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, mortality rates reach 600,000 annually and vaccination is an important preventive measure. For the first objective, gnotobiotic pigs were vaccinated priming with a peroral (PO) live attenuated human rotavirus (AttHRV) and boosting (2x) with 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. Vaccination with 2/6VLP alone conferred no protection 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. 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 moderate numbers of intestinal IgA antibody secreting cells but low VN antibody titers in serum. This study confirmed that RV neutralizing antigens are needed for a non-replicating HRV vaccine formulation to induce protective immune responses in neonatal pigs. For the third objective, dendritic cells (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 cytokine producing DCs, their uptake/binding of fluorescent 2/4/6/7VLPs and membrane bound TGFbeta1-latency associated peptide (LAP) CD4+ regulatory T cells known to be induced by pDCs using flow cytometry. At post-inoculation day (PID) 2, only the high dose pigs developed diarrhea and induced significantly lower frequencies of intestinal IFNalpha+ pDCs, lower uptake/binding of 2/4/6/7VLP-GFP by intestinal and splenic pDCs and lower frequencies of regulatory T cells compared to a lower dose. Titers of infectious virus shed were similar. Cell-damage byproducts are known to inhibit pDCs. Higher early rates of diarrhea, possibly associated with enterocyte damage byproducts may decrease pDC function thereby preventing induction of regulatory T cells and facilitating adaptive immune responses to HRV.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:rotavirus non replicating vaccines porcine dendritic cell
Date of Publication:01/01/2007