Peter Valent

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grasspollen allergens as well as recombinant B cell epitope‐containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen‐specific immunotherapy. We found that the administration of aluminium hydroxide‐adsorbed grass pollen extract induced complex changes in allergenu2009/u2009epitope‐specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl pu20041 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl pu20041 epitopes was provided by the demonstration that preincubation of rPhl pu20041 with human serum containing therapy‐induced Phl pu20041‐specific IgG inhibited rPhl pu20041‐induced histamine release from basophils of a grass pollen‐allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component‐resolved immunotherapy.

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80u2009% of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B‐ and T‐cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site‐directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol pu20095 mutants with low IgE‐binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol pu20095 wild‐type allergen. In addition, Lol pu20095 mutants retained the ability to induce proliferation of group 5 allergen‐specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol pu20095 sequence yield mutants with reduced allergenic activitythat represent potential vaccine candidates for immunotherapy of grass pollen allergy.

Conversion of grass pollen allergen-specific human IgE into a protective IgG1 antibody

More than 100 million individuals exhibit IgE‐mediated allergic reactions against Phlu2004pu20042, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phlu2004pu20042‐specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen‐allergic patient. Recombinant Phlu2004pu20042‐specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N‐terminal amino acids of Phlu2004pu20042 and cross‐reacted with groupu20042 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG1 to obtain a complete, recombinant Phlu2004pu20042‐specific human IgG1. This antibody blocked the binding of grass pollen‐allergic patients IgE (n=26; mean inhibition: 58%) to Phlu2004pu20042 and caused a 100‐fold reduction of Phlu2004pu20042‐induced basophil histamine release. The recombinant human Phlu2004pu20042‐specific IgG1 may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.