Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome

Abstract

The development of new protein subunit vaccines has stimulated the search for improved adjuvants to replace traditional aluminum-containing products. We investigated the adjuvant effects of a synthetic Toll-like receptor 4 (TLR4) agonist on vaccine efficacy in an experimental model of toxic shock syndrome. The TLR4 agonist E6020 has a simplified structure consisting of a hexa-acylated acyclic backbone. The vaccine examined is a recombinantly attenuated form of staphylococcal enterotoxin B (STEBVax). Using cells stably transfected with TLRs, E6020 transduced signals only through TLR4, suggesting monospecificity, while Escherichia coli 055:B5 lipopolysaccharide activated both the TLR2/6 heterodimer and TLR4. Coadministration of E6020 with STEBVax, by the intramuscular or intranasal route, induced significant levels of immunoglobulin G (IgG) in BALB/c mice. Further, increased IgG production resulted from the combination of E6020 with aluminum hydroxide adjuvant (AH). The antibody response to the vaccine coadministered with E6020 was a mixed Th1/Th2 response, as opposed to the Th2-biased response obtained with AH. Mice vaccinated with STEBVax coadministered with AH, TLR4 agonists, or a combination of both adjuvants were protected from toxic shock. Our data demonstrate the effectiveness of the synthetic TLR4 agonist E6020 as an alternative adjuvant for protein subunit vaccines that may also be used in combination with traditional aluminum-containing adjuvants.

FIG. 1.

NF-κB activation in CD14⁺ monocytes is equivalently enhanced following treatment with a natural or a synthetic TLR4 agonist. Monocytes were isolated and treated either with solvent-extracted LPS, which stimulates only TLR4, or with E6020. Levels of NF-κB activation were measured at 1 and 12 h following treatment. Activation of NF-κB is expressed in relative light units, presented as arithmetic means. Error bars, standard deviations.

FIG. 2.

The synthetic TLR4 agonist E6020 exhibits dose-dependent signaling through the TLR4 receptor complex and does not cross-react with TLR2/6. HEK293 cells transfected with TLR4 (A), MD2/CD14 (B), TLR4/MD2/CD14 (C), or TLR2/6 (D) were treated with various concentrations of E. coli 055:B5 LPS or E6020, and secretion of the reporter gene (human placental alkaline phosphatase) was measured after 3 days. NF-κB-dependent activation of the reporter gene is expressed in relative light units.

FIG. 3.

Production of TNF-α, IL-10, and IL-4 was stimulated in CD14⁺ monocytes following treatment with a natural or a synthetic TLR4 agonist. Monocytes were isolated and treated either with solvent-extracted LPS, which stimulates only TLR4, or with E6020. Levels of TNF-α (A), IL-10 (B), IL-4 (C), and IL-5 (D) were measured at 1 and 12 h following treatment. Data are presented as arithmetic means. Error bars, standard deviations. Significant increases (P < 0.05) in TNF-α, IL-10, and IL-4 levels were observed by 12 h (and by 1 h for TNF-α) with both treatments.

FIG. 4.

Levels of SEB-specific IgG in serum were enhanced following i.m. or i.n. administration of various formulations of STEBVax. BALB/c mice were administered three doses of vaccine at 3-week intervals. Seroconversion occurred by day 14 for i.m. administration and by day 35 for i.n. administration.

FIG. 5.

Mice administered STEBVax vaccine containing AH and a TLR4 agonist, either LPS or E6020, had significantly higher SEB-specific serum IgM levels on day 49, 1 week following the final vaccine administration. Asterisks indicate IgM concentrations significantly greater (P < 0.05) than those observed for the saline control.

FIG. 6.

Mice that received STEBVax vaccine containing a TLR4 agonist, either LPS or E6020, responded with a mixed Th1/Th2 response, as opposed to the Th2-biased response obtained with AH. Levels of SEB-specific IgG1 and IgG2a were determined by enzyme-linked immunosorbent assays for sera obtained on day 49, 1 week after the final vaccination.