These findings will facilitate clinical trials of this vaccine candidate into a useful, safe, non-replicating, parental vaccine against RVs. Acknowledgments We thank Enqi Huang for preparation of the vaccines, Sherrie Clark-Deener, Kevin Pelzer, Julie Settlage and Amy Rizzo for veterinary services, Karen Hall, Rachel McNeil for animal care. feces, and significantly lower fecal cumulative consistency scores in post-challenge day (PCD) 1C7 among vaccinated pigs compared to the mock immunized controls. The P24-VP8* vaccine was highly immunogenic in Gn pigs. It induced strong VP8*-specific serum IgG and Wa-specific virus-neutralizing antibody responses from post-inoculation day 21 to PCD 7, but did not induce serum or intestinal IgA antibody responses or a strong effector T cell response, which are consistent with the immunization route, the adjuvant used, and MK-0812 the nature of the non-replicating vaccine. The findings are highly translatable and thus will facilitate clinical trials of the P24-VP8* nanoparticle vaccine. expression system. The distal surface of each P domain, corresponding to the outermost surface of the P particle, contains three surface loops, which can tolerate large sequence insertions. Based on this concept, a nanoparticle vaccine was developed by inserting the HRV VP8* antigen into the loop sections of the P domains. The P24-VP8* nanoparticle consists of a 24-valent core of NoV P particle and 24 surface-displayed HRV VP8*s. The P24-VP8* nanoparticle shares the features of the P24 particle in self-formation, easy production, and high stability over a wide range of temperatures [30]. Efficacy studies in mice revealed that the P24-VP8* nanoparticle vaccine is highly immunogenic and capable of inducing a significantly higher VP8* specific antibody response as compared with free VP8* particles even without adjuvant [30]. The main objectives of this study were to assess the immunogenicity and protective efficacy of a novel P24-VP8* nanoparticle vaccine using the gnotobiotic (Gn) pig model of human rotavirus infection and disease. The Gn pig model of HRV (Wa, G1P [8]) infection and diarrhea has been well established and used in the pre-clinical evaluation of HRV vaccine efficacies [31]. No other conventional lab animals develop diarrhea after HRV inoculation [32]. Pigs are genetically, physiologically, anatomically, and immunologically similar to humans [33,34,35], allowing data from Gn pigs to be translated to humans. The immunogenicity and protective efficacy of the P24-VP8* nanoparticle vaccine were determined using the Gn pig model of HRV infection and disease. High serum IgA, IgG, and virus-neutralizing (VN) antibody titers, as well as HRV-specific IFN- producing T cells, have been correlated with protection from HRV infection and disease, and data has been demonstrated to be comparable in Gn pigs and human studies [24,36,37]. 2. Materials and Methods 2.1. Human Rotavirus The virulent HRV (VirHRV) inoculum consisted of a pool composed of intestinal contents collected from the 27th passage in Gn pigs of the Wa strain HRV based MK-0812 on successive passages carried out in Gn pigs. A total of 1 1 105 fluorescent focus-forming units (FFUs) of VirHRV were diluted in 5 mL of Diluent #5 [minimal essential media (MEM, ThermoFisher Scientific); 100 IU of penicillin per mL, 0.1 mg of dihydrostreptomycin per ml; MK-0812 and 1% HEPES] for the inoculation of each Gn pig. The median infectious dose (ID50) and median diarrhea dose (DD50) of the VirHRV in Gn pigs were determined as approximately 1 FFU [38]. The cell culture-adapted HRV Wa strain (AttHRV), derived from the 35th passage in African green monkey kidney cells (MA104, ATCC# CRL-2378.1) [38,39], were used as the positive control for the assessment of RV antigens in feces using enzyme-linked immunosorbent assay (ELISA). The origination and passage history of the VirHRV and AttHRV have been explained by Wentzel et al. [40]. 2.2. Vaccine The P24-VP8* vaccine was comprised of 200 g of P24-VP8* proteins and 600 g Aluminum hydrogel (Al(OH)3) adjuvant. The vaccine was stored at 4 C (up to 8 months) until administered to Gn pigs (Supplementary Figure S1). The dosage of the P24-VP8* vaccine was selected based on a similar VP8* molar amount of the P2-VP8 subunit vaccine used in the clinical trial [19,27,29]. The VP8* region used Rabbit polyclonal to RAB14 in this vaccine was designed.