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J Antimicrob Chemother 2012; 67: 789 – 801 doi:10.1093/jac/dkr588 Advance Access publication 18 January 2012 Antiviral applications of Toll-like receptor agonists Nigel J. Horscroft*, David C. Pryde and Helen Bright Pfizer Global R & D, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK *Corresponding author. Tel: +41-788-84-95-60; E-mail: [email protected] In the past, antiviral research has focused mainly on viral targets. As the search for effective and differentiated antiviral therapies continues, cellular targets are becoming more common, bringing with them a variety of challenges and concerns. Toll-like receptors (TLRs) provide a unique mechanism to induce an antiviral state in the host. In this review we introduce TLRs as targets for the pharmaceutical industry, including how they signal and thereby induce an antiviral state through the production of type I interferons. We examine how TLRs are being therapeutically targeted and discuss several clinically precedented agents for which efficacy and safety data are available. We describe some of the chemistries that have been applied to both small molecule and large molecule leads to tune agonist potency, and offer a differentiated safety profile through targeting certain compartments such as the gut or the lung, thereby limiting systemic drug exposure and affecting systemic cytokine levels. The application of low-dose agonists of TLRs as vaccine adjuvants or immunoprotective agents is also presented. Some of the challenges presented by this approach are then discussed, including viral evasion strategies and mechanism-linked inflammatory cytokine induction. Keywords: TLR, immune modulation, therapy, virus Introduction In his 1908 Nobel lecture, Ilya Mechnikov commented on the use of subcutaneously applied nucleic acids to protect patients from infection during and after surgery. He reported that ‘the results achieved are so encouraging that it is possible to predict new progress in the approach to the dressing of wounds’.1 One hundred years later we have a better understanding of the role of pattern recognition receptors (PRRs) in sensing and responding to a variety of invading pathogens.2 The detection of exogenous nucleic acids by Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) is essential to mounting an innate response to viral infection.3,4 A deeper understanding of the biochemical pathways of the innate immune system has led to a number of innovative approaches to antiviral therapy.5,6 Prosecution of these targets has proven challenging for both biologists and chemists; nevertheless, this class of antiviral therapy has grown rapidly in recent years.7 The innate immune system PRRs are the sentinels of the innate immune system, recognizing pathogen-associated molecular patterns (PAMPs), the components of invading pathogens.8 PAMPs include various bacterial cell wall components such as lipopolysaccharides (LPSs), peptidoglycans (PGNs) and lipopeptides, as well as flagellin and viral nucleic acids. TLRs TLRs are the best-characterized PRRs and are evolutionarily conserved across a diverse range of species.9 They are homologues of the Drosophila Toll gene first identified as being essential for development and later for antifungal and antibacterial immunity.10,11 TLRs are type I transmembrane proteins featuring an extracellular leucine-rich domain and a cytoplasmic tail that contains a conserved Toll/IL-1 receptor (TIR) domain (Figure 1a).12,13 The leucine-rich domain consists of 19 –25 leucine-rich repeats (LRRs) made up of an a-helix and a b-strand connected by a loop. Each repeat contains the motif XLXXLXLXX as well as conserved hydrophobic residues. The threedimensional structure of the human TLR3 ectodomain revealed that this region forms a crescent shape, and, upon dimerization, a groove is formed on the concave surface where the two monomers meet.14,15 It is thought that negatively charged double-stranded RNA binds in this groove. TLRs are predominantly expressed in tissues involved in immune function as well as those exposed to the external environment, such as lung and the skin. Most TLRs are located on the plasma membrane, with the exception of TLR3, TLR7, TLR8 and TLR9, which are expressed intracellularly, predominantly in endosomes.16 – 18 This intracellular expression is thought to minimize the recognition of self RNA and DNA.19 To date, 11 human and 13 murine TLRs have been identified. However, only a subset of these is thought to recognize viral components (Table 1). TLR3, TLR7, TLR8 and TLR9 distinguish between