Mutagenesis to Orient Conjugation and Preserve Self-adjuvant Properties of Flagellin in Conjugates

Bacterial flagellins are unique for their capacity to activate both the innate and the adaptive immune response through a Toll-like receptor 5 (TLR5) signaling cascade. Used as a carrier protein in conjugate vaccines, it is crucial to preserve their self-adjuvant properties during the conjugation step. Considering the absence of cysteine in the Salmonella enterica flagellin FliC sequence, we have investigated the impact of five mutations (A2 C, K180 C, T240 C, D251 C and S306 C) alone or in combination on TLR5 activation. The FliC mutated at the four positions K180 C, T240 C, D251 C and S306 C displayed much the same activity as native flagellin whether the cysteine residues were free or conjugated. These results pave the way for the preparation of self-adjuvanting conjugate vaccines based on cysteine-mutated FliC as a carrier protein.