Figure 1. Construction of Ad26 vectors.

(a) Seven Ad26 vectors were produced expressing SARS-CoV-2 S protein variants: (i) tPA leader sequence with full-length S (tPA.S)¹², (ii) tPA leader sequence with full-length S with mutation of the furin cleavage site and two proline stabilizing mutations (tPA.S.PP)^13–15, (iii) wildtype leader sequence with native full-length S (S), (iv) wildtype leader sequence with S with deletion of the cytoplasmic tail (S.dCT)¹⁶, (v) tandem tPA and wildtype leader sequences with full-length S as a strategy to enhance expression (tPA.WT.S)¹², (vi) wildtype leader sequence with S with deletion of the transmembrane region and cytoplasmic tail, reflecting the soluble ectodomain, with mutation of the furin cleavage site, proline stabilizing mutations, and a foldon trimerization domain (S.dTM.PP)¹⁵, and (vii) wildtype leader sequence with full-length S with mutation of the furin cleavage site and proline stabilizing mutations (S.PP). Red triangle depicts tPA leader sequence, black triangle depicts wildtype leader sequence, red X depicts furin cleavage site mutation, red vertical lines depict proline mutations, open square depicts foldon trimerization domain. S1 and S2 represent the first and second domain of the S protein, TM depicts the transmembrane region, and CT depicts the cytoplasmic domain. (b) Western blot analyses for expression from Ad26 vaccine vectors encoding tPA.S (lane 2), tPA.S.PP (lane 3), S (lane 4), S.dCT (lane 5), tPA.WT.S (lane 6), S.dTM.PP (lane 7), or S.PP (lane 9) under non-reduced conditions in MRC-5 cell lysates using a human monoclonal antibody (CR3046). This experiment was repeated three times. For gel source data, see Supplementary Figure 1.