*3.6. Induction of Anti-Fel d 1 IgG Antibody Responses in Cats upon Vaccination with Fel-CuMV*

Subcutaneous injection of Fel-CuMV vaccine was considered to be well-tolerated and safe, as only mild and transient side reactions were noted. Cat sera were assessed for Fel d 1-specific IgG antibody responses throughout the course of the study (Figure 5). Participant #8 had three cats, identified as cats 8.1, 8.2, and 8.3. There are no serological data available for cat 8.3 because it rejected the sampling procedure. There was a significant increase in anti-Fel d 1 IgG in all vaccinated cats (*p* < 0.001) detectable after two immunizations at week 10. By the end of the study at week 27, the anti-Fel d 1-specific antibody response in sera (*p* = 0.001) was still significantly increased compared to baseline.

The antibody responses of the study cats were compared with the OSSS and petting time of the provocation test and the GWSS of the participants at the corresponding timepoints (Figure 5C–E). The parameters for assessing the allergic symptoms of the participants correlated well with the antibody

responses in cats. Petting time showed a direct correlation with the anti-Fel d 1 IgG titers, whereas the OSSS and the GWSS measurements were inversely correlated with the anti-Fel d 1 IgG titers.

**Figure 5.** Antibody responses in cat sera. (**A**) Anti-Fel d 1 IgG antibody titers in cats before and after immunization, *n* = 12. (**B**) Mean anti-Fel d 1 IgG antibody titers with SEM, *n* = 12. (**C**) Anti-Fel d 1 IgG antibody titers in cats at baseline (before immunization), week 10 and 27 vs. OSSS at corresponding timepoints baseline (before immunization), week 8 and 24. (**D**) Anti-Fel d 1 IgG antibody titers in cats at baseline (before immunization), week 10 and 27 vs. petting time at corresponding timepoints baseline (before immunization), week 8 and 24. (**E**) Anti-Fel d 1 IgG antibody titers in cats at baseline (before immunization), week 10 and 27 vs. GWSS at corresponding timepoints baseline (before immunization), week 8 and 25. (C-E) Data included from participants (*n* = 9) with corresponding study cats (*n* = 9). Statistical significances were obtained by an exact Wilcoxon matched-paired signed rank test.

#### *3.7. Data of the Extension Study*

After completion of the main study, an extension study was conducted with seven of the original participants (Figure 6A). Reasons for the discontinuation of three participants were the exclusion of the urticaria patient, the cat of one participant died due to a study-unrelated icterus, and personal preferences of one participant.

The analysis presented in Figure 6 includes the data obtained from the seven participants and their respective cats (seven study cats plus two cats; one participant had three cats), who participated in both studies.

As described above, there was a reduction in the OSSS from week 8 to 24 (mean OSSS ranging from 4.3 to 5.6) compared to baseline (mean OSSS 12.3). The mean OSSS increased to 9.3 in the intervening period between both studies (i.e., from week 24 to 54). After the boost immunization in week 56, there was a small decrease in mean OSSS observed in study weeks 58 to 98 ranging from 6.0 to 7.4. Although the reduction in the OSSS over both study periods compared to baseline was not statistically significant, a trend of reduced OSSS was observed over the entire study period of almost 2 years (94 weeks) after vaccination of the cats.

**Figure 6.** Extension study: Changes in allergic symptoms of participants after booster injection of cats. (**A**) Study design of the main and extension study including seven participants and nine cats. In addition to the main study, the participants performed a provocation test before the boost (week 56) followed by four additional provocation tests in study weeks 58, 62, 78, and 98. Participants also recorded their general organ specific symptoms (GSS) without provocation at monthly intervals during the extension study. The cats received a boost immunization of 100 μg Fel-CuMV subcutaneously in week 56. In addition to the collected serum samples of the main study, cat sera were also collected in study weeks 56, 62, and 78. (**B**) Mean OSSS with SEM over the course of the main and extension study of seven participants. (**C**) Mean petting time over the course of the main and extension study of seven participants. (**D**) Mean general symptoms (G(W)SS) over the course of the main (assessed weekly) and extension (assessed monthly) study of seven participants. (**E**) Measurement of anti-Fel d 1 IgG in cat sera of nine cats participating in the main and extension study. (**F**) Mean anti-Fel d 1 IgG antibody titers with SEM in cat sera. (**G**) Mean anti-Fel d 1 IgG antibody titers in cats vs. mean OSSS over the course of the main (baseline (before immunization), week 8/10 and 24/27) and extension study (week 54/56 (before boost), week 62 and 78). (**H**) Mean anti-Fel d 1 IgG antibody titers in cats vs. mean petting time

