Demographic and Clinical features
A total of 37 COVID-19 patients were included in the current study, including 21 females and 16 males. These patients were divided into severe and non-severe (mild or moderate) group based on the disease severity according to the China National Health Commission Guidelines for Diagnosis and Treatment of SARS-CoV-2. The median age of the patients was 53.5 years, ranging from 27 to 76 years. There were no significant differences in age and gender between two groups (Supplementary Table 1). A total of 119 serum samples from the 37 patients were serially collected, ranging from 6 days after symptom onset to 45 days during hospitalization. The median sample days in severe and non-severe group were 18.5 and 19 days after symptoms onset.
For clinical manifestations, common symptoms in our cohort included fever, cough, fatigue, sore throat and chest tightness (Supplementary Table 1). High grade fever (P<1×10-4), chest tightness (P=0.007), shortness of breath (P=6×10-4), nausea or vomiting (P=9×10-4) were reported significantly more in severe group compared to non-severe group. Severe group also had more comorbidities such as diabetes (P=1.9×10-4) (Supplementary Table 1).
As shown in Table 1, blood examination results showed that both absolute count and percentage of leukocyte and neutrophil were significantly higher in severe group than non-severe group (P<0.05), while the percentage of lymphocyte and monocyte were significantly lower in severe group. Serum biochemical study showed that the severe cases had significantly higher levels of lactate dehydrogenase (LDH) (P=0.005), total bile acid (TBA) (P<1×10-4), r-glutaminase (P=0.011), adenosine deaminase (P<1×10-4), procalcitonin (P=0.004), C-reactive protein (P<1×10-4) and D-dimer (P=0.049) compared to non-severe cases (Table 1). Lower percentage of CD3+ T cell, CD3-CD16/56+ NK cell and higher CD3-CD19+ B cell percentage in severe groups were also observed, but due to the limited flow cytometry analysis data (severe case N=7, non-severe case N=17), the differences were not statistically significant (P>0.05). These results suggest increased systemic inflammation, dysfunction of liver and compromised T cell response are associated with the severity of COVID-19 patients.
Anti-SARS-CoV-2 antibody responses
Serum anti-SARS-CoV-2 S-specific IgG, IgA and IgM antibodies were detected in all samples (Fig 1a), and antibody level in severe group were all significantly higher than non-severe group (P<0.001). The four IgG subclasses targeting SARS-CoV-2 S protein were detected in all samples, with overall IgG1 and IgG3 responses higher than IgG2 and IgG4 responses. The severe group also showed higher IgG1-IgG4 levels than non-severe group (P< 1×10-4) (Fig 1b). Serum antibodies titers against SARS-CoV-2 S, RBD, receptor binding motif (RBM), N terminal domain (NTD), and C-terminal domain (CTD) were measured by ELISA. S-targeting antibody titer in severe group ranged from 4818 to 65392 (median 17803), followed by RBD-specific antibody titers (233 - 4871, median 1406), NTD-specific antibody titers (111 - 4795, median 579), CTD-specific antibody titers (66 - 1038, median 247), and RBM-specific antibody titers were the lowest (67 - 438, median 228) (Fig 1c). The similar trend of antibody titers was observed in non-severe group. As expected, the S-targeting antibody titer is the highest, ranging from 889 to 36571 (median 8282), followed by RBD-specific antibody titers (123 - 2574, median 437), NTD-specific antibody titers (67 - 2448, median 192), CTD-specific antibody titers (50 - 1353, median 125), and RBM-specific antibody titers were the lowest (55 - 754, median 153). Similar to Ig isotypes and IgG subclasses, the IgG titers of antibody targeting different domains of S protein were also significantly higher in severe group (P<0.001).
Serum antibody blocking RBD binding to ACE2
To examine whether the serum could result in antiviral activity, we next detected whether the serum antibody could block SARS-CoV-2 RBD to bind the ACE2 receptor, which will exert potential neutralizing activity of SARS-CoV-2 in infected patient. In severe group, the blocking percentages ranged from -20.4% to 94.7% (median 7.3%), which was significantly higher than non-severe group (-20.8%-65.9%, median -2.7%, P=5×10-4) (Fig 2a). While only some samples exhibited good inhibitory effect, other samples did not block RBD-ACE2 engagement and seemed the ACE-2 binding-enhanced signal. Obviously, the severe group showed higher positive blocking rate (75.0%) than non-severe group (42.3%) (Fig 2b). Positive correlations were found between antibody titers and blocking percentage. In severe group, the blocking percentage were positively correlated with S-IgG1 (r=0.372, P=0.009), S-IgG3 (r=0.594, P<1×10-4), S-IgG (r=0.454, P=0.001), NTD-IgG titer (0.414, P=0.004), especially strongly correlated with RBD-IgG (r=0.803, P<1×10-4) and CTD-IgG titer (r=0.802, P<1×10-4) (Fig 3a-3d, 3f and 3g). In non-severe group, IgG3 (r=0.364, P=0.002), RBD-IgG (r=0.331, P=0.005), and NTD-IgG titer (r=0.480, P<1×10-4) were positively associated with blocking percentage (Fig 3b, 3d and 3f).
