Comparative Longitudinal Serological Study of Anti-SARS-CoV-2 Antibody Profiles in People with COVID-19

Abstract

Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.

Keywords: SARS-CoV-2; antibody profile; polyclonal durability; serology.

Figure 1

Dot blot analysis to assess patient antibody specificities for S1, RBD, S2 protein domains, and N protein. (A) Representative dot blots. Serum samples were derived from patients with mild (patients #4 and #31), moderate (patients #43, #44, #45, #46, #47 and #39), and severe (patient #63) COVID-19-related diseases. For sera from patients with mild and moderate disease, the first (strips 10, 92, 126, 129, 132, 135, 138 and 114), second (11, 93, 127, 130, 133, 136, 139, and 115), and third (12, 94, 128, 131, 134, 137, 140 and 116) serum samples were taken within the time periods of 0–100, 101–200, and 201–300 days, respectively. For patient #63 with severe disease, sera were taken on days 21, 23, and 29 days (strips 173, 174, and 175, respectively) after disease onset. Supplementary Table S1 shows a summary of all patient serum samples, with a total of 63 patients and 175 sera. Nitrocellulose-enhanced membranes were spotted with the individual proteins as indicated (A), incubated with 1:100 dilution of patient serum samples from mild, moderate, and severe COVID-19 patients, and then anti-human IgG-HRP secondary antibody was added. Dots were developed by chemiluminescence and images were taken. Integrated density was calculated by measuring the intensity of each dot minus the background. P1 = positive control 1 (1:100 dilution); P2 = positive control 2 (1:300 dilution); N = negative control; FLAG = strip probed with anti-FLAG antibody. (B) Antibody levels against individual RBD, S1, S2 protein domains, and N protein in relation to the different disease categories. The values plotted are the integrated density of each dot per antigen regardless of time points, age, and gender. The heights of the bar graphs represent the mean value of all individual measurements. Kruskal–Wallis test was conducted to compare the mean values of unpaired samples. p value: **** < 0.0001; *** < 0.0002; ** < 0.002; not significant (ns) < 0.12.

Figure 2

Presence of anti-N, anti-S1, anti-RBD, or anti-S2 antibodies (IgG) (panels (A–D), respectively) in patients with mild, moderate, or severe disease at different time periods post-symptom onset, as detected by dot blot analysis. Box plots were used to show the spread of the data. Data were analyzed using the analysis of variance (ANOVA) for paired samples. Mean values were compared using Dunn’s multiple comparisons test; p value: *** < 0.0002; * < 0.0332; not significant (ns) < 0.1234.

Figure 3

Surrogate virus neutralization test (sVNT) inhibition activities of COVID-19 patients with broad, restricted, and no anti-S antibody levels as measured by dot blot analysis. The sVNT inhibition of sera was taken at the indicated time periods post-symptom onset. The heights of the bar graphs represent the mean values of all individual measurements. Data were analyzed using the analysis of variance (ANOVA) for unpaired samples. Mean values were compared using Dunn’s multiple comparisons test; p value: **** < 0.0001; ** < 0.0021; * < 0.0332; not significant (ns) < 0.1234. sVNT: surrogate virus neutralization test (GenScript). Data are also presented in Supplementary Table S3.

Figure 4

Presence of neutralizing antibodies (microneutralization test, MN) and antibodies inhibiting RBD binding to the ACE2 receptor (surrogate virus neutralization test, sVNT). Comparison of MN titres and binding inhibition (sVNT) in patients with broad anti-S1, -RBA, -S2, and -N antibody responses (patients #4, #47, and #44), narrow antibody responses (patients #43, #45, and #46), and undetected responses (patients #39 and #63). (A) The table summarizes the inhibition of RBD binding to ACE2 receptor (sVNT, % inhibition) and titres to neutralize SARS-CoV-2 (MN test). ND: not detected. (B). Data are graphed as microneutralization titres. Patient and serum numbers are provided. DPSO: Days after symptom onset indicating when the serum samples were taken (see also legend to Figure 1).