Genetic engineering of allergens: future therapeutic products

Genetic engineering of allergens for specific immunotherapy should aim at the production of modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). Common approaches for engineering of hypoallergens usually require knowledge of T and B cell epitopes and involve changing specific base pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). DNA family shuffling has the advantage that it does not require a priori knowledge of structural and functional properties for efficient generation of hypoallergens. The combination of the hypoallergen concept with the Th1-inducing genetic immunization approach might be an attractive alternative for protein-based immunotherapy.     

Targeting the MHC class II pathway of antigen presentation enhances immunogenicity and safety of allergen immunotherapy

Background:  Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects.
Methods:  The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients.
Results:  MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-γ and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1.
Conclusion:  MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.     

Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines

BACKGROUND: The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability. OBJECTIVE: To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1. METHODS: One point (Y141A) and two deletion (135Delta10 and 140Delta5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures. RESULTS: The 135Delta10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135Delta10 as the variant with the lowest beta-hexosaminidase-releasing capacity. Furthermore, the same 135Delta10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen. CONCLUSION: The 135Delta10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy.     

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.     

In vitro and in vivo responses to the recombinant bovine dander allergen Bos d 2 and its fragments

BACKGROUND: About one in every four cases of occupational rhinitis recorded in Finland is animal-induced. Bovine allergens are the most important in this respect and the largest patient group consists of dairy farmers. Allergen immunotherapy, if proven effective, safe and feasible, would be ideal for their treatment. The development of recombinant allergens has offered new potential therapeutic prospects. Fragments of recombinant Bos domesticus (Bos d 2) allergen could be suitable for this purpose because they are recognized by T cells but their IgE-binding capacity is attenuated. OBJECTIVE: The aim of this study was to verify whether the potential of the two fragments of recombinant Bos d 2 (corresponding to amino acids 1-131 and 81-172) to induce immediate allergic reaction in a shock organ (nose) was decreased compared to the complete recombinant allergen, which would be an advantageous property for a preparation intended for allergen immunotherapy. METHODS: The study group consisted of 10 dairy farmers with cow-induced allergic rhinitis. We used the IgE titres against native Bos d 2 measured by indirect IgE ELISA to characterize the level of sensitization and compared the IgE titres in the rhinitis patients with 12 cow-sensitized asthmatic farmers and 12 healthy students. In vitro reactivity against recombinant Bos d 2 and its two fragments was studied by indirect IgE ELISA and in vivo reactivity by nasal provocation tests. RESULTS: The IgE titres against native Bos d 2 of patients with rhinitis tended to be lower than the titres of asthmatics. The healthy students did not exhibit any detectable IgE reactivity to native Bos d 2. In the patients with rhinitis, there was no statistically significant difference between IgE responses against native and recombinant Bos d 2, whereas with both in vitro and in vivo, the reactivity to both fragments of recombinant Bos d 2 was lower than the reactivity to the complete recombinant allergen. CONCLUSIONS: Due to the decreased in vivo capacity to induce immediate allergic reactions, the fragments may be better tolerated in allergen immunotherapy than the complete allergen.     

Phl p 3: Structural and immunological characterization of a major allergen of timothy grass pollen

BACKGROUND: The relevant importance of individual allergens for allergic sensitization is only partially understood. More detailed information on allergen structure and how it influences immunological responses can lead to better diagnosis of disease and improved preparations for allergen-specific immunotherapy. Grass pollen contains several different allergens, and although the group 3 allergens have been classified long ago, their structure and allergenicity have been poorly investigated. OBJECTIVE: To characterize Phl p 3 from timothy grass pollen and compare it with Phl p 2 with respect to biochemical structure and allergenicity. METHODS: Natural Phl p 2 and Phl p 3 were separated from a pollen extract by chromatography and characterized by 2D electrophoresis and protein sequencing. The complete sequences were determined by DNA cloning and detected in natural pollen extracts by mass spectrometry. Further comparisons of the allergens were made for IgE-binding and cross-reactivity, allergenicity was determined by basophil CD203c activation and skin prick test and 3D structures were compared by molecular modelling. RESULTS: Phl p 3 reveals molecular masses of 10.958 and 10.973 kDa and pIs of 8.9 and 9.3, respectively, Phl p 2 a molecular mass of 10.816 kDa and a pI of 4.6. The sequence identity is 58%. In spite of these differences in the primary structures, both allergens reveal similar conformational structures, resulting in similar immunological and allergological moieties. CONCLUSIONS: The group 3 and group 2 allergens are major allergens with similar 3D structures. Although they differ considerably in their protein sequences and their pIs, they show only a slightly higher immunological reactivity for Phl p 3 on the B-cell level (conformational epitopes). But distinct differences between the sequences may influence reactivity at the T cell level.     

