A chimeric human-cat Fcgamma-Fel d1 fusion protein inhibits systemic, pulmonary, and cutaneous allergic reactivity to intratracheal challenge in mice sensitized to Fel d1, the major cat allergen

Co-aggregation of FcepsilonRI with FcgammaRIIb can block FcepsilonRI-mediated reactivity and Fc gamma:allergen chimeric proteins, by co-crosslinking FcgammaRIIb to allergen-specific IgE bound to the FcepsilonRI can block allergen-specific reactivity. We evaluated whether a human cat chimeric fusion protein (GFD) composed of part of the human Ig G1 Fc fused to the major cat allergen (Fel d1) would function as allergen immunotherapy while not inducing acute allergic reactivity in mice sensitized to Fel d1. Injection of GFD 6 h prior to Fel d1 challenge acutely blocked systemic and skin reactivity to Fel d1 challenge while mice given subcutaneous immunotherapy with GFD at days 37, 38, and 39 showed inhibition of systemic, lung, and cutaneous reactivity to Fel d1 2 weeks later. GFD immunotherapy did not induce systemic reactivity. Overall, the Fcgamma-Fel d1 chimeric fusion protein blocked Fel d1-induced IgE-mediated reactivity but did not induce in vivo mediator release on its own; suggesting that this approach using allergen combined with Fc gamma1 so as to achieve inhibitory signaling may provide an enhanced form of allergen immunotherapy.     

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy

Background:  Allergen-specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long-lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate-based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy.
Methods:  BALB/c mice were subcutaneously immunized with the recombinant (r) cat allergen Fel d 1 followed by intranasal challenge with cat dander extract (CDE). The sensitized mice were therapeutically treated with rFel d 1 covalently coupled to CBPs (CBP-rFel d 1). Airway hyper-reactivity (AHR), infiltration of leucocytes in bronchoalveolar lavage (BAL) fluid, allergen-specific serum immunoglobulin levels and in vitro splenocyte responses were evaluated.
Results:  Mice treated with CBP-rFel d 1 showed reduced features of allergic inflammation. They responded with (i) significantly decreased AHR and infiltration of eosinophils in BAL fluid after CDE challenge, (ii) the serum level of rFel d 1-specific IgE was reduced and the level of IgG₂a was more pronounced after CBP-rFel d 1 treatment, and (iii) there was also a tendency of decreased allergen-specific cellular response.
Conclusions:  Carbohydrate-based particles are effective tools as adjuvant and allergen carriers for use in ASIT and constitutes a promising strategy to improve allergy treatment.     

Chimeric human fcgamma-allergen fusion proteins in the prevention of allergy

Allergic responses are strongly associated with Th2-type immune responses, and modulation of the skewed Th2 response toward a more balanced response is the major goal of allergen immunotherapy (IT) in allergic disorders. To achieve this goal, several approaches have been tested. The authors previously showed that a human immunoglobulin (Ig) Fcgamma-Fcvarepsilon fusion protein (GE2) that directly cross-links FcvarepsilonRI and FcgammaRIIb on human mast cells and basophils was able to inhibit degranulation, and they reasoned that human gamma-allergen fusion protein would achieve a similar inhibitory effect in an allergen-specific fashion while preserving the immunogenicity of the allergen component. Therefore, the authors constructed and developed a human-cat chimeric fusion protein composed of the human Fcgamma1 and the cat allergen Fel d1 (Felis domesticus) for cat allergen-specific IT. This article summarizes the therapeutic features and potential of this novel fusion protein for allergic IT.     

Human T and B cell immune responses to Fel d 1 in cat-allergic and non-cat-allergic subjects

Background In allergic individuals exposure to allergen leads to the induction of allergen-specific IgE which, upon binding to its high affinity receptors on mast cells and basophils. primes these cells for degranulation. This degranulation. a result of specific IgE allergen-interaction, initiates the debilitating symptoms of allergy and the potentially life-threatening symptoms of anaphylaxis. The lack of symptoms following antigen encounter by non-allergic individuals is probably due to the undetectable levels of allergen-specific IgE in the plasma of non-allergic individuals. Objective To compare the immune responses of allergic and non-allergic individuals. Method We compared the immune responses of 42 cat-allergic subjects with 16 nem-cat-allergic subjects to the major cat allergen. Fel d I. We have measured plasma immunoglobulin levels and the proliferative responses of fel d 1 primed T cell lines to Fel d 1 peptides. Results While these two groups have similar levels of Fel d 1 specific IgG. only subjects in the cat-allergic group have detectable Fel d 1 specific IgE. Affinity purified Fel d 1 was used to generate T cell lines from peripheral blood mononuclear cells of these same subjects. The proliferative responses of these T cell lines to intact Fel d 1 and a set of overlapping peptides covering the entire sequence of the molecule demonstrated that the pattern of epitope recognition was similar in both groups. Conclusion Our data suggest that factors other than T cell recognition of specific epitopes are responsible for the nature of allergic immune responses generated when allergen is encountered.     

