Fig. 1.

Structure, function and immunogenicity of self-replicating RNA vaccines. a, RNA constructs used for immunization. m⁷G, ‘cap’ at the 5′ end of the mRNA. b–f, RNA constructs transcribed and capped in vitro were transfected into BHK21 cells; 24 h later, cells were tested for β-gal expression using the X-gal assay. Data are representative of three independent experiments with similar results. g, Quantitative analysis of RNA replication using TaqMan real-time RT–PCR. Total cellular RNA was isolated at 6, 10, 14, 24 and 48 h after transfection with the Rep-LacZ RNA. Using vector-specific primers, the copy number of the mRNA Rep-LacZ was then quantified relative to 18s ribosomal RNA. This experiment was done three independent times with similar results. h, Induction of antigen-specific antibody responses in mice by self-replicating RNA vaccines. Sera obtained from mice 21 d after immunization with RNA were tested by ELISA for the presence of IgG antibodies against the recombinant β-gal protein. i, Induction of CD8⁺ T-cell responses in mice by self-replicating RNA vaccines. Splenocytes obtained from mice 21 d after immunization with RNA were re-stimulated in vitro in the presence of β-gal_876-884, then cultures were assayed for β-gal-specific CD8⁺ T-cell recognition by measuring IFN-γ release after exposure to CT26 alone (□) or pulsed with the synthetic peptides derived from β-gal_876-884 (▨) or P815A_35-43 ( ) or transduced with a retrovirus encoding LacZ (■).