title: 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.
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA.
authors.forename: Ayesha
authors.initials: A
authors.lastname: Misquith
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA.
authors.forename: H W Millie
authors.initials: HW
authors.lastname: Fung
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA.
authors.forename: Quinton M
authors.initials: QM
authors.lastname: Dowling
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Guderian
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Vedvick
authors.affiliation: Infectious Disease Research Institute (IDRI), 1124 Columbia Street, Ste 400, Seattle, WA 98104, USA. Electronic address: cfox@idri.org.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Fox
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry* / Q000494:pharmacology*; D010316:Particle Size; D051197:Toll-Like Receptor 4 / Q000819:agonists*
chemical_list: D000276:Adjuvants, Immunologic; D051197:Toll-Like Receptor 4
keywords: Glucopyranosyl lipid adjuvant; In vitro bioactivity; Particle size; Physicochemical characterization; TLR4 agonist; Vaccine adjuvant formulation