Baseline Serum Vitamin A and D Levels Determine Benefit of Oral Vitamin A&D Supplements to Humoral Immune Responses Following Pediatric Influenza Vaccination

Abstract

Maximizing vaccine efficacy is critical, but previous research has failed to provide a one-size-fits-all solution. Although vitamin A and vitamin D supplementation studies have been designed to improve vaccine efficacy, experimental results have been inconclusive. Information is urgently needed to explain study discrepancies and to provide guidance for the future use of vitamin supplements at the time of vaccination. We conducted a randomized, blinded, placebo-controlled study of influenza virus vaccination and vitamin supplementation among 2 to 8 (inclusive) year old children over three seasons, including 2015-2016 (n = 9), 2016-2017 (n = 44), and 2017-2018 (n = 26). Baseline measurements of vitamins A and D were obtained from all participants. Measurements were of serum retinol, retinol-binding protein (RBP, a surrogate for retinol), and 25-hydroxyvitamin D (25(OH)D). Participants were stratified into two groups based on high and low incoming levels of RBP. Children received two doses of the seasonal influenza virus vaccine on days 0 and 28, either with an oral vitamin supplement (termed A&D; 20,000 IU retinyl palmitate and 2000 IU cholecalciferol) or a matched placebo. Hemagglutination inhibition (HAI) antibody responses were evaluated toward all four components of the influenza virus vaccines on days 0, 28, and 56. Our primary data were from season 2016-2017, as enrollment was highest in this season and all children exhibited homogeneous and negative HAI responses toward the Phuket vaccine at study entry. Responses among children who entered the study with insufficient or deficient levels of RBP and 25(OH)D benefited from the A&D supplement (p < 0.001 for the day 28 Phuket response), whereas responses among children with replete levels of RBP and 25(OH)D at baseline were unaffected or weakened (p = 0.02 for the day 28 Phuket response). High baseline RBP levels associated with high HAI titers, particularly for children in the placebo group (baseline RBP correlated positively with Phuket HAI titers on day 28, r = 0.6, p = 0.003). In contrast, high baseline 25(OH)D levels associated with weak HAI titers, particularly for children in the A&D group (baseline 25(OH)D correlated negatively with Phuket HAI titers on day 28, r = -0.5, p = 0.02). Overall, our study demonstrates that vitamin A&D supplementation can improve immune responses to vaccines when children are vitamin A and D-insufficient at baseline. Results provide guidance for the appropriate use of vitamins A and D in future clinical vaccine studies.

Keywords: antibody response; baseline; influenza virus vaccine; pediatric; supplement; vitamins A and D.

Figure 1

Estimated fold change in hemagglutination inhibition (HAI) responses compared between placebo (black) and vitamin A and D supplement (A&D, red) study groups at different time points of the study in season 2016–2017. Participants were placed into one of four groups based on vitamin levels. Cut-offs for sufficiency (termed “high”) were ≥ 22,000 ng/mL for retinol-binding protein (RBP) and ≥ 30 ng/mL for 25(OH)D. Groups were “Low A/Low D”, n = 10; “High A/Low D”, n = 12; “Low A/High D,” n = 11; “High A/High D,” n = 11 (see Table 2). For each of the four viruses, the GEE model was constructed with log2-transformed HAI titers as the response, age, race, and a three-way interaction among time, incoming vitamin levels, and study groups as covariates, and the first order autoregressive (AR1) as the working correlation structure. Estimated mean fold changes are plotted with 90% confidence intervals. p-values were obtained from post hoc comparisons from GEE models (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Relationships between baseline vitamin levels and HAI responses in season 2016–2017. Spearman correlation coefficients are plotted for comparisons between baseline vitamin levels and HAI titers among participants enrolled during the 2016–2017 season. R values are plotted on the Y axis in A–D. Positive correlations are indicated in green and negative correlations are indicated in red. The top row shows RBP correlations with HAI titers in placebo (A) and A&D (B) groups. Specifically, correlations are shown between baseline RBP and day 0 HAI, baseline RBP and day 28 HAI, baseline RBP and day 56 HAI, baseline RBP and peak HAI, baseline RBP and changes in HAI between days 0 and 28, and baseline RBP and changes in HAI between days 0 and 56. * p < 0.05; ** p < 0.01; *** p < 0.001. The middle row substitutes 25(OH)D for RBP in placebo (C) and A&D (D) groups. In the bottom row, detailed correlative data are shown for RBP versus B/Phuket/3073/13 HAI titers on day 28 in the placebo group (n = 22, some values overlap) (E), and 25(OH)D versus B/Phuket/3073/13 HAI titers on day 28 in the A&D group (n = 22, some values overlap) (F).