For DNA immunization

against HIV or HPV it was shown that codon-optimization of the antigen encoding expression plasmids enhanced the immunogenicity of the vaccines, primarily through increased antigen expression [9] and [10]. The impact of codon-optimization has also been demonstrated for viral vector systems [11] and [12]. Particularly for A-1210477 mw RNA viruses replicating by a viral RNA-dependent RNA polymerase instead of the cellular transcription machinery, codon-optimization may overcome additional restrictions on protein expression. For several proteins (F, P, N) of respiratory syncytial virus (RSV), expression of wildtype sequences under the control of eukaryotic promoters was shown to be largely inhibited by premature polyadenylation [13] and [14]. In a comparative study, DNA vaccination with a codon-optimized

expression plasmid coding for the F-protein increased the protective efficacy against RSV challenge by 1–2 orders of magnitude compared to wildtype plasmids [15]. Since expression of influenza virus proteins also depends on a viral RNA polymerase, we decided to compare the immunogenicity of DNA vaccines based on codon-optimized and wildtype sequences. The vaccines used in this study are based on the pVAX expression plasmid, where the antigen expression is controlled by a CMV promoter. The wildtype sequence of the HA of the virus strain A/Texas/05/2009 (H1N1) was synthesized by Geneart (Regensburg, Germany), followed VE-821 by PCR amplification and cloning into the pVAX backbone. The resulting plasmid, pV-Texas, is referred to here as HAwt. The plasmid inhibitors pTH-HAco also synthesised by Geneart, carries a codon-optimized sequence for the

HA followed by a C-Terminal V5 tag (HAco) and the open reading frame was cloned into pVAX (pV-HAco) to eliminate possible differences in expression levels and immune responses resulting from different plasmid backbones. An Non-specific serine/threonine protein kinase alignment of the two nucleotides is shown in supplementary Fig. 1. DNA for immunization was prepared using the NucleoBond® Xtra Maxi EF Kit (Macherey-Nagel, Düren, Germany) and tested for endotoxin levels with the LAL quantification assay (Cambrex Bio Science, Verviers, Belgium), confirming that the dose used for immunization of mice contained less than 0.1 EU (Endotoxin Units). Some of the control animals received a VSV-G expressing plasmid, pHIT-G [16] as an irrelevant DNA control. 6–8-Week-old female Balb/cJRj mice were purchased from Janvier (Le Genest-ST-Isle, France) and housed in singly ventilated cages in accordance with the national law and institutional guidelines. The DNA was diluted in PBS and 30 μg were used for one intramuscular immunization followed by electroporation. The injection and electroporation procedure was performed consistent with previous reports [17] and in accordance to the manual supplied by the manufacturer (Ichor Medical Inc., San Diego, USA).