Increased Expression and Immunogenicity of Sequence-Modified Human Immunodeficiency Virus Type 1 gag Gene

Abstract

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cellmediated 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 expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1 SF2 p55Gag were modified extensively. First, the viral codons were changed to conform 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 p55Gag protein expression to levels that ranged from 322-to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8 ؉ cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with 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 results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

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.…”

Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense

“…Another approach to obtain Rev-independence is the introduction of silent mutations in the coding region of Gag-Pol in order to eliminate the instability sequences (INS) that are responsible for the Rev-dependent character of the mRNAs coding for Gag-Pol. [28][29][30][31][32] Using this Rev-independent version of Gag-Pol in combination with a Rev-dependent or with a Rev-independent transfer vector yielded titers of approximately 5 × 10 5 tu/ml and 1 x 10 5 tu/ml, respectively. 28 Titers similar to these ones are expected to be obtained when using this system to package Rev-independent HIV-based vectors constitutively expressing TdRev.…”

Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells

“…Using a plasmid encoding the HIV p55 gag protein under the control of the cytomegalovirus early promoter (pCMVKm2.GagMod.SF2), 25 BMDC on day 6 of culture were treated with the naked plasmid, or the same plasmid formulated on PLG-CTAB microparticles. Messenger RNA was isolated 24 h later, and transgene expression was analyzed by RT-PCR.…”