Phenotypic Screening Funnel for Evaluation of Therapies Against BSL-3 Neurotropic Alphaviruses

Rapid drug discovery is crucial for neglected and emerging viral pathogens such as Ebola virus, Middle Eastern Respiratory Syndrome virus (MERS), Lassa virus, Chikungunya virus, and West Nile virus. The recent outbreaks of Ebola virus disease in West Africa in 2014-2016 and Zika virus epidemics in Brazil and other parts of South and North America in 2015-2016 demonstrated the scarcity of general anti-viral treatments efficient against these surfacing viral threats.

With changes in the global climate, many mosquito-borne diseases are both emerging and moving into new areas. Three of these are arboviruses of the alphavirus genus and family of Togaviridae that are currently geographically restricted to the Americas and cause severe and potentially fatal encephalitis in humans when they emerge from enzootic transmission: eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), and Venezuelan encephalitis virus (VEEV). All three of these viruses are listed as CDC category B bioterrorism agents. In humans, EEEV infection rapidly progresses to severe neurological disease with the possibilities of paralysis, seizure, and death in 30-80% of cases, with long-term neurological defects in 35% of survivors. Currently, there are no efficient anti-viral therapies or licensed human vaccine approved by the FDA for any of these infections.

The real power of phenotypic screening resides in the disease-relevance of the in vitro models chosen for the screening funnel. All three arboviruses are known to infect both neuronal cells and astrocytes. The development of the Stemonix® microBrain® Assay Ready platform, 384-well plates with a fully functional mixture of differentiated cortical neurons and astrocytes, allowed us to simultaneously evaluate the potency of anti-viral therapies against neurotropic viruses in both neurons and astrocytes. Because VEEV has the ability to infect additional cell lineages, including myeloid type cells, VEEV viral inhibition studies require a more extended portfolio of in vitro tests to develop the full characterization of potential inhibitors in all different cellular backgrounds.

In this work, we show the development of a phenotypic screening funnel to provide an efficient primary filter for down selection of potent molecules for further evaluation in PK/PD and Tox models and/or efficacy in rodent models.

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