Balanced immunogenicity upon vaccination with DNA.HTI in mice. (A) Vaccination schedule. Groups of mice (n = 5) were vaccinated twice by IM injection followed by in vivo electroporation (EP) using 20 μg DNA.HTI and a mixture of 3 plasmids encoding for full-length p55gag, Pol and a Nef-Tat-Vif fusion protein (DNA.COMB). Cellular immune responses were measured from thawed splenocytes by IFN-γ ELISPOT 2 weeks after the 2nd vaccination using 8 peptide pools covering the HTI sequence. (B) The number of HTI positive responses (reactive pools) in mice immunized with 20 μg DNA.HTI or DNA-COMB is shown. (C) Total magnitude of the HTI-specific IFN-γ responses is depicted from the mice shown in panel B. (D) Comparison of the IFN-γ responses in mice vaccinated with DNA.COMB targeting the regions included in the HTI (grey bars) and the total IFN-γ specific response to peptide pools spanning the complete Gag, Pol, NTV. (E) Distribution of HTI-specific induced IFN-γ responses against Gag, Pol, Vif and Nef in mice from panel B is shown (DNA.HTI in mice 1–5; DNA.COMB in mice 6–10) using peptide pools spanning the protein sequences included in HTI. (F) Binding antibodies to Gag were detected by Western immunoblot. The membranes contain Gag proteins from HEK293 cells transfected with 1 μg of p55gag plasmid producing the unprocessed p55gag protein or transfected with a Gag-Prt plasmid expressing p55gag, the processing intermediate p37gag (p24gag and p17gag) and processed p24gag proteins. Membranes were probed with human sera from an HIV-infected individual or pooled plasma from mice immunized with 20 μg of DNA.HTI and DNA.COMB (at a 1:100 dilution). (G) Anti-HIV-1 p24gag antibodies were measured in pooled plasma from DNA.HTI and DNA.COMB vaccinated C57BL/6 mice by a standard clade B p24gag ELISA. The graph shows absorbance (optical density, OD).