Calcifediol boosts the efficacy of ChAdOx1 nCoV-19 (COVISHIELD) vaccine via upregulation of key genes associated with memory T cell responses

doi:10.21203/rs.3.rs-3939229/v1

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Calcifediol boosts the efficacy of ChAdOx1 nCoV-19 (COVISHIELD) vaccine via upregulation of key genes associated with memory T cell responses

https://doi.org/10.21203/rs.3.rs-3939229/v1

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published 20 Jun, 2024

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The ChAdOx1 nCoV-19 (COVISHIELD) vaccine has emerged as a pivotal tool in the global fight against the COVID-19 pandemic. In our previous study eligible subjects were supplemented with calcifediol, a direct precursor to the biologically active form of vitamin D, calcitriol with an objective to enhance the immunogenicity of the COVISHIELD vaccine. Herein we investigated the effects of calcifediol supplementation on gene expression profiles in individuals who received the COVISHIELD vaccine. Peripheral blood mononuclear cells were isolated from vaccinated individuals with and without calcifediol supplementation at baseline, 3 and 6 months, and the gene expression profiles were analyzed using high-throughput sequencing. The results revealed distinct patterns of gene expression associated with calcifediol supplementation, suggesting potential molecular mechanisms underlying the beneficial effects of calcifediol in improving the efficacy of COVISHIELD vaccine via augmentation of T cell memory responses, innate immune mechanisms such as NOD signaling pathway, JAK/STAT and TGF beta pathways. Calcifediol supplementation in vaccinated individuals also downregulated the pathways related to the Coronavirus disease. Taken together, our findings provide valuable insights into the interplay between vitamin D receptor (VDR) signaling and vaccine-induced immune responses and offer another approach in improving vaccination induced antiviral responses.

Health sciences/Medical research/Outcomes research

Biological sciences/Immunology/Vaccines/Adjuvants

ChAdOx1nCov-19

COVISHIELD

Calcifediol

Vitamin D

Coronavirus

Gene Expression

Vitamin D is a fat-soluble vitamin, which has both skeletal as well as extra skeletal benefits, holds a crucial place in maintaining an individual's holistic health and wellness. It is synthesized by the non-enzymatic conversion of 7-dehydrocholesterol upon exposure of the inner layers of the epidermis to ultraviolet (UV-B) rays of wave-length 290–320 nm from the sun. Vitamin D undergoes a two-step hydroxylation process for conversion to its active form, the first hydroxylation, facilitated by 25-hydroxylase, occurs in the liver, resulting in 25-hydroxyvitamin D (calcifediol); the second hydroxylation, takes place in the kidneys and certain other tissues, yielding 1,25-dihydroxyvitamin D (Calcitriol) through the catalytic activity of enzyme 25(OH)D3-1α-hydroxylase which is encoded by the gene Cyp27b1 (cytochrome P450 family 27 subfamily B member 1) (1). Upon binding of 1,25(OH)2D to its ligand, vitamin D receptor (VDR), the VDR associates with its heterodimeric partner, retinoid X receptor, and subsequently interacts with vitamin D response elements (VDREs) present in the target genes (2). Initially, vitamin D was well known for its role in regulating calcium homeostasis and promoting the development of strong and healthy bones. However, studies in past few years have demonstrated that benefits of vitamin D extend beyond bone health and include the maintenance of a healthy immune system as most of the immune cells express VDR and VDREs (3). Vitamin D influences the differentiation and function of various immune cells, including T cells, B cells, dendritic cells, and macrophages. Vitamin D can suppress the differentiation of Th1 and Th17 cells, which are involved in pro-inflammatory responses while promoting the Th2 and Treg cell differentiations, which are important in maintaining immune tolerance (5). Additionally, vitamin D enhances production of antimicrobial peptides that can kill viruses and bacteria via triggering genes downstream of VDR like cathelicidins, Cyp24, and DEFB4 (6). In addition, vitamin D has been found to suppress the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), while increasing the expression of anti-inflammatory cytokines, such as interleukin-10 (IL-10) (4).

