suppression by dendritic cells. Science 2003; 299:1033-6. 201. Fletcher S, Steffy K, Averett D. Masked oral prodrugs of toll-like receptor 7 agonists: a new approach for the treatment of infectious disease. Curr Opin Investig Drugs 2006; 7:702-8. 202. Horsmans Y, Berg T, Desager JP, Mueller T, Schott E, Fletcher SP, et al. Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection. Hepatology 2005; 42:724-31. 203. Xiang AX, Webber SE, Kerr BM, Rueden EJ, Lennox JR, Haley GJ, et al. Discovery of ANA975: an oral prodrug of the TLR-7 agonist isatoribine. Nucleosides Nucleotides Nucleic Acids 2007; 26:635-40. 204. Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol 2004; 25:381-6. 205. Jiang W, Pisetsky DS. Enhancing immunogenicity by CpG DNA. Curr Opin Mol Ther 2003; 5:180-5. 206. Bauer S, Wagner H. Bacterial CpG-DNA licenses TLR9. Curr Top Microbiol Immunol 2002; 270:145-54. 207. Ashkar AA, Rosenthal KL. Toll-like receptor 9, CpG DNA and innate immunity. Curr Mol Med 2002; 2:545-56.

Human Vaccines



208. Vollmer J. Progress in drug development of immunostimulatory CpG oligodeoxynucleotide ligands for TLR9. Expert Opin Biol Ther 2005; 5:673-82. 209. Vollmer J, Weeratna RD, Jurk M, Davis HL, Schetter C, Wullner M, et al. Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity. J Leukoc Biol 2004; 76:585-93. 210. Vollmer J. CpG motifs to modulate innate and adaptive immune responses. Int Rev Immunol 2006; 25:125-34. 211. Jurk M, Kritzler A, Debelak H, Vollmer J, Krieg AM, Uhlmann E. Structure-activity relationship studies on the immune stimulatory effects of base-modified CpG toll-like receptor 9 agonists. ChemMedChem 2006; 1:1007-14. 212. Gursel M, Verthelyi D, Gursel I, Ishii KJ, Klinman DM. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J Leukoc Biol 2002; 71:813-20. 213. Gursel M, Verthelyi D, Gursel I, Ishii KJ, Klinman DM. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J Leukoc Biol 2002; 71:813-20. 214. Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman DM. Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. J Immunol 2001; 166:2372-7. 215. Verthelyi D, Zeuner RA. Differential signaling by CpG DNA in DCs and B cells: not just TLR9. Trends Immunol 2003; 24:519-22. 216. Uhlmann E, Vollmer J. Recent advances in the development of immunostimulatory oligonucleotides. Curr Opin Drug Discov Devel 2003; 6:204-17. 217. Vollmer J. TLR9 in health and disease. Int Rev Immunol 2006; 25:155-81. 218. Davis HL, Weeratna R, Waldschmidt TJ, Tygrett L, Schorr J, Krieg AM. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J Immunol 1998; 160:870-6. 219. Weeratna RD, McCluskie MJ, Xu Y, Davis HL. CpG DNA induces stronger immune responses with less toxicity than other adjuvants. Vaccine 2000; 18:1755-62. 220. Sugai T, Mori M, Nakazawa M, Ichino M, Naruto T, Kobayashi N, et al. A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine. Vaccine 2005; 23:5450-6. 221. Cooper CL, Davis HL, Morris ML, Efler SM, Adhami MA, Krieg AM, et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J Clin Immunol 2004; 24:693-701. 222. Siegrist CA, Pihlgren M, Tougne C, Efler SM, Morris ML, AlAdhami MJ, et al. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response. Vaccine 2004; 23:615-22. 223. Tross D, Klinman DM. Effect of CpG oligonucleotides on vaccine-induced B cell memory. J Immunol 2008; 181:5785-90. 224. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 2006; 5:471-84. 225. Klinman DM, Xie H, Ivins BE. CpG oligonucleotides improve the protective immune response induced by the licensed anthrax vaccine. Ann N Y Acad Sci 2006; 1082:137-50. 226. Klinman DM. CpG oligonucleotides accelerate and boost the immune response elicited by AVA, the licensed anthrax vaccine. Expert Rev Vaccines 2006; 5:365-9. 227. Shaw MH, Reimer T, Kim YG, Nunez G. NOD-like receptors (NLRs): bona fide intracellular microbial sensors. Curr Opin Immunol 2008; 20:377-82. 228. Rietdijk ST, Burwell T, Bertin J, Coyle AJ. Sensing intracellular pathogens-NOD-like receptors. Curr Opin Pharmacol 2008; 8:261-6. 229. Franchi L, Park JH, Shaw MH, Marina-Garcia N, Chen G, Kim YG, et al. Intracellular NOD-like receptors in innate immunity, infection and disease. Cell Microbiol 2008; 10:1-8. 230. Girardin SE, Philpott DJ. Mini-review: the role of peptidoglycan recognition in innate immunity. Eur J Immunol 2004; 34:1777-82. 231. Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity 2008; 28:246-57. 232. Uehara A, Fujimoto Y, Kawasaki A, Kusumoto S, Fukase K, Takada H. Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1. J Immunol 2006; 177:1796-804. 233. Girardin SE, Travassos LH, Herve M, Blanot D, Boneca IG, Philpott DJ, et al. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J Biol Chem 2003; 278:41702-8. 234. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003; 300:1584-7. 235. Roychowdhury A, Wolfert MA, Boons GJ. Synthesis and proinflammatory properties of muramyl tripeptides containing lysine and diaminopimelic acid moieties. Chembiochem 2005; 6:2088-97. 236. Uehara A, Sugawara Y, Kurata S, Fujimoto Y, Fukase K, Kusumoto S, et al. Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells. Cell Microbiol 2005; 7:675-86. 237. Fujimoto Y, Inamura S, Kawasaki A, Shiokawa Z, Shimoyama A, Hashimoto T, et al. Chemical synthesis of peptidoglycan fragments for elucidation of the immunostimulating mechanism. J Endotoxin Res 2007; 13:189-96. 238. Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, et al. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 2003; 4:702-7.

