Salivary anti-SARS-CoV-2 IgA as an accessible biomarker of mucosal immunity against COVID-19

Abstract

Background: Mucosal immunity, including secretory IgA (sIgA), plays an important role in early defenses against respiratory pathogens. Salivary testing, the most convenient way to measure sIgA, has been used to characterize mucosal immune responses to many viral infections including SARS, MERS, influenza, HIV, and RSV. However, its role has not yet been characterized in the COVID-19 pandemic. Here, we report development and validation of a rapid immunoassay for measuring salivary IgA against the SARS-CoV-2 virus, and report quantitative results in both pre-COVID-19 and muco-converted subjects.

Methods: We developed and refined a specific test for salivary IgA against SARS-CoV-2 on the Brevitest platform, a rapid immunoassay system designed for point-of-care use. A qualitative test was validated as per FDA guidelines with saliva obtained from subjects prior to the emergence of COVID-19, and from PCR-confirmed COVID-19 patients. We also generated a quantitative measure of anti-SARS-CoV-2 salivary IgA. Time taken for saliva self-collection was measured and its ease-of-use assessed.

Results: We successfully validated a qualitative salivary assay for SARS-CoV-2 IgA antibodies, with positive and negative predictive values of 92% and 97%, respectively, and no observable cross-reactivity with any of seven potential confounders. Pre-COVID-19 saliva samples showed an 8-fold range of IgA concentrations, suggesting a broad continuum of natural antibody resistance against the novel virus, though at levels lower than that observed in COVID-19 PCR-confirmed subjects. Samples from muco-positive subjects also shown a ~9-fold variation in salivary IgA levels, with elevated salivary IgA observed beyond three months after onset of symptoms. We observed a correlation (r=0.4405) between salivary IgA levels and COVID-19 disease severity. In anecdotal observations, we observed individuals who exhibited antibodies early in the course of their disease, contemporaneously with a positive PCR test, as well as individuals who muco-converted despite no known direct exposure to a COVID-19 patient, no symptoms, and negative molecular and/or serum antibody tests. Salivary collection took 5-10 minutes, and was reported as being easy (mean of 1.1 on a scale of 1 to 10).

Implications: Mucosal immunity, including secretory IgA, plays an important role in host defense against respiratory pathogens, and our early data suggest it may do so in COVID-19. Salivary IgA, an accessible marker of mucosal immunity, may be a useful indicator of several key parameters including individual and community immune response, disease severity, clinical risk, and herd immunity. The non-invasive nature and ease of saliva collection facilitates its potential use as a biomarker for ongoing patient assessment and management, as well as a community surveillance tool. By measuring mucosal immune responses directly and systemic immune responses indirectly, salivary IgA could be useful in developing and deploying a vaccine(s) against COVID-19. Quantitative IgA assessment could also potentially serve as a tool to segment the population into different risk categories and inform individual and collective decisions relating to appropriate activities and vaccine prioritization/delivery. These data reinforce the importance of further investigation into the role of mucosal immunity and IgA in host responses against COVID-19.

Figure 1.

Increasing concentrations of commercially available anti-S1 and anti-NP IgA antibodies were spiked into simulated saliva in order to mimic COVID-19 patient samples. These samples were loaded onto the BRAVO assay and run via the Brevitest analyzer. Results indicated a broad linear range of acceptable antibody concentrations that could be robustly detected with BRAVO assay. A simple linear regression was utilized to demonstrate the linear range of the BRAVO assay (r²=0.9706, p<0.0001).

Figure 2.

True negative (pre-COVID, acquired prior to December 2019) patient saliva samples were sourced from a commercial vendor and assessed using the BRAVO assay. Although variation in detectable salivary IgA existed across the samples (potentially due to polyreactive IgA or cross-reactivity with prior coronavirus exposure), the results clustered around a mean of 43.2 AU. We set a threshold cut-off to separate negative from positive samples where 95% of samples were negative. This value was set at 75 AU, or 1.65 standard deviations above the negative value mean (indicated by the broken red line).

Figure 3.

(A) The BRAVO clinical validation study included pre-COVID samples (collected prior to December 2019) as negative controls and saliva from PCR-confirmed COVID-19 patients as the positive control. The 30 negative control samples used to determine a cut-off for muco-positivity as described in the methods. One false positive (green dot) had readings above the cut-off. Of the 38 COVID-19 subjects, three (red dots) has salivary IgA levels below the cut-off and were deemed to be False Negative results.

Figure 4.

A) The number of days since onset of symptoms or the number of days since a positive PCR result (whichever was greater) was plotted against the BRAVO result. No correlation was identified. B) At the time of sample collection, patients were asked to rate their COVID-19 disease severity by using the WHO Ordinal Scale, where 0–4 indicates mild disease (increasing in severity) and >5 indicates severe disease. We observed a correlation between disease severity and BRAVO result, with a two-tailed Pearson Correlation of 0.4405 and a p-value=.0044. (C) For patients who were able to record the number of days of symptoms, we assessed if IgA levels (as determined via BRAVO score) correlated with duration. We did not identify any correlation.