Due to erroneous beliefs that a normal diet and a few minutes in the sun are sufficient sources of vitamin D, dietary deficiencies are consistently underdiagnosed (3). High altitudes, seasonal variations, avoidance or inadequate sun exposure, dark skin, aging, and excessive sunscreen use are the factors causing vitamin D insufficiency by reducing the amount of UV radiation that reaches the skin. Low levels of vitamin D have been linked to diseases like inflammatory bowel disease (7) and a higher risk of respiratory infections, such as COVID-19, influenza as well as autoimmune diseases (8, 9). Various studies and meta-analysis during COVID-19 pandemic indeed found an association between low vitamin D levels and a higher risk of severe COVID-19 outcomes, such as hospitalization and death (10–13). The proposition has been put forward that activation of VDR may have the capacity to decrease the likelihood of acute respiratory distress syndrome (ARDS), cardiac complications, coagulopathy, and mortality in patients with COVID-19 (10–14). Calcifediol, an immediate precursor of calcitriol, represents a potential option available for mitigating the adverse effects of vitamin D deficiency, including functioning of the immune system (15). In comparison to cholecalciferol, calcifediol has a longer half-life, better absorption and is more effective in raising 25-hydroxyvitamin D levels (16, 17). Calcifediol can be administered orally as a dietary supplement, making it more convenient and accessible than other drugs that require injection or intravenous administration.

In a recent study, we conducted an open-label, placebo-controlled, interventional trial to investigate whether calcifediol (25(OH)D3) supplementation boosts the efficacy of the ChAdOx1 nCoV-19 vaccine. Herein we observed that ChAdOx1 nCoV-19 vaccine recipients receiving oral calcifediol for 6 months attained more circulating 1,25(OH)2D levels as compared to those on placebo. Calcifediol supplementation resulted in a decline in the cumulative percentage of probable COVID-19 disease in all the groups. Calcifediol supplementation also resulted in a higher in vitro proliferation of lymphocytes stimulated with SARS-CoV-2 S-peptide pool coupled with a higher IL-10 and TGF-β and decreased expression of IL-6. The results suggested that calcifediol supplementation possibly triggered memory T cell responses and promoted hybrid immunity with a Treg or Th2 phenotype (18).

The present study was planned to unravel how calcifediol supplementation influences the gene expression profile of immune cells in vaccinated individuals. Our rationale was to understand the crosstalk between the VDR signaling and vaccine-mediated immune responses. Therefore, we further isolated the peripheral blood mononuclear cells (PBMCs) of the representative subjects and performed RNA-seq analysis to distinguish variations in gene expression between subjects who received calcifediol versus those on placebo (control group), at three distinct time points: baseline, 3rd month, and 6th month. To the best of our knowledge, there is no earlier study on the role of Calcifediol or Vitamin D supplementation in COVID vaccine recipients, analyzing changes at gene level in the immune cells. The results of RNA-seq analysis of calcifediol subjects provided valuable insights into the differentially regulated genes and molecular pathways regulated by vitamin D and the potential pathways involved in improving the efficacy of COVISHIELD vaccine and augmentation of genes associated with innate and adaptive immune responses in conferring protection from COVID-19.

Subject Recruitment

The study was approved by the institutional ethics committee (IEC) of PGIMER, Chandigarh. The participants recruited were healthy individuals aged, 18–60 years who had received two doses of the ChAdOx1 nCoV-19 vaccine (COVISHIELD ™, Serum Institute of India, Pune, India) with a 12-week interval at the COVID-19 vaccination center of PGIMER. This was an open-label, placebo-controlled, interventional trial with two pre-specified groups: Calcifediol supplemented and a placebo group (CTRI/2021/08/035709). Of the 24 subjects who underwent screening, 12 received 50 µg calcifediol (2 capsules of 25µg)/day (Calcifediol-supplemented group), and the other 12 received placebo (2 capsules/day) (Placebo group). Both groups received the intervention for 6 months, starting from the first dose of the vaccine. The team reinforced the importance of daily supplementation during each follow-up visit. Compliance was measured by collecting empty capsule bottles during each visit. Any negative reactions resulting from the use of calcifediol supplementation, including high levels of calcium in the blood or urine and renal calculi, were also noted. Blood samples were taken using three types of vials: plain (3ml), EDTA (4ml), and heparin (12ml). The subjects were asked to return for follow-up appointments at 1, 3, and 6 months after receiving the vaccination. The SF-36 health survey questionnaire was administered to assess their quality of life at the start of the study and the final follow-up.

Biochemical analyses:

Serum calcium, inorganic phosphate, alkaline phosphatase, and albumin levels were measured to assess the calcemic profile using an automated biochemical analyzer (COBAS 8000, Roche Diagnostics, Germany). Electrochemiluminescence immunoassay (ECLIA) (COBAS e801, Roche Diagnostics, Germany) was used to measure plasma 25(OH)D (D represents either or both D2 and D3) and intact parathyroid hormone (iPTH) levels. Chemiluminescence immunoassay (CLIA, Diasorin Liason) was used to measure plasma 1,25(OH)2D levels.

