Abstract
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.
Highlights
The expression of sufficient amounts of transgenic protein in a gene-modified cell is crucial in molecular biology and clinical biotechnology
Codon optimization has become a readily available tool to increase the expression of transgenes in basic research as well as clinical settings, but codon optimization may affect polypeptide sequences translated from the +2 and +3 alternative reading frames (ARFs)
We report that codon-optimization renders the HPV16 E7 oncogene artificially immunogenic via the generation of a cryptic epitope from an ARF, which does not exist in the wild type sequence
Summary
The expression of sufficient amounts of transgenic protein in a gene-modified cell is crucial in molecular biology and clinical biotechnology. Since gene synthesis has become a time- and cost-efficient method for the design of nucleotide sequences, codon optimization has been established as a standard tool to maximize protein expression in a desired system. The genetic code for translating nucleotide sequences to proteins uses 64 nucleotide triplets (codons), which encode 20 amino acids and three translational stop signals. Cryptic T Cell Epitope by Codon Optimization design, data collection and analysis, decision to publish, or preparation of the manuscript
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