Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M607938200 on December 14, 2006

J. Biol. Chem., Vol. 282, Issue 6, 3778-3787, February 9, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/6/3778    most recent
M607938200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gafvelin, G.
Right arrow Articles by Punnonen, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gafvelin, G.
Right arrow Articles by Punnonen, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens*

Guro Gafvelin{ddagger}12, Stephen Parmley§1, Theresa Neimert-Andersson{ddagger}, Ulrich Blank||, Tove L. J. Eriksson{ddagger}, Marianne van Hage{ddagger}, and Juha Punnonen§

From the {ddagger}Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, 17176 Stockholm, Sweden, §Maxygen Inc., Redwood City, California 94063, Inserm U699, F-75018 Paris, France, and the ||FacultédeMédecine, Site Xavier Bichat, Université Paris 7-Denis Diderot, F-75018 Paris, France

Received for publication, August 18, 2006 , and in revised form, December 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The large increase in allergic diseases observed during the past decades has urged the development of safe and efficient treatments of allergy. Many new concepts based on targeting the underlying immunological causes of IgE-mediated allergy have been suggested (1–5). However, the only treatment that causes a long-lasting relief of symptoms is allergen-specific immunotherapy (ASIT).3 Today ASIT is based on the repeated administration of allergen extracts prepared from the allergen source. Although successful clinical outcomes are well documented, several problems are associated with allergen extract-based ASIT, including the risk of inducing local and systemic side effects and induction of new sensitizations (6, 7).

Large batches of well defined single allergen components can be produced using recombinant techniques, making it feasible to solve problems linked to allergen extract-based ASIT. The most severe side effects of ASIT are caused by the binding of injected allergen to allergen-specific IgE on high affinity Fc{epsilon}RI receptor-bearing effector cells, leading to cross-linking of the Fc{epsilon}RI receptors, degranulation, and release of anaphylactogenic mediators. Therefore, allergens with reduced IgE binding capacity have been proposed to improve the safety of ASIT (8, 9). Recently birch pollen allergic patients were treated with such hypoallergenic molecules for the first time (10).

Numerous strategies have been used to generate hypoallergens. The most straightforward approach is to target specific IgE-binding B-cell epitopes and disrupt them by site-directed mutagenesis (11–15). However, this requires knowledge of the most important IgE epitopes, which is seldom available. Another problem is that even when B-cell epitopes of an allergen have been thoroughly analyzed, mutations at the critical sites do not necessarily lead to reduced IgE reactivity in all patients (16, 17). Alternatively, molecular modifications that change the overall three-dimensional structure of the protein can be performed. Such modifications include disruption of disulfide bonds (18–20), oligomerization (21, 22), duplication of amino acid sequences (23), fragmentation (24, 25), and introduction of prolines (16) or point mutations in known functional protein domains (25). These approaches have proven successful for many allergens, but it is often difficult to predict modifications that result in hypoallergenic properties while retaining proper conformational structure and T-cell epitopes that are important for induction of allergen-specific IgG.

Directed molecular evolution by DNA shuffling and screening offers a way of introducing a desired property to a protein without detailed structural or functional knowledge of the protein (26–28). These techniques enable the generation of large libraries of genes from which encoded proteins can be selected on the basis of acquisition or improvement of a certain quality. In a common format, DNA from two or more homologous genes are fragmented and then reassembled by primerless PCR resulting in a large pool of chimeric genes. The variants in the resulting library are then screened for candidate genes with a given biochemical or structural property, often based upon functional assays. To improve a desired property, multiple rounds of DNA shuffling and screening may be carried out to select highly improved variants of the starting genes (29). This approach has previously been successfully applied on, for example, cytokines, antibodies, co-stimulatory molecules, promoters, viruses, and viral antigens (30–35). Given the difficulties in predicting how to construct hypoallergens, directed molecular evolution provides promising approaches to create modified allergens for use in ASIT (29).

