Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. MTT Assay
2.3. Treatments
2.4. Viruses
2.5. Time Course and MOI for VSV-SARS-CoV-2-S∆21
2.6. Viral Titration Protocol for SARS-CoV-2
2.7. Models of Infection
- Pre-treatment model: Vero E6 cells were pre-treated with the specific hormone, seeded in 96-wells plate, and the day after the treatment was removed, the virus was added in presence of the compounds; after 3 h of viral adsorption, the viral inoculum was removed, cells were washed, and then medium without treatments was added.
- Pre–post-treatment model: Infection was performed as in pre-treatment model, but after the 3 h of viral adsorption, medium with the different treatments was added.
2.8. Real Time qPCR
2.9. Bio-Plex Multiplex System
2.10. Statistical Analysis
3. Results
3.1. MTT E2 and Calcitriol
3.2. VSV-Based Pseudovirus SARS-CoV-2 Infection: MOI and Time Course
3.3. Models of Infection and Plaque Assay: VSV-Based Pseudovirus SARS-CoV-2
3.4. Models of Infection and Viral Titre (TCID50/mL) SARS-CoV-2
3.5. VSV-Based Pseudovirus SARS-CoV-2 and SARS-CoV-2 Entry: Gene Expression of ACE2 and VDR
3.6. Cytokines
3.7. SARS-CoV-2 Infection and PD-L1 Expression
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
SARS-CoV-2 | Severe Acute Respiratory Syndrome Coronavirus-2 |
COVID-19 | Coronavirus Disease 19 |
E2 | 17,β-Estradiol |
Calcitriol | 1,25 α Dihydroxyvitamin D3 |
ACE2 | Angiotensin-Converting Enzyme 2 |
VDR | Vitamin D Receptor |
PD-L1 | Programmed Death-Ligand 1 |
WHO | World Health Organization |
SARS-CoV | Severe Acute Respiratory Syndrome Coronavirus-1 |
MERS-CoV | Middle East Respiratory Syndrome |
+ssRNA | Positive Single-Stranded RNA |
TMPRSS2 | Transmembrane Serine Protease 2 |
CSS | Cytokine Storm Syndrome |
sPD-L1 | Soluble Programmed Death-Ligand 1 |
PD-1 | Programmed Death-1 |
TGFβ | Transforming Growth Factor-β |
NO | Nitric Oxide |
CVDs | Cardiovascular Disease |
E1 | Estrone |
E3 | Estriol |
E4 | Estetrol |
ERs | Estrogen Receptors |
EREs | Estrogen Response Elements |
Ap1 | Activator Protein-1 |
Sp1 | Specificity Protein-1 |
PI3K | Phosphatidylinositol 3-Kinase |
MAPK | Mitogen-Activated Protein Kinase |
RXRs | Retinoid X Receptors |
VDRE | Vitamin D Receptor Element |
DMEM | Dulbecco’s Modified Eagle’s Medium |
FBS | Fetal Bovin Serum |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide |
VSV | Vesicular Stomatitis Virus |
GFP | Green Fluorescent Protein |
FFU | Foci Forming Unit |
CPE | Cytopathic Effect |
TCID 50 | 50% Tissue Culture Infectious Dose |
TEM | Transmission Electronic Microscope |
ON | Overnight |
LL-37 | Antimicrobial Peptide Cathelicidin |
VEGF | Vascular Endothelial Growth Factor |
ARDS | Acute Respiratory Distress Syndrome |
Appendix A
Treatment | ||||
---|---|---|---|---|
Pre-Treatment | ||||
CT INF | E2 INF | CALCITRIOL INF | E2 + CALCITRIOL INF | |
130 | 105 | 117 | 108 | |
158 | 95 | 107 | 145 | |
313 | 101 | 110 | 122 | |
