Polymerized Allergen Research Articles

Polymerization and fractionation of house dust mite allergen.

Glutaraldehyde polymerization and appropriate fractionation of allergens result in significantly decreased allergenicity with retained immunogenicity. In a manner similar to that employed with other allergens, we have prepared monomer dust mite (MDM) and polymerized dust mite (PDM). As with other polymerized allergens such as polymerized ragweed, PDM is more than 2 logs less allergenic than MDM as determined by cutaneous end point titration in sensitive patients. Moreover, PDM and MDM are similarly immunogenic in rabbits. As determined by inhibition studies (with rabbit antisera) PDM is antigenically complete and contains no new epitopes against which antibody was produced. In summary, we have prepared PDM which has the desired characteristics of reduced allergenicity, retained immunogenicity, antigenic completeness, and absence of new antigenic determinants.

IMMUNOTHERAPY WITH MODIFIED ALLERGENS

Efforts to improve conventional immunotherapy by allergen modification have generally followed one of three approaches. (1) Impedance of allergen release from site of injection, an example being alum-precipitation, has been somewhat successful. (2) Suppression of specific IgE has been unsuccessful in human trials. (3) Reduction of allergenicity with retention of immunogenicity appears to be the most promising, polymerized allergens and allergoid being examples. There are some reports of combining modifications. Modified extracts can be standardized. The specific tests to be used depend, in part, on characterization of the unique modification of the allergen.

Comparison of parallel line skin test assay (PLST) and ELISA inhibition (EI) methods for determination of relative potency of polymerized grass and ragweed antigenic extracts

Relative potency estimates were performed by parallel line skin test assay (PLST) and ELISA inhibition methods for three polymerized allergen extracts (Bermuda grass, orchard grass, and a copolymer of short and giant ragweed) versus four unmodified RAST standardized reference extracts (Bermuda grass, orchard grass, and giant and short ragweed) in nine subjects. One subject experienced a systemic reaction, requiring treatment at the end of the PLST assay. Another subject had a systemic reaction during limited skin testing performed approximately 72 hours after completion of PLST. Relative potency values for the polymerized extracts obtained by PLST were much lower than those obtained by ELISA inhibition, but results were significantly (r = 0.95; p < 0.01) correlated. Because polymerized allergen extracts are designed to be hypoaller genie, a skin test assay may underestimate their potency relative to an unmodified reference extract.

Standardization of modified allergens using polymerized ragweed as a model system

Polymerized allergens are manufactured by crosslinking allergen molecules with glutaraldehyde and then selecting a desired molecular weight range by gel filtration chromatography. Polymerized allergen extracts have retained antigenicity but reduced allergenicity when compared with conventional allergenic extracts. Thus, the important parameters to standardize in polymerized extracts are allergenicity, antigenicity, and molecular weight range. As our initial studies have demonstrated that allergenicity correlates with antigenicity and molecular weight, only the latter are necessary to measure. In short, polymerized allergen extracts could be standardized simply in terms of in vitro reactivity and chromatographic profile.

Modified forms of allergen immunotherapy

There have been many attempts to modify allergens and thus to improve upon conventional immunotherapy, which has been proved effective but has several drawbacks. These include the risk of systemic reactions and the time and cost involved. The modifications have taken three major approaches. One approach, to impede allergen release from the site of deposition, has met with limited success. An example is alum precipitation, which has modestly reduced the number of injections and incidence of systemic reactions. A second approach, to suppress specific IgE production and to induce specific tolerance, has not been successful in man. The third approach, to reduce allergenicity while retaining immunogenicity of allergens, has been the most successful and appears to have the most promise. One example is polymerized allergens, which have been shown to be safe, efficacious, and immunogenic in multiple clinical trials. Other examples include allergoids and glutaraldehyde-treated, tyrosine-adsorbed allergens.

64 Lymphocyte responses to pollen allergens: Effect of polymerized allergen immunotherapy

64 Lymphocyte responses to pollen allergens: Effect of polymerized allergen immunotherapy

Persistence of efficacy after a brief course of polymerized ragweed allergen: A controlled study

Immunotherapy with PRW has been demonstrated to be safe and effective. To determine whether the efficacy would remain in successive ragweed seasons without further therapy, a trial was conducted comparing PRW to histamine placebo therapy in patients that had received courses of PRW previously. Those patients were also compared to previously untreated ragweed-sensitive patients. In a double-blind fashion, 21 previously treated patients were treated before the 1982 ragweed season with four weekly injections of PRW therapy, whereas 21 previously treated patients were treated with four injections of placebo therapy. An additional control group of 21 previously untreated ragweed-sensitive patients received no injections. Daily symptom and medication score sheets were completed by patients each day of the ragweed season. Blocking antibody rose elevenfold with treatment ( p < 0.0001) in the PRW group. There was a statistically significant difference in symptom score mean between untreated patients (1007 ± 174) and previously treated patients whether they received supplemental injections (554 ± 180) ( p < 0.01) or whether they did not (650 ± 168) ( p < 0.03). In summary the efficacy of 15 injections of PRW immunotherapy persists at least several years without need for supplemental immunotherapy.

Reduced allergenicity of high molecular weight ragweed polymers

Polymerized ragweed antigens of different molecular weight ranges were studied to determine if the degree of allergenicity was dependent on molecular size of the polymers. In this study, allergenicity is defined as the ability to elicit IgE-mediated skin reactivity. Ragweed antigen treated with glutaraldehyde was fractionated into one preparation with a molecular weight range of 200,000 to 20,000,000 and another with molecular weights less than 200,000. Except for the difference in molecular sizes of the two preparations, all ragweed polymers had been treated in an identical fashion. The low molecular weight fraction had cutaneous endpoint reactivity greater than 10 3 to 10 8 times that of the high molecular weight materials. In these and other studies, immunogenicity defined as the ability to induce an IgG antibody response was retained by the high molecular weight material. The results are consistent with the hypothesis that allergenicity decreases as the molecular weight of the polymerized allergen increases.

Polymerization of mixtures of grass allergens

Mixed grass pollen allergens were precipitated from crude grass extract by 90% saturation with ammonium sulfate. The precipitate was dissolved and polymerized with glutaraldehyde. Polymerized allergens with a molecular weight range of 200,000 to 4,000,000 were isolated. The resultant grass allergen polymers had reduced allergenicity but were capable of absorbing out IgG antibody from sera of 2 patients treated with crude grass allergens. This alteration of allergenicity was intended to reduce the ability of allergens to react with IgE-sensitized cells rather than to modify or destroy antigenic determinants. Prior exposure of allergens to phenol used as a preservative inhibited the polymerization process probably by blocking combining sites with which glutaraldehyde reacts.