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Effect of calcifediol supplementation as add-on therapy on the immune repertoire in recipients of the ChAdOx1 nCoV-19 vaccine: A prospective open-label, placebo-controlled, clinical trial
Correspondence to: Department of Endocrinology, R 3024, Research Block-B, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 160012, India.
Correspondence to: Society of Endocrine Health Care for Elderly, Adolescents and Children (SEHEAC), 92E/I, Ground Floor, Munirka Market, New Delhi 110067, India.
Department of Endocrinology, International Life Sciences Institute (ILSI) and Society of Endocrine Health Care for Elderly, Adolescents and Children (SEHEAC), New Delhi, India
Coronavirus disease 2019 (COVID-19) has caused formidable global health crisis, and multiple vaccination programs have been rolled out to curb the spread of the pandemic. Genetic disorders like primary immunodeficiency diseases severely compromise the generation of protective immunity following vaccinations. The spectrum also includes those genetic disorders that have a lesser impact on immunological memory such as Down Syndrome (DS). In a recent study, Valentini et al.
Factor associated with SARS-CoV-2 vaccination serological efficacy in adolescents and adults with Down syndrome: data from an international, collaborative initiative of the Trisomy 21 Research Society.
confirmed that the serological efficacy of COVID-19 vaccination is least affected by DS. The authors included a substantial number of subjects and further observed that the older subjects exhibit lower anti-SARS-CoV-2 antibody titers 6 months after vaccination. Earlier, Yarci-Carrion et al.
had also reported that DS and non-DS subjects develop similar anti-SARS-CoV-2 titers after the third dose of vaccine that decline with age. However, the same group demonstrated that DS subjects develop milder T cell responses to the standard 2-dose vaccine.
Development of an effective immune response in adults with down syndrome after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination.
Factor associated with SARS-CoV-2 vaccination serological efficacy in adolescents and adults with Down syndrome: data from an international, collaborative initiative of the Trisomy 21 Research Society.
further denote that the factors modifying immune responses to vaccination in such disorders are less explored. This may include nutritional deficiencies like vitamin D [25(OH)D]. Indeed, reports have shown that hypovitaminosis D is quite frequent in subjects with DS.
It is also known that individuals with low 25(OH)D levels have a higher risk of acquiring severe COVID-19 and stimulation of vitamin D receptor can reduce COVID-19-associated hospitalization and mortality.
Vitamin D receptor stimulation to reduce acute respiratory distress syndrome (ARDS) in patients with coronavirus SARS-CoV-2 infections: revised Ms SBMB 2020_166.
However, studies on vitamin D supplementation in COVID-19 vaccination have yielded conflicting results with some reporting improvement, while others observing lack of association between 25(OH)D status and antibody titers post vaccination.
Vitamin D supplementation does not influence SARS-CoV-2 vaccine efficacy or immunogenicity: sub-studies nested within the CORONAVIT randomised controlled trial.
Most of these studies, however, did not assess cellular immune responses, which are more likely to be influenced by vitamin D. Calcifediol [25(OH)D3], the immediate precursor of 1,25(OH)2D3 leads to a faster achievement of desired levels and biodistribution of circulating 1,25(OH)2D3 as compared to cholecalciferol (vitamin D3).
but there are no data on its utility in improving efficacy of COVID-19 vaccines. Therefore, we evaluated the efficacy of calcifediol supplementation on cell-mediated immune responses besides anti-SARS-CoV-2 titers post COVID-19 vaccination. We carried an open-label, placebo-controlled clinical trial to assess the impact of calcifediol supplementation on the efficacy of ChAdOx1 nCoV-19 vaccine in terms of, protection from breakthrough infection and COVID-19 disease, anti-SARS-CoV-2 antibody titers, SARS-CoV-2 specific lymphocyte proliferation and cytokine secretion.
Herein 580 adult subjects receiving ChAdOx1 nCoV-19 vaccine were recruited at the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. The subjects received either calcifediol (50 µg/day) or placebo, orally for 6 months (Supplementary Fig. 1, Supplementary Table 1) and divided in 3 groups [SARS-CoV-2 unexposed, SARS-CoV-2 exposed and those initiating intervention at the 2nd dose of vaccine (2nd dose group)]. The details of follow-up, blood sampling and various assays are mentioned in supplementary materials.
At first, we observed that calcifediol supplementation led to a rise in circulating 1,25(OH)2D levels during follow-up, with 25(OH)D and 1,25(OH)2D levels showing a positive association [adjusted β (95% C.I) 18.4 (6.3, 30.6), p = 0.003] (Table 1). During follow-up, the cumulative percentage of probable COVID-19 disease was lower in the calcifediol recipients (59/262, 22.5%) versus placebo (52/152, 34.2%) in all the groups (p = 0.009) (OR, 95% CI 0.56, 0.36–0.87) (Supplementary Table 2), with no significant difference in anti-spike (S) antibody titers between the calcifediol and placebo groups (Supplementary Fig. 2, Supplementary Table 3). The SARS-CoV-2-specific lymphocyte proliferation indices and cytokine levels were evaluated in representative samples. Calcifediol supplementation resulted in higher SARS-CoV-2-specific proliferation indices particularly at the last follow-up (Fig. 1, Supplementary Table 4) with a positive correlation with 25(OH)D levels [adjusted β (95% C.I) 0.22 (0.087, 0.36), p = 0.001] (Table 1). In the SARS-CoV-2 unexposed group, IL6 levels were lower at 4th month (p = 0.016) whereas, levels of IL10 were higher at 4th (p = 0.0002) and 6th month (p = 0.0001) in the calcifediol group. The levels of TGF-β also increased in the same group at 4th month (p = 0.0002) (Fig. 1, Supplementary Fig. 3, Supplementary Table 5). In the SARS-CoV-2 exposed and 2nd dose subjects, there was no significant difference in the concentration of cytokines between the calcifediol and placebo groups. (Supplementary Figs. 4–5) The regression analysis yielded a negative association of 25(OH)D with IFN-γ [adjusted β (95% C.I) −156.9 (−307.1, −6.6), p = 0.041] (Table 1). The levels of CRP, iPTH and other biochemical parameters remain unchanged throughout the study across all the groups (Supplementary Tables 6–8).
