In vitro evaluation of TLR4 agonist activity: Formulation effects

Abstract

Effective in vitro evaluation of vaccine adjuvants would allow higher throughput screening compared to in vivo studies. However, vaccine adjuvants comprise a wide range of structures and formulations ranging from soluble TLR agonists to complex lipid-based formulations. The effects of formulation parameters on in vitro bioactivity assays and the correlations with in vivo adjuvant activity is not well understood. In the present work, we employ the Limulus amebocyte lysate assay and a human macrophage cellular cytokine production assay to demonstrate the differences in in vitro bioactivity of four distinct formulations of the synthetic TLR4 agonist GLA: an aqueous nanosuspension (GLA-AF), an oil-in-water emulsion (GLA-SE), a liposome (GLA-LS), and an alum-adsorbed formulation (GLA-Alum). Furthermore, we demonstrate the importance of the localization of GLA on in vitro potency. By comparing to previous published reports on the in vivo bioactivity of these GLA-containing formulations, we conclude that the most potent activators of the in vitro systems may not be the most potent in vivo adjuvant formulations. Furthermore, we discuss the formulation considerations which should be taken into account when interpreting data from in vitro adjuvant activity assays.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL‐18, and IFN‐γ

“…Typically, in vitro systems are employed to evaluate inflammasome activity. Interestingly, the in vitro activity of GLA-SE is lower than that of the aqueous formulation of GLA-the reverse of the in vivo hierarchy [61]. Here, we saw no evidence for caspase activation by in vitro stimulation with GLA-SE indicating that the models used here were insufficient to replicate in vivo processes.…”

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach

“…CpG concentration was measured by UV absorbance at 260 nm after 1:20 dilution into ethanol:HCl. GLA concentration was measured by reverse-phase HPLC with an C18 column (Atlantis T3 or Agilent XBridge) and charged aerosol detection (CAD) using a methanol:chloroform:water mobile gradient as described previously [20]. To detect unbound TLR ligand, alum-containing formulations were centrifuged briefly as indicated and the supernatant assayed by UV absorbance or HPLC-CAD.…”

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

“…A major issue that has arisen previously in designing a Pfs25-based vaccine is the need for a strong yet human-use-compatible adjuvant to overcome the inherent lack of antigenicity of Pfs25; in a phase I clinical trial, erythema nodosum associated with the Montanide ISA 51 oil-in-water adjuvant was observed in a small number of subjects, which led to cessation of that vaccine formulation (21). New adjuvants based on squalene-oil-in-water emulsion in the context of a nontoxic lipid A-like moiety, glucopyranosyl lipid A (GLA), have been developed in extensive animal/in vitro experimentation (22)(23)(24)(25) and used in human experimentation (26), and they have been demonstrated to have the desired effects of low reactogenicity, induction of high levels of effector antibodies, and optimized affinity maturation.…”