key: cord-0961016-i1921zaa
authors: Toraih, Eman Ali; Sedhom, Jessica Ashraf; Dokunmu, Titilope Modupe; Hussein, Mohammad Hosny; Ruiz, Emmanuelle ML; Muthusamy, Kunnimalaiyaan; Zerfaoui, Mourad; Kandil, Emad
title: Hidden in plain sight: The effects of BCG vaccination in COVID-19 pandemic
date: 2020-06-12
journal: bioRxiv
DOI: 10.1101/2020.06.09.142760
sha: e09faeb9067a2052bb96f7842dfe3a8bc8de55aa
doc_id: 961016
cord_uid: i1921zaa

To investigate the relationship between BCG vaccination and SARS-CoV-2 by bioinformatic approach. Two datasets for Sars-CoV-2 infection group and BCG-vaccinated group were downloaded. Differentially Expressed Genes were identified. Gene ontology and pathways were functionally enriched, and networking was constructed in NetworkAnalyst. Lastly, correlation between post-BCG vaccination and COVID-19 transcriptome signatures were established. A total of 161 DEGs (113 upregulated DEGs and 48 downregulated genes) were identified in the Sars-CoV-2 group. In the pathway enrichment analysis, cross-reference of upregulated KEGG pathways in Sars-CoV-2 with downregulated counterparts in the BCG-vaccinated group, resulted in the intersection of 45 common pathways, accounting for 86.5% of SARS-CoV-2 upregulated pathways. Of these intersecting pathways, a vast majority were immune and inflammatory pathways with top significance in IL-17, TNF, NOD-like receptors, and NF-κB signaling pathways. Our data suggests BCG-vaccination may incur a protective role in COVID-19 patients until a targeted vaccine is developed. Supplementary Materials (https://drive.google.com/open?id=15Na738L282XNaQAJUh0cZf1WoG9jJfzJ)

The coronavirus pandemic now affects over 2 million people with nearly 130,000 deaths reported thus far, resulting in unprecedented ramifications on global health, social infrastructure, and economic trade. Since its emergence in Wuhan, China in 2019, SARS-coronavirus-2 (SARS-CoV-2), the causative agent of the disease, has spread rapidly across the globe to over 185 countries. Curiously, the degree of intensity has varied markedly between countries -even those with similar climates, geography, and/or healthcare infrastructure. The transmission pattern does not appear to follow previous SARS-CoV virus transmission 1 or climatic zones based on human transmission 2-4 as temperate and tropical countries have reported the disease 5 . The incidence of COVID-19 pandemic varies widely with the highest number of cases in the United States, Italy, China and lowest in regions of the world historically vulnerable to infectious disease such as sub-Saharan Africa. The disproportionately high morbidity and mortality of COVID-19 infection in some countries has been linked to several factors including, but not limited to differences in efforts to mitigate the disease spread (i.e. social distancing, travel restrictions), circulating SARS-CoV strains, and vaccination policies.

In order to confront COVID-19 disease, it is critical to characterize the virus' biological and immunological pathways.

Recent studies have uncovered the mechanism of viral entry of the SARS-CoV-2 relies on human angiotensin converting enzyme 2 (hACE2) acting as the host cell receptor. hACE2 interacts with the glycoprotein spike (S) of SARS-CoV2 in order to form a complex structure, a receptor-binding mode motif similar to some related viruses, including the one responsible for Severe Acute Respiratory Syndrome (SARS) [6] [7] [8] [9] [10] [11] . Another study, which analyzed the transcriptional changes in the immune genes of 3 COVID patients, reported increased inflammatory responses to the virus, revealing increased Tcell activation and cytokine expression, indicating pro-inflammatory pathways may be prognostic markers and/or serve as potential targets in COVID-19 disease 12 . Increased IL-6 expression and tumor necrosis factor (TNF-α) has also been reported, furthering the notion that cytokines are playing a key role in COVID-induced pneumonia 13 . Other studies have hypothesized that protection by the Bacille Calmette-Guérin (BCG) vaccination, which was originally developed to protect against Tuberculosis (TB) disease, may be playing a critical immunological role against CoV-Sars-2 [14] [15] [16] [17] [18] [19] .

