Introduction and Background of the Project

1.Introduction
The Filoviridae family comprises elongated filamentous viruses with genomes composed of negative-strand single-stranded RNA. Typically harbored in reservoir species like bats, these are zoonotic viruses, opportunistically crossing over to infect humans and other mammals, leading to severe and often fatal haemorrhagic fever. Among its most notorious members are the Ebola virus (EBOV), Marburg virus (MARV), and Sudan Ebola virus (SUDV), all associated with recent outbreaks in Africa. The EBOV outbreak spanning from 2014 to 2016 in West Africa stands as the most severe on record, with over 28,000 suspected cases and more than 11,000 fatalities ¹. MARV has also caused sporadic outbreaks in Uganda, the Republic of Congo, Guinea, Ghana, and other African nations, with mortality rates ranging from 24 to 88% ².

2.Project objective
The project aims to use a multi-filovirus "rainbow" system that can simultaneously test for EBOV, MARV, and SUDV using a single cell line to identify potential treatments against filoviruses using the RIKEN NPDepo library. Additionally, the project seeks to study the effectiveness of selected confirmed hits against other filoviruses (Tai Forest, Reston, Bundibogyo, Lloviu) in order to identify potential broad-spectrum antiviral compounds. This collaboration leverages the screening capabilities and drug development expertise of Japan’s largest comprehensive institution, PDP, and academic investigators to achieve its goals.

3.Project design
The primary screen will be performed on a subset of the RIKEN NPDepo library (20,000 compounds). The screen will be in a 384-well plate format with a single compound concentration of 10 µM. Compounds will be evaluated for their antiviral activity and impact on cell viability. Approximately 200 compounds will be selected for further confirmation studies based on specific criteria. Confirmed actives will undergo broad-spectrum antiviral testing and prioritization for further profiling against live Ebola virus. Selected hits meeting specific criteria will be considered for future development.

How can your partnership (project) address global health challenges?

Currently a vaccine against Ebola has been approved by FDA, but information about the duration of protection, vaccine effectiveness in outbreak situations, and the need for booster doses are still lacking³. Prophylactic measures are limited to isolating infected individuals, and patient care focuses primarily on managing symptoms, as there's no specific treatment available. The urgency for pandemic preparedness is underscored by the high mortality rates associated with filoviruses and the lack of therapeutic options. To address this threat, MMV and RIKEN aim to identify potential broad-spectrum anti-filovirus compounds, which can potentially lead to the future development of effective medical interventions for this disease, contributing to global health security and preparedness for future outbreaks.

What sort of innovation are you bringing in your project?

There is an unmet need for drugs active against filoviruses, Ebola and Marburg in particular. Our project aims at identifying new starting points for drug discovery against for which there are currently no drug candidates. The RIKEN NPDepo library is a depository of small molecules (druglike compounds, pure natural products and derivatives) and has never been screened against filoviruses.

Role and Responsibility of Each Partner

As the designated grantee for this project, the MMV is responsible for delivering the work plan within the agreed timeline and budget and for GHIT reporting.

The project will be carried out by a team consisting of scientists and project managers from RIKEN and MMV. RIKEN will provide a library of 20,000 compounds that have not yet been tested against the target virus of this proposal. The library will be tested at KU Leuven in the Maes’ lab, who has extended experience working with filoviruses. The studies will be conducted in collaboration between RIKEN, MMV, and MMV partner test centers.

All project data will be registered in a shared database managed by MMV and accessible to both partners.

Others (including references if necessary)

1. Malvy D., (2019). Ebola virus disease. The Lancet 393, 936–948.

2. Hampton L.M. (2023). Ebola outbreak detection and response since 2013. Lancet Microbe 4, e661–e662

3. Kieh M, et al. (2022). Randomized Trial of Vaccines for Zaire Ebola Virus Disease. N Engl J Med. 29;387(26):2411-2424.

Final Report

1. Project objective
The objective of this project was to identify novel compounds with antiviral activity against Ebola (EBOV), Sudan Ebola (SUDV), and Marburg (MARV) viruses. Through a collaborative screening effort between MMV and RIKEN, supported by the Global Health Innovative Technology Fund (GHIT), the project aimed to screen a diverse compound library to uncover and prioritize active hits.

2. Project design
This project was designed as a collaborative antiviral screening effort between MMV and RIKEN, supported by GHIT. The RIKEN NP Depo library (20,036 compounds) was screened, at the Laboratory of Clinical and Epidemiological Virology, Rega Institute, KU Leuven at Piet Maes laboratory, in collaboration with the Virology, Antiviral Drug ＆ Vaccine Research Group, Rega Institute, KU Leuven. Data generated through the screening cascade were jointly reviewed to prioritize compounds for downstream confirmatory activities and selection of the most attractive confirmed hit compounds.

3. Results, lessons learned
Through this project, MMV and RIKEN successfully established a collaborative antiviral screening workflow with KU Leuven, using a multi-filovirus "rainbow" system that can simultaneously test for EBOV, MARV, and SUDV using a single cell line. A total of 20,036 compounds were screened, from which 253 primary hits were selected based on higher than 90% inhibition of the probe activity (80% for EBOV), and less than 30% reduction in DAPI signal compared to vehicle-treated controls.
These primary screening hits were tested in dose-response against the triple assay, as well as individually against each of the three viruses. This resulted in 11 confirmed hits (EC50 ＜10 µM and selectivity index ＞5) against 2 viruses, and 5 confirmed hits against all 3 viruses.
Subsequent testing of the confirmed hits against Tai Forest, Reston and Bundibugyo  viruses allowed to assess further the potential for broad-spectrum activity across filoviruses.
Overall, the project demonstrated the feasibility and value of leveraging internal collaboration to rapidly generate antiviral hit matter for emerging and neglected viral pathogens.
The screening campaign yielded a set of validated antiviral hits that can serve as a foundation for future optimization efforts by downstream partners. In summary, the project achieved its primary objective of identifying novel compounds with activity against Ebola, Sudan Ebola and Marburg viruses. It also provided practical insights to inform the design and execution of future GHIT supported antiviral screening initiatives.