Table 1Regression analysis for the association between 25(OH)D levels with various study parameters.
Variables
Adjusted* β (95% C.I)
P-value
IL2
-105.4 (−249, 38.2)
0.149
IL6
-1607.3 (−3848.4, 633.9)
0.159
IFN-γ
-156.9 (−307.1, −6.6)
0.041
IL4
-17.6 (−179.5, 144.2)
0.83
IL10
260.7 (−7.0, 528.4)
0.056
TGF-β
80.2 (−878.5, 1038.8)
0.869
LPA
0.22 (0.087, 0.36)
0.001
1,25(OH)2D
18.4 (6.3, 30.6)
0.003
*Adjusted for all parameters given in the table including age and sex.
Abbreviations: IL; Interleukin, IFN-γ; Interferon gamma, TGF-β; Transforming growth factor beta, LPA; Lymphocyte proliferation indices. The β (beta) values represent the estimated coefficients for each variable and the 95% confidence intervals (C.I). The adjusted β values and p-values account for the influence of other variables in the model.
Fig. 1(A-C) Plasma 25(OH)D levels at baseline, 3rd month, and 6th month in SARS-CoV-2 unexposed, exposed, and 2nd dose subjects. (D-F) The lymphocyte proliferation indices of peripheral blood mononuclear cells co-cultured with SARS-CoV-2 S-protein peptide pool cocktail are shown in the representative subjects of all the groups. The selection of subjects was arbitrary. (G-I) The cytokine levels (IL6, IL10, and TGF-β) in the plasma derived from whole blood cultures stimulated with SARS-CoV-2 S-protein peptide pool are shown in the representative subjects in the SARS-CoV-2 unexposed group. The representative subjects with calcifediol supplementation had plasma 25(OH)D levels> 40 ng/ml. Data are represented as median, IQR and range with mean represented as a dot. [*] represents P = <0.05, [**] represents P = <0.01, [***] represents P = <0.001, [****] represents P = <0.0001.
Our findings are in slight contrast with another large study where vitamin D supplementation did not have any benefit on the rates of breakthrough infection, antibody titers or SARS-CoV-2 peptides stimulated IFN-γ levels.
Vitamin D supplementation does not influence SARS-CoV-2 vaccine efficacy or immunogenicity: sub-studies nested within the CORONAVIT randomised controlled trial.
However, our subjects had much lower 25(OH)D levels at baseline and with calcifediol supplementation, most attained higher levels (>50 ng/ml), which might explain better outcome of active vitamin D mediated effects. Post stimulation with SARS-CoV-2 peptides, higher levels of anti-inflammatory cytokines (IL-10 and TGF-β) and lower levels of IL-6 were released in SARS-CoV-2 unexposed group receiving calcifediol, with higher rates of lymphocyte proliferation. Taken together, these data indicate that the proliferating T cells possibly have a Treg or Th2 phenotype, which could be confirmed by immunophenotyping of proliferating cells. Esparcia-Pinedo et al.
Development of an effective immune response in adults with down syndrome after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination.
had observed lower SARS-CoV-2 specific T cell responses with a standard 2-dose vaccine regimen in DS subjects. In this context, our data showing improvement in SARS-CoV-2 specific T cell responses with calcifediol supplementation becomes relevant in conditions with vitamin D deficiencies. Additionally, the lymphocyte proliferation data from the exposed group, indicates enhancement of hybrid immunity by calcifediol supplementation.
To conclude, ours is the first interventional study with calcifediol reporting multiple outcomes in COVID-19 vaccination. The limitations include low number of subjects in the unexposed group due to high exposure of SARS-CoV-2 in the community, suboptimal follow-up owing to the surge of Omicron variant during the study, lack of a group with cholecalciferol supplementation. Nevertheless, the study presents a novel and safe approach to improve the efficacy of COVID-19 vaccines in vitamin D-deficient states that offers additional skeletal benefits.
The sponsors were not involved in study design, collection, analysis and interpretation of data or the writing of the manuscript including the decision to submit the paper for publication.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors acknowledge the support of all the technical staff and volunteers including Kasmeen, Shivani Verma, Priya Hitaishi, Shallu Singhmar, Shallu, Himanshu, Vivek Sharma, Mudasir Hasan, Pintoo Kumar, Ajay Kumar, Rajdavinder, Harmanpreet, Aparna, Neetika in completing the study. We also express our special thanks to Peter M. Mueller, Scott A. Miller and Deep K. Patel for their useful suggestions and support throughout the study. The study was funded by Dishman Carbogen Amcis Ltd, Bubendorf, Switzerland.
Factor associated with SARS-CoV-2 vaccination serological efficacy in adolescents and adults with Down syndrome: data from an international, collaborative initiative of the Trisomy 21 Research Society.
Development of an effective immune response in adults with down syndrome after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination.
Vitamin D receptor stimulation to reduce acute respiratory distress syndrome (ARDS) in patients with coronavirus SARS-CoV-2 infections: revised Ms SBMB 2020_166.
Vitamin D supplementation does not influence SARS-CoV-2 vaccine efficacy or immunogenicity: sub-studies nested within the CORONAVIT randomised controlled trial.
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