cDNA Library Preparation and RNA-Sequencing

Total RNA was extracted from PBMCs by using the RNeasy Mini Kit (Qiagen, Germany) as per the manufacturer’s instructions. The concentration and integrity of total RNA were checked using the Qubit RNA Assay Kit in a Fluorometer (Qubit 4.0, Thermo Fisher Scientific, IN) while RNA quality was assessed with the Tapestation 4200™ (Agilent Technologies, Santa Clara, USA). RNA samples were taken with an initial RNA input of 500ng. “KAPA mRNA Capture kit” was used for mRNA capture by oligo mag beads, followed by mRNA fragmentation using heat and magnesium. The KAPA RNA Hyper prep kit for Illumina sequencing was used for the construction of mRNA-seq libraries. Next, the cDNA was synthesized using a reverse transcriptase and random hexamers in a first-strand synthesis reaction. Subsequently, the cDNA was converted to double-stranded cDNA where Uracil is added instead of Thymine leading to the addition of dAMP to the 3′ ends of the resulting dscDNA. In adapter ligation, dsDNA adapters with 3′ dTMP overhangs were ligated to library insert fragments followed by library amplification, to amplify library fragments carrying appropriate adapter sequences at both ends using a high-fidelity, low-bias PCR. The library was sequenced using an Illumina NextSeq 2000 platform to generate 60M, 2x150 bp reads/sample.

Differentially Expressed Genes (DEGs) Identification

The raw data was evaluated for quality control using FastQC (version 0.11.9). Subsequently, Trim Galore (version 0.6.6) was utilized for the removal of poor-quality sequences and trimming of adapters. These processed reads were mapped to the reference transcriptome using the STAR (version 2.7.10a) alignment tool and then quantified using FeatureCounts (version 2.0.3), a module of the R package called R subread (version 2.0.1). For the annotation of Ensembl gene identifiers with gene symbols, descriptions, genomic locations, and biotypes, the R package BiomaRt (version 2.46.0) was employed to access the Ensembl database (version 103). To check for ribosomal contamination rRNA contamination detection was run against the SILVA database. Less than 1% of reads were annotated as rRNA which were filtered out from the datasets. The expression profiles of individual transcripts were normalized to transcripts per million (TPM). The count-based technology DESeq2 package (v.3.6.1) was operated in R (version 4.2.2) using Ubuntu (22.04.1 LTS) to conduct the differential expression analysis. Genes exhibiting a Log2 fold change at a cut off of one with Benjamini–Hochberg corrected p-value (false discovery rate, FDR) of less than 0.05 were considered significant vitamin D targets. Volcano plots and heatmaps were generated with the Enhanced Volcano (version 1.10.0) and Pheatmap (version 1.0.12) R packages respectively.

Enrichment Analyses and Network Visualization

Enrichment analysis and visualization of genes was performed using ‘Shiny GO (version 0.77)’ (18) and ‘PathfindR (version 2.2.0)’ Gene Ontology (GO). GO enrichment analysis annotated the mechanism of action of target genes in three parts: biological process (BP), cellular composition (CC) and molecular function (MF). Enrichment analysis was performed to obtain bubble plots to annotate the upregulated and signaling pathways in which the target genes were involved, using the Kyoto Encyclopedia of Genes and Genomes (KEGG). For gene set enrichment analysis, GSEA (version 4.3.2 - build 13) was used to generate GSEA Plots. Protein-protein interaction networks were generated using STRING, a web-based tool.

Statistical Analysis

All statistical analyses were performed using Graphpad Prism (version 8.0). Normality of the data was checked by D’Agostino and Pearson normality test. Normally distributed data was compared using a two-sided t-test, whereas Group analysis was performed by two-way ANOVA. The significance level was set at p < 0.05.

Influence of Calcifediol supplementation on plasma 25(OH)D, iPTH, calcitriol, and serum calcium levels

Participants were recruited to assess the impact of calcifediol supplementation on the efficacy of ChAdOx1 nCoV-19 vaccine. The subjects were divided into 2 groups, the calcifediol arm and the placebo arm. After consideration of the criteria for inclusion and exclusion, a total of 24 adult subjects were selected and the transcriptomic analysis was performed at 3 time points; baseline, 3rd month, and 6th month as shown in the flow diagram (Fig. 1). Plasma levels of 25(OH)D, calcitriol, iPTH, and serum calcium at all time points were recorded (Table 1).

At first, we assessed the impact of calcifediol supplementation on plasma 25(OH)D levels. Baseline plasma 25(OH)D levels were similar in both the treated (mean ± SD, ng/ml) (13.91 ± 5.98) and the placebo cohort (11.39 ± 6.9). In the treated group, the plasma 25(OH)D levels (mean ± SD, ng/ml) increased at the 3rd month (134.9 ± 42.8) (p < 0.001) and then plateaued at the 6th month (85.01 ± 34.49) (p < 0.001) post calcifediol supplementation, indicative of the effectiveness of calcifediol in boosting Vitamin D levels. In the placebo arm, the plasma 25(OH)D levels remained lower throughout the study period (Fig. 2A).

