Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein

doi:10.1128/JVI.75.22.10991-11001.2001

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Journal of Virology, November 2001, p. 10991-11001, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10991-11001.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein

Ludwig Deml,¹ Alexandra Bojak,¹ Stephanie Steck,¹ Marcus Graf,¹ Jens Wild,¹ Reinhold Schirmbeck,² Hans Wolf,¹ and Ralf Wagner¹^,^*

Institute of Medical Microbiology, University of Regensburg, 93053 Regensburg,¹ and Institute of Medical Microbiology and Immunology, University of Ulm, 89069 Ulm,² Germany

Received 18 April 2001/Accepted 7 August 2001

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encodingthe human immunodeficiency virus type 1 (HIV-1) group-specificantigen (Gag). In the presence of Rev, an expression vector containingthe wild-type (wt) gag gene flanked by essential cis-acting sitessuch as the 5'-untranslated region and 3'-Rev response elementsupported substantial Gag protein expression and secretion inhuman H1299 and monkey COS-7 cells. However, only weak Gag productionwas observed from the murine muscle cell line C2C12. In contrast,optimization of the Gag coding sequence to that of highly expressedmammalian genes (syngag) resulted in an obvious increase in theG+C content and a Rev-independent expression and secretion ofGag in all tested mammalian cell lines, including murine C2C12muscle cells. Mice immunized intramuscularly with the syngag plasmidshowed Th1-driven humoral and cellular responses that were substantiallyhigher than those obtained after injection of the Rev-dependentwild-type (wt) gag vector system. In contrast, intradermal immunizationof both wt gag and syngag vector systems with the particle guninduced a Th2-biased antibody response and no cytotoxic T lymphocytes.Deletion analysis demonstrated that the CpG motifs generated withinsyngag by codon optimization do not contribute significantly tothe high immunogenicity of the syngag plasmid. Moreover, low dosesof coadministered stimulatory phosphorothioate oligodeoxynucleotides(ODNs) had only a weak effect on antibody production, whereasat higher doses immunostimulatory and nonstimulatory ODNs showeda dose-dependent suppression of humoral responses. These resultssuggest that increased Gag expression, rather than modulationof CpG-driven vector immunity, is responsible for the enhancedimmunogenicity of the syngag DNAvaccine.

^* Corresponding author. Mailing address: Institute for Medical Microbiology, Klinikum Regensburg, Franz-Josef-Strauss Allee11, 93053 Regensburg, Germany. Phone: 49 (0) 941-944-6452. Fax:49 (0) 941-944-6402. E-mail: ralf.wagner{at}klinik.uni-regensburg.de.

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