New Strategies for Prevention and Treatment of Insect Bite Hypersensitivity in Horses

Treatment of equine insect bite hypersensitivity (IBH) needs to be improved. Allergen-specific immunotherapy (ASIT), the only curative treatment of allergy, currently has only a limited efficacy for treatment of IBH. This review highlights the latest findings in prophylactic and therapeutic strategies. Prophylactic vaccination against IBH using recombinant Culicoides allergen has been developed in unexposed Icelandic horses and is ready to be tested. Therapeutic virus-like particle (VLP)–based vaccines targeting equine interleukin- (IL-) 5 or IL-31 improved clinical signs of IBH by induction of anti-cytokine antibodies thus reducing eosinophil counts or allergic pruritus, respectively. First studies for development of ASIT using pure r-Culicoides allergens have yielded promising results and need now to be tested in clinical studies for both prevention and treatment of IBH. Therapeutic vaccines inducing neutralizing antibodies against IL-5 or IL-31 will be valuable future treatments for reduction of clinical signs of IBH.


Introduction
Equine insect bite hypersensitivity (IBH), also known as sweet itch, Queensland itch, summer eczema, or Kasen is the most common allergic skin disease of horses [1]a n di s clinically manifested as a chronic relapsing seasonal dermatitis [reviewed in 2]. IBH is initially presented as pruritic dermatosis frequently affecting the mane and tail area followed by self-trauma leading to hair loss and excoriations, which contribute to development of secondary bacterial infections [2][3][4]. It is caused by bites of insects of the genus Culicoides, known as biting midges [5,6]. The prevalence correlates with geographical distribution of the midges [7], i.e. depends on environmental factors [8,9] as well as on genetic factors, such as breed, lineage or family [reviewed in 2]. IBH has been described worldwide, except in Iceland, and affects approximately 10% of horses of all breeds [7].
While feeding, the midges inject a pool of various salivary gland proteins leading to sensitization and allergy in predisposed horses [10,11]. Studies in humans have shown that during the sensitization process, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) are released from inflamed or injured epithelial cells, inducing type 2 innate lymphoid cells (ILC2) to produce IL-5 and IL-13 [12,13]. Under the influence of these cytokines and TSLP, antigen-presenting cells (APCs) direct the immune response towards Th2 with production of IL-4 and IL-13. Subsequently, B cells undergo class switch and produce allergen-specific IgE that binds to high affinity FcεRI receptors on basophils and mast cells, leading to sensitization (Fig. 1A) [14,15]. In sensitized, allergic individuals, re-exposure to the allergen causes cross-linking of IgE bound to high-affinity FcεRI receptors on mast cells and basophils and subsequent immediate release of effector cell mediators. Vasoactive amines, lipid mediators, granule enzymes, and cytokines cause the clinical signs of immediate hypersensitivity (Fig. 1B). Late-phase reaction occurs 2-4h after the exposure with a peak after 24 h. The release of inflammatory mediators by the activated mast cell causes infiltration of leukocytes, mostly Th2 cells and eosinophils at the site of the allergic reaction (Fig. 1B) [14,15].
Similarly to human allergy, various studies in horses have shown an imbalance between a Th2 and T regulatory (Treg)/ Th1 immune response in IBH [16][17][18][19], with a relative increase of the Th2 response. The involvement of IgE-mediated, type I hypersensitivity reactions to Culicoides allergens with activation of mast cells, followed by a late-phase reaction characterized by infiltration of lymphocytes and eosinophils is well established [reviewed in [7,20,21]. Few studies suggest a possible involvement of delayed-type hypersensitivity (DTH; type IV) in the pathogenesis of IBH [22]. Type IV hypersensitivities can be further divided into types IVa, IVb, IVc, and IVd based on predominant cell types. Type IVb of DTH in drug hypersensitivity, is strongly associated with IL-5 producing Th2 cells and eosinophilia [23]. Eosinophils are thus a common feature of both late-phase reaction of type I hypersensitivities and type IV hypersensitivities. Moreover, they seem to play a crucial role in IBH, as the allergic lesions are often characterized by infiltration with eosinophils [7]and eosinophilia is associated with severity of IBH [24••]. Furthermore, studies by Fettelschloss-Gabriel et al. indicate an important role for IL-5 in the pathogenesis of IBH [24••, 25••]. IL-5 is a cytokine that displays multiple effects on eosinophils and affects their differentiation, migration, activation and survival. Once activated by IL-5, eosinophils release granule enzymes and effector molecules such as leukotrienes and (1) Upon re-exposure to Culicoides allergens, (2) the allergens bind to the mast cell-bound allergen-specific IgE antibodies, causing cross-linking between the receptors. (3) Mast cells are thus activated and release inflammatory mediators, such as histamine, leukotrienes, prostaglandins, and PAF, (4) leading to development of edema, erythema, and itch, within minutes of allergen re-exposure. (5) Activated mast cells also release chemokines and cytokines responsible for the recruitment of effector cells to the allergy site. (6) These effector cells are mainly eosinophils, Th2 cells, and basophils, causing the latephase reaction, which starts 2-4 h after exposure and reaches its peak at 24 h. (7) Th2 cells produce cytokines, in particular IL-5 that further recruits eosinophils to the site of allergic inflammation major basic protein that in turn trigger degranulation of mast cells and basophils, thus forming a vicious circle of allergic inflammation.
Although IBH is the most common skin disease of horses, the only efficient treatment available is, beside reduction of exposure to Culicoides by stabling, use of blankets or repellents, symptomatic therapy using glucocorticoids [7,26]. Antihistamines seem to be of limited efficacy in the treatment of IBH [27]. In this review we will highlight the most recent developments in preventive options and treatments for the equine IBH.

