Working with a ryegrass protein -- one of the most potent environmental allergens known -- the team, from the University of Melbourne’s Plant Molecular Biology and Biotechnology Group, has modified the gene responsible for its production.

The modified gene produced a protein that has a significantly reduced allergenic response, yet one still capable of boosting a person’s immune response. These twin features are the key to a potential vaccine, or effective immunotherapy -- the practice of giving allergic patients small, but increasing doses of allergen-containing extracts to boost their immunity to the allergen.

“Skin prick tests to assess the protein’s safety suggest it is a significantly safer and more effective alternative to the current immunotherapy used to treat grass pollen allergies,” says Associate Professor Prem Bhalla, joint leader of the group.

Their research is published in the latest European Journal of Immunology.

Current immunotherapy carries a significant risk of lethal side effects that include anaphylactic shock, a condition in which the body rapidly becomes hypersensitive to an allergen.

“The symptoms of allergenic disease, like asthma, hayfever, allergic dermatitis and conjunctivitis, can be controlled with drugs, but specific immunotherapy is the only way to treat the causes of allergies that produce a potentially life-threatening hypersensitivity (Type 1 allergies)," says Bhalla.

Since its introduction in 1911, immunotherapy has been subject to criticism and debate, largely because the underlying immunological mechanisms of the human body are still unknown and because there has been no way of controlling the contents of the allergen-containing extracts.

To boost their immune response to an allergen, patients suffering severe allergic responses are usually given crude extracts of the allergen to which they are allergic.

“In addition to the allergen, the extracts contain unknown and virtually impossible to standardize combinations of proteins and other ingredients. It is these unknown components that place the patient at risk of anaphylactic shock. The therapy also runs the risk of the patient developing allergies to the unknown proteins in the extract because they are going back for this treatment and exposing their immune system to these proteins year after year,” says Bhalla.

“We had to find a way of changing the ryegrass protein so it would lose its allergenic properties, yet retain its ability to boost the body’s immune response.

“The mutated gene is placed in a bacterium, which can then produce the modified protein in large quantities. In its purified form, the protein becomes a potential vaccine.

“The modified protein, delivered to a patient in its pure form without other contaminants, reduces the risk of a patient going into anaphylactic shock by a possible factor of ten and increases the efficacy of immunotherapy.”

To reach this stage of the research took 6 years. The team initially isolated the ryegrass protein known as Lol p 5 and identified the gene responsible for its production. They then made nine different mutant forms of the original wild type protein by making a series of small, but specific modifications to the gene.

One of these mutations (mut 9) produced a protein that produced an allergic response at least tenfold reduced in all patients compared to the wild type protein. Other forms produced varied results. Some caused a 100-fold reduction in allergic reaction in some patients. In others, the same mutant protein caused an increase in allergic reaction.

“Mut 9 was the universal vaccine. But the discovery that some of the modified proteins can have even better results with some patients suggests that patients could be matched up with a vaccine tailored to their immune system or individual sensitivity,” says co-group leader Associate Professor Mohan Singh.

Analysis of the mutant proteins’ structure showed they exhibited a similar structure to the wild type allergen -- an important factor if the body is to build an appropriate immune response to the allergen.

Further analysis showed the body’s immune system retained its ability to respond to and build immunity against the potent group 5 allergens to which the ryegrass protein belongs.

“Our results contribute to the concept of using major pollen allergens for allergen-specific immunotherapy and to the safe and effective forms of allergy treatment,” says Singh.

[Contact: Jason Major]

07-Feb-2002