Original article Optimization of codon usage is required for effective genetic immunization against Art v 1, the major allergen of mugwort pollen Pollen from mugwort (Artemisia vulgaris) is one of the main allergen sources in central Europe and parts of Asia during late summer. Among patients suffering from pollinosis, the incidence of allergic disease caused by mugwort pollen is between 10 and 14% (1, 2). As the major allergen of mugwort pollen, Art v 1 (3) represents an important target for the conventional specific immunotherapy (SIT) as well as for novel therapy concepts such as genetic desensitization. The latter has emerged as an important new vaccin- ation strategy and a powerful alternative to protein-based vaccination against pathogens, tumors and even allergic diseases (4, 5). Intramuscular or intradermal injection of plasmid DNA encoding clinically relevant allergens can induce immune responses with a Th1 bias and promote the formation of IFN-c producing CD4+ T cells (6) thus stimulating B cells to synthesize immunoglobulin G (IgG)2a antibodies and inhibit IgE production (7–11). Furthermore, genetic immunization offers a palette of modulating possibilities to enhance the immunogenicity of plasmid constructs (12). One of these modulating approaches is based on the fact that most amino acids are encoded by more than one codon, and codon usage varies from organism to organ- ism. Therefore, differences in the codon usage concerning a heterologous gene and the transfected cell can have a Background: As the major allergen of mugwort pollen, Art v 1 is an important target for specific immunotherapy. However, both recombinant protein as well as a gene vaccine for Art v 1 failed to be immunogenic in mice. In order to improve immunogenicity we focused on genetic immunization because inter- specific differences of codon usage have been shown as an obstacle for effective induction of immune responses with gene vaccines encoding infectious patho- gens. Objective: In order to find out, whether codon usage might also be used to improve genetic immunization with allergen genes, the response against a gene vaccine expressing the wild-type gene of Art v 1 (pCMV-wtArt) was compared with a synthetic codon-optimized vector with human codon usage (pCMV- humArt). Methods: Balb/c mice were injected intradermally with pCMV-wtArt or pCMV- humArt. In vitro expression levels of both constructs were compared in trans- fection experiments. Total immunoglobulin G (IgG), IgG1, IgG2a and IgE antibodies were analyzed by enzyme-linked immunosorbent assay and the anaphylactic activity of the sera was determined by allergen-specific degranu- lation of rat basophil leukemia-2H3 cells. Results: No immune response was detectable with the gene vaccine expressing the wildtype Art v 1, but immunization with pCMV-humArt revealed a strong and allergen-specific induction of antibody responses. The antibodies recognized both the recombinant as well as the purified natural (glycosylated) Art v 1 molecule. The response type was Th1-biased, as indicated by high levels of IgG2a antibodies. Expression analysis with B16 mouse melanoma cells trans- fected with pCMV-humArt or pCMV-wtArt revealed an impaired expression of the wild-type vector but normal translation after recoding. Conclusion: The results demonstrate that optimization of codon usage offers a simple way to improve immunogenicity and therefore should be routinely con- sidered in the development of gene vaccines for the treatment of allergy. R. Bauer 1 , M. Himly 2 , A. Dedic 2 , F. Ferreira 2 , J. Thalhamer 1 , A. Hartl 1 1 Institute of Chemistry and Biochemistry, Immunology Group, University of Salzburg; 2 Institute of Genetics and General Biology, University of Salzburg, Salzburg, Austria Key words: allergy; Art v 1; codon usage; DNA vaccines; genetic immunization; mugwort. Dr J. Thalhamer University of Salzburg Institute of Chemistry and Biochemistry Immunology Group Hellbrunnerstr. 34, A-5020 Salzburg, Austria Accepted for publication 12 February 2003 Allergy 2003: 58: 1003–1010 Printed in UK. All rights reserved Copyright Ó Blackwell Munksgaard 2003 ALLERGY ISSN 0105-4538 1003