Response to Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen.

The authors of J. Immunol. 184, 725-735 respond to the article by Rupp (2012), Acta Cryst. F68, 366-376.

Antigen aggregation decides the fate of the allergic immune response.

Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level.

Ragweed is one of the most important pollen allergens in North America and parts of Europe. Although the major allergen Amb a 1 was isolated and cloned in 1991, recombinant Amb a 1 was not explored further to improve diagnosis and specific immunotherapy of ragweed-pollen allergy. In the present study the immunological properties of natural Amb a 1 and its proteolytical cleavage products was investigated in detail and compared with recombinant produced Amb a 1 variants. Characterization of natural Amb a 1 and the identification of its proteolytic fragments, designated Amb a 1 alpha and Amb a 1 beta, was performed by N-terminal sequencing and mass spectroscopy. Amb a 1 and fragments were further produced in Escherichia coli, purified, and immunologically characterized. Amb a 1-specific T-cell cultures were used to compare the T-cell response to the different Amb a 1 variants. Divergent immunological properties of Amb a 1 alpha (aa 181-396) and Amb a 1 beta (aa 26-180) were revealed. Amb a 1 beta contained important IgE epitopes, whereas Amb a 1 alpha showed low IgE binding. When compared to natural Amb a 1, all recombinant variants possessed >100-fold reduced IgE-mediated mediator release activity. At the T-cell level recombinant and natural Amb a 1 stimulated comparable T-cell responses and the T-cell reactivity was largely directed to the C-terminal part. The results demonstrated that recombinant Amb a 1 alpha behaves as hypoallergen with reduced IgE binding but preservation of the major T-cell reactivity. In addition, recombinant Amb a 1 alpha can be easily purified to homogeneity in large quantity and therefore represents an ideal candidate for specific immunotherapy.

Identification of small molecule inhibitors of anthrax lethal factor.

The virulent spore-forming bacterium Bacillus anthracis secretes anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease that inactivates key signaling molecules, such as mitogen-activated protein kinase kinases (MAPKK), to ultimately cause cell death. We report here the identification of small molecule (nonpeptidic) inhibitors of LF. Using a two-stage screening assay, we determined the LF inhibitory properties of 19 compounds. Here, we describe six inhibitors on the basis of a pharmacophoric relationship determined using X-ray crystallographic data, molecular docking studies and three-dimensional (3D) database mining from the US National Cancer Institute (NCI) chemical repository. Three of these compounds have K(i) values in the 0.5-5 microM range and show competitive inhibition. These molecular scaffolds may be used to develop therapeutically viable inhibitors of LF.