Allergen-Specific Immunotherapy
Allergen-specific immunotherapy (ASIT) is the only allergy modifying treatment for type I hypersensitivities [28] and has been shown to reduce progression of the disease as well as to decrease the risk of development of new allergic conditions in atopic patients [29]. ASIT has been used for therapy of human allergies for over 100 years and was first applied by Noon et al. who treated hay fever patients with pollen extract [30]. The mechanism underlying ASIT is thought to be a shift of the immune response from Th2 towards a regulatory immune response. In response to ASIT, IgG antibodies are produced, in particular IgG4 that block the binding of allergen-specific IgE antibodies to the allergens. Additionally, ASIT is associated with decreased production of IL-4 and IL-5, the signature cytokines of Th2 CD4 + Tc e l l s [ 31], an increase of the allergen-specific Treg with the production of regulatory cytokines, IL-10 and TGF-β [32,33], as well the induction of allergen-specific Breg [34].
ASIT has proven to be relatively inexpensive, highly effective, and long-lasting when high-quality antigens are used [35]. Allergen extracts have been used with a good outcome. However, the success of therapy depends on the quality of the extracts used as reviewed in Zhernov et al. On top of that, standardization of allergen extracts is difficult. They can vary greatly in the amount, potency and immunogenicity of individual allergens and major allergens can be lacking [36]. As IgE-mediated type I allergies are a global health problem affecting around 30% of human population in industrialized countries, it is vital to further develop ASIT with focus on safety and clinical improvement using well-defined standardized allergens [36]. The only way forward is by means of molecular applications, and presently different strategies to produce recombinant allergens are in development [35,36] with the aim to reduce their allergenicity but maintain immunogenicity [37].
Another approach to control allergies is the use of preventive immunotherapy, which can be applied in high-risk individuals before sensitization occurs, or in individuals with increased allergen-specific IgE before development of clinical signs [38]. To date, there are only few studies on preventive immunotherapy. Campana et al. showed that vaccination of non-allergic adults with recombinant hypoallergen derivatives of the major birch pollen allergens, Bet v 1 in Alum decreased the risk of developing birch allergy [39•].

Allergen-Specific Immunotherapy in Equine IBH
To our knowledge, three attempts to treat IBH with ASIT have been published [40][41][42]. All three have used whole body extract (WBE) of Culicoides midges. These crude WBE consist of a mixture of hundreds of proteins and other substances, whereby the salivary gland proteins, i.e. the allergens for IBH, only represent a minute fraction of the extract. Furthermore, the extracts were not derived from the most abundant Culicoides species present in the environment of horses. This might be important as van der Meide et al. have shown that IBH-affected horses have higher IgE levels to midges caught in the environment of the horses as compared with laboratory-bred species [43].
In two placebo-controlled studies IBH-affected horses treated with Culicoides whole body extract (WBE) showed no significant improvement in clinical signs when compared with the placebo-control group [40,42]. However, in both groups, clinical signs after vaccination improved when compared with signs before vaccination, probably because of a better insect control [42]. Anderson et al. obtained a significant improvement of clinical signs in IBH-affected horses treated with Culicoides WBE; however, a control group was not included [41]. The low efficacy of ASIT with insect WBE in early studies in humans [44,45] as well as the controversial non-conclusive results of these studies show the necessity of using pure, well-defined allergens instead of crude WBE, ideally by first defining the sensitization profile of each horses. To achieve this goal, molecular approaches had to be applied for identification and production of Culicoides salivary allergens.

