**4. Discussion**

Allergy is the most common chronic disease in Europe and U.S and often manifests with chronic conditions including rhinitis, conjunctivitis and asthma, which is associated with individual morbidity and high socio-economic costs. In fact, more than 150 million Europeans are affected by chronic allergic diseases (EAACI, 2016). The danger of developing chronic asthma is particularly high, especially in allergies, which are caused by air-borne allergens such as hay fever and cat allergy. One in five patients with allergies lives with the constant fear of getting an asthmatic or anaphylactic shock

at any time or even dying as a result of a severe allergic reaction upon allergen encounter. The World Health Organization estimates that approximately 300 million individuals currently suffer from asthma worldwide and expect an increasing incidence to 400 million by 2025 [21,40]. This development is worrisome, threatening health and economies alike, and demands action of the global community for the development of new therapies, medications and diagnostic tools to address this major challenge [7,41]. Cat allergy contributes to a significant proportion of the allergic disease burden.

There are several new therapeutic developments to treat cat allergy that are currently pursued by various research groups and clinicians. Our novel approach of vaccinating the cat against Fel d 1, offers a cost-effective therapy without the risk of inducing severe side effects in cat-allergic patients, as they often occur during AITs, and without adversely affecting the cat. The vaccine targets Fel d 1, the major cat allergen for humans. By immunizing cats, the reactive allergen level can be lowered, thereby alleviating the symptoms of cat allergic patients. Here, we present clinical data of a first combined human and animal field trial conducted as an open-label, exploratory methodological study. The aim of the study was to assess different methods of measuring the allergic symptoms and determine if those methods could detect changes in allergic symptoms after vaccination of cats with Fel-CuMV (HypoCatTM).

The collection of clinical research data must be reproducible, valid and traceable using standardized procedures [42,43]. However, the selection and combination of various tests and scoring systems for monitoring allergic symptoms and their changes usually have a significant impact on the outcome of clinical trials and the development of innovative medications. Therefore, it is advisable to select as few questionnaires, tests and scoring systems as possible to capture the changes that best reflect the effect of the treatment [44,45]. To this end, we developed a new method to record allergic symptoms by a self-assessed, home-based symptom score, the provocation test. The validity of the test was evaluated in a previously conducted human clinical trial involving non-immunized cats (NCT02399579). The test consists of two elements: an assessment of organ-specific symptom score (OSSS) after petting—a standardized score—and measurement of the time of petting the cat until a defined level of allergy symptom was reached (level of 5 on a VAS). Both parameters, the OSSS and the time, are analyzed separately. In addition to the provocation test, another self-assessed, home-based standard method was investigated in this study: a general symptom score (G(W)SS) assessing the allergic symptoms without provocation.

The mean OSSS as part of the provocation test at week 24 was reduced in seven of nine participants compared to baseline. Moreover, the change in the OSSS was apparent throughout the course of the main study (weeks 8, 12, 16, 20 and 24 vs. baseline) and showed a variable yet sustained reduction in seven of nine participants. It should be noted that a reduction in the OSSS was not necessarily expected to be large, since the cat owners were required to record their symptoms after petting the cat until they had reached a symptom strength of 5 on the VAS. Hence, upon cessation of the provocation test (i.e., petting), the OSSS would have been similar on each occasion. A reason why a substantial decrease in OSSS in the main study was achieved may have been due to the fact that three participants, at week 24, were able to pet their cat for the maximum time of 45 min. In this circumstance, the VAS score of 5 was not reached, and thus the OSSS was lower. Regarding the extension study, several participants at several occasions could pet their cat to the maximum petting time of 45 min and did not reach the symptom strength of 5 on a VAS. Thus, the allergic symptoms were not as pronounced, resulting in lower OSSS.

The second parameter of the provocation test was the petting time. Relative to baseline, an increased time of petting was observed in eight of the nine participants at week 24 and over the entire period of the main study in seven of the nine. The increase in petting time was even more pronounced in the extension study. Several participants could pet their cat to the maximum time of 45 min on several occasions. The average petting time increased by a factor of two upon immunization of the cats and showed a sustained improvement over the period of the main and extension studies, demonstrating that the participants could interact longer with their cats.

A change in general allergic symptoms without provocation assessed by the G(W)SS was noted upon vaccination of the cats. Relative to baseline, a reduction in the GWSS at week 24 and over the period of the main study was observed in eight of nine participants. Moreover, the GSS measured throughout the course of the extension study showed a variable yet sustained reduction in seven of seven participants. Of note, the improvement in general allergic symptoms of the participants recorded without direct interaction with their cats at all timepoints after immunization of the cats over the period of the main and extension studies indicates that the participants generally felt better and suffered less from allergic symptoms.

