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Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides.

Pellaton C, Perrin Y, Boudousquié C, Barbier N, Wassenberg J, Corradin G, Thierry AC, Audran R, Reymond C, Spertini F - Clin Transl Allergy (2013)

Bottom Line: Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen.The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, Lausanne, 1011, Switzerland. francois.spertini@chuv.ch.

ABSTRACT

Background: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.

Methods: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.

Results: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.

Conclusions: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.

Trial registration: ClinicalTrials.gov NCT01719133.

No MeSH data available.


Related in: MedlinePlus

Human basophil degranulation assay. Panel A: Basophils from two subjects allergic to birch (circle and square symbols) were activated by rBet v 1, AllerT, T4-T5 or T6-T7-T8 mixes at indicated concentrations. Panel B: Basophils from a non-allergic donor were pre-incubated with sera from 3 subjects allergic to birch (circles, squares and triangles) and incubated with the indicated concentrations of rBet v 1 and AllerT. Degranulation is expressed as the percentage of CD63 positive basophils. Panel C: Rat basophils expressing hu FcϵRI were pre-incubated with sera from 10 subjects allergic birch and reacted with either rBet v 1 or AllerT (filled and open symbols respectively). Results were grouped by IgE class levels as determined by the ImmunoCAP assay (classes 4 to 6; n= 3 or 4 sera per class). Hexosaminidase release is expressed as the percentage of release induced by anti-total IgE antiserum.
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Figure 4: Human basophil degranulation assay. Panel A: Basophils from two subjects allergic to birch (circle and square symbols) were activated by rBet v 1, AllerT, T4-T5 or T6-T7-T8 mixes at indicated concentrations. Panel B: Basophils from a non-allergic donor were pre-incubated with sera from 3 subjects allergic to birch (circles, squares and triangles) and incubated with the indicated concentrations of rBet v 1 and AllerT. Degranulation is expressed as the percentage of CD63 positive basophils. Panel C: Rat basophils expressing hu FcϵRI were pre-incubated with sera from 10 subjects allergic birch and reacted with either rBet v 1 or AllerT (filled and open symbols respectively). Results were grouped by IgE class levels as determined by the ImmunoCAP assay (classes 4 to 6; n= 3 or 4 sera per class). Hexosaminidase release is expressed as the percentage of release induced by anti-total IgE antiserum.

Mentions: In preparation for application to human subjects, AllerT COPs and rBet v 1 were compared for their capacity to induce human basophil degranulation. In a direct Basotest™ assay, blood was collected from patients allergic to birch pollen and degranulation due to IgE crosslinking was tested in response to COPs mixes, namely T1-T2-T3, T4-T5 or T6-T7-T8 (Figure 4A). None of the COP mixes was able to induce functional IgE crosslinking at any concentration tested (up to 1 μM), whereas rBet v 1 induced 50% degranulation already at about 0.1 nM. Thus AllerT displays an at least 104-fold lower capacity to activate basophils from allergic patients.


Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides.

Pellaton C, Perrin Y, Boudousquié C, Barbier N, Wassenberg J, Corradin G, Thierry AC, Audran R, Reymond C, Spertini F - Clin Transl Allergy (2013)

Human basophil degranulation assay. Panel A: Basophils from two subjects allergic to birch (circle and square symbols) were activated by rBet v 1, AllerT, T4-T5 or T6-T7-T8 mixes at indicated concentrations. Panel B: Basophils from a non-allergic donor were pre-incubated with sera from 3 subjects allergic to birch (circles, squares and triangles) and incubated with the indicated concentrations of rBet v 1 and AllerT. Degranulation is expressed as the percentage of CD63 positive basophils. Panel C: Rat basophils expressing hu FcϵRI were pre-incubated with sera from 10 subjects allergic birch and reacted with either rBet v 1 or AllerT (filled and open symbols respectively). Results were grouped by IgE class levels as determined by the ImmunoCAP assay (classes 4 to 6; n= 3 or 4 sera per class). Hexosaminidase release is expressed as the percentage of release induced by anti-total IgE antiserum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672070&req=5

Figure 4: Human basophil degranulation assay. Panel A: Basophils from two subjects allergic to birch (circle and square symbols) were activated by rBet v 1, AllerT, T4-T5 or T6-T7-T8 mixes at indicated concentrations. Panel B: Basophils from a non-allergic donor were pre-incubated with sera from 3 subjects allergic to birch (circles, squares and triangles) and incubated with the indicated concentrations of rBet v 1 and AllerT. Degranulation is expressed as the percentage of CD63 positive basophils. Panel C: Rat basophils expressing hu FcϵRI were pre-incubated with sera from 10 subjects allergic birch and reacted with either rBet v 1 or AllerT (filled and open symbols respectively). Results were grouped by IgE class levels as determined by the ImmunoCAP assay (classes 4 to 6; n= 3 or 4 sera per class). Hexosaminidase release is expressed as the percentage of release induced by anti-total IgE antiserum.
Mentions: In preparation for application to human subjects, AllerT COPs and rBet v 1 were compared for their capacity to induce human basophil degranulation. In a direct Basotest™ assay, blood was collected from patients allergic to birch pollen and degranulation due to IgE crosslinking was tested in response to COPs mixes, namely T1-T2-T3, T4-T5 or T6-T7-T8 (Figure 4A). None of the COP mixes was able to induce functional IgE crosslinking at any concentration tested (up to 1 μM), whereas rBet v 1 induced 50% degranulation already at about 0.1 nM. Thus AllerT displays an at least 104-fold lower capacity to activate basophils from allergic patients.

Bottom Line: Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen.The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois (CHUV), Rue du Bugnon, Lausanne, 1011, Switzerland. francois.spertini@chuv.ch.

ABSTRACT

Background: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.

Methods: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.

Results: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.

Conclusions: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.

Trial registration: ClinicalTrials.gov NCT01719133.

No MeSH data available.


Related in: MedlinePlus