In this study we applied multigene recombination to three group 2 mite allergen genes, two isoforms of the major allergen from the dust mite Lepidoglyphus destructor, Lep d 2 (36) and a group 2 allergen from the related dust mite Glycyphagus domesticus, Gly d 2 (37). The two Lep d 2 isoforms, Lep d 2.01 and Lep d 2.02, share 89.6% identity at the amino acid level and Gly d 2 share 79.2% identity with Lep d 2.01 and 78.4% with Lep d 2.02. Thus, these three dust mite group 2 allergens are well suited as model genes to test the concept of directed molecular evolution for generating hypoallergens.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sera and Peripheral Blood Mononuclear Cells (PBMC)— Blood samples were drawn from eight patients who had earlier participated in a study on storage mite allergy (38). They all had IgE to Lep d 2 (39) and positive skin prick tests to recombinant (r) Lep d 2.01 (40). The ImmunoCAP values for L. destructor (Pharmacia CAP System, Phadia AB, Uppsala, Sweden) ranged between 0.35–12.6 kUA/liter, median 2.1 kUA/liter. In addition, all eight patients had been shown to respond to rLep d 2 in lymphoproliferation tests (41). PBMC from seven of these patients were prepared by Ficoll-Paque (Amersham Biosciences) gradient centrifugation as earlier described (41). The PBMCs were frozen at 107 cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 25 µg/ml gentamicin (Invitrogen), 50% (v/v) heat-inactivated fetal calf serum (Hyclone, Logan, UT), and 10% dimethyl sulfoxide (Merck, Darmstadt, Germany), initially 1 °C/min in freezing containers (Nalgene Cryo 1o, Nalge Company) to -80 °C and then transferred to -150 °C where the cells were stored until use. A serum pool was prepared for screening, consisting of sera from all eight patients, and a ninth patient (27.8 kUA/liter to L. destructor and positive to rLep d 2 in immunoblotting). The study was approved by the regional ethics committee at the Karolinska Institute, and informed consent was given by each patient.

Animals—Female AJ mice (12 weeks of age) were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed with food and water ad libitum. The experiments were approved by the Swedish local ethics committee for animal welfare (N348/05).

DNA Shuffling of Allergen Genes—DNA encoding the two isoforms of Lep d 2, Lep d 2.01, and Lep d 2.02 (36), and Gly d 2 (37) served as templates for the DNA shuffling of Group 2 mite allergens. The allergen genes were originally cloned into pET vectors (Novagen Inc., Madison, WI) including His6 tag-encoding sequences at the 3'-ends. The three allergen genes were subsequently cloned into the lac promoter construct pCK200P comprising a pelB leader fused to the N terminus of the allergen inserts and a His6 tag at the C terminus. A chimeric construct was made, which carries the first 20 codons of Gly d 2 and codons 21–125 of Lep d 2.01. The resulting chimeric gene was shuffled with full-length Lep d 2.02 and Gly d 2 genes using multigene recombination as previously described (28, 30, 32, 33). The shuffled library of DNA inserts was cloned into pCK200P. Parental constructs and selected clones were sequenced on an ABI DNA sequencer (ABI, Foster City, CA).

Protein Expression and Purification—Escherichia coli harboring pCK200P containing wild-type allergens or shuffled clones were induced to express soluble protein. Briefly, overnight cultures grown at 37 °C in 2x YT medium with chloramphenicol (100 µg/ml) were diluted 5-fold into fresh 2x YT, chloramphenicol 100 µg/ml, soluble protein expression was induced with 1 mM isopropyl 1-thio-beta-D-galactopyranoside at 22 °C, and bacteria were grown for 4 h with vigorous shaking. The cells were pelleted, and soluble proteins were released from the periplasm by incubation with lysozyme and DNase I. Cells, cellular debris, and insoluble material were removed by centrifugation and filtration in a Steriflip® filtration device (Millipore, Billerica, MA). Recombinant proteins in the soluble extracts were purified on cobalt-Sepharose (Talon®, Clontech Laboratories, Palo Alto, CA) according to the manufacturer's recommendations.

Wild-type genes and shuffled inserts were also cloned into vector pCK700i, a lac promoter construct for cytoplasmic expression, which lacks a leader sequence at the N terminus and carries a His6 tag at the C terminus. Recombinant proteins were induced at 37 °C, cells lysed as above, and the insoluble fraction dissolved in extraction buffer (8 M urea, 25 mM HEPES pH 7.5, 300 mM NaCl). Extracts were loaded on nickel-Sepharose (Qiagen, Valencia, CA), washed with extraction buffer, and eluted in extraction buffer containing 250 mM imidazole. Fractions were diluted 10-fold in PBS and refolded overnight at 4 °C. Proteins were dialyzed in PBS, clarified by centrifugation, and passed over a Detoxi-gel® endotoxin removal column (Pierce). The endotoxin levels were analyzed using a Limulus Amebocyte Lysate Endochrome assay (Charles River Endosafe, Charleston, SC).

