Immunogenicity of Phleum pratense depigmented allergoid vaccines: experimental study in rabbits.

BACKGROUND: Immunogenicity studies are based on accurate preclinical and clinical assessment of pharmaceutical products. The immunogenicity of modified allergen vaccines has not been fully elucidated, and the mechanisms involved are not well understood. Animal and human models have recently shown that depigmented allergoids induce specific immunoglobulin (Ig) G against individual allergens, thus supporting the clinical efficacy of these vaccines. OBJECTIVE: The aim of this study was to investigate the production of specific IgG against individual antigens and their isoforms in rabbits injected with depigmented allergoid extracts of Phleum pratense pollen. METHODS: Two New Zealand rabbits were immunized with depigmented-polymerized extracts adsorbed onto aluminum hydroxide (Depigoid) of P pratense. Rabbits were injected 3 times (35 microg Phl p 5). Specific IgG titers against native, depigmented, and depigmented-polymerized extracts and individual allergens (rPhl p 1 and rPhl p 5a) were analyzed by direct enzyme-linked immunosorbent assay. The capacity of these synthesized antibodies to recognize individual native and depigmented allergens and different isoforms was evaluated by immunoblot and 2-D analysis. RESULTS: All rabbits produced high titers of specific IgG against the 3 extracts. Rabbits injected with depigmented allergoids produced similar specific antibody titers against native, depigmented, and depigmented-polymerized extracts. Serum samples recognized individual allergens and their isoforms in the nonmodified extracts. CONCLUSION: Vaccines containing depigmented allergoid extracts of P pratense induce immunogenicity in vivo. The antibodies produced after injection of these extracts clearly recognized allergens and different isoforms in their native configuration.

Depigmented and polymerised house dust mite allergoid: allergen content, induction of IgG4 and clinical response.

BACKGROUND: Polymerised allergenic extracts (allergoids) are commonly used in allergen immunotherapy. Clinical efficacy and safety of these extracts have been demonstrated. Recently, allergen sequences have been identified by mass spectrometry in depigmented and polymerised (Dpg-Pol) extracts. The objectives of this study were to investigate the presence of allergens in Dpg-Pol extracts of house dust mite and to analyze the immunological changes induced by these extracts in asthmatic patients enrolled in a double-blind, placebo-controlled study. METHODS: Dpg-Pol extracts were manufactured and vaccines with a composition of 50% Dermatophagoides pteronyssinus and 50% D. farinae (100 HEPL/ml) were prepared. Allergen composition was analyzed by mass spectrometry. Patients with asthma and rhinoconjunctivitis were treated in a 1-year, double-blind, placebo-controlled, parallel-group study with 6 up-dosing and monthly maintenance injections. Specific IgE and IgG4 titres to D. pteronyssinus, Der p 1 and Der p 2 were measured in patients' sera using the CAP system and direct ELISA experiments. RESULTS: Sequences from the major allergens Der p 1 and Der p 2 and from other allergens were identified in native and Dpg-Pol extracts. There was a statistically significant increase in specific IgG4, a decrease in the ratio of IgE/IgG4 to D. pteronyssinus and a significant increase in specific IgG4 to Der p 1 and Der p 2 in the patients allotted to active treatment. CONCLUSIONS: The detection of allergen sequences suggests preservation of major and minor allergens in Dpg-Pol allergoids from house dust mites. Efficacy in asthma treatment and the increase in specific IgG4 seem to be associated with the presence of major allergens in Dpg-Pol allergen extracts.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Extraordinary stability of IgE-binding Parietaria pollen allergens in relation to chemically bound flavonoids.

It is known that the skin-active and IgE-binding components in Parietaria pollen extracts are not restricted to the predominant protein allergens of M(r) 12000-15000, but are present as well among the naturally occurring constituents of M(r) < 10000. Indeed, the IgE-binding Parietaria pollen components are quite heterogeneous, ranging from high- to low-molecular mass, whereby the IgE-binding epitopes display an unusual chemical stability. Furthermore, the pollen of Parietaria species demonstrably contain a high proportion of flavonoid pigments. Since these pollen grains cannot be collected entirely free from non-pollen plant parts, the usual allergenic extracts of Parietaria encompass both the polyphenolic substrate molecules and the enzyme polyphenoloxidase as ingredients for the oxidative generation of flavonol-protein conjugates during the extraction process. In the present work this is illustrated by spectroscopic analyses of the free and bound flavonoids in Parietaria pollen extracts, as well as of the peptide fragments produced from the allergenic proteins by enzymatic or chemical hydrolysis. None of these relatively harsh treatments had a significant effect on the IgE-binding properties of the allergenic (sub-)components, even though detectable proteins in isoelectric focusing and immunoblotting were lost. It is proposed that the extraordinary stability of IgE-binding Parietaria components over a wide molecular range may be attributed to chromophoric flavonoid side-chains as (parts of) the corresponding B-cell epitopes.

