Detection of SARS-CoV-2 and Its Related Factors on the Mucosal Epithelium of the Tongue

SARS-CoV-2 infects a variety of tissues, including the oral cavity. However, there are few reports examining the association of SARS-CoV-2 with tongue mucosal tissues with sticky tongue debris. This study investigated the presence of SARS-CoV-2 and its associated molecules by dissecting tongue tissue from autopsy specimens of 23 patients who died of COVID-19-related illness (pneumonia). Immunohistochemical staining, electron microscopy, and PCR analysis were performed on the tongue tissue specimens. The mucosal epithelium of the tongue formed a very thick keratinized with well-developed filiform papillae in all cases. ACE2 and TMPRSS2 were consistently co-expressed in all samples in the epithelium. The S-protein was strongly expressed in basal cells and the epithelial surface. S-protein-positive viral particles were detected in the tongue's stratified squamous epithelium via an immunoelectron microscope. Based on PCR amplification of the N1 and N2 regions, the SARS-CoV-2 gene was detected on the tongue epithelium, tongue submucosa, and in tongue debris. This suggests that tongue debris, including the squamous epithelial tissue, could be a source of SARS-CoV-2 in saliva. Furthermore, removing tongue debris may decrease the amount of SARS-CoV-2 in the oral cavity.

Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary IgA: 58-870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

Prevalence of saliva immunoglobulin A antibodies reactive with severe acute respiratory syndrome coronavirus 2 among Japanese people unexposed to the virus.

While the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to public health as the number of cases and COVID-19-related deaths are increasing worldwide, the incidence of the virus infection is extremely low in Japan compared with many other countries. To explain this uncommon phenomenon, we investigated the prevalence of naturally occurring ("natural") antibodies, focusing on those of the secretory immunoglobulin A (sIgA) form, reactive with SARS-CoV-2 among Japanese people. One hundred and eighty healthy Japanese volunteers of a wide range of age who had been considered to be unexposed to SARS-CoV-2 participated in this study. Saliva samples and blood samples were collected from all of the 180 participants and 139 adults (aged ≥ 20 years) included therein, respectively. The determination of saliva IgA antibodies, mostly comprising sIgA antibodies, as well as serum IgA and immunoglobulin G antibodies, reactive with the receptor binding domain of the SARS-CoV-2 spike-1 subunit proteins was conducted using an enzyme-linked immunosorbent assay. The major findings were that 52.78% (95% confidence interval, 45.21%-60.25%) of the individuals who had not been exposed to SARS-CoV-2 were positive for saliva IgA antibodies with a wide range of levels between 0.002 and 3.272 ng/mL, and that there may be a negative trend in positivity for the antibodies according to age. As we had expected, a frequent occurrence of assumable "natural" sIgA antibodies reactive with SARS-CoV-2 among the studied Japanese participant population was observed.

Detection of cross-reactive immunoglobulin A against the severe acute respiratory syndrome-coronavirus-2 spike 1 subunit in saliva.

Abundant secretory immunoglobulin A (SIgA) in the mucus, breast milk, and saliva provides immunity against infection of mucosal surfaces. Pre-pandemic breast milk samples containing SIgA have been reported to cross-react with SARS-CoV-2; however, it remains unknown whether SIgA showing the cross-reaction with SARS-CoV-2 exists in saliva. 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 involved 137 (men, n = 101; women, n = 36; mean age, 38.7; age range, 24-65 years) dentists and doctors from Kanagawa Dental University Hospital. Saliva and blood samples were analyzed by polymerase chain reaction (PCR) and immunochromatography for IgG and IgM, respectively. We then identified patients with saliva samples that were confirmed to be PCR-negative and IgM-negative for SARS-CoV-2. The cross-reactivity of IgA-positive saliva samples with SARS-CoV-2 was determined by enzyme-linked immunosorbent assay using a biotin-labeled spike recombinant protein (S1-mFc) covering the receptor-binding domain of SARS-CoV-2. The proportion of SARS-CoV-2 cross-reactive IgA-positive individuals was 46.7%, which correlated negatively with age (r = -0.218, p = 0.01). The proportion of IgA-positive individuals aged ≥50 years was significantly lower than that of patients aged ≤49 years (p = 0.008). SIgA was purified from the saliva of patients, which could partially suppress the binding of SARS-CoV-2 spike protein to the angiotensin converting enzyme-2 receptor. This study demonstrates the presence of SARS-CoV-2 cross-reactive SIgA in the saliva of individuals who had never been infected with the virus, suggesting that SIgA may help 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.

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 tongue and gingival mucosa, the oral cavity is a potential entry point for SARS-CoV-2. This study evaluated the inhibitory effects of general ingredients of toothpastes and mouthwashes on the spike protein-ACE2 interaction and the TMPRSS2 protease activity using an in vitro assay. 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 inhibitor-binding site of ACE2. Furthermore, tranexamic acid exerted inhibitory effects on TMPRSS2 protease activity. Our findings suggest that these toothpaste and mouthwash ingredients could help prevent SARS-CoV-2 infection.

Existence of SARS-CoV-2 Entry Molecules in the Oral Cavity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, which promote entry of the virus into the host cell, have been identified as determinants of SARS-CoV-2 infection. Dorsal tongue and gingiva, saliva, and tongue coating samples were examined to determine the presence of these molecules in the oral cavity. Immunohistochemical analyses showed that ACE2 was expressed in the stratified squamous epithelium of the dorsal tongue and gingiva. TMPRSS2 was strongly expressed in stratified squamous epithelium in the keratinized surface layer and detected in the saliva and tongue coating samples via Western blot. Furin was localized mainly in the lower layer of stratified squamous epithelium and detected in the saliva but not tongue coating. ACE2, TMPRSS2, and furin mRNA expression was observed in taste bud-derived cultured cells, which was similar to the immunofluorescence observations. These data showed that essential molecules for SARS-CoV-2 infection were abundant in the oral cavity. However, the database analysis showed that saliva also contains many protease inhibitors. Therefore, although the oral cavity may be the entry route for SARS-CoV-2, other factors including protease inhibitors in the saliva that inhibit viral entry should be considered.