Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein

We have analyzed the influence of codon usage modifications on the expressi on levels and immunogenicity of DNA vaccines, encoding the human immunodefi ciency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence o f Rev, an expression vector containing the Mid-type (wt) gag gene flanked b y essential cis-acting sites such as the 5'-untranslated region and 3'-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production w as observed from the murine muscle cell line C2C12. In contrast, optimizati on of the Gag coding sequence to that of highly expressed mammalian genes ( syngag) resulted in an obvious increase in the G+C content and a Rev-indepe ndent expression and secretion of Gag in all tested mammalian cell lines, i ncluding murine C2C12 muscle cells. Mice immunized intramuscularly with the syngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-depende nt wild-type (wt) gag vector system. In contrast, intradermal immunization of both wt gag and syngag vector systems with the particle gun induced a Th 2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysi s demonstrated that the CpG motifs generated within syngag by codon optimiz ation do not contribute significantly to the high immunogenicity of the syn gag plasmid. Moreover, low doses of coadministered stimulatory phosphorothi oate oligodeoxynucleotides (ODNs) had only a weak effect on antibody produc tion, whereas at higher doses immunostimulatory and nonstimulatory ODNs sho wed a dose-dependent suppression of humoral responses. These results sugges t that increased Gag expression, rather than modulation of CpG-driven vecto r immunity, is responsible for the enhanced immunogenicity of the syngag DN A vaccine.