Complement inactivation by allergenic plant pollen extracts.

Dialyzed aqueous extracts of plant pollen are widely used in the clinical practice of allergy for diagnostic and therapeutic purposes. The present investigation shows that such allergenic extracts are capable of consuming complement in every human serum, independent of the clinical condition. Complement is engaged by way of the first component C1, but without the participation of allergen-specific antibodies. The capacity of distinct pollen extracts to inactivate haemolytic complement was found to depend on the plant species, the most potent extracts being from the pollen of the weeds and trees. Analysis by UV-spectroscopy of the flavonoids remaining firmly bound to the proteins gives rise to the proposal that complement inactivation by allergenic and non-allergenic pollen extracts is due to polyphenolic (flavonoid) structures complexed with, or chemically conjugated to, the pollen proteins.

Investigation of acid phosphatase as a possible IgE-binding marker for pollen allergens and their polymerized derivatives.

The enzyme acid phosphatase represents an important component among the allergenic proteins in most pollen extracts. However, determination of the level of this enzyme in extracts of various pollen species, as well as protein separation by molecular sieving shows that acid phosphatase cannot be used as a marker for IgE-binding protein allergens. A conspicuous discrepancy was observed between the heat- and acid-sensitivities of the phosphatase enzyme relative to the overall IgE-binding properties of the pollen extracts. Remarkably, the enzyme remained unaffected by cross-linking the pollen proteins with glutardialdehyde, whereas this process of polymerization caused the IgE-binding potency to diminish considerably. It is concluded that the enzyme acid phosphatase is not a suitable marker for the potency assessment of pollen allergens, but may be useful for monitoring the production process of polymerized allergoids.

Evaluation of the potency of allergenic extracts by inhibition of IgG-antibody binding.

A method is described for the in vitro potency evaluation of allergenic extracts by using their capacity of binding to specific human IgG antibodies. The results obtained with this inhibition-type enzyme immunoassay are compared with the analyses by the customary method of IgE antibody binding. A large series of allergenic protein fractions from the pollen of the Gramineae, Olea europea, Parietaria judaica, and from the dust mite Dermatophagoides pteronyssinus as well as from cat dander and peanuts were examined for inhibition of specific antibodies of both IgG and IgE isotypes. Potency evaluation by inhibition assays for IgE- and IgG-binding showed a significant correlation for the points of 50% inhibition (r = 0.92, p = 0.0001), but not for the slopes of the inhibition curves, i.e. the respective antibody avidities. Evidence is provided that the strict relationship between IgE- and IgG-inhibition by allergens could not be explained by possible cross-contaminations of the anti IgE- or anti IgG-reagents employed in the immunoassays. It is concluded that the inhibition of IgG-antibody binding presents a fast, reliable and low-cost alternative for the potency control of allergens used in clinical practice.

Pru p 3 (LTP) content in peach extracts.

BACKGROUND: Lipid transfer proteins are molecules widely distributed in fruits. Sensitization to LTP is frequent in fruit sensitive patients. The aims of this study were to purify LTP and to assess the content of LTP in ripe peach peel and pulp extracts by ELISA inhibition using polyclonal antibodies. METHODS: LTP was purified from ripe yellow peach peel by two different column chromatography methods. A polyclonal antibody was produced by injecting purified LTP into two New Zealand white rabbits. ELISA inhibition and rabbit monospecific polyclonal antibody were used to calculate the LTP content in Springcrest and Miraflores varieties of peach peel and pulp extracts. Purified LTP (2.5 mg/ml) was used to skin test 24 peach-sensitive patients. RESULTS: The purified LTP showed a single band at approximately 9 kDa. The polyclonal antibody raised anti LTP recognized only the LTP molecule in the peach extracts. LTP content, expressed in micro g/mg of freeze-dried extract in four extracts were: yellow peach peel, 15.48; yellow peach pulp 2.25; red peach peel 14.67 and red peach pulp 1.84. Twenty patients (83.3%) had a positive skin test with purified LTP. CONCLUSIONS: We have developed a system to determine the concentration of LTP in peach extracts. LTP in peel extracts is approximately seven times greater than in pulp.