1: Int Arch Allergy Immunol. 2003 Feb;130(2):119-24.

Oral immunization with a recombinant major grass pollen allergen induces blocking antibodies in mice.

de Weerd N, Bhalla PL, Singh MB.

Plant Molecular Biology and Biotechnology Laboratory, Institute of Land and Food Resources, University of Melbourne, Parkville, Vic., Australia.

BACKGROUND: Immunotherapy for the treatment of pollen allergies traditionally involves a series of parenteral injections of a crude pollen extract. Successful application of this treatment results in the development of systemic tolerance tothe sensitizing allergens, including the induction of blocking
antibodies.

OBJECTIVE: We sought to investigate whether oral immunization with a recombinant pollen allergen could induce a systemic immune response, and the production of systemic blocking antibodies in mice.

METHODS: C57BL/10 mice were orally  administered rLol p 5 or sodium chloride solution via gavage.

RESULTS: We report that the oral administration of rLol p 5 induced a systemic immune response,
including the induction of both blocking and interspecific cross-reactive antibodies. CONCLUSION: Our results suggest that oral administration of a major grass pollen allergen can induce the development of a systemic immune responseincluding the production of systemic blocking and cross-reactive antibodies, a response that may offer immunological protection upon subsequent
allergen exposure.

Copyright 2003 S. Karger AG, Basel

PMID: 12673065 [PubMed - indexed for MEDLINE]

2: Eur J Immunol. 2002 Jan;32(1):270-80.

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

Swoboda I, De Weerd N, Bhalla PL, Niederberger V, Sperr WR, Valent P, Kahlert H, Fiebig H, Verdino P, Keller W, Ebner C, Spitzauer S, Valenta R, Singh MB.

Plant Molecular Biology and Biotechnology Laboratory, Institute of Land and Food Resources, The University of Melbourne, Parkville, Australia.

More than 400 million individuals are sensitized to grass pollen allergens. Group

5 allergens represent the most potent grass pollen allergens recognized by morethan 80 % of grass pollen allergic patients. The aim of our study was to reducethe 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 ofLol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with lowIgE-binding capacity and reduced allergenic activity as determined by basophihistamine 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 p 5 wild-type allergen. In addition, Lol p 5mutants retained the ability to induce proliferation of group 5 allergen-specificT cell lines and clones. Our results demonstrate that a few point mutations inthe Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

Publication Types:
Research Support, Non-U.S. Gov't
PMID: 11782018 [PubMed - indexed for MEDLINE]

3: Int Arch Allergy Immunol. 2001 Jan-Mar;124(1-3):51-4.

Reduction in allergenicity of grass pollen by genetic engineering.

Bhalla PL, Swoboda I, Singh MB.

Plant Molecular Biology and Biotechnology Laboratory, Institute of Land and Food

Resources, University of Melbourne, Parkville, Australia.

p.bhalla@landfood.unimelb.edu.au

BACKGROUND: Hay fever and allergic asthma triggered by grass pollen allergensaffect approximately 20% of the population in cool temperate climates. Ryegrassis the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grasspollen allergen because it reacts with IgE antibodies of more than 90% of grasspollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made indeveloping diagnostic and therapeutic reagents for designing new and moreeffective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of
allergen content in the source plant.

METHODS: High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one ofmajor allergen, Lol p 5, was introduced. The expression of antisense RNA wasregulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity.

RESULTS: Analysis of proteins with allergen-specific monoclonal and polyclonalantibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plantsin which Lol p 5 gene expression is perturbed showed normal fertile
pollendevelopment.

CONCLUSIONS: Our studies showed that it is possible to selectively'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity. Copyright 2001 S.

Karger AG, Basel

Publication Types:

Research Support, Non-U.S. Gov't

PMID: 11306924 [PubMed - indexed for MEDLINE]

4: Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11676-80.

Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

Bhalla PL, Swoboda I, Singh MB.

Plant Molecular Biology and Biotechnology Laboratory, Institute of Land and Food

Resources, University of Melbourne, Parkville, Victoria 3052, Australia.

p.bhalla@landfood.unimelb.edu.au

Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source ofallergens because of its prodigious production of airborne pollen. Lol p 5 is themajor allergenic protein of ryegrass pollen, judging from the fact that
almost all of the individuals allergic to grass pollen show presence of serum IgEantibodies against this protein. Moreover, nearly two-thirds of the IgEreactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly
reduce the allergic potential of ryegrass pollen. Here, we report down-regulationof Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter.Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reducedallergenicity as reflected by low IgE-binding capacity of pollen extract ascompared with that of control pollen. The transgenic ryegrass plants in which Lolp 5 gene expression is perturbed showed normal fertile pollen development,indicating that genetic engineering of hypoallergenic grass plants is
possible.