Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0

Oh, Seo-ho; Kim Cho, Young-Saeng; Lee, Ho-Bin; Lee, Sang-Mok; Kim, Whee-Soo; Hong, Liang; Cho, Chong-Su; Choi, Yun-Jaie; Kang, Sang-Kee

The cells of the innate immune system are equipped with two germline-encoded receptors to detect flagellin present on flagellated bacteria. The transmembrane receptor TLR5 detects extracellular flagellin, thus activating a number of innate and adaptive immune cells via methods such as secretion of proinflammatory cytokines. Injection of virulence factors containing flagellin from invading microbes results in flagellin being found within the cytosol of macrophages and being recognized by the cytosolic receptor NLRC4/NAIP5, which supports a stronger pathogen killing environment through IL-18, IL-1β and pyroptosis [20] . The role of these two receptors on, in particular, flagellin's adjuvant effect is suggested to be complementary, as a single knockout of either pathway did not interfere with the adjuvancy of flagellin, while a double-knockout resulted in a significant reduction of the adjuvancy of flagellin [21, 37] . However, the NLRC4/NAIP5 pathway may interfere with flagellin's adjuvancy in the presence of intact TLR5. Li W et al. [22] demonstrated that FliC-L3A flagellin that lacks the ability to activate NLRC4/NAIP5 due to a point mutation, induced a significantly higher antigen-specific serum IgG titer than wild-type flagellin when used as an adjuvant with the model antigen p24 in mice. A/J mice, which are naturally partially deficient in Naip5, displayed higher IgG responses to ovalbumin (OVA) than C57BL/6 mice after immunization with a mixture of OVA and flagellin [37] . Our data revealed that S0-F treatment induced significantly higher levels IL-18, a representative cytokine of the NLRC4/NAIP5 pathway, than F-S0 or F treatment in mice (Fig. 6B) , suggesting that the superior ability of S0-F to activate the NLRC4/NAIP5 pathway may interfere with flagellin's adjuvancy and reduce serum S0specific IgG despite its advantage in the conjugated form.