Inhibition of SARS-CoV-2 Infection in Vero Cells by Bovine Lactoferrin under Different Iron-Saturation States

Alves et al., Pharmaceuticals, doi:10.3390/ph16101352

Sep 2023

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In Vitro study showing that iron-free and iron-saturated bovine lactoferrin inhibited SARS-CoV-2 ancestral and omicron Vero cell infection. Treatment before or during infection was effective, while treatment after infection showed no significant effect. Saturated lactoferrin showed improved efficacy, with 70% vs. 55% reduction for treatment before infection. Electron microscopy confirmed improved efficacy with saturated lactoferrin.

The results suggest that lactoferrin treatment may be most effective if given prophylactically or immediately at the onset of COVID-19 symptoms. While both iron-free and iron-saturated lactoferrin inhibited infection, the saturated form appeared more effective, suggesting that iron supplementation may improve lactoferrin's efficacy against COVID-19.

13 preclinical studies support the efficacy of lactoferrin for COVID-19:

2 In Silico studies Cutone, Miotto

10 In Vitro studies Alves, Andreu, Cutone, Kobayashi-Sakamoto, Mirabelli, Ostrov, Piacentini, Salaris, Yathindranath, Yazawa

1 In Vivo animal study Berkowitz

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1. Alves et al., Inhibition of SARS-CoV-2 Infection in Vero Cells by Bovine Lactoferrin under Different Iron-Saturation States, Pharmaceuticals, doi:10.3390/ph16101352.mdpi.com, doi.org.

2. Andreu et al., Liposomal Lactoferrin Exerts Antiviral Activity against HCoV-229E and SARS-CoV-2 Pseudoviruses In Vitro, Viruses, doi:10.3390/v15040972.mdpi.com, doi.org.

3. Berkowitz et al., Sigma Receptor Ligands Prevent COVID Mortality In Vivo: Implications for Future Therapeutics, International Journal of Molecular Sciences, doi:10.3390/ijms242115718.mdpi.com, doi.org.

4. Cutone et al., Lactoferrin binding to Sars-CoV-2 Spike glycoprotein protects host from infection, inflammation and iron dysregulation., Research Square, doi:10.21203/rs.3.rs-1605740/v1.researchsquare.com, doi.org.

5. Kobayashi-Sakamoto et al., Bovine lactoferrin suppresses the cathepsin-dependent pathway of SARS-CoV-2 entry in vitro, International Dairy Journal, doi:10.1016/j.idairyj.2023.105805.sciencedirect.com, doi.org.

6. Miotto et al., Molecular Mechanisms Behind Anti SARS-CoV-2 Action of Lactoferrin, Frontiers in Molecular Biosciences, doi:10.3389/fmolb.2021.607443.frontiersin.org, doi.org.

7. Mirabelli et al., Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19, Proceedings of the National Academy of Sciences, doi:10.1073/pnas.2105815118.pnas.org, doi.org.

8. Ostrov et al., Highly Specific Sigma Receptor Ligands Exhibit Anti-Viral Properties in SARS-CoV-2 Infected Cells, Pathogens, doi:10.3390/pathogens10111514.mdpi.com, doi.org.

9. Piacentini et al., Lactoferrin Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain, International Journal of Molecular Sciences, doi:10.3390/ijms23105436.mdpi.com, doi.org.

10. Salaris et al., Protective Effects of Lactoferrin against SARS-CoV-2 Infection In Vitro, Nutrients, doi:10.3390/nu13020328.mdpi.com, doi.org.

11. Yathindranath et al., Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection, International Journal of Nanomedicine, doi:10.2147/IJN.S448005.dovepress.com, doi.org.

12. Yazawa et al., Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19, Research Square, doi:10.21203/rs.3.rs-2676422/v1.researchsquare.com, doi.org.

Alves et al., 25 Sep 2023, peer-reviewed, 16 authors.

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

This Paper Lactoferrin All

Inhibition of SARS-CoV-2 Infection in Vero Cells by Bovine Lactoferrin under Different Iron-Saturation States

Nathalia S Alves, Adriana S Azeve, Brenda M Dias, Ingrid S Horbach, Bruno P Setatino, Caio B Denani, Wales D Schwarcz, Sheil Maria B Lima, Sotiris Missailidis, Ana Paula, D An Bom, Andréa M V Silva, Débora Barreto Vieira, Marcos Alexandr, N Silva, Caroline A Barros, Alberto M Carlos, Carvalho, Rafael B Gonçalves

doi:10.3390/ph16101352

Despite the rapid mass vaccination against COVID-19, the emergence of new SARS-CoV-2 variants of concern, such as omicron, is still a great distress, and new therapeutic options are needed. Bovine lactoferrin (bLf), a multifunctional iron-binding glycoprotein available in unsaturated (apo-bLf) and saturated (holo-bLf) forms, has been shown to exert broad-spectrum antiviral activity against many viruses. In this study, we evaluated the efficacy of both forms of bLf at 1 mg/mL against infection of Vero cells by SARS-CoV-2. As assessed with antiviral assays, an equivalent significant reduction in virus infection by about 70% was observed when either form of bLf was present throughout the infection procedure with the SARS-CoV-2 ancestral or omicron strain. This inhibitory effect seemed to be concentrated during the early steps of virus infection, since a significant reduction in its efficiency by about 60% was observed when apo-or holo-bLf were incubated with the cells before or during virus addition, with no significant difference between the antiviral effects of the distinct iron-saturation states of the protein. However, an ultrastructural analysis of bLf treatment during the early steps of virus infection revealed that holo-bLf was somewhat more effective than apo-bLf in inhibiting virus entry. Together, these data suggest that bLf mainly acts in the early events of SARS-CoV-2 infection and is effective against the ancestral virus as well as its omicron variant. Considering that there are no effective treatments to COVID-19 with tolerable toxicity yet, bLf shows up as a promising candidate.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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