Borhani, Shayan G.Levine, Max Z.Krumpe, Lauren H.Wilson, JenniferHenrich, Curtis J.O’Keefe, Barry R.Lo, DonaldSittampalam, G. SittaGodfrey, Alexander G.Lunsford, R. DwayneMangalampalli, VenkataTao, DingyinLeClair, Christopher A.Thole, AaronFrey, DouglasSwartz, JamesRao, Govind2023-01-062023-01-062022-12-20https://doi.org/10.1101/2022.12.19.521044http://hdl.handle.net/11603/26586This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.21 pagesen-USThis work was written as part of one of the author's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.Public Domain Mark 1.0http://creativecommons.org/publicdomain/mark/1.0/Text