double-stranded RNA, single-stranded RNA and CpG-containing DNA, facilitating the recognition of all viral species and inducing the expression of type I interferons (IFNs). The signalling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. Nevertheless, a wide variety of TLR agonists have been shown to inhibit hepatitis B virus (HBV) and hepatitis C virus # The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] 789 Review (a) (b) EXTRACELLULAR 5’-ppp-ssRNA RIG-I TLR7/8/9 Leucine-rich repeats TLR3 Mitochondrion RIG-I TRIF MyD88 IRAK4 TRAF6 TRAF6 IRAK1 MAVS TRAF3 FADD TRAF6 1-2 cysteine-rich regions IRF3 NF-κB IRF3 NF-κB IRF7 IRF7 NF-κB Transmembrane domain Cytoplasmic domain (highly conserved TIR domain) NF-κB NF-κB IRF7 IRF7 IRF7 IRF3 IRF3 IRF3 INTRACELLULAR IFN beta, TNF IFN alpha IFN beta IL-10, ISG15 Figure 1. (a) Schematic of a TLR type 1 transmembrane protein. (b) TLR signalling. Viral nucleic acids are primarily detected by either endosomal or cytoplasmic PRRs. The resulting signalling cascades converge at the transcription factors NF-kB and IRFs 3 and 7. This leads to the production of a number of cytokines, including the type 1 IFNs that initiate the innate response. Table 1. PRRs involved in the recognition of viral components PRR Virus Component recognized haemagglutinin protein Reference TLR2 MV 124 TLR3 RNA and DNA viruses dsRNA 125–128 TLR4 RSV, VSV envelope proteins, glycoproteins 129–132 TLR7/8 RNA viruses: HCV, FluV ssRNA 3, 22, 133–136 TLR9 DNA viruses: HSV CpG DNA 137–140 RIG-I RNA viruses: NDV, SV, dsRNA, ssRNA with a VSV, FluV, MV 5′ -triphosphate 4, 141– 143 MV, measles virus; RSV, respiratory syncytial virus; VSV, vesicular stomatitis virus; HCV, hepatitis C virus; FluV, influenza virus; HSV, herpes simplex virus; NDV, Newcastle disease virus; SV, Sendai virus; ds, double-stranded; ss, single-stranded. (HCV) (Table 2) and are discussed later.20 – 22 Based on a combination of cellular expression patterns and type I IFN signalling pathways, TLR3, TLR7 and TLR9 are considered the best targets for antiviral therapy.23 Signalling TLR signalling consists of two distinct pathways (for review see Brikos and O’Neill24). TLR7, TLR8 and TLR9 signal via the myeloid differentiation primary response gene 88 (MyD88)- 790 dependent pathway, with the adaptor protein MyD88 playing a pivotal role. Upon activation by single-stranded RNA or CpG DNA these TLRs induce the recruitment of the adapter protein MyD88 via its TIR domain. TIR domains initiate the signalling cascade through TIR adapters, leading to downstream responses tailored to specific pathogens. This leads to the activation of the transcription factors nuclear factor-kB (NF-kB) and IFN regulatory factors (IRFs) 3 and 7 (Figure 1b). These in turn induce the production of pro-inflammatory cytokines such as tumour necrosis factor-a (TNF-a), IL-1 and IL-6 (where IL stands for interleukin), and type 1 IFNs, respectively. Signalling via TLR3 is initiated by double-stranded RNA. TLR3 activation is MyD88 independent and relies on the adaptor molecule TIR-domain-containing adaptor-inducing IFN-b (TRIF). However, the TLR3 signalling cascade also results in the activation of NF-kB, IRF3 and IRF7, and the production of pro-inflammatory cytokines and IFN. RLR signalling, on the other hand, proceeds via the interaction of the caspase activation and recruitment domain (CARD) of the RLR with the CARD of the mitochondrial antiviral signalling protein (MAVS), also known as IPS-1, VISA and Cardif.25 MAVS is anchored to the mitochondrial outer membrane, a feature essential for function (Figure 1b).26 In a similar fashion to TLR signalling, a downstream cascade results in the activation of NF-kB, IRF3 and IRF7. From a therapeutic point of view, the key outcome of this early signalling cascade is the production of type I IFNs. These IFNs are then secreted and function in both an autocrine and paracrine manner to produce more IFN, effectively amplifying their own production. Interaction with the type I IFN receptor leads to the induction of numerous genes that code for proteins that are directly or indirectly antiviral. JAC Review Table 2. Clinically precedented TLR agonists Compound Structure Imiquimod Target, intended viral disease NH2 Company Reference TLR7, hepatitis C 3M 144 TLR7, hepatitis C 3M 38 TLR7, hepatitis C Pfizer 43 TLR9, hepatitis C Coley 55 TLR7, hepatitis C Anadys 48 N N N Resiquimod NH2 N N N O HO PF-4878691 NH2 N N N NH O O Actilon S oligonucleotide Isatoribine O HO HO S O OH N OH N N NH2 GS9620 structure not disclosed TLR7, hepatitis B and hepatitis C Gilead Sciences 65 –68 