A Novel Adjuvant Allergen Complex, CBP-Fel d 1, Induces Upregulation of CD86 and Cytokine Release in Human Dendritic Cells

Allergen-specific immunotherapy (SIT) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). Drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. An alternative carrier is 2 μm carbohydrate-based particles (CBPs). In mouse, allergen-coupled CBPs have been demonstrated to skew the allergen-specific immune response towards a Th1-like activity (Grönlund et al. Immunology, 2002). We here coupled the recombinant major cat allergen Fel d 1 to CBPs (CBP-Fel d 1) by cyanogen-bromide activation, resulting in covalent binding. The effect of CBP-Fel d 1 on monocyte-derived dendritic cells (MDDCs) from healthy human blood donors was studied. We found that the majority of the CD1a⁺ MDDCs were capable of taking up FITC-labelled CBP-Fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. Furthermore, incubation with CBP-Fel d 1 resulted in an upregulation of the costimulatory molecule CD86 on the MDDCs, which was not observed with Fel d 1 or CBPs alone. Finally, CBP-Fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and a fourfold increase in the release of the chemokine interleukin-8 from MDDCs. Taken together, the effects CBPs possess make them interesting as novel allergen carriers for SIT.     

Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses in sensitized mice

Background:  Defined particles carrying tightly bound allergens at high density have been suggested as alternatives in allergy vaccination. Carbohydrate based particles (CBP), sized 2 μm, provide a platform for covalent coupling of allergens.
Objective:  To investigate the mechanisms of antigen presentation by CBP, as well as cellular and humoral responses after vaccination with the major cat allergen Fel d 1, covalently coupled to CBP.
Methods:  Mice ( n  = 10/group) were subcutaneously vaccinated with CBP-rFel d 1, CBP or phosphate buffer saline (PBS) before sensitization with rFel d 1 and challenged with cat dander extract. Fluorescent and ⁷⁵Se-radiolabeled tracking of allergens and particles were performed with flow cytometry and whole-body autoradiography. Humoral, cellular and regulatory immune responses were analyzed by ELISA and flow cytometry. Cytokines were measured in bronchoalveolar lavage fluid and splenocyte cultures.
Results:  CBP-rFel d 1 prevented induction of airway inflammation and induced allergen-specific T-cell anergy. CBP-rFel d 1 also induced rapid IgM and IgG1-responses compared with soluble rFel d 1. Particles were phagocytosed by antigen-presenting cells and transported to draining lymph nodes and spleen. Moreover, antigen coupled to CBP remained longer at the injection site compared with alum.
Conclusions:  Covalent coupling of rFel d 1 to CBP induces rapid antibody production, prevents induction of allergic immune responses and systemic allergen spreading. Thus, CBP comprise several attractive adjuvant features for use in allergy vaccination.
Clinical Implications:  Prolonged allergen exposure through covalent coupling to particles suitable for phagocytosis, provides an adjuvant for safer and efficient allergy vaccination.     

Epicutaneous allergen administration: is this the future of allergen‐specific immunotherapy?

IgE-mediated allergies, such as allergic rhinoconjunctivitis and asthma, have become highly prevalent, today affecting up to 30% of the population in industrialized countries. Allergen-specific immunotherapy (SIT) either subcutaneously or via the sublingual route is effective, but only few patients (<5%) choose immunotherapy, as treatment takes several years and because allergen administrations are associated with local and, in some cases, even systemic allergic side-effects because of allergen accidentally reaching the circulation. In order to resolve these two major drawbacks, the ideal application site of SIT should have two characteristics. First, it should contain a high number of potent antigen-presenting cells to enhance efficacy and shorten treatment duration. Secondly, it should be nonvascularized in order to minimize inadvertent systemic distribution of the allergen and therefore systemic allergic side-effects. The epidermis, a nonvascularized multilayer epithelium, that contains high numbers of potent antigen-presenting Langerhans cells (LC) could therefore be an interesting administration route. The present review will discuss the immunological rational, history and actual clinical experience with epicutaneous allergen-specific immunotherapy.     