The BCG is a century-old vaccine that is given as an attenuated live strain of Mycobacterium bovis used to confer immunity against some strains of TB. Various studies have indicated that BCG vaccination has a role extending far beyond TB treatment alone, eliciting non-specific effects (NSEs) within the innate immune system alongside the adaptive immune response 20 . BCG vaccination has been identified for its protective role specifically against respiratory viral infections, including Influenza A and respiratory syncytial virus (RSV) 21 , and is the standard therapy for certain types of bladder cancer. Although the exact mechanism remains elusive, BCG-protection is thought to be conferred by epigenetic and immunological moderation of the immune response through the release of pro-inflammatory cytokines (TNF-α, IL-6, and IFNγ) and the role of Vitamin D [22] [23] [24] . One such proposed mechanism behind the protective effects of BCG-vaccination heavily implicates the role of Vitamin D. During antimicrobial response (such as in M.tb infection), toll-like receptors (TLRs) upregulate the expression of VDR on immune cells and pulmonary epithelial cells. VDR then binds to calcitriol, the active form of Vitamin D, and together regulates the transcriptional activity of several antimicrobial peptides such as cathelicidin and beta-defensin [25] [26] [27] [28] . Vitamin D alters T-cell activation and IFN-gamma stimulation, increasing the expression of several pro-inflammatory cytokines. Overall, the non-specific effects following BCG vaccination are 3|P a g e conferred by epigenetic and transcriptional modulation of the innate immune system as evidenced by its far-reaching role in several viral infections.

Furthermore, it has been hypothesized that countries with nationalized BCG vaccination policies show decreased morbidity and mortality to COVID-19 when compared to those where no such uniform policy exists (such as the United States or Italy); however, these preliminary data is limited given it is yet to be peer-reviewed and fails to account for several confounding factors such as age, testing rates 29, 30 , and the accuracy of the BGC World Atlas 31 Overall, the far-reaching effects of BCG vaccination testify to its dynamic role: eliciting NSEs, curbing inflammation in cancer models, and reducing viremia in several distinct pathogens, including RSV, Influenza A, yellow fever, and herpes simplex virus 21, 32 . Our inclination is that BCG vaccination may provide putative immunity against COVID-19 until a targeted vaccine can be well studied and mass produced. Altogether, this study aims to elucidate the relationship between BCG vaccination and SARS-CoV-2 through bioinformatic analysis of biological and immunological pathways underlying both.

The microarray data analyzed in this study were obtained from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/), accession number GSE147507, published on March 25 th , 2020. This data set analyzed gene expression profile of a normal human bronchial epithelial (NHBE) cell line, derived from a 79-year-old Caucasian female, after SARS-CoV-2 viral infection. The following samples were analyzed; SARS-CoV-2 infected NHBE cells (GSM4432381, GSM4432382, GSM4432383) compared to mock treated NHBE cells (GSM4432378, GSM4432379, GSM4432380).

To compare the transcriptomic alterations in SARS-CoV-2 viral infection with host immune response following administration of BCG vaccine, the RNA sequencing data (GSE87186) was retrieved from the SRA database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87186) in Biojupies Analysis Notebook (https://amp.pharm.mssm.edu/biojupies/analyze) and processed by ARCHS4 (all RNA-seq and ChIP-seq sample and 5|P a g e

Through KEGG pathway enrichment analysis, down-regulated KEGG signaling pathways following BCG vaccination were intersected with up-regulated pathways in SARS-CoV-2 infection using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/). Deregulated genes in each pathway were explored and the direction of expression were compared between SARS-CoV-2 and BCG-vaccinated experiments in KEGG mapper (https://www.genome.jp/kegg/mapper.html).

Raw data for the reads is provided in Supplementary Table S1 . After normalization, the quality was checked in density and box plots (Supplementary Figure 1A 