ANA-773 structure not disclosed TLR7, hepatitis C Anadys 50 IMO-2125 modified oligonucleotide TLR9, hepatitis C Idera Pharmaceuticals 73,74 Antiviral effector molecules Imiquimod Of the hundreds of genes up-regulated by IFN stimulation, few have been thoroughly characterized. It is presumed that many are involved in the establishment of an antiviral state. Well-characterized examples of IFN-induced antiviral proteins include protein kinase R (PKR), the myxovirus resistance (Mx) GTPases, the RNA-specific adenosine deaminase (ADAR1), the ubiquitin-like modifier ISG15 and 2′ -5′ oligoadenylate synthetase (OAS) that activates endoribonuclease L (RNase L).27 – 30 Imiquimod (Figure 2a) is an example of the imidazoquinoline class of small molecule TLR7 agonists that have been the subject of extensive research activity over the past three decades, primarily by 3M Pharmaceuticals. Imiquimod,32 commercialized under the brand name Aldara, was initially launched in 1997 for the topical treatment of genital and perianal warts resulting from human papillomavirus (HPV) infection.33,34 In 2004 the product was commercialized in the USA for the topical treatment of actinic keratosis and superficial basal cell carcinoma. Phase II/III trials are currently under way for the treatment of Bowen’s disease. At the time of launch, imiquimod was shown to be a topical immune response modifier, capable of inducing the synthesis of type I IFNs and other cytokines in various cell types, although the precise mechanism of action of the compound was only confirmed to be TLR7 agonism some years later.35 A number of studies have shown imiquimod to be poorly tolerated upon oral dosing, reportedly through a Therapeutics There are few TLR-targeted agents in current clinical development or launched for the treatment of viral infections (Table 2).31 Those agents are described below, and on-going preclinical work with TLR ligands directed towards antiviral applications is detailed. 791 Review (a) (b) N N (c) NH2 NH2 NH2 N N N N N N O S HO resiquimod imiquimod (d) (f) (e) NH2 NH2 N N N O O N N N N NH2 O N N NH O OH S NH O 3M-003 S O 3M-011 852A (PF-4878691) (g) (h) O O S HO O S HO O OH N N O HO N N OH O NH2 O NH2 ANA-245 N N O ANA-975 (i) NH2 N N H (j) NH2 H N N O N N O H N O N N N MeO2C SM-324405 SM-276001 (k) (l) NH2 N MeO O H N O N N NH2 N F F H N O N F SM-360320 PF-4171455 Figure 2. Small molecule TLR7, TLR8 and mixed TLR7/8 agonists. Examples of imidazoquinolines (a)–(f), nucleosides (g) and (h) and 8-hydroxyadenine derivatives (i)– (l). 792 JAC Review centrally mediated mechanism, based on studies carried out in ferrets and possibly involving cytokine release triggered by imiquimod,36 although later studies with more potent non-emetic agents would appear to call this hypothesis into question (see the 8-Hydroxyadenine derivatives section below). Other compounds from the imidazoquinoline series include resiquimod (Figure 2b),37 a mixed TLR7/8 agonist that reached Phase III for the topical treatment of genital herpes before being discontinued due to a lack of adequate efficacy. Resiquimod has also been investigated as an oral treatment of HCV infection in a Phase IIa study in which patients received a 0.01 mg/kg dose of resiquimod twice weekly for 4 weeks.38 At this dose level the compound was well tolerated, but did not result in meaningful effects on viral load or significant changes in systemic levels of cytokines or biomarkers. Subsequent publications and patent applications have detailed further examples of imidazoquinoline series variants in the peripheral functionality around this core and covering a range of indications including viral infection.39 – 41 Modest structural modifications have transformed these TLR7-specific agonists into TLR8-specific agonists (Figure 2c) or mixed TLR7/8 agonists (Figure 2d and Figure 2e). Using these tools, 3M has demonstrated TLR8 agonism correlates well with higher levels of proinflammatory TNF-a induction than IFN-a induction, while TLR7 agonism is biased towards greater induction of IFN-a.39 This indicates that a selective TLR7 agonist could be preferable for antiviral indications. In 2007 3M sold the majority of its TLR portfolio to the Coley Pharmaceutical Group, which was itself later acquired by Pfizer.42 While Pfizer has continued to use oligonucleotides acquired from Coley as vaccine adjuvants, further development of the Coley small molecule TLR agonist collection appears to have been limited to the most advanced agent, which is discussed in the following section. PF-4878691 852A (PF-4878691, Figure 2f) is an imidazoquinoline analogue that was investigated by Coley for the treatment of cancers. It is a potent TLR7 agonist modelled to dissociate its antiviral and inflammatory activities. Following the Pfizer acquisition, 852A was repurposed for the potential treatment of HCV infection and progressed to a Phase I proof-of-mechanism study in healthy volunteers. 