Epitope mapping with truncated E7co microgene fragments.

<p>(a) Scheme of truncated E7co microgenes (grey bars) and full-length E7wt (white bar) that were stably expressed in K562-HLA-B*27:05 target cells through MP71 retrovirus transduction. Microgenes were coupled to mCherry expression marker via an IRES element to confirm transgene expression. (b) TCR-transduced T cells (B21, B23, S16 and S51) were cocultured with microgene-expressing target cells and supernatant was tested for IFNγ by ELISA. Untransduced T cells were used as negative control. Results are shown as mean +/− SEM of duplicates. n.d., not detectable.</p

Redesign of +3 ARF prevents E7co from recognition by TCR-transduced T cells.

<p>(a–c) Exchanging wobble nucleotides (bold) of the primary +1 ORF leads to (a) introduction of stop codons or (b–c) amino acid exchanges (1mut, 2mut) at anchor residue positions only in the +3 ARF (bold amino acids) without changing the polypeptide sequence from +1 ORF translation. (d) TCR-tranduced T cells (B21, B23, S16 and S51) were cocultured with K562-B*27:05 target cells that express one of the mutant microgene constructs (1–30 nt) of E7co. Untransduced T cells were used as a negative control. Reactivity was assessed by IFNγ ELISA. Results are shown as mean +/− SEM of duplicates.</p

Expression of HPV16 E7 from codon-optimized (co) and wild type (wt) sequences.

<p>(a) The myelogenous leukemia cell line K562 was transfected via electroporation with 15 μg of E7wt (thin line) or E7co (bold line) ivtRNA, stained intracellularly for E7 protein expression, analyzed by flow cytometry and depicted on a bi-exponential scale of a histogram. Non-electroporated cells (gray area) serve as negative control. MFI (median fluorescence intensity) values have been calculated by FlowJo8.7. (b) DCs from two healthy donors were electroporated with E7co ivtRNA and E7 protein expression was confirmed by flow cytometric analysis (bold line). Non-electroporated DCs (gray area) were used as a control.</p

Epitope binding prediction to HLA-B*27:05.

<p>Epitope binding prediction to HLA-B*27:05.</p

Epitope mapping identifies a cryptic epitope from +3 ARF of E7co as TCR target.

<p>(a–b) The +2 and +3 ARFs of E7co translate for cryptic peptide sequences (bold italic) not encoded by the +2 and +3 ARFs of E7wt. (c) T cells transduced with TCRs B21, B23, S16 and S51 were cocultured with K562-B*27:05 cells pulsed with candidate epitopes from the +1 ORF and the +3 ARF of E7co and a selection of control peptides (Ctrl.). Untransduced T cells were used as negative control. Reactivity was asessed by IFNγ ELISA. Results are shown as mean +/− SEM of duplicates.</p

TCRs are specific for HLA-B*27:05 and E7co but not E7wt.

<p>TCR-transduced T cells were tested for HLA restriction and antigen specificity by coculture with different target cells to measure IFNγ release by ELISA. Untransduced (ut) T cells were used as negative control. Results are shown as mean +/− SEM of duplicates. (a) Restriction mapping of four different TCRs (B21, B23, S16 and S51) was perfomed using K562 target cells carrying E7co and one of the six cognate MHC class I molecules of the original donor. (b) HLA-B*27:05-engineered target cell lines of different origin (CaSki, HT-3 and K562) were tested for recognition by TCR-transduced T cells.</p

Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine