Fig 1: Immunogenicity of ChAdOx1/MVA in different mouse strains. Mice were immunized intramuscularly either prime alone with ChAdOx1 or prime-boost with ChAdOx1/MVA, following different regimens outlined in table 1. Data for (A) CD-1 prime alone, (B) CD-1 prime and boost, (C) C57BL/6, (D) HLA-A2 transgenic and (E) BALB/c were generated in studies 1, 2, 3, 6 and 5, respectively. At the end of each study, mice were terminated, and spleens were harvested. Splenocytes in single cell suspension were applied to IFN? ELISpot at 200 000 per well and restimulated with 1 µg/peptide/mL of PSA, PAP, 5T4 or STEAP1 overlapping 15-mer peptide pools for 18 hours. 0.4% DMSO and ConA were used as assay negative and positive controls. Top row: stacked bar graphs show average SFC/106 splenocytes generated in response to each peptide pool; bottom row: scatter graphs show responses produced by individual animals to each peptide, line indicates mean. Data are displayed following subtraction of DMSO background control and application of assay positivity threshold (max of 2×SD DMSO control or 25 spot-forming cells (SFC)/106 splenocytes). MVA, modified vaccinia Ankara.
Fig 2: Confirmation of antigen expression by western blot. Cells were transduced with ChAdOx1 (lane 1) or ChAdOx1 (negative control, lane 2) expressing prostate immunogens, and cell lysates were probed with antibodies against PSA, PAP, STEAP1 and 5T4 as shown. The respective commercially available recombinant proteins were included as positive controls (lane 3). No signal was detectable for the 5T4 positive control protein.
Fig 3: Comparing T cell immunogenicity of ChAdOx1 prime administered IM followed by 2× MVA boost administered IM or intravenouos: intracellular cytokines. Study design: refer to figure 3A. Spleens samples were collected and prepared into single cell suspensions for application into flow cytometry assays. Splenocytes (1×106 cells per well) were stained for surface markers, fixed and permeabilized then stained for intracellular markers following an 18-hour stimulation with 2× prostate immunogen peptide pools; PSA+5T4 and PAP+STEAP1, 1 µg/peptide/mL. (A) Bar graph panel shows mean±SD monofunctional CD8+ T cell responses to PSA+5T4 and PAP+STEAP1 peptide pools at days 42 and 85, with IM/IM and IM/intravenous prime–boost regimens. (B) Bar graph panel shows mean polyfunctional CD8+ T cell responses to PSA+5 T4 peptide pool at days 42 and 85, in IM/IM and IM/intravenous prime–boost regimens. dots represent individual mice. IM, intramuscular.
Fig 4: Comparing T cell immunogenicity of ChAdOx1 prime administered IM followed by 2× MVA boost administered IM or intravenous: IFN? ELISpot. Study design (A): C57BL/6 mice were primed with ChAdOx1 intramuscularly and boosted on days 14 and 28 on with MVA either intramuscularly (group 1) or intravenously (group 2). Mice were sacrificed on day 42 or day 85 (n=6 per timepoint, per group), and spleens were harvested and prepared into single cell suspensions for immunogenicity analysis. (B) Splenocytes were applied to IFN? ELISpot at 200 000 per well and restimulated with 1 µg/peptide/mL of PSA, PAP, 5T4 or STEAP1 overlapping 15-mer peptide pools for 18 hours. 0.4% DMSO and ConA were used as assay negative and positive controls. Bar graph shows average±SD of the total magnitude of response for groups 1 and 2 at day 42 and day 85, calculated as the sum of SFC/106 splenocytes generated in response to 4× prostate immunogen antigen peptide pools; dots represent individual mice. (C) stacked bar graphs show average SFC/106 splenocytes generated in response to individual peptide pools. (D) Scatter graphs show responses produced by individual animals to each peptide pool; line indicates mean. Data are displayed following subtraction of DMSO background control and application of assay positivity threshold (max of 2xSD DMSO control or 25 SFC/106 splenocytes). IM, intramuscular; MVA, modified vaccinia Ankara.
Supplier Page from Sino Biological, Inc. for Human TPBG / 5T4 Protein (His Tag)