Characterization of the protective and therapeutic efficiency of a DNA vaccine encoding the major birch pollen allergen Bet v 1a

BACKGROUND: An estimated 100 million individuals suffer from birch pollen allergy. More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen Bet v 1a, and almost 60% of them are sensitized exclusively to this allergen. OBJECTIVE: DNA immunization using the Bet v 1a gene was evaluated with respect to its prophylactic and therapeutic efficacy. METHODS: A DNA vaccine containing the entire Bet v 1a cDNA under the control of a CMV-promoter was constructed. In order to estimate the protective efficiency, animals received three injections of this vaccine prior to sensitization with recombinant Bet v 1a. Vice versa, in a therapeutic approach, sensitization was followed by treatment with the DNA vaccine. RESULTS: The Bet v 1a DNA vaccine induced strong Bet v 1-specific antibody responses with a Th1-biased response type. Animals which received the DNA vaccine were protected against a following allergic sensitization with Bet v 1a. The protective effect was characterized by suppression of Bet v 1-specific immunoglobulin (Ig)E production, lack of basophil activation and enhanced interferon (IFN)-gamma expression. In a therapeutic situation, treatment of sensitized animals with DNA vaccines decreased IgE production, IgE-mediated basophil release and drastically reduced anaphylactic activity as measured by passive cutaneous anaphylaxis assays. Concerning the cellular immune response, DNA immunization induced a sustaining and dominant shift from a Th2 type response towards a balanced Th1/Th2 type response as indicated by increased IFN-gamma but unchanged IL-5 levels in lymphoproliferation assays. CONCLUSION: The results demonstrate the allergen-specific protective and therapeutic efficacy of a DNA vaccine encoding the clinically highly relevant allergen Bet v 1a indicating the suitability of this concept for the treatment of allergic diseases.     

Generation of hypoallergenic DNA vaccines by forced ubiquitination: preventive and therapeutic effects in a mouse model of allergy

BACKGROUND: Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T(H)1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. OBJECTIVE: A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. METHODS: Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG(1), IgG(2a), and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-gamma and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. RESULTS: Prevaccination with ubiquitinated Bet v 1-stimulated T(H)1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. CONCLUSION: The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. CLINICAL IMPLICATIONS: Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.     

Cytokine and antibody responses in birch-pollen-allergic patients treated with genetically modified derivatives of the major birch pollen allergen Bet v 1

BACKGROUND: Recently, recombinant hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, were used to treat birch-pollen-allergic patients in a double-blind, placebo-controlled, multi-centre immunotherapy study. The aim of this study was to evaluate the effects of vaccination with aluminium-hydroxide-adsorbed recombinant Bet v 1 derivatives versus placebo on T-cell, cytokine and antibody responses in a subgroup of patients. METHODS: Blood was drawn from patients of the Swedish centre (n = 27; rBet v 1 fragments: n = 10; rBet v 1 trimer: n = 8, and placebo-aluminium hydroxide: n = 9) before the start and after completion of the treatment. PBMC were stimulated with rBet v 1 and analysed for cytokine (IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-gamma)-secreting cells by ELISpot. Bet v 1-specific antibody levels in serum (IgG(1-4), IgE and IgA) were measured by ELISA. Skin prick tests with defined Bet v 1 concentrations were performed before and 10-11 months after the beginning of the study. RESULTS: Bet v 1-specific IgG levels, consisting of IgG(1), IgG(2) and IgG(4), were significantly increased after treatment with recombinant allergen derivatives. Treatment with rBet v 1 trimer led to a significant (p < 0.05) reduction of Bet v 1-reactive IL-5- and IL-13-producing cells, reflecting a reduced Th2 response. In addition, a decreased number of Bet v 1-reactive IL-4 producing (p = 0.07) and an increase of IL-12-producing (p = 0.06) cells was noted in the trimer-treated patients. In contrast to placebo, active treatment resulted in significantly reduced immediate-type skin reactions to Bet v 1 even 10-11 months after treatment. CONCLUSION: Vaccination with recombinant hypoallergenic Bet v 1 derivatives induces a Bet v 1-specific IgG response and leads to reduced skin reactivity in allergic patients. A reduction of Bet v 1-specific Th2 responses was observed in trimer-treated patients, which may reflect the intrinsic property of this allergen derivative.     

