Subbalakshmi S. Malladi

Comparison of the immunostimulatory and proinflammatory activities of candidate Gram-positive endotoxins, lipoteichoic acid, peptidoglycan, and lipopeptides, in murine and human cells

The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.

Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinoline Analogues

Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N(1)-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N(1) and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC(50) value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-alpha induction activities in whole human blood models.

Potential adjuvantic properties of innate immune stimuli.

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-acid-inducible 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, up-regulation 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.

Structure−Activity Relationships in Toll-like Receptor-2 Agonistic Diacylthioglycerol Lipopeptides

The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.

Toll-Like Receptor (TLR)-7 and -8 Modulatory Activities of Dimeric Imidazoquinolines

Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific molecular patterns present in molecules that are broadly shared by pathogens but are structurally distinct from host molecules. The TLR7-agonistic imidazoquinolines are of interest as vaccine adjuvants given their ability to induce pronounced Th1-skewed humoral responses. Minor modifications on the imidazoquinoline scaffold result in TLR7-antagonistic compounds which may be of value in addressing innate immune activation-driven immune exhaustion observed in HIV. We describe the syntheses and evaluation of TLR7 and TLR8 modulatory activities of dimeric constructs of imidazoquinoline linked at the C2, C4, C8, and N(1)-aryl positions. Dimers linked at the C4, C8, and N(1)-aryl positions were agonistic at TLR7; only the N(1)-aryl dimer with a 12-carbon linker was dual TLR7/8 agonistic. Dimers linked at C2 position showed antagonistic activities at TLR7 and TLR8; the C2 dimer with a propylene spacer was maximally antagonistic at both TLR7 and TLR8.

Immunoprofiling toll-like receptor ligands: Comparison of immunostimulatory and proinflammatory profiles in ex vivo human blood models.

There is a pressing need for the development of novel, safe, and effective adjuvants. The recent discovery and characterization of pathogen-associated molecular pattern (PAMP)-recognizing elements such as the Toll-like, NOD-like, and RIG-like receptors, has brought into sharp focus the role of PAMPs in bridging the innate and adaptive immune responses, and a detailed understanding of the immunostimulatory vis-à-vis proinflammatory activities could lead to the development of effective adjuvants, monophosphoryl lipid A being an excellent example. We describe in this paper a series of hierarchical assays that were employed to characterize TLR agonists in vitro including primary TLR-reporter assays, secondary indices of immune activation, and tertiary screens characterizing transcriptomal activation patterns to identify optimal immunostimulatory chemotypes. The evaluation of representative members of known human TLR agonists demonstrate that TLR2, -4, -5, and -7 agonists were immunostimulatory. TLR7 agonists were extremely immunostimulatory, stimulating nearly all subsets of lymphocytes without inducing proinflammatory cytokine responses. The TLR5 agonist, flagellin, while immunostimulatory, was also highly proinflammatory. These results suggest that TLR agonists other than lipid A-like chemotypes could be developed into potential adjuvants, and that this series of hierarchical assays could be adapted to rapidly identify in large libraries, compounds with adjuvantic potential that lack proinflammatory responses.

Regioisomerism-dependent TLR7 agonism and antagonism in an imidazoquinoline.

Chronic immune activation is a hallmark of progressive HIV infection. Recent reports point to the engagement of toll-like receptor 7 (TLR7) and -9 by viral RNA as contributing to the activation of innate immune responses, which drive viral replication leading to immune exhaustion. The only known class of TLR7 antagonists is single-stranded phosphorothioate oligonucleotides, which has been demonstrated to inhibit immune activation in human and Rhesus macaque in vitro models. The availability of a selective and potent small-molecule TLR7 antagonist should allow the evaluation of potential benefits of suppression of TLR7-mediated immune activation in HIV/AIDS. Gardiquimod is a known N(1)-substituted 1H-imidazoquinoline TLR7 agonist, the synthesis of which has not been published. We show that the 3H regioisomer is completely inactive as a TLR7 agonist and is weakly antagonistic. A des-amino precursor of the 3H regioisomer is more potent as a TLR7 antagonist, with an IC(50) value of 7.5 microM. This class of compound may serve as a starting point for the development of small-molecule inhibitors of TLR7.

Potent Adjuvanticity of a Pure TLR7-Agonistic Imidazoquinoline Dendrimer

Engagement of toll-like receptors (TLRs) serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. We synthesized a dendrimeric molecule bearing six units of a potent TLR7/TLR8 dual-agonistic imidazoquinoline to explore if multimerization of TLR7/8 would result in altered activity profiles. A complete loss of TLR8-stimulatory activity with selective retention of the TLR7-agonistic activity was observed in the dendrimer. This was reflected by a complete absence of TLR8-driven proinflammatory cytokine and interferon (IFN)-γ induction in human PBMCs, with preservation of TLR7-driven IFN-α induction. The dendrimer was found to be superior to the imidazoquinoline monomer in inducing high titers of high-affinity antibodies to bovine α-lactalbumin. Additionally, epitope mapping experiments showed that the dendrimer induced immunoreactivity to more contiguous peptide epitopes along the amino acid sequence of the model antigen.

Structure–Activity Relationships in Human Toll-like Receptor 2-Specific Monoacyl Lipopeptides

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS) compounds are potential vaccine adjuvants. We had previously determined that at least one acyl group of optimal length (C(16)) and an appropriately orientated ester carbonyl group is essential for TLR2-agonistic activity. We now show that these structurally simpler analogues display agonistic activities with human, but not murine, TLR2. SAR studies on the monoacyl derivatives show that the optimal acyl chain length is C(16), and aryl substituents are not tolerated. A variety of alkyl and acyl substituents on the cysteine amine were examined. All N-alkyl derivatives were inactive. In contradistinction, short-chain N-acyl analogues were found to be highly active, with a clear dependence on the chain length. A cysteine N-acetyl analogue was found to be the most potent (EC(50): 1 nM), followed by the N-butyryl analogue. The N-acetyl analogue is human TLR2-specific, with its potency comparable to that of PAM(2)CS.

Structure‐Based Design of Novel Human Toll‐like Receptor 8 Agonists

Toll‐like receptor (TLR)‐8 agonists activate adaptive immune responses by inducing robust production of T helper 1‐polarizing cytokines, suggesting that TLR8‐active compounds might be promising candidate vaccine adjuvants. Recently, a C2‐butyl furo[2,3‐c]quinoline was reported with purely TLR8 agonistic activity. This compound was successfully co‐crystallized with the human TLR8 ectodomain, and the co‐crystal structure revealed ligand‐induced reorganization of the binding pocket of TLR8. The loss of a key hydrogen bond between the oxygen atom of the furanyl ring of the agonist and Thr 574 in TLR8 suggested that the furan ring is dispensable. Employing a disconnection strategy, 3‐ and 4‐substituted aminoquinolines were investigated. Focused structure‐based ligand design studies led to the identification of 3‐pentyl‐quinoline‐2‐amine as a novel, structurally simple, and highly potent human TLR8‐specific agonist (EC50=0.2 μM). Preliminary evaluation of this compound in ex vivo human blood assay systems revealed that it retains prominent cytokine‐inducing activity. Together, these results indicate the suitability of this compound as a novel vaccine adjuvant, warranting further investigation.