over the course of the main (baseline (before immunization), week 8/10 and 24/27) and extension study (week 54/56 (before boost), week 62 and 78). (**I**) Mean anti-Fel d 1 IgG antibody titers in cats vs. mean G(W)SS over the course of the main (baseline (before immunization), week 8/10 and 24/27) and extension study (week 54/56 (before boost), week 62 and 78). (**G**-**I**) Data included from participants (*n* = 7) with corresponding study cats (*n* = 7). Statistical significances were obtained by an exact Wilcoxon matched-paired signed rank test.

The GSS followed a consistent trend of lower symptoms from week 5 to 25 after the initial cat immunizations in weeks 4, 7 and 10 (Figure 6D). The mean GSS at baseline (weeks 1–3) of 7.5 improved to a mean score of 6.8, which was already detectable in week 5, and further improved to 2.3 in study week 14. By the end of the main study in week 25, the mean GSS of 4 was still lower compared to baseline (i.e., GSS 7.5). Before cats received the boost injection in week 56, the mean GSS increased to 6.5 but was still lower compared to baseline (i.e., GSS 7.5 of weeks 1–3). Upon booster injection, allergic symptoms improved again, reaching the lowest mean GSS of 3 in week 70. Clinical improvement was still evident by the end of the study in week 98 (i.e., GSS 4.8). Notably, the participants had reduced GSS at any timepoint after the first vaccination of their cats in study week 4, as the mean GSS over both study periods was always lower compared to baseline (weeks 1–3).

Cat sera were assayed for Fel d 1-specific IgG antibodies throughout both study periods (Figure 6E,F). The mean Fel d 1-specific IgG titers increased ~300-fold measured after two injections in week 10 and were still on a high level at the end of the main study at week 27 (Figure 6F). Specific antibodies declined between weeks 27 and 56 (before the boost) compared to the peak response detected in week 10. Following administration of a booster injection in week 56, the specific IgG levels increased again in eight of nine cats when measured by week 62, 6 weeks after the boost. Almost 4 months later, by the end of the study in week 78, the anti-Fel d 1 IgG responses were still significantly higher compared to baseline and were slightly above the antibody level measured before boost (week 56).

The antibody responses were inversely related to the OSSS and G(W)SS over the period of the main and extension studies (Figure 6G,I). In contrast, antibody responses directly correlated to the petting time of the provocation test (Figure 6H), indicating that specific antibodies reduced symptoms and increased petting time. Specifically, after the induction of anti-Fel d 1 antibodies detected in week 10, the OSSS decreased from a mean score of 12.5 at baseline to 5.1 and 5.4, measured in week 8 and 24, respectively. During the intervening phase of the main and extension study, the antibody titers declined and the mean OSSS increased to 9.3, detected in week 54. After the boost injection which caused an increase in specific antibodies, the mean OSSS decreased to a mean score of 6.0 and 6.6 in study weeks 62 and 78, respectively. Similar observations were made for G(W)SS.

The antibody titers also showed a correlation with petting time. Relative to baseline (week 1–3), mean petting time and anti-Fel d 1 IgG, determined in week 8 and 10, increased the mean petting time by a factor of 2, demonstrating that owners were found to be able to interact with their cats twice as long before they developed an allergic symptom strength of 5 according to VAS. The subsequent decrease in specific antibodies was associated with a decrease in petting time observed in weeks 24 and 54, although still above baseline. Upon the increase in specific antibodies after the booster injection, petting time increased again by a factor of 2.3 compared to baseline.
