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

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.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.