m-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides.

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

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.

Comparison of three clinical scoring systems for Culicoides hypersensitivity in a herd of Icelandic horses.

BACKGROUND: Culicoides hypersensitivity (CH), an intensely pruritic and seasonal allergic dermatitis, is a common allergic disease affecting horses worldwide. Currently, there is no validated clinical scoring system for the quantification of clinical signs associated with CH. OBJECTIVES: To (i) determine the best cut-off point of three scoring systems, (ii) test the accuracy of each system when compared to the clinical diagnosis of an experienced veterinarian and (iii) assess agreement between systems. ANIMALS: Icelandic horses (n = 20); eight with CH and 12 unaffected, from a research herd receiving no treatments for allergic dermatitis. METHODS AND MATERIALS: Lesion scores were recorded biweekly from April until September with three clinical scoring systems (A, B and C) by a single observer initially blinded to CH status. Separate logistic regression analyses for each time point were used to determine appropriate cut-offs for CH classification. Spearman's rho and Cohen's kappa were calculated to analyze correlation of scores and agreement of CH categorization between systems, respectively. RESULTS: The best allergic cut-off scores for system A, B and C were determined to be three, eight and 12, respectively. For each system median areas under the curve (>0.85) were excellent and discriminatory ability for correctly classifying CH status was strong. Excellent correlation between scores for each system (Spearman's rho > 0.96) and excellent intersystem agreement for CH categorization (kappa ≥ 0.73) were found across scoring time points. CONCLUSION AND CLINICAL IMPORTANCE: Results support the use of these scoring systems as templates for the future standardization of a CH clinical scoring system.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects.

BACKGROUND: Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. OBJECTIVE: We developed a new strategy to treat Fel d 1-induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. METHODS: A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin-derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. A first tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. RESULTS: The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti-Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both in vitro and in vivo. A reduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. CONCLUSION: Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

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.

The Prospects of an Active Vaccine Against Asthma Targeting IL-5.

Allergen-specific T helper type 2 (Th2) responses followed by eosinophilic inflammation of the lung are important causes of allergic asthma. Interleukin-5 (IL-5) is a master regulator of eosinophil differentiation as well as activation. Blocking IL-5 using monoclonal antibodies (mAbs) against IL-5 is a powerful way to improve asthmatic symptoms in patients with an eosinophilic component of the disease. We have previously shown that vaccination against IL-5 can abrogate eosinophilic inflammation of the lung in allergic mice. More recently, we have demonstrated that eosinophil-mediated skin disease in horses with insect bite hypersensitivity can be strongly reduced by vaccination against IL-5. Here we would like to propose the development of a similar vaccine for the treatment of asthma in 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.