Culicoides Allergens in IBH
Over the last decade, extensive work has been put into identifying the causative allergens for IBH and producing them as pure recombinant proteins. Allergens from the three Culicoides species C. nubeculosus [46,47], C. sonorensis [48], and C. obsoletus [49,50] have been identified and produced. All of them have been expressed in Escherichia coli, some in insect cells and barley [51], and one in Pichia pastoris

Preventive and Curative ASIT
Having access to pure Culicoides allergens opens the possibility for development of both prophylactic and therapeutic ASIT. Ideally, preventive immunotherapy should be carried out prior to exposure to the allergens. Iceland is free of Culicoides that feed on horses, and therefore horses in Iceland do not suffer from IBH [55]. However, prevalence of IBH in Icelandic horses exported to Culicoides endemic area is high, as more than 50% of them get affected if not protected from the midges [56,57]. Since Icelandic horses are only sensitized after the export, they offer a unique opportunity for development and study of preventive immunotherapy.
For development of prophylactic vaccine, not only the source of allergens and type of adjuvants have to be considered, but also the injection route may be of a great importance. Studies by Senti et al. in human grass-pollen allergic patients indicate that intralymphatic application of the allergens is more efficient and does not have more side effects than the subcutaneous route, commonly used in ASIT. Compliance to ASIT is often low because of the long duration of treatment and the many injections needed. The most important advantage of the intralymphatic application compared with the conventional treatment was that only 3 injections versus 54 were required.
With the aim to develop a prophylactic ASIT against equine IBH (Fig. 2), application route and adjuvants had first to be evaluated. Two vaccination experiments using pure r-Culicoides allergens were carried out in healthy Icelandic horses not exposed to Culicoides. The aim was to determine which immunization protocol would lead to the induction of a Th1/Treg immune response with production of IgG-blocking antibodies, an important feature of successful ASIT. In the first study, horses were immunized 3 times at 4-week intervals, receiving 10 μg of each allergen/injection either intradermally or intralymphatically. This study showed that the adjuvant IC31®, a TLR-9 agonist, was essential to induce a strong antibody response. The induced antibodies, mainly IgG1 and IgG4/7, were partly able to inhibit binding of IgE to the allergens, i.e. blocking antibodies were induced. Importantly, no allergen-specific IgE were induced by this prophylactic vaccination, indicating no risk of sensitization. The intralymphatic route resulted in a slightly stronger antibody response than the intradermal application [58••] and was used in the second experiment. Unfortunately, IC31® was no more available for use in horses; thus, in the second study, Alum alone, an adjuvant often used in ASIT, was compared with a combination of Alum and monophosphoryl lipid A (MPLA, a TLR-4 agonist). Culicoides r-allergens mixed in Alum alone and Alum/MPLA induced similar levels of allergen-specific IgG1 and IgG4/7 antibodies with strong blocking capacity. However, PBMC from horses vaccinated with Alum/MPLA, but not those from horses vaccinated with Alum only, produced significantly more IFN-γ and IL-10 upon allergenspecific in vitro re-stimulation as compared with unvaccinated control horses [59••]. To summarize, intralymphatic injections with small amounts of recombinant allergens in Alum/MPLA resulted in a Th1/Treg immune response with induction of high specific IgG antibodies levels, displaying a strong blocking capacity. This indicates that such an immunization protocol would be suitable for prophylactic ASIT. A challenge experiment needs now to be performed to know whether this type of prophylactic vaccination can protect Icelandic horses against IBH after exposure to Culicoides.
As an alternative to subcutaneous immunotherapy (SCIT), sublingual immunotherapy (SLIT) has also been shown to be effective in human patients [29] with the advantage that it can be applied by the patients at home, reducing the number of visits to the doctor [29]. We are presently developing a version of SLIT for horses, where a porridge of transgenic barley expressing Culicoides allergens is brought in contact with the oral mucosa using a special bit [52••]. This system has the advantage that the barley grains are used directly, thus bypassing protein purification, which is cumbersome and expensive. In a pilot study, four horses were treated 6 times with this transgenic barley over a period of 20 weeks. After the treatment, a weak IgG1 and IgG4/7 antibody response was seen. Following one boost 8 months after the last treatment, a stronger allergen-specific IgG1 and IgG4/7 antibody response was achieved and the serum had some blocking activity. Allergen-specific antibodies could also be detected in the saliva. This pilot experiment suggests that this approach might be a useful option for ASIT in horses. Transgenic barley expressing various Culicoides allergens is now being produced with the aim to perform clinical trials in IBH-affected horses.
Therapeutic Vaccine Against IL-5 Eosinophils play a significant role in the pathogenesis of IBH, making them important therapeutic targets. Besides accumulating in IBH skin lesions, eosinophil counts were also found to be enhanced in peripheral blood. In fact, blood eosinophils were shown to correlate to IBH severity score [24••]. In humans affected with hyper-eosinophilic conditions, humanized monoclonal antibodies (mAb) targeting the IL-5 or IL-5 receptor α have proven to be safe and beneficial for the patients [60,61]. This approach, however, will probably not be available for equine patients due to high production costs. A new approach has thus been developed: In contrast to passive immunization approach with mAb, horses were actively immunized against the eosinophilic master-molecule IL-5. Linkage of equine IL-5 to VLPs of the cucumber mosaic virus (CuMV) generated polyclonal antibody responses against IL-5 in vaccinated horses [24••, 25••]. Since this approach is limited by the horse's own immune system, effective antibody titer induction is ensured by the VLP design. Besides their small size of 20-200 nm that enables free draining into the lymphatic systems and mediates binding of native protein to B cell receptors, the VLP was optimized for effective stimulation of the innate and the adaptive immune system. The surface of the VLPs is highly repetitive allowing B cell receptor (BCR) cross-linking inducing potent antibody responses in absence of T cells. This leads to complement fixation via complement receptor 2 and subsequent follicular dendritic cell activation driving T celldependent differentiation of germinal center B cells into class-switched affinity-matured long-lived plasma cells [62]. In addition, the VLP contains non-coding E. coli RNA activating innate immune receptors for pathogenassociated molecular patterns (PAMPs) such as toll-like receptor (TLR)-7 [63]. Finally, an integrated universal T cell epitope of tetanus toxin (CuMV TT ) suggests to boost the immune response towards the vaccine in all tetanusvaccinated individuals [64]. Thus, the eIL-5-CuMV TT vaccine has been shown to induce effective antibody titers against IL-5 that led to significantly reduced numbers of eosinophils in blood of IBH-affected horses when comparing to placebo treatment (Fig. 3)[25••]. The first treatment year with two initial injections induced only short-lived antibody titers, hence a mid-season booster injection was required. However, once immunity was established by three basic vaccination shots, a single annual booster injection prior to IBH season was sufficient to maintain antibody titer throughout the following IBH season. The vaccine-induced anti-IL-5 antibody titers were shown to be reversible, an important safety criterion [25••].
Moreover, studies in mouse models indicate that the endogenous protein was not able to boost antibody titers in absence of VLP-linkage, because the induced immune response is dependent on the VLP-specific T cell help [65]. This represents an important autoimmunity safety criterion. If confirmed in a clinical setting, this would represent an advantage of the active vaccination over the passive mAb approach, where anti-therapeutic antibodies can be induced, limiting their use in affected patients [66]. Furthermore, the active immunization strategy described here requires substantially less frequent injections and the administered dose is significantly lower in the vaccine compared with doses of therapeutic mAb. With regards to clinical benefit, two placebo-controlled double-blind randomized clinical trials with IBH-affected Icelandic horses showed that the eIL-5-CuMV TT vaccine significantly reduced IBH lesion scores in line with decreased eosinophil blood counts. The IBH lesion reduction was more pronounced in the second treatment year due to more stable antibody responses [24••, 25••].

