Exploring Spike-Dependent and ACE2-Independent SARS-CoV-2 Interactions with Salivary Epithelial Cells in the Absence of ACE2.

Salivary gland infection by SARS-CoV-2 requires viral entry via routes and mechanisms that remain unresolved. This study examined the expression of the angiotensin-converting enzyme 2 (ACE2) receptor in salivary tissues and basal cell-derived human salivary progenitor cells (hS/PCs), an unstudied potential entry point for SARS-CoV-2. Multiple detection modalities, including immunocytochemistry, Western blotting, flow cytometry and RT-PCR, demonstrated a consistent lack of ACE2 protein and transcript in both tissue specimens and primary salivary epithelial cells. Antigen retrieval at pH 9 was determined to be optimal for immunodetection protocols, yet ACE2 remained undetectable. Small intestine tissue served as a positive control, confirming the validity of the methods and reagents we used. Considering there can be other receptors for SARS-CoV-2, flow cytometric analyses demonstrated that recombinant SARS-CoV-2 spike protein failed to bind to salivary epithelial cells, in contrast to HEK293 cells engineered to overexpress ACE2, which showed robust spike binding. Additional studies showed that patient-derived salivary cells, negative for ACE2, are not infected by the SARS-CoV-2 pseudovirus, while ACE2-positive cells are readily infected. These findings strongly support our conclusion that salivary cells do not serve as major targets for SARS-CoV-2 infection via ACE2, spike protein, or an alternate receptor. Thus, salivary cells are unlikely major targets for SARS-CoV-2 infection, either through direct exposure to viral particles in ductal fluids or via access to basal cells across the basement membrane.