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

Abstract

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by 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 was observed from the murine muscle cell line C2C12. In contrast, optimization 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-independent expression and secretion of Gag in all tested mammalian cell lines, including 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-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wt gag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of the syngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of the syngag DNA vaccine.

Efficient induction of T‐cell responses to carcinoembryonic antigen by a heterologous prime‐boost regimen using DNA and adenovirus vectors carrying a codon usage optimized cDNA

“…This observation is in line with data obtained in mice and nonhuman primates with genetic vaccines for HCV, HIV and HPV, whereby vectors carrying codon sequence optimized cDNA are characterized by increased expression and enhanced immunogenicity of the viral polypeptides. [26][27][28][29][30][31][32] Although it is reasonable to assume that the higher expression of CEA will lead to enhanced presentation by the MHC of CEA-derived epitopes, an enhanced immune response can also be ascribed, at least in part, to an increase in CpG motifs in the synthetic gene administered. The role of immune modulation of unmethylated CpG in governing the immune response has been described both in rodents and in nonhuman primates.…”

“…[26][27][28][29][30][31][32] To compare the efficiency of expression of the CEAopt to that of CEA, groups of 10 C57Bl/6 mice were injected into the quadriceps with different doses of the AdCEAopt vector ranging from 1 x 10 7 to 1 3 10 4 pfu. Three days post injection, CEA protein levels in the mice sera were determined and compared to those of control groups that had been injected with the same doses of Ad-CEA.…”

“…justified by the observation that for highly expressed genes, G or C is generally preferred over A or T. In fact, in a variety of experimental systems, optimizing the codon usage of the target gene has been shown to enhance expression and increase immunogenicity. [26][27][28][29][30][31][32] In our study, the immunogenic properties of a genetic vaccine based on plasmid DNA-EP and Ad vector have been examined by monitoring the amplitude and type of immune responses to CEA. In addition, the immunogenicity of a codon usage optimized cDNA encoding CEA was examined both in C57Bl/6 and in CEA transgenic mice.…”

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

“…This is even true for viral genes from viruses that infect humans as the natural host. 25,26 One would assume that such viral genes would already be humanized due to the co-evolution with the human host. However, the virus may have for certain reasons evolved not to have all or certain genes codon optimized since this might help the virus to avoid the immune response.…”

Influence of polypeptide size and intracellular sorting on the induction of epitope-specific CTL responses by DNA vaccines in a mouse model

“…The supernatant (10 ml) was cleared by centrifugation and subsequently pelleted through a 30% (w/v) sucrose cushion at 120,000 × g at 16 • C for 2.5 h. The pellet was resuspended in 50 l PBS and subsequently analysed by immunoblotting using an anti-p24 mab (16/4/2) [28] as described previously in detail [29]. The protein content was determined using a commercial p24 capture assay (Abbott, Wiebaden, Germany).…”