Figure 1.
The poly-antigen DNA vaccine activates antigen-specific CD8 and CD4 T cells in vivo. (a) Schematic representation of the neoantigen DNA vaccine and the resulting poly-epitope peptide sequence. Direction of the open reading frame (ORF) is indicated. The individual CD8 and CD4 epitopes in the peptide are encircled in dark or light blue, respectively. For each of the three neoepitopes, the amino acid (aa) change resulting from somatic mutation is highlighted in red. (b) Upper panel: Quantification of the mean fluorescence intensity (MFI) using 25-D1.16 antibody of the SIINFEKL peptide presentation on MHC I molecule after transfecting MC38 cells in vitro with either the GFP plasmid (negative control) or the neoantigen plasmid. Lower panel: Activation of SIINFEKL-specific T cell hybridoma B3Z cells by MC38 cells transfected with the neoantigen DNA vaccine. The SIINFEKL synthetic peptide (1 µM) was added as a positive control (orange bars), and a plasmid coding for GFP was used as a negative control. Statistical significance was determined by one-way ANOVA followed by multiple comparison, *** p < 0.001 (c) Proliferation of adoptively transferred OT-I and OT-II cells, 3 days after intradermal injection of 10 µg of the neoantigen DNA vaccine. (d) Kinetics of in vivo antigen presentation to OT-I or OT-II cells after injection of the DNA construct. Proliferation of the OT-I (blue lines) and OT-II cells (red lines) measured by CFSE dilution in the inguinal lymph nodes of C57BL/6 mice immunized with either neoantigen DNA vaccine (left panel) or 50 µg of OVA CTL and helper peptides (right panel), used as positive controls for OT-I and OT-II cells proliferation, respectively. Error bars indicate mean ± SD, N = 2. (e) Proliferation of adoptively transferred OT-I and OT-II cells, 3 days after intradermal injection of 10 µg of different variants of neoantigen DNA vaccine. Error bars indicate mean ± SD, N = 2. Significance in relation to the GFP plasmid negative control was determined by one-way ANOVA followed by multiple comparisons. * p < 0.05, ** p < 0.01.