Prevention of allergen-specific IgE production and suppression of an established Th2-type response by immunization with DNA encoding hypoallergenic allergen derivatives of Bet v 1, the major birch-pollen allergen



In atopic patients, programming towards a preferential Th2 immunity leads to IgE antibody production and cellular Th2 immunity against otherwise harmless antigens. We report the development ofprophylactic and therapeutic DNA vaccines for the major birch-pollen allergen, Bet v 1. We constructed three DNA vaccines, coding for the complete cDNA, coding for two hypoallergenic fragments or coding for a hypoallergenic Bet v 1 mutant. The protective effect was studied in mice pretreated by intradermal DNA injections, then sensitized with Bet v 1 protein. Mice pretreated with any of the three Bet v 1-specific DNA vaccines were protected against allergic sensitization to Bet v 1. Protection was characterized by a lack of Bet v 1-specific IgE production, a lack of basophil activation and an enhanced IFN-γ expression. DNA vaccines with wild-type Bet v 1 induced strong Bet v 1-specific antibody responses whereas DNA vaccines with hypoallergenic Bet v 1 derivatives induced no (fragments) or only transient (mutant) Bet v 1-specific antibody responses. A therapeutic approach with the fragment-DNA vaccine reduced IgE production and stimulated a sustained Th1 cytokine milieu. Our results demonstrate that DNA vaccines with hypoallergenic forms of the allergen specifically protect against sensitization and suppress established Th2-type responses. This concept may be applied for the development of safe and specific DNA vaccines for the prophylaxis and therapy of allergic diseases.


Recombinant Bet v 1a


Rat basophil leukemia

1 Introduction

More than 25% of the population in civilized countries suffers from IgE-mediated allergies 1. Because of complex genetic and environmental factors, atopic persons are prone to mount IgE antibody responses and Th2-like cellular responses to otherwise harmless environmental antigens 2. Sensitization to allergens, a process that takes place early in childhood, requires IL-4-triggered class-switch to IgE production and leads to the establishment of specific IgE antibody responses, which are boosted by repeated allergen contact 35. At present, allergen-specific immunotherapy represents the only curative approach towards therapy of IgE-mediated allergies 6, 7.

Genetic immunization has been demonstrated as a powerful alternative to protein-based vaccination in the past few years 810. It has been shown that injection of plasmid DNA encoding clinically relevant allergens can induce immune responses with a Th1 bias and promote the formation of IFN-γ-producing CD4+ T cells 11. These IFN-γ-secreting Th1 cells can stimulate B cells to synthesize IgG2a antibodies and inhibit IgE production 1116.

To develop strategies for minimizing the risk of anaphylactic side-effects resulting from the potential expression of biologically active allergens following DNA vaccination, we have studied the prophylactic and therapeutic effects of vaccines based on cDNA coding for T cell epitope-containing allergen variants with reduced allergenic activity after sensitization with the active wild-type allergen 17, 18. The major birch-pollen allergen Bet v 1, representing one of the most frequent environmental allergens and against which more than 100 million allergic patients are sensitized, was used as a model 19. Recently, recombinant hypoallergenic versions consisting of two fragments comprising aa1–74 and aa75–160 of Bet v 1 as well as a Bet v 1 mutant have been developed 17, 18, which exhibited a strongly reduced allergenic activity in birch-pollen-allergic patients but retained Bet v 1-specific T cell epitopes.

Here we constructed DNA vaccines containing the cDNA coding for the hypoallergenic Bet v 1 derivatives and compared them with a vaccine based on the Bet v 1 wild-type DNA regarding their ability to specifically prevent allergic sensitization to the Bet v 1 allergen and to convert an existing Th2 type response. Our finding that DNA vaccines formulated on the basis of hypoallergenic T cell epitope-containing allergen-derivatives can prevent and modulate allergic sensitization to the wild-type allergen is discussed regarding the use of such DNA vaccines for specific prophylaxis and therapy of allergic diseases.

