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Kuan-Wei Chen, PhD, Katharina Blatt, MSc, Wayne R

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1 Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy 
Kuan-Wei Chen, PhD, Katharina Blatt, MSc, Wayne R. Thomas, PhD, Ines Swoboda, PhD, Peter Valent, MD, Rudolf Valenta, MD, Susanne Vrtala, PhD  Journal of Allergy and Clinical Immunology  Volume 130, Issue 2, Pages e4 (August 2012) DOI: /j.jaci Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Construction of Der p 2/Der p 1 mosaic proteins. Three fragments of Der p 1 (1.1, 1.2, and 1.3) comprising amino acids 1 to 84, 85 to 143, and 144 to 222 and 2 fragments of Der p 2 (2.1 and 2.2) comprising amino acids 1 to 53 and 54 to 129 were reassembled in the order 1.3, 2.2, 1.2, 2.1, and 1.1. Der p 2/1C contains the original amino acid sequence of Der p 1 and Der p 2, including the 12 cysteine residues (dashed vertical lines), whereas in Der p 2/1S the cysteine residues were exchanged for serine residues. Both proteins contain a hexahistidine tag at the C-terminus. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Expression and purification of rDer p 2/Der p 1 mosaic proteins. Coomassie-stained SDS-PAGE–containing protein extracts of BL21 (DE3) expressing Der p 2/1C and Der p 2/1S (lanes 1), purified Der p 2/1C and Der p 2/1S (lanes 2; A), and purified Der p 2/1C and Der p 2/1S separated under reducing (lanes r) and nonreducing (lanes nr) conditions (B) and molecular weight markers (lanes M) are shown. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Far-UV CD spectra of nDer p 1, rDer p 2, rDer p 2/1C, and rDer p 2/1S. The mean residue ellipticity (y-axis) at given wavelengths (x-axis) is shown for each protein. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IgE reactivity of nDer p 1, rDer p 2, and rDer p 2/1 mosaic proteins. Dot-blotted nDer p 1, rDer p 2, the 2 Der p 2/1 mosaic proteins, and BSA were tested for IgE reactivity with sera from 32 patients with HDM allergy (lanes 1-32), serum from a nonallergic subject (NC), or buffer without serum (BC). Bound IgE was detected with iodine 125–labeled anti-human IgE antibodies and visualized by using autoradiography. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Allergenic activity of nDer p 1, rDer p 2, and rDer p 2/1 mosaic proteins. Basophils from 10 patients with HDM allergy were stimulated with various concentrations of nDer p 1, rDer p 2, rDer p 2/1C, and rDer p2/1S (x-axes). Expression of CD203c was determined by using fluorescence-activated cell-sorting analysis, and stimulation indices (SI) are shown (y-axes). Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Reactivity and epitope specificity of IgG antibodies induced by immunization of rabbits with nDer p 1, rDer p 2, and rDer p2/1 mosaic proteins. Sera obtained from rabbits before (pre) or after (immune) immunization with nDer p 1, rDer p 2, rDer p 2/1C, and rDer p 2/1S were tested for IgG reactivity to dot-blotted nDer p 1, rDer p 2, rDer p 2/1 mosaic proteins, and BSA or with synthetic Der p 1– and Der p 2–derived peptides (Der p 1 P1-P8 and Der p 2 P1-P5). Bound IgG antibodies were detected with iodine 125–labeled donkey anti-rabbit IgG antibodies. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Reduced allergenicity of the 2 rDer p 2/1 mosaic proteins. RBL cells were loaded with sera from mice obtained before (preimmune) or after (immune) immunization with nDer p 1, rDer p 2, or the 2 mosaic proteins (x-axes). nDer p 1–specific (A) and rDer p 2–specific (B) release of β-hexosaminidase are displayed as percentages of total β-hexosaminidase release (medians ± SDs for the 5 sera from each group of mice, y-axes). Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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