April 2020 Nsp3 abcam ab181620 Nucleocapsid Sino Biological 40143-MM05 Validation Commercial antibodies validated as per manufacturers website: Beta actin Sigma A5441 Immunoblot on chicken fibroblast cell extracts Spike Abcam Ab252690 Validated by ELISA on free peptide from SARS-CoV-1 Nsp3 abcam ab181620 Validated by western blot on SARS-CoV-1 infected cells Nucleocapsid Sino Biological 40143-MM05 Validated by western blot with corresponding viruses Eukaryotic cell lines Policy information about cell lines Cell line source(s) VeroE6 cells were obtained from ATCC, Calu-3 cells were obtained from Manfred Frey, originally from ATCC. Authentication Cells were not further authenticated Mycoplasma contamination Cells have been tested and are free of mycoplasma.

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Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.