Salivary Exosome Proteomics and Bioinformatics Analysis in 7,12-Dimethylbenz[a]anthracene-induced Oral Cancer with Radiation Therapy—A Syrian Golden Hamster Model

Exosomes carry cellular proteins and contain molecules that can be potential biomarkers of diseases. This study used a Syrian golden hamster model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinoma with radiation therapy to exclude the confounding factors that may affect outcomes in clinical studies, and re-examine the role of exosomes during tumorigenesis. We used data-dependent acquisition-based quantitative proteomics and bioinformatics analyses and found unique proteins present (desmocollin-2) or absent (Glucagon-cAMP-PKA-CREB pathway-related proteins) in the salivary exosomes of the pre-radiation DMBA-treated group (PreD). Comparing our data to other studies, salivary exosomes in the PreD group were found carrying proteins that the tumor mass does not express and lacking the proteins needed during tumorigenesis. Immunohistochemistry staining showed p53 expression but a negative apoptotic signal in the PreD tumor tissue. We thus suggest that inhibition of desmocollin-2 expression in tumor tissue may impede the activation of cell apoptosis. However, both the origin of the salivary exosomes and main role of the salivary exosome proteins should be clarified in future studies.

Dietary-Induced Elevations of Triglyceride-Rich Lipoproteins Promote Atherosclerosis in the Low-Density Lipoprotein Receptor Knockout Syrian Golden Hamster

Elevated triglycerides are associated with an increased risk of cardiovascular disease (CVD). Therefore, it is very important to understand the metabolism of triglyceride-rich lipoproteins (TRLs) and their atherogenic role in animal models. Using low-density lipoprotein receptor knockout (LDLR−/−) Syrian golden hamsters, this study showed that unlike LDLR−/− mice, when LDLR−/− hamsters were fed a high cholesterol high-fat diet (HFD), they had very high plasma levels of triglycerides and cholesterol. We found that LDLR−/− hamsters exhibited increased serum TRLs and the ApoB100 and 48 in these particles after being fed with HFD. Treatment with ezetimibe for 2 weeks decreased these large particles but not the LDL. In addition, ezetimibe simultaneously reduced ApoB48 and ApoE in plasma and TRLs. The expression of LRP1 did not change in the liver. These findings suggested that the significantly reduced large particles were mainly chylomicron remnants, and further, the remnants were mainly cleared by the LDL receptor in hamsters. After 40 days on an HFD, LDLR−/− hamsters had accelerated aortic atherosclerosis, accompanied by severe fatty liver, and ezetimibe treatment reduced the consequences of hyperlipidemia. Compared with the serum from LDLR−/− hamsters, that from ezetimibe-treated LDLR−/− hamsters decreased the expression of vascular adhesion factors in vascular endothelial cells and lipid uptake by macrophages. Our results suggested that in the LDLR−/− hamster model, intestinally-derived lipoprotein remnants are highly atherogenic and the inflammatory response of the endothelium and foam cells from macrophages triggered atherosclerosis. The LDL receptor might be very important for chylomicrons remnant clearance in the Syrian golden hamster, and this may not be compensated by another pathway. We suggest that the LDLR−/− hamster is a good model for the study of TRLs-related diseases as it mimics more complex hyperlipidemia.

577. COVI-VAC™, a Live Attenuated COVID-19 Vaccine, Provides Single Dose Protection Against Heterologous Challenge with SARS-CoV-2 Beta (B.1.351) in the Syrian Golden Hamster Model

Background Although multiple COVID-19 vaccines are currently in use, emergence of novel SARS-CoV-2 variants with reduced neutralization raises concern of future vaccine escape. COVI-VAC™ is a live attenuated SARS-CoV-2 strain based on WA/1 being developed as an intranasal COVID-19 vaccine. COVI-VAC is attenuated through removal of the furin cleavage site and introduction of 283 silent, deoptimizing mutations that maintain viral amino acid sequence but slow viral replication in vivo by up to 5 logs. Notably, COVI-VAC presents all viral antigens in their native conformation and is not limited to spike. COVI-VAC demonstrated attenuation, immunogenicity and single dose protection in both the Syrian golden hamster and non-human primate models and currently in Phase 1 clinical trials. In this study, we evaluated efficacy of COVI-VAC against challenge with the Beta/B.1.351 variant in Syrian golden hamsters. Methods Syrian golden hamsters, 7-10 weeks of age were, vaccinated intranasally with 8.25x104 PFU COVI-VAC (n=28) or vehicle control (n=16). Twenty seven days post-vaccination, animals were challenged intranasally with 3x104 PFU of wildtype (WT) SARS-CoV-2 Beta. Animals were weighed daily. Further analysis is being conducted with serum and key tissues from pre and post challenge timepoints to include neutralizing antibody, biodistribution (subgenomic qPCR) and histopathology. Results COVI-VAC prevented weight loss following challenge with the heterologous variant of SARS-CoV-2, B.1.351/Beta (Figure). Results of additional analyses will be available before the IDWeek meeting. Change in Weight following SARS-CoV-2 Beta Challenge Conclusion COVI-VAC is protective against heterologous challenge with SARS-CoV-2 Beta. By presenting all viral antigens, COVI-VAC may be less affected by viral evolution than spike-based vaccines. Disclosures Anna Kushnir, PHD, Codagenix Inc (Employee) Steffen Mueller, PhD, Codagenix Inc (Board Member, Employee, Shareholder) Sybil Tasker, MD, MPH, FIDSA, Codagenix Inc (Employee, Shareholder) J. Robert Coleman, PhD, Codagenix Inc. (Board Member, Employee, Shareholder)

Tissue Distribution of ACE2 Protein in Syrian Golden Hamster (Mesocricetus auratus) and Its Possible Implications in SARS-CoV-2 Related Studies

The Syrian golden hamster (Mesocricetus auratus) has recently been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers these models’ optimal use. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin-converting enzyme 2, a proven functional receptor for SARS-CoV-2 in different organs of the hamster. Using two different antibodies (MA5-32307 and AF933), we have conducted immunoblotting, immunohistochemistry, and immunofluorescence analysis to evaluate the ACE2 expression in different tissues of the hamster. Further, at the mRNA level, the expression of Ace2 in tissues was evaluated through RT-qPCR analysis. Both the antibodies detected expression of ACE2 in kidney, small intestine, tongue, and liver. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine were negative for ACE2 expression. Analysis of tissues from different age groups and sex didn’t show any obvious difference in ACE2 expression pattern or level. Together, our findings corroborate some of the earlier reports related to ACE2 expression patterns in human tissues and contradict others. We believe that this study’s findings have provided evidence that demands further investigation to understand the predominant respiratory pathology of SARS-CoV-2 infection and disease.

Tissue distribution of ACE2 protein in Syrian golden hamster (Mesocricetus auratus) and its possible implications in SARS-CoV-2 related studies

Recently, the Syrian golden hamster (Mesocricetus auratus) has been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers optimal use of these models. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin□converting enzyme 2 (ACE2), a proven functional receptor for SARS-CoV-2 in different organs of the hamster. We have adapted immunoblot analysis, immunohistochemistry, and immunofluorescence analysis techniques to evaluate the ACE2 expression pattern in different tissues of the Syrian golden hamster. We found that kidney, small intestine, esophagus, tongue, brain, and liver express ACE2. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections for ACE2 showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine (caecum, colon, and rectum) were negative for ACE2 expression. Together, our findings corroborate some of the earlier reports related to ACE2 expression pattern in human tissues and also contradicts some others. We believe that the findings of this study will enable the appropriate use of the Syrian golden hamster to carryout SARS-CoV-2 related studies.