COVID-19 serology at population scale: SARS-CoV-2-specific antibody responses in saliva

Pranay R Randad, Nora Pisanic, Kate Kruczynski, Yukari C Manabe, David Thomas, Andrew Pekosz, Sabra L Klein, Michael J Betenbaugh, William A Clarke, Oliver Laeyendecker, Patrizio P Caturegli, H Benjamin Larman, Barbara Detrick, Jessica K Fairley, Amy C Sherman, Nadine Rouphael, Srilatha Edupuganti, Douglas A Granger, Steve W Granger, Matthew Collins, Christopher D Heaney, Pranay R Randad, Nora Pisanic, Kate Kruczynski, Yukari C Manabe, David Thomas, Andrew Pekosz, Sabra L Klein, Michael J Betenbaugh, William A Clarke, Oliver Laeyendecker, Patrizio P Caturegli, H Benjamin Larman, Barbara Detrick, Jessica K Fairley, Amy C Sherman, Nadine Rouphael, Srilatha Edupuganti, Douglas A Granger, Steve W Granger, Matthew Collins, Christopher D Heaney

Abstract

Non-invasive SARS-CoV-2 antibody testing is urgently needed to estimate the incidence and prevalence of SARS-CoV-2 infection at the general population level. Precise knowledge of population immunity could allow government bodies to make informed decisions about how and when to relax stay-at-home directives and to reopen the economy. We hypothesized that salivary antibodies to SARS-CoV-2 could serve as a non-invasive alternative to serological testing for widespread monitoring of SARS-CoV-2 infection throughout the population. We developed a multiplex SARS-CoV-2 antibody immunoassay based on Luminex technology and tested 167 saliva and 324 serum samples, including 134 and 118 negative saliva and serum samples, respectively, collected before the COVID-19 pandemic, and 33 saliva and 206 serum samples from participants with RT-PCR-confirmed SARS-CoV-2 infection. We evaluated the correlation of results obtained in saliva vs. serum and determined the sensitivity and specificity for each diagnostic media, stratified by antibody isotype, for detection of SARS-CoV-2 infection based on COVID-19 case designation for all specimens. Matched serum and saliva SARS-CoV-2 antigen-specific IgG responses were significantly correlated. Within the 10-plex SARS-CoV-2 panel, the salivary anti-nucleocapsid (N) protein IgG response resulted in the highest sensitivity for detecting prior SARS-CoV-2 infection (100% sensitivity at ≥10 days post-SARS-CoV-2 symptom onset). The salivary anti-receptor binding domain (RBD) IgG response resulted in 100% specificity. Among individuals with SARS-CoV-2 infection confirmed with RT-PCR, the temporal kinetics of IgG, IgA, and IgM in saliva were consistent with those observed in serum. SARS-CoV-2 appears to trigger a humoral immune response resulting in the almost simultaneous rise of IgG, IgM and IgA levels both in serum and in saliva, mirroring responses consistent with the stimulation of existing, cross-reactive B cells. SARS-CoV-2 antibody testing in saliva can play a critically important role in large-scale 'sero'-surveillance to address key public health priorities and guide policy and decision-making for COVID-19.

Conflict of interest statement

Conflict of interest

In the interest of full disclosure, D.A.G. is founder and Chief Scientific and Strategy Advisor at Salimetrics, LLC and Salivabio, LLC and these relationships are managed by the policies of the committees on conflict of interest at Johns Hopkins School of Medicine and the University of California at Irvine. N.R. received funds from Sanofi Pasteur, Quidel, Merck and Pfizer.

Figures

Figure 1.
Figure 1.
Correlation between saliva and serum SARS-CoV-2 antigen-specific IgG among matched saliva and serum samples (n=28). Pearson correlation coefficient is provided for each antigen-specific IgG. p values are provided for statistically significant correlations only (p<0.05). Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; MFI=mean fluorescence intensity.
Figure 2.
Figure 2.
Correlation between saliva and serum SARS-CoV-2 antigen-specific IgA among matched saliva and serum samples (n=26). Pearson correlation coefficient is provided for each antigen-specific IgA. p values are provided for statistically significant correlations only (p<0.05). Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; MFI=mean fluorescence intensity.
Figure 3.
Figure 3.
Correlation between saliva and serum SARS-CoV-2 antigen-specific IgM among matched saliva and serum samples (n=26). Pearson correlation coefficient is provided for each antigen-specific IgM. p values are provided for statistically significant correlations only (p<0.05). Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; MFI=mean fluorescence intensity.
Figure 4.
Figure 4.
The sensitivity and specificity of each SARS-CoV-2 antigen-specific IgG, IgA, and IgM in saliva. Samples collected from individuals with RT-PCR confirmed prior SARS-CoV-2 infection are stratified into samples collected Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; Se: Sensitivity; Sp: specificity; MFI=mean fluorescence intensity. &Not all samples were tested for all isotypes due to limited saliva volume
Figure 5.
Figure 5.
The sensitivity and specificity of each SARS-CoV-2 antigen-specific IgG, IgA, and IgM in serum. Samples collected from individuals with RT-PCR confirmed prior SARS-CoV-2 infection are stratified into samples collected Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; Se: Sensitivity; Sp: specificity; MFI=mean fluorescence intensity. &Not all samples were tested for all isotypes due to limited serum volume
Figure 6.
Figure 6.
Comparison of saliva and serum SARS-CoV-2 antigen-specific IgG (red), IgA (blue), and IgM (green) responses vs. days post-COVID-19 symptom onset. The trajectories of IgG (red), IgA (blue), and IgM (green) responses are estimated using a LOESS curve. Dashed lines indicate cut off values for IgG (red), IgA (blue), and IgM (green). Note. Sino Biol.: Sino Biological; NAC: Native Antigen Company; N: nucleocapsid protein; ECD: S1: S1 subunit of spike protein; S2: S2 subunit of spike protein; ectodomain (S1 subunit+S2 subunit of the spike protein); RBD: receptor binding domain; (h): produced in human cell; (i): produced in insect cell; MFI=mean fluorescence intensity.

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