Of 23710 genes screened in the experiment, 7107 genes with constant values were removed. A total of 161 annotated genes were identified to be differentially expressed, which included 113 upregulated DEGs and 48 downregulated genes (Supplementary Figure 1D) . The top variable genes between the SARS-CoV-2 and mock treated samples is shown in is an important structural protein that provides a protective barrier in stratified squamous epithelium and is associated with anti-microbial response; (b) Granulocyte-colony stimulating factor (CSF3), a proinflammatory cytokine, has been shown to impair CD8+ T cell functionality and act as a modulator of T-Cell and dendritic cell functions, (c) Intercellular adhesion molecule 2 (ICAM2) is expressed on bronchial epithelial, mediates adhesive interactions important for antigen-specific immune response, NK-cell mediated clearance, and lymphocyte recirculation, (d) S100A7A, immunogenic-related calcium binding protein, regulated by TLR4, and associated with psoriasis, (e) TNFAIP3-interacting protein 3 (TNIP3), which binds to zinc finger protein and inhibits NF-kappa-B activation induced by tumor necrosis factor, Toll-like receptor 4 (TLR4), and (f) PCNA-AS1, the antisense and regulator for Proliferating Cell Nuclear Antigen gene.

Of the down-regulated genes, (a) hes related family bHLH transcription factor with YRPW motif 2 (HEY2) is a nuclear transcription factor that represses DNA and negatively regulates miR-146a, IL-6, IL-1β, and TNF-α expression, (b) Protein Table S2 .

6|P a g e In order to analyze the aberrant gene expression pattern in SARS-CoV-2 infection, further functional analysis and annotation for DEGs were performed ( Figure 1C) . Thirteen lncRNAs were deregulated following SARS-CoV-2 treatment.

Annotation analysis revealed that PPT2-EGFL8 readthrough (PPT2-EGFL8, FC=-3.5169|p=5.86E-06) was previously associated with circulating phospho-and sphingolipid concentrations, some other were associated with gastric, breast, and prostate cancer as INHBA-antisense RNA 1 (INHBA-AS1, FC=4.0|p=2.41E-07), RHPN1 antisense RNA 1 (RHPN1-AS1, Table S2 ).

To explore the functions of the DEGs, the 113 upregulated and 48 downregulated genes were subjected to GO and KEGG pathway enrichment analysis. As shown in Figure 2A Table S4 ). In contrast, no significant GO terms or pathways were significant for the downregulated genes.

As depicted in Figure 2D , upregulated pathways are highly connected with inflammatory cytokines exhibiting significant crosstalk in particular. IL-6 (also known as B-stimulatory factor-2) is involved in the final differentiation of B cells into immunoglobulin-secreting cells and the induction of acute-phase reactants. IL-6 has sequence similarity with granulocyte colony-stimulating factors 2 and 3 (CSF2 and CSF3), which are also highly expressed following SARS-CoV-2 infection.

Further, several interleukins (IL17C, IL19, IL36A, and IL36G) noted serve as proinflammatory cytokines for the regulation of dendritic cells and T cells, as well as Interleukin-2 Receptor Subunit Gamma (IL2RG), which encodes a common gamma chain essential for IL-receptor function. Overexpression of 2 members of the CC chemokine family were observed: C-C Motif Chemokine Ligand 2 (CCL2) and C-C Motif Chemokine Ligand 2 (CCL20), which function to induce the migration of monocytes and lymphocytes, respectively (www.GeneCards.org)

Gene-miRNA interactions network was constructed ( Figure 2E ). It consisted of 77 seeds (significant genes), 1136 nodes, and 1776 edges mapped to the corresponding molecular interaction databases. KEGG pathway enrichment analysis of the network was significant for four pathways; namely IL-17 signaling pathway (p=7E-05, Hits=5/93), TNF signaling pathway (p=0.0017, Hits=4/110), cytokine-cytokine receptor pathway (p=0.012, Hits=5/294), and Influenza A (p=0.044, Hits=3/167). After extraction of nodes relevant to these immune-related pathways, the densely connected microRNAs in the cluster module included: miR-26b-5p, miR-26a-5p, miR-124-3p, miR-7-5p, miR-17-5p, miR-335-5p, mirR-24-3p, miR-203a-3p, and miR-122-5p.