852A induced biomarkers of the immune and IFN responses in a dose-dependent and dose-frequency-related manner.43 However, two subjects in the top (9 mg) dose group experienced serious adverse events, characterized by flu-like symptoms, hypotension and lymphopenia, leading to termination of the study and further clinical development of the compound. Isatoribine Isatoribine (ANA-245, Figure 2g),44 and its masked prodrug ANA-975 (Figure 2h),45 is the most clinically studied oral TLR7 agonist reported to date, having entered Phase II studies for the treatment of HCV infection. Similarly to imiquimod, this agent was known to be an immune response modifier before its mechanism of action was found to be through activation of the TLR7 receptor.46 Anadys initially carried out a proof-of-concept study in HCV patients with intravenous isatoribine administered once or twice daily for 7 days or three times weekly for 14 days.47 The treatment was well tolerated, and in the highest once-daily dose group, receiving an 800 mg dose, 8 of 12 patients showed a statistically significant .0.5 log drop in viral load. This viral load reduction was accompanied by a dose-dependent systemic increase in the IFN-stimulated gene products 2,5-OAS and ISG15. This study provided proof of principle that a systemically dosed TLR7 agonist can produce viral load reductions in hepatitis C patients. The observed decreases in viral load were modest, considering the relatively high dose used, and could reflect weak primary pharmacology at the TLR7 receptor. Whether efficacy in patients would increase with a more potent compound while avoiding unacceptable increases in systemic cytokine levels, or how this antiviral profile would perform as part of a combination of therapies, remains to be seen. Anadys partnered with Novartis to develop ANA-975 as an oral agent, which was also found to be well tolerated when administered to healthy volunteers, with efficient generation of isatoribine in the systemic circulation through a sequence of ring oxidation and ester hydrolysis. Since then, Anadys suspended a 28 day Phase Ib study in HCV patients due to observations of dose-dependent ‘intense immune stimulation’ accompanied by polyclonal B-cell expansion in a concurrent 13 week animal toxicity study.48 Development of a reformulated ANA-975 was later discontinued following the results of a further 13 week animal toxicity study. Analysis of this study indicated that daily dosing of the compound was unlikely to support an adequate therapeutic index. Interestingly, Anadys rationalized their prodrug approach primarily on the basis of avoiding exposure of gut immune tissue to a TLR7 agonist,49 which they asserted would produce localized gut-related side effects such as nausea, emesis and gastroenteritis, all symptoms of some of the known TLR7 agonists, which are poorly tolerated by the oral route (see the Imiquimod section above).47 ANA-975 also addressed the poor oral bioavailability of isatoribine. Another prodrug of isatoribine, ANA-773, whose structure has not been disclosed, is currently being developed by Anadys for HCV. ANA-773 demonstrated an antiviral response in HCV patients in a Phase I clinical trial that appeared to exceed the efficacy seen previously with ANA-975. A Phase II trial was expected to start in 2011, and it will be critical to observe if the cytokine stimulation observed with ANA-975 is replicated with ANA-773 in longer-term dosing, or if there is an inherent design in the prodrug strategy of ANA-773 that limits systemic exposure, and thus cytokine release.50 However, Roche recently announced plans to acquire Anadys, and it is not currently clear what projects will survive the acquisition. CPG10101 Short sequences of synthetic oligodeoxynucleotides (ODNs) have been shown to possess broad, potentially therapeutic applications as anti-infective, antitumour and antiallergic agents and as Th1 vaccine adjuvants.51,52 ODNs containing sequencespecific unmethylated CpG motifs are abundantly present in the DNA of several pathogens, but not in mammals, in which their presence primes a host immune response through activation of the TLR9 receptor. This results in the activation and proliferation of immune cells to mount an immune response to the pathogen