The potential of COVID-19 patients’ sera to cause antibody-dependent enhancement of infection and IL-6 production

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35–40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Five different and DC-differentiated Mylc lines (3×10 4 /well, orange circles) and Vero cells (4×10 4 /well, blue circles) were infected with the titrated amount of dengue virus type 2, strain 16681, in 96-well plates. After incubation at 37°C for 3 days, the SNs were harvested. Vero cells were cultured with the undiluted SNs, and cells were stained with 4G2 Ab (anti-Flavivirus Envelope protein Ab) three days later. The number of stained focuses per well was counted.
More than 100 focuses are plotted as >100. The assays were performed in duplicate, and the result is expressed as an average.

Supplemental Figure 3
Changes in the expression of the CD14 cell surface marker.
K-ML2 and K-ML2 (AT) clone 35 cells before and after DC-differentiation were stained with FITC-conjugated anti-CD14 Ab in the presence of Fc-blocker (eBioscience). As a negative control, FITC-conjugated isotype-matched Ab was used.
Supplemental Figure 4 The expression of ACE2 and TMPRSS2 in Mylc cell lines.
K-ML2 and K-ML2 (AT) cells before and after DC-differentiation were analyzed for the expression of ACE2 and TMPRSS2 by qPCR. Error bars indicate SD. N=3.
Supplemental Figure 6 Comparison of experimental protocols in SARS-CoV-2 infection. in the presence or absence of Fc-blocker (eBioscience). As a negative control, unstained cells were used.
Supplemental Figure 8 Infection-enhancing activity of serum from mice immunized with SARS-CoV-2.
K-ML2 (AT) clone 35 cells (2×10 4 /well) were cultured with a constant amount of SARS-CoV-2 (1,250 copies/μL) in the presence or absence (control culture) of serially-diluted serum from (a) a normal mouse or (b) mice immunized with SARS-CoV-2. Three days later, the culture SNs were harvested, and the amount of virus was determined by qPCR (N=3). The increment of viruses is expressed as the fold increase compared with the amount of viruses in control culture (N=6). Red or black lines on the y-axis indicate the mean of control culture + a three or six SD cut-off, respectively. The black dotted line indicates fold increase = 1. (c) The region indicated with a dotted line in (b) is shown on a log-log scale.
Supplemental Figure 9 Infection-enhancing activity of serum derived from patients infected with SARS-CoV-2.
As shown in the Fig. 3 legend, the results from ADE assays were classified into (a) Apparent  Patient-derived sera #32, #38, and #99 belong to the A-2 subgroup in ADE assay, and serum #62 belongs to the No ADE group. (c) Clone 35-40 cells were cultured with SARS-CoV-2 (1×10 4 copies/μL) and serially-titrated serum from SARS-CoV-2-immunized or normal mice. The amount of IL-6 in each SN after three days' culture was measured. The amount of IL-6 is plotted as the OD value in the ELISA assay. Error bars indicate SD. N=3.
Supplemental Figure 13 IL-6 production-enhancing activity of sera derived from patients infected with SARS-CoV-2.
K-ML2 (AT) clone 35-40 cells (2×10 4 /well in a 96-well flat plate) were cultured with (blue circles) or without (orange circles) SARS-CoV-2 (1×10 4 copies/μL) in the presence or absence of serially-diluted serum from COVID-19 patients (N=3). Three days later, the amount of IL-6 in culture SNs was measured by ELISA. Error bars indicate SD. The position of each result corresponds to that in Fig. 3a and Supplemental Fig. 9.
Supplemental Figure 14 Enhanced IL-6 production observed by sera derived from COVID-19 patients depends on FcR.
K-ML2 (AT) clone 35-40 cells were cultured with a constant dose of patient's serum and SARS-CoV-2 in the presence or absence of the titrated amount of 4G2 Ab. N=3. Serum from patient #24, #73 and #78 represents IL-6 production-enhancing serum. 4G2 Ab was used as FcRbinding competitor reagent. Three days later, the amount of IL-6 in culture SNs was measured by IL-6 ELISA. The amount of IL-6 in the absence of 4G2 or 4G2 and patient's serum was regarded as 100% or 0% IL-6 production, respectively. The percent inhibition of IL-6 production by 4G2 Ab is plotted in y-axis.
Supplemental Table 1 Information about COVID-19 patients (sex and age).
Supplemental Table 2 The amount of anti-SARS-CoV-2 IgG in each serum sample from COVID-19 patients.
Supplemental Table 3 Raw data of control cultures in qRT-PCR experiments.
In one qRT-PCR experiment to measure the amount of SARS-CoV-2, the control cultures (cells plus SARS-CoV-2 without serum) were set in 6-9 wells. This Table summarizes