The broad objective of this proposal is to develop an alternative therapy for the control and prophylaxis of equine herpesvirus type 1 (EHV-1) infections. Our group has recently shown that equine mesenchymal stem cell (MSC) secreted factors significantly reduce EHV-1 replication in infected cells, and can enhance the protective responses of host cells in the context of an EHV-1 infection in vitro. These observations led to our specific hypothesis, namely that equine MSC secreted factors can control EHV-1 infection by directly inhibiting viral replication and/or preventing EHV-1-induced damage to host cells. As such, MSC secreted factors, either used as a short-term prophylactic and/or during outbreaks to protect contact animals, can serve as a therapy or therapeutic adjunct to control EHV-1 infections. To test this hypothesis, we propose 2 specific aims:

Aim 1: Test the efficacy of MSC secreted factors against EHV-1 in vitro. Our preliminary data show antiviral activity of MSC secreted factors against EHV-1 in 2D cell culture systems. These experiments will be repeated using additional EHV-1 pathotypes (ie. neuropathogenic and non-neuropathogenic strains). Results will then be validated in equine nasal mucosa explants; physiologically relevant 3D respiratory culture models that closely mimic the in vivo environment, and which we have used successfully in the past for EHV-1 studies. These 3D cultures will be analyzed for EHV-1 replication in both epithelial cells and resident immune cells, and epithelial integrity will be analyzed. Completion of this aim will provide us with in-depth information regarding the direct antiviral effects of MSC secreted factors as well as indirect effects of factors secreted by MSC on EHV-1-induced host cell damage in vitro.

Aim 2: Test the efficacy of MSC secreted factors against EHV-1 in vivo. We found that we can lyophilize MSC secreted factors without losing activity upon reconstitution. This provides us with a readily available, shelf-stable product that can be applied to horses intranasally. To begin testing the prophylactic and therapeutic efficacy of MSC secreted factors in horses, we will apply them intranasally at 24h before, 24h after, and at the time of EHV-1 infection. A vehicle control will also be included. Efficacy will be evaluated based on clinical and virological parameters including nasal virus shedding, induction of fever, and viremia, as well as local immunological parameters. Completion of this aim will provide us with the first in vivo data on the efficacy of factors secreted by MSC to control EHV-1 infections.

This is the first study to focus on the antiviral properties of equine MSC secreted factors against EHV-1. The in vitro and in vivo data generated will provide the scientific foundation for developing and implementing strategies to reduce the negative impacts of EHV-1 infections in horses. In the long term, and if the proof-of-concept in vivo study proposed here is successful, the equine MSC secreted factors have potential as a cost-effective, off-the-shelf, biological alternative therapy or therapeutic adjunct to control EHV-1, and possibly also other infections important to the equine industry.