Upstream regulatory cell signaling layers responsible for the observed pattern in gene expression following SARS-CoV-2 infection is depicted in Figure 2F -H. Network included integrated transcription factors and kinases with PPI. The top enriched transcription factors were RELA proto-oncogene (p=1.55E-06), BCL3 (p=0.0014), TP63 (p=2.9E-05) and VDR (p=0.0051). RELA, a NF-KB Subunit, has a key role in mediating inflammation, differentiation, cell growth, tumorigenesis and apoptosis (www.GeneCards.org). It is associated with 12 overlapping targets in the DEGs list (IER3, STAT1, NFKB2, ICAM1, DRAM1, NEDD9, IL32, NFKBIA, TNFAIP3, NFKBIZ, TNIP1). Another NF-kB regulator, BCL3, which has paradox effect according to its subcellular localization; regulates transcriptional activation of NF-kappa-B target gene while 8|P a g e being in the nucleus and inhibits the nuclear translocation of the NF-kappa-B p50 subunit when existed in the cytoplasm (www.GeneCards.org). From the DEGs defined following SARS-CoV-2 infection, six genes (IER3, STAT1, NFKB2, NFKBIA, IRF9, and BIRC3) were targeted by BCL3. The nuclear receptor for calcitriol, VDR, was also significantly associated with the up-regulated DEGs (NFKB2, NFKBIA, and IRAK2).

Gene set enrichment analysis (GSEA) showed over-representation of SARS-CoV-2 genes in Tuberculosis pathway (ID: hsa05152, p=6.09E-04). In this particular pathway, 116 DEGS (of 179 genes) were upregulated (Supplementary Figure   S2 ). IL6, TLR9, and TNF were at the top of that list. VDR and CEPB are further noted for their critical regulation of inflammatory and immune responses, including SARS-CoV-2 infected cells. (Degree=221) and AKT2 (Degree=48) were involved in 6 (out of 11) pathways.

A total of 52 enriched KEGG pathways were upregulated in SARS-CoV-2 infection. Cross-reference of these upregulated pathways with downregulated KEGG pathways in BCG vaccination experiment, 45 common pathways were intersected accounting for 86.5% of SARS-CoV-2 upregulated pathways (Figure 4A-C) . These pathways were categorized into the following groups: (1) cellular processes, (2) organismal systems, (3) environmental information, and (4) human diseases. 

To gain insight into the engendering mechanism of the SARS-CoV-2 infection, the gene expression profiles of the virus were systematically analyzed through bioinformatic techniques. In this study, a total of 161 DEGs, including 113 upregulated and 48 downregulated, were screened. The biological functions of these DEGs were explored based on GO function and pathway enrichment data. Further analysis indicated significant upregulation in 45 distinct KEGG pathways in SARS-CoV-2 infection overlapped with pathways down-regulated following the BCG vaccination. In addition to the pathway analysis, this study describes potential kinases, transcription factors, miRNAs, and lncRNAs differentially expressed in Sars-CoV-2 infection (Figure 2 and 5) . Several candidate DEGs, including IL-6, CCL20, CSF2, ICAM, and CXCL1/2 were highlighted for their key roles in respiratory viral infection, tuberculosis, and overall immune function. To elucidate the relationship between COVID-19 infection and BCG-vaccination, the common pathways were categorized into the following groups: inflammatory and immunoregulatory, signaling, and infectious pathways.

Of SARS-CoV-2 infected cells, analysis implicates several upregulated genes enriched in inflammatory pathways, most significantly in the IL-17 and NOD-like receptor pathways. Conversely, these same pathways were downregulated in the BCG-vaccinated group, suggesting BCG vaccination may compensate for aspects of pathway dysregulation in SARS-CoV-2 infection. The family of IL-17 mediates protective innate immunity against external pathogens and plays a central role in the self-clearance of intracellular pathogens 37, 38 . Additionally, T helper cells (Th17), which themselves produce Il-17, are known to be key players in the pathogenesis of chronic inflammatory diseases and autoimmune tissue destruction 39 .

11|P a g e Elevated Th17 responses and IL-17 pathways are seen in COVID-19 patients and have been linked to 'cytokine storms', a surge of pro-inflammatory molecules that are associated with the Acute Respiratory Distress Syndrome (ARDS) typically seen in these patients 40 . BCG vaccination has been shown to confer protective immunity in the Rhesus macaque model by decreasing Th17 cell maturation, thus suppressing Th17 response and preventing production of IL-17 23, 41 . Further, IL-17A and IL-17F induce proinflammatory gene expression of NF-B, MAPK, in synergy with TNF-and IL-6. IL-17 gene family also eliminates self-reactive T cells through production of granulocyte colony-stimulating factor (G-CSF) and several chemokines upregulated in Sars-CoV-2 infected cells, including CXCL1, CXCL2, CCL20 and IL-17 39, 42 (Figure 9 ).