Intranasal treatment with a recombinant hypoallergenic derivative of the major birch pollen allergen Bet v 1 prevents allergic sensitization and airway inflammation in mice

BACKGROUND: The major birch pollen allergen Bet v 1 represents one of the most prevalent environmental allergens responsible for allergic airway inflammation. OBJECTIVE: In the present study we sought to compare the complete recombinant Bet v 1 allergen molecule with genetically produced hypoallergenic fragments of Bet v 1 regarding mucosal tolerance induction in a mouse model of allergic asthma. METHODS: BALB/c mice were intranasally treated with recombinant Bet v 1 or with two recombinant Bet v 1 fragments (F I: aa 1-74; F II: aa 75-160) prior to aerosol sensitization with birch pollen and Bet v 1. RESULTS: Intranasal application of F II, containing the major T cell epitope, led to significant reduction of IgE/IgG1 antibody responses, in vitro cytokine production (IL-5, IFN-gamma, IL-10) and negative immediate cutaneous hypersensitivity reactions comparable to the pretreatment with the complete rBet v 1 allergen. Moreover, airway inflammation (eosinophilia, IL-5) was inhibited by the pretreatment with either the complete Bet v 1 or F II. However, for prevention of airway hyperresponsiveness the complete molecule was required. The mechanisms leading to immunosuppression seemed to differ in their dependence on the conformation of the molecules, since tolerance induced with the complete Bet v 1, but not with F II, was transferable with spleen cells and associated with increased TGF-beta mRNA levels. CONCLUSION: We conclude that mucosal tolerance induction with recombinant allergens and genetically engineered hypoallergenic derivatives thereof could provide a convenient and safe intervention strategy against type I allergy.     

Mucosal tolerance induction with hypoallergenic molecules in a murine model of allergic asthma

Type I allergy, frequently elicited by airborne allergens, has constantly increased within recent years. Birch pollen and its major allergen Bet v 1 represent a major source of type I allergens. By genetic engineering hypoallergenic Bet v 1 fragments were produced, which lost the IgE binding capacity but retained the T cell epitopes. We have established a murine model of aerosol sensitization to birch pollen and its major allergen Bet v 1, leading to type I allergic immune responses and airway hyperresponsiveness. In the present study we demonstrate that mucosal administration of recombinant Bet v 1 prior to sensitization led to allergen-specific suppression of B and T cell responses in vivo and in vitro, reduction of eosinophilic infiltration in the lungs and inhibition of airway hyperresponsiveness. Intranasal pretreatment with the nonanaphylactic fragments of Bet v 1 prevented allergic immune responses and airway inflammation to the same degree as the pretreatment with the complete molecule. We conclude from our studies that mucosal tolerance induction with hypoallergenic molecules could provide a safe and convenient treatment strategy against type I allergies.     

Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.

Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain Th1-based autoimmune diseases. We have established a model of aerosol sensitization leading to Th2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence Th2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4, but not IFN-gamma, production were markedly decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens--in conjugated as in unconjugated form--had different effects on the Th2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB--and probably also other antigen-delivery systems--strongly depend on the nature of the coupled antigen-allergen.     

Effects of adjuvants on the immune response to allergens in a murine model of allergen inhalation: cholera toxin induces a Th1-like response to Bet v 1, the major birch pollen allergen