2 Results

2.1 Genetic immunization with hypoallergenic Bet v 1 derivatives induces no or only transient antibody production specific for recombinant Bet v 1a

Animals immunized with recombinant Bet v 1a (rBet) protein as well as those genetically immunized with the gene vaccine encoding the entire Bet v 1a coding region (pCMV-Beta) produced a strong humoral immune response (Fig. 1A and B). The subclass response after protein immunization was restricted mainly to IgG1, whereas DNA immunization induced the expression of high levels of allergen-specific IgG1 and IgG2a antibodies. Genetic immunization with the Bet v 1 mutant (pCMV-Betmut) elicited a delayed onset and only transient production of both antibody subclasses specific for Bet v 1a (Fig. 1C). No antibody response against rBet was induced by the DNA vaccine encoding the fragments (pCMV-Bet1/74 and pCMV-Bet75/160) of the allergen (Fig. 1D).

Figure 1.

Bet v 1-specific IgG1 and IgG2a immune responses. BALB/c mice (n=9) were immunized with rBet protein or plasmid DNA, and sensitized with rBet protein (n=5) or left untreated (n=4; white symbols). Antibody responses are displayed as kilophoton counts / sec, immunizations/sensitizations are indicated by arrows.

2.2 Genetic vaccination modulates the subsequent immune response induced by sensitization with recombinant allergen

To investigate whether the Th1-type stimulus (see Table 1 for secretion of IFN-γ in supernatants of stimulated spleen cells) induced by intradermal genetic immunization has any effect on the subsequent response induced by protein immunization, all three groups (pCMV-Beta, pCMV-Betmut, and pCMV–Bet1/74 and 75/160) were boosted twice with 5 μg rBet 6 weeks after the last DNA immunization. In the pCMV-Beta group the rBet boost induced a further increase of IgG1 and IgG2a antibodies (Fig. 1B). The antibody levels in animals without protein boosting reached a peak at 60 days after the first injection and then declined (Fig. 1B).

The groups pre-immunized with the Bet v 1 mutant or with the Bet v 1a-fragment gene vaccines showed a strong induction of IgG1 and IgG2a after boosting with rBet (Fig. 1C and D). The influence of the DNA-pre-immunization can also be seen by comparing Fig. 1A and D, which demonstrates that DNA immunization with fragments of Bet v 1a does not induce Bet v 1a-specific antibodies but modulates the subsequent response against rBet by inducing elevated levels of allergen-specific IgG2a antibodies (p=0.024 compared with protein immunization). In contrast, protein immunization of a group of animals without DNA pre-immunization primarily induced IgG1 antibodies and only very low levels of IgG2a antibodies (Fig. 1A). Pre-immunization with plasmid DNA encoding the irrelevant allergen Phl p 5 did not influence the response against rBet (Fig. 1B).

Table 1. Secretion of IFN-γ in supernatants of stimulated spleen cells (protective approach)a)
Antigen used for stimulationTreatment
 rBet pCMV-BetapCMV-Beta + sensitizationpCMV-BetmutpCMV-Betmut + sensitizationpCMV-Bet1/74 and pCMV-Bet75/160pCMV-Bet1/74 and pCMV-Bet75/160 + sensitization
  1. a) Spleen cells of mice (n=3) were harvested after various immunization ± sensitization treatments — 6 months after the last DNA immunization or 5 months after the second protein immunization — and cultured in the presence of various antigens used as stimulants. IFN-γ levels are expressed as pg/ml. n.d., not determined.

rBet117.3 ± 2.3 312.5 ± 35.0 367.1 ± 85.9 422.9 ± 98.4346.1 ± 79.3614.8 ±113.3512.9 ± 204.8
rBet mutant126.9 ± 5.2 349.6 ± 43.7 491.6 ± 164.1460.9 ± 76.7395.0 ± 73.8n.d.n.d.
rBet aa1–74134.6 ± 4.8 404.9 ± 116.6410.3 ± 82.4 n.d.n.d.601.0 ±29.1 442.0 ± 120.3
rBet aa75–160130.4 ± 5.7 484.5 ± 44.8 389.1 ± 18.8 n.d.n.d.n.d.339.7 ± 36.2 
Birch-pollen extract187.2 ± 22.9341.4 ± 24.2 426.9 ± 127.0527.3 ± 63.8580.5 ± 98.6 642.3 ± 153.7572.5 ± 108.7
Medium control124.0 ± 1.8 128.0 ± 6.9 117.7 ± 6.7 114.5 ± 7.8 118.9 ± 9.8 116.9 ± 8.9 115.7 ± 14.4 

2.3 Genetic immunization specifically inhibits the induction of Bet v 1-specific IgE responses and Bet v 1-induced immediate reactions

Fig. 2 shows the levels of specific anti-Bet v 1a IgE antibodies in the serum 4 weeks after protein administration. Genetic pre-immunization with any of the three constructs was able to prevent the induction of IgE antibodies induced by protein sensitization (p ranging between <0.001 and <0.006) (Fig. 2A). In contrast, pre-immunization with pCMV-Phlp5 and subsequent sensitization with rBet did not prevent the induction of anti-Bet v 1a IgE antibodies (Fig. 2B).