High-dose allergen exposure leads to tolerance

Reports of decreased sensitization to cat allergen (Fel d 1) among individuals living with a cat or subjects exposed to high-dose cat allergen may be explained by the development of a form of high-dose tolerance resulting from natural exposure to an inhalant allergen. Although the epidemiological data regarding the relationship between exposure and sensitization to Fel d 1 are conflicting, the ability for high-dose Fel d 1 to induce a characteristic nonallergic immune response with a distinctive serum antibody profile has been established. Definition of this modified T-helper (Th)2 response to cat allergen, coupled with the renewed interest in regulatory T cells within the immunology field, has provided an avenue for exploring the mechanism by which IgE antibody-mediated responses are controlled. There is mounting evidence to suggest that the modified Th2 response is a variation of the allergic response and that the modified Th2-allergic axis is influenced by allergen dose and genetics. This article discusses putative immune mechanisms of tolerance within the context of an allergen-specific system. The relevance of high-dose allergen exposure and alternate factors such as endotoxin to the development of tolerance is considered. Fel d 1 exhibits unique molecular and immunological characteristics that may contribute to its tolerogenic properties. Major T-cell epitopes of Fel d 1 that preferentially induce regulatory factors have been defined. Furthermore, hightiter IgE antibody responses associated with atopic dermatitis are characterized by a defect in the T-cell repertoire that is specific to these epitopes. Identification of Fel d 1 epitopes that induce interleukin-10 may provide new targets for treatment.     

Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination

Background:  The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.
Methods:  A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.
Results:  Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.
Conclusion:  We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.     

Vaccination for birch pollen allergy: comparison of the affinities of specific immunoglobulins E, G1 and G4 measured by surface plasmon resonance

BACKGROUND: Allergen-specific immunotherapy (SIT) is associated with increased levels of allergen-specific IgG in serum. However, it is not clear to what extent qualitative changes in the allergen binding capacity of IgG may be induced as well. OBJECTIVE: The purpose of this study was to investigate the influences of SIT on antibody affinity. METHODS: The binding affinity of purified serum IgG1, IgG4 and IgE to the major allergen in birch (Betula verrucosa) pollen, Bet v 1, was analysed by surface plasmon resonance. The antibodies were obtained from 10 birch pollen-allergic patients receiving SIT and from 10 patients with no SIT. RESULTS: The patients having received SIT have a significant higher titre of anti-Bet v 1 antibodies in their blood, but the affinity to Bet v 1 of allergen-specific IgE, IgG1 and IgG4 does not differ between the two groups. For IgG1 and IgG4, correlations between less allergic symptoms and affinity of the antibodies were observed both in the SIT group and to a smaller extent in the non-SIT group. CONCLUSION: SIT has no effect on antibody affinity of allergen-specific IgE, IgG1 or IgG4. Allergic patients with high-affinity IgG1 and IgG4 antibodies report less symptoms than patients with low-affinity antibodies.     

Subcutaneous and sublingual immunotherapy:Where do we stand?

Though symptoms of allergic diseases can be reduced by the use of drugs such as corticosteroids,antihistamines or leukotrien antagonists,the only treatment directed to change the natural course of allergic disease is allergen-specific immunotherapy(SIT). Its efficacy can last years after the cessassion of the treatment.SIT brings on regulatory T cells with the capacity to generate interleukin-10 and transforming growth factor-b,restricts activation of mast cells and basophils,and shifts antibody isotype from Ig E to the noninflammatory type immunoglobulin G4. Subcutaneous(SCIT)and sublingual(SLIT) immunotherapy are the two most used ways at the present for applying SIT. These two treatments were demonstrated to be effective on reducing symptoms and medication use,in prevention of new sensitizations and in protecting from progression of rhinitis to asthma. The safety of SLIT appears to be better than SCIT although there have been a few head to head comparisons. In order to overcome compliance problems or possible systemic side effects which maybe faced during this long-term treatment,recent investigations have been focused on the implementation of allergens in quite efficacious and safer ways.     

