2D). The adjuvant activity of the cleavage product NSP4(112–175) was tested using KLH. Similar to full-length NSP4, either 10 μg or 20 μg of the cleavage product NSP4(112–175) enhanced KLH-specific serum IgG (5-fold) and fecal IgA (30-fold) (Fig. 3A and B) to levels higher than those observed in mice that received KLH alone (p < 0.05, Mann–Whitney U). As both doses induced equivalent antibody titers we chose the lowest dose to perform the subsequent experiments. These data indicate that the adjuvant domain of this protein is located in the C-terminus of NSP4 and that 10 μg of the cleavage product is optimal to elicit this effect. To test whether NSP4 from other rotavirus strains besides

the simian SA11 Cl3 NSP4 can also function as adjuvants, we tested the adjuvant activity of NSP4 from Anti-cancer Compound Library in vivo both a virulent and tissue culture-attenuated pair of porcine

rotavirus strains, OSU-v and OSU-a, respectively. As shown in Fig. 4, both OSU-a (GMT = 14,703) and OSU-v (GMT = 14,703) NSP4 induced an enhanced (8-fold increase) TT-specific serum, but not fecal, antibody response compared to the group receiving TT antigen alone. In addition, the levels of antibody induced by OSU-a and OSU-v NSP4 were similar to that induced by SA11 Cl3 NSP4. We next determined if NSP4, localized within a VLP, retained adjuvant activity. NSP4(112–175) was genetically fused to the inner core protein VP2 and when co-expressed with VP6 in insect cells VLPs (NSP4-2/6) were produced which were morphologically

indistinct from 2/6 VLP (Fig. 5A). Significantly increased (12-fold) TT-specific serum antibody was induced Adriamycin in the group of mice that received NSP4-2/6 intranasally with TT (GMT = 1838) compared to the TT alone group (GMT = 159) (Fig. 5B). In addition, despite the inability of the soluble NSP4 to enhance humoral response against TT, NSP4 internalized within 2/6-VLPs elicited significantly increased fecal IgA levels (p ≤ 0.05) compared to the co-administered antigen ( Fig. 5C). This adjuvant effect was due to the presence of NSP4 since 2/6 VLPs given with TT did not increase antigen-specific antibody responses and the level of antibody was comparable to the group receiving second TT alone In this study we demonstrated the mucosal adjuvant activity of rotavirus nonstructural protein NSP4 using model antigens. Full-length NSP4 from the SA11 rotavirus strain as well as a cleavage product NSP4 (112–175) were able to function as intranasal adjuvants and enhanced both serum and mucosal antibody responses specific to the co-administered antigen. In addition, an attenuated NSP4 from an avirulent porcine OSU-a rotavirus as well as NSP4 delivered inside a rotavirus VLP can efficiently enhance antigen-specific antibody responses. The adjuvant property of NSP4 varied depending upon the co-administered antigen suggesting that the outcome of adjuvanticity is affected by the nature of the antigen tested.