To determine the extent to which genetic immunization can prevent immediate-type reactions, an effector-cell test was used. The measurement of allergen-specific degranulation of rat basophil leukemia (RBL)-2H3 cells passively sensitized with murine sera was performed by the detection of β-hexosaminidase release after cross-linking of receptor-bound antibodies with birch-pollen extract (Fig. 3). Sera from rBet-sensitized mice induced Bet v 1-specific basophil degranulation (Fig. 3A). By contrast, sera from mice pretreated with Bet v 1-specific genetic immunization did not induce degranulation regardless of a subsequent Bet v 1 protein injection was given (dark-gray bars) or not (light-gray bars) (Fig. 3A). Preimmunization with Phl p 5 plasmid DNA and boosting with rBet did not inhibit Bet v 1-specific degranulation (Fig. 3B, right-hand histogram compared with left-hand histogram).

Figure 2.

Levels of Bet v 1a-specific IgE 4 weeks after sensitization. (A) IgE responses of mice pretreated with plasmid DNA (pCMV-Beta, pCMV-Betmut or pCMV-Bet1/74 and 75/ 160) and sensitized with rBet. (B) IgE response of mice pretreated with pCMV-Phlp5 plasmid DNA and sensitized with rBet. Control = untreated animals, rBet = sensitization without DNA pre-immunization. RLU, relative light units.

Figure 3.

RBL-cell release assay performed with sera obtained 4 weeks after sensitization. RBL-2H3 cells were passively sensitized with murine sera obtained on day 91 and cross-linking of receptor-bound antibodies was performed with birch-pollen extract. (A) Release values induced with sera from mice immunized with plasmid DNA alone (n=4; light-gray shading) or immunized, then sensitized with rBet (n=5: dark-gray shading). (B) Basophil release with sera of mice immunized with pCMV-Phlp5 alone (n=3) or immunized with pCMV-Phlp5 plasmid DNA, then sensitized with rBet (n=3). Control (n=6) = sera of untreated animals, rBet (n=6) = sera of mice sensitized without DNA immunization.

2.4 Genetic immunization establishes a long-lasting antigen-specific Th1 response associated with IFN-γ production

To investigate whether a long-lasting and allergen-specific Th1 memory was induced by DNA pre-immunization, in vitro stimulation of spleen cells was performed with rBet and birch-pollen extract (Fig. 4). The antigen-specificity of each group was proven by stimulation with ovalbumin, which gave values comparable to the non-immunized control group. Lymphoproliferation assays performed 230 days after the last injection of the gene vaccine still revealed a significantly elevated stimulation index (Fig. 4B) compared with non-immunized animals (Fig. 4A). Protein boost of DNA-preimmunized animals did not enhance the proliferative activity of spleen cells (Fig. 4C).

The analysis of the culture supernatants revealed that in all groups pre-immunized with the gene vaccine, IFN-γ was significantly raised (p<0.024) in comparison with mice who had only received protein (Table 1). The Bet v 1a-specific T cell response was not restricted to pCMV-Beta, but was also detected in mice treated with pCMV-Betmut and pCMV-Bet1/74 and 75/160. No statistically significant differences could be found between the groups treated by DNA vaccination. Determination of IL-5 revealed no statistically significant differences between all groups (data not shown).

Figure 4.

Allergen-specific proliferation of isolated spleen cells. Splenocytes of control and immunized animals (n=3) were cultured with rBet (white histograms) or birch-pollen extract (shaded histograms), and [3H]thymidine uptake was measured. (A) Non-immunized animals. (B) Stimulation index of the groups immunized with plasmid DNA without subsequent sensitization. (C) Stimulation index of the groups immunized with plasmid DNA followed by sensitization.

2.5 Genetic immunization with hypoallergenic Bet v 1 fragments modulates an established Th2 response

To address the therapeutic potential of the hypoallergenic DNA vaccine encoding the fragments of Bet v 1a, a Th2 type response was established by sensitizing mice with rBet followed by a treatment with the vaccine. Two groups of animals — one injected with recombinant allergen but not with plasmid DNA and one group receiving a mock-vector lacking the Bet v 1a gene — served as control groups.