Dose dependence and time course of the immunologic response to administration of standardized cat allergen extract

BACKGROUND: The immunologic response to allergen immunotherapy with 3 serial 5-fold doses of cat extract has been studied after approximately 5 weeks of immunotherapy. The highest dose containing 15 mug of Fel d 1 produced the most consistent and favorable response. It is unknown whether the comparative response on reaching a maintenance dose is maintained with long-term maintenance therapy. OBJECTIVE: The purpose of this investigation was to evaluate the immunologic responses with these 3 serial doses of cat hair and dander extract at baseline, after reaching the maintenance dose (approximately 5 weeks), and after 1 year of maintenance immunotherapy. METHODS: Twenty-eight patients with cat allergy randomized in a double-blind study were assigned to one of 4 treatment groups: placebo or cat hair and dander extract containing 0.6 mug of Fel d 1, 3 mug of Fel d 1, and 15 mug of Fel d 1 at maintenance. Studies included skin prick tests and late cutaneous reactions with cat hair and dander extract, titrated nasal challenges with the extract, serum cat allergen-specific IgG4 and IgE measurement, and flow cytometric and ELISA analysis of whole blood and intranasal cytokines (TGF-beta, IL-10, IFN-gamma, IL-4, and IL-5). RESULTS: Twenty-six subjects completed the study. After both 5 weeks and 1 year, significant and dose-dependent differences were seen with total symptom scores on nasal challenge ( P < .0001), with titrated skin prick testing with cat dander extract at 5 weeks ( P = .014) and 1 year ( P < .0001), and with cat-specific IgG4 measurement at 5 weeks ( P = .004) and 1 year ( P = .003). At 1 year, neither flow cytometry of whole blood nor ELISA evaluation of nasal cytokines demonstrated any significant differences among the treatment groups. CONCLUSION: The response to titrated nasal allergen challenge, titrated skin prick testing, and allergen-specific IgG4 measurement to cat immunotherapy at 5 weeks is predictive of the response at 1 year.     

Immunological mechanisms of allergen-specific immunotherapy

The studies on the mechanisms of specific immunotherapy (SIT) point out its targets that decide on the efficacy of SIT and hence might be used for its further improvement. Several mechanisms have been proposed to explain the beneficial effects of immunotherapy. The knowledge of the mechanisms underlying allergic diseases and curative treatment possibilities has experienced exciting advances over the last three decades. Studies in several clinical trials in allergen-SIT have demonstrated that the induction of a tolerant state against allergens in many ways represents a key step in the development of a healthy immune response against allergens. Several cellular and molecular mechanisms have been demonstrated: allergen-specific suppressive capacities of both inducible subsets of CD4(+) CD25(+) forkhead box P3(+) T-regulatory and IL-10-secreting type 1 T-regulatory cells increase in peripheral blood; suppression of eosinophils, mast cells, and basophils; Ab isotype change from IgE to IgG4. This review aims at the better understanding of the observed immunological changes associated with allergen SIT.     

Mechanisms of allergen-specific immunotherapy

Allergen-specific immunotherapy (SIT) has been used for almost a century as a desensitizing therapy for allergic diseases and represents the only curative and specific method of treatment. Administration of appropriate concentrations of allergen extracts has been shown to be reproducibly effective when patients are carefully selected. The mechanisms by which allergen-SIT has its effects include the modulation of T-cell and B-cell responses and related antibody isotypes as well as effector cells of allergic inflammation, such as eosinophils, basophils, and mast cells. The balance between allergen-specific T-regulatory (Treg) and T(H)2 cells appears to be decisive in the development of allergic and healthy immune responses against allergens. Treg cells consistently represent the dominant subset specific for common environmental allergens in sensitized healthy individuals. In contrast, there is a high frequency of allergen-specific T(H)2 cells in patients with allergy. The induction of a tolerant state in peripheral T cells represents an essential step in allergen-SIT. Peripheral T-cell tolerance is characterized mainly by generation of allergen-specific Treg cells leading to suppressed T-cell proliferation and T(H)1 and T(H)2 cytokine responses against the allergen. This is accompanied by a significant increase in allergen-specific IgG(4), and also IgG(1) and IgA, and a decrease in IgE in the late stage of the disease. In addition, decreased tissue infiltration of mast cells and eosinophils and their mediator release including circulating basophils takes place. Current understanding of mechanisms of allergen-SIT, particularly the role of Treg cells in peripheral tolerance, may enable novel treatment strategies.     