The other major upregulated inflammatory pathway, NOD-Like receptor signaling pathway, detects various pathogens and stimulates innate immune responses against them, driving the subsequent activation of cytokine, NF-B, and MAPK pathways. Upregulation of NOD-like receptor signaling without appropriate negative feedback regulation can contribute to pathological tissue damage 43 . Innate and adaptive immune response to an invading pathogen relies on the ability of the body to recognize foreign elements and is stimulated by inflammasomes, which are intracellular multi-protein complexes such as NOD-like receptors (NLR) 44 . NLRC5 (short for NOD-like receptor family CARD domain containing 5) regulates major histocompatibility complex class I expression in the course of an infection. In accordance with this, a study reported knockdown of NLRC5 resulted in decreased levels of CD8+ cells in influenza A virus 45 . Our study identifies significant upregulation of IL-17 and NOD-like receptor signaling pathways in the immune response of COVID-19, pathways that were markedly downregulated in BCG-vaccination.

Similarly, several signal transduction pathways were up-regulated in Sars-CoV-2 and inversely downregulated in the BCGvaccinated group with TNF, NF-κB, MAPK and JAK/STAT signaling pathways conferring the most statistical significance.

TNF signaling upregulation is heavily involved in the complex regulation of immune cells. Following trimerization with either TNFR1 (expressed nearly ubiquitously) or TNFR2 (expressed mostly in immune cells), the TNF signaling pathway can activate NF-κB and MAPK signaling pathways downstream of it. Upon viral infection, NF-κB proteins regulate the transcription of many genes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and antiapoptotic proteins 46 . In a study of Middle Eastern Respiratory Syndrome (MERS), a viral infection caused by a member of the coronavirus family, researchers found the virus downregulated antiviral cytokines (TNF-alpha) as it induced proinflammatory cytokines (Il-1 beta, Il-6, Il-8) during initial infection 47 . In a study of Severe Acute Respiratory Syndrome (SARS), yet another member of the coronavirus family, TNF-alpha was similarly downregulated and induced NF-κB activation 46 . In the SARS pathogen, ACE2 was determined to be the functional receptor of the virus through the regulation of TNF-alpha, which ultimately permitted viral entry and promoted respiratory pathogenesis. Altogether, the literature on other members of the coronavirus family indicates a disruption of TNF-signaling pathway, which is in accordance with the marked pathway upregulation of TNF pathways signaling noted in our findings. In BCG vaccinated models in vitro, exposure of human peripheral blood mononuclear cells (PBMCs) to BCG treatment boosted IL-6 and TNF-α expression in response to lipopolysaccharide stimulation 22 . Similarly, BCG-immunized adults produced high TNF-α and IL-1β expression 3-month post-vaccination 23 . In addition to sustained cytokine response at 3 months, the production of heterologous Th1 and Th17 remained elevated 1-year post-BCG vaccination, concluding the vaccine created long-term immune responses to pathogens besides M.tb alone 23 .

12|P a g e Downstream of TNF, the NF-κB signaling pathway similarly affects a broad range of biological processes, including adaptive immune, inflammatory, and stress responses. Upon viral infection, NF-κB proteins regulate the transcription of many genes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and anti-apoptotic proteins 46 . Of the COVID-19 upregulated genes involved in the NF-κB pathway were CSF-2 and IL-6, transcription factors and innate immune system mediators 55, 56 58 . Several studies have recently advocated for the potential benefit of commercially available Jak 1 and 2 inhibitors as anti-inflammatory treatment in COVID-19 cases 58 ; however, there is concern from the scientific community that JAK inhibitors may promote the evolution of SARS-CoV-2 virus as this has been reported in herpes viruses. BCG vaccination may be key in attenuating the JAK/STAT pathway without entirely impairing interferon-mediated response. In vitro experiments show that BCG vaccination induces 2 members of the suppressor of cytokine signaling (SOCS) family, SOCS1/3, eliciting a negative feedback regulator of the JAK/STAT signaling cascade via IFN-gamma regulation 59 .

Overall, although TNF, NF-κB, MAPK, and JAK/STAT signaling were upregulated in SARS-CoV-2 infection, they were markedly downregulated in BCG, suggesting BCG vaccination may be targeting these same pathways. The exact mechanism remains elusive, but identification of these pathways and their targets are key to mitigating pathogenesis and merit further investigation.

