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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

Degranulation of rat basophil lines induced by AllerT and its respective individual COPs T1, T2 and T3. Beta-hexosaminidase release was measured from RBL-2H3 cells loaded with sera obtained from r Bet v 1-sensitized mice and stimulated in panel A. with 5.7 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=10; in panel B: with 57 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=3; panel C: Beta-hexosaminidase release from RBL-2H3 cells loaded with 2 sera (dilutions 1:10, 1:50 and 1:100) obtained from r Bet v 1-sensitized mice and stimulated with serial concentrations of Bet v 1. Degranulation was measured using a colorimetric assay and is expressed in OD. Median and quartiles are indicated as horizontal bars. Data were analyzed by a non-parametric paired t test and compared to rBetv1. **p <0.01.
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Figure 2: Degranulation of rat basophil lines induced by AllerT and its respective individual COPs T1, T2 and T3. Beta-hexosaminidase release was measured from RBL-2H3 cells loaded with sera obtained from r Bet v 1-sensitized mice and stimulated in panel A. with 5.7 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=10; in panel B: with 57 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=3; panel C: Beta-hexosaminidase release from RBL-2H3 cells loaded with 2 sera (dilutions 1:10, 1:50 and 1:100) obtained from r Bet v 1-sensitized mice and stimulated with serial concentrations of Bet v 1. Degranulation was measured using a colorimetric assay and is expressed in OD. Median and quartiles are indicated as horizontal bars. Data were analyzed by a non-parametric paired t test and compared to rBetv1. **p <0.01.

Mentions: The capacity of COPs to induce basophil degranulation was characterized using rat basophil leukemia cells (RBL) [28]. Upon stimulation with rBet v 1, hexosaminidase accumulated significantly in the supernatant from RBL cells primed with sera from Bet v 1-sensitized mice (see below). On the contrary, AllerT as well as individual COPs were unable to induce hexosaminidase release even at ten-fold (5.7 nM, panel A) and hundred-fold (57 nM, panel B) higher concentrations than optimal rBet v 1 (0.57 nM, panel C) (Figure 2).


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)

Degranulation of rat basophil lines induced by AllerT and its respective individual COPs T1, T2 and T3. Beta-hexosaminidase release was measured from RBL-2H3 cells loaded with sera obtained from r Bet v 1-sensitized mice and stimulated in panel A. with 5.7 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=10; in panel B: with 57 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=3; panel C: Beta-hexosaminidase release from RBL-2H3 cells loaded with 2 sera (dilutions 1:10, 1:50 and 1:100) obtained from r Bet v 1-sensitized mice and stimulated with serial concentrations of Bet v 1. Degranulation was measured using a colorimetric assay and is expressed in OD. Median and quartiles are indicated as horizontal bars. Data were analyzed by a non-parametric paired t test and compared to rBetv1. **p <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Degranulation of rat basophil lines induced by AllerT and its respective individual COPs T1, T2 and T3. Beta-hexosaminidase release was measured from RBL-2H3 cells loaded with sera obtained from r Bet v 1-sensitized mice and stimulated in panel A. with 5.7 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=10; in panel B: with 57 nM rBet v 1, T1, T2, T3, AllerT or left unstimulated, n=3; panel C: Beta-hexosaminidase release from RBL-2H3 cells loaded with 2 sera (dilutions 1:10, 1:50 and 1:100) obtained from r Bet v 1-sensitized mice and stimulated with serial concentrations of Bet v 1. Degranulation was measured using a colorimetric assay and is expressed in OD. Median and quartiles are indicated as horizontal bars. Data were analyzed by a non-parametric paired t test and compared to rBetv1. **p <0.01.
Mentions: The capacity of COPs to induce basophil degranulation was characterized using rat basophil leukemia cells (RBL) [28]. Upon stimulation with rBet v 1, hexosaminidase accumulated significantly in the supernatant from RBL cells primed with sera from Bet v 1-sensitized mice (see below). On the contrary, AllerT as well as individual COPs were unable to induce hexosaminidase release even at ten-fold (5.7 nM, panel A) and hundred-fold (57 nM, panel B) higher concentrations than optimal rBet v 1 (0.57 nM, panel C) (Figure 2).

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