Based on the fact that type I allergies are frequently elicited by inhalant allergens, we have established a model of aerosol inhalation leading to allergic sensitization in BALB/c mice. Using this model we studied the effects of aluminium hydroxide (Al(OH)₃), known to enhance IgE antibody responses, compared with cholera toxin (CT), a potent mucosal adjuvant, on the immune response to birch pollen (BP) and its major allergen Bet v 1. Two groups of BALB/c mice were either systemically immunized with recombinant Bet v 1 in Al(OH)₃ and subsequently aerosol exposed to BP allergen, or aerosolized with BP and CT. IgE-mediated skin reactions were only elicited in the mice which had received Bet v1/Al(OH)₃. Allergen-specific serum IgE and IgG1 antibodies dominated in the Al(OH)₃ group, IgG2a antibody levels to BP and rBet v 1 were markedly higher in the sera of mice exposed to CT with the allergen. IgA antibodies were only detected in the bronchial lavage of the CT-treated group. Moreover, the latter group displayed consistently higher T cell proliferative responses to BP and interferon-gamma production in vitro. Thus, the systemic immunization with rBet v 1 in Al(OH)₃ before inhalation of the BP extract promoted a Th2-like immune response, while CT mixed with the aerosolized BP extract rather induced a Th1-like immune response. In an attempt to reverse these ongoing immune responses we could achieve a shift towards a Th0 response. Immunization with BP extract without adjuvant treatment led to undetectable antibody or cellular immune responses. We conclude from the present study that the induction of an immune response to BP allergen after aerosol inhalation can be directed towards a Th1- or a Th2-like response. Once established, the immune response can be modulated.     

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1

BACKGROUND: We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. OBJECTIVE: The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. METHODS: BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. RESULTS: In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. CONCLUSION: Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.     

Non-anaphylactic surface-exposed peptides of the major birch pollen allergen, Bet v 1, for preventive vaccination

BACKGROUND: Almost 100 million allergic patients are sensitized to the major birch pollen allergen, Bet v 1, a 17 kDa protein containing most of the IgE epitopes present in pollens of trees belonging to the Fagales order and plant-derived food. OBJECTIVE: Our aim was to develop an approach for the rational design of B cell epitope-derived, non-allergenic peptide allergy vaccines. METHODS: According to the three-dimensional (3-D) structure of birch pollen allergen, Bet v 1, six peptides comprising 25-32 preferably solvent-exposed amino acids were synthesized. RESULTS: Because of lack of secondary structure, the peptides showed no allergenic activity in allergic patients. In a mouse model of birch pollen allergy, peptide vaccination induced Bet v 1-specific IgG and prevented IgE-mediated allergic sensitization to Bet v 1. The protective role of peptide-induced blocking antibodies is demonstrated by inhibition of allergic patients IgE binding to the allergen and by blocking of allergen-induced basophil degranulation. CONCLUSION: Our results indicate the mechanistic importance of blocking antibodies for allergy vaccination and present a B cell epitope-based approach for the rational design of safe peptide allergy vaccines whenever the structure of the disease-eliciting allergen is known.     

Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level

Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type I hypersensitivity reactions to celery tuber. We evaluated the T cell response to the major allergen in celeriac, Api g 1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet v 1. Api g 1-specific T cell lines (TCL) and clones (TCC) were established from peripheral blood mononuclear cells of allergic patients. Epitope mapping of Api g 1 with overlapping Api g 1-derived peptides revealed one dominant T cell-activating region, Api g 1(109-126). TCL and TCC generated with Api g 1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet v 1 were stronger than to Api g 1. Epitope mapping with Bet v 1-derived peptides revealed that T cells specific for several distinct epitopes distributed over the complete Bet v 1 molecule could be activated by Api g 1. Bet v 1(109-126) was identified as the most important T cell epitope for cross-reactivity with Api g 1. This epitope shares 72% amino acid sequence similarity with the major T cell-activating region of the food allergen, Api g 1(109-126). Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet v 1-specific Th2 cells by Api g 1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals.     

