key: cord-0798061-ls8ia96u
authors: Tsukinoki, K.; Yamamoto, T.; Handa, K.; Iwamiya, M.; Ino, S.; Sakurai, T.
title: Detection of cross-reactive IgA in saliva against SARS-CoV-2 Spike1 subunit
date: 2021-04-01
journal: nan
DOI: 10.1101/2021.03.29.21253174
sha: 17168c09b2e39ad49313ee4d609a77fa3ef3e1b0
doc_id: 798061
cord_uid: ls8ia96u

Abundant secretory IgA (sIgA) in mucus, breast milk, and saliva provides immunity that prevents infection of mucosal surfaces. sIgA in pre-pandemic breast milk samples have been reported to cross-react with SARS-CoV-2, but whether it also occurs in saliva and, if so, whether it cross-reacts with SARS-CoV-2, has remained unknown. We aimed to clarify whether sIgA in saliva cross-reacts with SARS-CoV-2 spike 1 subunit in individuals who have not been infected with this virus. The study included 137 (male, n = 101; female, n = 36; mean age, 38.7 [from 24 to 65] years) of dentists and doctors in the Kanagawa Dental University Hospital. Saliva and blood samples were analyzed by PCR and immunochromatography for IgG and IgM, respectively. We then identified patients with saliva samples that were confirmed as PCR- and IgM-negative for COVID-19. Proportions of SARS-CoV-2 cross-reactive IgA-positive individuals were determined by enzyme-linked immunosorbent assay using a biotin-labeled spike S1-mFc recombinant protein covering the receptor-binding domain of SARS-CoV-2. The proportion of SARS-CoV-2 cross-reactive IgA-positive individuals was 46.7%, and this correlated negatively with age (r = -0.218, p = 0.01). The proportion of IgA-positive individuals [≥] 50 y was significantly lower than that of patients aged [≤] 49 y (p = 0.005). sIgA was purified from the saliva of all patients, and the salivary sIgA was found to suppress the binding of SARS-CoV-2 spike protein to the ACE-2 receptor. We found SARS-CoV-2 cross-reactive sIgA in the saliva of some participants who had never been infected with the virus, suggesting that sIgA helps prevent SARS-CoV-2 infection

glands onto mucosal surfaces, where it plays a central role in preventing the entry of 48 antigens from the mucosa [1] . Severe acute respiratory syndrome coronavirus 2 (SARS-49

CoV-2) infects humans via the oral and nasal cavities, and the lungs [2]. The squamous 50 cells of the tongue and periodontal tissues express angiotensin-converting enzyme-2 51 (ACE-2), a SARS-CoV-2 receptor, transmembrane protease serin 2 (TMPRSS2), and furin, 52 which are proteases that promote infection [3] , and saliva can harbor SARS-CoV-2 [4] . 53

Saliva also contains several substances that suppress infection (such as lactoferrin, 54 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 1, 2021. ; https://doi.org/10.1101/2021.03.29.21253174 doi: medRxiv preprint lysozyme, and sIgA, which is the most abundant) and prevent the virus from entering 55 into the oral cavity [5] . 56

Cross-reactive sIgA (CRsA) against SARS-CoV-2 was identified in breast milk before 57 the COVID-19 pandemic [6, 7] . Furthermore, SARS-CoV-2-reactive CD4+ T cells were 58 also detected in about 40%-60% of unexposed individuals before the pandemic, 59

suggesting that T cells have cross-reactivity to common cold coronaviruses and SARS-60

CoV-2 [8]. Later findings [9, 10] suggested that prior infection with a coronavirus creates 61 immunological memory that is associated with sIgA cross-reactivity . 62

Infection with SARS-CoV-2 causes COVID-19, which manifests with a unique 63 spectrum of symptoms ranging from asymptomatic to fatal acute respiratory failure [11] . 64

The severity and prevalence of SARS-CoV-2 infection noticeably differs among age 65 groups and countries, respectively [12] . Immune mechanisms might explain this wide 66 disparity, but they are not yet fully understood. Immunoglobulin G (IgG) can eliminate 67 SARS-CoV-2, and vaccine development against this virus is urgently needed [13] . 68 However, mucosal immunity conferred by sIgA has not been investigated from the 69 viewpoints of recovery from SARS-CoV-2 infection and its prevention. New findings in 70 this area might facilitate deeper understanding of COVID-19 characteristics. 71

Therefore, we aimed to create an enzyme-linked immunosorbent assay (ELISA) for 72 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 1, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 1, 2021. ; https://doi.org/10.1101/2021.03.29.21253174 doi: medRxiv preprint 6 Saliva collection for ELISA 91 We collected samples using Salivettes ® (Sarstedt AG & Co. KG, Nümbrecht, Germany) 92 at a fixed room of the hospital between 9 a.m. and 12 p.m. in August 2020, under the 93 infection control. The participants were instructed to refrain from eating, drinking, and 94 brushing their teeth for at least 1 hour before sample collection. The saliva samples were 95 immediately centrifuged at 2,000 × g for 15 minutes and then stored at -80ºC. Laboratories, Kumamoto, Japan). Biotin-labeled Spike 1 were added to a concentration 108 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 1, 2021. showed an absorbance of 2.402, which was 2.155 or higher, the binding between ACE-2 189 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 1, 2021. Participants with saliva samples that tested negative for COVID-19 determined by 205 PCR and antibody tests did not develop COVID-19 at the time of this article submission. 206

Thus, we believe that they had not been exposed to SARS-CoV-2. As such, we revealed 207 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 1, 2021. nucleocapsid proteins, it is reasonable that saliva contains antibodies such as salivary 219 sIgA with cross-reactivity to SARS-CoV-2. However, the epitope behind salivary CRsA 220 cross-reactivity remains to be clarified. 221

The present study found that CRsA levels decreased with advancing age. This might 222 be explained by the fact that IgA levels decrease with age [21]. In addition, cross-reactive 223

IgG antibodies were identified in 62%, 43.75%, and 5.72% of serum samples from 224 individuals aged 1-16, 17-25, and ≥ 26 years, respectively, before the SARS-CoV-2 225 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 1, 2021. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 1, 2021. ; https://doi.org/10.1101/2021.03.29.21253174 doi: medRxiv preprint

Intake of 283 indigestible carbohydrates influences IgA response and polymeric Ig receptor 284 expression in the rat submandibular gland

An overview of COVID-19

Existence 288 of SARS-CoV-2 entry molecules in the oral cavity

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted April 1, 2021. ; https://doi.org/10.1101/2021.03.29.21253174 doi: medRxiv preprint