ARS-CoV-2 is the causal agent for COVID-19, and the World Health Organization classifies this virus as an airborne pathogen transmitted by asymptomatic, pre-symptomatic and symptomatic individuals through close contact via exposure to infected droplets and aerosols 1,2 . Although SARS-CoV-2 transmission can occur by activities involving the oral cavity, such as speaking, breathing, coughing, sneezing and even singing [3][4][5] , most attention has focused on the nasal-lung axis of infection 6 . Oral manifestations, such as taste loss, dry mouth and oral lesions, are evident in about half of COVID-19 cases [7][8][9] , although it remains unknown whether SARS-CoV-2 can directly infect and replicate in oral tissues, such as the salivary glands (SGs) or mucosa. This is critical because, if these are sites of early infection, they could play an important role in transmitting the virus to the lungs or the gastrointestinal tract via saliva, as has been suggested for other microbial-associated diseases, such as pneumonia 10 and inflammatory bowel diseases 11,12 (Extended Data Fig. 1a).SARS-CoV-2 uses host entry factors, such as ACE2 and TMPRSS family members (TMPRSS2 and TMPRSS4) 13,14 , and understanding the cell types that harbor these receptors is important for determining infection susceptibilities throughout the body [15][16][17] . ACE2 and TMPRSS2 expression has been reported in oral tissues 18,19 ; however, there are no comprehensive descriptions of viral entry factor expression nor direct confirmation of SARS-CoV-2 infection in oral tissues. We hypothesized that SGs and barrier epithelia of the oral cavity and oropharynx can be infected by SARS-CoV-2 and contribute to the transmission of SARS-CoV-2. To test this, we generated two human oral single-cell RNA sequencing (scRNA-seq) atlases to predict cell-specific susceptibilities to SARS-CoV-2 infection. We confirmed ACE2 and TMPRSS expression in SGs and oral mucosa epithelia. SARS-CoV-2 infection was confirmed using autopsy and outpatient samples. Saliva from asymptomatic individuals with COVID-19 demonstrated the potential for viral transmission. In a prospective clinical cohort, we found a positive correlation between salivary viral load and taste loss; we also demonstrated persistent salivary antibody responses to SARS-CoV-2 nucleocapsid and spike proteins. ResultsOral tissue atlases reveal resident immune cells and niche-specific epithelial diversity. The SGs and the barrier mucosa of the oral cavity and oropharynx are likely gateways for viral infection, replication SARS-CoV-2 infection of the oral cavity and saliva

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing datasets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection andimplicates saliva in viral transmission.

ObjectivesSjögren’s syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS.MethodsRetroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically.ResultsFollowing LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs.ConclusionsThese results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.