Immunological properties of conjugates of ragweed pollen antigen E with methoxypolyethylene glycol or a copolymer of D-glutamic acid and D-lysine.

Abstract

Chemically modified allergens may be useful immunotherapeutic reagents in man. What is required of such reagents is that the modified allergens show reduced allergenic activities yet retain the immunosuppressive properties of the native molecule. It is with this objective that we have prepared conjugates of antigen E with two polymers of large molecular weights. Antigen E is the major allergen of ragweed pollen and it is an acidic protein of ~38,000 daltons (1). The two polymers are methoxypolyethylene glycol (MPEG) 1 of ~5,000 daltons and a copolymer of Dglutamie acid and D-lysine (DGL) of ~34,000 daltons. MPEG was chosen because MPEG protein conjugates are known to have reduced allergenic and immunogenic activities (2, 3). DGL was chosen because DGL conjugates with haptens of low molecular weight are known to possess hapten-specific immunosuppressive properties (4, 5). To study the influence of the bulky MPEG groups on the immunochemical properties of the conjugate, we also prepared a conjugate using methanol in place of MPEG.In this paper we report comparative studies on the immunological properties of antigen E and its conjugates in the mouse as the experimental animal. The mouse system was chosen since it is generally accepted that the IgE response in this system is the best model for reaginic antibody formation in man (6). MPEG and methoxy antigen E conjugates were prepared by coupling the protein with cyanuric chloride activated MPEG (2) or methanol (7) as shown in Fig. 1. DGL antigen E conjugate was prepared via an intermolecular disulfide bond formation (8). As shown in Fig. 2, antigen E and DGL were first modified to contain reactive thiol and 4-dithiopyridyl groups, then they were coupled via these groups. Materials and Methods Materials.Antigen E was isolated from ragweed pollen as described (9); its isoelectric forms B and C were used interehangeably in this study. MPEG of average mol wt of 5,000 was a gift of Union Carbide Corp., New York. A random copolymer of DGL in a molar ratio of 6:4, having an average mol wt of 34,000, was a gift from Miles

Recombinant Allergens with Reduced Allergenicity but Retaining Immunogenicity of the Natural Allergens: Hybrids of Yellow Jacket and Paper Wasp Venom Allergen Antigen 5s

“…A second approach to reduce the accessibility of B cell epitopes of allergen is by polymerization on formaldehyde or glutaraldehyde treatment (9 -11) or by attachment of nonimmunogenic polymers (12). One limitation of this approach is that near-complete loss of the discontinuous B cell epitopes usually occurred when allergens were modified with Ͼ100-fold reduction in allergenicity.…”

“…The high concentrations of hybrids required for half-maximal inhibition relative to that of Ves v 5 are probably related to their epitope densities, because epitope density is known to strongly influence the affinity constant of a multivalent Ag and a bivalent Ab (28,29). This may explain why the longest N-terminal hybrid, PV , showed the lowest concentration for half-maximal inhibition, and it is followed by the next longest hybrids, PV , PV , PV , and PV [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] .…”

“…At the highest concentration of 50 or 500 g/ml inhibitor tested, the two N-terminal hybrids, EA-PV 1-46 and EA-PV 1-155 , showed maximal inhibition approaching 100%, as EA-or KR-Ves v 5 did. Two other N-terminal hybrids, KR-PV and EA-PV , had maximal inhibition of ϳ60%, and the shortest N-terminal hybrid, EA-PV [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] , had maximal inhibition of ϳ20%. The C-terminal hybrid EA-PV 156 -204 had maximal inhibition of ϳ15%, and the shorter C-terminal hybrid, EA-PV 195-204 , was not tested.…”

“…The data in set A of Table III indicate that, of the three N-terminal hybrids, PV 1-155 was as immunogenic as Ves v 5 was, PV 1-46 was half as imunogenic, and PV 1-18 was approximately one-ninth as immunogenic. The data in set B indicate that PV and PV were more immunogenic than PV 1-24 and PV [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] . More importantly, the data from both sets suggest that the longer N-terminal hybrids PV and PV 1-32 stimulate higher contents of Ves v 5-specific Abs and lower contents of Pol a 5-specific Abs than the two shorter hybrids, PV and PV [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] , did.…”

“…EA-PV and EA-PV [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] showed geometric mean reductions of 126-and 583-fold, respectively, in group A patients and 0.7-and 24-fold decreases, respectively, in group B patients. The two C-terminal hybrids, EA-PV 156 -204 and EA-PV [195][196][197][198][199][200][201][202][203][204] , had reductions of 1139-and 3207-fold in group A patients, respectively, and 3-and 32-fold in group B patients, respectively.…”