Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy

Abstract

Innate immune system activation is a critical step in the initiation of an effective adaptive immune response; therefore, activation of a class of innate pathogen receptors called pattern recognition receptors (PRR) is a central feature of many adjuvant systems. It has recently been shown that one member of an intracellular PRR, the NLRP3 inflammasome, is activated by a number of classical adjuvants including aluminum hydroxide and saponins [Eisenbarth SC, Colegio OR, O'Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008;453(June (7198)):1122-6; Li H, Willingham SB, Ting JP, Re F. Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol 2008;181(July (1)):17-21]. Inflammasome activation in vitro requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. Here we present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using a simple approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester, poly(lactic-co-glycolic acid) (PLGA), loaded with antigen. We demonstrate that LPS-modified particles are preferentially internalized by dendritic cells compared to uncoated nanoparticles and the system, when administered to mice, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild-type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1beta consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1beta. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as 'danger signals' [Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008;9(August (8)):847-56]. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to stimulate two potent innate immune pathways represents a potent new approach to simultaneous antigen and adjuvant delivery.

Figure 1

Characterization of nanoparticles. A) Schematic of biodegradable particles with lipopolysaccharide modification. B) Controlled release profile of OVA from LPS/OVA (●) and -/OVA (■) particles. Figure represents release from nanoparticles at 37°C in phosphate-buffered saline in triplicate over 3 weeks. C) Scanning Electron Microscopy (SEM) image of LPS-modified nanoparticles. Scale bar is 2 μm. D) Size distribution profiles for LPS-modified and unmodified OVA-loaded nanoparticles.

Figure 2

Evaluation of uptake efficiency. A) Dendritic cells were incubated with nanoparticles encapsulating rhodamine B and analyzed by confocal microscopy (green: phalloidin-FITC, blue: DAPI, and red: rhodamine B). A representative image is shown. B) Flow cytometry analysis of dendritic cells pulsed with Nile red-loaded nanoparticles: LPS/Nile red (■) and -/Nile red (□). Mean fluorescence in FL2 is shown.

Figure 3

LPS-modified nanoparticles activated the NLRP3 inflammasome. A) Proposed mechanism of activation. Signal 1: LPS triggers Toll-like receptor 4 (TLR4) signaling, which ultimately activates the transcription factor, NF-κB. Pro-IL-1β is upregulated and is cleaved by caspase-1 in an inflammasome complex. Signal 2: Nanoparticles are phagocytosed by cells, disrupt lysosomal compartments, and activate the NLRP3 receptor. NLRP3 recruits ASC, an adapter molecule, which binds caspase-1. B) Macrophages from WT (□), NLRP3-deficient () and caspase-1-deficient (■) mice were isolated and incubated with LPS-modified and unmodified OVA-loaded particles with and without prior stimulation with soluble LPS. ATP with LPS pre-stimulation was used as a positive control. After 24 h, supernatant was analyzed for IL-1β by ELISA. C) WT macrophages and D) BMDCs were pre-stimulated with LPS and then treated with cytochalasin D (cytoD), to inhibit internalization; CA-074 Me, to inhibit cathepsin B; or media alone. After incubation with particles or alum for 24 h or ATP for 20 min, supernatant was analyzed for IL-1β.

Figure 4

In vitro vaccination with nanoparticles. A) Dendritic cells were pulsed with −/OVA, LPS/OVA, −/− with soluble OVA and LPS, soluble OVA and soluble LPS, or soluble OVA at an OVA concentration of 50 μg/ml. Cells were then co-cultured with OT-1 transgenic splenocytes. Supernatant IFN-γ was quantified by ELISA after 48 hours. B) Dendritic cells were pulsed with soluble OVA at high concentrations and then co-cultured with OT-1 splenocytes in order to achieve similar levels of T cell activity. Dotted line represents IFN-γ levels from cells pulsed with LPS/OVA from (A).

Figure 5

OVA-specific immune responses 2 weeks after subcutaneous vaccination. Animals received a single vaccination by s.c. injection with particle formulations, alum, or CFA as adjuvants. A) Serum antibody titers were analyzed by ELISA. Data shown are mean OVA-specific IgG titer. B) Splenocytes were stimulated ex vivo with OVA and supernatant was analyzed for IFN-γ secretion at 48 h by ELISA. For all groups: n=5. Experiments were repeated 2 more times (with n=4) with similar results.

Figure 6

Antigen-specific IgG and survival in mice after vaccination with encapsulated West Nile virus envelope protein antigen. Mice (n=20) were vaccinated with LPS/rWNVE subcutaneously (s.c.), orally, or intranasally (i.n.). As a negative control, mice were subcutaneously administered LPS/OVA particles. A) Anti-rWNVE IgG titers in the serum pre-challenge were quantified by ELISA. B) Mice were challenged with 1000 PFU of isolate 2741 and monitored for mortality for 21 days. LPS/OVA (●); rWNVE alone (); LPS/rWNVE s.c. (); LPS/rWNVE oral (); and LPS/rWNVE nasal (;). Results are combined data from two experiments (n=10).