Prevalence of Salivary IgA Reacting with SARS-CoV-2 among Japanese People Unexposed to the Virus

While the COVID-19 pandemic caused by SARS-CoV-2 has posed a threat to public health as the number of cases and COVID-19-related deaths are increasing worldwide, the incidence of the virus infection are extremely low in Japan compared with many other countries. To explore the reason for this strange phenomenon, we hypothesized the high prevalence of "natural" secretory IgA in saliva as mucosal IgA reacting with SARS-CoV-2, and thus surveyed the positivity for, as well as levels of, such reactive salivary IgA in a cohort of Japanese people of a wide range of age. The major findings were that 95/180 (52.78 %) of overall individuals who had not been exposed to SARS-CoV-2 were positive for salivary IgA with the levels ranging from 0.002 to 3.272 ng/ml, and that there may be a negative trend in positivity for salivary IgA according to age. These results suggest a role of mucosal IgA in host defense against SARS-CoV-2 infection. One Sentence Summary"Natural" secretory immunoglobulin A autoantibodies may play a role in mucosal defense against SARS-CoV-2.

Detection of cross-reactive IgA in saliva against SARS-CoV-2 Spike1 subunit

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 [24-65] 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 = 0.008). 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.

The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2, in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the mucosa of the tongue and gingiva, the oral cavity seems like it is an entry point for SARS-CoV-2. Daily oral care using mouthwash seems to play an important role in preventing SARS-CoV-2 infection. However, the relationship between daily oral care and the mechanisms of virus entry into host cells is unclear. In this study, we evaluated the inhibitory effects of ingredients that are generally contained in toothpaste and mouthwash on the interaction between the spike protein and ACE2 and on the serine protease activity of TMPRSS2 using an enzyme-linked immunosorbent assay and in vitro enzyme assay, respectively. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to the inhibitor-binding site of ACE2. In addition, tranexamic acid and 6-aminohexanoic acid, which act as serine protease inhibitors, exerted inhibitory effects on TMPRSS2 protease activity. Further experimental and clinical studies are needed to further elucidate these mechanisms. Our findings support the possibility that toothpaste and mouthwash contain ingredients that inhibit SARS-CoV-2 infection.

Effects of Stress on Mouse beta-Defensin-3 Expression in the Upper Digestive Mucosa

PURPOSE: Gastrointestinal integrity and immune surveillance are affected by stress. Stress also adversely affects mucosal barrier function. beta-defensins constitute an integral component of the innate immune system as antimicrobial peptides, serving as the first line of defense against microbial pathogens at the epithelial surfaces of the upper digestive mucosa. The primary objective of this study was to determine the effects of stress on the expression profile of mouse beta-defensin-3 in the upper digestive mucosa of mice with diabetes. MATERIALS AND METHODS: We established a mouse model of restraint stress by using NSY/Hos mice with type 2 diabetes mellitus. We used real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry to investigate the effects of stress and glucocorticoid administration on mouse beta-defensin-3 expression in the upper digestive mucosa of the gingiva, esophagus, and stomach. RESULTS: Mouse beta-defensin-3 mRNA expression was higher in the esophagus than in the gingiva or stomach (p<0.05). In the esophagus, mouse beta-defensin-3 mRNA expression was lower in stressed mice than in non-stressed mice (p<0.05). Furthermore, immunoreactivity to mouse beta-defensin-3 protein was lower in the esophagus of stressed mice than non-stressed mice, consistent with the results of mRNA expression analysis. Systemic glucocorticoid administration also downregulated esophageal mouse beta-defensin-3 mRNA expression. CONCLUSION: Our novel findings show that stress decreases mouse beta-defensin-3 expression in the esophagus of mice with diabetes, possibly due to increased endogenous glucocorticoid production. It appears to be highly likely that stress management may normalize mucosal antimicrobial defenses in patients with diabetes.

Chronic Stress Induces Neurotrophin-3 in Rat Submandibular Gland

PURPOSE: Plasma neurotrophin-3 (NT-3) levels are associated with several neural disorders. We previously reported that neurotrophins were released from salivary glands following acute immobilization stress. While the salivary glands were the source of plasma neurotrophins in that situation, the association between the expression of neurotrophins and the salivary gland under chronic stress conditions is not well understood. In the present study, we investigated whether NT-3 levels in the salivary gland and plasma were influenced by chronic stress. MATERIALS AND METHODS: Expressions of NT-3 mRNA and protein were characterized, using real-time polymerase chain reactions, enzyme-linked immunosorbent assay, and immunohistochemistry, in the submandibular glands of male rats exposed to chronic stress (12 h daily for 22 days). RESULTS: Plasma NT-3 levels were significantly increased by chronic stress (p<0.05), and remained elevated in bilaterally sialoadenectomized rats under the same condition. Since chronic stress increases plasma NT-3 levels in the sialoadenectomized rat model, plasma NT-3 levels were not exclusively dependent on salivary glands. CONCLUSION: While the salivary gland was identified in our previous study as the source of plasma neurotrophins during acute stress, the exposure to long-term stress likely affects a variety of organs capable of releasing NT-3 into the bloodstream. In addition, the elevation of plasma NT-3 levels may play important roles in homeostasis under stress conditions.