137 | 118 | 125 | 97 | |
130 | 70 | 103 | 65 | |
114 | 70 | 98 | 54 | |
138 | 87 | 81 | 112 | |
130 | 77 | 83 | 75 | |
151 | 67 | 80 | 70 | |
Pre–Post-Treatment | ||||
146 | 84 | 133 | 128 | |
194 | 97 | 121 | 126 | |
131 | 90 | 115 | 127 | |
114 | 94 | 94 | 115 | |
118 | 108 | 128 | 104 | |
115 | 90 | 142 | 130 | |
180 | 84 | 119 | 102 | |
119 | 94 | 80 | 110 | |
153 | 90 | 104 | 77 |
Cytokines/Chemokines and Growth Factors Average Concentrations (pg/mL) | |||||||||
---|---|---|---|---|---|---|---|---|---|
Eotoxin | IL-5 | IL-6 | IL-8 | IL-10 | IL-12 | IL-15 | Rantes | VEGF | |
Pre-Treatment | |||||||||
E2 INF | 0.34 | 5.6 | 0.07 | 0.23 | 0.00 | 1.43 | 13.04 | 0.57 | 33.36 |
CALCITRIOL INF | 0.40 | 7.01 | 0.17 | 0.12 | 0.26 | 2.09 | 16.38 | 0.68 | 23.08 |
E2 + CALCITRIOL INF | 0.38 | 8.16 | 0.08 | 0.05 | 0.00 | 2.09 | 10.40 | 0.32 | 24.05 |
CT INF | 0.36 | 5.84 | 0.07 | 0.10 | 0.05 | 2.26 | 12.98 | 0.5 | 19.31 |
E2 | 0.34 | 3.00 | 0.96 | 0.26 | 0.00 | 1.76 | 9.87 | 0.57 | 0.00 |
CALCITRIOL | 0.38 | 4.45 | 4.11 | 0.13 | 0.00 | 2.09 | 12.07 | 0.45 | 47.25 |
E2 + CALCITRIOL | 0.38 | 6.16 | 10.95 | 0.05 | 0.00 | 1.43 | 14.02 | 0.44 | 33.87 |
CT | 0.37 | 4.44 | 3.32 | 0.12 | 0.02 | 1.93 | 11.72 | 0.62 | 27.00 |
Pre–Post-Treatment | |||||||||
E2 INF | 0.34 | 5.3 | 0.13 | 0.00 | 0.00 | 1.76 | 6.56 | 0.57 | 0.00 |
CALCITRIOL INF | 0.38 | 5.31 | 0.39 | 0.05 | 0.00 | 2.09 | 7.75 | 0.45 | 11.65 |
E2 + CALCITRIOL INF | 0.38 | 3.54 | 0.08 | 0.17 | 0.00 | 1.76 | 12.07 | 0.62 | 14.93 |
CT INF | 0.37 | 4.72 | 0.05 | 0.08 | 0.02 | 2.01 | 8.14 | 0.59 | 11.05 |
E2 | 0.41 | 6.44 | 10.98 | 0.18 | 0.00 | 2.42 | 13.11 | 0.57 | 48.09 |
CALCITRIOL | 0.27 | 6.17 | 2.22 | 0.12 | 0.00 | 2.42 | 11.03 | 0.38 | 20.66 |
E2 + CALCITRIOL | 0.38 | 4.16 | 4.44 | 0.05 | 0.00 | 1.76 | 10.91 | 0.57 | 31.32 |
CT | 0.39 | 2.97 | 6.62 | 0.11 | 0.00 | 1.84 | 12.74 | 0.50 | 42.78 |
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Vercellino, N.; Ferrari, A.; Sammartino, J.C.; Bellan, M.; Iskandar, E.; Lilleri, D.; Minisini, R. Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry. Pathogens 2025, 14, 765. https://doi.org/10.3390/pathogens14080765
Vercellino N, Ferrari A, Sammartino JC, Bellan M, Iskandar E, Lilleri D, Minisini R. Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry. Pathogens. 2025; 14(8):765. https://doi.org/10.3390/pathogens14080765
Chicago/Turabian StyleVercellino, Nicole, Alessandro Ferrari, José Camilla Sammartino, Mattia Bellan, Elizabeth Iskandar, Daniele Lilleri, and Rosalba Minisini. 2025. "Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry" Pathogens 14, no. 8: 765. https://doi.org/10.3390/pathogens14080765
APA StyleVercellino, N., Ferrari, A., Sammartino, J. C., Bellan, M., Iskandar, E., Lilleri, D., & Minisini, R. (2025). Influence of Sex and 1,25α Dihydroxyvitamin D3 on SARS-CoV-2 Infection and Viral Entry. Pathogens, 14(8), 765. https://doi.org/10.3390/pathogens14080765