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239. Fritz JH, Girardin SE, Fitting C, Werts C, Mengin-Lecreulx D, Caroff M, et al. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4. Eur J Immunol 2005; 35:2459-70. 240. Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands. Immunity 2008; 28:246-57. 241. Tada H, Aiba S, Shibata K, Ohteki T, Takada H. Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells. Infect Immun 2005; 73:7967-76. 242. Wolfert MA, Murray TF, Boons GJ, Moore JN. The origin of the synergistic effect of muramyl dipeptide with endotoxin and peptidoglycan. J Biol Chem 2002; 277:39179-86. 243. Kobayashi KS, Chamaillard M, Ogura Y, Henegariu O, Inohara N, Nunez G, et al. Nod2dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005; 307:731-4. 244. Fritz JH, le Bourhis L, Sellge G, Magalhaes JG, Fsihi H, Kufer TA, et al. Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 2007; 26:445-59. 245. Shuang C, Wong MH, Schulte DJ, Arditi M, Michelsen KS. Differential expression of Tolllike receptor 2 (TLR2) and responses to TLR2 ligands between human and murine vascular endothelial cells. J Endotoxin Res 2007; 13:281-96. 246. Keestra AM, de Zoete MR, van Aubel RA, van Putten JP. Functional characterization of chicken TLR5 reveals species-specific recognition of flagellin. Mol Immunol 2008; 45:1298-307. 247. Lundberg AM, Drexler SK, Monaco C, Williams LM, Sacre SM, Feldmann M, et al. Key differences in TLR3/poly I:C signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems. Blood 2007; 110:3245-52. 248. Nahori MA, Fournie-Amazouz E, Que-Gewirth NS, Balloy V, Chignard M, Raetz CR, et al. Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells. J Immunol 2005; 175:6022-31. 249. Roberts TL, Sweet MJ, Hume DA, Stacey KJ. Cutting edge: species-specific TLR9mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides. J Immunol 2005; 174:605-8. 250. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Speciesspecific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004; 303:1526-9. 251. Edwards AD, Diebold SS, Slack EM, Tomizawa H, Hemmi H, Kaisho T, et al. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unresponsiveness to imidazoquinolines. Eur J Immunol 2003; 33:827-33. 252. Rehli M. Of mice and men: species variations of Toll-like receptor expression. Trends Immunol 2002; 23:375-8. 253. Takada H, Uehara A. Chemical structures of bacterial peptidoglycan and immunomodulation in mammals. Tanpakushitsu Kakusan Koso 2004; 49:1161-7. 254. Magalhaes JG, Philpott DJ, Nahori MA, Jehanno M, Fritz J, le Bourhis L, et al. Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin. EMBO Rep 2005; 6:1201-7. 255. Takeuchi O, Akira S. MDA5/RIG-I and virus recognition. Curr Opin Immunol 2008; 20:17-22. 256. Yoneyama M, Onomoto K, Fujita T. Cytoplasmic recognition of RNA. Adv Drug Deliv Rev 2008; 60:841-6. 257. Kawai T, Akira S. Antiviral signaling through pattern recognition receptors. J Biochem 2007; 141:137-45. 258. Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science 2006; 314:997-1001. 259. Sumpter R Jr, Loo YM, Foy E, Li K, Yoneyama M, Fujita T, et al. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 2005; 79:2689-99. 260. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006; 441:101-5. 261. Cui S, Eisenacher K, Kirchhofer A, Brzozka K, Lammens A, Lammens K, et al. The C-terminal regulatory domain is the RNA 5'-triphosphate sensor of RIG-I. Mol Cell 2008; 29:169-79. 262. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, et al. 5'-Triphosphate RNA is the ligand for RIG-I. Science 2006; 314:994-7. 263. Fujita T. Virology. Sensing viral RNA amid your own. Science 2006; 314:935-6. 264. Cui S, Eisenacher K, Kirchhofer A, Brzozka K, Lammens A, Lammens K, et al. The C-terminal regulatory domain is the RNA 5'-triphosphate sensor of RIG-I. Mol Cell 2008; 29:169-79. 265. Byrd-Leifer CA, Block EF, Takeda K, Akira S, Ding A. The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol. Eur J Immunol 2001; 31:2448-57. 266. Bryant CE, Ouellette A, Lohmann K, Vandenplas M, Moore JN, Maskell DJ, et al. The cellular Toll-like receptor 4 antagonist E5531 can act as an agonist in horse whole blood. Vet Immunol Immunopathol 2007; 116:182-9. 267. Omueti KO, Beyer JM, Johnson CM, Lyle EA, Tapping RI. Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination. J Biol Chem 2005; 280:36616-25. 268. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003; 299:2076-9.

Human Vaccines

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269. Misch EA, Hawn TR. Toll-like receptor polymorphisms and susceptibility to human disease. Clin Sci (Lond) 2008; 114:347-60. 270. Texereau J, Chiche JD, Taylor W, Choukroun G, Comba B, Mira JP. The importance of Toll-like receptor 2 polymorphisms in severe infections. Clin Infect Dis 2005; 41:408-15. 271. Schroder NW, Schumann RR. Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis 2005; 5:156-64.

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