Rationally Designed TLR4 Ligands for Vaccine Adjuvant Discovery

Abstract

Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A) are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4) activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD). The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs), and human monocyte-derived dendritic cells (DCs). This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates.

Bordetella pertussis Whole Cell Immunization, Unlike Acellular Immunization, Mimics Naïve Infection by Driving Hematopoietic Stem and Progenitor Cell Expansion in Mice

“…We hypothesize that the use of adjuvants that induce IFN responses and/or TLR4 signaling will enhance ACVs by influencing HSPC priming and expansion. Our future work with the use of rationally designed, functionally diverse lipid A adjuvants ( 63 ) will provide answers as to how induction of HSPC expansion in ACV-immunized mice may contribute to downstream immune responses. Once we have elucidated how these adjuvants may enhance vaccine-induced immunity in mice and other animal models, we expect that this knowledge will lead to improved ACV vaccines for use in humans.…”

Genomic and Phenotypic Diversity among Ten Laboratory Isolates of Pseudomonas aeruginosa PAO1

“…LPS was prepared from 1 liter of culture grown at 37°C in LB (with 1 mM MgCl 2 ) using a phenol-water extraction followed by removal of contaminating phospholipids, as previously described (75)(76)(77). TLR4 stimulation was conducted as previously described (76). Lipid A was prepared from the LPS preparations using mild acid hydrolysis as previously described (78).…”

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate

“…In fact, a chemically modified LOS, monophosphoryl lipid A (MPL®), that has diminished reactogenicity but potent adjuvanticity [28,29] is used as a vaccine adjuvant in GlaxoSmithKline papillomavirus (HPV) vaccine Cervarix®. Recently, rationally designed lipid A-based Toll-like receptor 4 (TLR4) ligands have been reported for vaccine adjuvant discovery and modulating the innate immune response [30,31]. Given both the potential use of core OS as a vaccine and a modified lipid A as a vaccine adjuvant, LPS structure elucidation capability to support these efforts is critical.…”