Treating insect-bite hypersensitivity in horses with active vaccination against IL-5 Antonia Fettelschoss-Gabriel, PhD, Victoria Fettelschoss, MSc, Franziska.

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Treating insect-bite hypersensitivity in horses with active vaccination against IL-5 Antonia Fettelschoss-Gabriel, PhD, Victoria Fettelschoss, MSc, Franziska Thoms, PhD, Christoph Giese, PhD, Michelle Daniel, BSc, Florian Olomski, MSc, Jivko Kamarachev, MD, Katharina Birkmann, DVM, Maya Bühler, DVM, Martin Kummer, DVM, Andris Zeltins, PhD, Eliane Marti, DVM, Thomas M. Kündig, MD, Martin F. Bachmann, PhD Journal of Allergy and Clinical Immunology Volume 142, Issue 4, Pages 1194-1205.e3 (October 2018) DOI: 10.1016/j.jaci.2018.01.041 Copyright © 2018 The Authors Terms and Conditions

Journal of Allergy and Clinical Immunology 2018 142, 1194-1205 Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 1 Diagnostics and characterization of IBH. A-D, Correlation of IBH lesion scores to blood parameters. IBH lesion scores were determined in biweekly to 4-week intervals during a whole season (April until October). Average lesion scores per horse were generated. EDTA blood and serum were taken once during the season with high symptoms (August). Eosinophil levels (EDTA blood) and Culicoides species–specific IgE levels (serum) were quantified by IDEXX Diavet. Correlation between average lesion scores and Culicoides species–specific IgE levels (IgE levels >150 ELISA absorbance units [EA] are considered allergic; dotted gray line; Fig 1, A), blood eosinophil count (Fig 1, B), serum IL-5 levels (quantified by means of ELISA; Fig 1, C), and blood eosinophil counts in a larger cohort (Fig 1, D) are shown. E and F, IBH skin biopsy. A representative skin biopsy specimen of a fresh lesion from an IBH-affected horse. Fig 1, E, Eosinophils in an IBH skin biopsy specimen. Hematoxylin and eosin staining was used. An overview (scale bar = 800 μm, upper panel) and enlarged section (scale bar = 200 μm, lower panel) are shown. Eosinophil-rich regions in the enlarged section are marked by circles. Fig 1, F, T cells in an IBH biopsy specimen. CD3 staining was used. An overview (scale bar = 300 μm) is shown. A CD3+-rich region is marked by a circle. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 2 Production and characterization of eIL-5 protein. A, SDS-PAGE analysis of purified eIL-5–C-His. Samples from various stages of the inclusion body preparation and purification were applied to a 4-12% B/T gel and run under reducing conditions. Proteins were stained with Coomassie Blue. Lane M, Size marker; lane 1, lysate (sample A); lane 2, soluble fraction (sample B); lane 3, solubilized inclusion bodies (sample C); lane 4, flow through (unbound material, sample D); and lane 5, pooled eIL-5 monomer (eIL-5, m) eluate from the Ni-NTA column (sample E). B, SDS-PAGE of refolded recombinant eIL-5–C-His. An aliquot of purified eIL-5–C-His was separated on a 4-12% B/T gel and run under nonreducing conditions (+SDS, no dithiothreitol, no heating). Proteins were stained with Coomassie Blue. eIL-5, d, eIL-5 dimer; eIL-5, m, eIL-5 monomer. Lane M, Size marker; lane 1, pooled denatured eluate from the Ni-NTA column; lane 2, refolded and homodimer enriched eIL-5–C-His. C, Far-UV circular dichroism spectrum of purified eIL-5–C-His. The spectrum demonstrates that eIL-5–C-His was folded. D, Detection of purified eIL-5–C-His with commercial anti–eIL-5 antibody. Anti-His antibody–coated ELISA plates were incubated with purified eIL-5–C-His and detected with a commercially available anti–eIL-5 antibody. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 3 Vaccine production and analysis of the coupling reaction of eIL-5–C-His to CMVTT. A and B: Lane M, Size marker; lane 1, tri(2-carboxyethyl)phosphine hydrochloride (TCEP)–activated eIL-5–C-His; lane 2, CMV-VLP after derivatization with the chemical cross-linker succinimidyl-6(β-maleimidopropionamido)hexanoate (SMPH); lane 3, eIL-5–C-His–CMVTT coupling reaction. eIL-5–C-His, CMVTT, and eIL-5–CMVTT were loaded in an equimolar manner to compare coupling efficacy. A, SDS-PAGE. Proteins were stained with Coomassie Blue. c, Coupling; CMV, m, CMVTT monomer; eIL-5, d, eIL-5 dimer; eIL-5, m, eIL-5 monomer. B, Western blotting with α-His antibody staining. c, Coupling; eIL-5, d, eIL-5 dimer; eIL-5, m, eIL-5 monomer. