In a recent study in Pharmaceutics, researchers screened various compounds for anti-viral properties against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Additionally, in vivo and cell-based studies were adopted to investigate further the anti-viral properties of the active form of vitamin D, calcitriol.
Study: Evaluation of In Vitro and In Vivo Antiviral Activities of Vitamin D for SARS-CoV-2 and Variants. Image Credit: Kateryna Kon/Shutterstock
Background
The coronavirus disease 2019 (COVID-19) pandemic accelerated the research on developing anti-virals, vaccines, and monoclonal antibody therapies. COVID-19 vaccines have effectively limited the severity of SARS-CoV-2 infections, and anti-viral drugs such as remdesivir continue to treat COVID-19 patients. Despite this, the emergence of new SARS-CoV-2 variants with increased transmissibility and immune evasive abilities continues to be a cause for concern.
Therefore, there is a need to develop newer, more effective vaccines and anti-virals and to explore the anti-viral efficacy of existing therapeutics that can be repurposed against the emergent SARS-CoV-2 variants.
About the study
The present study used human nasal epithelial cells, Vero E6 African green monkey kidney cells, and human hepatoma cells for cell-based studies. The nasal epithelial cells were obtained through air-liquid interface cultures of nasal epithelial progenitor cells. These cells were procured from healthy humans undergoing plastic surgery of the septa.
Nasopharyngeal swabs of COVID-19 patients were used to obtain wild-type SARS-CoV-2, validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Once obtained, the SARS-CoV-2 was propagated in the Vero E6 cells.
Four compound libraries were screened to identify novel compounds with potential anti-viral properties against SARS-CoV-2. These included a 462-compound angiotensin converting enzyme-2 (ACE-2)-targeted compound library (CADD), a natural product library containing 57 compounds, a flavonoids library with 500 compounds, and a drug library containing 1,172 compounds. The latter was approved by the United States (U.S.) Food and Drug Administration (FDA).
The CADD compounds and the compounds in the natural product library were screened for efficacy in pre-infection treatment, while the flavonoids and the FDA-approved drugs were screened for post-infection treatment efficacy. Cell viability at four days after infection based on virus-induced cytopathic effects or toxicity to the compound was used to assess the primary screening results.
Dose-dependent inhibition assays and cell viability tests were also used to validate these results. Furthermore, primary human nasal epithelial cell lines were used to validate calcitriol, imatinib mesylate, and citicoline results.
The messenger ribonucleic acid (mRNA) expression levels of the vitamin D receptor, cathelicidin, and 24-hydroxylase were measured in uninfected and SARS-CoV-2 infected cells using qRT-PCR. Furthermore, untreated Vero E6 cells and cells treated with calcitriol were subjected to Western blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This was done to determine SARS-CoV-2 protein expression levels.
Keratin 18-human angiotensin converting enzyme-2 (K18-hACE2) mouse models were used for the in vivo tests involving treatment with different concentrations of calcitriol and subsequent intranasal infection with SARS-CoV-2. Viral titer assessments and histological analyses were conducted four days after infection.
Results
The results from the cell-based assays indicated that calcitriol exhibited strong anti-SARS-CoV-2 potency by increasing the expression of cathelicidin, an antimicrobial peptide, through the modulation of the vitamin D receptor. However, the in vivo tests using K18-hACE2 mice showed negligible changes in parameters such as viral titers, survival rate, weight, histological scoring, and physiological conditions in mice treated with calcitriol pre- and post-infection and challenged with SARS-CoV-2.
Furthermore, analysis of the expression levels of the vitamin D receptor mRNA in SARS-CoV-2-infected cells based on calcitriol treatment revealed that SARS-CoV-2 affects the vitamin D receptor pathway. In turn, meaning it can cause alterations in vitamin D metabolism. The upregulation of genes involved in controlling viral replication, such as 24-hydroxylase and cathelicidin, upon the exogenous addition of calcitriol, indicated that the active form of vitamin D does have an anti-viral role.
However, the inability of the in vivo test results to support the findings from the in vitro cell-based assays suggested that the utilization of calcitriol in the body differs from one species to another. Thus, explaining the differences between the studies in the mice models versus the Vero E6 and human epithelial cells or that cathelicidin is not regulated by vitamin D in mice. Additionally, the calcitriol dosage administered to the mice could have been insufficient to elicit protective anti-viral effects.
Furthermore, based on the dosage administered to the mice, the human equivalent dosage would be five micrograms of calcitriol per kilogram or more, which suggests that treatment with calcitriol in the usual dosage might not exhibit any anti-viral protective effects against SARS-CoV-2 in humans. Higher doses of calcitriol could also be harmful to patients with hyperparathyroidism.
Conclusions
The results indicated that cell-based assays using Vero E6 and human epithelial cells showed that calcitriol treatment before SARS-CoV-2 infection could exhibit protective effects. However, the in vivo tests using mice models did not corroborate those findings. The authors believe that additional studies on the pharmacokinetics of calcitriol are required to determine its prophylactic use against SARS-CoV-2.
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