Standardized Two-Step Testing of Antibody Activity in COVID-19 Convalescent Plasma

The COVID-19 pandemic revealed an urgent need for rapid profiling of neutralizing antibody responses and development of antibody therapeutics. The current Food and Drug Administration-approved serological tests do not measure antibody-mediated viral neutralization, and there is a need for standardized quantitative neutralization assays. We report a high-throughput two-step profiling approach for identifying neutralizing convalescent plasma. Screening and down-selection for serum antibody binding to the receptor-binding domain are followed by quantitative neutralization testing using a chimeric vesicular stomatitis virus expressing spike protein of SARS-CoV-2 in a real-time cell analysis assay. This approach enables a predictive screening process for identifying plasma units that neutralize SARS-CoV-2. To calibrate antibody neutralizing activity in serum from convalescent plasma donors, we introduce a neutralizing antibody standard reagent composed of two human antibodies that neutralize SARS-CoV strains, including SARS-CoV-2 variants of concern. Our results provide a framework for establishing a standardized assessment of antibody-based interventions against COVID-19. Funding Information: This work was supported by the National Institute of Health (NIH) National Center for Advancing Translational Sciences (NCATS) grant 3UL1TR002243-04S4, Vanderbilt Institute for Infection, Immunology, and Inflammation (VI4), the Hays Foundation COVID-19 Research Fund (to I.T.), Defense Advanced Research Projects Agency (DARPA) grant HR0011-18-2-0001, U.S. N.I.H. contract 75N93019C00074, the Dolly Parton COVID-19 Research Fund at Vanderbilt, and a grant from Fast Grants, Mercatus Center, George Mason University. Declaration of Interests: I.T. reports grants from NIH/NIAID, during the conduct of the study, and has served as a consultant for Nashville Biosciences and Horizon Therapeutics. J. D. C., L.J.S., and M.R.D. report grants from NIH/NCATS, during the conduct of the study. T.W.R. reports grants from NIH/NCATS, during the conduct of the study; personal fees from Cumberland Pharmaceuticals, Inc, personal fees from Sanofi Pharma, and personal fees from Cytovale, outside the submitted work. T.G.S. reports grants from NIH, during the conduct of the study. W.H.S. reports grants from NCATS of the NIH, during the conduct of the study. M.S.D. is a consultant for Inbios, Vir Biotechnology, Fortress Biotech, and Carnival Corporation and on the Scientific Advisory Boards of Moderna an Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology and Emergent BioSolutions. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca and grants from NIH, and DARPA during the conduct of the study. Vanderbilt University has applied for patents related to antibodies described in this paper. All other authors declare no competing interests. Ethics Approval Statement: The studies were approved by the Vanderbilt University Medical Center Institutional Review Board, and samples were collected after written informed consent was obtained by research personnel.