Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model

<div><p>Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.</p> </div

Vaccinated VAS groups had higher frequencies of B cells in ileum (pre-challenge) and duodenum (post-challenge).

<p>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 (<i>p</i> ≤ 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.</p

Vitamin A status affected HRV IgA antibody responses post-virulent HRV challenge.

<p>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, <i>p</i> ≤ 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 (<i>p</i> ≤ 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).</p

Vitamin A deficient vaccinated groups had lower HRV IgG antibody responses post-virulent HRV challenge.

<p>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, <i>p</i> ≤ 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.</p

Summary of pilot data for different regimens for vitamin A supplementation.

<p>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.</p

Summary of innate and Th17 cytokine responses.

<p>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<i>, p</i> ≤ 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.</p

Vitamin A deficiency induced pro-inflammatory immune responses.

<p>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<i>, p</i> ≤ 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.</p

Skin Vaccination against Rotavirus Using Microneedles: Proof of Concept in Gnotobiotic Piglets.

Live-attenuated oral rotavirus (RV) vaccines have lower efficacy in low income countries, and additionally are associated with a rare but severe adverse event, intussusception. We have been pursuing the development of an inactivated rotavirus vaccine (IRV) using the human rotavirus strain CDC-9 (G1P[8]) through parenteral immunization and previously demonstrated dose sparing and enhanced immunogenicity of intradermal (ID) unadjuvanted IRV using a coated microneedle patch in comparison with intramuscular (IM) administration in mice. The aim of this study was to evaluate the immune response and protection against RV infection and diarrhea conferred by the administration of the ID unadjuvanted IRV using the microneedle device MicronJet600® in neonatal gnotobiotic (Gn) piglets challenged with virulent Wa G1P[8] human RV. Three doses of 5 μg IRV when administered intradermally and 5 μg IRV formulated with aluminum hydroxide [Al(OH)3] when administered intramuscularly induced comparable rotavirus-specific antibody titers of IgA, IgG, IgG avidity index and neutralizing activity in sera of neonatal piglets. Both IRV vaccination regimens protected against RV antigen shedding in stools, and reduced the cumulative diarrhea scores in the piglets. This study demonstrated that the ID and IM administrations of IRV are immunogenic and protective against RV-induced diarrhea in neonatal piglets. Our findings highlight the potential value of an adjuvant sparing effect of the IRV ID delivery route