challenge. Due to evolutionary divergence, the precise sequence motifs that optimally stimulate immune cells from one species may weakly stimulate the immune cells of 793 Review another. For example, mouse TLR9 responds optimally to the sequence GACGTT, and human TLR9 to GTCGTT. One major drawback of natural CpG ODNs is the susceptibility of the phosphodiester backbone to serum and cellular nucleases, which can be addressed by using phosphorothioate backbones, which are nuclease resistant. Several CpG-containing phosphorothioate ODNs are in clinical development, including several from the CpG platform of Coley Pharmaceuticals. CpG-7909 (ProMune),53 a 24-mer B-class CpG ODN, is the most advanced agent. Licensed to Pfizer, the compound entered Phase III trials for the treatment of non-small cell lung cancer and breast cancer, but current development has been halted, as it did not improve survival when added to the current standard of care.54 Coley was also developing CPG10101 (Actilon) for the treatment of chronic HCV.55 CPG10101 had progressed to a Phase IIa trial in combination with pegylated IFN (PEG-IFN) and ribavirin in HCV patients who had not previously responded to treatment, but it failed to significantly reduce viral load. Coley decided to discontinue the development of CPG10101 in order to focus its resources on other product candidates in the company’s portfolio and to seek a licensing partner for CPG10101. 8-Hydroxyadenine derivatives Dainippon Sumitomo Pharmaceuticals Company Ltd has partnered with AstraZeneca to investigate a series of 8-hydroxyadenine derivatives that strongly activate the TLR7 receptor and are structurally related to the base component of isatoribine.56 SM-276001 (Figure 2i) is undergoing preclinical investigation as a potential treatment for both HCV and HBV infection, and is one of a series of disclosed adenine derivatives that demonstrate both high potency of IFN induction in vitro in mouse splenocytes and in vivo in the mouse following oral dosing. In cynomolgus monkeys, SM-276001 was shown to elicit a superior systemic IFN induction response to resiquimod.57 – 59 Interestingly, Dainippon Sumitomo has shown that SM-276001 is non-emetic in the ferret model at doses up to 30 mg/kg,57,60 while resiquimod is strongly emetic at doses that achieve some 10-fold lower Cmax values. The researchers at Dainippon Sumitomo postulate that the emetic behaviour of resiquimod reflects a greater extent of CNS penetration.60 The results of more detailed analysis of compound levels in the brain or comparative cytokine profiles induced by different TLR7 ligands have not been released. Recent disclosures claim extended ester prodrugs of this series61,62 for intratracheal administration to inhibit allergen-induced airway inflammation, but importantly did not induce any systemic IFNs. A key feature of this class of compounds is the ‘antedrug’ concept, in which an active ester (e.g. Figure 2j) is rapidly metabolized in plasma to the much less active carboxylic acid equivalent. How efficacious such an inhaled administration approach is for respiratory tract infections remains unexplored. Analogous functionality at the C2 position has also shown potent TLR7 agonism,63 such as the related SM-360320 (Figure 2k) that inhibits HCV replication in hepatocytes both dependently and independently of its ability to induce type I IFNs.22 The Pfizer group has disclosed a potent series of 8-oxo-deazapurines, such as PF-4171455 (Figure 2l), that are being developed for the treatment of HCV.64 794 GS9620 In a recent disclosure by Gilead Sciences,65 the selective TLR7 agonist GS9620 was shown to have a good pharmacology profile in vitro and pharmacokinetic profile in animals. The compound has a reported in vitro PBMC stimulation EC50 of 291 nM and a 30-fold selectivity over the TLR8 receptor. It shows good solubility and has a modest pharmacokinetic half-life in preclinical species. Notably the hepatic extraction of the compound is high, and the authors claimed that systemic IFN levels appeared not to be driven by systemic compound exposure, but primarily by engagement of gut-associated lymphoid tissue (GALT). This was supported by orally administered GS9620 driving higher systemic IFN levels in cynomolgus monkeys than an intravenous dose of the same compound, despite the latter achieving a higher systemic exposure of the compound. The compound induced cytokines and IFN-stimulated genes at low doses in animals, and was well tolerated in monkeys when dosed every other day at 1.5 mg/kg for 4 weeks. It induced a protective antiviral response in woodchucks