FIG 1.
Construction and rescue of rVSV Delta SP and influenza M2e bivalent vaccines. (A) Schematic diagram of the Delta SPΔC and EboGPΔM-tM2e immunogens present in the bivalent vaccines. (a) SARS-CoV-2 Delta-SPΔCA742 (SPΔC1), containing a C-terminal 17 aa (DEDDSEPVLKGVKLHYT) deletion and an I742A mutation as indicated. The nine mutations in Delta SP are listed in the lower part. (b) Delta SPΔC2, containing the C-terminal 17 aa deletion and another 381 aa (encompassing aa744 to aa1124) deletion in the S2 domain. The eight mutations in SPΔC2, are listed in lower part. (c) EboGPΔM-RBD, the RBD of SARS-CoV-2 was used to replace the MLD domain in EboGP. (d) EboGPΔM-tM2e, four copies of influenza virus M2 ectodomain (24 aa) polypeptide (tM2e) replaced the MLD domain in EboGP. (B) The attenuated virus entry of SPΔC1. A549ACE2 cells were infected with equal amounts of SPΔCDelta-PVs or SPΔC1-PVs (adjusted by P24) carrying the Gluc gene, as indicated. At 48 h postinfection, the Gluc activity in the supernatant of different infected cultures was measured. Data represent the mean ± SD of two replicates from a representative experiment out of three performed. (C and D) The attenuated cell-to-cell fusion ability of SPΔCDelta- or SPΔC1-mediated syncytia formation was analyzed by coculturing the SPΔCDelta- or SPΔC1-expressing 293T cells with A549ACE2 cells. The amounts of syncytia were counted after 24 h in five different views of the microscope (C) and was also imaged under bright-field microscopy (D). (E) Schematic diagram of V-EM2e/SPΔC1, V-EM2e/SPΔC2 and V-EM2e/ERBD and the virus rescuing procedures. 293T and Vero E6 coculture cells were cotransfected with V-ΔG-EM2/SPΔC1, V-ΔG-EM2/SPΔC or V-ΔG-EM2/RBD and helping plasmids (T7, N, L, P plasmids). The supernatants containing V-EM2e/SPΔC1, V-EM2e/SPΔC2, and V-EM2e/ERBD viruses were used to infect Vero E6 cells to generate the rVSV stocks.