Elevated plasma iPTH levels are indicative of calcium deficiency, triggering processes to increase blood calcium, often at the expense of bone mineralization. Calcifediol supplementation may contribute in maintaining adequate calcium levels, potentially reducing the need for heightened iPTH secretion. At baseline, the plasma iPTH levels (mean ± SD, ng/ml) in the treated group (60.34 ± 28.67) were similar to the placebo cohort (53 ± 19.97). Post calcifediol supplementation, the iPTH levels in the calcifediol cohort dipped (38.1 ± 13.3 ng/ml) with a slight increase in the placebo cohort (65.65 ± 37.08 ng/ml) at 3rd month although the changes were non-significant. At 6th month as well, there was no significant change in the plasma iPTH levels in both the groups (Fig. 2B).

Similarly, in the treated group, calcifediol supplementation did not alter the serum calcium levels (mg/dl) (9.55 ± 0.5) at 3rd month and (9.61 ± 0.3) level at 6th month (Fig. 2C). Nevertheless, this finding does not undermine the importance of vitamin D in maintaining optimal calcium levels for overall health, bone density, and proper immune function.

Calcitriol Concentrations and Vitamin D Signaling:

Calcitriol is the active hormonal form of vitamin D, and plays a pivotal role in regulating calcium and phosphorus levels, bone mineralization, and immune responses. Individuals supplemented with calcifediol demonstrated elevated 25(OH)D levels and exhibited a significant increase in mean (± SD) calcitriol levels (pmol/l) (165.04 ± 53.24) (adjusted p = 0.04) at 3rd month and (155.6 ± 50.6) (adjusted p = 0.03) concentration at 6th month (Fig. 2D) when the delta increase in calcitriol levels was compared with baseline levels. Further, there was a positive correlation between 25(OH)D and calcitriol levels (R = 0.31, R² = 0.09) (p = 0.007). This relationship underscores the importance of calcifediol as a precursor to calcitriol, emphasizing the superiority of calcifediol over native vitamin D and the clinical relevance of maintaining optimal vitamin D status.

Table 1

Clinical and Biochemical characteristics of the study subjects at all time points
	Baseline		3rd Month		6th Month
Characteristic	Placebo (N = 12)	Calcifediol (N = 12)	Placebo (N = 12)	Calcifediol (N = 12)	Placebo (N = 12)	Calcifediol (N = 12)
Sex (female)	4	6	4	6	4	6
Age (years)	28.2 ± 9.1	35.8 ± 10	28.2 ± 9.1	35.8 ± 10	28.2 ± 9.1	35.8 ± 10
25(OH)D (ng/ml)	13.91 ± 5.98	11.39 ± 6.9	15.71 ± 6.95	134.9 ± 42.8	13.4 ± 5.94	85.01 ± 34.49
Calcitriol (pmol/l)	134.84 ± 34.1	113.5 ± 32	132.14 ± 43.38	165.04 ± 53.24	120.5 ± 47.2	155.6 ± 50.6
iPTH (pg/ml)	60.34 ± 28.67	53 ± 19.97	65.65 ± 37.08	38.1 ± 13.3	59.72 ± 26.16	52.5 ± 12.1
Calcium (mg/dl)	8.89 ± .08	9.37 ± 0.3	9.39 ± 0.5	9.55 ± 0.5	9.38 ± 0.6	9.61 ± 0.3

Table 1

The 25(OH)D, intact parathyroid hormone (iPTH) and calcitriol levels were measured in plasma while the calcium levels were determined in serum specimens. The data is shown as mean ± standard deviation (SD).

Outcome of calcifediol supplementation on the gene expression profile of PBMCs

The raw count data of the subjects was normalized with variance stabilizing transformation (VST) method of DESeq2 (Fig. 3A) and Principal Component Analysis (PCA) plot (Fig. 3B) showing the calcifediol intervention arm in different clusters at each time-point and indicating a time-dependent effect of the calcifediol supplementation. The PCA of the placebo group was also plotted (Supplementary Fig. 1). Since the placebo subjects did not receive any intervention, the PCA plot did not show any pattern at different time points.

A subgroup analysis of gene expression from treated baseline to treated 3rd and treated 6th month revealed a total number of 1725 genes and 10353 genes respectively being expressed differentially at log2 fold change of 1 with a significance level of p < 0.05 (Fig. 3C). Upon comparing the differentially expressed genes (DEGs) of this subgroup, we found that 649 genes (Fig. 3D) were consistently upregulated and 270 genes (Fig. 3E) were consistently downregulated at both 3rd and 6th month during calcifediol supplementation. Since the comparison of the treated group at 6th month versus baseline demonstrated the maximum number of DEGs and majority of those DEGs also appeared in the 3rd month, this comparison was chosen primarily for downstream analysis.