infected with woodchuck HBV and produced a reduction in viral load when administered to chimpanzees infected with HBV.66,67 Most notably, GS9620 was safe and well tolerated when administered to healthy human volunteers at single ascending doses of 0.3 – 12 mg.68 Treatment was accompanied by a dose-dependent increase in cytokines and in IFN-stimulated genes, but IFN elevations were only detected with the 12 mg dose. The most common adverse events seen with GS9620 were headaches, chills and fever, but there were no serious adverse events or discontinuations and GS9620 continues to be progressed for the treatment of HBV and HCV. Its structure has not as yet been disclosed. This study indicates that some of the toxicities observed above with previous clinical studies using TLR7 agonists may be surmountable. The observation that efficacy is preserved in monkeys despite low systemic exposure may offer a means of driving sufficient cytokine induction to be efficacious without the intense immune system induction seen with some of the previously investigated compounds. Further details of the Gilead clinical programme are eagerly anticipated. Immunomodulatory oligonucleotides The next generation of oligonucleotide TLR agonists are exemplified by the immunomodulatory ODNs (IMOs) of Idera Pharmaceuticals (formerly Hybridon) that combine a novel synthetic DNA structure, called an immunomer, and a synthetic YpR motif, where Y and R are synthetic analogues of natural bases.69,70 Naturally occurring CpG DNA (Figure 3a) forms double-stranded helical structures; immunomers consist of two 3′ -3′ -linked identical DNA fragments, which are still able to present two 5′ -termini to the TLR9 receptor. These secondgeneration ODNs have the advantage of greater metabolic stability than their natural nucleotide predecessors, especially when they incorporate a phosphorothioate backbone, and the ability to induce a different cytokine profile depending on the precise structure and sequence of the immunomer. Early CpR IMOs (e.g. Figure 3b) in which the G base is replaced by a synthetic analogue (R) demonstrated the same speciesspecific immunomodulatory activity seen with natural ODNs. Interestingly, later YpG motifs (Figure 3c and d) in which the C JAC Review (a) (b) O S – P O O O N S S N O P O P O N N S O N P O O O NH N O – N O O O O NH2 O O O O – – NH2 O N NH N O NH2 NH2 CpG CpR (c) (d) O S – P O O O S P N – P O NH2 O N O O O – S N OH O NH O O O O O O N S O N N – P N O O O NH O NH2 N O N N NH NH2 Figure 3. Immunomodulatory oligonucleotides based on CpG sequences on a phosphorothioate backbone. base is replaced with a synthetic analogue show speciesindependent activity, and different immunomers show a clear structure–activity relationship depending on the exact structure of the base used. For example, A-rich sequences at the 5′ -end of the sequence confer TLR7 selectivity on the sequence, while 5-substituted C bases of YpG motifs can switch sequences to being antagonistic. IMOs have been shown to be well tolerated in mice at doses up to 10 mg/kg following intraperitoneal, subcutaneous, intravenous or intratumoral administration, and have been optimized further through modifications to linker structure and length.71 The most advanced molecule in the Idera portfolio is IMO-2055,72 being developed for the treatment of cancer and as a vaccine adjuvant. In a Phase I study in treatment-naive HCV patients, IMO-2125 demonstrated similar efficacy to the standard of care and was well tolerated.73 However, a Phase II study has been delayed due to atypical lymphocytic proliferation observed in a 26 week non-clinical rodent study.74 The company awaits the outcome of a non-clinical non-human primate study. Oligoribonucleotide analogues have also been developed as TLR7/8 agonists.75 Poly-ICLC Poly-ICLC (Hiltonol) is a polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose. It is a very stable double-stranded RNA and potent TLR3 agonist with a strong IFN-inducing ability. Preclinical studies in mice suggest poly-ICLC and liposome-encapsulated poly-ICLC are safe and offer broad-spectrum protection against influenza viruses as well as other respiratory viruses, including respiratory syncytial virus (RSV) and severe acute respiratory syndrome (SARS) virus (reviewed in Wong et al.76). However, in a cotton rat model of RSV or flu infection, efficacious doses of poly-ICLC were also associated with augmented lung inflammation.77 Nevertheless, Phase I/II clinical trials have shown poly-ICLC to be safe and efficacious when given to glioblastoma patients, improving their survival time when added