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Allergen Gene Vaccination. Approach for allergen specific immunotherapy in severe food allergy Anne Behnecke, MD Department of Clinical Immunology and Allergy David Geffen School of Medicine at UCLA. Food Allergy. Peanut, tree nut, egg, milk, fish, soy, wheat, shellfish

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Allergen gene vaccination l.jpg

Allergen Gene Vaccination

Approach for allergen specific immunotherapy in severe food allergy

Anne Behnecke, MD

Department of Clinical Immunology and Allergy

David Geffen School of Medicine at UCLA


Food allergy l.jpg
Food Allergy

  • Peanut, tree nut, egg, milk, fish, soy, wheat, shellfish

  • Increasing prevalence/incidence of food allergies :

    6-8% of children, 3-4% of adults1

  • 2-fold increase in reported peanut allergy, 3-fold increase in sensitization in UK (1989/1996) 2

  • U.S. Europe

  •  1% of the US and UK population affected by peanut allergy2

  • Peanut allergy resolves for only 20% of young children by school age2

  • Food anaphylaxis fatality in the U.S.: in 59% of 63 deaths peanut as a trigger2

  • Peanut is a ubiquitious food: accidental ingestion is common, annual incidence rate of 14.3% reported in Canada

    1Sampson HA. Food allergy - accurately identifying clinical reactivity. Allergy. 2005;60 Suppl. 79:19-24.

    2 Sicherer SH, Sampson HA, Peanut allergy: Emerging concepts and approaches for an apparent epidemic. Current revies of allergy and clinical immunology, July 2007

 NO effective therapy for food allergies


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IgE-based allergen DNA vaccine

  • consisting of cell targeting protein bound to allergen gene DNA

  • specifically designed to target the allergen DNA to high affinity FcRI bearing Antigen-presenting cells (APC)

  • targeting Dendritic cells additionally by use of DC-specific promoter


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RationaleIgE-based allergen DNA vaccine

high affinity FcRI receptor on Antigen-presenting cells

 genetically modified hFc chain(genetically attached to 60 Lysine residues in a fusion gene =EPL) ionically bound to peanut allergen gene (e.g.Ara h2)


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RationaleIgE-based allergen DNA vaccine

- affinity to human IgE 2-3 logs higher than most ligand receptor interaction

- APCs of atopic patients express much higher FcRIlevels than non-allergic individuals

 Endocytosis of the FcRI bound DNA complexes  partly released in the cytosol or nucleus compartments to be expressed  FcRI chains recycled to the surface

 conventional mouse APCs do not express FcRI


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Murine model

hFcRI+ Tg mice

hIgE+ hFcRI+ Tg mice


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RationaleIgE-based allergen DNA vaccine

  •  Dendritic cell specific expression



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TAT sequence and expression

Nuclear localization signal (NLS)

Expression plasmid

a. fascin-promotor

b. gene cDNA

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CHe2-4 of hFce

Polylysine (60)

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FceRI

Dendritic Cells


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Experimental design and expression


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Experimental design and expression

  • Laboratory outcomes

  • Allergen specific humanIgE

  • Allergen specific murine IgG1, 2a, 2b, 3, IgA, IgM

  • Cytokine mRNA expression by RT-PCR (IL-4, IL-5, IL-10, IL-12, IL-13, TGF-b and IFN-g)

  • Cytokine production (IL-4, IL-5, IL-10, IL-12, IL-13, TGF-b and IFN-g) in response to allergen in vitro

  • IL-4 and IFN-g expressing cell frequency (Elispot) in response to allergen in vitro

  • T cell response: key cytokine production that reflects characteristic Th1/Th2 and T regulatory responses

     harvested cells from spleen and lymph nodes will be cultured and pulsed with purified allergen for 48 hours to induce memory T cell cytokine production (IL-4, IL-5, IL-10, IL-12, IL-13, TGF-b and IFN-g)

     measured by cytokine specific ELISA assays and the cytokine mRNA expression profiles by the quantitative real-time RT-PCR


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Experimental design and expression

  • Anaphylactic clinical index:

     scoring system: 0 = no symptoms

    1 = scratching and rubbing aroundthe nose and head

    2 = puffiness around the eyes and mouth, diarrhea,pilar erecti, reduced activity, and/or decreased activity withincreased respiratory rate

    3 = wheezing, labored respiration,cyanosis around the mouth and the tail

    4 = no activity afterprodding, or tremor and convulsion

    5 = death

  • Serum histamine levels and core body temperature changes:

    used as parameters for systemic anaphylaxis

  • Mast cell degranulation: during systemic anaphylaxis, assessed byhistologic examination of ear tissues


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Conclusion and expression

  • Allergen gene vaccination represents a promising alternative to the protein-based allergen-specific IT in terms of safety and efficacy especially in food allergy

  • Allergen gene vaccination can modulate allergic responses but the current state of the art requires far improved targeting

  • FcRI on APCs in humans provides a mechanism to target an Fc protein allergen DNA complex for IT

  • Use of Dendritic cell type specific promoter allows targeted DNA vaccine to be expressed in DC with cell type restricted fashion

  • even the best murine protocol will not directly translate to a human protocol

     Pilot investigation to test the general applicability and potency of this new platform as therapeutic strategy for treatment of food allergy


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Peanut Butter Mice and expression

  • INGREDIENTS:

  • 1/2 cup butter, room temperature

  • 1 cup creamy peanut butter

  • 1/2 cup packed light brown sugar

  • 1/2 cup granulated sugar

  • 1 egg

  • 1 teaspoon vanilla extract

  • 1/2 teaspoon baking soda

  • 1 1/2 cups all-purpose flour

  • 1 cup peanut halves

  • chopped red or green candied cherries or cinnamon red hots

  • 60 3-inch pieces red or black shoestring licorice

  • PREPARATION:

  • Beat butter and peanut butter in a large bowl with electric mixer until creamy. Add sugars and beat until fluffy. Beat in egg, vanilla and baking soda until well blended. Beat flour in slowly until blended.

  • Cover and chill dough for about 1 hour until firm enough to handle. Heat oven to 350°.

  • Shape level tablespoons of dough into 1-inch balls. Taper each ball by one end into tear drop shape. Press one side flat. Place flat sides down 2 inches apart on ungreased cookie sheet. Press in sides of balls to raise backs of mice. The dough spreads slightly as it bakes. Gently push 2 peanut halves in head of each for ears and 2 pieces of green or red candy for eyes. With a wooden pick make a 1/2-inch deep hole at tail end. Bake 8-10 minutes until firm. Remove to rack, insert licorice tails and cool completely.Makes about 60 mice.


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Preliminary work and expression

  • PCA assay

  • dose dependent PCA reaction to the peanut allergic patient’s serum (from a to f), but no reaction to the serum from a healthy donor (g) or to saline (h)

  •  FceRIa Tg mice express skin reactivity to human IgE in vivo challenge

  •  ability to do allergen specific graded skin testing in actively sensitized

  • FceRIa Tg mice


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