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

Flagellin has been shown to be a highly potent adjuvant through TLR5 in a number of innate and adaptive immune cells [19] . To interpret different S0-specific IgG outcomes among various groups receiving the flagellin adjuvant, we used the epithelial cell line Caco-2, which expresses TLR5 constitutively (Supplementary Fig. 3 F-S0 and F effectively induced IL-8 more significantly than S0 alone at 10 and 100 nM. At 1 nM, treatment with F induced IL-8 more significantly than treatment with S0-F or F-S0. Similar patterns were observed for MCP-1 (Fig. 5B) . There was no evident difference in the ability of S0-F and F-S0 to induce IL-8 or MCP-1. For more in-depth analysis, we verified each recombinant protein's ability to activate the TLR5 pathway in vivo. Mice were injected intraperitoneally with equivalent molar values of each recombinant protein, and sera were analyzed by ELISA to detect IL-6 as a marker of TLR5 activation. The data revealed that the F treatment induced stronger serum IL-6 levels when compared to S0-F or F-S0 treatments at 4 h (Fig. 6A) . Consistent with the in vitro data, S0-F and F-S0 had a tendency to induce higher levels of serum IL-6 than S0, but the levels were lower than for F. TLR5 stimulation profile of each recombinant protein was also confirmed by TLR5 reporter system and similar tendency was once again confirmed (Supplementary Fig. 2 Flagellin is recognized not only by the transmembrane receptor TLR5 on the cell surface but also by the cytosolic receptor NLRC4/NAIP5 [20] . Flagellin's adjuvant effect on the humoral immune response can be influenced by activation of both receptors [21, 22] . Therefore, we also sought to determine the ability of each recombinant protein to activate the cytosolic receptor NLRC4/NAIP5 by measuring its responsive cytokine IL-18 from sera. Interestingly, the S0-F-injected group had serum IL-18 levels that were significantly higher than all other groups at 1 h (Fig. 6B) . Similar patterns were also observed at 4 h.