to standard care.78,79 Thus poly-ICLC has a promising role to play as a safe, effective and broadspectrum antiviral and vaccine adjuvant; as such, a number of Phase I studies in healthy volunteers are currently under way (clinicaltrials.gov trial identifiers NCT00773097, NCT01127464, NCT01299662 and NCT00646152). Immunoprotection using TLR agonists Prophylactic administration of a TLR agonist can also be beneficial by promoting an enhanced protective immune response. For example, a single intranasal dose of an imidazoquinoline mixed TLR7/8 agonist was shown to significantly reduce nasal viral titres when given 72 h pre-challenge or 6 h post-challenge with influenza virus in a rat model.80 Viral inhibition correlated with 795 Review dose and the level of type I IFN induced. Interestingly, a second pre-infection dose did not offer any additional viral suppression, and prophylactic administration of the small molecule agonist was found to be superior to using rat recombinant IFN-a. The same group have shown that TLR8 agonism can enhance immune responses in human neonatal antigen-presenting cells.81 Corixa has published patent applications that detail several members of their TLR4 receptor agonist platform as potential immunostimulants,82 at least some of which have been shown to offer protection against viral challenge. For example, a GlaxoSmithKline group has shown that pre-treatment of mice with CRX-527 was found to enhance both CD4+ and CD8+ T cell responses via induction of significant amounts of IL-12, which offered protection against a challenge by influenza virus and RSV.83 There have been similar reports of TLR3,84 TLR985 and TLR486 agonists also offering protection in murine models of influenza. A group at the University of California has synthesized conjugates of SM-360320 (Figure 2k) in which the parent compound is covalently attached to mouse serum albumin. The presence of the conjugated group was found to enhance cytokine induction in vitro, and was designed to keep the TLR7 agonist agent within the lung when inhaled, thereby reducing the risk of systemic cytokine induction. Pre-treatment with the conjugate in an infectious model of influenza virus produced significantly delayed mortality.87 Vaccine adjuvant/immunostimulation using TLR agonists Several of the agents described above are either being investigated or developed as vaccine adjuvants or general immune system activators, in addition to several more that are described briefly below. Eisai has published on several lipid A analogues that are agonists of the TLR4 receptor. E-6020 is currently in preclinical development for use as a vaccine adjuvant.88,89 Corixa has taken a non-toxic, truncated lipid A-based TLR4 agonist, monophosphoryl lipid A (MPL),90 into Phase III development as a vaccine adjuvant.91 The TLR7 ligand, loxoribine, was being developed by Johnson and Johnson as an immunostimulant,92 but its development appears to have been discontinued. Idera’s IMO-2055, known as Amplivax in adjuvant applications, is being used by Immune Response Corporation alongside its investigational HIV vaccine technology in Phase II trials in HIV patients.72 IC-31 is a TLR9 receptor agonist that is being used as an adjuvant in several prophylactic vaccines (partnered with Novartis). It is currently in Phase I trials.93,94 Dynavax has developed a vaccine technology based on a phosphorothioate oligodeoxyribonucleotide vaccine containing a short DNA immunostimulatory sequence (ISS) that targets the TLR9 receptor.95 Their most advanced vaccine is Heplisav (ISS-1018), which recently completed a successful Phase III trial for the prevention and immunotherapy of HBV.96 – 98 ISS-1018 also successfully completed a Phase II clinical study for the treatment of B-cell non-Hodgkin’s lymphoma.99 Following promising safety trials, Dynavax has partnered with AstraZeneca to evaluate ISS-1018 in Phase II trials for asthma.100 The ISSs can be used alone or linked with antigens. For example, Dynavax’s Phase III trials are being conducted with ISS-1018 combined with either a major ragweed allergen or a hepatitis B surface antigen. 