A Trans -Complementation System for SARS-CoV-2

The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development. Funding: P.-Y.S. was supported by NIH grants AI142759, AI134907, AI145617, and UL1TR001439; awards from the Sealy & Smith Foundation, Kleberg Foundation, John S. Dunn Foundation, Amon G. Carter Foundation, Gilson Longenbaugh Foundation, and Summerfield Robert Foundation; and fund in sponsored research agreement from Q2 Solutions. M.S.D. was supported by R01 AI157155. V.D.M. was supported by NIH grants U19AI100625, R00AG049092, R24AI120942, and a STARs Award from the University of Texas System. S.C.W. was supported by NIH grant R24 AI120942. J.L. is supported by the postdoctoral fellowship from the McLaughlin Fellowship Endowment at UTMB. P.R. and X.X. were partially supported by the Sealy & Smith Foundation. Conflict of Interest: X.Z., X.X., and P.-Y.S. have filed a patent on the trans-complementation system of SARS CoV-2. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. Ethical Approval: Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). The research protocol for use of human serum specimens was approved by the University of Texas Medical Branch (UTMB) Institutional Review Board (IRB protocol number 20-0070). All human serum specimens were obtained at the UTMB with patient information de-identified. All aspects of this study were approved by the Institutional Biosafety Committee of the University of Texas Medical Branch at Galveston before the initiation of this study.