Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection

Abstract

Background: Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect.

Methods: We systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects.

Results: Using trimeric spike glycoprotein, rather than nucleocapsid enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting.

Conclusions: Detecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Funding: This work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

Figure 1.. Hospitalised patients respond strongly to multiple viral proteins.

Serological responses from hospitalised (HS, n=6), non-hospitalised convalescents (NHC, n=6), RT-PCR+ asymptomatic subjects (AS, n=6) or pre-2019 normal donors (Pre19, n=6) as determined by ELISA using HRP-labelled anti-IgG, IgA and IgM, against 0.1μg purified A) viral spike protein S1 fragment (S1), B) Receptor Binding Domain (RBD) or C) Nucleocapsid (N). D) Area Under the Curve (AUC) of responses shown in A-C. The mean ± standard deviation from the mean (SD) is plotted.

Figure 2.. Stabilised, trimeric S antigen is a superior antigen to detect Ab in NHC.

A) Size exclusion chromatogram (SEC) for SARS-CoV-2 S protein fractions collected for further use and denoted by dashed grey lines. B) Coomassie stained SDS-PAGE gel for two separate expressions of SARS-CoV-2 (left) and silver stain of batch one under reducing and non-reducing conditions (right). C) Surface plasmon resonance (SPR) characterizing the interaction between SARS-CoV-2 S protein and Ace2. The plotted lines represent the averages of three analytical repeats at each concentration. D) Serological responses from hospitalised (HS, n=9), or pre-2019 normal donors (Pre19, n=10) as determined by ELISA using HRP-labelled anti-IgG represented as absorbance values or E) Signal:noise ratio at each serum dilution against 0.1μg purified viral trimeric spike protein (S) or the S1 fragment (S1). F) Mean absorbance values of 6 sera per group against 0.1 or 0.2μg S or nucleocapsid (N). G) Signal:Noise ratio at each serum dilution against 0.1 or 0.2 μg of S or N. Error bars represent standard deviation from the mean (SD).

Figure 3.. Antigen targeting and antibody isotypes do not differ depending upon the severity of disease.

Serological responses from hospitalised (H, n=3), non-hospitalised convalescent (NHC, n=3) or pre-2019 donors (Pre19, n=3) as determined by ELISA using HRP-labelled anti-IgG1, IgG2, IgG3, or IgG4 against 0.1μg A) trimeric spike protein (S), B) Receptor Binding Domain (RBD) or C) Nucleocapsid (N). D) Area Under the Curve (AUC) of IgG1 and IgG3 responses as shown in A-C. The mean ± standard deviation from the mean (SD) is plotted.

Figure 4.. Combined detection of IgG, IgA and IgM enhances discrimination of infected and pre19 groups.

Serological responses from non-hospitalised convalescents (NHC, n=20) or pre-2019 donors (Pre19, n=4) as determined by ELISA using HRP-labelled anti-IgG, IgA and IgM or combined GAM, against 0.1μg purified viral spike protein (S). A) Absorbance values of 20 NHC and Pre19 sera against S. B) Absorbance values of 14 low positive NHC sera with or without amplification, pre-2019 controls (n=4). C) Signal:noise ratio of multiple dilutions of anti-S IgG, IgA, IgM or GAM before and after amplification.

Figure 5.. Amplification of antibody responses in saliva enables detection of S-specific, but not N-specific IgG, IgA and IgM.

Salivary antibody responses from self-reported symptomatic subjects (SRSS, n=8) or pre-2019 negative controls (Pre19, n=4) as determined by ELISA using HRP-labelled anti-IgG, IgA and IgM or combined GAM, against 0.1μg purified viral spike protein (S). A) Absorbance values of NHC and Pre19 saliva against S before amplification and B) after amplification. C) Absorbance values of the same saliva samples against N before or D) after amplification. E) Signal:noise ratio of multiple dilutions of anti-S salivary IgG, IgA, IgM or GAM before and after amplification.

Figure 6.. Serum and salivary anti-SARS-CoV-2 antibody responses do not always correlate.

Absorbance values of paired serum diluted 1:40 and saliva diluted 1:2 from RT-PCR negative health care workers (n= 39) that were asymptomatic at the time of testing serum, as determined by ELISA using combined HRP-labelled anti-IgG, IgA and IgM (GAM) against 0.1μg trimeric spike protein (S) with signal amplification. A) Correlation between paired serum and saliva absorbance values with percentages of samples positive for anti-S antibodies in either serum, saliva or both. Positivity was determined by cut-offs for each fluid (dotted lines) based on the mean + 3 standard deviations of 6 pre-2019 (Pre19) negative samples. Solid line represents simple linear regression of all samples. B) Correlation of the same paired serum and saliva absorbance values coded by self-reported historic SAR-CoV-2-like symptoms (blue) or no historic symptoms (yellow).