Exploratory analyses of leukocyte responses in hospitalized patients treated with ozanimod following a severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infection

Abstract

Sphingosine-1-phosphate receptor 1 (S1P₁) ligands effectively reduce immunopathological damage in viral pneumonia models. Specifically, S1P₁ ligands inhibit cytokine storm and help preserve lung endothelial barrier integrity. We recently showed that the S1P receptor ligand ozanimod can be safely administered to hospitalized patients with coronavirus disease 2019 (COVID-19) exhibiting severe symptoms of viral pneumonia, with potential clinical benefits. Here, we extend on this study and investigate the impact of ozanimod on key features of the immune response in patients with severe COVID-19. We quantified circulating cytokine levels, peripheral immune cell numbers, proportions and activation status; we also monitored the quality of the humoral response by assessing anti-severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) antibodies. Our findings reveal that patients receiving ozanimod during acute SARS-CoV-2 infection exhibit significantly reduced numbers of circulating monocytes compared with those receiving standard care. Correspondingly, in the ozanimod-treated group, circulating levels of C-C motif ligand 2 (CCL2) were decreased. While treatment with ozanimod negatively impacted the humoral response to COVID-19 in unvaccinated patients, it did not impair the development of a robust anti-SARS-CoV-2 antibody response in vaccinated patients. These findings suggest that ozanimod influences key immune mechanisms during the acute phase of SARS-CoV-2 infection.

Keywords: COVID‐19; S1P; anti–SARS‐CoV‐2 antibodies; clinical trial; monocytes.

Figure 1

Impact of ozanimod on circulating lymphocyte proportions and CD69 surface expression in patients with coronavirus disease 2019 (COVID‐19). Peripheral blood mononuclear cells (PBMCs) from patients with severe COVID‐19 were isolated for flow cytometry analyses. Gating was performed as shown in Supplementary figure 1a and the proportion of (a) B cells, (b) T cells and (c) natural killer (NK) cells were quantified in both standard of care (SOC) and ozanimod (OZA) groups. (d) CD4 and CD8 T cells are shown as a ratio. Median fluorescence intensities (MFIs) for CD69 on (e) CD4 T cells, (f) CD8 T cells and (g) B cells are depicted. Baseline fluorescence using fluorescent minus one control was removed from each patient on each day. Each result was baseline‐corrected by dividing the MFI on each day by the MFI for the same patient on day 1. Averages ± standard errors of the mean are shown for each tested day for each group. n = 11–22 for SOC and 11–18 for OZA. *P < 0.05.

Figure 2

Circulating monocyte levels are lower in the ozanimod (OZA) group compared with the standard of care (SOC) group. (a) Complete blood counts were performed to assess total circulating monocytes. (b–k) Peripheral blood mononuclear cells (PBMCs) from patients with severe coronavirus disease 2019 (COVID‐19) were isolated and monocytes were analyzed by flow cytometry. Gating was performed as described in Supplementary figure 1b. (b) Classical monocyte and (c) intermediate monocyte frequencies were computed at day 7, that is, when significant differences were observed between both groups for total monocyte numbers. Median fluorescence intensities (MFIs) on classical monocytes (d–g) and intermediate monocytes (h–k) were measured for CD86, human leukocyte antigen (HLA)‐DR, CD163 and CD69. Baseline fluorescence from fluorescence minus one control was removed for every patient. cMono, classical monocytes; iMono, intermediate monocytes. Averages ± standard errors of the mean are shown for each tested day for each group. n = 11–22 for SOC and 11–18 for OZA. *P < 0.05.

Figure 3

Longitudinal comparison of specific circulating cytokines between the standard of care (SOC) and ozanimod (OZA) groups. The serum sample was collected at baseline (day 1; before ozanimod administration) and during treatment (on days 3 and 7). (a–f) A Luminex assay (23‐plex; Bio‐Techne) was performed on serum to evaluate levels of various cytokines in both groups. (g) P‐values for the group–time interaction for each circulating mediator are indicated. Averages ± standard errors of the mean are shown for each tested day for each group. n = 11–22 for SOC and 11–18 for OZA. CCL2, C–C motif ligand 2; CXCL13, C–X–C motif ligand; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor. *P < 0.05.

Figure 4

Circulating mediators at the follow‐up visit. The serum sample was collected on a follow‐up visit on day 90. (a–f) A Luminex assay was performed to evaluate the levels of various circulating mediators in both groups. (g) P‐values for each circulating mediator are indicated. Individual patient values (dots) and averages (horizontal line) ± standard errors of the mean are shown. n = 14–18 for the standard of care (SOC) group and 15–18 for the ozanimod (OZA) group. EpCAM, epithelial cellular adhesion molecule; IFN, interferon; IL, interleukin; TREM‐1, triggering receptors expressed on myeloid cells 1; VEGF, vascular endothelial growth factor. *P < 0.05.

Figure 5

Ozanimod hinders the primary humoral response to severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). The serum sample was collected at baseline [day 1 (D1)] on day 5 (D5) and on a follow‐up visit on day 90 after hospital admission to measure the impact of ozanimod on the concentration of anti–SARS‐CoV‐2 immunoglobulin Gs. (a) Relative antibody‐level progression is shown for each eligible patient between D1 and D5 in the standard of care (SOC) and the ozanimod (OZA) groups. Paired t‐tests were used between D1 and D5 to assess changes in relative antibody unit mL⁻¹ in both groups. (b) The mean for unvaccinated (black dots) and vaccinated (red dots) patients from each group is depicted with a horizontal line and the 80% protection threshold against symptomatic infection as shown by Feng et al. is delimited by a blue dashed line. 50% of patients received at least one vaccine shot before the follow‐up visit. t‐tests were performed to assess whether SARS‐CoV‐2–specific antibody levels were different between the two groups when vaccinated (red) and unvaccinated (black; P = 0.06). n = 19 (8 vaccinated) for the SOC group and n = 17 (10 vaccinated) for the OZA group. ns, not significant. *P < 0.05.