Impact of Vitamin D Supplementation on Influenza Vaccine Response and Immune Functions in Deficient Elderly Persons: A Randomized Placebo-Controlled Trial

Abstract

Background: Immunosenescence contributes to reduced vaccine response in elderly persons, and is worsened by deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can both potentiate the innate immune response and inhibit the adaptive system, and so modulate vaccination response. Objective: This randomized placebo-controlled double-blind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response. Design: Deficient volunteers (Vit-D serum <30 ng/mL) were assigned (V1) to receive either 100,000 IU/15 days of cholecalciferol (D, n = 19), or a placebo (P, n = 19), over a 3 month period. Influenza vaccination was performed at the end of this period (V2), and the vaccine response was evaluated 28 days later (V3). At each visit, serum cathelicidin, immune response to vaccination, plasma cytokines, lymphocyte phenotyping, and phagocyte ROS production were assessed. Results: Levels of serum 25-(OH)D increased after supplementation (D group, V1 vs. V2: 20.7 ± 5.7 vs. 44.3 ± 8.6 ng/mL, p < 0.001). No difference was observed for serum cathelicidin levels, antibody titers, and ROS production in D vs. P groups at V3. Lower plasma levels of TNFα (p = 0.040) and IL-6 (p = 0.046), and higher ones for TFGβ (p = 0.0028) were observed at V3. The Th1/Th2 ratio was lower in the D group at V2 (D: 0.12 ± 0.05 vs. P: 0.18 ± 0.05, p = 0.039). Conclusions: Vit-D supplementation promotes a higher TGFβ plasma level in response to influenza vaccination without improving antibody production. This supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response. A deeper characterization of metabolic and molecular pathways of these observations will aid in the understanding of Vit-D's effects on cell-mediated immunity in aging. This clinical trial was registered at clinicaltrials.gov as NCT01893385.

Keywords: aging; cathelicidin; cytokine; influenza vaccination; leukocyte phenotyping; randomized trial; vitamin-D deficiency.

Figures

Figure 1

CONSORT flow chart. The flow of participants through the trial is represented by a diagram, as suggested by the CONSORT group. P group, Placebo supplementation; D group, Vit-D supplementation.

Figure 2

Vitamin D serum levels. V1, inclusion; V2, end of supplementation and vaccination; V3, 28 days post-vaccination. Data are expressed as mean ± SD. Statistical analysis was performed by two-way ANOVA for the supplementation and time effect followed by Bonferroni post hoc test (p < 0.05); *Significantly different between groups at the same visit (p < 0.05); a,b Significantly different between visits in the same group (p < 0.05).

Figure 3

Cathelicidin serum levels. V1, inclusion; V2, end of supplementation and vaccination; V3, 28 days post-vaccination. Data are expressed as mean ± SD. No statistical difference was observed by two-way ANOVA (p < 0.05).

Figure 4

Correlation between 25-(OH)D and cathelicidin serum levels. The relationship between cathelicidin and 25-(OH)D serum levels was estimated using Pearson correlation. (A) Correlation at inclusion (V1) for all volunteers (star P group, circle D group) (r = −0.24, p = 0.14). (B) Correlation before (V1: dark circle) and after the supplementation period (V2: light circle) for D group (r = −0.10, p = 0.53).

Figure 5

Post-vaccination seroconversion and seroprotection rates. (A) Seroconversion rate: percentage of subjects achieving at least a 4-fold increase or an increase from >10 to 40 in Ab titer for seronegative subjects; (B) Seroprotection rate: percentage of subjects reaching an Ab titer 40. *Significantly different between P and D group using Mann-Whitney U-test (p < 0.05).

Figure 6

HAI antibody titer response to inactivated influenza virus vaccine in seronegative and seropositive volunteers. HAI antibody titers stratified by pre- and post-vaccination status. (A) Antibody titers for H1N1 pandemic influenza A (A/California/7/2009, H1N1pdm09); (B) Antibody titers for H3N2 pandemic influenza seasonal influenza A (A/Texas/50/2012, H3N2); (C) Antibody titers for Yamagata seasonal influenza B (B/Massachusetts/2/2012, Yamagata lineage). Results are expressed as GMT (95% IC); a,bSignificantly different between pre- and post-vaccination titers in the same group using paired Wilcoxon test (p < 0.05); *Significantly different for a same period (pre- or post-vaccination) between P group and D group using Mann-Whitney U-test (p < 0.05).

Figure 7

Phenotyping of peripheral blood CD3+CD4+ T cells. (A) Gating strategy of a representative sample used to identify T helper cells (Th. CD3+CD4+), Th1 cells (CD4+, CD 183+), Th2 cells (CD4+,CD294+), Th17 cells (CD4+, CD196+), and Treg cells (CD25+, CD127−); (B) Proportion from the different T helper and Treg cells in D group compared to controls. V1, inclusion; V2, end of supplementation and vaccination; V3, 28 days post-vaccination; Data are expressed as mean ± SD. No statistical difference was observed by two-way ANOVA (p < 0.05); *Determined using Mann-Whitney U-test between P and D groups (p < 0.05).