Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge

Abstract

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

FIG. 1.

General structure of AGPs.

FIG. 2.

(A) Induction of nonspecific resistance to challenge with Listeria monocytogenes by intravenous pretreatment of TLR4 mutant C3H/HeJ mice (± standard errors of the means [SEM]; hatched bars) or wild-type C3H/HeOUJ mice (plus SEM; filled bars) with CRX-524 (five mice per group). The data are presented as log₁₀ protection versus vehicle control on the y axis. The average log₁₀ numbers of splenic CFU in vehicle-treated control mice ± SEM were 7.46 ± 0.08 for C3H/HeJ and 7.61 ± 0.08 for C3H/HeOuJ (no significant difference between C3H/HeJ and C3H/HeOuJ in this regard by Student's t test). Compared to vehicle-treated mice, the number of splenic CFU was significantly lower in CRX-524-treated C3H/HeOuJ mice (all doses tested) but not in CRX-524-treated C3H/HeJ mice (P < 0.05; Student's t test). (B) Induction of nonspecific resistance to intranasal influenza virus challenge by intranasal pretreatment of TLR4 mutant C3H/HeJ mice or wild-type C3H/HeOuJ mice with CRX-524 (five mice per group). Anesthetized mice received 5 μg of CRX-524 in a 20-μl volume via the intranasal/intrapulmonary route. Filled bars, total weights; hatched bars, disease index scores. Percent survival for each group 21 days after infection is presented at the bottom of the graph. Treatment with CRX-524 resulted in a significant increase in survival of C3H/HeOuJ mice but not C3H/HeJ mice, relative to vehicle treatment (P < 0.05; Fisher's exact test).

FIG. 3.

(A) Induction of nonspecific resistance to challenge with Listeria monocytogenes (plus SEM [error bars]) by intravenous pretreatment with AGP compounds (hatched bars, 1-μg dose; filled bars, 20-μg dose) that differ in acyl chain length at positions R1, R2, and R3 (five BALB/c mice per group). The average log₁₀ number of splenic CFU in vehicle-treated control mice ± SEM was 7.51 ± 0.13. At either dose tested, only CRX-526 and CRX-554 failed to result in significantly lower splenic CFU than the vehicle control (P < 0.05; Student's t test). (B) Induction of nonspecific resistance to intranasal influenza virus challenge by intranasal pretreatment with AGP compounds that differ in acyl chain lengths at positions R1, R2, and R3 (five BALB/c mice per group). Filled bars, total weights; hatched bars, disease index scores. Percent survival for each group 21 days after infection is presented at the bottom of the graph. Treatment with AGPs having secondary fatty acids containing nine or more carbon atoms resulted in a significant increase in survival relative to vehicle treatment (P < 0.05; Fisher's exact test).

FIG. 4.

(A) Induction of nonspecific resistance to challenge with Listeria monocytogenes (plus SEM [error bars]) by intravenous pretreatment with AGP compounds (1-μg dose) that have one or two inactive six-carbon acyl chains at positions R1, R2, and/or R3 (five BALB/c mice per group). The average log₁₀ number of splenic CFU in vehicle-treated control mice ± SEM was 7.38 ± 0.17. Relative to vehicle-treated mice, splenic CFU were significantly lower in mice treated with AGPs containing zero or one six-carbon secondary fatty acid but not in mice treated with compounds containing two six-carbon secondary fatty acids (P < 0.05; Student's t test). (B) Induction of nonspecific resistance to intranasal influenza virus challenge by intranasal pretreatment with AGP compounds that differ in acyl chain lengths at positions R1, R2, and R3 (five BALB/c mice per group). Filled bars, total weights; hatched bars, disease index scores. Percent survival for each group 21 days after infection is presented at the bottom of the graph. Only CRX-527 treatment resulted in a significant increase in survival relative to vehicle treatment (P < 0.05; Fisher's exact test). No treat, no treatment.

FIG. 5.

Induction of nonspecific resistance to challenge with Listeria monocytogenes (plus SEM [error bars]) by intravenous pretreatment with AGP compounds (1-μg dose) that have various functional groups at position R4 on the aglycon unit (five BALB/c mice per group). The average log₁₀ number of splenic CFU in vehicle-treated control mice ± SEM was 7.98 ± 0.13. All compounds tested resulted in significantly lower numbers of splenic CFU than the vehicle control (P < 0.05 using Student's t test).

FIG. 6.

Induction of nonspecific resistance to challenge with Listeria monocytogenes (plus SEM [error bars]) by intravenous pretreatment with AGP compounds (1-μg dose) that have different spacer lengths between the sugar moiety and aglycon moieties (five BALB/c mice per group). The average log₁₀ number of splenic CFU in vehicle-treated control mice ± SEM was 7.03 ± 0.07. Only CRX-571 treatment did not result in significantly lower numbers of splenic CFU relative to vehicle treatment (P < 0.05; Student's t test).