Differentially Expressed Genes (DEG) analysis

DEG analysis of the calcifediol supplemented (treated) group at 6th month in comparison to baseline of the same group revealed that at the 6th month of calcifediol supplementation, a total of 10,353 genes were differentially regulated of which 6389 genes were upregulated and 3964 genes were downregulated. Whereas 1064 genes were found to be upregulated and 661 genes were downregulated at 3rd month as compared to baseline. In comparison between the treated 6th month with the placebo 6th month, 49 genes were upregulated and 324 genes were downregulated as depicted in the respective volcano plots the significant differentially expressed genes with fold change more than 1 and having adjusted p value < 0.05 are marked in bright red whereas the non-significant genes with fold change less than 1 and having adjusted p value > 0.05 are marked in light blue (Fig. 4A, B and C) and the top 50 DEGs whose expression had most variance in these 3 sub-analysis were depicted in heatmaps (Fig. 4D, E and F) respectively. Genes involved in multiple biological processes appeared to be involved and the key genes of this comparison included PTGS2, EREG, RSG1, AREG, NR4A3, EGR3, and GSTM1. As a proof of concept, we also checked the expression of 114 genes related to VDR signaling and found that the expression of these genes increased at 3rd month and plateaued at 6th month of the calcifediol supplementation (Supplementary Fig. 2).

Upregulation of innate and adaptive immune signaling pathways upon calcifediol supplementation

The role of vitamin D in enhancing innate immunity is well known. When we compared the treated 6th month subjects versus their baseline data with, the KEGG Gene set enrichment analysis (GSEA) revealed genes involved in the NOD, as well as JAK/STAT, TGF BETA signaling pathways, were upregulated at 6th month (Fig. 5), indicating higher induction of VDR signaling in DCs and macrophages potentially inducing antimicrobial or antiviral proteins like β-defensins and cathelicidins. While defensins protects the host cells by disrupting microbial membranes including SARS-CoV-2, cathelicidins attract various immune cells via chemotaxis and also enhance phagocytosis by macrophages, increasing vascular permeability and ultimately activation of B and proliferation of T cells (12), thus strengthening innate immune responses that appear to be augmented by calcifediol supplementation.

Downregulation of Coronavirus Disease and Inflammatory Pathways

Upon comparing the treated 6th month with the treated baseline, the downregulated DEGs function was explored using KEGG pathway enrichment analysis. The enrichment analysis indicated that Coronavirus disease, Neutrophil extracellular trap formation (NET), Systemic lupus erythematosus (SLE), and Necroptosis were among the topmost down-regulated pathways. Notably, the Coronavirus disease pathway was one of the most consistently down-regulated pathways, not just in the comparison between the treated 6th month vs treated baseline (Supplementary Fig. 6) but also in other comparisons, mainly, treated 6th month vs treated 3rd month (Supplementary Fig. 7) and treated 6th month vs placebo 6th month (Supplementary Fig. 8).

Next, the protein-protein interaction (PPI) network of the genes related to Coronavirus disease showed significant enrichment (p < 1.0e-16). Neutrophil extracellular trap formation (NET) and Systemic lupus erythematosus (SLE) also showed significant enrichment in PPI analysis (p < 1.0e-16) in the STRING database (Fig. 6). The upregulated pathways shown by KEGG pathway analysis in the treated 6th month vs treated baseline included JAK-STAT signaling, TGF beta signaling, NF-kappa B signaling pathway and cell cycle (Supplementary Fig. 3) some of which were also seen in comparison of treated 3rd month versus baseline (Supplementary Fig. 4). Since a very few numbers of genes were differentially expressed in comparison between treated 6th month vs placebo 6th month, a smaller number of pathways appeared upregulated, due to which no meaningful conclusion could be drawn (Supplementary Fig. 5).

Gene ontology analysis on the role of calcifediol supplementation in biological processes

The Gene ontology analysis revealed the role of calcifediol in upregulating about 376 biological processes (Supplementary Table 1) in comparison between treated 6 months versus treated baseline samples. Some of the relevant pathways included genes involved in the activation and regulation of innate immune responses, memory T cell responses, positive regulation of T cell chemotaxis signaling, T cell receptor signaling pathway, B cell receptor signaling, Notch signaling pathway, cytokine mediated signaling pathway, defense and cellular response to virus, and calcium ion transmembrane transport, many of which are important in generating antiviral responses. Of note, many genes that are related to generation and maintenance of memory T cells responses were found to be upregulated, indicating the positive impact of calcifediol supplementation in improving efficacy of COVISHIELD vaccine. The genes corresponding to these pathways are shown in Table 2. Calcifediol also downregulated nearly 157 biological processes (Supplementary Table 2). Some of these processes included mRNA splicing, via spliceosome, rRNA processing, and transcription by RNA polymerase II.

