High levels of specific antibodies against S protein are associated with disease severity
Using a flow cytometry-based assay to detect antibodies against full length S protein , we first examined if specific IgM, IgA and IgG against the S protein were associated with COVID-19 disease severity (n=81; Table S1) over the early course of infection, at time points of median 5 days, 10 days and 23 days post-illness onset (pio). The patients were classified into three groups: mild (no pneumonia), moderate (pneumonia with no hypoxia), or severe (pneumonia with hypoxia) . At median 5 days pio, the S protein antibody responses were low, regardless of the isotype (Fig. 1). We did not observe any significant difference between the three disease severity groups. At median 10 days and 23 days pio, S protein IgM, IgA and IgG responses were associated with disease severity (Fig. 1). Patients in the severe group had higher IgM, IgA and IgG responses at median 10 days and 23 days pio. Various groups of controls (Table S1) were also assessed in parallel (Fig. S1): (1) recovered SARS individuals (n=20), (2) healthy controls (n=22), and (3) seasonal human CoV patients (n=20).
We further examined if the S protein antibody responses were associated with clinical outcomes (Fig. S2) such as pneumonia, requirement for supplemental oxygen and intensive care unit (ICU) admission. S protein antibody responses were associated with pneumonia at median 23 days pio, while all three isotypes, IgM, IgA and IgG responses were associated with more severe clinical outcomes, requirement for supplemental oxygen and ICU admission, at a median of 10 days pio.
We proceeded to study if the disease severity was associated with a particular IgG subclass. At median 5 days pio, IgG subclass responses were low (Fig. 2). At median 10 and 23 days pio, where the IgG subclass responses were higher, we found an association between all IgG subclasses and the disease severity at both median 10 days and 23 days pio (Fig. 2). We also observed an association between all IgG subclasses and specific clinical outcomes such as pneumonia, requirement for supplemental oxygen and ICU admission (Fig. S3). Higher IgG1, IgG2, IgG3 and IgG4 responses were detected in patients with pneumonia at median of 23 days pio, but not at median of 10 days. In contrast, the association between higher responses for all IgG subclasses and requirement for supplemental oxygen was found as early as 10 days pio.
IgG subclass imbalance is associated with disease severity
We investigated whether disease severity was associated with IgG subclass imbalance. To this end, we combined the IgG1 and IgG3 responses, typically indicative of Th1 responses, and IgG2 and IgG4 responses, typically indicative of Th2 responses, and computed the ratio of the combined IgG1 and IgG3 response to the combined IgG2 and IgG4 responses,13/24 ratio (Fig. 3A). A ratio of 1 indicates a balanced IgG subclass response, where the combined IgG1 and IgG3 response is similar to the combined IgG2 and IgG4 response. A ratio higher than 1 indicates IgG subclass imbalance, where the responses were skewed towards a more dominant IgG1 and IgG3 response. Comparing the 13/24 ratio across the different severity groups, we did not observe any association at the earlier time points, median 5 days and 10 days pio. However, at median 23 days pio, the 13/24 ratio was associated with disease severity, where the skew towards IgG1 and IgG3 Th1 response was greater in patients with mild disease than patients with moderate and severe disease. The mild group have a mean 13/24 ratio of 22.39, compared with 1.56 and 1.38 in the moderate and severe groups, respectively. We also computed the ratio of the IgG1 response to the IgG4 response, 1/4 ratio (Fig. 3B). Similar to the 13/24 ratio, we found an association between the 1/4 ratio and disease severity. Interestingly, the association between the 1/4 ratio and disease severity was more pronounced – the 1/4 ratio was significantly higher in the mild group (58.88) than the moderate group (5.88), which, in turn, was significantly higher than the severe group (2.36).
We then investigated if patients with no symptom also have an IgG subclass imbalance with a dominant IgG1 and IgG3 response. Asymptomatic patients (n=50; Table S1) developed specific antibodies, and IgG1 is the dominant IgG subclass response (Fig. 3C). Indeed, the asymptomatic patients were found to have a IgG subclass imbalance skewed towards a dominant IgG1 and IgG3 response (mean 13/24 ratio: 75.41; mean 1/4 ratio: 252.1, Fig. 3D).
