Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

Abstract

Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1β and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1β/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1β in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigen-specific responses in vivo.

Keywords: NLRP3; Toll-like receptor 4 (TLR4); adjuvants*; caspase 1 (CASP1); human immunodeficiency virus (HIV); inflammasome; monophosphoryl lipid A; saponin; vaccine.

FIGURE 1.

QS-21 structure. Structure of QS-21 showing three key structural domains and the aldehyde site that has been identified as an adjuvant-active site.

FIGURE 2.

QS-21 induces caspase-1/11-dependent IL-1β secretion. Murine BMDCs (A) or macrophages (B) from WT or caspase-1/11-deficient mice were stimulated for 6 h with alum (100 μg/ml) or QS-21 (10 or 2 μg/ml) with or without MPLA (5 μg/ml). IL-6 and IL-1β were measured in culture supernatants by ELISA. The data are triplicates ± S.E. and are representative of two or more independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate a significant difference detected between WT and caspase-1/11 cells determined by Student's t test.

FIGURE 3.

QS-21-MPLA induced IL-1β secretion requires TLR signaling and activates the NLRP3 inflammasome. A–G, murine BMDCs (A–C) or macrophages (D–G) from WT or ASC-, NLRC4-, TLR4-, MyD88-, Trif-, GBP5-, or NLRP3-deficient mice were stimulated for 6 h with the indicated stimulants with or without MPLA (5 μg/ml); LPS was at 10 ng/ml, AbISCO-100® was 5 μg/ml, and QS-21 was at 2 μg/ml unless otherwise indicated. IL-18 (C), IL-6 (A, B, D, E, and G), and IL-1β (A, B, D, E, and G) were measured in culture supernatants by ELISA. The data are triplicates ± S.E. and are representative of two or more independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 indicate a significant difference detected between WT and deficient cells determined by Student's t test (B–E and G) or by analysis of variance followed by Tukey's multiple comparisons test (A). F, Western blot indicated cleaved caspase-1 p20 and IL-1β p17 and full-length caspase-1 p45 and β-actin p 45 as a loading control.

FIGURE 4.

QS-21-induced cell death under serum-free conditions is independent of caspase-1 and rescued by the addition of exogenous cholesterol. Murine BMDCs (B) or macrophages (A and C–G) from WT or ASC-, caspase-1/11-, NLRP3-, RIP3-, or RIP3/caspase-8-deficient mice were stimulated for 1 (A) or 6 h (A–G) with the indicated stimulants with or without MPLA (5 μg/ml). In A and D, S. enterica serovar Typhimurium used a MOI of 1 bacterium per cell. In E, QS-21 was used at 10 μg/ml. In F, cholesterol was added at 30 μg/m l. Cell viability was determined with calcein AM (A–C and E–G) or LDH (D) and is represented as the percentage of dead cells relative to unstimulated cells and a positive control. IL-6 and IL-1β (E) were measured in culture supernatants by ELISA. Experiments were performed in serum-free medium. The data are triplicates ± S.E. and are representative of two or more independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 indicate a significant difference determined by Student's t test.

FIGURE 5.

Phagocytosis, lysosomal acidification, and caspase-1 activity are required for QS-21-induced IL-1β secretion. Murine macrophages from WT or GBP5- or NLRP3-deficient mice were stimulated for 6 h with the indicated stimulants (A–C). Cytochlasin D (5 μm) and bafilomycin A (83 μm) inhibitors (A) or 20 μm caspase-1 and cathepsin B inhibitors (B) were added 1 h before the cells were stimulated. QS-21 was used at 2 μg/ml unless otherwise indicated. In C, cells were treated with 100 units/ml of IFN-γ for 14 h prior to stimulation to induce maximal levels of Gbp5; Yersinia species were used at an MOI of 10 bacteria per cell and S. enterica serovar Typhimurium was used at an MOI of 1. IL-1β and IL-6 were measured in culture supernatants by ELISA. The data are triplicates ± S.E. and are representative of two or more independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 indicate a significant difference determined by Student's t test.

FIGURE 6.

Other QS-21-containing saponins activate IL-1β secretion through NLRP3. Murine macrophages from WT or NLRP3-deficient mice were stimulated for 6 h with the indicated stimulants (5 μg/ml for all except 2 μg/ml for QS-21) in the presence or absence of MPLA (5 μg/ml) (A and B). IL-1β and IL-6 were measured in culture supernatants by ELISA. The data are triplicates ± S.E. and are representative of two or more independent experiments. *, p < 0.05 indicates a significant difference determined by Student's t test.

FIGURE 7.

NLRP3 suppresses antigen-specific responses by a QS-21 adjuvanted vaccine in vivo. A and B, WT mice were immunized with QS-21, gp120, or a combination of QS-21/gp120 two times, with the second immunization 4 weeks after the first. C–E, WT and NLRP3-deficient mice were immunized with QS-21 with or without HIV gp120 two times, with the second immunization 4 weeks after the first. Seven days after the second immunization (B and C), IL-2, IFN-γ (C), IL-6, and IL-4 ELISpots were performed on splenocytes and anti-gp120 ELISAs were performed on serum (A and D). For ELISpots, splenocytes were seeded into 96-well plates in triplicate and stimulated for 18 h with twogp120-specific peptide pools (G pool or V3) or media alone (Mock). Cytokine spots were quantified using CTL software; the data are triplicates ± S.E. E, the muscle at the injection site was removed and homogenized, and the supernatants were analyzed by ELISA for IL-1β. Values were normalized to the total amount of tissue removed from each animal. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate a significant difference determined by Student's t test. The data represent one of three experiments with five mice per group.