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.

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Helicobacter pylori causes a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease. Despite an intensive proinflammatory response, the immune system is not able to clear the organism. However, the immune escape mechanisms of this common bacterium are not well understood. We investigated the interaction between H. pylori and human dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system. Stimulation of DCs with different concentrations of H. pylori for 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production. Lipopolysaccharide (LPS) from Escherichia coli, a known DC maturation agent, was used as a positive control. The cytokine release after stimulation with LPS was comparable to that induced by H. pylori except for IL-12. After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced by H. pylori. We further investigated the potential of H. pylori to induce maturation of DCs. Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation with H. pylori or LPS. We found no significant differences between H. pylori seropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules. These data clearly demonstrate that H. pylori induces a strong activation and maturation of human immature DCs.Helicobacter pylori is a gastric pathogenic gram-negative bacterium that colonizes the gastric mucus layer but does not invade the mucosal epithelium. This bacterial colonization leads to a cellular infiltrate of polymorphonuclear leukocytes, an acute immune response, followed by the migration of macrophages, lymphocytes, and plasma cells in the gastric mucosa, resulting in chronic gastritis. This chronic inflammation does not necessarily produce symptoms but does increase the risk of developing peptic ulcer disease, adenocarcinoma of the distal stomach (antrum and fundus), and primary non-Hodgkin's lymphoma of the stomach (MALTomas) (8,42,43).Although H. pylori induces an immune response involving both the innate and the acquired immune systems, the host is unable to clear the organism from the mucosa, resulting in lifelong infection. This inability to eliminate the bacterium may be due to immune-evasive strategies. Possible mechanisms were investigated, with emphasis on the acquired immune response. Several studies have shown inhibitory effects of H. pylori on cell proliferation (11, 24-26, 60), and the induction of H. pylori-specific regulatory T cells that actively suppress T-cell response have been described (31).Recent studies have investigated possible impairment of antigen presentation. VacA, an H. pylori virulence factor, was reported to interfere with proteolytic processing of tetanus toxoid and was shown to inhibit the Ii-depen...

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal immune response with elevated levels of the Th1 cytokines TNF, IL‐12 and IFN‐γ. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of chronic intestinal inflammation by as yet unknown mechanisms. Bacterial DNA contains unmethylated cytosine‐guanosine dinucleotides (CpG) which strongly activate Th1‐mediated immune responses. To test whether these CpG‐motifs contribute to intestinal inflammation we treated mice with dextran‐sulfate‐sodium (DSS)‐induced acute or chronic colitis for 5 days with CpG‐containing oligodeoxynucleotides (CpG‐ODN). Colonic inflammation was assessed by histological scoring. Colonic cytokine RNA was quantified by reverse transcription‐PCR and cytokine secretion from mesenterial lymph node cells by ELISA. In chronic colitis, CpG‐ODN treatment severely aggravated inflammation by 50%. Colonic expression of IFN‐γ and TNF was elevated (200‐ and 150‐fold, respectively) and IFN‐γ and IL‐12 secretion from lymph node cells was increased 5,000‐ and 8‐fold, respectively, compared to GpG‐ODN‐treated controls. Similar effects were obtained in acute colitis. In conclusion, CpG‐motifs of bacterial DNA have proinflammatory activity by strengthening the Th1 arm of immunity in DSS‐induced colitis, and might therefore play asignificant role in the initiation and perpetuation of inflammation in IBD.

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...