The modulatory action of C-Vx substance on the immune system in COVID-19

Abstract

The modulatory effect of C-Vx, a novel therapeutic agent, on the immune system of COVID-19 patients was investigated. The functions of T and NK cells of COVID-19 patients with different disease severity were evaluated by flow cytometry in response to C-Vx stimulation. The levels of pro- and anti-inflammatory cytokines were detected by multiplex assay in supernatants after cell culture with C-Vx. Bradykinin, IRF3, and IFN-α levels were also measured by ELISA in the presence or absence of C-Vx stimulation. As a result, increased CD107a expression was observed on NK cells in response to C-Vx addition. The proliferation of T cell subsets was increased by C-Vx, decreasing by disease severity. IL-4 and IL-10 levels were elevated while IFN-γ and IL-17 levels were reduced in T cells following C-Vx stimulation. However, the levels of pro-inflammatory IL-1β, IL-6, IL-8, IFN-γ and GM-CSF were significantly increased upon C-Vx stimulation. IFN-α levels tended to increase after incubation with C-Vx. These findings support an immunomodulatory action of C-Vx on the immune system of patients with a mild and moderate phase of COVID-19.

Keywords: C-Vx; COVID-19; cytokines; cytotoxicity; immune system; inflammation; lymphocytes; proliferation.

Figure 1.

The evaluation of the effect of C-Vx on T and NK cell ratios and NK cell cytotoxicity. (A) CD3⁺ total T, (B) CD3⁻CD16⁺CD56⁺ NK, (C) CD4⁺, (D) CD8⁺ T cell frequencies, and (E) CD107a expression of NK cells in patient groups and healthy donors with and without C-Vx. (*p < 0.05, **p < 0.001, ***p < 0.0001) (US: unstimulated, HD: healthy donors).

Figure 2.

The alterations in intracellular cytokine levels of T cell subsets in response to C-Vx. IFN-γ, IL-17, TNF-α, IL-4, and IL-10 levels of (A) CD4⁺ and (B) CD8⁺ T cells after culture of PBMCs stimulated or not with C-Vx. (*p < 0.05, **p < 0.001, ***p < 0.0001) (US: unstimulated, HD: healthy donors).

Figure 3.

Determination of lymphocyte proliferation in response to C-Vx. The proliferation capacities of (A) total lymphocytes, (B) CD3⁺ T cells, (C) CD3⁻CD16⁺CD56⁺ NK cells, (D) CD4⁺, and (E) CD8⁺ T cell subsets after cell culture with/without PHA stimulation with or without C-Vx. (*p < 0.05, **p < 0.001, ***p < 0.0001) (US: unstimulated, HD: healthy donors).

Figure 4.

Bradykinin, IRF3, and IFN-α levels in response to C-Vx. The effects of C-Vx addition on (A) IRF3, (B) Bradykinin, and (C) IFN-α levels in plasma and culture supernatants were measured by ELISA method (*p < 0.05, **p < 0.001, ***p < 0.0001) (US: unstimulated, HD: healthy donors).

Figure 5.

The alterations in cytokine levels after C-Vx addition. IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, TNF-α, and GM-CSF levels were measured by multiplex assay in supernatants of cultured PBMCs of patients and healthy donors with/without C-Vx. (*p < 0.05, **p < 0.001, ***p < 0.0001) (US: unstimulated, HD: healthy donors).

Figure 6.

The network analysis of immune parameters in COVID-19 patients with/without C-Vx. Blue and red lines indicate positive and negative correlations. The significance of correlations increases in proportion with increased darkness of line colours. (US: unstimulated).