Ragweed Pollen Antigens

Essentially all of the peptides and proteins in pollen extracts can elicit the formation of IgG antibodies in animals. Therefore, they are antigens. Only some of these antigens, however, are allergens [i.e., stimulate and bind IgE (in humans)]. Crossed immunoelectrophoresis shows that short ragweed pollen extract contains at least 52 antigens (as recognized by the rabbit antisera), but only 22 of these are allergens, as shown by their binding of specific IgE from the sera of ragweed-sensitive patients (41). Use of sophisticated biochemical methods has resulted in the isolation of ragweed fractions of up to 300 times the potency of crude ragweed extracts, as measured by the ability to induce positive skin test results in appropriate subjects and the ability to cause histamine release from their peripheral blood leukocytes in vitro.

Several investigators have studied purified ragweed antigens. The early work of King and Norman ( 41,42) laid the foundation for the purification and analysis of allergens. Two major allergens, Amb a 1 (antigen E) and Amb a 2 (antigen K), were isolated by gel filtration and ion exchange chromatography. These have certain immunologic and chemical properties in common, but differ in molecular weight and biologic activity. Recently, sophisticated molecular biology techniques have enabled workers to isolate and clone DNA sequences (cDNA) for many other ragweed allergens. This has allowed comparisons of DNA sequences. DNA sequences showing similarity (homology) are likely to correspond to proteins with similar function and structural antigenicity. In addition to the two major ragweed allergens, eight intermediate or minor allergens have been isolated. These are Amb a 3 through Amb a 7 and cystatin.

Amb a 1 is a protein contained primarily in the intine of the pollen grain ( 13). It accounts for about 6% of the total protein of whole ragweed extract. Quantitative studies of ragweed-sensitive patients with Amb a 1 have shown a positive correlation with skin test reactivity and leukocyte histamine release, but no correlation with protein-nitrogen content in six commercial preparations of ragweed extract (43). Techniques are available, however, such as radial immunodiffusion, that allow direct quantitation of Amb a 1 in allergenic extracts, and, by use of RAST inhibition, the potency of ragweed allergenic extracts can be assessed. The U.S. Food and Drug Administration (FDA) requires that ragweed allergenic extracts be labeled with their Amb a 1 content.

Amb a 1 consists of two fragments, named A and B. These fragments are not bound covalently and are dissociated readily, which results in a significant loss of allergenic activity. Recombination of these polypeptide chains does not restore allergenic activity, presumably because the steric conformation is not readily restored. Amb a 1 is resistant to enzymatic degradation, suggesting that readily accessible amino or carboxyl groups are not the principal immunologic determinants. Interestingly, 10-fold more Amb a 1 is extractable in vitro at the pH of nasal secretions from patients with allergic rhinitis (pH 7 to 8), than at the pH of nasal secretions from nonatopic individuals (pH 6.3) (13).

Four isoallergenic variants have been demonstrated for Amb a 1, both by physiochemical studies and recent cDNA analyses (44). Isoallergens have the same immunologic properties and similar chemical structures, but differ in some way such as isoelectric point, carbohydrate content, or amino acid composition ( 12).

It has been calculated that the maximal amount of ragweed Amb a 1 that a person breathing outdoor air in southeastern Minnesota would inhale is approximately 0.2 pg in a season (45). The amount of Amb a 1 produced by an individual ragweed plant appears to be determined genetically. There is considerable variation in the amount extractable by standard methods from pollen from plants grown under identical conditions (59-468 pg/mL) (46).

Amb a 2 (antigen K) constitutes about 3% of extractable ragweed pollen protein. Approximately 90% to 95% of ragweed-sensitive subjects show skin reactivity to this antigen. Amb a 2 may cross-react slightly with Amb a 1, a finding reinforced by a 68% sequence homology at a DNA level (47).

Since the isolation of Amb a 1 and 2, additional minor allergens designated Amb a 3 (Ra 3), Amb a 4 (Ra 4), Amb a 5 (Ra 5) (48,49. and 50), Amb a 6 (Ra 6) (51), Amb a 7 (Ra 7) (52), and cystatin (53) have been identified. In contrast to Amb a 1, these low-molecular-weight fractions are rapidly extractable (<10 minutes) from pollen and have basic isoelectric points (54). Amb a 3 has a relatively high carbohydrate content, making it similar to certain grass pollen antigens. It consists of a single peptide chain of 102 amino acids. Two variants of Amb a 3 differing by a single amino acid residue have been described; however, this difference does not alter the allergenic specificity (55). This gene has not been cloned. Amb a 5 consists of a single polypeptide chain whose 45 amino acids have been sequenced. The two isoallergenic forms differ at the second position by the substitution of leucine for valine in about 25% of samples. The frequency of positive skin test results to these antigens in ragweed-sensitive subjects demonstrates that approximately 90% to 95% react to Amb a 1 and Amb a 2, 20% to 25% react to Amb a 3 and Amb a 6, and about 10% to Amb a 5. The frequency of reaction to Amb a 4 is not known. A small fraction (10%) of ragweed-sensitive patients are more sensitive to Amb a 3 and 5 than to Amb a 1.

Amb a 6 and Amb a 7 show sequence homology to other plant proteins involved in lipid metabolism and electron transport, respectively ( 52,56). Cystatin, the most recent ragweed allergen to be cloned, shows homology to a family of cysteine protease inhibitors found in other plants ( 53).

These various allergens have made it possible to study genetic responses in the ragweed-sensitive population. A complex antigen such as Amb a 1 appears unrelated to total serum IgE or to any specific HLA phenotype, whereas subjects who respond to the lower molecular weight allergens such as Amb a 3 have elevated total serum IgE levels (57). Response to Amb a 5 requires an immune response (Ir) gene usually associated with HLA DW2 (57). Sensitivity to Amb a 3 has been associated with increased frequency of the HLA-A2 and HLA-B12 phenotype (13). When a group of highly pollen-sensitive patients were skin-prick tested with individual purified ragweed and ryegrass allergens, each patient reacted in a distinctive pattern. This pattern was undoubtedly genetically programmed ( 50).

In addition to the short ragweed allergens just described, an allergen from giant ragweed (A trifida), Amb t V (Ra 5G), has been identified (58). Other allergens that cause allergic rhinitis have been purified from additional weeds. These include Sal p 1 from S pestifer (Russian thistle) (59), Par j 1 and Par j 2 from Parietaria judaica pollen (Coccharia) (60,61), and Par o 1 from Parietaria officinalis (62). The cDNA for Par j 1 and Par o 1 also have been described (63,64). Art v 1 and Art v 2 from A vulgaris (mugwort) also have been purified (65). Mugwort has shown significant cross-reactivity with ragweed, including Art v1 and recombinant Bet v 1 (66).

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