Exploration of the Mechanisms of Adjuvanticity for Toll-like Receptor Agonists
Salyer, Alex Christopher Dean
University of Kansas
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Small-molecule agonists have been identified for Toll-like Receptors (TLR) 2, TLR4, TLR7 and TLR8 thus far, and chemotypes other than those of canonical ligands are yet to be explored for a number of innate immune receptors. The discovery of novel immunostimulatory molecules would enhance the repertoire of tools available for interrogating innate immune effector mechanisms, and provide additional venues for vaccine adjuvant development. It is with this in mind that we aimed to identify novel immunostimulatory compounds by high-throughput screening, characterize transcriptomal ‘signatures’ of innate immune stimulation and explore mechanisms of adjuvanticity for TLR2, TLR2/7 and TLR8 agonists. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, nucleotide-binding oligomerization domain-like receptors (NOD) 1 and NOD2 was utilized in screening 123,943 compounds, in which amphotericin B (AmpB) and nystatin were identified as prominent hits. The polyene antifungal agents act as TLR2- and TLR4-agonists. The TLR4-stimulatory activity of AmpB was similar to that of monophosphoryl lipid A, suggestive of TRIF-biased signaling. The adjuvantic activity of AmpB, at a dose of 100 micrograms, was comparable to several other candidate adjuvants in rabbit models of immunization. (Chapter 2) We sought to identify transcriptomal signatures of innate immune stimulating molecules using next-generation RNA sequencing with the goal of being able to utilize such signatures in identifying novel immunostimulatory compounds with adjuvantic activity. We observed that the CC family of chemokines, particularly CC chemokines 1, 2, 3, 4, 7, 8, 17, 18, 20, and 23, were broadly upregulated by most TLR and nucleotide-binding domain and leucine-rich repeat–containing receptors (NLR) stimuli, while the CXC chemokine family appeared to show distinctions in upregulation. Extracellular receptors such as TLR2, TLR4 and TLR5 induced the transcription of CXC chemokines including CXCL5, CXCL6 and CXCL8, whereas intracellular receptors such as TLR7 and TLR8 upregulated CXC chemokines 11 and 12. A comparison of a variety of TLR agonists in a standardized rabbit immunization model indicated prominent adjuvantic activity for TLR2 agonists. Strong chemokine induction by TLR2 agonists was observed in human peripheral blood mononuclear cells. In addition, human foreskin fibroblasts stimulated with TLR2/6 agonists, but not TLR1/2 agonists resulted in chemokine production, which was consistent with strong expression of TLR2 and TLR6, but not of TLR1, in fibroblasts. TLR2/6 stimulated fibroblasts demonstrated functional chemotactic responses to human T cell and natural killer cells subsets. (Chapter 3) We hypothesized that an ESAT-6-based subunit vaccine adjuvanted with a TLR2/7 hybrid would induce balanced T helper (Th) 1/Th2 responses capable of conferring protection against M. tuberculosis. We therefore covalently linked a potent TLR2 agonist with a dual TLR7/8 agonist, and observed that the resulting TLR2/7 hybrid molecules remained active, though less potent, against TLR2 and TLR7. The TLR2/7 hybrid was equipotent to the two individual TLR agonists in a standardized rabbit immunization model, but induced higher ‘quality’ antibodies as measured by surface plasmon resonance. Linear epitope mapping revealed that the hybrid induced immunoreactivity to more contiguous epitopes in a model antigen. The hybrid molecule was able to induce increases in ESAT-6-specific interferon-γ spot-forming units in the lungs of mice, and reduce the mycobacterial burden in the lungs following M. tuberculosis challenge. (Chapter 4) Part-structures of the 2-aminobenzimidazole scaffold were examined with a view to identifying structural requisites corresponding to the smallest possible fragment of the benzimidazole core that would allow for retention of TLR8-agonistic activity. TLR8-specific agonistic activity was retained in 1-pentyl-4-phenyl-1H-imidazol-2-amine. The crystal structure of this compound bound to TLR8 ectodomain displayed binding interactions that are common to other TLR8 agonists. This compound showed markedly attenuated proinflammatory properties in ex vivo human blood models. Structure-activity relationship (SAR) studies revealed that 4-(2-(benzyloxy)phenyl)-1-pentyl-1H-imidazol-2-amine inhibited TLR signaling in a variety of TLR reporter cell lines, as well as in pharmacologically-relevant human blood model systems. A kinase screen of this compound showed relative specificity for calmodulin kinases. (Chapter 5) The effects of TLR8 agonists on innate immune function suggest that these compounds could potentially be useful as vaccine adjuvants in neonatal vaccines. We examined how TLR8 agonists influence processing of soluble antigens by antigen presenting cells. TLR8-active compounds were unique in inducing pyroptosis-like death in monocytes, leading to the formation of CD14+ extracellular vesicles (ECV) of 100-400 nm diameter. ECV formation was dependent on myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-associated kinases (IRAK) 1 and 4, and p38 mitogen-activated protein kinase (MAPK). The monocyte-derived ECVs contain near-intact soluble antigens, and stimulate antigen-specific recall responses in autologous CD4+ T lymphocytes. The formation of antigen-loaded, monocyte-derived ECVs may be a distinct mechanism underlying the adjuvantic activities of TLR8 agonists. (Chapter 6) The results presented here highlight the applicability of high-throughput screens for the identification of novel innate immune stimuli, and identified transcriptomal profiles to aid in determining adjuvanticity of new compounds, as well as aiding in target identification. The insight gained into mechanisms of adjuvanticity for the TLR2, TLR2/7, and TLR8 agonists highlights the utility of TLR agonists as vaccine adjuvants, and justifies the continued study of small-molecule innate immune stimuli for applications in vaccines.
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