Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

Abstract

We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8 þ CTLs, but was independent of B and CD4 þ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6-12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.

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.…”

Improved osteogenic vector for non-viral gene therapy

“…Several studies have successfully exploited codon optimisation to increase yield in eukaryotic cells for in vitro production of recombinant proteins (interleukin 2 (Ou et al, 2014), coagulation factor VIII (Ward et al, 2011)), vaccination (Frelin et al, 2004;Ko et al, 2005), and gene replacement therapy such as microdystrophin (Foster et al, 2008) and PhiC31 recombinase (Raymond and Soriano, 2007). However, codon optimisation on growth factor genes for application in tissue regeneration has great potential, but has mostly been unexploited yet.…”

“…Therefore, delivery of up to mg dosages at once are required to provide a sufficient stimulus. As a result, these supraphysiological local dosages lead to increased occurrence of protein diffusion and side effects (Carragee et al, 2011;Glassman et al, 2011;Shimer et al, 2009). As a promising alternative, gene therapy allows local and sustained release of growth factors.…”