The primary sensitization with rBet induced a predominant Th2 type response as indicated by high levels of IgG1 and IgE and only marginal IgG2a titers (Fig. 5). Two injections of DNA vaccine significantly reduced the IgE titers and IgE-mediated release (p<0.05). IgE-reduction could also be observed with the mock vector (statistically not significant) pointing to the well-known non-specific Th1 effect 20 of plasmid DNA (the "CpG effect"). Immunization with the fragment vaccine or the mock-vector decreased the IgG1 and IgG2a antibody subclass levels, with a more pronounced effect for IgG1, but none of the differences proved to be statistically significant because of high standard deviations. An additional sensitization with one injection of rBet followed by one injection of DNA further boosted the response of all antibody classes, but the IgE-suppressive effect of the DNA vaccine was maintained. Concerning the IgE-mediated cell release, the therapeutic effect of the mock-vector almost completely disappeared after the second sensitization.

Analysis of a panel of cytokines in supernatants of proliferation cultures from experimental day 195 (Table 2) elicited an increase of IFN-γ (p=0.02) and a decrease of IL-5 (p=0.02), indicating that the IgE-down-regulating effect may be a consequence of the DNA-vaccine-induced Th1 shift. Furthermore, neither IL-2 (p=0.06) nor IL-10 production (p=0.33) was significantly changed.

Figure 5.

Course of RBL-cell release and antibody-subclass levels after sensitization with rBet and treatment with a DNA vaccine encoding hypoallergenic fragments of Bet v 1a. BALB/c mice (n=6 for each group) were sensitized with rBet followed by alternate injections of plasmid DNA and protein (indicated by arrows). A mock-vector group served as indicator for antigen-independent CpG effects. A second control group (rBet) was injected with recombinant allergen according to the same immunization-schedule but received no plasmid DNA. Significant differences (p<0.05) between the rBet group and the DNA vaccine group are indicated with asterisks, differences between the DNA-vaccine group and the mock-vector group are indicated with x.

Table 2. Secretion of cytokines in supernatants of stimulated spleen cells (therapeutic approach)a)
 IFN-γ IL-5IL-2IL-10
  1. a) Spleen cells of mice (n=6) were harvested on day 195 of the therapeutic schedule (see experimental design). Cytokine levels are expressed as mean ± SEM pg/ml. pCMV-Bet1/74 Bet75/160 (Group 2) = animals sensitized with rBet and treated with Bet v 1a fragments, mock-vector (Group 3) = animals sensitized with rBet and treated with mock-vector, rBet (Group 1) = animalssensitized with rBet according to the same immunization-schedule but without treatment with plasmid DNA. p values are shown for comparisons between groups as indicated.

Group 1: rBet 28.69 ± 19.66 474.75 ± 196.331083.39 ± 163.06627.19 ± 408.54
Group 2: pCMV-Bet1/74 Bet75/160 112.8 ± 75.05 229.71 ± 94.17  388.5 ± 169.99 989.9 ± 777.34
Group 3: mock-vector160.70 ± 174.22344.65 ± 158.21 772.14 ± 575.77793.14 ± 995.66
p Group 1 vs. Group
p Group 1 vs. Group
p Group 2 vs. Group 30.550.150.290.71

3 Discussion

On the basis of the fact that immunization with allergen-encoding plasmids established an allergen-specific Th1 response and was able to antagonize established allergen-specific Th2 responses in experimental animal models, genetic vaccination has been suggested for immunotherapy of IgE-mediated allergies 1116.

In this study we have evaluated the protective and therapeutic capacity of DNA vaccines encoding derivatives of the birch-pollen allergen Bet v 1 and addressed the issues of allergen-specificity and the hypoallergenic nature of the gene product. DNA vaccines based on hypoallergenic Bet v 1 fragments and a Bet v 1 mutant containing the T cell epitope repertoire of the wild-type allergen were compared with a DNA vaccine based on the Bet v 1 wild-type allergen.

