Development and characterization of synthetic glucopyranosyl lipid adjuvant system as a vaccine adjuvant

Abstract

Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative T(H)1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants.

Conflict of interest statement

Competing Interests: Dr. Reed is a founder of, and holds an equity interest in, Immune Design Corp., a licensee of certain rights associated with GLA. This does not alter the authors' adherance to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Stability of GLA adjuvant formulations.

(A) Differences between the synthetic GLA (structure in grey) and naturally occurring endotoxins. (i) – The GLA has no attached residues on the hydroxyl; endotoxins have some or many sugars attached to this site. (ii) – There is no second phosphate on GLA; naturally occurring lipid A cores have two phosphates with one attached to site “ii”. (iii) – The chain attachment positions, number, and lengths are defined. Naturally occurring species can have a variety of attachment sites, number of chains and the number of carbons within the chains vary. (B) HPLC chromatograms of GLA and MPL. Arrows indicate the predominant acylated form: hexa- (GLA) and pentaacylated form (MPL). (C) Mass spectrometry profiles of GLA and MPL. (D) Particle size over time of GLA-AF and GLA-SE. As controls, stability of -SE and MPL-SE are shown. Data presented are the mean ± SD of 3–4 different lots of adjuvant formulations.

Figure 2. BMDC gene expression in response to TLR4 agonists.

BMDC were stimulated for 4 h with GLA-AF (0.5 µg/mL), MPL-AF (0.5 µg/mL), or LPS (40 ng/mL). Total RNA was extracted, amplified, biotinylated, and hybridized to the mouse inflammation 4x2K CustomArray (Combimatrix). Comparative analysis of treated versus untreated samples was done using BRB-ArrayTools software (NCI). Statistical significance was determined by a random-variance t-test and a p-value <0.001. A 2.5-fold change above untreated samples was considered significant upregulation. Selected genes were partitioned into distinct categories and displayed in the heat-map for each set.

Figure 3. Dose-dependent activation and maturation of BMDC in response to GLA stimulation.

BMDC were stimulated with 0.01–1000 ng/mL of GLA-AF, MPL-AF, LPS or equivalent volumes of AF. (A) IL-12p40, TNF, and IL-6 cytokine levels in culture supernatants after 24 h. (B) Percentage of CD86+, CD40+, and MHC Class II+ DC cells determined within the CD11c+ gate after 48 h of incubation with the TLR4 agonists (C57BL/6, left panels; BALB/c, right panels). (C) DC were pulsed with media or ID83 antigen (50 ng/mL), without or with 1 ng/mL of TLR4 agonist, washed and further incubated with antigen-specific CD4 T cells for 48 h. IFN-γ levels in culture supernatants were measured by ELISA. Data shown are representative of two independent experiments.

Figure 4. Rapid activation of dendritic cells in response to GLA.

C57BL/6 mice (n = 6) were immunized 3x, 2 wks apart with ID83 antigen co-administered with 1, 5 or 20 µg of GLA-SE or MPL-SE. Control groups included saline, ID83, and ID83+SE. (A) Mice were bled before (time 0) and 4 h after the first injection. Innate cytokine levels of IL-12p40, TNF, IL-6, MCP-1, CCL5 and CXCL10 were determined by Luminex.

Figure 5. GLA promotes a TH1-type response. C57BL/6 mice (n = 6) were immunized 3x, 2 wks apart with ID83 antigen co-administered with 1, 5 or 20 µg of GLA-SE or MPL-SE. Control groups included saline, ID83, and ID83+SE.

Splenocytes were harvested 10 days following the third immunization and stimulated in vitro with ID83 for 72 h. Levels of TH1 (IFN-γ, TNF), TH2 (IL-5, IL-13) and regulatory (IL-10) cytokines in culture supernatants were determined by multiplex ELISA (Luminex). Data shown are mean ± SD of triplicate wells, and are representative of two independent experiments. * P<0.05, ** P<0.01.

Figure 6. Kinetic of human DC gene expression in response to TLR4 agonist stimulation.

DC were stimulated with 1 µg/mL of GLA-AF, MPL-AF, or LPS. (A) RNA transcripts for a panel of MyD88- and/or TRIF-dependent genes (upper panel), TRIF-dependent only genes (middle panel), or genes associated with DC maturation (lower panel) were captured after 2, 4, and 8 h of stimulation using the QuantiGene multiplex assay. (B) Protein levels were determined after 4, 8, and 24 h stimulation for a subset of molecules in culture supernatants by ELISA. Data shown are the mean ± SD of duplicate wells, and are representative of DC from two blood donors. Difference significance between GLA-AF and MPL-AF groups at a given time point were determined by unpaired Student's t-Test, P<0.05 (*).

Figure 7. Dose-dependent activation and maturation of human DC in response to GLA stimulation.

Human monocyte-derived DC were incubated with 1, 10, 100, and 1000 ng/mL of GLA-AF or MPL-AF, or 1000 ng/mL of LPS. Levels of cell surface co-stimulatory molecules and intracellular cytokines were determined by flow cytometry. (A) IL-12p70 and TNF mean fluorescence fold (MFI) change over AF control at 12 h post-stimulation. (B) HLA-DR, CD40, CCR7, CD83, and CD86 co-stimulatory molecules MFI change over AF control at 48 h post-stimulation. (C) CD86 expression on DC stimulated with 100 ng/mL GLA-AF or -AF for 48 h in the presence of 1 µg/mL of anti-TLR-2, anti-TLR4, or IgG2a isotype control antibody. Data shown are the mean ± SD (n = 3, DC cultures from different blood donors), and representative of two independent experiments.