Practice question

DNA vaccines third-generation platform delivering plasmid DNA encoding microbial antigen rather live attenuated organism purified protein circumventing biosafety level concerns anti-vector immunity. Plasmid constructed E. coli DH5 alpha containing bacterial origin ColE1 antibiotic resistance beta-lactamase ampicillin selection strong eukaryotic expression cassette CMV immediate early enhancer promoter high constitutive transcription Pol II chimeric intron A human beta-globin improving mRNA stability splicing removing premature terminator target gene viral spike bacterial protective antigen tumor neoantigen optimized human codon usage removing rare codons increasing translation bovine GH polyA terminating transcription adding polyA tail aiding nuclear export. Additional unmethylated CpG immunostimulatory motifs GTCGTT enriched activating TLR9 plasmacytoid dendritic MyD88 IRF7 type I interferon alpha endogenous adjuvant stimulating Th1. Intramuscular injection 1 mg plasmid saline followed electroporation needle electrodes 100V 50ms pulses increasing myocyte membrane permeability transient pores enhancing uptake 100-fold plasmid enters nucleus nucleopore remains episomal circular monomer not integrating <1 per 10000 cells transcribed mRNA capped polyadenylated exported NXF1 translated ribosomes producing antigenic protein. Endogenously synthesized degraded immunoproteasome 20S generating peptides 8-11 aa loaded MHC I H2Kb HLA-A2 presenting CD8 CTL endogenous pathway perforin granzyme killing while secreted captured APC phagocytosed MHC II activating