Subcutaneous injection of three doses of Fel-CuMV vaccine followed by a single boost injection a year later was considered to be well tolerated in several breeds of privately owned adult cats. No serious adverse event occurred during the study. The clinical signs and reactions upon vaccination with Fel-CuMV were mild and reversible. No related effects on body weight, food consumption, behavior and appearance were observed. Moreover, the intended immunological response in cats immunized with Fel-CuMV, i.e., induction of anti-Fel d 1 IgG antibodies, was achieved. Anti-Fel d 1 antibodies were measured in sera at week 10, three weeks after the second immunization. Previous studies in cats (*n* > 60) with Fel-CuMV have shown that about 50% of immunized animals achieved peak titers 2–3 weeks after the second immunization [36]. This would correspond to week 10 of the current study. These findings support the observation of the improved symptoms assessed by the provocation test and G(W)SS of the participants from week 8 and 6, respectively onwards, as Fel d 1-specific antibodies were present and had the potential to neutralize Fel d 1 as previously shown [36]. The improvement of symptoms had lessened by the end of the main study, which may be related to the kinetics of the antibody response, which showed a decline from week 10 to week 27. Upon administration of a booster injection, the symptoms and petting time improved again.

However, the results and conclusions of this open label, exploratory methodology study with a small sample size (*n* = 9 in main and *n* = 7 in extension study) must be taken with caution. There was no pre-existing information available regarding the expected change in symptoms of the participants before and after immunization of their cats. As a consequence, no sample size calculation could be done beforehand. Nevertheless, it is interesting to note that several parameters measured in the study were suggestive that targeting the major cat allergen Fel d 1 by immunization of cats with Fel-CuMV had a positive impact on the allergic sensation of the participant. In particular, the time that participants were able to interact with their cats before a particular level of allergic symptoms was reached increased significantly.

Improvements of the organ specific symptoms with (OSSS) and without provocation (G(W)SS) over the course of the study were also noted. Notably, it was observed that the improvement in these measures had lessened at the end of the main study but increased again after the booster injection. This observation of the kinetics is valuable for planning and conducting further studies. Therefore, all measured parameters (i.e., provocation test and GSS) are suitable to assess changes in allergic symptoms of the cat owners.

Finally, from a veterinary perspective, targeting Fel d 1, the major cat allergen in humans, by active vaccination has, to date, been well-tolerated. The general health status of the cats was not compromised by the induction of auto-antibodies against Fel d 1. However, allergic symptoms of the cat allergic owners were alleviated. As a result of the owner being less burdened by their allergy, the quality of life of their cat may be improved. The ability of allergic cat owners to better tolerate and increase the duration of their interactions with their pet can benefit the animal through better training and socialization and awareness of the animals' overall health. Moreover, the likelihood of abandonment and subsequent euthanasia in animal shelters could be decreased.

**Author Contributions:** Conceptualization, F.T., G.T.J., S.H., G.S., T.M.K. and M.F.B.; methodology, F.T., S.H., G.T.J., G.S., T.M.K. and M.F.B.; software, F.T., S.H., N.G.; validation, F.T., S.H., G.T.J., T.M.K. and M.B.F.; formal analysis, F.T., S.H., A.E., N.G., G.T.J. and M.F.B.; investigation, G.S., T.M.K. and M.F.B.; resources, F.T., G.T.J., S.H., G.S., T.M.K., A.S., G.P., T.L., M.C.F., L.A.T., C.S.N., S.R., and M.F.B.; data curation, F.T., S.H., N.G., G.T.J. and M.F.B.; writing—original draft preparation, F.T., G.T.J., S.H., G.S., C.S.N., S.R., N.G., T.M.K. and M.F.B; writing—review and editing, A.S., G.P., T.L., M.C.F. and L.A.T.; visualization, F.T. and S.H.; supervision, N.G., G.T.J., T.M.K. and M.F.B.; project administration, F.T., G.T.J.; funding acquisition, M.C.F., L.A.T., G.S., T.M.K. and M.F.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work in this manuscript was funded by Benchmark Animal Health, UK and supported by the Swiss Commission for Technology and Innovation, (CTI project 16015.2 PFLS-LS to M.F.B., T.M.K. and G.S.).

**Acknowledgments:** We thank Antonia Gabriel-Fettelschoss, Monique Vogel and Pål Johansen for scientific discussion or careful reading of the manuscript.

**Conflicts of Interest:** Authors F.T., G.T.J., S.H., G.S., T.M.K. and M.F.B. have a financial relationship with HypoPet AG involving employment, stock ownership or payments for research activities. Authors A.S., G.P., T.L., M.C.F. and L.A.T. have a financial relationship with Benchmark Animal Health involving employment, or stock ownership. F.T., G.T.J., G.S., T.M.K., and M.F.B. own a patent for "Compositions against cat allergy". A.E., C.N., S.R., and N.G. have no conflict of interest.
