The serological diversity of serum IgG/IgA/IgM against SARS‐CoV‐2 nucleoprotein, spike, and receptor‐binding domain and neutralizing antibodies in patients with COVID‐19 in Japan

Abstract Background We compared the temporal changes of immunoglobulin M (IgM), IgG, and IgA antibodies against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleoprotein (N), spike 1 subunit (S1), and receptor‐binding domain (RBD), and neutralizing antibodies (NAbs) against SARS‐CoV‐2 in patients with coronavirus disease 2019 (COVID‐19) to understand the humoral immunity in COVID‐19 patients for developing drugs and vaccines for COVID‐19. Methods A total of five confirmed COVID‐19 cases in Nissan Tamagawa Hospital in early August 2020 were recruited in this study. Using a fully automated chemiluminescence immunoassay analyzer, we measured the levels of IgG, IgA, and IgM against SARS‐CoV‐2 N, S1, and RBD and NAbs against SARS‐CoV‐2 in COVID‐19 patients' sera acquired multiple times in individuals from 0 to 76 days after symptom onset. Results IgG levels against SARS‐CoV‐2 structural proteins increased over time in all cases but IgM and IgA levels against SARS‐CoV‐2 showed different increasing trends among individuals in the early stage. In particular, we observed IgA increasing before IgG and IgM in some cases. The NAb levels were more than cut‐off value in 4/5 COVID‐19 patients some of whose antibodies against RBD did not exceed the cut‐off value in the early stage. Furthermore, NAb levels against SARS‐CoV‐2 increased and kept above cut‐off value more than around 70 days after symptom onset in all cases. Conclusion Our findings indicate COVID‐19 patients should be examined for IgG, IgA, and IgM against SARS‐CoV‐2 structural proteins and NAbs against SARS‐CoV‐2 to analyze the diversity of patients' immune mechanisms.

among individuals in the early stage. In particular, we observed IgA increasing before IgG and IgM in some cases. The NAb levels were more than cut-off value in 4/5 COVID-19 patients some of whose antibodies against RBD did not exceed the cut-off value in the early stage. Furthermore, NAb levels against SARS-CoV-2 increased and kept above cut-off value more than around 70 days after symptom onset in all cases.

Conclusion:
Our findings indicate COVID-19 patients should be examined for IgG, IgA, and IgM against SARS-CoV-2 structural proteins and NAbs against SARS-CoV-2 to analyze the diversity of patients' immune mechanisms. However, the chronological measurements of antibody isotypes against SARS-CoV-2 structural proteins and NAbs against SARS-CoV-2 in Japanese individuals have not been performed. Therefore, in this study, we measured temporal changes in the IgG, IgA, and IgM antibodies against SARS-CoV-2 N, S1, and receptor-binding domain (RBD) and NAbs against SARS-CoV-2.
A total of five men in their 20-50 s with COVID-19 confirmed in Nissan Tamagawa Hospital (Table 1), Tokyo, Japan, in August 2020 were enrolled in this study. At least seven serum samples for each patient were collected from 0 to 76 days after symptom onset. Levels of IgG, IgA, and IgM against SARS-CoV-2 N, S1, and RBD and NAb against SARS-CoV-2 were measured using a fully automatic CLIA analyzer, iFlash3000 (kits and an analyzer from Shenzhen YHLO Biotech Co.). The cut-off value for indicating a positive test result as used by the manufacturer for all kits was 10 AU/ml. IgG levels against SARS-CoV-2 increased after symptom onset in all patients with COVID-19 but levels of IgM and IgA against N and S1 exhibited different increasing trends among patients ( Figure 1A-F). For example, in Patient 5, IgM and IgG levels were low but IgA levels were high on Day 11. A recent study reported that IgA levels in serum increased soon after symptom onset with mild symptoms while that a case with the severity of symptoms showed a delayed but very strong IgA response against SARS-CoV-2. 3 Furthermore, measurement of serum IgA, besides IgM and IgG, improved diagnostic accuracy for SARS-CoV-2 infections. 4 We also observed higher NAb levels in patients with severe symptoms than NAbs in patients with the mild symptoms. The NAb levels were reported to increase after SARS-CoV-2 infection in most individuals 5 and be associated with clinical disease severity, 6,7 confirming the results of our studies. IgA and NAb levels against SARS-CoV-2 could be biomarker for COVID-19 severity.
The NAb kit has the advantages of detecting angiotensinconverting enzyme 2 (ACE2) competitively binding to RBD-coated particles with all antibodies having neutralization activity against SARS-CoV-2 structural proteins while a typical antibody kit against components detects each isotype, not reflecting total NAb levels. In fact, we observed NAb levels keeping above the cut-off value more than around 70 days after symptom onset in all COVID-19 patients some of whose antibody levels against RBD were lower than the cutoff value (Patient 2, Patient 3, and Patient 5). Furthermore, some of the antibody isotypes against RBD fall below 10 AU/ml in early stage, while NAb levels exceed the cut-off value in 4/5 patients. For example, in Patient 3, the levels of any antibody isotypes against RBD were less than 10 AU/ml when NAb levels over the cut-off value from Day 9 to Day 11 after symptom onset. The NAb kit could be as useful as IgG, IgA, and IgM kit against S1 and RBD for the accurate T A B L E 1 Symptoms exhibited by the patients in this study. Antibodies against the S1 region except for RBD of SARS-CoV-2 were reported to have neutralizing activity. 8,9 We should detect NAbs by using S1-coated particles, including the RBD region for analyzing NAbs.
Second, we found the persistence of NAbs in patients with COVID-19 around 70 days after symptom onset but we have not measured the neutralization activity of antibodies for a longer time yet. Some longitudinal studies have reported that neutralization activity against SARS-CoV-2 significantly declined as early as 6 weeks and that persisted as late as 5 months after symptom onset. 10,11 Furthermore, the binding surface in SARS-CoV-2 RBD to ACE2 is reported to be less antigenic than that of other S regions 12 so the antibody levels against RBD could go down earlier than antibody levels against S1, which may affect chronological changes in NAb levels.

DATA AVAILABILITY STATEMENT
Data available on request from the authors.

ETHICS STATEMENT
This study was performed at the University of Tokyo and Nissan Tamagawa Hospital approved by their ethics committee (protocol number R2-05 and Tama2020-003), and informed consent was obtained from all participants individually.

TRANSPARENCY STATEMENT
The corresponding author, Takeshi Kawamura confirms that manuscript is an honest, accurate, and transparent account of the study being reported.