Further, in the current study, BCG vaccination down regulated several pathways related to viral and bacterial inflammatory disease pathways including influenza A, measles, Herpes simplex virus 1, Legionellosis, and tuberculosis, that were up-13|P a g e regulated in SARS-CoV-2 infection. Prior studies have demonstrated non-specific effects of BCG against viral infection 21 , and bacterial infections 32, 60 . In one study, mice inoculated with BCG displayed overall increased resistance to encephalomyocarditis, murine hepatitis, type 1 and 2 herpes simplex, foot-and-mouth disease and A0 and A2 influenza viruses 61 . Intercellular adhesion molecules (or CD54), included in the upregulated DEGs, are receptors utilized for pathogen entry into host cells that promote intercellular signaling and survival of virus in the host cell. In human rhinovirus infection and influenza virus, ICAM-1 is involved in viral protein uncoating, delivering the viral RNA genome into the cytoplasm of host cells across a lipid bilayer 62, 63 . Other studies corroborate this showing that most viruses interact with and induce ICAM-1 expression, including HIV, Human Parainfluenza virus, and rhinovirus infection [64] [65] [66] . Presence of cytokines such as IFN-γ, TGF-beta, and TNFα induces expression of ICAM on macrophage for activation of T cells, this was observed in human TB infected macrophages 67 . BCG immunotherapy of bladder tumors cells showed mycobacteria infection-induced expression of ICAM-1 molecules, thereby eliciting immune response through improved antigen presentation to T lymphocytes 68 . BCG is thought to inhibit inflammasome activation via Zinc metalloproteases in order to improve immunogenicity 60, 69 and remains the current standard therapy for non-muscle invasive bladder cancer.

Compared to mock-treated cells, SARS-CoV-2 infected cells exhibited significant enrichment of VDR transcription factor, one of the major downstream signaling transduction nuclear receptor in tuberculosis KEGG pathway, and was significantly associated with the up-regulated DEGs, namely NFKB2, NFKBIA, and IRAK2, which are related to NF-κB and Toll-like receptor signaling pathways. Normally, VDR binds to calcitriol, the active form of Vitamin D. The VDR-calcitriol complex interacts with retinoid-X-receptor to form a heterodimer capable of regulating transcriptional activity, leading to several pleiotropic transcriptional effects. Similarly, VDR gene expression was continuously upregulated during HIV 70 and influenza virus infection 71 . Previously reported, VDR signaling may repress cytokine gene expression in activated T-cells, consequently reducing inflammatory response 28, 72 . Part of the mechanism of action of BCG vaccine against tuberculosis is through increased production of Vitamin D, which binds with its nuclear receptor VDR, resulting in the generation of antimicrobial peptides (cathelicidin and beta-defensin) and death of intracellular M.tb 44 . In short, Vitamin D plays an important role in modulating the innate and adaptive immune response alongside its classically characterized role in bone health. Long before the scientific basis was understood, Vitamin D was unknowingly and empirically being used to help tuberculosis patients who were sent to sanatoriums for sun-light exposure and prescribed cod-liver -remedies rich in Vitamin D 73 . Since then, Vitamin D has been well characterized for its immuno-modulatory properties, including the regulation of IFN-gamma, a key activator of macrophage response 74 and promoting the production of regulatory T cells 75 .

