Characterization of SARS CoV 2 specific antibodies in COVID 19 patients reveals highly potent neutralizing IgA
Dear Editor, The coronavirus disease 2019 (COVID 19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV 2) is an ongoing threat to public health. Due to the presence of neutralizing antibodies in convalescent patients, convalescent plasma transfusion has been tested as a promising therapy for severe COVID 19 patients. We and others previously found a surprisingly early serum IgA antibody response in the COVID 19 patients. However, the relative contribution of the IgA antibody towards virus neutralization with respect to other antibody isotypes is unknown. Highly purified SARS CoV 2 spike receptor binding domain (RBD) and Nucleocapsid protein (NP) (Supplementary Fig. S1) were employed to develop affinity immobilized columns, respectively. Serum antibodies specific to RBD and NP were purified from 90mL virus inactivated sera collected from 50 COVID 19 convalescent patients using the columns prepared above, respectively (Supplementary Fig. S2a, e) NP or RBD specific IgG was then purified by using a protein G column, which only binds to IgG (Supplementary Fig. S2b, f) The NP or RBD specific IgM were purified by using an antiIgM affinity column and IgA was further purified by pull down using anti IgA affinity beads (Supplementary Fig. S2c, g) respectively (see Supplemental Methods for details) The purified NP or RBD IgA, IgM, and IgG were confirmed by western blotting with isotype specific antibodies (Supplementary Fig. S3) and mass spectrometry (data not shown) The amount of RBDor NP specific antibodies is in the order of IgG IgA IgM (Supplementary Table S1) Absolute concentrations of each antibody isotypes provide important humoral immune response information in COVID 19 patients. Using these highly purified antibodies as standards, we generated standard curves (Supplementary Fig. S4) to convert the relative light unit (RLU) measured with RBD and NP based chemiluminescence diagnostic kits into absolute antibody concentrations (mg per mL) for 216 serum samples from 87 COVID 19 patients. To simplify plots from a large number of samples, median values with an interquartile range of antibody concentrations were plotted vs. time windows. As shown in Fig. 1a c, the median concentration of RBD IgA reached the highest (8.84 mg/mL) from 16 to 20 days after illness onset, declining subsequently but maintaining at about 3.62 mg/mL until 41 days. The median concentration of RBD IgG was the lowest in the early stages but rise after the 15th day after the illness onset. RBD IgG concentration reached the peak on the 21st 25th days after illness onset at 16.47 mg/mL and stayed at a relatively high concentration (11.40 mg/mL) until the 41st day. The trend of RBD IgM was very similar to that of RBD IgA. As expected, the trend of NP specific antibodies was roughly the same as that of RBDspecific antibodies (Fig. 1d f) for each isotype. The median concentration of NP IgA reached the highest (10.07 mg/mL) level on the 11th 15th days after illness onset. The median concentration of NP IgG reached the peak on the 21st 25th days after the illness onset as 149.37 mg/mL. Unlike NP IgA, the highest median concentration of NP IgM (6.72 mg/mL) appeared on the 16th 20th days after the illness onset. The absolute concentration of the three isotypes of antibody in 216 COVID 19 patients' sera provides a guide for convalescent serum therapy and vaccine development. Biolayer interferometry (BLI) was performed to further characterize the binding properties of the purified antibodies. As shown in Supplementary Fig. S5d f, the purified SARS CoV 2 NP IgA, IgM, and IgG exhibited a high cross reactivity with the SARS CoV 1 NP, probably due to the high identity between SARS CoV 2 NP and SARS CoV 1 NP. By contrast, RBD IgA, IgM, and IgG specifically bound to SARS CoV 2 RBD but not SARS CoV 1 RBD (Supplementary Fig. S5a c) These results indicate that RBD is more suitable than NP as an antigen for specific testing kits. SARS CoV 2 virus entry into host cells requires the interaction between RBD and Angiotensin converting enzyme 2 (ACE2) on the cell surface, so the RBD antibodies may block virus infection. Competitive ELISA was performed to assess the RBD ACE2 blocking abilities of the RBD specific antibodies. As shown in Fig. 1g i, the three isotypes of RBD specific antibodies block the binding of ACE2 to the RBD immobilized on plate with different strengths. Among them, IgA exhibited the strongest competitive capacity with IC50 of 4.68 0.46 mg/mL, which was better than that of IgG (IC50= 9.36 1.001 mg/mL) and IgM (IC50= 22.65 0.04 mg/ mL) Furthermore, the SARS CoV 2 neutralizing assay was performed to evaluate the neutralizing potency of the RBDspecific antibodies. The sera pool of COVID 19 convalescent patients was tested first, which exhibited an 80% neutralizing ability of SARS CoV 2 virus infection of Vero E6 cells (Fig. 1j) Subsequently, the purified RBD specific antibodies were tested separately. RBD IgA, IgM, and IgG showed different SARS CoV 2 infection neutralizing potency. Consistent with the results of competitive ELISA, IgA exhibited a higher neutralizing potency than IgM and IgG. The ND50 value of IgA was 2.42 0.89 mg/mL, while ND50 values of IgM and IgG were nearly 20.31 mg/mL and 39.73 mg/mL, respectively (Fig. 1k m) Even though IgA is only 2fold stronger than IgG in competing ACE2 to bind with RBD, IgA is about 10 fold more potent than IgG in the neutralization assay. These results showed that the IgA antibodies present in convalescent patients' sera play non neglectable roles in neutralizing SARS CoV 2 in addition to the traditionally thought IgG. Studies on humoral responses to infections mediated by IgA are receiving increasing attention. It was proposed that secretory IgA might be a therapeutic strategy against SARS CoV 2. However, there is no experimental data on anti SARS CoV 2 efficacy by IgA in serum. Here, we purified NPand RBD specific antibodies directly from convalescent patients' serum pool, and determined the absolute mass concentration of all types of serum antibodies in COVID 19 patients. RBD specific antibodies are able to block ACE2 binding to RBD and neutralize live SARS CoV 2. RBD IgA exhibited an impressive neutralizing potency, with an ND50 value of 2.42 0.89 mg/mL, which is 10 fold better than that of RBD IgG. This study provides direct evidence for the potent antiviral activity of serum derived RBD IgA. Furthermore,
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