FIG 1.
Enhanced expression, plaque size phenotype, and innate immune profile of NYVAC-C-KC vectors. (A) Expression of HIV-1 gp140 determined by Western blotting. Monolayers of HeLa cells were mock infected or infected with NYVAC-WT, NYVAC-KC, NYVAC-gp140, NYVAC-KC-gp140, or NYVAC-KC-gp140-ΔB19R at 5 PFU/cell. At 6 or 24 h postinfection (h.p.i.), cells were lysed in Laemmli buffer, and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blotting using the rabbit polyclonal anti-gp120 antibody. (B) Analysis of plaque size. Monkey BSC-40 cells were infected with serial dilutions of NYVAC-WT, NYVAC-KC, NYVAC-gp140, NYVAC-KC-gp140, or NYVAC-KC-gp140-ΔB19R. At 48 h postinfection, cells were fixed and stained with crystal violet to visualize virus plaques. (C) Analysis of the innate immune profile in infected human THP-1 macrophages. Human THP-1 macrophages were mock infected or infected with NYVAC-WT, NYVAC-KC, NYVAC-gp140, NYVAC-KC-gp140, or NYVAC-KC-gp140-ΔB19R at 5 PFU/cell. At 3 and 6 h postinfection, RNA was extracted, and the mRNA levels of IFN-β, MIP-1α, IL-8, IL-1β, and hypoxanthine phosphoribosyltransferase (HPRT) were analyzed by RT-PCR. Results are expressed as the ratio of the mRNA level of the gene of interest to HPRT mRNA levels. A.U., arbitrary units. P values indicate significantly higher responses in comparisons of NYVAC-KC-gp140 to NYVAC-KC-gp140-ΔB19R at the same hour (*, P < 0.05; **, P < 0.005; ***, P < 0.001).