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.

Based on the human immunodeficiency virus type 1 (HIV-1) gag gene, subgenomic reporter constructs have been established allowing the contributions of different cis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimized gag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.Late human immunodeficiency virus type 1 (HIV-1) gene expression depends on cis-acting elements and the interaction of Rev with its cognate RNA recognition site, the Rev responsive element (RRE) (reviewed in reference 25). Nuclear retention of late HIV-1 unspliced and singly spliced mRNAs in the absence of Rev has been explained in many reports by inefficient splicing of the viral transcripts (4,15,16,20,26). Furthermore, the binding of U1 small nuclear RNP to an upstream splice donor site seemed to be required for Revdependent Env expression (18), whereas efficient splicing achieved by positioning a functional intron upstream of the env gene yielded Rev-independent expression (14). Experiments employing Rev-dependent ␤-globin reporter constructs suggested that inefficient splicing is essentially required for nuclear retention, which in turn represents a prerequisite for timely regulated 20). In view of the fact that many HIV-1 splice sites are suboptimal (22), it was speculated that Rev promotes the export of late HIV-1 RNAs entrapped within the splicing machinery (4, 13, 15).However, the Rev-mediated nuclear export process seems not to be directly related to splicing, as shown by the observation of Fischer and colleagues that Rev can also export RNAs retained in the nucleus for entirely unrelated reasons, such as U-rich U6 RNAs (10). Accordingly, env mRNA has been reported to remain Rev dependent also in the absence of any functional splice sites (21). It has been postulated that these RNAs contain cis-active inhibitory sequences (INS) within their coding regions negatively regulating their expression (19,21,22,31). Fusion of proposed INS-containing fragments to a chloramphenicol acetyltransferase gene reporter resulted in decreased expression and Rev responsiveness (6,28,31). Consequently, low-level gene expression of gag and pol open reading frames in the absence of Rev was overcome by clustered point mutations within the wobble positions of the coding DNA sequence (29,30).The scope of this study was to determine, based on a subgenomic Rev-dependent gag reporter construct, the critical contribution of proposed INS elements within the gag coding region and the 5Ј untranslated region (UTR) including the major splice donor...

In this study, we analyzed the in vitro expression, potency and longevity of immune responses induced in a Balb/c mouse model by a synthetic HIV-1 gag gene exhibiting a codon usage that was adapted to that of highly expressed mammalian genes (syngag). In contrast to a vector containing the wild-type (wt) gag gene, the syngag construct enabled highly efficient Gag expression in both human and rodent cell lines in complete absence of Rev and Rev-responsive element. Immunization of Balb/c mice with the wt gag plasmid DNA induced only weak and inconsistent humoral immune responses. Mice vaccinated by syngag but not wt gag developed substantial and highly consistent Gag-specific antibody titers showing a clear T helper 1 polarization even with low doses of DNA. Moreover, vaccinated mice developed a strong Gag-specific cellular immune response, including cytotoxic T cells, which was not observed in wt gag-immunized animals. Both humoral and cellular immunity were efficient and lasted for more than 20 weeks. Furthermore, the induction of the humoral as well as the cellular immune response was independent of the immunization route (intramuscular or subcutaneous). These results clearly show the advantages of codon-optimized genes with respect to the expression and immunogenicity of plasmid DNA constructs, making them promising vaccine candidates for further studies.

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