Background: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen–specific immunotherapy. Objective: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. Methods: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV–Beta) and Bet v 1d (pCMV–Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN–γ and IL–4 levels were measured by ELISA. Allergen–specific proliferation was determined by incorporation of [3H]–thymidine. Results: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th–1–type response. The antisera against both isoforms were highly cross–reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN–γ and IL–4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV–Beta, pCMV–Betd caused no significant allergen–specific proliferation and induced only marginal levels of the key cytokines. Conclusions: Based on the assumption that the induction of a strong Th–1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.

The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.

Simplified models and empirical potentials are being increasingly used for the analysis of proteins, frequently augmenting or replacing molecular mechanics approaches. Recent folding simulations have employed potentials that, in addition to terms assuring proper polypeptide geometry, include only two noncovalent effects - hydrogen bonding and hydrophobicity, with extremely simple approximations to the latter. The potentials that have been used in the free-energy ranking of protein-ligand complexes have generally been more involved. These potentials have more detailed solvation models and account for both local (hydrophobic and polar) solute-solvent phenomena and long range electrostatic solvation effects. The models of solvation that have been used most frequently are surface area related atomic parameters, knowledge-based models extracted from protein-structure data, and continuum electrostatics with an additional area-related parameter. The knowledge-based approaches to solvation, although convenient and accurate enough, are suspect of double counting certain free-energy terms.

The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 → Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.