Identification of Adjuvantic Activity of Amphotericin B in a Novel, Multiplexed, Poly-TLR/NLR High-Throughput Screen

Abstract

Small-molecule agonists have been identified for TLR7, TLR8, TLR4 and TLR2 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. A multiplexed, reporter gene-based high-throughput assay capable of detecting agonists of TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1 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. These results point to its potential applicability as a safe and effective adjuvant for human vaccines.

The Role of Signaling via Aqueous Pore Formation in Resistance Responses to Amphotericin B

“…In fact, the differential role of K ϩ efflux in the secretion of IL-1ß compared to TNF-␣ is shown clearly in the data obtained in THP-1 cells that indicated that when the AmB concentration was raised from 0.625 M to 5 M, the secretion of TNF-␣ increased by about 30-fold whereas the secretion of IL-1ß increased by only 4-fold (94). Such significant differences between the stimulatory effects that are exerted by the two types of AmB channels in immune cells are thus important to take into account for the development of more-specific targeted approaches for stimulation of AmB immune and adjuvant responses (96).…”

Better Adjuvants for Better Vaccines: Progress in Adjuvant Delivery Systems, Modifications, and Adjuvant–Antigen Codelivery

“…Some ligands of these receptors have been studied as vaccine adjuvants. The in vitro screening evaluation models of these targets can be used as a powerful tool for adjuvant discovery and evaluation [76]. Table 2 lists selected adjuvant targets, their cellular distribution, and agonists.…”

BBIQ, a pure TLR7 agonist, is an effective influenza vaccine adjuvant

“…16 Given these differences between the effects of TLR7 and TLR8 agonists, 2,17,18 we wished to evaluate the adjuvant activity of a pure TLR7 agonist, 1-benzyl-2-butyl-1H-imidazo [4,5-c]quinolin-4-amine (BBIQ). As depicted in Figure 2, BBIQ 15 was reported to be the most active TLR7 agonist, 7,[19][20][21] whereas the related derivative 17 was confirmed to be a dual TLR7/8 agonist. 22,23 The other closely related imidazoquinoline 16 22 with additional aminomethyl functionality as well as the tricyclic oxazoloquinoline 18, 24 pyrazoquinoline 19 24 was dual TLR7/8 agonists.…”