BACKGROUND:The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.METHODS:A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.RESULTS:Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.CONCLUSION:We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.

Abstract The Laue method in combination with a synchrotron source can produce large amounts of data very rapidly. This technique offers fast data collection which in many cases eliminates the time dependent component from radiation damage. The method suffers from two principal problems: The difficulty in obtaining low resolution data from Laue photographs. This arises from geometrical restrictions inherent in Laue diffraction, and from computational difficulties when separating harmonic overlaps and applying wavelength dependent corrections to low resolution singlets. The consequence of this is a “low resolution hole” in the data set. The loss of low resolution data poses difficulties in identifying atoms with high B-factors in the structure.

Xenon gas can be used as a heavy atom for determining phases in a protein. We demonstrate that an interpretable electron density map can be obtained for sperm whale metmyoglobin from a single xenon derivative using iterative single isomorphous replacement with the anomalous scattering method.