Modulation of an allergic immune response via the mucosal route in a murine model of inhalative type-I allergy

A murine model of aerosol inhalation, leading to sensitization to birch pollen (BP) and its major allergen Bet v 1, was established in order to try to influence type-I allergic immune responses via the mucosal route. We previously demonstrated that simultaneous inhalation of BP and cholera toxin, a potent mucosal adjuvant, induced a Th1-like immune response to the allergen in naive mice and modulated allergic immune responses in sensitized mice. In contrast to cholera holotoxin, mucosal application of the cholera B subunit (CTB) conjugated to antigen has been shown to induce peripheral tolerance in certain models of Th1-based autoimmune diseases. In the present study we investigated the potential of such an antigen delivery system to suppress Th2-based, allergic immune responses. Mucosal administration of CTB/Bet v 1 conjugates prior to sensitization led to significantly increased allergen-specific IgE/IgG1 and IgG2a antibody levels and cytokine production (IL-5, IFN-gamma) in vitro. Thus, CTB coupled to Bet v 1 acted as an adjuvant rather than a tolerogen. On the other hand we noted that mucosal application of CTB coupled to ovalbumin led to marked suppression of antigen-specific IgE antibody levels and IL-5 production in vitro and thereby restricted allergic sensitization. These results indicated that the effects of CTB/antigen conjugates depended on the nature of the antigen. In contrast to Bet v 1 coupled to CTB, nasal as well as oral application of low doses of unconjugated, Bet v 1 prior to aerosol sensitization inhibited allergen-specific antibody responses of all isotypes, cutaneous type-I skin tests in vivo as well as allergen-specific lymphoproliferative responses and cytokine production (IL-4, IL-5, IL-10, IFN-gamma) in vitro, suggesting that both T- and B-cell tolerance to the allergen were induced. Taken together, mucosal tolerance induction as well as the use of certain transmucosal antigen delivery systems might be promising new strategies to modulate type-I allergic immune responses     

Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.

Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain T(h)1-based autoimmune diseases. We have established a model of aerosol sensitization leading to T(h)2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence T(h)2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4 production were decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens-in conjugated as in unconjugated form-had different effects on the T(h)2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB-and probably also other antigen-delivery systems-strongly depend on the nature of the coupled antigen-allergen.     

Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen Bet v 1

BACKGROUND: Probiotic lactic acid bacteria (LAB) are able to modulate the host immune system and clinical trials have demonstrated that specific strains have the capacity to reduce allergic symptoms. Therefore, we aimed to evaluate the potential of recombinant LAB producing the major birch pollen allergen Bet v 1 for mucosal vaccination against birch pollen allergy. METHODS: Recombinant Bet v 1-producing Lactobacillus plantarum and Lactococcus lactis strains were constructed. Their immunogenicity was compared with purified Bet v 1 by subcutaneous immunization of mice. Intranasal application of the live recombinant strains was performed to test their immunomodulatory potency in a mouse model of birch pollen allergy. RESULTS: Bet v 1 produced by the LAB was recognized by monoclonal anti-Bet v 1 and IgE antibodies from birch pollen-allergic patients. Systemic immunization with the recombinant strains induced significantly lower IgG1/IgG2a ratios compared with purified Bet v 1. Intranasal pretreatment led to reduced allergen-specific IgE vs enhanced IgG2a levels and reduced interleukin (IL)-5 production of splenocytes in vitro, indicating a shift towards non-allergic T-helper-1 (Th1) responses. Airway inflammation, i.e. eosinophils and IL-5 in lung lavages, was reduced using either Bet v 1-producing or control strains. Allergen-specific secretory IgA responses were enhanced in lungs and intestines after pretreatment with only the Bet v 1-producing strains. CONCLUSIONS: Mucosal vaccination with live recombinant LAB, leading to a shift towards non-allergic immune responses along with enhanced allergen-specific mucosal IgA levels offers a promising approach to prevent systemic and local allergic immune responses.     

