Studies of human rotavirus candidate non-replicating vaccines and innate immunity in a gnotobiotic pig model of human rotavirus disease

Rotavirus is the major cause of severe dehydrating diarrhea in children and young infants worldwide. The mortality rates reach 600,000 annually, mainly in developing countries and vaccination is an important preventive measure. The first two objectives of my PhD research were to produce and test a combination of replicating and non-replicating human rotavirus (HRV) vaccines or non-replicating HRV vaccines in the gnotobiotic pig model to minimize or avoid the use of more reactogenic live HRV vaccines. The third objective was to assess the mucosal and systemic dendritic cell responses after RV infection because these responses are largely uncharacterized but are important in understanding immunity induced after infection and for design of vaccines. The neonatal gnotobiotic pig is susceptible to HRV for more than 8 weeks and their gnotobiotic status assures that wild type rotavirus infection does not occur during vaccination. Additionally gnotobiotic pigs are optimal for the study of innate immune responses to HRV in-vivo by excluding any confounding factors (e.g. commensal flora, other pathogens etc). For the first objective, gnotobiotic pigs were vaccinated priming with a peroral (PO) live attenuated human rotavirus (AttHRV) and boosting (2x) with a non-replicating 2/6 virus-like particles (VLPs) intranasally (IN) using ISCOM as adjuvant. High protection rates against diarrhea and shedding (71%) were induced which coincided with higher IgA antibody titers in small intestinal contents and serum virus neutralizing (VN) antibody responses. In contrast, vaccination with 2/6VLP alone conferred no protection against diarrhea or shedding suggesting that neutralizing antigens, VP4 and VP7 were needed as part of a non-replicating vaccine formulation to induce protective immune responses in neonatal pigs. Consequently the second objective was to test a non-replicating vaccine that included RV neutralizing antigens. A combination of semipurified VP4 and 2/6/7VLP PO followed by VP4+2/6VLP IN using ISCOM as adjuvant was tested. A 67% protection rate against diarrhea and 33% protection rate against shedding were elicited with high to moderate numbers of IgA antibody secreting cell responses in the gut but low VN antibody titers in serum. Vaccination with 2/6/7VLP PO and 2/6VLP IN (that lacked the semipurified VP4) conferred low protection rates (33% against diarrhea and no protection against shedding) suggesting that VP4 was an essential component of a non-replicating rotavirus vaccine. This study confirmed that RV neutralizing antigens are needed for a non-replicating HRV vaccine formulation to induce protective immune responses in neonatal pigs. Additionally, to improve protection rates against shedding, VN titers likely need to be enhanced by giving more potent adjuvants and/or a higher vaccine concentrations. For the third objective, viral dose effects on DCs ex-vivo and the association with clinical outcome were examined in gnotobiotic pigs after a high or low dose of HRV. We assessed intestinal and splenic activated (CD80/86+) IFNalpha, IL-12, IL-10, IL-6 and TNFalpha producing DCs and their uptake/binding of fluorescent 2/4/6/7VLPs by flow cytometry and serum/intestinal cytokines by ELISA. Because infection with HRV induced mainly intestinal plasmacytoid DCs (pDCs), we studied membrane bound TGFbeta1-latency associated peptide (LAP) CD4+ regulatory T cells known to be induced by pDCs. At post-inoculation day (PID) 2, a high HRV dose induced significantly lower frequencies of intestinal activated IFNalpha + pDCs (and lower IL-12, IL-6 and TNFalpha+ pDCs) than a low dose. The frequencies of intestinal IFNalpha+ pDCs correlated with serum IFNalpha concentrations (r=0.73 p<0.01) suggesting that the pDCs were activated in-vivo. Furthermore a high HRV dose induced lower uptake/binding of 2/4/6/7VLP-GFP by intestinal and splenic pDCs and lower frequencies of circulating membrane bound TGFbeta LAP+ CD4+(SWC3-CD8-), T cells compared to a lo...