Neoantigen Expression in HCMV Vectors

As a neoantigen for expression in TB40/E-BAC derived vectors, we used human papillomavirus type 16 (HPV-16) consensus E6/E7 fusion protein (ConE6E7, GenBank accession number: FJ229356) (46 (link)). In addition, the HLA-A2-binding peptide E6₂₉₋₃₈ (TIHDIILECV) derived from the E6 protein of HPV-16 (47 (link)) was fused with an AA-linker (AATIHDIILECV) to the C-terminus of HCMV IE1 (E6peptideIE1) or HCMV UL83 (E6peptideUL83). The corresponding sequences were synthesized and verified by Integrated DNA Technologies (IDT). The synthesized E6/E7 encoding sequence was digested with EcoRI and Kpn-I and cloned into the expression vectors pEF6/V5-His A and pcDNA™3.1 (+). These constructs were named pEF6E6/E7EcoRI and pcDNAE6/E7Kpn-I, respectively. Recombinant HCMV was generated using BAC technology as previously described (48 (link)). All recombinant BAC clones were confirmed by PCR and DNA-sequencing of the target area. Viruses were reconstituted from BACs by electroporation of 1 × 10⁶ Fi301 cells using program A24 of the Nucleofector II (Amaxa) and a basic Nucleofector kit (Lonza), according the manufacturer's instructions.