**Immunization of Cats against Fel d 1 Results in Reduced Allergic Symptoms of Owners**

**Franziska Thoms 1,2, Stefanie Haas 1,2, Aline Erhart 3, Claudia S. Nett 4, Silvia Rüfenacht 5, Nicole Graf 6, Arnis Strods 7, Gauravraj Patil 7, Thonur Leenadevi 7, Michael C. Fontaine 7, Lindsey A. Toon 7, Gary T. Jennings 1,2, Gabriela Senti 8, Thomas M. Kündig <sup>9</sup> and Martin F. Bachmann 10,11,\***


Received: 28 January 2020; Accepted: 26 February 2020; Published: 6 March 2020

**Abstract:** An innovative approach was tested to treat cat allergy in humans by vaccinating cats with Fel-CuMV (HypoCatTM), a vaccine against the major cat allergen Fel d 1 based on virus-like particles derived from cucumber mosaic virus (CuMV-VLPs). Upon vaccination, cats develop neutralizing antibodies against the allergen Fel d 1, which reduces the level of reactive allergen, thus lowering the symptoms or even preventing allergic reactions in humans. The combined methodological field study included ten cat-allergic participants who lived together with their cats (*n* = 13), that were immunized with Fel-CuMV. The aim was to determine methods for measuring a change in allergic symptoms. A home-based provocation test (petting time and organ specific symptom score (OSSS)) and a general weekly (or monthly) symptom score (G(W)SS) were used to assess changes in allergic symptoms. The petting time until a pre-defined level of allergic symptoms was reached increased already early after vaccination of the cats and was apparent over the course of the study. In addition, the OSSS after provocation and G(W)SS recorded a persistent reduction in symptoms over the study period and could serve for long-term assessment. Hence, the immunization of cats with HypoCatTM (Fel-CuMV) may have a positive impact on the cat allergy of the owner, and changes could be assessed by the provocation test as well as G(W)SS.

**Keywords:** cat allergy; vaccination; Fel d 1; HypoCat™; virus-like particle

#### **1. Introduction**

Cats are among the most popular and common pets worldwide and are a significant source of indoor allergens [1]. Hence, allergies to cats are widespread, with a prevalence of 10%–30% in the Western population [2]. A total number of 10 *Feline domesticus* (Fel d) allergens, that are recognized by human IgEs, have been identified [3–9]. Fel d 1, an uteroglobin-like protein, is considered to be the major cat allergen. In fact, 94% of patients allergic to cats have Fel d 1-specific IgE [10]. Fel d 1 belongs to the family of secretoglobins with homologies to uteroglobin. Its function is unknown but it has been postulated to play a potential role in skin protection and pelt conditioning or have an involvement in the transport of steroids, hormones and pheromones [11,12]. Fel d 1 is produced in sebaceous, salivary, lacrimal, and anal glands and is present in the saliva, tears, skin and fur [13–16]. It is shed from the cat to the environment through airborne dander and if inhaled by humans may result in sensitization and induction of cat allergy [17].

The immune response against innocuous cat allergens is characterized as type I and IV hypersensitivity, involving Th2 cells shaping the environment for production of IgE antibodies by B and plasma cells and recruitment of additional inflammatory cells [18–20]. Affected patients suffer from mild symptoms, e.g., sneezing, itchiness of skin and eyes, to severe symptoms ranging from conjunctivitis, rhinitis to asthma, which, upon direct exposure to cats, can lead to life-threatening conditions. There are several recommendations for dealing with cat allergy [21]. Allergic people are advised to avoid allergen exposure by removal of all potential allergen-containing or contaminated objects in the households, e.g., pillows, blankets, carpets, rugs. Environmental cleaning and the use of air humidifiers and HEPA filters can also contribute to the relief of symptoms. Another approach is to remove the cat. However, the bond between owners and their cats is often so strong that they are more likely to accept the risk to their health, which they may not even be fully aware of, than give up their pet [22].

Cat allergic subjects usually treat their allergic symptoms with antihistamines and corticosteroids. Another possibility is allergen specific immunotherapy (AIT), which is the only disease-modifying option, but carries the risk of inducing serious side effects and may take years. In fact, AIT can require 30–80 injections over a duration of three to five years with a low chance of success. New approaches explore different routes of administration (e.g., epicutaneous, sublingual, intralymphatic), different formulations of allergens with adjuvants (e.g., MPL, MCT), the introduction of mutations into the protein sequence which delete T cell or IgE epitopes, and finally the use of short peptides instead of full-length allergens [23–31]. The challenge for the development of new therapies is exemplified by the recent failure of a phase III clinical study testing a peptide-based vaccine to treat cat allergy [32]. Orengo et al. are developing a monoclonal IgG antibody therapy targeting Fel d 1 in humans that aims to increase the allergen-specific IgG/IgE ratio and relieve symptoms and showed good clinical impact [33].

An alternative approach to the problem of cat allergy, and one that does not involve separation of the cat from its owner, is to lower Fel d 1 on the animal itself. One recently described method is the addition of anti-Fel d 1 IgY harvested from chicken eggs to cat food. A reduction in active Fel d 1 in saliva and fur has been reported but whether this will result in clinically significant reductions in allergy still needs to be addressed [34,35].

Another approach to lowering allergenic Fel d 1 levels on the cat is active immunization with the aim of inducing anti-Fel d 1 antibodies in the animal itself. Towards this end, a feline vaccine targeting Fel d 1 in cats to treat cat allergy in humans is being developed [36]. The vaccine is based on a recombinantly expressed Fel d 1 protein covalently linked to a virus-like particle (VLP) derived from the Cucumber mosaic virus (CuMV) [37]. The VLP consists of the CuMV coat protein, without any viral genetic information, which serves as a carrier and induces, due to its particulate and repetitive structure, strong and sustained antibody responses, even against self molecules like the Fel d 1 protein in cats [38,39]. To date, vaccination with Fel-CuMV (HypoCatTM) has been tested in 70 cats and was well tolerated without short- or long-term (two years) side effects. Furthermore, vaccination induced strong neutralizing anti-Fel d 1 IgG responses lowering levels of reactive allergen in tear extracts of study cats tested with human basophils from cat allergic subjects [36].

In the current manuscript, we report the results of a first field trial with ten cat allergic participants living together with their cats, and the cats were vaccinated with Fel-CuMV. The aim of this exploratory methodology study was 1) to determine a suitable method for measuring a change in the allergic symptoms of the owner and 2) quantify changes in the interactions between the cat and the owner. These methods may then be used in a larger trials in the future. Three parameters were monitored over a duration of almost two years. A home-based provocation test determined the petting time, defined as the time during which the owner was able to interact with the cat until a certain level of symptoms using a visual analogue scale (VAS score of 5) was reached and their organ-specific symptoms score (OSSS) after petting their cats. In addition, a general weekly or monthly symptom score (GWSS and GSS, respectively) assessed overall changes in allergic symptoms in human subjects without interaction with the cat. The provocation test was assessed as a parameter of the acute allergic reaction, comparable to a hospital-based provocation test (e.g., nasal or conjunctival test), whereas the G(W)SS served as readout of chronic symptoms. As observed in previous studies [36], vaccination with Fel-CuMV was well-tolerated by cats and induced strong serum antibody responses. Changes in symptoms of cat owners and interaction times with their cats were observed after vaccination of the pets. Thus, both tests, the provocation test and the G(W)SS, are suitable to detect changes in the symptoms of cat allergic patients. In particular, the petting time may serve as an early efficacy read-out following vaccination of the cats, whereas the OSSS and G(W)SS seem to be more appropriate for long-term monitoring.
