Vitamin D3 Supplementation Enhances Symptom Relief and Modulates Microbiome and Immune Responses in Pediatric Post-Acute Sequelae of COVID-19: A Randomized Controlled Trial

Background :Post-acute sequelae of COVID-19 (PASC) in children present persistent symptoms that impair quality of life. Vitamin D’s immunomodulatory and microbiome-modulating properties suggest its potential to alleviate PASC symptoms. This study evaluated the efficacy of Vitamin D3 supplementation in alleviating symptom severity and to explore associated immunological and microbiome alterations in children with PASC. Methods: In a double-blind randomized controlled clinical trial, 33 children with PASC were assigned to receive 2,000 IU/day of oral vitamin D (Group A, n=21) or placebo (Group B, n=12) for 6 months. Serum 25-hydroxyvitamin D [25(OH)D] levels, symptom severity (Children’s Somatic Symptom Inventory-24, CSSI-24), immune cytokines, and checkpoint proteins were assessed at baseline (M0) and 6 months (M6). Nasal and rectal microbiota were analyzed using 16S rRNA sequencing to evaluate composition and predict functional pathways. Results: Serum 25(OH)D increased significantly in Group A compared with Group B (p < 0.01). Group A demonstrated significant reductions in CSSI-24 scores (p < 0.05), with improvements in neuropsychiatric, respiratory and cardiovascular symptoms. Pro-inflammatory cytokines including IFN-γ (FDR = 0.01) and MIP-1α (FDR = 0.0004) decreased, while checkpoint proteins TIM-3 and HVEM increased (both FDR < 0.05). Nasal microbiota in Group A at M6 showed increased richness and enrichment of Sphingomonas, Paenibacillus, Ralstonia, and Sphingobium, with functional pathways related to xenobiotic metabolism altered after supplementation. Coprobacillus abundance in rectal samples positively correlated with 25(OH)D levels and negatively with IFN-γ (p = 0.02). Pooling participants from both time points across group A indicated that higher 25(OH)D levels were associated with lower symptom scores. Conclusions: Vitamin D3 supplementation improved symptom severity, modulated immune responses, and altered microbiota profiles in children with PASC. These findings support Vitamin D3 as a potential adjunctive therapy for pediatric PASC and highlight host–microbe–immune interactions as therapeutic targets.