Figure 1.
Vitamin A deficient control groups had higher fecal diarrhea scores and fecal HRV shedding post-virulent HRV challenge.
Mean HRV fecal shedding (A,B) and fecal scores (C,D) post-virulent HRV challenge in vaccinated and control vitamin A deficient (VAD) and sufficient (VAS) groups. Different lower-case letters indicate statistically significant difference (one-way ANOVA followed by Duncan’s multiple range test, p ≤ 0.05) among the treatment groups belonging to VAD and VAS pigs at the same time-point. Bars at each time-point indicate standard error of the means. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.
Figure 2.
Vitamin A status affected HRV IgA antibody responses post-virulent HRV challenge.
Geometric mean serum (A) and intestinal (B) HRV IgA antibody titers and mean intestinal HRV IgA antibody secreting cells (C,D) in vitamin A deficient (VAD) and sufficient (VAS) gnotobiotic pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation at pre- (PID26/PCD0) and post (PID36/PCD10)-HRV challenge time-points. Data shown as mean values ± standard error of the mean. Bars with different lower-case letters for HRV IgA antibody titers (A,B) at each time-point (pre- or post-challenge) differ significantly between groups (Duncans multiple range t test on log10 transformed data, p ≤ 0.05). Significant differences between groups for HRV IgA ASC are indicated by capped lines as determined by non-parametric Kruskal-wallis rank sum test (p ≤ 0.05). The arrow (C) indicates increased duodenal IgA ASC in Vac+VitA group compared to Vac group for both VAD and VAS pigs at PID26/PCD0 (pre-challenge). Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only. Pre-challenge: n=8-13 (serum IgA antibodies), 3-7 (intestinal IgA antibodies and ASC), Post-challenge: n=4-7 (serum and intestinal IgA antibodies and ASC).
Figure 3.
Vitamin A deficient vaccinated groups had lower HRV IgG antibody responses post-virulent HRV challenge.
Geometric mean serum HRV IgG (A) titers in vitamin A deficient (VAD) and sufficient (VAS) gnotobiotic pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation at pre- (PID26/PCD0) and post (PID36/PCD10)-HRV challenge time-points. Data shown as mean values ± standard error of the mean. Bars with different lower-case letters for HRV IgG antibody titers (A) at each time-point (pre- or post-challenge) differ significantly between groups (Duncans multiple range t test on log10 transformed data, p ≤ 0.05). (B) shows the HRV IgG1:IgG2 ratios for different treatment groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only. Pre-challenge: n=8-13, Post-challenge: n=4-7.
Figure 4.
Vaccinated VAS groups had higher frequencies of B cells in ileum (pre-challenge) and duodenum (post-challenge).
Mean frequency of B lymphocytes (CD21+CD3-) among mononuclear cells in vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation in ileum (A), duodenum (B), blood (C) and spleen (D) at pre- (PID26/PCD0) and post (PID36/PCD10)-virulent HRV challenge. Bars represent mean values and standard error of the mean. Significant differences between groups are indicated by capped lines as determined by non-parametric Kruskal-wallis rank sum test (p ≤ 0.05). The dotted rectangles (A) indicate trend for higher frequencies of B cells in vaccinated VAS groups compared to vaccinated VAS groups at pre-challenge. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only, Pre = pre-challenge (number), Post = post-challenge (number). Pre-challenge: n= 3-7, Post-challenge: n=4-7.
Figure 5.
Vitamin A sufficient vaccinated groups had higher intestinal and systemic T regulatory cells post-virulent HRV challenge.
Mean frequencies CD3+CD4+ (A) and CD3+CD8+ (B) T cells among mononuclear cells (MNCs) and CD4+CD25+Foxp3+ (C,D) among CD4 T cells in vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation in blood, spleen, ileum and duodenum post-virulent HRV challenge Bars represent mean values and standard error of the mean. Significant differences between groups are indicated by capped lines as determined by non-parametric Kruskal-wallis rank sum test (p ≤ 0.05). Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.
Figure 6.
Vitamin A deficiency induced pro-inflammatory immune responses.
Mean concentrations (± SEM) of IL12 (A,D; Th1), IFNγ (B,E; Th1) and IL10 (C,F; anti-inflammatory) cytokines in serum of vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation. Data shown as mean values ± standard error of the mean. Different lower-case letters indicate statistically significant difference (non-parametric Kruskal-wallis rank sum test, p ≤ 0.05) among the treatment groups belonging to VAD and VAS pigs at the same time-point for each cytokine. The circles (C,F) indicate 2-3 fold higher IL10 in VAS control groups compared to VAD control groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.
Figure 7.
Summary of innate and Th17 cytokine responses.
Mean concentrations (± SEM) of IFNα (A,D; innate), IL8 (B,E; innate) and IL17 (C,F; Th17) cytokines and chemokines in serum of vitamin A deficient (VAD) and sufficient (VAS) pigs vaccinated with AttHRV vaccine or placebo with or without vitamin A supplementation. Data shown as mean values ± standard error of the mean. Different lower-case letters indicate statistically significant difference (non-parametric Kruskal-wallis rank sum test, p ≤ 0.05) among the treatment groups belonging to VAD and VAS pigs at the same time-point for each cytokine. The circle (f) indicates increase in IL17 in vaccinated VAS groups compared to VAS control groups. Vac = 3X AttHRV vaccinated only, Vac+VitA = 3X AttHRV vaccinated + 100,000 IU of vitamin A, Ctrl = non-vaccinated and non-vitamin A supplemented, Ctrl+VitA = 3X 100,000 IU of vitamin A only.
Figure 8.
Summary of pilot data for different regimens for vitamin A supplementation.
Geometeric mean titers of serum (A) and intestinal (B) HRV IgA antibody titers and mean intestinal HRV IgA antibody secreting cells (C) in AttHRV vaccinated pigs with supplemented of 50,000 IU for 11 days (Vac+VitA multiple doses, n=2) or 100,000 IU at each vaccine time-point (Vac+VitA, n=2). Data shown as mean values ± standard error of the mean. Arrows in a, b, c and d indicate increase in these parameters for Vac+VitA multiple dose compared to Vac+VitA group. Vac+VitA = 3X AttHRV vaccinated + supplemental vitamin A. No statistical analysis was done because of low numbers of pigs tested per treatment in this pilot study.