Vaccination against IL-31 for the treatment of atopic dermatitis in dogs  Martin F. Bachmann, PhD, Andris Zeltins, PhD, Gints Kalnins, Dr med vet, Ina.

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Vaccination against IL-31 for the treatment of atopic dermatitis in dogs  Martin F. Bachmann, PhD, Andris Zeltins, PhD, Gints Kalnins, Dr med vet, Ina Balke, PhD, Nina Fischer, MSc Biol, Ana Rostaher, Dr med vet, Kaspars Tars, PhD, Claude Favrot, Dr med vet  Journal of Allergy and Clinical Immunology  Volume 142, Issue 1, Pages 279-281.e1 (July 2018) DOI: 10.1016/j.jaci.2017.12.994 Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Generating of canine IL-31-CMVTT and antibody responses. A, SDS page analysis of coupling reaction before and after removal of free IL-31. Lane 1: coupling reaction of IL-31 and CMVTT before purification. Lane 2: free IL-31; Lane 3: coupling product after purification. *Presence of extra bands due to coupling of canine IL-31 to monomers of CMVTT VLPs. B, Outline of the study showing time points of immunization and experimental challenges with HDM. Six dogs were immunized with the vaccine in alum, whereas the other 6 were treated with alum alone. C and D, Antibody responses were measured in the sera against IL-31 (Fig 1, C) or CMV (Fig 1, D) by ELISA at the indicated time points. Arrows in C and D indicate time points of vaccination. The numbers indicate individual dogs. E, A correlation between IL-31 and CMV titers is shown. ELISA titers are expressed as log3 of 10-fold prediluted sera leading to half-maximal absorbance in the ELISA. The graph includes all 4 time points after vaccination and does not take into account the placebo dogs. Journal of Allergy and Clinical Immunology 2018 142, 279-281.e1DOI: (10.1016/j.jaci.2017.12.994) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Pruritus in vaccinated and placebo dogs and correlation with antibody responses. A and B, Dogs allergic to HDM were immunized with placebo (placebo) or vaccine (active) and challenged at the indicated time points with HDM extract. Pruritus was quantified by counting the number of scratches over 6 hours. Day 0 depicts baseline before immunization. Fig 2, A, Average number of scratching and SEM. Fig 2, B, The number of vaccine responders. Dogs showing more than 50% reduced scratching compared with the average of the placebo dogs were counted as responders. A 1-way ANOVA was performed in Fig 2, A, and chi-square tests in Fig 2, B. C and D, Scratching is correlated to antibody titers in immunized and placebo dogs. Antibody levels and scratching were correlated 35 days (Fig 2, C) and 75 days (Fig 2, D) after initial vaccination. ELISA titers are expressed as log3 of 10-fold prediluted sera leading to half-maximal absorbance in the ELISA. Circled dogs are on active. *A placebo dog that was likely injected because it also has anti-CMV titers at this time point. Journal of Allergy and Clinical Immunology 2018 142, 279-281.e1DOI: (10.1016/j.jaci.2017.12.994) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Properties of refolded canine IL-31. A, Elution profile from gel-filtration chromatography on HiLoad 16/600 Superdex 200 PG column. B, Circular dichroism analysis of refolded canine IL-31. Theoretical curves for helix, sheet, and random coil are shown as well. Journal of Allergy and Clinical Immunology 2018 142, 279-281.e1DOI: (10.1016/j.jaci.2017.12.994) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions