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Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Published on: March 8, 2012

Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not

Felix K M Lorenz¹, Susanne Wilde², Katrin Voigt¹

¹Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

|March 24, 2015

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.

Insights

Codon optimization can unexpectedly create new immune targets. This study found a hidden epitope in optimized genes, potentially impacting gene therapy and vaccine design by causing immune rejection.

Area of Science:

Immunology
Molecular Biology
Gene Therapy

Background:

Codon optimization is standard for enhancing transgene expression in research and clinical applications.
Immunological consequences of codon optimization have not been previously documented.

Purpose of the Study:

To investigate potential immunological side effects of codon optimization.
To determine if codon optimization can induce T cell responses against transgenes.

Main Methods:

T cells were incubated with dendritic cells pulsed with RNA encoding codon-optimized E7 oncogene.
T cell receptor recognition of target cells expressing wild-type versus codon-optimized E7 was assessed.
Epitope mapping was performed to identify recognized epitopes.
A stop codon was introduced into the alternative reading frame to assess its protective effect.

Main Results:

T cell clones recognized target cells expressing codon-optimized E7 but not wild-type E7.
A cryptic epitope in the +3 alternative reading frame of codon-optimized E7 was identified as the target.
This epitope is not present in the wild-type E7 sequence.
Introducing a stop codon in the +3 alternative reading frame prevented T cell recognition.

Conclusions:

Codon optimization can artificially induce immunogenicity in transgenes by creating dominant cryptic epitopes.
This finding is crucial for gene therapy to prevent rejection of corrected cells.
Codon optimization strategies for DNA- and RNA-based vaccines need careful consideration due to potential artificial immunogenicity.

Read full analysis

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