Other Treatment Options for IBH
Anecdotal reports indicated that treatment of horses with a vaccine against dermatophytosis reduced clinical signs of IBH, possibly by redirecting the immune response from Th2 towards Th1. A placebo-controlled study could, however, not demonstrate a significant improvement of clinical signs after three injections of Insol®-Dermatophyton [70]. Interestingly, an increase in serum IFN λ ,T N F α and IL-10 could only be observed in the treated but not in the placebo group, indicating the induction of a Th1/Treg immune response [70]. Additionally, topical treatments can also alleviate clinical signs of IBH. A cream containing omega-3-fatty acids, humectants, and emollients has been shown to improve clinical lesions of IBH, but did not influence the pruritic score [71].
JAK kinase inhibitors, as used for treatment of atopic dermatitis in dogs [72], are not registered for treatment of horses. Nevertheless, anecdotal reports indicate that the JAK kinase inhibitor oclacitinib may have some effect for symptomatic treatment of IBH.

Conclusions
Biologicals are increasingly used for treatment of allergy, and the IL-5 and IL-31 therapeutic vaccines represent innovative advances for symptomatic treatment of IBH. Further studies are now needed to determine for which patient groups the respective therapeutic vaccines are most suited and whether their combination might further improve clinical response.
The availability of a large panel of pure recombinant Culicoides allergens relevant for IBH open new options for preventive and therapeutic ASIT. Suitable application routes and adjuvants have been studies in horses not exposed to Culicoides. A clinical trial for testing the efficacy of a preventive ASIT needs to be performed. Finally, therapeutic ASIT with the pure Culicoides allergens and the newly developed immunization protocols can now be tested in IBH patients, ideally by selecting the relevant Culicoides allergens for each IBH patients, whereby the recently developed microarray represents an efficient tool for selection of the required Culicoides allergens.  Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.