Distinguishing Incubation and Acute Disease Stages of Mild-to-Moderate COVID-19

Abstract

While numerous studies have already compared the immune responses against SARS-CoV-2 in severely and mild-to-moderately ill COVID-19 patients, longitudinal trajectories are still scarce. We therefore set out to analyze serial blood samples from mild-to-moderately ill patients in order to define the immune landscapes for differently progressed disease stages. Twenty-two COVID-19 patients were subjected to consecutive venipuncture within seven days after diagnosis or admittance to hospital. Flow cytometry was performed to analyze peripheral blood immune cell compositions and their activation as were plasma levels of cytokines and SARS-CoV-2 specific immunoglobulins. Healthy donors served as controls. Integrating the kinetics of plasmablasts and SARS-CoV-2 specific antibodies allowed for the definition of three disease stages of early COVID-19. The incubation phase was characterized by a sharp increase in pro-inflammatory monocytes and terminally differentiated cytotoxic T cells. The latter correlated significantly with elevated concentrations of IP-10. Early acute infection featured a peak in PD-1⁺ cytotoxic T cells, plasmablasts and increasing titers of virus specific antibodies. During late acute infection, immature neutrophils were enriched, whereas all other parameters returned to baseline. Our findings will help to define landmarks that are indispensable for the refinement of new anti-viral and anti-inflammatory therapeutics, and may also inform clinicians to optimize treatment and prevent fatal outcomes.

Keywords: COVID-19; IP-10; SARS-CoV-2; acute infection; antibodies; cytotoxic T cells; disease phases; plasmablasts.

Figure 1

Multidimensional flow cytometry and phosphoprotein analyses revealed minor changes of bulk immune cell response during mild-to-moderate COVID-19. (A) UMAP projection of flow cytometry data show the topological distribution of immune cell populations based on differentially expressed surface antigen patterns between healthy controls and COVID-19 patients. (B) Heatmaps of normalized protein phosphorylation data from sorted CD14+ monocytes (left) and CD19+ B lymphocytes (right) demonstrated contingent hierarchical clustering of healthy controls and COVID-19 patients.

Figure 2

Kinetics of plasmablast proportions and SARS-CoV-2 specific immunoglobulin enrichment delineated disease stages during mild-to-moderate COVID-19. (A) Representative pseudocolor plots of CD38 and CD27 expression data of CD19⁺CD45RA⁺ B lymphocytes showed the disease stage-dependent increase of plasmablast proportions (rectangular gate). (B) Top left: Quantitative data of plasmablast abundances at different disease stage. Top right, bottom left and bottom right: Semi-quantitative data of SARS-CoV-2 specific IgM, IgG and IgA titers in plasma samples. The data for anti-RBD, -S1, -S2 and –N were combined for IgM and IgG, respectively.* p < 0.05; ** p < 0.01; *** p < 0.001; Tukey-Kramer test for plasmablasts, Kruskal-Wallis test with Dunn’s correction for multiple comparisons for Antibody isotypes. MFI: Median fluorescence intensity. A₄₅₀: Absorbance at 450 nm.

Figure 3

Pro-inflammatory monocytes were more abundant during the incubation phase. (A) Representative pseudocolor plots of CD14 and CD16 expression data of SSC^med monocytes demonstrated kinetics for the enrichment of pro-inflammatory monocytes (polygonal gate). (B) Quantitative data of pro-inflammatory monocyte proportions. * p < 0.05; ** p < 0.01; Tukey-Kramer test.

Figure 4

The portion of CD16⁻CD177⁺ granulocytes gradually increased until the late acute infection phase. (A) Representative pseudocolor plots of CD16 and CD177 expression data of SSC^hi leukocytes showed that CD16⁻CD177⁺ granulocytes specifically emerged at the late acute infection phase (Q3 gate). (B) Quantitative flow cytometry data indicated gradually increasing abundances of CD16⁻CD177⁺ granulocytes. ** p < 0.01; Tukey-Kramer test.

Figure 5

The scope of cytotoxic T lymphocytes activation depended on the respective disease stage during mild-to-moderate COVID-19. (A) Representative pseudocolor plots of CD38 and CD27 expression data of CD8⁺ T cells demonstrated the accumulation of the CD38⁺CD27⁻ subpopulation (Q3 gate) during the incubation and early acute infection phases. (B) Quantitative flow cytometry data of CD8⁺CD38⁺CD27⁻ T cell proportions. (C) CD38 expression by CD8⁺ T cells at the different disease stages. (D) Representative plots of PD-1 and CD27 expression data showed an increase of PD-1⁺ T cells that are either CD27⁺ (Q2 gate) or CD27⁻ (Q3 gate). (E) Quantitative data of CD8⁺PD-1⁺ T cell proportions showed their enrichment during the incubation and acute infection phases. * p < 0.05; ** p < 0.01; *** p < 0.001; Tukey-Kramer test.

Figure 6

Quantitative data of IP-10 concentrations in plasma samples at different disease stages. * p < 0.05; ** p < 0.01; Tukey-Kramer test.

Figure 7

Mild-to-moderate COVID-19 induced apoptosis-independent CD4⁺ and CD8⁺ T lymphopenia at the incubation and early infection phases. (A) Total cell numbers of cytotoxic T cells (left) and T helper cells (right). (B) Quantitative data of apoptotic cell proportions among cytotoxic T cells (left) and T helper cells (right). * p < 0.05; ** p < 0.01; *** p < 0.001; Tukey-Kramer test.

Figure 8

Differential kinetics of immune cell populations along distinct disease stages during mild-to-moderate COVID-19.