Targeting eosinophils by active vaccination against interleukin-5 reduces basophil counts in horses with insect bite hypersensitivity in the 2nd year of vaccination.

Previously, virus-like particle (VLP)-based self-vaccinations targeting interleukin (IL)-5 or IL-31 have been suggested to treat equine insect bite hypersensitivity (IBH), a seasonal recurrent allergic dermatitis in horses. The IL-5-targeting equine vaccine significantly reduced blood eosinophil counts in horses, similar to human monoclonal antibodies targeting IL-5 or the IL-5 receptor alpha (IL-5Rα). Previous studies in humans have also reported an additional effect on reduction of basophil counts. The aim of the present study was to evaluate whether an equine anti-IL-5 vaccine affected blood basophil counts. Horses with IBH were followed in a 3-year trial consisting of a placebo administered in the 1st year, followed by vaccination using an equine (e)IL-5-VLP vaccine in the 2nd and 3rd years. There was a strong reduction in circulating eosinophil counts after vaccination against IL-5. Additionally, there were reduced basophil counts, but only in the 3rd year of the study, suggesting a bystander effect of the anti-IL-5 vaccine on basophil counts.

Molecular mechanisms and treatment modalities in equine Culicoides hypersensitivity.

Equine Culicoides hypersensitivity (CH) is the most common allergic condition in horses affecting the skin. This review focuses on immunopathology and molecular mechanisms of equine CH. The role of eosinophils is emphasized, as well as disease severity and the influence of long-term chronic allergen exposure on T helper (Th) 2 cells. Using current knowledge from human allergic disorders, similar effects are hypothesized in equine patients. Key aspects of CH diagnosis and treatment are discussed, focusing on allergen specific immunotherapy and allergen-independent approaches, such as targeting hypereosinophilia through interleukin-5 and allergic non-histaminic pruritus though interleukin-31.

Safety Profile of a Virus-Like Particle-Based Vaccine Targeting Self-Protein Interleukin-5 in Horses.

：Background: Insect bite hypersensitivity (IBH) is an eosinophilic allergic dermatitis of horses caused by type I/IVb reactions against mainly Culicoides bites. The vaccination of IBH-affected horses with equine IL-5 coupled to the Cucumber mosaic virus-like particle (eIL-5-CuMVTT) induces IL-5-specific auto-antibodies, resulting in a significant reduction in eosinophil levels in blood and clinical signs. Objective: the preclinical and clinical safety of the eIL-5-CuMVTT vaccine. Methods: The B cell responses were assessed by longitudinal measurement of IL-5- and CuMVTT-specific IgG in the serum and plasma of vaccinated and unvaccinated horses. Further, peripheral blood mononuclear cells (PBMCs) from the same horses were re-stimulated in vitro for the proliferation and IFN-γ production of specific T cells. In addition, we evaluated longitudinal kidney and liver parameters and the general blood status. An endogenous protein challenge was performed in murine IL-5-vaccinated mice. Results: The vaccine was well tolerated as assessed by serum and cellular biomarkers and also induced reversible and neutralizing antibody titers in horses and mice. Endogenous IL-5 stimulation was unable to re-induce anti-IL-5 production. The CD4+ T cells of vaccinated horses produced significantly more IFN-γ and showed a stronger proliferation following stimulation with CuMVTT as compared to the unvaccinated controls. Re-stimulation using E. coli-derived proteins induced low levels of IFNγ+CD4+ cells in vaccinated horses; however, no IFN-γ and proliferation were induced following the HEK-eIL-5 re-stimulation. Conclusions: Vaccination using eIL-5-CuMVTT induces a strong B-cell as well as CuMVTT-specific T cell response without the induction of IL-5-specific T cell responses. Hence, B-cell unresponsiveness against self-IL-5 can be bypassed by inducing CuMVTT carrier-specific T cells, making the vaccine a safe therapeutic option for IBH-affected horses.

Interleukin 31 in insect bite hypersensitivity-Alleviating clinical symptoms by active vaccination against itch.

