SARS-CoV-2 Latency in Oral and Salivary Tissues

The premise of our proposal for project SARS-CoV-2 Latency in Oral and Salivary Tissues was to demonstrate that SARS-CoV-2 infections of the salivary glands, or other oral tissues such as tongue or tonsil, may enter a state of non-replicative latency, undetected by current testing of nasopharyngeal secretions, and contribute to false negatives and carrier spread upon viral reactivation. In conducting this study, the project team comprised of Dr. Mary C. Farach-Carson (UTSD, Houston, TX), Dr. Simon Young (UTSD, Houston, TX), Dr. Danielle Wu (UTSD, Houston, TX) and Dr. Robert L. Witt (CCHS, Newark, DE) made some progress toward the three original outlined aims and those advances how been detailed below:

Aim 1. Genotype ACE2 variants present in cells. To sequence the various ACE2 polymorphisms present in our salivary cell populations, the project team is utilizing the services of the Baylor College of Medicine (BCM) Genomic and RNA Profiling Core. In collaboration with the core staff, we identified the Illumina Ampliseq platform as the optimal choice for sequencing ACE2. Following an initial delay in having samples sequenced because of issues involving supply lines for critical reagents, staffing issues during the phased reopening of the Texas Medical Center, and sample quality issues, we now have begun regular sequencing. We learned that the BCM Core needed to use a fast ramp thermocycler to generate the PCR libraries that could be successfully sequenced. Together, with the Core and Illumina, we successfully worked out the details of the sequencing protocol and sequenced three samples. We are now rapidly expanding cells from a diverse cohort of patients to finish sequencing. We are using FASTQ files generated from the sequencer to verify the polymorphisms in our patient populations and expect to have a set of cells with different versions of ACE2 for use in Aim 2 shortly.

Aim 2. Use salivary cells representing various genotypes of ACE2 cultured in 3D for study of SARS-CoV2 infection efficiency. The project team has been expanding the hS/PCs sent for sequencing for ductal differentiation in our 3D hydrogel systems. In parallel, they are producing our pseudo-typed SARS-CoV-2 virus. The team has successfully created lentiviral particles pseudotyped with the spike protein of SARS-CoV2 and for proof-of-concept infected ACE2 expressing HEK293T cells with p-SARS-CoV-2. Currently, we are finishing a large batch and titering the lentiviral particles for use in infectivity studies. While working through the sequencing issues in aim 1, the project team also validated rt-qPCR for ACE 2. In doing so, we discovered that our undifferentiated hS/PCs do not express ACE2, thus require ductal differentiation for infectivity studies. Current work focuses on driving ductal differentiation in hydrogels or matrix-coated dishes by supplying appropriate cues favoring this phenotype and we hope to continue this with a no-cost extension on the current funding that has been generously provided.

It is known from previous work that ductal differentiation of salivary hS/PCs requires special signals from extracellular matrix in combination with soluble factors produced by tissue stroma and nerves. Initial approaches used employed tissue culture dishes coated with either Matrigelâ„¢ or laminin 5, a major component of the ductal basement membrane. We observed a modest increase in ACE2 transcript levels in cultured, differentiating hS/PCs by day 13 compared to day 6 (Figure 3), but we expect to greatly improve on this by adding stromal factors FGF7 and FGF10. Earlier work showed that branching and expression of the ductal marker K19 was increased by a protocol using a hyaluronic acid/laminin1-containing (1:1) culture system (Figure 4). Cells were encapsulated as single cells into gels and allowed to assemble into microstructures over 7 days. On day 8, 100 ng/ml of FGF10 was added and culture continued until day 10, at which time 100 ng/ml of FGF7 was added for an additional day. On day 11, cells in gels were fixed and stained. The project team, will use this system, or a variation of it, for continuing studies in Aim 2. We are testing if addition of FGF7/FGF10 are sufficient to induce a ductal phenotype expressing ACE2. In addition, we will test if co-culture with nerve cells, present in close proximity to ductal tissues in salivary glands, or factors secreted by nerve cells will differentiate hS/PCs into ACE2 expressing ductal-like tissues suitable for SARS-CoV2 infectivity studies.

Aim 3. Examine oral tissues from asymptomatic or recovered COVID-19 patients for presence of SARS-CoV2 transcripts. As a result of The DTA Foundation’s generous support, the project team was able to receive and have frozen over 30 salivary gland patient tissues that were collected during the pandemic. Because of hospital policies on surgeries during the pandemic, all patients undergoing surgery were negative for SARS-CoV2 by PCR assay at the time of surgery. In later phases of the project, the research team intends to isolate RNA from these specimens and determine if any possess transcripts for SARS-CoV2 despite testing negative. Given the broad infectivity of Omicron in late 2021 and early 2022, we expect that future tissue collections will provide a better source of recovered patients for testing. At present, we are more focused on completing the studies in Aims 1 and 2, due to the unforeseen delays and impacts of an ongoing pandemic. Since submitting this proposal, the research team has learned through analysis of oral tissues that the receptor for SARS-CoV2, ACE2, is located almost exclusively along the ductal lining in human salivary glands. Thus, tissue engineered models to test infectivity must include replicates of the ductal lining and be induced to express ACE2. We have learned that our human salivary stem/progenitor cell (hS/PC) population does not express ACE2, as expected for an undifferentiated cell population. Achieving the proposal objectives thus required two technical innovations upon which we have made great progress that paves the way forward: 1) creating a hydrogel system that will support ductal cell differentiation from hS/PCs, and 2) creating a pseudovirus to mimic SARS-CoV2 (various subtypes) that will infect ductal-like cells expressing various polymorphisms of ACE2.