(752e) Cell Free Production of Flagellin As a Vaccine Adjuvant and Its Ordered Display On Virus-Like Particles

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this report, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. Especially when flagellin is urgently needed for new and highly potent vaccines at short notice, for example, to combat pandemic influenza threats, CFPS can provide such a means for rapid, cost-effective and high-level production of protein products in a few hours. The synthesized flagellin product had high affinity for the Toll-Like Receptor 5 (hTLR5) (EC50 = 2.4 ± 1.4 pM); approximately 10 to 50 fold higher than previously reported. Observations from protein purification, SDS-PAGE, alanine scanning and multiple mutations indicated that the C-terminal D0 domain of flagellin has disordered tertiary structure, making it susceptible to proteolytic degradation. This proteolysis could be reduced by protease inhibitors, the use of protease-deficient cell extracts, or deletion of the flagellin D0 domain.  

   We hypothesized that the TLR5 receptor activation could be enhanced if flagellin were presented in an ordered array on the surface of a virus-like particle (VLP). We incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particles (VLPs) using bioorthogonal azide-alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold.

    In this study, we set out to produce more potent vaccine designs.  Unexpectedly, we also gained an important insight into our innate immune systems. Cell-free protein synthesis overcame serious proteolytic challenges, and the intact flagellin then exhibited unexpectedly high affinity to the toll-like receptor, TLR5. It stimulated cellular responses at 2 pM (10^-12M) concentrations, at least a thousand fold lower than for most biological interactions.  When attached to VLPs, the flagellin was even more effective. When bacteria invade our bodies, they find an extremely hospitable environment.  In order to overcome this threat, our immune systems must respond very rapidly and very strongly.  Our measurements indicate that immune cells can detect incredibly low concentrations of flagellin so they can quickly mobilize to win the battle.