Innate and adaptive defenses against SARS-COV-2 in the oral cavity during acute unvaccinated and breakthrough infections

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Background. Vaccines against SARS-CoV-2 (CoV-2) do not prevent infection and subsequent transmission. Strong evidence now implicates the oral cavity as a primary site of acute CoV-2 infection It is therefore incumbent upon us to search for evidence as to why primary mucosal sites, such as the tongue, remain vulnerable to CoV-2 infection despite vaccination. Our goal. Immune responses are both innate, which are not specific to an invading pathogen, and adaptive, which are specific antibody responses. We hypothesize that interrogating oral epithelial cells and saliva collected from CoV-2 acutely infected unvaccinated and breakthrough-infected subjects will reveal early and specific innate and adaptive immune responses that vary with viral load, viral variants, viral clearance, and patient sex and age. We will collect samples at two time points from acutely infected unvaccinated participants; one within 7 days and the second within 14 days of testing positive. We will evaluate the relationship among measures of oral mucosal viral replication, innate and adaptive factors from salivary, cytological, and serological sources and determine their associations with quantitative viral measures in the oral mucosa. Innate factors to be analyzed in the saliva include, but not limited to, cytokines, antimicrobial peptides, interferons (IFN) and secretory IgA that are correlated with early infection events. To cross validate our results we will also characterize viral strains and develop novel PCR-based assays for intracellular CoV-2 RNA. Studies will include single cell RNA sequencing to analyze virus-induced gene expression changes in infected and bystander cells from the tongue. Full genome sequencing will be used to determine if viral variants are present and correlate oral immune profiles with variants. Finally we will develop digital PCR assays to selectively amplify sub genomic CoV-2 RNA as a rapid measure of infected cells. Once this baseline information is established, we will compare the adaptive and innate oral immune factors of CoV-2 vaccinated and breakthrough study participants, respectively, to the oral immune responses in natural infection. How will we advance the field? This study will allow us to chronicle for the first time the impact of vaccination on oral immune responses of healthy individuals and compare them to immune responses from COVID-19-exposed individuals. We anticipate that our integrated accounting of the oral immune responses during the natural history of SARS-CoV-2 infections will provide an invaluable reference data set for patients, healthcare professionals and those measuring the efficacy of antiviral treatments, immunotherapies and vaccines.