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201 Codon-Usage Optimization of gag, pol, env and nef Genes of an HIV-1 Subtype C Strain
F. Gao*, Y. Li, C.M. Rodenburg, Y. Chen, D.R. Shaw, A.J. Zajac, and B.H. Hahn
Univ. of Alabama at Birmingham, USA
Background: To improve HIV-1 protein expression in the context of a DNA vaccine, we codon-usage optimized gag, pol, env, and nef genes of a prototypic HIV-1 subtype C strain (96ZM651).
Methods: Genes were synthesized by ligating overlapping oligonucleotides that encoded wild-type (wt) 96ZM651 protein sequences but reflected the codon usage of highly expressed human genes. These were cloned into eukaryotic expression vectors and tested for protein expression in vitro and in vivo.
Results: In vitro transcription/translation yielded similar amounts of expressed protein for all constructs. However, optimized gag, pol, env, and nef genes expressed 24 to 532 times more protein following transfection into 293T cells. One set of env constructs (gp140) was then selected for intramuscular DNA immunization of mice. Groups of 5 mice were injected 5 times with 50 mg of optimized gp140, wt gp140, optimized gp140 plus mouse GM-CSF DNA, and vector control, respectively. Anti-gp140 antibodies were detected after the first boost in 3 of 5 mice immunized with the optimized gp140 DNA construct (with titers peaking after the third boost), but none of the 5 mice injected with wt gp140 developed detectable antibody responses. GM-CSF did not enhance antibody responses. Two weeks after the last boost, mice were sacrificed, spleen cells harvested, and cellular immune responses determined by intracellular cytokine staining following restimulation with gp140 expressing A20 cells. In all mice immunized with the optimized gp140 greater than 0.5% of splenic CD4 T cells produced IFN-gamma in response to gp140 stimulation, whereas antigen-specific CD4 responses were not above background levels in mice immunized with noncodon-optimized constructs. Although less pronounced, similar trends were also observed for gp140-specific CD8 T-cell responses.
Conclusions: These results indicate that codon-usage optimization increases HIV-1 gene expression in vitro and appears to markedly enhance both humoral and cellular immune responses upon DNA vaccination in vivo.
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