The rationale of the first approach was that gene products of the Bet v 1a fragments should lack three-dimensional structures related to the native Bet v 1a, thus lacking cross-reacting B cellepitopes. Like the situation after protein immunization, genetic immunization leads to effective processing of the fragments by APC and presentation of epitopes via MHC class II molecules. However,genetic immunization activates not only Th2 cells but also a substantial number of Th1 cells as indicated by IgG2a antibody response, and additionally stimulates a Th1-biased cytokine milieu. With respect to their Bet v 1a T cell epitope-specificity, these helper cells may also interact with B cells that had recognized and processed native allergen during sensitization. Therefore, the distribution of Th1 and Th2 cells after DNA immunization will influence the milieu and response-type after a sensitization with the native allergen. The increase of IgG2a antibodies in the groups vaccinated with plasmid DNA and boosted with protein could be interpreted in that way and, obviously, DNA immunization had created sufficient Bet v 1a-specific Th1 cells that were able to modulate a subsequent immune reaction against the native allergen.

This assumption was confirmed by the lymphoproliferation assays and cytokine measurements. Six months after the third injection of plasmid DNA encoding Bet1/74 and Bet75/160 (without subsequent sensitization) in vitro stimulation of cultured spleen cells elicited allergen-specific proliferation induced by rBet and the recombinant fragments. Fragment 75/160 was superior to fragment 1/74, which is also in agreement with data, obtained by protein immunization with identical fragments, that reveal the C-terminal fragment as the immunodominant one 17, and with the results from epitope mapping, which found the immunodominant helper epitopes for BALB/c in the C-terminal region 21.

Moreover, using the DNA vaccines encoding the hypoallergenic fragments in a therapeutic approach revealed that stimulation of an allergen-specific Th1 type response can also suppress an established IgE response. The IgE-suppressing effect could be maintained after repeated sensitization. This second round of sensitization and DNA immunization apparently reduced the antigen-independent processes (or enhanced the antigen-specific ones) as indicated by the decreased influence of the mock-vector treatment on IgE-mediated cell release. Increased IFN-γ and decreased IL-5 levels in supernatants of proliferation cultures from cells harvested 52 days after the last treatment indicate a sustained Th1 effect of the DNA vaccines.

The second type of DNA-based vaccine developed for decreasing the risk of anaphylactic side-effects of immunotherapeutic approaches utilized a hypoallergenic Bet v 1 mutant 18. As with the allergen fragments, the basic intention was to investigate whether the advantage of the Th1-inducing genetic immunization could be combined with the hypoallergenic property to createa safe and effective vaccine. For this purpose we selected a recently designed hypoallergenic mutant of Bet v 1 with low IgE-binding but normal T cell activation 18. The DNA-based response to this Bet v 1 mutant was strikingly different from that against the native Bet v 1a, with a very unusual course of the humoral response thus pointing to the altered immunogenicity and possibly hypoallergenic properties also in the mouse. Nevertheless, the Bet v 1 mutant vaccine induced protection from an allergic response against Bet v 1a wild-type protein.

The mechanism for the altered humoral immunogenicity of hypoallergens escapes us at present. An altered affinity-maturation of IgE antibodies induced by the hypoallergenic molecules may be oneexplanation. In the case of the hypoallergen Bet v 1d gene, which displays 95% homology with the sequence of pCMV-Beta, the number of mutations is small, and computer-aided structural analysis gaveno hint for drastic changes of the three-dimensional protein structure 12. Antibodies produced against Bet v 1a recognize the hypoallergenic isoform 12 and theBet v 1 mutant (data not shown). Nevertheless, our data indicate that a genetic vaccine coding for a hypoallergen can fulfill the criterion of weak antibody production but antigen-specific Th1 induction and protection against an allergic immune response.

Among the three different approaches presented in this work, namely a DNA vaccine encoding the native allergen, the fragmented allergen or a hypoallergenic mutant, we consider the fragments asthe best alternative. This approach ideally fulfills the requirements for lacking anaphylactic side-effects and the induction of anti-allergic immune responses.

In summary, we anticipate that the use of DNA vaccines based on hypoallergenic allergen derivatives may help to advance clinical evaluation and application of allergen-specific prophylactic and therapeutic DNA vaccination strategies.

4 Materials and methods

4.1 Experimental design

Groups of nine female BALB/c mice received three intradermal injections of 100 μg plasmid DNA coding for the major birch-pollen allergen Bet v 1a (pCMV-Beta), a Bet v 1 mutant (pCMV-Betmut) or a plasmid mix encoding two fragments of Bet v 1a (pCMV-Bet1/74 and pCMV-Bet75/160) at 1-week intervals. At days 57 and 64 after the first injection, five animals of each group were sensitized with rBet v 1a. The remaining four animals served as non-sensitized controls. In addition a group of six untreated animals was sensitized with rBet v 1.

