Detection of allergen composition and in vivo immunogenicity of depigmented allergoids of Betula alba.
BACKGROUND
Chemical modification of allergen vaccines to reduce IgE binding improves safety while maintaining clinical efficacy. However, this also complicates the characterization of allergoids using techniques as for native allergen extracts.


OBJECTIVE
The objective of this study was to analyse the molecular size of Betula alba depigmented allergoids, conservation of major allergens in the allergoids and in vivo antibody response to immunization.


METHODS
The molecular size of depigmented allergoids was evaluated by high performance-size exclusion chromatography and light scattering techniques. Protein composition was compared with native extracts by capillary liquid chromatography-tandem mass spectrometry based peptide mapping. Rabbits were immunized with depigmented allergoid of Betula pollen adsorbed onto aluminium hydroxide (Depigoid). IgG antibodies against individual allergens were determined by ELISA and immunoblot.


RESULTS
Depigmented allergoids contained a range of high molecular weight particles, approximately 60% of which had a molecular weight of 1-3 MDa. Peptide sequencing confirmed the preservation of five isoforms of Bet v 1, as well as Bet v 2, Bet v 6 and Bet v 7. Sera from immunized rabbits showed high levels of specific IgG to rBet v 1.0101 and rBet v 2.


CONCLUSIONS
The mean protein content was 544+/-106 microg per mg of freeze-dried material for depigmented allergoids and 434+/-71 for native extracts. They retain the capacity to induce specific IgG antibodies against individual allergens present in the native extract. These findings confirm the immunogenicity of depigmented allergoids and may explain why patients treated with these vaccines are protected against the native allergens. Analysis of molecular size and allergen content may be useful techniques for characterization and standardization of allergoid products.
Carnés
J
J
Research & Development Department, Laboratorios LETI S.L., Calle del Sol no. 5, 28760 Tres Cantos, Madrid, Spain. jcarnes@leti.com
Himly
M
M
Gallego
M
M
Iraola
V
V
Robinson
D S
DS
Fernández-Caldas
E
E
Briza
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D052179:Antigens, Plant / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D029662:Betula / Q000737:chemistry* / Q000276:immunology*; D002850:Chromatography, Gel / Q000379:methods; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010860:Pigments, Biological / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D011058:Pollen / Q000737:chemistry* / Q000276:immunology; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D011817:Rabbits; D053719:Tandem Mass Spectrometry; D014611:Vaccination
D052179:Antigens, Plant; D007074:Immunoglobulin G; D010860:Pigments, Biological; D010936:Plant Extracts; D010940:Plant Proteins; D020033:Protein Isoforms