Jens Wild

Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein

ABSTRACT 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 essentialcis-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 thesyngag 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 wtgag 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 thesyngag 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 thesyngag DNA vaccine.

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen

Murine MHC class I‐restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL‐defined epitopes of this viral envelope protein are the Ld ‐binding 12‐mer S28 – 39 peptide IPQSLDSWWTSL in H‐2 d mice, and the Kb ‐binding 8‐mer S208 – 215 peptide ILSPFLPL in H‐2b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H‐2 class I‐binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the Ld ‐binding peptide S28 – 39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the Kb ‐binding peptide S208 – 215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I‐binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.

Ongoing Murine T1 or T2 Immune Responses to the Hepatitis B Surface Antigen Are Excluded from the Liver that Expresses Transgene-Encoded Hepatitis B Surface Antigen

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.

Potential of Equine Herpesvirus 1 as a Vector for Immunization

ABSTRACT Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.

Fusion of Epstein-Barr Virus Nuclear Antigen-1-Derived Glycine–Alanine Repeat to Trans-Dominant HIV-1 Gag Increases Inhibitory Activities and Survival of Transduced Cells In Vivo

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

Impact of ETIF Deletion on Safety and Immunogenicity of Equine Herpesvirus Type 1-Vectored Vaccines

ABSTRACT Heterologous gene transfer by viral vector systems is often limited by factors such as preexisting immunity, toxicity, low packaging capacity, or weak immunogenic potential. A novel viral vector system derived from equine herpesvirus type 1 (EHV-1) not only overcomes some of these obstacles but also promotes the robust expression of a delivered transgene and the induction of antigen-specific immune responses. Regarding an enhanced safety profile, we assessed the impact of the gene encoding the sole essential tegument protein, ETIF, on the replication and immunogenicity of recombinant EHVs. The deletion of ETIF severely attenuates replication in permissive RK13 cells and a human lung epithelial cell line but without influencing transgene expression. Whereas the intranasal administration of a recombinant luciferase EHV in BALB/c mice resulted in transgene expression in nasal cavities and lungs for 5 to 6 days, the ETIF deletion limited expression to 2 days and resulted in 30-fold-less luminescence. Attenuated replication was accompanied by a decreased capacity to induce CD8+ T cells against a delivered HIV Gag transgene in BALB/c mice following repeated intranasal application. However, a single subcutaneous immunization with a gag DNA vaccine primed specific T cells for substantial expansion by two subsequent intranasal booster immunizations with either the gag recombinant ETIF mutant or the parental virus. In addition to inducing Gag-specific serum antibodies, this prime-boost strategy clearly outperformed three sequential immunizations with the parental or EHV-ΔETIF virus or repeated DNA vaccination by inducing substantial specific secretory IgA (sIgA) titers.