Table 2

List of Biological processes and the genes involved that were found differentially upregulated upon calcifediol supplementation
Biological Processes	Upregulated Genes
Activation of innate immune response	PRKDC, SFPQ, MATR3, SIN3A
Regulation of innate immune response	XIAP, CASP8, LRP8, N4BP1, SAMHD1, APPL1, APPL2
Positive regulation of T cell chemotaxis	ADAM10, ADAM17, OXSR1, WNK1
T cell receptor signaling pathway	PDE4D, PIK3CA, PLCG2, PTPRJ, TRAF6, TXK, MALT1, PTPN22, RC3H2, WNK1, RC3H1, DENND1B
Defense response to virus	BCL2, LYST, DHX15, IFNAR2, NCBP1, EIF2AK2, RNASEL, PLA2G10, G3BP1, ZMYND11, CARD8, DDX58, SAMHD1, SETD2, TLR7, IFIH1, TRIM56, TRIM52, SLFN11, MLKL, PDE12, SERINC5, NLRP9, GBP7
Cellular response to virus	CHUK, MAPK14, DDX3X, FMR1, HIF1A, IL12A, LGALS8, RIOK3, IFIH1
Cytokine-mediated signaling pathway	CBL, IL6ST, JAK2, KIT, PTPN11, PTPRJ, SOS1, IRAK3, IL31RA
Notch signaling pathway	ADAM10, NOTCH2, MAML2
B cell receptor signaling pathway	BCL2, CD38, CTLA4, NFATC2, PLCG2, TEC, VAV3, BANK1, PLEKHA1
Calcium ion transmembrane transport	ATP2A2, ATP2B4, ITGAV, P2RX7, PKD2, RYR3, SLC8A1, TRPC1, TRPC5, SLC24A1, TRPM7, MCOLN3, TRPM6, ANO6, MCOLN2
Memory T cell responses	EOMES, ID3, ADAM23, EPHA4, ITK, BCL11B, PRDM1, CCR6, ZNF365, TGFBR3, DOCK9, INPP4B, EPHA4, ITK, BCL11B, PRDM1, PPP2R2B, SLFN5, FBXO32

Genes related to memory T cells are upregulated among the differential expressed genes

In the gene ontology analysis, many genes related to the memory T cell function were found to be upregulated. Since the main objective of the intervention (calcifediol) was to improve the efficacy of the COVISHIELD vaccine, therefore, we further tried to validate the expression of key genes required for the maintenance of memory T cell responses in representative samples. The expression of EOMES, ID3, IL7R, and BLC6 was confirmed by RT-PCR using TaqMan probes (Supplementary Table 3) and the results were similar with RNA transcriptome analysis. We observed that the expression of all 4 genes, particularly, ID3, IL7R, and BLC6 increased in 3rd month (p < 0.05) post calcifediol supplementation (Fig. 7). However, in the 6th month, the expression levels of these genes returned to near baseline levels. Taken together, the results from RT-PCR indicate improvement in memory T cell responses post-calcifediol supplementation in the subjects.

In our earlier study, we observed that calcifediol supplementation augmented protection from severe COVID-19 by promoting memory T cell responses and anti-inflammatory cytokines (18). As mentioned previously, this was the first interventional trial with calcifediol supplementation in the recipients of ChAdOx1 nCoV-19 vaccine. The observed improvement in efficacy of COVISHIELD vaccination could be attributed to calcifediol supplementation but it can be scientifically proven by either immunophenotyping of SARS-Cov-2 specific T cells or by in vivo animal studies or by assessing the whole gene expression of immune cells of the recipients. Since VDR is expressed by almost all immune cells, we chose the latter approach and analyzed the gene expression profile of the treated and placebo subjects at regular time intervals. Moreover, hydroxylase activities especially those of 25(OH)D3-1α-hydroxylase are high in monocytes and macrophages and VDR signaling is active in lymphocytes. Therefore, the current study undertook an important and critical question about the role of calcifediol on the gene expression profile in PBMCs of ChAdOx1 nCoV-19 vaccine recipients to understand the potential factors that can enhance the innate and adaptive immunity, improve memory T cell responses, reduce cytokine storm during viral exposure and ultimately boost vaccine efficacy.