Inhibition of CD23‐dependent facilitated allergen binding to B cells following vaccination with genetically modified hypoallergenic Bet v 1 molecules

Background Vaccination with hypoallergenic recombinant Bet v 1 derivatives (Bet v 1 fragments and Bet v 1 trimer) is associated with the induction of IgG antibodies specific to natural Bet v 1. Objective To investigate whether IgG antibodies induced following vaccination with genetically modified hypoallergenic Bet v 1 derivatives are able to inhibit IgE‐facilitated binding of allergen‐IgE complexes to B cells. Methods Sera from 46 patients obtained before and after subcutaneous vaccination with Bet v 1 trimer (n=14), Bet v 1 fragments (n=11) or placebo (n=21) were incubated with recombinant (r) Bet v 1 and an indicator serum (IS) from a birch pollen‐allergic patient with high CD23 binding capacity. Bet v 1 immune complexes were added to a CD23‐expressing B cell line and co‐operative binding of Bet v1‐IgE complexes to CD23 was measured with a polyclonal anti‐IgE FITC antibody using a bio‐functional cellular flow cytometric assay. Results When sera from patients vaccinated with rBet v 1 derivatives were incubated with Bet v 1 and the IS, a reduction of IgE binding to CD23 was observed. This effect was not seen when sera from placebo‐treated patients were used. The decrease in CD23/IgE binding was statistically significant in the trimer group [pre‐ vs. post‐specific immunotherapy (SIT): P=0.02; trimer vs. placebo: P<0.04] but not in the Bet v 1 fragments‐treated group. Trimer‐treated patients had higher levels of Bet v 1‐specific IgG than fragment‐treated patients. The degree of inhibitory activity of IgE‐facilitated allergen binding correlated with Bet v 1‐specific IgG levels following SIT (R=0.492; P=0.012). Conclusion Vaccination with both recombinant Bet v 1 derivatives induces Bet v 1‐specific IgG antibodies, which are able to inhibit the co‐operative binding of allergen‐IgE complexes to CD23, and may thereby reduce allergen‐specific T cell responses. Cite this as: I. Pree, M. H. Shamji, I. Kimber, R. Valenta, S. R. Durham and V. Niederberger, Clinical & Experimental Allergy, 2010 (40) 1346–1352.     

Isolation of a high‐affinity Bet v 1‐specific IgG‐derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments

Background

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild‐type allergens. However, so far no treatment‐induced IgG antibodies have been characterized.

Objective

To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject.

Methods

A phage‐displayed combinatorial single‐chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1‐reactive antibody fragments. ScFvs were tested for specificity and cross‐reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross‐reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients’ IgE binding to ELISA plate‐bound allergens and allergen‐induced basophil activation was assessed.

Results

A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3‐1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients’ polyclonal IgE binding to Bet v 1, and partially suppressed allergen‐induced basophil activation.

Conclusion

Immunization with unfolded hypoallergenic allergen derivatives induces high‐affinity antibodies even in nonallergic subjects which recognize the folded wild‐type allergens and inhibit polyclonal IgE binding of allergic patients.     

Strategies for the development of safe and effective DNA vaccines for allergy treatment

During the past ten years, a great number of studies have demonstrated that injection of plasmid DNA coding for certain genes results in the induction of humoral and cellular immune responses against the respective gene product. The features of DNA vaccines enable a broad range of applications, including the induction of protective immunity against viral, bacterial, and parasitic infections, and open up new perspectives for the treatment of cancer. Furthermore, based on their Th1-promoting properties, DNA vaccines also turned out to balance Th2-mediated immune reactions, a quality which renders them a promising alternative for immunotherapy against allergy. Their unique immunological properties offer new possibilities for the development of vaccines, which do not cause anaphylactic side effects, a major drawback of specific immunotherapy (SIT). In this review, we present approaches to avoid the translation of native allergenic determinants, thus preventing release of allergy mediators stimulated by crosslinking of pre-existing or vaccine-induced IgE antibodies on mast cells. Three approaches are described, which fulfill these requirements: (i) cutting the allergen gene into overlapping fragments, which lack any antigenic determinant of the native allergen, but display the original repertoire of T cell epitopes, (ii) using hypoallergenic derivatives or (iii) fusing the allergen with ubiquitin, thus fragmenting the antigen and destroying its native structure. The presented experiments demonstrate that DNA vaccines are suitable to balance an allergic response in a protective as well as a therapeutic experimental design, thus demonstrating their potential for allergy treatment. In addition to conventional plasmid DNA vaccines, aspects and perspectives of replicon-based DNA vaccines will be discussed.     