Side-effects of allergen-specific immunotherapy. A prospective multi-centre study

BACKGROUND AND OBJECTIVE: The safety of allergen-specific immunotherapy (SIT) is a parameter of great interest in the overall assessment of the treatment. A clinical database was developed in order to obtain early warnings of changes in the frequency and severity of side-effects and sufficient data for the evaluation of possible risk factors. METHODS: During a 3-year period, four allergy centres in Copenhagen, Denmark, included data from all patients initiating SIT to a common database. Information on initial allergic symptoms, allergens used for treatment, treatment regimens and systemic side-effects (SSEs) during the build-up phase was collected. RESULTS: A total of 1,038 patients received treatment with 1,709 allergens (timothy, birch, mugwort, house dust mite (HDM), cat, and wasp and bee venom), 23,047 injections in total. Most SIT patients completed the updosing phase without side-effects, but there was a significant difference between allergens: wasp (89%), birch (82%), HDM (81%), cat (74%) and grass (70%) (P=0.004). A total of 582 SSEs were registered in 341 patients. Most side-effects were mild grade 2 reactions (78%). A difference in severity between allergens was observed (P=0.02), with grass giving most problems. The type of allergen but not patient- or centre-related parameters seemed predictive of side-effects. CONCLUSIONS: Allergen extracts differ in their tendency to produce side-effects. Multi-centre studies like the present one allow more patients to be evaluated, and thereby provide a more efficient surveillance of side-effects. Online Internet-based registration to a central national database of every allergen injection would be an even more powerful tool for evaluation of risk factors and surveillance of side-effects.     

Oligoclonal analysis of the atopic T cell response to the group 1 allergen of Cynodon dactylon (bermuda grass) pollen: pre- and post-allergen-specific immunotherapy

BACKGROUND: Bermuda grass pollen (BGP) is an increasingly important seasonal aeroallergen in Australia and other subtropical and temperate regions. BGP shares minimal allergenic cross-reactivity with pollens of rye grass or other Pooideae grasses often used for desensitization regimens in grass pollen allergy. Current allergen immunotherapy is seldom used in asthmatic patients due to IgE-mediated side effects. Since clinically effective immunotherapy is linked with altered allergen-specific T cell response, characterisation of human T cell reactivity to Cyn d 1, the major B cell allergen of BGP, should permit the design of effective and safe immunotherapy for BGP allergy. METHODS: Short-term BGP-specific CD4+ T cell lines were established from peripheral blood of 14 BGP-sensitive patients before and after conventional 50% BGP and 50% 7-grass mix subcutaneous specific allergen immunotherapy (SIT). T cell diversity of antigen specificity and function was assessed by proliferation and cytokine production to BGP, Cyn d 1 and Cyn d 1 peptides. RESULTS: Three highly immunogenic regions of Cyn d 1 were identified in 13/14 patients pre-SIT: Cyn d 1 (109-128), (181-209) and (217-241). The SIT regimen was clinically efficacious. Following SIT, decreased proliferation to BGP, Cyn d 1 and Cyn d 1 peptides was observed with a marked decrease in the IL-5:IFN-gamma ratio. CONCLUSIONS: Cyn d 1 is a major T cell allergen of BGP. Decreased Cyn d 1-specific IL-5 dominant T cell responses were observed in association with clinically effective treatment with the 50% BGP and 50% 7-grass mix. Identified dominant T cell regions of Cyn d 1 should facilitate safer vaccine development for BGP-induced asthma in addition to rhinitis.     

Rational design of hypoallergens applied to the major cat allergen Fel d 1

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.     

Mutant derivatives of the main respiratory allergen of cow are less allergenic than the intact molecule.

BACKGROUND: Allergen immunotherapy offers an alternative for drug treatment in the management of allergic diseases. Because immunotherapy often induces side-effects, less allergenic preparations would be beneficial. OBJECTIVE: The purpose of this study was to examine whether the allergenicity of a cow-derived lipocalin allergen, Bos d 2, could be diminished by substituting or deleting carboxy-terminal amino acids including the cysteine which forms a disulphide bond with a cysteine inside the molecule. METHODS: Four recombinant mutants of Bos d 2 were created by substituting or deleting the four most carboxy-terminal amino acids. The immunological characteristics of the mutant preparations were compared with the unmodified rBos d 2 by Western blotting, ELISA inhibition, skin prick tests, and the proliferative responses of allergen-specific T-cell clones. RESULTS: In Western blot, one of the two monoclonal antibodies showed reduced binding to the preparations without the terminal cysteine. In contrast, the other monoclonal antibody, human IgE and rabbit immune serum bound equally well to all the preparations. ELISA inhibition analyses revealed, however, that the preparations without the terminal cysteine bound antibody less efficiently. They were needed 15-38 times more than the unmodified rBos d 2 to cause the same level of inhibition. Surprisingly, one of the mutants with the terminal cysteine but a mutated adjacent amino acid turned out to be the weakest in inducing skin reactivity. All the preparations stimulated well allergen-specific T-cell clones. CONCLUSIONS: The results show that the allergenicity of a lipocalin allergen, Bos d 2, can be diminished by modifying the carboxy-terminal end of the molecule. Modifications in the area which encompasses a disulphide bond impaired the antibody binding without affecting the T-cell stimulatory capacity. It was also shown that in vivo tests are necessary for determining the allergenicity of a modified allergen.     