Serum antibody binding titers with Fc receptors
To detect whether the difference of serum samples in inhibition or enhancement RBD binding with ACE2 was non-specifically induced by Fc function of serum antibodies, we examined the binding activity of serum sample to Fc receptors, which included an activating receptor FcγRⅡa and an inhibitory receptor FcγRⅡb. The binding titer of serum antibody to FcγRⅡa ranged from 635 to 345005 (median 12953) in severe group and 437-94649 (median 2653) in non-severe group, while binding titers to FcγRⅡb ranged from 111 to 8375 (median 276) in severe group and 111 to 3287 (median 204) in non-severe group. Notably, both FcγRⅡa and FcγRⅡb binding titer were significantly higher in severe group than non-severe group (P<1×10-4 and P=0.030, respectively)(Fig 4a). However, no correlation was found between the blocking rate and FcγRⅡtiter in both severe group (r=0.053, P=0.723) and non-severe group (r=-0.082, P=0.498) (Fig 4c), nor was the correlation between blocking rate and FcγRⅡb titer in severe group (r=0.113, P=0.444) and non-severe group (r=-0.161, P=0.180) (Fig 4d). In addition, we performed an analysis using the ratio of FcγRⅡa and FcγRⅡb binding titers in severe group and non-severe group. Consistent with the binding titers in the separate groups, this ratio in severe group is significantly higher than non-severe group (P<1×10-4), and no correlation with ACE2-blocking was found (r=0.059, P=0.690 and r=-0.049, P=0.685, respectively) (Fig 4b-4e), indicating severe group’s Igs FcγR-binding activity is much stronger.
Differential expression profile of cytokines in severe and non-severe case
To assess other immune factors in blood samples, we continued to analyze the profile of cytokines in COVID-19 patients. Elevated level of nine pro- and anti-inflammatory cytokines were observed in the severe cases as compared with that of the non-severe cases. For severe group, IL-6, IL-8, IP-10, MCP-3, and MIG showed the most significant elevation (P<1×10-4), followed by MCP-1 (P=3×10-4), GRO-α (P=0.006) and BAFF (P=0.003). Differences of IFN-γ, IL-1β, IL-1Ra, IL-15, VEGF-A, and APRIL between two groups were not statistically significant (P>0.05) (Fig 5). These results suggest that significantly higher inflammation responses in severe group than non-severe group infected by SARS-CoV-2.
Specificity and Correlation of antibody responses in severe and non-severe group
The results above indicated that the severe group’ antibody level were much higher than non-severe group‘s. To investigate the feature of Ig, we analyzed the correlations of Ig isotypes and IgG titers of different domain targeting antibody. As shown in Fig 6a, significant correlation between S-IgG and S-IgM was observed both in severe group (r=0.499, P=3×10-4) and non-severe group (r=0.584, P<1×10-4), S-IgA was positively correlated with S-IgM (r=0.786, P=0.040) and S-IgG (r=0.528, P<1×10-4) only in non-severe group. Notably, RBD-IgG were strongly correlated with S-IgG both in severe group (r=0.676, P<1×10-4) and non-severe group (r=0.665, P<1×10-4) (Fig 6b). Besides, RBM-IgG were positively correlated with S-IgG in severe group (r=0.365, P=0.011), while CTD-IgG were positively correlated with S-IgG in non-severe group (r=0.648, P<1×10-4). In addition, strong correlation between RBD-IgG and CTD-IgG were found in both severe group (r=0.657, P<1×10-4) and non-severe group (r=0.586, P<1×10-4), RBD-IgG were positively correlated with RBM-IgG in severe group (r=0.248, P=0.037), while NTD-IgG with RBD-IgG (r=0.315, P=0.008), NTD-IgG with CTD-IgG (r=0.306, P=0.030) showed significant correlation only in non-severe group (Fig 6c). Together, these results indicated that RBD domain was the main target on S for SARS-CoV-2 specific antibody in severe group, whereas RBD and CTD were both frequently targeted in non-severe group.
Correlations between antibody responses and days after symptoms onsets in two groups
In analyzing the specificity of antibody responses, RBD was the main domain for SARS-CoV-2’ S spike specific antibody in severe group. We continued to investigate the correlations between antibody responses and days since symptom onset against two groups. S-IgG titer increased significantly with longer days after symptom onsets in non-severe group (r=0.451, P<1×10-4) (Fig 7a). Similarly, accompanied with more time after symptom onsets, CTD-IgG titer were higher when symptom lasts (r=0.385, P=0.007), while that correlation with RBD-IgG titer or NTD-IgG titer were not significant (r<0.3) (Fig 7b-d). In addition, S-IgM were negatively correlated with days after symptom onset only in severe group (r=- 0.511, P=2×10-4) (Fig 7e), while correlations between S-IgA and days after symptom onset were not statistically significant (Fig 7f). Notably, S-IgG1 was negatively associated with illness days in severe group (r=- 0.434, P=0.002), while S-IgG3 in severe group (r=0.363, P=0.011) and S-IgG1 (r=0.417, P=3×10-4) in non-severe group was positively associated with days after symptom onset (Fig 7g and 7h). These results suggest that different antibody dynamics between the severe group and non-severe group induced by SARS-CoV-2 infection.