Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses

Fig. 1.. In vitro characterization of rRRV-HuNoV-VP1. (A) Electrophoretic analysis of viral dsRNA. Viral dsRNAs were purified from rRRV and different passages of rRRV-HuNoV-VP1, separated by RNA-PAGE, and visualized by silver staining. The modified gene segment 7 is indicated by a red asterisk. (B and C) Growth kinetics of rRRV and rRRV-HuNoV-VP1 in MA104 cells. MA104 cells were infected with either rRRV or rRRV-HuNoV-VP1 at an MOI of (B) 1 FFU/cell or (C) 0.01 FFU/cell. The samples were frozen at each indicated time point and the virus titers were determined by a focus forming unit (FFU) assay. Representative data of two independent experiments with triplicate samples are shown as mean with SD. Statistical analysis was performed by Student’s t test for each time point. Statistical significance is indicated as *P < 0.05, **P < 0.01. (D) Immunostaining analysis of protein expression by rRRV-HuNoV-VP1 in MA104 cells. MA104 cells were infected with the rRRV or rRRV-HuNoV-VP1 at MOI of 1 FFU/cell and fixed at 24 h postinfection, or the cells were transfected with pCAG-HuNoV-VP1 plasmid and fixed at 3 d posttransfection. The cells were stained with antibodies specific to RV-VP6 (red),…

Fig. 2.. In vivo characterization of rRRV-HuNoV-VP1. (A and C) Diarrhea rate by rRRV and rRRV-HuNoV VP1 in (A) 129sv or (C) Stat1−/− mice. Five-day-old 129sv pups were orally inoculated with rRRV (n = 8) or rRRV-HuNoV-VP1 (n = 8) (3.9 × 105 FFU/pup), and 5-d-old Stat1−/− pups were orally inoculated with rRRV (n = 7) or rRRV-HuNoV-VP1 (n = 8) (3.9 × 105 FFU/pup). Diarrhea was monitored daily following gentle abdominal palpation until 10 d postinfection. Pups were regarded as positive for diarrhea if liquid or unformed stools were observed. The daily percentage of diarrhea is shown. (B and D) Fecal shedding curve in (B) 129sv or (D) Stat1−/− mice after rRRV or rRRV-HuNoV-VP1 inoculation. Fecal RV antigens in stools collected from 1 to 10 d postinfection were measured by ELISA. Data are shown as mean scores of net optical density at 450 nm (OD450) with SD. The dotted lines show background signals measured by stools from uninfected pups. Statistical analysis was performed with two-way ANOVA. Statistical significance is indicated as n.s.: not significant.

Fig. 3.. Systemic antibody responses against RV and HuNoV VLPs in 129sv and Stat1−/− mouse following rRRV-HuNoV-VP1 infection. (A) Schematic presentation of the infection and immunization experiments. Five-day-old 129sv (n = 6) or Stat1−/− (n = 11) pups were orally inoculated with rRRV-HuNoV-VP1 (3.9 × 105 FFU/pup) at 0 WPI and intraperitoneally boostered with the virus (3.9 × 105 FFU/pup) at 9 WPI. Blood and stool samples were collected at 4, 6, 8, and 10 WPI. (B and C) Temporal dynamics of serum IgG titers against RV and HuNoV VLP. Mouse sera were serially diluted and tested in ELISA for (B) anti-RV IgG or (C) anti-HuNoV VLP. The IgG titer was defined as the highest serum dilution at which the OD score is higher than an uninfected mouse serum. Data are shown as mean with SD. Statistical analysis was performed by two-way ANOVA. Statistical significance is indicated as **P < 0.01. (D) Serum neutralization activities against RV. Mouse sera were serially diluted, mixed with RRV, and incubated at 37 °C for 1 h. The virus titer in the mixture of the serum and RRV was determined using MA104 cells in an FFU assay. Data are shown as mean scores of the percentage reduction of the focus number…

Fig. 4.. Mucosal antibody responses against RV and HuNoV VLPs in 129sv and Stat1−/− mouse following rRRV-HuNoV-VP1 infection. (A and B) Temporal dynamics of fecal IgA responses to RV. The amount of fecal IgA against RV in (A) 1% (w/v) of fecal suspension from 129sv or (B) 0.1% (w/v) of fecal suspension from Stat1−/− was determined by ELISA. Data are shown as the mean score of the net OD450 with SD. The dotted lines show the limit of detection by stools from uninfected pups. A stool with a higher OD score than the dotted line was considered positive. The number of positive stools and total stools are shown below the graphs. (C and D) Temporal dynamics of fecal IgA responses to HuNoV VLP. The amount of fecal IgA against HuNoV VLP in 10% (w/v) of fecal suspension from (C) 129sv or (D) Stat1−/− pups were determined by ELISA. Data are shown as the mean score of net OD450 with SD. The dotted lines show background signals by stools from uninfected pups. A stool with a higher OD score than the dotted line was considered positive. The number of positive stools and total stools are shown below the graphs. (E) Fecal neutralization activities against RV. Mouse stool suspensions were serially diluted,…

Fig. 5.. Genome stability and antigenicity of a recombinant RRV expressing HuNoV P protein. (A) Electrophoretic analysis of viral dsRNA. Viral dsRNAs were purified from rRRV and different passages of rRRV-HuNoV-P, separated by RNA-PAGE, and visualized by silver staining. The modified gene segment 7 is indicated by a red asterisk. (B) Temporal dynamics of serum IgG titer against HuNoV P particle. Mouse sera were serially diluted and tested in ELISA for anti-HuNoV P particle. The IgG titer was defined as the highest serum dilution at which the OD score is higher than an uninfected mouse serum. Data are shown as mean with SD. Statistical analysis was performed by two-way ANOVA. Statistical significance is indicated as *P < 0.05. (C) Temporal dynamics of fecal IgA responses to HuNoV P particle. The amount of fecal IgA against HuNoV P particle in 10% (w/v) of fecal suspension from 129sv or Stat1−/− pups were determined by ELISA. Data are shown as the mean scores of net OD450 with SD. The dotted lines show background signals by stools from uninfected pups.