A double-blind,placebo-controlled immunotherapy dose-response study with standardized cat extract

BACKGROUND: Allergen immunotherapy with doses of cat extract containing approximately 15 microg of the major allergen, Fel d 1, have been proved clinically effective in several double-blind, placebo-controlled studies. However, the maintenance doses used in allergy practice in the United States are often considerably less than this proven dose. OBJECTIVE: The purpose of this investigation was to determine whether maintenance immunotherapy with cat dander extract containing 0.6 microg or 3.0 microg of Fel d 1 was more effective than placebo and similar in efficacy to treatment with extracts containing 15.0 microg Fel d 1, immunologic parameters being used as the outcome. METHODS: Twenty-eight cat-allergic patients were randomly entered, 7 in each group, into a double-blind, placebo-controlled comparison of the immunologic response to treatment with placebo or cat dander extract containing 0.6 microg, 3.0 microg, or 15.0 microg of Fel d 1. Maintenance doses were achieved in 8 visits over a period of 4 weeks through use of a cluster regimen; each subject then received 1 weekly maintenance injection before posttreatment measurements were made. The response to immunotherapy was assessed before immunotherapy and after the first weekly maintenance injection. Studies included responses to titrated skin prick tests to cat extract and an unrelated allergen and serum allergen-specific IgE and IgG4. Titrated nasal challenges were performed with cat extract; measurement of mRNA and secreted cytokines (IL-4, IL-5, and IFN-gamma) was done at 6 hours. Serum cytokines (IL-4, IL-5, IFN-gamma) were measured, and flow cytometric analysis of intracellular cytokines (IL-4, IL-5, IFN-gamma) was performed. Cat allergen-stimulated lymphocyte proliferation was performed with measurement of cytokines in the supernatant (IL-4, IL-5, IFN-gamma). RESULTS: All 28 subjects completed the study. Significant and dose-dependent differences were encountered in the titrated skin prick tests (P =.008), the cat-specific IgG(4) (P =.01), and the reduction in CD4+/IL-4+ PBMCs on flow cytometry (P =.03). There were no significant differences between placebo and cat dander extract containing Fel d 1 0.6 microg. Both extracts containing 3.0 microg and 15.0 microg produced significant decreases in skin prick test sensitivity (P =.02 and P =.002, respectively). The extracts containing 3.0 microg and 15.0 microg produced significant increases in cat-specific IgG4 (P =.01 and P =.006, respectively). Only the 15.0-microg-per-dose extract produced a significant reduction in the percent of CD4+/IL-4+ PBMCs (P =.003). CONCLUSION: In this double-blind, placebo-controlled study, a maintenance dose of cat dander extract containing 15.0 microg Fel d 1 produced the most consistent immunologic response. A dose of 3.0 microg reduced skin prick test sensitivity and increased cat-specific IgG4 but did not reduce the circulating CD4+/IL-4+ PBMCs, a change that is likely related to the clinically significant response to allergen immunotherapy. These findings suggest that a maintenance dose of 15.0 microg of Fel d 1 is most apt to be clinically effective for allergen immunotherapy.     

Mechanisms of subcutaneous allergen immunotherapy

Allergen-specific immunotherapy (SIT) is the only curative approach in the?treatment of allergic diseases defined up-to-date. Peripheral T-cell tolerance to allergens, the goal of successful allergen-SIT, is the primary mechanism in healthy immune responses to allergens. By repeated administration of increased doses of the causative allergen, allergen-SIT induces a state of immune tolerance to allergens through the constitution of T regulatory (Treg) cells, including allergen-specific interleukin (IL)-10-secreting Treg type 1 cells and CD4(+)CD25(+)Treg cells; induction of suppressive cytokines, such as IL-10 and transforming growth factor β; suppression of allergen-specific IgE and induction of IgG4 and IgA; and suppression of mast cells, basophils, eosinophils, and inflammatory dendritic cells. This review summarizes the current knowledge on the mechanisms of allergen-SIT with emphasis on the roles of Treg cells in allergen-SIT.