*2.8. Vaccination of Cats*

The vaccine Fel-CuMV and the vaccine production was described previously [36]. A vaccine dose contained 100 μg Fel-CuMV (HypoCatTM) formulated in 1 mL aqueous buffer solution. Subcutaneous injections were applied in study weeks 4, 7 and 10 (main study) and 56 (extension study). The vaccine used in this study was produced by Benchmark Vaccines Limited, UK using a GMP-like production process. Prior to each vaccination, the cats were thoroughly examined by one of the participating veterinarians for their general health status, including a routine physical exam with measurement of the body weight and temperature, checking the site of injection, pulse and breathing rate, abdomen palpation and general appearance. During the course of the study, the owners regularly checked the health of their animal. Twenty-four hours after every injection the owners were called by the study personnel to enquire after the well-being of the cat.

## *2.9. Antibody Responses in Cats*

Blood for serum antibody measurements was collected from the vena jugularis or cephalica using serum tubes (Sarstedt, Germany). Sampling was done in the study weeks 4, 7, 10, 27, 56, 62, and 78. After clotting (30 min) and centrifugation (1000 ×*g*, 5 min) of blood samples, sera were transferred to labeled polypropylene tubes (Eppendorf tubes, 1.5 mL, Germany) and stored at ≤ −15 ◦C until thawed for antibody analyses.

A validated ELISA (developed by HypoPet AG) was performed to determine anti-Fel d 1 IgG in cat sera previously described [36]. ELISA titers are given as the reciprocals of the dilutions needed to achieve 50% of the optical density of the maximal signal measured at saturation (OD50). The geometric mean titers were calculated from the individual titers of the cats from each group.

## *2.10. Data and Statistical Analyses*

The analysis for the human part included data from 9 out of 10 participants and was performed according to the "per protocol" (PP) analysis. Analyses were conducted in this way because one participant had urticaria, accompanied by pruritus and wheal formation, on two occasions during the study. The participant was unable to distinguish urticaria symptoms from the symptoms of cat allergy and was thus excluded from analysis. The analyses of cat data included all 13 cats, whereas the correlation of antibody responses in cats with measurements of human allergic symptoms included data from 9 out of 10 participants with their respective study cats (*n* = 9).

Statistical analyses were performed with Excel, Graph Pad Prism and R. Data were summarized by descriptive statistics: mean, standard deviation, median, first and third quartile, minimum, and maximum. This study was classified as a pilot study because there was no pre-existing information available concerning the expected change in symptoms of allergic cat owners before and after immunization of their cats (the effect size). The changes in the symptoms of the main study were evaluated using an exact Wilcoxon matched-paired signed rank test. Hypotheses were tested at a two-sided significance level of α = 0.05. For comparison between baseline and course of study, the mean of all values was computed.
