Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medicine

Major Professor

Shyam Mohapatra, Ph.D.

Committee Member

Burt Anderson, Ph.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Bala Chandran, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Keywords

COVID-19, fatty acids, Metabolites, RSV, SARS-CoV-2

Abstract

Coronavirus disease-19 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is an ongoing pandemic-endemic. In the United States, it remains the third leading cause of death in 2022. SARS-CoV-2, an RNA virus, has a genome prone to rapid mutation, generating variant species capable of resisting therapies and vaccination efforts. However, it has been estimated that more than 40% of SARS-CoV-2 infections may be asymptomatic, which calls into question any biological differences between the asymptomatic and afflicted groups. Additionally, Respiratory Syncytial Virus (RSV) is the most significant cause of bronchiolitis and asthma in neonates, with an increasing burden on the elderly. Concurrent outbreaks of SARS-CoV-2 and RSV remain a considerable problem. The microbiome and dietary practices are important factors impacting patient health and immune system homeostasis. The microbiome has an intimate relationship with the host’s immune system, providing both tempering and stimulating factors in the form of metabolites, which can act on distal organ sites, forming multiple axes. One class of metabolites is short-chain fatty acids (SCFAs), produced from dietary fiber fermentation. Metabolites, including SCFA, can cross the gut lumen into circulation and other organ systems, including the lungs, referred to as the “gut-lung axis.” The Free fatty acid receptor (FFAR) 2 is the receptor for SCFA acetate, which signals for the upregulation of toll-like receptors (TLRs), improving interferon responsiveness and an antiviral phenotype. However, the half-life of SCFAs is 1-2 hours in serum. Another fatty acid class is the Omega-3 polyunsaturated long-chain fatty acids (LCFAs), which have an affinity for the spike protein of SARS-CoV-2 at the receptor binding domain. LCFA interaction with SARS-CoV-2 has been shown to antagonize viral binding and has a ~16-hour half-life. Further, the Omega class of LCFA activates FFAR4, which inhibits IL-6 and IL-1β induction, reducing inflammation associated with the COVID-19 cytokine storm. From these observations, we sought to study the relationship between these two fatty acid classes in inducing an antiviral phenotype and their synergistic potential. Specifically, we found that SCFA acetate and LCFA linolenic acid act synergistically, reducing SARS-CoV-2 nucleoprotein (CoV-2 N) expression by qPCR. The concentrations derived by the synergy screen were used to investigate the downstream effects, in vitro. The results displayed reduced CoV-2 N expression, IL-6 expression, and IL- 1β and increased IFN- β expression by qPCR while showing reduced virus plaque-forming units (PFU). We also observed a similar phenotypic response in A549 cells treated with these combinations that had been infected with RSV. Interestingly, using FFAR2 and FFAR4 inhibitors reversed this phenotype on cytokines but did not affect viral titers when linolenic acid was used. Since acetate has a short half-life, we hypothesized that a liposomal nanoformulation incorporating linolenic acid could encapsulate acetate and be delivered intranasally during COVID-19 and may ablate infection. Lastly, we demonstrated that liposomal nanoformulations with linolenic acid could deliver a payload upon interaction with SARS-CoV-2, which has a dual effect for drug release, as well as the particle itself, hampering the virus directly by linolenic acid. We tested this formulation encapsulating acetate and found that it ablated infection in a murine model of SARS-CoV-2 and increased FFAR2 and FFAR4 expression in the lungs by qPCR and IHC. Even though vaccines and therapies have come into use for the prevention and treatment of COVID-19, their efficacy has waned over time. By studying how acetate, linolenic acid, and FFARs contribute to the survival of COVID-19, we were able to better understand how SARS-CoV-2 infection and host factors are interrelated for disease outcome and resistance. These findings allow us to strategize potential ways of combatting COVID-19 in the future by considering host factors as targets via nanoformulations.

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