Potent SARS-CoV-2-Specific T Cell Immunity and Low Anaphylatoxin Levels Correlate With Mild Disease Progression in COVID-19 Patients

Abstract

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4⁺ and CD8⁺ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8⁺ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8⁺ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.

Keywords: COVID-19; ELISpot assay; SARS-CoV-2; T cell immunity; anaphylatoxin; neutralizing Abs.

Figure 1

IFNγ production in SARS-CoV-2 specific PBMCs from COVID-19 patients upon activation with virus-specific peptides. PBMCs from COVID-19 patients and HD were isolated from whole EDTA-blood using red blood cell lysis solution. ELISpot Multiscreen^® plates were coated overnight with anti-human IFNγ mAb 1-D1K (2 µg/ml). On the following day, PBMCs were stimulated overnight with stimuli as indicated. Spots were revealed by subsequent incubation with biotinylated anti-human IFNγ mAb 7-B6-1 (1 µg/ml), Streptavidin-Alkaline Phosphatase (1:1,000) and ready to use BCIP^®/NBT. Results are shown as median IFNγ spots normalized by 1 × 10⁶ cells from the different groups, Mild (n = 11), Severe (n = 15), Critical (n = 11), HD (n = 21). Error bars represent interquartile range. (A) IFNγ ELISpots of patient PBMCs stimulated with a cell activator cocktail (1:500) are shown. (B) IFNγ ELISpots of patient PBMCs stimulated using peptide pools covering the complete SARS-CoV-2 proteins M (left), N (middle) or S (right) are presented. Statistical differences were determined using one-way ANOVA with Dunnett´s post test. ns, not significant.

Figure 2

SARS-CoV-2-specific CD8⁺ T cells response in patients with mild, severe or critical COVID-19. PBMCs from COVID-19 patients and HD were stimulated 6 h at 37°C using either cell activator cocktail or SARS-CoV-2 PepTivator^® peptide pools. Cells were fixed after extracellular staining for CD8⁺ expression and then permeabilized. Intracellular staining was performed to determine polyfunctionality of T cells by measuring IL-2, IFN-γ, TNF-α and MIP-1β levels. Cells were analyzed by flow cytometry and activation was assessed by comparison of cytokine production with unstimulated cells. T cell activation was increased in all three COVID-19 groups compared to HD group. (A) Percentages of activated SARS-CoV-2-specific CD8⁺ T cells upon stimulation with cell activator cocktail are shown for each donor and median values are represented as red lines. (B) Percentages of SARS-CoV-2-specific CD8⁺ T cells upon stimulation with peptide pools covering the complete SARS-CoV-2 proteins M (left), N (middle) or S (right) are illustrated. Statistical variability is presented as interquartile range. Statistical differences were determined using the two-tailed unpaired student’s t-test. ns, not significant.

Figure 3

SARS-CoV-2-specific CD4⁺ T cells response in patients with mild, severe or critical COVID-19. PBMCs from COVID-19 patients and HD were stimulated for 6 h at 37°C using either cell activator cocktail or SARS-CoV-2 PepTivator^® peptide pools. Cells were fixed after extracellular staining for CD4⁺ expression and then permeabilized. Intracellular staining was performed to determine polyfunctionality of T cells by measuring IL-2, IFN-γ, TNF-α and MIP-1β levels. Cells were analyzed by flow cytometry and activation was assessed by comparison of cytokine production with unstimulated cells. Polyfunctionality of T cells was increased in all three COVID-19 groups compared to HD group. (A) Percentages of activated SARS-CoV-2-specific CD4⁺ T cells upon stimulation with cell activator cocktail are shown for each donor and medians are represented as red lines. (B) Percentages of SARS-CoV-2-specific CD4⁺ T cells upon stimulation with peptide pools covering the complete SARS-CoV-2 proteins M (left), N (middle) or S (right) are illustrated. Statistical variability is presented as interquartile range. Statistical differences were determined using two-tailed unpaired student’s t-test. ns, not significant.

Figure 4

Detection of SARS-CoV-2-specific antibodies and characterization of the neutralizing ability by patient antibodies. Titers of SARS-CoV-2-specific IgG antibodies were determined from plasma samples of COVID-19 patients. (A) Antibodies ratios were grouped in four categories (quartiles) and assigned to an antibody category (1 = low, 2 = medium low, 3 = medium high and 4 = high IgG titer). Antibody categories of all mild, severe and critically ill patients and healthy donors, analyzed by the Euroimmun (left) or Roche test kit, are shown. (B) Neutralization plaque assays were performed by 1 h incubation of SARS-CoV-2 strains and 1:10 diluted plasma samples from COVID-19 patients or culture medium as control. After incubation plasma–virus mix was ultracentrifuged and resuspended in reduced culture medium and used for inoculation of VeroE6/TMPRSS2 cells. After 1 h inoculation, solution was removed and cells were overlayed with culture medium containing agarose. After 3 days plaques were counted and plaque forming units per ml (PFU/ml) were calculated. Neutralizing ability of patients plasma samples were assessed by the reduction of PFU/ml and compared to medium-treated virus controls. Percentages of SARS-CoV-2 neutralization using a 1:10 dilution of plasma samples from mild, severe and critical COVID-19 patients are shown. (C) Neutralization plaque assay over a 1:10 to 1:2,500 dilution range was performed using only IgG positive plasma samples of COVID-19 patients. To determine the neutralizing capacity half-maximal neutralizing titer values (IC50 values) from neutralization curves were calculated using four-parameter nonlinear regression. IC50 values from mild (n = 5), severe (n = 9) and critical patients (n = 11) are illustrated. (D) Normalized IC50 values were calculated using the IC50 values from neutralization curves and previously established SARS-CoV-2-specific IgG antibody category from the Euroimmun test kit, due to its specificity towards the viral spike protein. Normalized IC50 values from mild (n = 5), severe (n = 9) and critical (n = 11) COVID-19 patients are shown. Median levels of antibody categories, normalization ability, IC50 and normalized IC50 are represented as red lines and error bars represent interquartile range. Statistical differences were determined using Kruskal–Wallis and Dunn’s post tests. ns, not significant.

Figure 5

Determination of anaphylatoxin levels in COVID-19 patients. (A) C3a level determination in mild, severe and critical COVID-19 patients as well as healthy donors. Plasma samples were harvested and C3a levels were analyzed using a BD Biosciences OptEIA Human C3a ELISA kit. (B) C5a level determination in mild, severe and critical COVID-19 patients as well as healthy donors. Plasma samples were harvested and C5a levels were analyzed using a BD Biosciences OptEIA Human C5a ELISA kit. C3a and C5a levels in ng/ml were determined for all patients and healthy donors and plotted on a bar graph. ns, not significant.