BACKGROUND: Insect bite hypersensitivity (IBH) is the most common seasonal pruritic allergic dermatitis of horses occurring upon insect bites. In recent years, a major role for IL-31 in allergic pruritus of humans, monkeys, dogs, and mice was acknowledged. Here, we investigate the role of IL-31 in IBH of horses and developed a therapeutic vaccine against equine IL-31 (eIL-31). METHODS: IL-31 levels were quantified in allergen-stimulated peripheral blood mononuclear cells (PBMCs) and skin punch biopsies of IBH lesions and healthy skin from IBH-affected and healthy horses. The vaccine consisted of eIL-31 covalently coupled to a virus-like particle (VLP) derived from cucumber mosaic virus containing a tetanus toxoid universal T-cell epitope (CuMVTT). Eighteen IBH-affected horses were recruited and immunized with 300 µg of eIL-31-CuMVTT vaccine or placebo and IBH severity score was recorded. RESULTS: IL-31 was increased in PBMCs and exclusively detectable in skin lesions of IBH-affected horses. Vaccination against eIL-31 reduced delta clinical scores when compared to previous untreated IBH season of the same horses and to placebo-treated horses in the same year. The vaccine was well tolerated without safety concerns throughout the study. CONCLUSION: TH2-derived IL-31 is involved in IBH pathology and accordingly the immunotherapeutic vaccination approach targeting IL-31 alleviated clinical scores in affected horses.

Active vaccination against interleukin-5 as long-term treatment for insect-bite hypersensitivity in horses.

BACKGROUND: Insect-bite hypersensitivity (IBH) in horses is a chronic allergic dermatitis caused by insect bites. Horses suffer from pruritic skin lesions, caused by type-I/type-IV allergic reactions accompanied by prominent eosinophil infiltration into the skin. Interleukin-5 (IL-5) is the key cytokine for eosinophils and we have previously shown that targeting IL-5 by vaccination reduces disease symptoms in horses. OBJECTIVE: Here, we analyzed the potential for long-term therapy by assessing a second follow-up year of the previously published study. METHODS: The vaccine consisted of equine IL-5 (eIL-5) covalently linked to a cucumber mosaic virus-like particle (VLP) containing a universal T cell epitope (CuMVTT ) using a semi-crossover design to follow vaccinated horses during a second treatment season. Thirty Icelandic horses were immunized with 300 µg of eIL-5-CuMVTT without adjuvant. RESULTS: The vaccine was well tolerated and did not reveal any safety concerns throughout the study. Upon vaccination, all horses developed reversible anti-eIL-5 auto-antibody titers. The mean course of eosinophil levels was reduced compared to placebo treatment leading to significant reduction of clinical lesion scores. Horses in their second vaccination year showed a more pronounced improvement of disease symptoms when compared to first treatment year, most likely due to more stable antibody titers induced by a single booster injection. Hence, responses could be maintained over two seasons and the horses remained protected against disease symptoms. CONCLUSION: Yearly vaccination against IL-5 may be a long-term solution for the treatment of IBH and other eosinophil-mediated diseases in horses and other species including humans.

Treating insect-bite hypersensitivity in horses with active vaccination against IL-5.

BACKGROUND: Insect-bite hypersensitivity is the most common allergic dermatitis in horses. Excoriated skin lesions are typical symptoms of this seasonal and refractory chronic disease. On a cellular level, the skin lesions are characterized by massive eosinophil infiltration caused by an underlying allergic response. OBJECTIVE: To target these cells and treat disease, we developed a therapeutic vaccine against equine IL-5 (eIL-5), the master regulator of eosinophils. METHODS: The vaccine consisted of eIL-5 covalently linked to a virus-like particle derived from cucumber mosaic virus containing the tetanus toxoid universal T-cell epitope tt830-843 (CMVTT). Thirty-four Icelandic horses were recruited and immunized with 400 µg of eIL-5-CMVTT formulated in PBS without adjuvant (19 horses) or PBS alone (15 horses). RESULTS: The vaccine was well tolerated and did not reveal any safety concerns but was able to induce anti-eIL-5 autoantibody titers in 17 of 19 horses. This resulted in a statistically significant reduction in clinical lesion scores when compared with previous season levels, as well as levels in placebo-treated horses. Protection required a minimal threshold of anti-eIL-5 antibodies. Clinical improvement by disease scoring showed that 47% and 21% of vaccinated horses reached 50% and 75% improvement, respectively. In the placebo group no horse reached 75% improvement, and only 13% reached 50% improvement. CONCLUSION: Our therapeutic vaccine inducing autoantibodies against self IL-5 brings biologics to horses, is the first successful immunotherapeutic approach targeting a chronic disease in horses, and might facilitate development of a similar vaccine against IL-5 in human subjects.

Serum antibodies and DNA indicate a high prevalence of equine papillomavirus 2 (EcPV2) among horses in Switzerland.

