A Novel Toll-Like Receptor 2 Agonist Protects Mice in a Prophylactic Treatment Model Against Challenge With Bacillus anthracis

Abstract

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam₃CSK₄ (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

Keywords: Bacillus anthracis; TLR2; agonist; in vivo; prophylactic.

FIGURE 1

Schematic diagrams demonstrating the dose regimen of the studies. Detailed description of the study design can be referred to the “experimental design of the efficacy studies” in the materials and methods section.

FIGURE 2

Toll-like receptor (TLR) agonists induced various level of resistance to B. anthracis infection. Kaplan-Meier curves of C57BL/6 mice infected with B. anthracis. Mice are pre-treated with indicated benchmark TLR agonists followed with B. anthracis infection. Statistically significant differences (Log-rank test) in time to death (days) between the treated verses the control are indicated. *** indicates p-value < 0.001. ** indicates p-value < 0.05.

FIGURE 3

Activity of a novel TLR1/2 agonist, DA-98-WW07. (A) Structure of DA-98-WW07 (IUPAC Name) (4R,7S,10R,14R)-4-carbamoyl-14-(dodecanoyloxy)- 7-methyl-6,9,17-trioxo-10-palmitamido-16-oxa-12-thia-5,8-diazaoctacosanoic acid). (B) Luciferase expression in HEK293-cells stable transfected with FLAG-tagged human TLR2 and a NF-κB-luciferase reporter gene after stimulation with various doses of DA-98-WW07. Pam₃CSK₄ was used as a benchmark for the calculation of percent efficacy. The curves represent three independent experiments. The top curve was generated from only one data set, while the bottom curves were the best fit from two replicate data sets. (C) Murine splenocytes were stimulated with different concentration of DA-98-WW07. Secretion of IL-6 in the supernatants was measured as an indicator of DA-98-WW07 activity. The best fit curve from a single experiment is shown panel (D) Human PBMCs were stimulated with different concentrations of DA-98-WW07. Secretion of IFN-γ, TNF-α and IL-6 in the supernatants was measured as an indicator of DA-98-WW07 activity. The curves represent different donors.

FIGURE 4

DA-98-WW07 protected mice from B. anthracis infection. Mice survival following infection with B. anthracis. Kaplan-Meier curves of C57BL/6 mice infected with B. anthracis. (A) Mice are treated with indicated TLR agonists 2 h prior to infection and 24 or 48 h post infection. (B) Surviving mice were re-challenged with B. anthracis 20 day post initial infection. The number of mice that were re-challenged in the different groups are DA-98-WW07 –10 μg (4 mice), DA-98-WW07 –30 μg (5 mice), DA-98-WW07 –100 μg (5 mice), Pam₃CSK₄- 300 μg (5 mice), and PBS control (10 mice). Statistically significant differences (Log-rank test) in time to death (days) between treated vs. control groups are indicated. ^*** indicates p-value < 0.001. ^** indicates p-value < 0.05.

FIGURE 5

Ciprofloxacin, when treated in adjunct with sub-optimal doses of DA-98-WW07, augmented protective activity against B. anthracis infection. Kaplan-Meier curves of C57BL/6 infected with B. anthracis. Mice were treated with indicated TLR agonists 2 and 24 h prior to infection and 24 or 48 h post infection. Concomitantly, mice were not treated or treated with ciprofloxacin 24, 48, 72, and 96 h post infection. Statistically significant differences (Log- rank test) in time to death (days) between treated vs. control group are indicated. ^** indicates p-value < 0.05.