Salivary and serum IgA and IgG responses to SARS-CoV-2-spike protein following SARS-CoV-2 infection and after immunization with COVID-19 vaccines

Abstract

Background: Secretory immunoglobulin A (sIgA) plays an important role in antiviral protective immunity. Although salivary testing has been used for many viral infections, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), its use has not yet been well established with the SARS coronavirus 2 (SARS-CoV-2). Quantification of salivary IgA and IgG antibodies can elucidate mucosal and systemic immune responses after natural infection or vaccination. Here, we report the development and validation of a rapid enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 salivary IgA and serum IgG antibodies, and present quantitative results for immunized subjects both prior to or following COVID-19 infections. Objective: Total and serum SARS-CoV-2 spike-specific IgG responses were compared with salivary spike-specific IgA and IgG responses in samples obtained from patients recently infected with SARS-CoV-2 and from subjects recently immunized with COVID-19 vaccines. Methods: A total of 52 paired saliva and serum samples were collected from 26 study participants: 7 subjects after COVID-19 infection and 19 subjects who were uninfected. The ELISA results from these samples were compared with five prepandemic control serum samples. Total IgG and SARS-CoV-2 spike-specific IgG in the serum samples from the subjects who were infected and vaccinated were also measured in a commercial laboratory with an enzyme immunoassay. Results: A wide variation in antibody responses was seen in salivary and serum samples measured by both methods. Three groups of serum total and IgG spike-specific SARS-CoV-2 antibody responses were observed: (1) low, (2) intermediate, and (3) high antibody responders. A correlational analysis of salivary IgA (sIgA) responses with serum IgG concentrations showed a statistical correlation in the low and intermediate antibody responder groups but not in the high group (which we believe was a result of saturation). Conclusion: These preliminary findings suggest measuring salivary and serum IgG and IgA merit further investigation as markers of current or recent SARS-CoV-2 infections.

Figure 1.

Three groups of serum total and IgG SARS-CoV-2 spike antibody responses were observed by SARS-CoV-2; total antibody, spike, semiquantitative method (left, y-axis) or SARS-CoV-2 antibody (IgG), spike, semiquantitative spike-specific method (right, y-axis): (1) low, (2) intermediate, and (3) high. The cutoff points for the low group's total ab value ranged from 0 to 600 μg/mL and, for the IgG level, from 0 to 5 μg/mL; the cutoff points for the intermediate group's total ab value ranged from 601 to 2000 μg/mL and, for the IgG level, from 5 μg/mL to 17 μg; the cutoff points for the high group's total ab value ranged from 2001 to >2500 μg/mL and, for the IgG level, from 17.1 to >20 μg/mL. IgG = Immunoglobulin G; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; ab = antibody.

Figure 2.

(A) A comparison of SARS-CoV-2 total antibody (red lines) and SARS-CoV-2 spike antibody (IgG) (blue lines) after immunization with COVID-19 vaccine in subject no. 1–1 who had been infected with SARS-CoV-2 5 months previously compared with subject no. 2–1 who had not been infected. (B) A comparison of total antibody and IgG spike antibody responses in 7 subjects infected and 18 subjects vaccinated by using groups stratified by cutoff values of high, intermediate, or low for evaluation as described in the Fig. 1 legend. SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2; IgG = immunoglobulin G; COVID-19 = coronavirus disease 2019.

Figure 3.

A standard curve of anti-spike IgG in ELISA. IgG = Immunoglobulin G; ELISA = enzyme-linked immunosorbent assay.

Figure 4.

EIA IgA and IgG antibody responses found in saliva and serum for study (A) subject no. 1–1 and (B) subject no. 2–1. The error bars in this and subsequent figures 5 and 6 signify SD and represent the variability in replicate measurements that were performed at discrete time points for the measurement of IgG or IgA. EIA = Enzyme immunoassay; IgA = immunoglobulin A; SD = standard deviation.

Figure 5.

The sIgA concentrations are correlated with serum IgG concentrations in the (A) low IgG and in (B) intermediate IgG groups in contrast to the (C) high IgG group, in which no correlation was found. Multiple time points were included because some subjects had multiple specimens collected for analysis. Each dot represents the average of IgG or IgA values performed in triplicate determinations ± SD. sIgA = Secretory immunoglobulin A; SD = standard deviation.

Figure 6.

The different IgA and IgG responses between the subjects who were infected and the subjects who were not infected. Multiple time points were included because some subjects had multiple specimens collected for analysis. IgA = Immunoglobulin A.