Susceptibility to nasal and oral tolerance induction to the major birch pollen allergen Bet v 1 is not dependent on the presence of the microflora

The indigenous microflora plays an integrative role in the maintenance of immunological homeostasis. Several studies reported that immunological tolerance is dependent on microbial colonization of the gut. In the present study, we investigated whether the absence of the microflora influences the sensitization process to an allergen as well as the ability to develop mucosal tolerance in a mouse model of birch pollen allergy. Germ-free or conventional BALB/c mice were intranasally or intragastrically pre-treated with the major birch pollen allergen Bet v 1 prior to sensitization with this allergen. Both germ-free and conventional mice displayed comparable Th2 biased immune responses after allergic sensitization. Oral as well as intranasal tolerization led to suppression of allergen-specific serum antibodies (IgG1, IgE, IgA) as well as cytokine production by splenocytes (IL-5, IFN-gamma) in both germ-free and conventional animals. Peyer's patches of germ-free animals were approximately 20 times smaller than in conventional animals, but the relative distribution of lymphocyte subpopulations was equal. We conclude that the absence of the microflora does not influence the ability to mount Th2 responses nor to establish tolerance towards the aeroallergen Bet v 1. Our findings may challenge the view that the commensal microflora is a key factor for breakdown of physiological tolerance and allergy development.     

Suppression of allergic airway inflammation and IgE responses by a class I restricted allergen peptide vaccine

CD8 T cells are known to deviate CD4 T-cell responses from Th2 toward Th1. Reduction of Th2 cytokines and increased interferon-γ ameliorates allergic airway disease. We have developed a novel approach to the suppression of allergic airway inflammation, by designing a MHC class I-restricted allergen peptide vaccine, which induces potent and long-lived CD8 T-cell responses. Vaccination of C57BL/6 mice before allergen sensitization completely prevented allergen-specific immunoglobulin E (IgE) antibody responses. Vaccination after sensitization failed to suppress IgE, but inhibited accumulation of eosinophils and neutrophils in airways after subsequent allergen challenge. Vaccination suppressed Th2 airway infiltration and enhanced the lung Th1 response without inducing excessive CD8 cellular infiltration or interleukin-17, and the combination of class I peptide with adjuvant was more effective than adjuvant alone. Airway hyperreactivity was prevented by vaccination in an allergen-specific fashion. Class I peptide vaccines might therefore represent a robust and long-lasting immunotherapeutic strategy in allergic disease.     

Monoclonal IgE antibodies against birch pollen allergens: novel tools for biological characterization and standardization of allergens

BACKGROUND: IgE antibodies are key players in immediate hypersensitivity reactions. Allergen characterization and standardization is usually based on the sera of allergic patients, whereas monoclonal IgE antibodies specific for clinically relevant allergens are very rare. OBJECTIVE: The aim of this study was to establish IgE mAbs specific for birch pollen allergens, because these are important inhalant allergens. METHODS: IgE-producing hybridomas were identified by using the highly sensitive rat basophilic leukemia cell mediator release assay with enhanced allergen stimulation by additional cross-linking with birch pollen-specific IgG antibodies. The obtained IgE mAbs were characterized by immunologic methods and by cDNA sequencing. RESULTS: Seven IgE mAbs specific for the birch pollen allergens Bet v 1 or Bet v 6 were obtained and were all biologically active in mast cell-based assays. Mediator release experiments with mAb combinations indicated that 2 different epitope regions were recognized on Bet v 1, whereas the 2 Bet v 6-specific mAbs bound to the same epitope region. After sensitization of rat basophilic leukemia cells with IgE mAbs, different amounts of Bet v 1 or Bet v 6 were detected in commercial diagnostic allergen reagents, whereas sensitization with polyclonal IgE resulted in similar allergenic potency of all products. CONCLUSIONS: IgE mAbs represent promising novel tools for allergen characterization and component-resolved standardization of allergen extracts.