Serological investigation of asymptomatic cases of SARS-CoV-2 infection reveals weak and declining antibody responses

Abstract

Without an effective vaccine against SARS-CoV-2, the build-up of herd immunity through natural infection has been suggested as a means to control COVID-19. Although population immunity is typically estimated by the serological investigation of recovered patients, humoral immunity in asymptomatic subjects has not been well studied, although they represent a large proportion of all SARS-CoV-2 infection cases. In this study, we conducted a serosurvey of asymptomatic infections among food workers and performed serological and cellular response analyses of asymptomatic subjects in Wuhan, the original epicenter of the COVID-19 outbreak. Our data showed that up to 5.91% of the food workers carried SARS-CoV-2 IgG antibodies asymptomatically; however, in 90.4% of them, the antibody level declined over a 2-week period. IgM and IgG antibodies, including neutralizing antibodies, were significantly lower in asymptomatic subjects than in recovered symptomatic patients with similar disease courses. Furthermore, the asymptomatic subjects showed lymphopenia and a prominent decrease in the B-cell population, as well as a low frequency of antibody-secreting cells and a low cytokine response. These factors probably contributed to the low and unsustained antibody levels in asymptomatic subjects. Our results show that asymptomatic subjects are likely to be vulnerable to SARS-CoV-2 reinfection, and neither the proportion of population immunity nor the breadth of immune responses is sufficient for herd immunity.

Keywords: COVID-19; SARS-CoV-2; asymptomatic; serology; weak antibody response.

Figure 1.

Serological investigation of SARS-CoV-2 in food workers in Wuhan. (A) Enzyme-linked immunosorbent assay (ELISA) screening of SARS-CoV-2 nucleocapsid protein (NP) IgG antibodies (n=508 subjects). The cutoff (dashed line) was five times the mean value of negative samples. Serum samples from healthy donors were used as the control (n=30). (B) Same ELISA test using a commercial kit. (C) NP IgG antibody levels in two consecutive serological tests (n=21).

Figure 2.

Phylogenetic tree of SARS-CoV-2 genomes. Three full-length genomes were obtained from three asymptomatic subjects collected on May 30 (AP43) or June 1 (AP44 and AP45) 2020 and were clustered with 1,927 publicly available representative genomes downloaded from the GISAID database. Sampling time is indicated at the end of each genome.

Figure 3.

Asymptomatic subjects exhibit a weak serological response. (A, B) Comparison of IgG_RBD (A) and IgM_RBD (B) between asymptomatic subjects (n=24) and recovered symptomatic patients (n=60). Serum samples from healthy donors were used as controls (n=20). The median values are shown. Statistical significance was calculated using an unpaired Student’s t-test, **P < 0.01; ****P < 0.0001. (C) Neutralizing titers of selected serum samples from asymptomatic and symptomatic patients. IgG_RBD ELISA values and neutralizing antibody (nAb) titers are shown. The titer was tested using surrogate neutralization assays, shown as inhibition ratio (column C).

Figure 4.

Lymphocyte ratio and signature cytokine levels in blood. (A) Flow-cytometric analysis of major lymphocyte populations in peripheral blood cells (PBCs) in asymptomatic subjects (n=10), 2-month recovered symptomatic patients (n=5), symptomatic acute infection (n=9) and healthy individuals (n=8). Data are shown as percentages of PBCs. (B) Comparison of blood IL-6 (left) and TNF-α (right) concentrations between asymptomatic subjects (n=15), 2-month recovered symptomatic patients (n=14), and symptomatic acute infection (n=14) or healthy donors (n=7). Human sera were used at 1:4 as recommended by the ELISA kit manufacturer. Mann-Whitney tests were used. Means ± SDs are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.

Figure 5.

Frequency of CD3⁻CD19⁺CD27^hiCD38^hi antibody-secreting cells (ASCs). (A) Gating strategy for ASCs in flow cytometry. (B) Frequency of ASCs in asymptomatic subjects (n=10), compared to that in recovered symptomatic individuals (positive control, n=2) or healthy donors (negative control, n=2).