In addition to its role in M.tb 76 , cathelicidin is a highly conserved protein that has been shown to direct antiviral activity in several respiratory viral infections including respiratory syncytial virus, human Rhinovirus (HRV), and influenza A [77] [78] [79] [80] [81] [82] . In vitro, Vitamin D has been shown to counteract the M.tb-induced downregulation of cathelicidin by actually recovering cathelicidin levels and promoting Th1 cell differentiation. Following BCG vaccination, Vitamin D levels remain elevated, indicating Vitamin D is being upregulated long after the initial inoculation. It has been postulated that increased calcitriol recruits dendritic cells from the site of inoculation to the lymph nodes, accounting for the sustained response 24 . This serves as a possible mechanism of protective effects of BCG vaccination in relation to consistently elevated plasma Vitamin D concentration present for as long as 9 months as reported by the authors 24 . A major complication of COVID-19 infection is inflammation of the air sacs of the lungs resulting in pneumonia. Vitamin D deficiency contributed to the pathogenesis of 14|P a g e acute respiratory distress syndrome (ARDS) 83 . Biotransformation into the active form of vitamin D-1,25-dihydroxyvitamin D (calcitriol) by the 1-α-hydroxylase (CYP27B1) enzyme found in liver cells, and immune cells expression of VDR, confirms major role of Vitamin D in immune regulation; both in innate and adaptive immune responses 25-28,72. . Mutation in the VDR gene single nucleotide polymorphism reported in children was associated with response to viral infection in children 82 . Given these findings, the transcriptional upregulation of VDR in SARS-CoV-9 may be contributing to the dysregulation of this response by upregulating cytokine response, interrupting microbial peptides, and promoting immune invasion in the lungs; therefore, the well characterized role of VDR in BCG vaccination could serve as a potential biomarker against COVID-19 disease.

In addition to VDR, our bioinformatic analysis showed deregulation of multiple transcription factors in SARS-CoV-2 infection. Specifically, TAL2, DACH2, and AFF2 were upregulated whose functional role in viral immune response is unclear, while HEY2, KLF2, EGR2, and ZBTB16 were under-expressed and were previously reported to have eminent role in fine-tuning immune response. HEY2 was the most downregulated gene in our list. It is a known transcription repressor, modulating cardiovascular development, neurogenesis, and oncogenesis 84 . A recent study demonstrated the role of HEY2 in inflammation, namely chronic periodontitis, via negative regulation of IL-6, IL-1β, and TNF-α expressions 85 . KLF2 is a key player in T cell differentiation, trafficking, quiescence, and survival, in addition to the regulation of endothelial function.

Mice with KLF2 deficiency experienced an activated T cell phenotype with severely reduced T cells in the periphery and increased susceptibility to HIV-1 infection 86, 87 . EGR2 transcription factor can control adaptive innate immunity and lead to functional impairment of T cells 88 

In the present study, we observed some deregulated miRNAs and highlighted the relationship between DEGs and miRNAs networking which may account for the transcriptomic changes evident in SARS-CoV-2 expression. Of particular note, mir-26a-5p, miR-26b-5p, and miR-124-3p, which have all been well characterized for their involvement in viral and bacterial inflammatory pathways, particularly Influenza A, RSV, and M.tb, were noted here for their putative respiratory pathogenesis. In M.tb infection, miR-26a-5p can modulate macrophage IFN-gamma responsiveness 3 . In another study, this same microRNA was shown to be downregulated in Influenza A virus in humans, contributing to viral pathogenesis 4 . The other family member, miR-26b-5p, can inhibit viral replication in Vesicular stomatitis virus (VSV) and Sendai virus (SeV) by inducing type-1 IFN expression 5 . RSV acts to upregulate miR-26b, which in turn inhibits Toll-like receptor TLR4, a key sentinel in adaptive immune response 6 

To the best of our knowledge, our study is the first to dig into the regulatory mechanisms underlying the host immune response against SARS-CoV-2infection and to provide a putative protective role of BCG vaccination. Altogether, several DEGs with a high connectivity degree in the PPI network were identified in this study, which showed the strong inverse correlation in SARS-CoV-2 infection and BCG vaccination with upregulation in the former and downregulation in the latter. This inverse relationship suggests BCG vaccination may mediate key pathways in SARS-CoV-2 infection through NSEs. Nevertheless, it is essential to recognize some limitations of administering BCG vaccines, including, the heterogeneity of BCG response due to pharmacogenomic variation of patients, contraindications of BCG-vaccination for immunosuppressed patients, and the presence of different strains of BCG vaccines in the market 100 . Although the exact mechanisms with which BCG elicits protective effects are not entirely understood, its efficacy in infectious, oncogenic, and inflammatory disease are well documented and may confer similar effects if COVID-19.

