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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 IgE competition assays. rBet v 1-specific IgE were measured by ELISA in sera from individual birch pollen allergic patients pre-incubated with increasing concentrations of either rBet v 1 or COPs as indicated on the X axis. Panel A: Sera from six allergic patients (indicated by various symbol shapes) were pre-incubated with either rBet v 1 (black symbols), AllerT (grey symbols), T4-T5 (open symbols) and T6-T8 (crossed circles). Panel B: Individual COPs T1, T2 and T3 were tested separately with serum from a representative patient as indicated in the Figure (control rBet v 1, black circles). Panel C: Sera from three allergic individuals was reacted with either T4 or T5 (black & white symbols) alone or control rBet v 1 (black symbols). Inhibition of binding is expressed as the inverse of the percentage of residual IgE binding to Bet v 1.
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Figure 1: Human IgE competition assays. rBet v 1-specific IgE were measured by ELISA in sera from individual birch pollen allergic patients pre-incubated with increasing concentrations of either rBet v 1 or COPs as indicated on the X axis. Panel A: Sera from six allergic patients (indicated by various symbol shapes) were pre-incubated with either rBet v 1 (black symbols), AllerT (grey symbols), T4-T5 (open symbols) and T6-T8 (crossed circles). Panel B: Individual COPs T1, T2 and T3 were tested separately with serum from a representative patient as indicated in the Figure (control rBet v 1, black circles). Panel C: Sera from three allergic individuals was reacted with either T4 or T5 (black & white symbols) alone or control rBet v 1 (black symbols). Inhibition of binding is expressed as the inverse of the percentage of residual IgE binding to Bet v 1.

Mentions: COPs were first tested in vitro for birch specific IgE binding by competition ELISA. Soluble rBet v 1 inhibited serum IgE binding with EC50 ranging from 10-14 to 10-10 M, (Figure 1A). COPs T1, T2 and T3 either in equimolar combination (AllerT) (Figure 1A) or individually (Figure 1B) showed no competitive activity up to 10-5 M. The same result was obtained using COPs T6, T7 and T8 in combination (Figure 1A), indicating that the precise ending of the COPs was not essential for reducing IgE binding. Results were comparable with T4 and T5 individually as competitors (Figure 1C). Surprisingly, the combination of T4-T5 as an equimolar mix did inhibit IgE binding with EC50 in the range of 10-7-10-8 M, i.e. at least at concentration 104-fold higher than rBet v 1 (Figure 1A). The fact that competition reaches completion suggests partial refolding due to interaction of T4 and T5 in solution, thus re-creating the original IgE epitope(s).


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 IgE competition assays. rBet v 1-specific IgE were measured by ELISA in sera from individual birch pollen allergic patients pre-incubated with increasing concentrations of either rBet v 1 or COPs as indicated on the X axis. Panel A: Sera from six allergic patients (indicated by various symbol shapes) were pre-incubated with either rBet v 1 (black symbols), AllerT (grey symbols), T4-T5 (open symbols) and T6-T8 (crossed circles). Panel B: Individual COPs T1, T2 and T3 were tested separately with serum from a representative patient as indicated in the Figure (control rBet v 1, black circles). Panel C: Sera from three allergic individuals was reacted with either T4 or T5 (black & white symbols) alone or control rBet v 1 (black symbols). Inhibition of binding is expressed as the inverse of the percentage of residual IgE binding to Bet v 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Human IgE competition assays. rBet v 1-specific IgE were measured by ELISA in sera from individual birch pollen allergic patients pre-incubated with increasing concentrations of either rBet v 1 or COPs as indicated on the X axis. Panel A: Sera from six allergic patients (indicated by various symbol shapes) were pre-incubated with either rBet v 1 (black symbols), AllerT (grey symbols), T4-T5 (open symbols) and T6-T8 (crossed circles). Panel B: Individual COPs T1, T2 and T3 were tested separately with serum from a representative patient as indicated in the Figure (control rBet v 1, black circles). Panel C: Sera from three allergic individuals was reacted with either T4 or T5 (black & white symbols) alone or control rBet v 1 (black symbols). Inhibition of binding is expressed as the inverse of the percentage of residual IgE binding to Bet v 1.
Mentions: COPs were first tested in vitro for birch specific IgE binding by competition ELISA. Soluble rBet v 1 inhibited serum IgE binding with EC50 ranging from 10-14 to 10-10 M, (Figure 1A). COPs T1, T2 and T3 either in equimolar combination (AllerT) (Figure 1A) or individually (Figure 1B) showed no competitive activity up to 10-5 M. The same result was obtained using COPs T6, T7 and T8 in combination (Figure 1A), indicating that the precise ending of the COPs was not essential for reducing IgE binding. Results were comparable with T4 and T5 individually as competitors (Figure 1C). Surprisingly, the combination of T4-T5 as an equimolar mix did inhibit IgE binding with EC50 in the range of 10-7-10-8 M, i.e. at least at concentration 104-fold higher than rBet v 1 (Figure 1A). The fact that competition reaches completion suggests partial refolding due to interaction of T4 and T5 in solution, thus re-creating the original IgE epitope(s).

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