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 4 Antibody titer of vaccinated horses against eIL-5 and CMVTT-VLP. Time points of vaccinations are indicated by gray arrows. A, Mean antibody titer of anti–eIL-5 IgG. B, Mean antibody titer of anti-CMV IgG. C, Antibody titer of anti–eIL-5 IgG of individual horses. D, Antibody titer of anti-CMV IgG of individual horses. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 5 Lesion scores of eIL-5–CMVTT–vaccinated horses improved significantly compared with placebo-treated horses in clinical study. A and B, Mean lesion score of placebo-treated horses (n = 15; Fig 5, A) and eIL-5–CMVTT–vaccinated horses (n = 19; Fig 5, B) followed from March until October in the pre-evaluation year (gray symbols) and treatment year (black symbols). Months with more than 1 measurement show average values. C, Average lesion score per horse of the previous season (Pre) versus the treatment season (Treat; black: active, eIL-5–CMVTT vaccinated, n = 19; gray: placebo, n = 15). D, Percentage of horses with 50% (black columns) and 75% (gray columns) lesion score improvement in eIL-5–CMVTT–vaccinated (V) and placebo (P) group. E, Δ Mean average lesion score from eIL-5–CMVTT–vaccinated horses (black column, n = 19) versus placebo-treated horses (gray column, n = 15). **P < .01. F, Area under the curve (AUC) anti–eIL-5 antibody (Ab) titer versus Δ average lesion score of placebo-treated horses (gray symbols, n = 15) and eIL-5–CMVTT–vaccinated horses (black symbols, n = 19). AUC includes all values during the whole treatment season. All graphs include all horses (n = 34) analyzed according to intention to treat. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 6 Interrelation between blood eosinophil counts and lesion scores. A, Mean blood eosinophil counts (gray symbols and line, left y-axis) and mean lesion scores (black symbol and line, right y-axis) of placebo-treated horses (n = 15) determined in the treatment season from January until October or March until October, respectively. The normal maximum blood eosinophil count is indicated by a dotted gray line. Months with more than 1 measurement show average lesion score values. B, Eosinophil counts versus lesion scores of all 15 placebo-treated horses for 10 time points from each horse. C, Eosinophil counts versus lesion scores of all 19 vaccinated horses for 10 time points from each horse. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig 7 Side effect: parasitic load. A, Mean helminth burden in placebo-treated horses (gray columns, n = 15) and eIL-5–CMVTT–vaccinated horses (black columns, n = 19). No measureable helminths in excrement = 0, detected helminthes in excrement = 1. Excrement was taken in the treatment season before injections (pre) and at the study's end in autumn (post). B, Average seasonal change in helminth burden for placebo-treated (gray column) and vaccinated horses (black column) was calculated by subtracting the mean value determined at the beginning from that at the end of the treatment season. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig E1 Score sheet lesion scoring. Results are as described in the Methods section of the main article. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions

Fig E2 Liquid chromatography–MALDI-MS/MS of purified and trypsin-digested eIL-5–C-His. Dimers were linked intermolecularly through 2 disulfide bridges through Cys44 of one monomer and Cys86 of the other and vice versa. Journal of Allergy and Clinical Immunology 2018 142, 1194-1205.e3DOI: (10.1016/j.jaci.2018.01.041) Copyright © 2018 The Authors Terms and Conditions