To address the question whether the effects of DNA preimmunization was antigen-specific or due to the adjuvant effect of Th1-inducing CpG motifs present in the plasmid DNA, another group of six animals was preimmunized with a plasmid DNA-construct encoding the timothy-grass-pollen allergen Phl p 5 (pCMV-Phlp5) using the same immunization schedule.

To study the therapeutic efficacy of the Bet v 1a-fragment-vaccine, groups of six female BALB/c mice received three subcutaneous injections (days 0, 21 and 31) of rBet. In that manner, sensitized mice were treated with Bet v 1a-fragment-vaccine or mock-vector at days 55 and 62. At day 110 they again received one injection of rBet followed by a further treatment with plasmid DNA 14 days later. Another group of six animals was injected with recombinant allergen according to the same immunization-schedule but received no plasmid DNA.

4.2 Construction of expression vectors

Expression vectors pCMV-Bet1/74 and pCMV-Bet75/160 were constructed from pCI (Promega, Madison, WS, USA) and code for the amino acids 1–74 and 75–160 of Bet v 1a.

pCMV-Beta 13 was used as a template for the PCR amplification of the two Bet v 1 fragments. For pCMV-Bet1/74, the forward PCR primer, which incorporated an EcoRI site, was 5′-GGTTGGAGAATTCATGGGTGTTTTCAATTAC-3′. The reverse primer which incorporated a stop codon (italicized) and a XbaI site, was 5′-GTTGGATCTAGATTACTCATCAACTCTGTCCTT-3′.

The forward primer for pCMV-Bet75/160 5′-GTTGGAGAATTCATGGTGGACCACACAAACTTC-3′contained an EcoRI site and a ATG-start codon (italicized); the reverse PCR primer, which incorporated an XbaI site, was 5′-GGTTGGATCTAGATTAGTTGTAGGCATCGGAGTG-3′.

The EcoRI-XbaI-digested PCR amplicons were ligated into an EcoRI-XbaI-digested pCI vector.

For the hypoallergenic mutant pCMV-Betmut, PCR amplification of the coding region of pMW172-Betmut 18 was conducted using the forward primer 5′-GAATTCATGGGTGTTTTCAATTACGA-3′ and the reverse primer 5′-TCTAGATTAGTTGTAGGCATCGGAGT-3′. The resulting fragment was subcloned into an EcoRI-XbaI-digested pCI vector.

pCMV-Phlp5 was constructed by subcloning PCR fragments generated from lambda gt11-cDNA 22.

4.3 DNA preparation

Large-scale purification of the expression vectors was conducted with Endo Free Plasmid Giga kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The endotoxin level in the plasmid DNA was <3 EU/ml as measured by the Pyroquant (Limulus amebocyte lysate) assay (Pyroquant Diagnostik, Moerfelden, Germany). Plasmid DNA was then analyzed by agarose gel electrophoresis and quantified by spectrophotometry (OD260:OD280 ratio >1.8). The plasmid DNA was stored in endotoxin-free H2O at –20°C.

4.4 DNA immunization and antigen sensitization

Female, 6–10-week-old BALB/c mice were obtained from the animal breeding facilities in Himberg, Austria, and maintained according to the local guidelines for animal care.

Mice were immunized intradermally into the shaved back with a total of 100 μg of plasmid DNA (pCMV-Beta or pCMV-Betmut) in a volume of 200 μl sterile PBS, Bet v 1a fragments were injected in a mixture of 100 μg pCMV-Bet1/74 and pCMV-Bet75/160 each. For sensitization, 5 μg rBet adsorbed to 100 μl Al(OH)3 (Serva, Heidelberg, Germany; 2 mg/ml) was injected subcutaneously in a total volume of 150 μl sterile PBS. The protective and therapeutic approaches were performed according to the experimental design as described above.