Ca2+离子对榛子过敏原Cor h 1二级结构和抗原活性影响研究

目的:研究Ca2+离子对非CaBPs蛋白类过敏原二级结构和抗原活性的影响。方法:以重组榛子主要过敏原Cor h 1为研究对象,用圆二色谱(CD)研究了系列浓度的Ca2+和EDTA各自存在情况下,不同蛋白浓度溶液中rCor h 1二级结构的变化及规律,并以榛子过敏患者血清为一抗,用ELISA实验分析了Ca2+和EDTA对rCor h 1抗原活性的影响。结果:在高浓度rCor h 1溶液中Ca2+的加入可以引起椭圆率的增加,而在低浓度的rCor h 1溶液中则引起椭圆率的降低;EDTA在高浓度和低浓度的rCor h 1溶液中均能引起rCor h 1二级结构相对含量的降低;椭圆率增加(或降低)的幅度与Ca2+、EDTA终浓度成正比。ELISA实验表明Ca2+和EDTA都能显著降低rCor h 1的抗原活性。结论:Ca2+离子可以显著影响rCor h 1的二级结构,降低其抗原活性,为非CaBPs蛋白类低致敏原的研究奠定了较好的前期研究基础。     

A mutant of the major apple allergen, Mal d 1, demonstrating hypo-allergenicity in the target organ by double-blind placebo-controlled food challenge

BACKGROUND: Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution. OBJECTIVE: To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo. METHODS: A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC). RESULTS: RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules. CONCLUSION: A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.     

Regulation of specific immune responses by chemical and structural modifications of allergens

Specific immunotherapy (SIT) is an efficient treatment of allergic diseases to defined allergens. Despite being used in clinical practice since early in this century, more rational and safer regimens are required, because SIT is faced with the risk of anaphylaxis and standardization problems of allergen-extract-based treatments. A better understanding of the pathogenesis of allergy and of the mechanisms of SIT has led to various approaches to overcome these problems. Knowledge of the influence of IgE-facilitated antigen presentation on allergen-specific Th2 responses increased the efforts to generate non-IgE-binding allergens. The current principal approach to allergen modification is to modify B cell epitopes in order to prevent IgE binding and effector cell cross-linking while preserving T cell epitopes to retain the capacity of inducing tolerance. In this way, the modified allergen will be directed to T cells by a phagocytosis/pinocytosis-mediated antigen uptake mechanism, bypassing IgE cross-linking and IgE-dependent antigen presentation. Accordingly, a differential regulation of allergen-specific T cell cytokine patterns and IgE:IgG production was demonstrated by modifications of the three-dimensional structure of allergens because of linearity in T cell epitopes and conformation dependence in B cell epitopes. In this context, chemically modified allergen extracts with low IgE-binding capacity have been developed to reduce anaphylactic side effects since the early 1980s. The progress of recombinant techniques for producing allergens and allergen derivatives has led to a dramatic improvement in the ability of developing novel vaccines for the treatment of allergy. This has enabled mutation or deletion of decisive amino acids in B cell epitopes and fractionation or oligomerization of allergens by genetic engineering as fruitful approaches to generate hypoallergenic vaccines. Moreover, non-IgE-binding short T cell epitope peptides and single-amino-acid-altered peptide ligands represent potential candidates for future SIT.     

Allergy diagnosis, allergen repertoires, and their implications for allergen-specific immunotherapy

Immunotherapy for allergic diseases represents an important but largely unmet medical need. Conventional immunotherapy suffers from several breakdowns related to the quality of the extracts used, the risk of inducing anaphylactic reactions, and the extremely long treatment time. Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome using genetically engineered recombinant allergens. New therapeutic strategies based on recombinant technology include peptide-based vaccines, engineered hypoallergens with reduced IgE-binding properties, nucleotide-conjugated vaccines that promote Th1 responses, and the possibility of developing prophylactic allergen vaccines.     

Generation of a low Immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.     