In the gene expression analysis, the PCA plot of the treated group showed different clusters at each of the time points showing time dependent effect of the calcifediol supplementation as well as compliance of the subjects. Also, the number of the genes that were upregulated at the 6th month of supplementation was higher than other comparisons, indicating long-term impact of the supplementation. This observation could also be attributed to the mode of action of VDR and VDREs that act mainly via secondary messengers. Our major aim was to find out those genes that were associated with memory T cell responses, however, it must be noted that vitamin-D influences several biological pathways, therefore the results of DEG analysis showed genes involved in several biological processes. Several earlier reports have indicated that Vitamin D is involved both in the regulation of the innate immunity, as well as in the modulation of the adaptive immune response through direct effects on T cell activation and on the phenotype and function of antigen presenting cells. In our study of calcifediol supplementation in ChAdOX2 vaccinated individuals, based on the high-throughput RNA sequencing data we have also found that vitamin D influences several pathways of innate as well as adaptive immune system such as the NOD signaling pathway JAK/STAT and TGF beta pathways. Most importantly, the pathway enrichment analysis revealed that coronavirus disease was consistently downregulated in various comparisons including the treated 6th month vs treated baseline, treated 6th month vs treated 3rd month and even among treated 6th month and placebo 6th month. These findings further corroborate the observed clinical and immunological responses seen in subjects receiving calcifediol supplementation along with ChAdOx1 nCoV-19 vaccine (18). Additionally, NETs and SLE were downregulated in the treated 3rd and treated 6th month groups, clearly indicating that calcifediol had a direct impact on COVID-19 disease, inflammation and autoimmunity. Neutrophils are essential for innate immunity. However, when COVID-19 infection occurs, it results in high levels of circulating NETs causing a reduction in blood flow in the capillaries leading to build-up of NETs in the lungs and other organs causing multiple organ failures (19). Therefore, calcifediol appeared to modulate the process of NETosis which might be important in curbing exaggerated inflammatory responses during the initial stages of secondary COVID infection. Vitamin D has also been reported in other diseases to reduce the formation of NETs (19, 20) including the treatment of SLE (21).

One of the significant observation in our study was upregulation of several genes involved in the T cell activation and proliferation like CD25, CD69, CD71, and CD38, which is in line with our previous study where calcifediol supplementation significantly increased lymphocyte proliferation (18) Likewise, the genes involved in the JAK-STAT pathway were also found upregulated which is known to play a crucial role in augmenting a battery of pro-inflammatory cytokine response in activated immune cells. In addition, upregulation of both subunits of the IFN-α receptor, a type of receptor involving primary in antiviral defense and expressed nearly by all cell types including the endocrine, immune, and central nervous system was observed.

In our previous study calcifediol has already been shown to increase serum IL-10 and the same is being reflected in this study as well. IL17 was also downregulated which can be interpreted as ensuing tolerogenic milieu. Additionally, there was also upregulation of the suppressor of cytokine signaling (SOCS) family of genes like SOCS1, 3, 4, and 6 which are potent negative regulators of proinflammatory cytokine signaling via triggering a negative feedback loop on the JAK/STAT pathway, thus thwarting excess inflammation. Another molecule associated with inhibitory immune signals, Cytotoxic T lymphocyte-associated protein 4 (CTLA4) was also found upregulated in our RNAseq data. CTLA4 is also known to be constitutively expressed in Tregs and is regarded as a key molecule for cell-mediated immunosuppressive functions. CTLA-4 competitively binds to CD80/CD86 present on APCs, thereby impeding the binding of CD28 and thus subsequent prevention of secondary stimulus for T cell activation. In our study we also found moderate upregulation of CTLA4 ligands (CD80/CD86) which highlighted the probable immune modulation by calcifediol. The zinc-finger transcription factor, Helios, which is critical for maintaining the anergic phenotype and suppressive activity of regulatory T cells (Tregs) was upregulated reflecting that T cell mediated tolerogenic signals have been long delivered (at the time of infection or re-infection). CD46 and CD35 (CR1) that are expressed on activated T cells and lead to the development of Tregs in the presence of IL2 were also seen upregulated. These findings highlight another arm of calcifediol-VDR signaling mediated regulation of adaptive immune responses that works in parallel to prevent exaggerated inflammatory responses during secondary COVID 19 infections.