4.5 Analysis of serum antibody titers

Black 96-well high-bind immunoplates (Greiner, Kremsmünster, Austria) were coated by overnight incubation at 4°C with 100 ng rBet/well in PBS. Plates were blocked with blocking buffer (PBS + 0.5% BSA + 0.2% Tween-20) for 1 h at room temperature. Sera were diluted 1:100 in blocking buffer and incubated for 1 h at room temperature. Horseradish-peroxidase-conjugated detection antibodies were rat anti-mouse IgG1, rat anti-mouse IgG2a or rat anti-mouse IgE (Serotec, GB) and were diluted 1:1000 in blocking buffer and reacted for 1 h at room temperature. The assay was developed with luminol (BM chemiluminescence substrate, Boehringer Mannheim, Mannheim, Germany) diluted 1:1 in H2O. Chemiluminescence (photon counts/second) was quantified by using a Lucy I ELISA-plate luminometer (Anthos-Labtec, Salzburg, Austria).

4.6 Lymphoproliferation

Splenocytes were prepared 5 and 6 months after the last protein or DNA immunization, respectively, erythrocytes were lysed, cells resuspended in MEM supplemented with 100 U of penicillin and streptomycin/ml, 5% heat-inactivated fetal calf serum, 2×10–6 M β-mercaptoethanol, 1 mM sodium pyruvate, 2 mM L-glutamine, 20 mM Hepes and non-essential amino acids, and were plated into 96-well, flat bottom tissue culture plates (BD-Falcon, NJ, USA) at a density of 4×105 cells/well. Five replicate wells were stimulated with 20 μg/ml rBet or birch-pollen extract 23 for 4 days under conditions of 37°C, 95% relative humidity, and 7.5% CO2. Ovalbumin (20 μg/ml) and medium alone served as negative controls. Wells were pulsed with 2 5 μCi of [3H]thymidine (Amersham, GB)/ml for an additional 24 h and then harvested with a cell harvester (Harvester 96 Mach IIIM, Tomtec Inc., CT, USA). Thymidine incorporation was measured in a scintillation counter (Wallac MicroBeta TriLux, Perkin Elmer, Finland).

4.7 Quantification of cytokines in culture supernatants

For determination of the cytokine production of spleen cells, cells were cultured with or without 20 μg/ml Bet v 1a in 48-well plates (Falcon, Becton Dickinson Labware, NJ, USA) at a concentration of 2×106 cells/well. After 72 h, supernatants were harvested and stored at –20°C until examination.

Cytokine levels (protective approach, Table 1) were quantified by sandwich ELISA using the OptEIA system (PharMingen, NY, USA) according to the manufacturer's instructions.

The concentration of IL-2, IL-5, INF-γ and IL-10 (therapeutic approach, Table 2) was measured by Fluorescent Bead Immunoassay (Bender MedSystems, Vienna, Austria) on a Luminex100 LabMap System (Luminex, Austin, TX, USA) according to the manufacturer's instructions.

4.8 RBL-cell mediator release

RBL-2H3 cells were plated in 96-well tissue culture plates (4×104/well) and incubated for 24 h at 37°C using 7% CO2. Passive sensitization was performed by incubation with murine sera raised against the birch-pollen allergen Bet v 1a at a final dilution of 1:30 for 2 h. To remove unbound antibodies, the cell layer was washed twice in Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 5.6 mM D-glucose, 12 mM NaHCO3, 10 mM Hepes and 0.1% BSA, pH 7.2). Cross-linking of the FcϵR-bound IgE and subsequent degranulation of RBL cells was induced by adding 100 μl birch-pollen extract (c=1.5 μg/ml) in Tyrode's buffer for 30 min in a humidified atmosphere at 37°C. Supernatants were analyzed for β-hexosaminidase activity by incubation with 80 μM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma) in citrate buffer (0.1 M, pH 4.5) for 1 h at 37°C. The reaction was stopped by addition of 100 μl glycine buffer (0.2 M glycine, 0.2 M NaCl, pH 10.7) and fluorescence was measured at λex: 360/λem:465 nm using a fluorescence microplate reader (Spectrafluor, Tecan, Austria). Results are reported as percentage of total β-hexosaminidase released after addition of 1% Triton X-100.

4.9 Statistical analysis

Data are expressed as means±SEM. Statistical significance was assessed by the non-parametric Mann Whitney rank-sum test.


This work was supported in parts by the Fonds zur Förderung der wissenschaftlichen Forschung (P13827, S8802, S8811, S8813 and Y078GEN), the Ludwig-Boltzmann-Institute for Experimental Surgery and the ICP program of the Austrian Federal Ministry for Education, Science and Culture.


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