An observational study investigating soluble immune checkpoints as indicators of severe COVID-19

Abstract

This study aimed to investigate the immunomodulatory behavior of soluble immune checkpoints (sICPs) and other biomarkers in the pathophysiology of SARS-CoV-2 infection. The study included 59 adult participants, 43 of whom tested positive for SARS-CoV-2. Patients were divided into three cohorts: those with moderate disease (n = 16), recovered patients with severe disease (n = 13), and deceased patients with severe disease (n = 16). In addition, 16 participants were pre-pandemic subjects negative for SARS-CoV-2. The relative activity of neutralizing antibodies (rNAbs) against SARS-CoV-2 and the values of 14 sICPs in peripheral blood were compared between the four groups. Because the increase of markers values of inflammation [NLR > 12; CRP > 150 mg/L] and venous thromboembolism [D-dimer > 0.5 mg/L] has been associated with mortality from COVID-19, the total and differential leukocyte counts, the NLR, and CRP and D-dimer values were obtained in patients with severe disease. No differences in rNAbs were observed between the cohorts. Only the levels of five sICPs, sCD27, sHVEM sTIM-3, sPD-1, and sPDL-1, were significantly higher in patients with severe rather than moderate disease. The sPDL-2 level and NLR were higher in deceased patients than in recovered patients. However, there was no difference in CRP and D-dimer values between the two groups. Of the five soluble biomarkers compared among patients with severe disease, only sPDL-2 was higher in deceased patients than in recovered patients. This suggests that immuno-inhibitory sICPs might be used as indicators for severe COVID-19, with sPDL-2 used to assess individual risk for fatality.IMPORTANCECOVID-19, the disease caused by a SARS-CoV-2 infection, generates a broad spectrum of clinical symptoms, progressing to multiorgan failure in the most severe cases. As activation of the immune system is pivotal to eradicating the virus, future research should focus on identifying reliable biomarkers to efficiently predict the outcome in severe COVID-19 cases. Soluble immune checkpoints represent the function of the immune system and are easily determined in peripheral blood. This research could lead to implementing more effective severity biomarkers for COVID-19, which could increase patients' survival rate and quality of life.

Keywords: COVID-19; SARS-CoV-2; immune checkpoints; observational study.

Fig 1

Seroconversion and relative neutralization activity against SARS-CoV-2. (A) Qualitative detection of IgG antibodies against the SARS-CoV-2 S protein. Values greater than 11 RU/mL (dashed line) are considered a positive seroconversion against SARS-CoV-2. (B) Relative neutralization activity against SARS-CoV-2. A percentage of the inhibition of RBD-ACE2 interaction above 30% (dashed line) was interpreted as positive neutralizing antibody activity. The center horizontal lines of the violin plots represent the median, and the horizontal lines at the ends represent the lower and upper quartiles. (C) Percentage of vaccinated patients against COVID-19. The P-values were obtained by analyzing the variables with the Kruskal-Wallis test with Dunnett’s multiple comparisons. ***P ≤ 0.0004; ****P < 0.0001; ns, not significant. Ctrl (negative controls); Mod (patients with moderate disease); Rec-S (recovered patients with severe disease); Dec-S (deceased patients with severe disease).

Fig 2

Stimulatory soluble immune checkpoints in COVID-19 patients. Medians are plotted with the interquartile ranges. The P-values were obtained by analyzing the variables with the Kruskal-Wallis test with Dunnett’s multiple comparison. *P ≤ 0.0417; **P ≤ 0.0097; *** P = 0.0009.

Fig 3

Inhibitory soluble immune checkpoints in COVID-19 patients. Medians were plotted with interquartile ranges. The P-values were obtained by analyzing the variables with the Kruskal-Wallis test with Dunnett’s multiple comparison. *P ≤ 0.0470; **P ≤ 0.0029; *** P ≤ 0.0003; ***P < 0.0001.

Fig 4

Total and differential leukocyte counts in COVID-19 patients with severe disease. Medians are plotted with interquartile ranges. Analyses were performed using a paired Wilcoxon signed-rank test. The dashed lines indicate the means of healthy subjects (20). Forty-two WBC differential counts from Rec-S and 30 WBC differential counts from Dec-S *P ≤ 0.0117; **P = 0.0077; ns P > 0.0500.

Fig 5

Levels of acute inflammation and venous thromboembolism markers in COVID-19 patients with severe disease. Means and standard errors were plotted for parametric data (NLR and CRP). For D-dimers, medians were plotted with interquartile ranges. Analyses were performed using a paired t-test (NLR: 42 determinations from Rec-S and 30 from Rec-D and CRP: 18 determinations from Rec-S and 32 from Rec-D) or paired Wilcoxon signed-rank test (D-dimer: 58 determinations from Rec-S and 56 from Rec-D). The dashed lines indicate the NLR or CRP cut-off points for the association with COVID-19 mortality (21) or the D-dimer cut-off point for the exclusion of venous thromboembolism (22). *P ≤ 0.0117; **P = 0.0077; ns P > 0.0500.