In the gene ontology analysis various genes related to generation and maintenance of memory T cells responses were found to be upregulated. We further assessed a few genes that are expressed in lymphocytes and involved in the generation and maintenance of long-lived memory T cells (22). RT-PCR verified the genes associated with memory T cell responses including, ID3, IL7R, and EOMES showing a similar trend to transcriptome analysis. ID3hi memory T cells express higher levels of IL7R, enabling a greater responsiveness to IL7, and promoting memory T-cell survival and homeostasis (22, 23). Another gene, EOMES, required in function and homeostasis of effector and memory T cells (28), was significantly increased in the unexposed group, indicating a transition of hyper-immune responses towards generation of memory T cells. BCl6 expression is transient and is required for the generation, but not maintenance, of memory CD8 + T cells (24, 25). The expression of most of these genes including ID3, IL7R, EOMES, and BCl6 increased at 3rd month in the treated group which stabilized by the end of 6th month highlighting the role of vitamin D in promoting T cell memory.

The clinical implications of our findings extend to the realm of vaccine responses and overall immune health. A balanced calcium metabolism could create an environment conducive to optimal T cell activation, cytokine signaling, innate and adaptive immune responses, as highlighted in our gene expression analysis. Subjects receiving calcifediol sustained better calcium homeostasis, which in turn could positively influence immune responses. The mutuality observed between calcitriol, calcium and PTH levels during calcifediol supplementation underscored the potential clinical relevance of optimizing vitamin D status. Our observations on the impact of calcifediol supplementation on serum calcium levels were limited to a period of 6 months. However, similar studies in a larger number of cohorts might be useful in proving the superiority of calcifediol over vitamin D supplementation in facilitating calcium absorption and utilization, leading to better calcium homeostasis. From a clinical perspective, this could have implications for bone health, muscle function, and other calcium-dependent physiological functions. Taken together, the findings from gene expression analysis support a dual, pro as well anti-inflammatory role of vitamin D involving several factors of innate and adaptive immune signaling pathways. It can be inferred that owing to its immunomodulatory properties, vitamin D influences our immune system in a spatiotemporally controlled manner, by enhancing specific and regulated immunogenicity of SARS-CoV-2 vaccination in subjects with optimal vitamin D status thus, reducing the chances of immunological severity of SARS-CoV-2 infection caused by exaggerated inflammatory responses following infection or re-infection. Our results further necessitate the need to explore mechanisms by which calcifediol promotes memory T-cell survival and homeostasis at the molecular level.

There were many challenges associated with this study including the variable confounding factors like heterogeneity in subjects including genetic diversity, varying exposure to SARS-CoV-2 prior to intervention, diet, and lifestyle that were beyond our control and these factors ultimately make the analysis process challenging. The strengths included adequate sample size, compliance of subjects, and time points of the study. It may also be noted that since the study was conducted on a small population, the same conclusions cannot be generalized for a larger population that inherits different genetic backgrounds. Therefore, more studies are needed to identify the optimal doses and confirm the importance of calcifediol supplementation or maintaining adequate vitamin D levels.

To conclude, the study presents an efficacious approach to improve the efficacy of not just COVID-19 but other viral vaccines, which is highly safe and offers better calcium homeostasis important for various physiological functions.

FUNDING

Dishman Carbogen Amcis Ltd, Ahmedabad, India.

Acknowledgements: The authors acknowledge the support of all the technical staff and volunteers including Kasmeen, Shivani Verma, Priya Hitaishi, Shallu Singhmar, Shallu, 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, Ahmedabad, India.

Role of the funding source:

The sponsors were not involved in the 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

Michael F. Holick: Grants from Solius Inc. and Carbogen-Amcis, Consultant Biogena Inc. and Solius Inc., Speaker's Bureau Sanofi Inc., Abbott Inc. All other authors declare no competing interest.

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There is a conflict of interest Michael F. Holick: Grants from Solius Inc. and Carbogen-Amcis, Consultant Biogena Inc. and Solius Inc., Speaker's Bureau Sanofi Inc., Abbott Inc. All other authors declare no competing interest.

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Calcifediol boosts the efficacy of ChAdOx1 nCoV-19 (COVISHIELD) vaccine via upregulation of key genes associated with memory T cell responses

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Journal Publication

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Abstract

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INTRODUCTION

METHODS

Subject Recruitment

Biochemical analyses:

cDNA Library Preparation and RNA-Sequencing

Differentially Expressed Genes (DEGs) Identification

Enrichment Analyses and Network Visualization

Statistical Analysis

RESULTS

Calcitriol Concentrations and Vitamin D Signaling:

Outcome of calcifediol supplementation on the gene expression profile of PBMCs

Differentially Expressed Genes (DEG) analysis

Upregulation of innate and adaptive immune signaling pathways upon calcifediol supplementation

Downregulation of Coronavirus Disease and Inflammatory Pathways

Gene ontology analysis on the role of calcifediol supplementation in biological processes

Genes related to memory T cells are upregulated among the differential expressed genes

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

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