In summary, bioinformatic approach identified key differentially expressed genes involved in the pathogenesis of COVID-19. Significant nodes in the PPI network, including high degrees in IL-17 signaling, TNF signaling pathway, NOD-like receptor, and NF-κB give novel insight into the inflammatory and signal transduction pathways mediating SARS-CoV-2 viral infection. The 45 common pathways upregulated in SARS-CoV-19 and downregulated in BCG vaccination suggest BCG vaccination may help to mitigate this pathway dysregulation. Although further experimental validation is warranted to verify our discoveries, the results give insight into potential biomarkers and targeted therapy to address the ongoing COVID-19 pandemic. We sustain BCG vaccination may be a safe and cost-effective alternative in incurring partial protection against COVID-19 pandemic until more targeted measures can be produced and implemented.

16|P a g e ET and EK designed the study. ET and MH analyzed the data. ET and EK interpreted the data. ET, JS, and TD search literature and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

We declare no competing interests

No supporting fund for the current study.

17|P a g e 

Supplementary Table S1 . Raw expression data in SARS-CoV-2 infected human normal bronchial epithelial cell line compared to mock-treated cells. 

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The mitogen-activated protein kinase (MAPK) pathway: role in immune evasion by Trypanosomatids

Role of MAPK/MNK1 signaling in virus replication

Effect of p38 mitogen-activated protein kinase on the replication of encephalomyocarditis virus

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages

Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFNgamma-and CD40L-mediated costimulation

Clinical and immunological features of severe and moderate coronavirus disease 2019

Baricitinib as potential treatment for 2019-nCoV acute respiratory disease

Mycobacterium bovis bacillus Calmette-Gueérin infection promotes SOCS induction and inhibits IFN-γ-stimulated JAK/STAT signaling in J774 macrophages

Non-specific immunity of BCG vaccine: A perspective of BCG immunotherapy

Increased resistance to virus infections of mice inoculated with BCG (Bacillus calmetteguérin)

ICAM-1 regulates the survival of influenza virus in lung epithelial cells during the early stages of infection

ICAM-1 receptors and cold viruses

Circulating intercellular adhesion molecule-1 (ICAM-1) as an early and sensitive marker for virus-induced T cell activation

Human parainfluenza virus type 3 upregulates ICAM-1 (CD54) expression in a cytokine-independent manner

Antibody to ICAM-1 mediates enhancement of HIV-1 infection of human endothelial cells

Mycobacterium tuberculosisinfected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcgammaRII and the mannose receptor

Induction of ICAM 1 expression on bladder tumours by BCG immunotherapy

Mycobacterium tuberculosis prevents inflammasome activation

Activation of the human immunodeficiency virus type I long terminal repeat by 1 alpha, 25-dihydroxyvitamin D3

Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells

Vitamin D signaling, infectious diseases, and regulation of innate immunity

A short history of phototherapy, vitamin D and skin disease

Immunosuppressive actions of 1,25-dihydroxyvitamin D3: preferential inhibition of Th1 functions

Vitamin D and its analogs as regulators of immune activation and antigen presentation

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages

Cathelicidins display conserved direct antiviral activity towards rhinovirus

The human cathelicidin LL-37 has antiviral activity against respiratory syncytial virus

The role of vitamin D in prevention and treatment of infection

Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response

Association between vitamin D receptor gene polymorphisms and response to treatment of pulmonary tuberculosis

Confirmation of an association between single nucleotide polymorphisms in the VDR gene with respiratory syncytial virus-related disease in South African children

Vitamin D deficiency contributes directly to the acute respiratory distress syndrome (ARDS)

Hey genes in cardiovascular development

microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis

Krüppel-like transcription factors: a functional family

Kruppel-like factor 2 regulates thymocyte and T-cell migration

Egr2 and 3 control adaptive immune responses by temporally uncoupling expansion from T cell differentiation

Role for EGR2 in the T-cell response to influenza

Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases

Chandipura Virus Utilizes the Prosurvival Function of RelA NF-κB for Its Propagation

A. p63 is necessary for the activation of human papillomavirus late viral functions upon epithelial differentiation

Mycobacterium tuberculosis decreases human macrophage IFN-γ responsiveness through miR-132 and miR-26a

microRNAs in circulation are altered in response to influenza A virus infection in humans

MiR-26b inhibits virus replication through positively regulating interferon signaling

Respiratory syncytial virus infection inhibits TLR4 signaling via up-regulation of miR-26b

MiR-23a facilitates the replication of HSV-1 through the suppression of interferon regulatory factor 1

Regulatory role of small nucleolar RNAs in human diseases

A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics

Tuberculosis vaccines in clinical trials

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