Th1/Th2 cytokine imbalance is associated with disease severity
To determine if the skewed Th1 over Th2 response observed with IgG subclasses was also evident in the cytokine profile, we examined the cytokine profiles for the three severity groups (Fig. S4A). We did not find any association between Th1 cytokines, IL-2 and TNF-α, and disease severity (Fig. S4B). Higher IFN-g levels, another Th1 cytokine, were found in the severe group, compared to the mild group, at median 23 days pio (Fig. S4A). In contrast, IL-6, a Th2 cytokine, was found to be higher in the severe group, compared with the mild and moderate group at median 5 days and 10 days pio (Fig. S4B). Interestingly, when we compared the ratio of IL-2 to IL-6, the IL-2/IL-6 ratio was the smallest with the severe group, compared with the mild and moderate groups, at median 5 days and 10 days pio (Fig. 4). Similarly, we also observed an association between the IFN-g/IL-6 ratio and disease severity and an association between the TNF-α/IL-6 ratio and disease severity at median 5 days and 10 days pio (Fig. 4). The severe group exhibited the smallest ratio (4.9, 140.7 and 0.5 for IL-2/IL-6, IFN-g/IL-6 and TNF-α/IL-6 ratios respectively) at median 5 days pio, showing that the bias towards Th1 cytokines was much less pronounced in the severe group (compared to the mild and moderate groups). On the contrary, asymptomatic patients had ratios of 639.2, 500.7 and 119.1 for IL-2/IL-6, IFN-g/IL-6 and TNF-α/IL-6 ratios respectively (Fig. S4C), exhibiting a more prominent Th1 over Th2 response.
IgG subclass and cytokine imbalance over the course of infection
To understand if IgG subclass imbalance was sustained over the course of infection, we examined the IgG subclass response at later stages, median 101 days and 180 days pio. At these later stages, all patients, including those in the severe group, have recovered from the disease. At both time points, all patients retained substantial IgG1 response, and IgG1 remained the dominant IgG subclass (Fig. S5). IgG2, IgG3 and IgG4 responses were low across all three severity groups at both time points. When we compared the 13/24 and the 1/4 ratios across the three severity groups, the trend, where the ratio was higher with the mild severity group than the moderate and severe severity group, was still sustained at median 101 days pio (Fig. 5A and 5B). However, at median 180 days pio, there is no significant difference in the 13/24 and 1/4 ratios between the three severity groups.
Comparing between the different time points, we observed that both 13/24 and 1/4 ratios were the lowest at median 10 days pio for patients with mild symptoms (Fig. 5C and 5D). Patients with moderate and severe symptoms exhibited lowest 13/24 and 1/4 ratios at median 23 days pio. Both 13/24 and 1/4 ratios increased over the time points, suggesting that IgG subclass imbalance towards a greater Th1 dominance increased over the time points in line with clinical recovery.
We also looked at the cytokine profile of the patients at median 101 days and 180 days pio (Fig. S4A). All three IL-2/IL-6, IFN-g/IL-6 and TNF-α/IL-6 ratios were similar across the three severity groups (Fig. 6A). While we did not see any significant difference in the three ratios across the time points for the mild group, we observed that a lower IL-2/IL-6 at earlier time points (median 5 and 10 days pio) and a higher IL-2/IL-6 ratio at later time points (median 23, 101 and 180 days pio) (Fig. 6B). For the severe group, the trend was more pronounced, with all three ratios being lower at earlier time points (median 5 and 10 days pio) and higher at later time points (median 23, 101 and 180 days pio). In general, severe patients demonstrated smaller skew towards Th1 over Th2 response at the early disease phase and the skew towards Th1 response gradually increased to similar levels as mild and moderate patients at the later time points in line with clinical recovery. The skew towards a greater Th1 over Th2 cytokine response at later time points is in agreement with our IgG subclass data, suggesting that a dominance of Th1 over Th2 responses might create a more favourable environment for clinical recovery.