Biotechnology of grass pollen allergens for improved hay fever immunotherapy

This thesis describes the application of biotechnology to the generation of improved immunotherapeutic reagents, and novel methods for the delivery of such reagents, for the treatment of grass pollen allergy. Using site-directed mutagenesis, nine variants of the major rye grass pollen allergen, Lol p 5, were developed and tested for reduced IgE-reactivity. The results show that alteration of as few as one amino acid residue can reduce the human IgE-reactivity of the rLol p 5 protein. Furthermore, variant 9, which possessed four internal amino acid substitutions and was five amino acid residues shorter than rLol p 5 at the carboxyl-terminal, showed reductions in IgE-reactivity, the ability to induce histamine release from basophils and immediate skin reactivity. This protein also had similar secondary structural properties to rLol p 5 and retained the ability to stimulate T cell-proliferation. Low IgE-binding variants of the group 1 allergen from rye grass pollen were also generated. Site-directed mutagenesis of rLol p 1 showed that replacement of the cysteine residue at amino acid position 77 could significantly reduce the IgE-reactivity of this protein. However, the greatest reduction in IgE-reactivity of rLol p 1 was effected by the generation of truncation variants lacking internal and carboxyl-terminal regions which represented mapped linear IgE epitopes of LoI p 1. Hypoallergenic forms of the two major rye grass pollen allergens may aid the development of safer reagents for the treatment of grass pollen allergy. The mucosal administration of immunotherapeutic reagents for the treatment of human diseases has been shown to offer numerous immunological advantages and pose fewer risks than parenteral immunization. To demonstrate the applicability of mucosal immunization for the delivery of immunotherapeutic reagents for the treatment of pollen allergy, rLol p 5 and its hypoallergenic variant were orally administered to naive mice. Both proteins were shown to induce systemic immune responses when delivered directly into the stomach. However, the hypoallergenic variant was shown to induce a reduced systemic IgE response but a similar total IgG response in mice, as compared to mice administered rLol p 5. Both proteins also induced the production of systemic antibodies that could block an interaction between rLol p 5 and human IgE antibodies and which could recognize pollen proteins from other grass species. rLoi p 5 and its hypoallergenic variant also induced comparable levels of mucosal IgA antibodies after repeated immunization. As an innovative method of vaccine production and delivery, recent advances in recombinant DNA technology and plant transformation techniques have allowed proteins from numerous human pathogens to be expressed in transgenic plants. In an attempt to develop a novel method of recombinant protein expression for the production and delivery of grass pollen allergy immunotherapy, rLol p 1 and rLol p 5 were recombinantly expressed following Agrobacterium-mediated transformation of tobacco. Tobacco-expressed recombinant grass pollen allergens were shown to possess similar IgE binding epitopes to native rye grass pollen proteins and E. coli expressed rLol p 5