Ablation of ovomucoid-induced allergic response by desensitization with recombinant ovomucoid third domain in a murine model

Attempts to modulate the allergenic response by hypoallergens aimed at eliminating IgE-binding epitopes have been established recently for allergen immunotherapy. Desensitization offers an alternative approach to mounting a protective immune response. We have shown previously that mutation of the decisive amino acids in the B cell epitope of the ovomucoid third domain suppresses IgE binding reactivity against human patient sera and we hypothesize that this hypoallergenic variant could be a potential candidate molecule for specific immunotherapy against an ovomucoid-induced IgE reaction. The aim of this study was to investigate whether hyposensitization with the ovomucoid-modified isoform could desensitize ovomucoid-sensitized mice. We mapped the immunodominant B cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid mutant isoform, having ablated allergenicity against patient sera, was used to desensitize ovomucoid-sensitized Balb/c mice by intraperitoneal injection. Female Balb/c mice were sensitized with intact ovomucoid molecule (Fovm) and desensitized with the modified isoform of the third domain of ovomucoid (GMFA). Intact ovomucoid-sensitized mice desensitized with phosphate-buffered saline (PBS) served as positive controls to maintain hypersensitivity. To gain insight into the efficacy of the modified ovomucoid variant in desensitization, effects on hypersensitivity reactions and histamine levels, followed by its association with antibody levels and cytokine profiles, were measured. Abrogation of the allergic response with complete suppression of anaphylactic symptoms and lower serum histamine levels was observed in the desensitized group by GMFA, accompanied by significantly reduced ovomucoid-specific IgE and IgG1 levels and enhanced specific IgG and IgG2a levels. The sensitized group showed severe anaphylactic symptoms, enhanced serum histamine concentrations and increased levels of specific IgE and IgG1. The level of interleukin (IL)-4 was decreased dramatically in the desensitized group and higher levels of interferon (IFN)-gamma were found, whereas mice sensitized with intact ovomucoid exhibited significantly higher levels of IL-4 favouring a Th2 skewed pathway. We demonstrate clearly that GMFA is able to ablate ovomucoid-induced allergic reactions in sensitized mice. This occurs via a suppression of specific IgE accompanied by an increase in suppressor T cell activity. This approach offers some promise for the development of treatment against ovomucoid-induced allergic response.     

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.     

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.     

Reduction of the in vivo allergenicity of Der p 2, the major house-dust mite allergen, by genetic engineering

The major allergen of the house-dust mite Dermatophagoides pteronyssinus, Der p 2, is recognized by approximately 90% of mite-allergic patients. We have produced two recombinant fragments of Der p 2 comprising aa 1-53 and aa 54-129 and a hybrid molecule (aa 54-129+1-53), combining the two fragments in inverse order, by genetic engineering. The recombinant Der p 2 derivatives were expressed in E. coli and purified to homogeneity. rDer p 2 derivatives (fragments and hybrid) showed a considerably reduced beta sheet structure and IgE reactivity compared to the Der p 2 wild-type allergen. The allergenic activity of the Der p 2 derivatives was reduced more than tenfold as evaluated in vitro in basophil activation assays and in vivo by skin prick testing of mite-allergic patients. Immunization of mice and rabbits with rDer p 2 derivatives induced Der p 2-specific IgG antibodies, which inhibited the binding of allergic patients' IgE to Der p 2. Immunization of mice with rDer p 2 derivatives induced less allergenic IgE responses than immunization with rDer p 2. Thus the rDer p 2 derivatives exhibited less in vivo allergenic activity and allergenicity than the Der p 2 allergen but preserved immunogenicity and may hence represent candidates for specific immunotherapy of house-dust mite allergy.     

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy

BACKGROUND: Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. OBJECTIVE: The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. METHODS: By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). RESULTS: The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. CONCLUSION: The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.     

Fel d 4, a cat lipocalin allergen

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals. OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels. METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed. RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog. CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.     

A hybrid expressing genetically engineered major allergens of the Parietaria pollen as a tool for specific allergy vaccination

BACKGROUND: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. METHODS: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. RESULTS: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. CONCLUSION: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.     

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes

BACKGROUND: Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. OBJECTIVE: Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. METHODS: Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. RESULTS: The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. CONCLUSION: The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects.     

The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogeneously recognized among allergic subjects

Pollen allergens are multivalent proteins that cross-link IgE antibodies on mast or basophil cells, inducing secretion of biologic mediators, and resulting in various allergic symptoms. The IgE-binding regions of the Parietaria judaica (Pj) pollen major allergen rPar j 2 were investigated. Twenty-nine single sera from Pj-allergic subjects were tested by Western blot against five recombinant peptides. At least four putative IgE-binding epitopes were identified. The analysis of their diffusion suggested a heterogeneous IgE-binding response. In fact, 75% of the sera reacted with peptide 1-54, 48% with peptide 48-101, 24% with peptide 1-30, 7% with peptide 29-54, and none with peptide 48-76. These five peptides were analyzed with the histamine-release assay. Only peptide 48-101 was capable of inducing degranulation and release of histamine. These results suggest that the recombinant rPar j 2 allergen contains IgE epitopes that are heterogeneously recognized by sensitive patients, and that therefore the therapeutic approach based on the use of haptenic peptides needs a careful evaluation.