796 Challenges Viral evasion strategies Viruses have evolved multiple mechanisms to evade the host immune system. For example, the NS3/4A protease is a key protein in HCV immune evasion.101 The protein blocks TLR3-mediated signalling by cleavage of TRIF102 and RIG-I-mediated signalling by cleavage of IPS-1.103,104 Since inhibition of NS3/4A proteolytic activity directly blocks viral replication as well as restores immune function to infected cells, drugs targeting NS3/4A would have dual pharmacodynamics, and have indeed been shown to be highly efficacious in the clinic.105 Other viruses evade the immune system in different ways. Kaposi’s sarcoma-associated herpesvirus (KSHV), a tumour-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologues of the cellular IRFs.106 The vaccinia virus-encoded proteins A46R and A52R contain TIR domains that interact with MyD88 and TRIF to inhibit immune activation.107 Vaccinia virus also encodes N1L, a protein that blocks TLR signalling by targeting kinases involved in the cascade.108 The V proteins of paramyxovirus interact with MDA5 to block immune activation,109,110 while influenza virus uses its NS1 protein to block the production of IFN.111 While it is possible that these mechanisms could compromise the efficacy of any therapy that seeks to activate the IFN response, all of the evasion strategies characterized thus far act within a virus-infected cell and have no effect on bystander cells. It is likely that an exogenous immune activator could mount an antiviral response in uninfected cells, preventing the spread of the infection. Safety The immune system is a double-edged sword. Activating the immune system can have unforeseen and catastrophic consequences, as witnessed during the TeGenero drug trial.112 Although TGN1412 did not target a PRR, there is some evidence that might be of concern to companies developing drugs that induce enhanced immune responses. Phase I clinical studies with ANA-773 have demonstrated that a TLR7 agonist can be efficacious and well tolerated; however, it remains to be seen if this compound ultimately has sufficient potency to significantly reduce viral load. Clinical development of several TLR7 agonists has been suspended due to safety concerns. Anadys suspended a 28 day Phase Ib study of their TLR7 agonist ANA-975 in HCV patients due to ‘intense immune stimulation’,48 and have since halted all development. In mice, a single dose of resiquimod resulted in a rapid and almost complete depletion of leucocytes from the blood.113 This depletion lasted 24 h and was caused by the retention of peripheral blood leucocytes in peripheral organs. Leucopenia is a recognized feature of infections with viruses that produce single-stranded RNA (e.g. influenza virus),114 as is an increased susceptibility to bacterial superinfection in influenza patients.115 Lymphopenia, together with severe hypotension and flu-like symptoms, was reported by the Pfizer group in the recent Phase I trial of PF-4878691.43 Pharmacokinetic –pharmacodynamic Review (PK –PD) modelling and preclinical toxicology data suggested that daily dosing with TLR7 agonists was inappropriate due to exaggerated immune stimulation,48 thus the oral dosing regimen for PF-4878691 was set at twice weekly. Nevertheless, antiviral activity (measured using an in vitro HCV replicon bioassay) was only observed in the serum from volunteers who received doses of PF-4878691 that were associated with mechanism-related adverse events. The study concluded that increasing potency to achieve greater antiviral efficacy would also induce more severe side effects and that the therapeutic index for this mechanism, based on systemic exposure, was not sufficient to progress at a safe and effective dose. CpG ODNs, acting via TLR9, have been implicated in triggering autoimmune responses, including rheumatoid arthritis, systemic lupus erythematosus and diabetes.116 – 119 TLR3 activation has also been associated with lupus nephritis.120 Conclusions The rationale for targeting TLRs as a means of treating viral infections is based on the presence of naturally occurring agonist molecules within invading viruses. Numerous other diseases can potentially be treated with PRR agonists and antagonists,121 – 123 and the field is set to expand. However, further basic and applied research is required to develop our understanding of TLRs and their downstream pathways before therapeutic application can become a reality. It is clear that Phase I safety studies of any TLR agonists will be very closely examined for both short- and long-term effects. Alternative dosing regimens may need to be examined to determine whether dosing less frequently than once daily can maintain efficacy while increasing safety. Targeting compounds to different immune system compartments could also be beneficial in limiting safety concerns. In this regard, the emerging animal data with the Gilead Sciences agent GS9620, in which systemic efficacy is observed in monkeys despite low systemic drug exposure, is encouraging. 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