Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene

A major challenge for the next generation of human immunodeficiency virus ( HIV) vaccines is the induction of potent, broad, and durable cellular immun e responses. The structural protein Gag is highly conserved among the HIV t ype 1 (HIV-1) gene products and is believed to be an important target for t he host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expre ssion efficiency of viral proteins. In order to achieve high-level Rev-inde pendent expression of the Gag protein, the sequences encoding HIV-1(SF2) p5 5(Gag) were modified extensively. First, the viral codons were changed to c onform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient exp ression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and thes e showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demons trated that the modified gag and gagprotease genes efficiently expressed pa rticles with the density and morphology expected for HIV virus-like particl es. Mice immunized with DNA plasmids containing the modified gag shelved Ga g-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated w ith plasmids containing the native gag gene, Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These resu lts highlight the useful application of modified gag expression cassettes f or increasing the potency of DNA and other gene delivery vaccine approaches against HIV.