BACKGROUND: The DNA of equine papillomavirus type 2 (EcPV2) is consistently found in equine papillomas and squamous cell carcinomas, indicating a causal association of EcPV2 in the pathogenesis of these tumours; however, little is known about the prevalence of this virus. HYPOTHESIS/OBJECTIVES: The aim of this study was to determine the geno- and seroprevalence of EcPV2 in clinically healthy horses in Switzerland. ANIMALS: Fifty horses presented to the equine department of the university clinic, displaying no skin or mucous membrane lesions or severe signs of other diseases, were sampled. METHODS: Cytobrush samples from the penis or vulva and serum samples were collected. To determine the genoprevalence of EcPV2, DNA was extracted from cytobrush samples and tested for viral DNA with a PCR assay amplifying a 338 bp fragment of the E7/E1 region of the viral genome. Seroprevalence was tested using an enzyme-linked immunosorbent assay aimed to detect antibodies against the major capsid protein (L1) of EcPV2. RESULTS: In five of 50 horses (10%), EcPV2-specific DNA was amplified but no antibodies could be detected, whereas in 14 of 50 horses (28%), antibodies against EcPV2 but no DNA were demonstrated. Both antibodies and viral DNA were detected in four of 50 horses (8%). Neither antibodies nor viral DNA were found in 27 of 50 horses (54%). CONCLUSIONS AND CLINICAL IMPORTANCE: The seroprevalence suggests that EcPV2 is prevalent in the Swiss equine population, while the genoprevalence indicates that currently ongoing infections are less common. The discrepancy between geno- and seroprevalence probably indicates different stages of infection in the tested cohort.

Ocular manifestations of a metastatic adenocarcinoma in a horse.

A 10-year-old German Warmblood gelding was referred to the Equine Department of the Vetsuisse Faculty, University of Zurich, Switzerland, for an iris mass OD, lethargy, intermittent fever, and coughing. Ophthalmic examination revealed a 7 × 9 mm raised, fleshy, whitish to pinkish, vascularized iris mass at the 2 o`clock position OD. Fundic examination showed multifocal round, brown to black, slightly raised lesions with indistinct margins and a surrounding hyperreflective zone OU. Physical examination revealed a temperature of 39.2 °C, sinus tachycardia, preputial and ventral edema, and an enlarged right mandibular lymph node. Results of a complete blood count and plasma biochemical profile showed mild anemia, leukocytosis, and thrombocytopenia. Severe splenopathy, moderate splenomegaly, and severe pulmonary pathology with nodules and large areas of consolidated lung parenchyma were observed on abdominal ultrasound and thoracic radiographs, respectively. Fine needle aspirates of the enlarged mandibular lymph node showed malignant epithelial neoplastic cells. The horse was euthanized because of the poor prognosis and subsequently underwent postmortem examination. Macroscopic necropsy and histopathology revealed an adenocarcinoma of suspected pulmonary origin with involvement of eyes, heart, liver, kidneys, spleen, diaphragm, skeletal muscles, mandibular, pulmonary, and internal iliac lymph nodes. Metastatic adenocarcinoma should be considered as a differential diagnosis in horses with iris masses, multifocal chorioretinal infiltrates, and clinical signs that conform to a paraneoplastic syndrome.

Relationship of heart rate and electrocardiographic time intervals to body mass in horses and ponies.

OBJECTIVES: To investigate the relationship of heart rate (HR) and ECG time intervals to body weight (BWT) in healthy horses and ponies. We hypothesized that HR and ECG time intervals are related to BWT. ANIMALS: 250 healthy horses of >30 breeds; 5.5 (1-30) y [median (range)]; 479 (46-1018) kg. METHODS: Prospective study. Standard base-apex ECGs were recorded while the horses were standing quietly in a box stall. Mean HR over 15 s was calculated and RR interval, PQ interval, QRS duration, and QT interval were measured by a single observer.QT was corrected for differences in heart rate using Fridericia's formula (QT(cf) = QT/(3)√RR). The relationship between ECG variables and BWT, age, sex, and RR interval was assessed using multivariate backward stepwise regression analyses. Goodness of fit of the model was improved when using log(BWT) compared to BWT. Body weight was overall the strongest predictor of HR and ECG time intervals. Therefore, only log(BWT) was included as an independent variable in the final model. The level of significance was p = 0.05. RESULTS: HR (R(2) = 0.21) showed a significant negative relationship and PQ (R(2) = 0.53), QRS (R(2) = 0.23), QT (R(2) = 0.14), and QT(cf) (R(2) = 0.02) showed significant positive relationships to log(BWT). CONCLUSIONS: Small equine breeds undergoing routine ECG recordings have slightly faster heart rates and shorter ECG time intervals compared to larger equine breeds. Although the magnitude of absolute differences may be small, body weight needs to be considered among other factors when comparing HR and ECG time intervals to normal ranges in horses.