On the role of allergen-specific IgG subclasses for blocking human basophil activation.

Successful treatment of IgE mediated allergies by allergen-specific immunotherapy (AIT) usually correlates with the induction of allergen-specific IgG4. However, it is not clear whether IgG4 prevents the allergic reaction more efficiently than other IgG subclasses. Here we aimed to compare allergen-specific monoclonal IgG1 and IgG4 antibodies in their capacity to inhibit type I allergic reactions by engaging FcγRIIb. We found that IgG1, which is the dominant subclass induced by viruses, binds with a similar affinity to the FcγRIIb as IgG4 and is comparable at blocking human basophil activation from allergic patients; both by neutralizing the allergen as well as engaging the inhibitory receptor FcγRIIb. Hence, the IgG subclass plays a limited role for the protective efficacy of AIT even if IgG4 is considered the best correlate of protection, most likely simply because it is the dominant subclass induced by classical AITs.

Glycan-specific IgG anti-IgE autoantibodies are protective against allergic anaphylaxis in a murine model.

BACKGROUND: IgE causes anaphylaxis in type I hypersensitivity diseases by activating degranulation of effector cells such as mast cells and basophils. The mechanisms that control IgE activity and prevent anaphylaxis under normal conditions are still enigmatic. OBJECTIVE: We aimed to unravel how anti-IgE autoantibodies are induced and we aimed to understand their role in regulating serum IgE level and allergic anaphylaxis. METHODS: We immunized mice with different forms of IgE and tested anti-IgE autoantibody responses and their specificities. We then analyzed the effect of those antibodies on serum kinetics and their in vitro and in vivo impact on anaphylaxis. Finally, we investigated anti-IgE autoantibodies in human sera. RESULTS: Immunization of mice with IgE-immune complexes induced glycan-specific anti-IgE autoantibodies. The anti-IgE autoantibodies prevented effector cell sensitization, reduced total IgE serum levels, protected mice from passive and active IgE sensitization, and resulted in cross-protection against different allergens. Furthermore, glycan-specific anti-IgE autoantibodies were present in sera from subjects with allergy and subjects without allergy. CONCLUSION: In conclusion, this study provided the first evidence that in the murine model, the serum level and anaphylactic activity of IgE may be downregulated by glycan-specific IgG anti-IgE autoantibodies.

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.

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

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.

CD23 provides a noninflammatory pathway for IgE-allergen complexes.

BACKGROUND: Type I hypersensitivity is mediated by allergen-specific IgE, which sensitizes the high-affinity IgE receptor FcεRI on mast cells and basophils and drives allergic inflammation upon secondary allergen contact. CD23/FcεRII, the low-affinity receptor for IgE, is constitutively expressed on B cells and has been shown to regulate immune responses. Simultaneous binding of IgE to FcεRI and CD23 is blocked by reciprocal allosteric inhibition, suggesting that the 2 receptors exert distinct roles in IgE handling. OBJECTIVE: We aimed to study how free IgE versus precomplexed IgE-allergen immune complexes (IgE-ICs) target the 2 IgE receptors FcεRI and CD23, and we investigated the functional implications of the 2 pathways. METHODS: We performed binding and activation assays with human cells in vitro and IgE pharmacokinetics and anaphylaxis experiments in vivo. RESULTS: We demonstrate that FcεRI preferentially binds free IgE and CD23 preferentially binds IgE-ICs. We further show that those different binding properties directly translate to distinct biological functions: free IgE initiated allergic inflammation through FcεRI on allergic effector cells, while IgE-ICs were noninflammatory because of reduced FcεRI binding and enhanced CD23-dependent serum clearance. CONCLUSION: We propose that IgE-ICs are noninflammatory through reduced engagement by FcεRI but increased targeting of the CD23 pathway.

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.

Virus-Specific Secondary Plasma Cells Produce Elevated Levels of High-Avidity Antibodies but Are Functionally Short Lived.

Most vaccines aim at inducing durable antibody responses and are designed to elicit strong B cell activation and plasma cell (PC) formation. Here we report characteristics of a recently described secondary PC population that rapidly originates from memory B cells (MBCs) upon challenge with virus-like particles (VLPs). Upon secondary antigen challenge, all VLP-specific MBCs proliferated and terminally differentiated to secondary PCs or died, as they could not undergo multiple rounds of re-stimulation. Secondary PCs lived in bone marrow and secondary lymphoid organs and exhibited increased production of antibodies with much higher avidity compared to primary PCs, supplying a swift wave of high avidity antibodies early after antigen recall. Unexpectedly, however, secondary PCs were functionally short-lived and most of them could not be retrieved in lymphoid organs and ceased to produce antibodies. Nevertheless, secondary PCs are an early source of high avidity antibodies and induction of long-lived MBCs with the capacity to rapidly differentiate to secondary PCs may therefore be an underestimated possibility to induce durable protection by vaccination.

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.

RNA and Toll-Like Receptor 7 License the Generation of Superior Secondary Plasma Cells at Multiple Levels in a B Cell Intrinsic Fashion.

Secondary plasma cells (PCs) originate from memory B cells and produce increased levels of antibodies with higher affinity compared to PCs generated during primary responses. Here we demonstrate that virus-like particles (VLPs) only induce secondary PCs in the presence of toll-like receptor (TLR) 7 and if they are loaded with RNA. Furthermore, adoptive transfer experiments demonstrate that RNA and TLR7 signaling are required for secondary PC generation, both at the level of memory B cell as well as PC differentiation. TLR7-signaling occurred in a B cell intrinsic manner as TLR7-deficient B cells in an otherwise TLR7-competent environment failed to differentiate into secondary PCs. Therefore, RNA inside VLPs is essential for the generation of memory B cells, which are competent to differentiate to secondary PCs and for the differentiation of secondary PCs themselves. While we have not tested all other TLR or non-TLR adjuvants with our VLPs, these data have obvious implications for vaccine design, as RNA packaged into VLPs is a simple way to enhance induction of memory B cells capable of generating secondary PCs.

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.