Characterization of the biological activity of a panel of TLR5 agonist

BIEDMA, ME; MORENO G; ROSSI AH; TABAREAU, J; BERGUER P; SIRARD, JC; RUMBO M

Buenos Aires

Congreso; I Meeting LASID-FAIC-SAI; 2015

Flagellin (FliC) activatesinnate immune responses via its recognition by Toll-like receptor 5(TLR5) and adaptive immunity in the host. TLR5 is areceptor widely distributed in mucosal epithelia and FliC, has beenproposed as mucosal adjuvant. The structure of FliC includes fourdomains, D0 and D1 are involved in TLR5 activation, whereas D2 and D3contain the main antigenic sites of the molecule. However, thecontribution of D2 and D3 to TLR5 activation has not been fullyelucidated. In this study, we evaluated 11 different deletionvariants of FliC affecting D2 and D3 domains using wild type FliC as reference. We found that the deletions affect differentially the capacity totrigger TLR5 in vitro. Circular dichroism (CD) analysisindicates that FliC mutants conserved mostly the secondary structurepresent in the wt molecule and underwent a reversible thermaltransition between 31 and 43 °C. The deletions in D2-D3 domainsresulted in low capacity to elicit anti-flagellin antibodies,however, all variants were able to trigger in vivo innateresponse activation at dose of 1 ug. Furthermore, to characterize theadjuvant properties, we assessed antibodies responses to OVAfollowing i.n. co-immunizations. Recombinant FliCs present mucosaladjuvant activity that correlates with TLR5 activating capacity. Thehighest adjuvant capacity was shown by variants which have a potencyto trigger TLR5 activation comparable to flagellin wt. Ourresults indicate that flagellin deletion variants showing maximalTLR5 activating capacity have the most promising features to be usedas mucosal adjuvants.