High Throughput (HTP) Expression—For HTP screening, E. coli harboring the plasmid were picked with a Q-BOT robotic colony picker (Genetix Pharmaceuticals, Tarrytown, NY) into 96-deep well plates containing 800 µl of 2x YT, chloramphenicol 100 µg/ml per well, and grown at 37 °C. Overnight cultures were diluted 10-fold into fresh 2x YT, chloramphenicol 100 µg/ml, 1 mM isopropyl 1-thio-beta-D-galactopyranoside, and soluble protein expression was induced at 22 °C and grown for 4 h with vigorous shaking. The cells were pelleted, and soluble proteins were released from the periplasm by incubation with lysozyme. Cells, cellular debris, and insoluble material were removed by centrifugation, and filtration of the soluble material through a Multiscreen® 96-well filter plate (Millipore).

Protein Quantification in Crude Lysates—The quantity of soluble, recombinant protein in crude extracts from HTP expression was indirectly estimated using a competitive immunoassay that detects the His6 tag on each protein. Briefly, a previously determined optimal concentration of biotinylated His6 peptide was preincubated with crude extracts for 30 min. A previously determined optimal quantity of anti-His6 antibody-HRP conjugate (Serotec, Raleigh, NC) was added and incubated for 45 min. The mixture was transferred to streptavidin-coated microtiter plates (Sigma) and incubated for 15 min. Following six washes with phosphate-buffered saline pH 7.4/0.1% Tween 20 (PBS-T), the captured complex was detected with Supersignal® chemiluminescent substrate (Pierce) and read on a Luminskan Ascent plate reader (Thermo Electron, Waltham, MA). Increasing amounts of purified His6-tagged rLep d 2.02 were added to compete with the peptide for binding by the antibody, to prevent the capture of the antibody-HRP conjugate to the plate and to calibrate the competition assay with a standard curve of known protein concentrations. For HTP screening, 100 µl of soluble, crude lysate from cells expressing rLep d 2.02 or shuffled allergens were preincubated with a fixed concentration of biotinylated peptide, and the assay was performed as described above. In addition, each 96-well plate in the HTP screening carried a standard curve of purified His6-tagged rLep d 2.02 for comparison.

IgE Bead-based Binding Assay—Purified rLep d 2.02 was biotinylated using Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's instructions and purified by gel filtration. Mouse monoclonal anti-human IgE-antibodies (Accurate Chemical, Westbury, NY) were cross-linked to 4.5 micron-activated magnetic beads (Dynal Biotech, Oslo, Norway) according to the manufacturer's instructions. Beads were blocked with bovine serum albumin and then incubated with a serum pool of sera from nine patients with storage mite allergy (as described above). Beads were washed five times with PBS-T to remove nonspecifically bound serum proteins and other antibody isotypes. The resulting "IgE beads" were incubated with a fixed concentration of biotinylated rLep d 2.02 for 1 h in 96-well round bottom plates, washed on a magnetic plate washer, incubated with streptavidin HRP (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 15 min, and washed on a plate washer. The captured allergen was detected with Supersignal® chemiluminescent substrate and read on a Luminskan Ascent plate reader (Thermo Electron). Increasing concentrations of purified rLep d 2.02 (not biotinylated) were preincubated with a fixed concentration of biotinylated rLep d 2.02 to compete for binding to IgE beads and to generate a standard curve reflecting the maximum IgE binding of the wild-type Lep d 2.02.

HTP screening was performed on crude lysates of cells expressing wild-type rLep d 2.02 or shuffled allergens as described above. For characterization of shuffled proteins, the pattern of IgE binding over a large concentration range was performed and compared with wild-type rLep d 2.02. Purified shuffled allergens or wild-type rLep d 2.02 were diluted 5-fold starting at 80–120 µg/ml, preincubated with a fixed concentration of biotinylated rLep d 2.02, and assayed as described above.

Lymphoproliferation Assay—Lymphoproliferation of PBMCs was assayed by incorporation of [3H]thymidine essentially as previously described (41). The cells were thawed, and 2 x 105 cells in 200 µl (>90% viability, as determined by trypan blue exclusion) were cultured per well in 96-well flat-bottomed plates (Falcon®, Becton Dickinson) in complete RPMI (cRPMI) consisting of RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated bovine growth serum (HyClone), gentamicin (25 µg/ml), L-glutamine (2 mM), penicillin (100 international units/ml), streptomycin (100 µg/ml; all from Invitrogen), and 2-mercaptoethanol (20 µM; Kebo-lab, Spånga, Sweden). Cells from each of seven donors were stimulated for 7 days with two concentrations (1 and 10 µg/ml) of rLep d 2.01, rLep d 2.02, rGly d 2, rGly d 2-Lep d 2 fusion protein, nine expressed shuffled allergens (L4 (cytoplasmic and periplasmic protein), L1, L2, L3, R1, R2, R3, R4, and R5) and as positive controls with 10 µg/ml tetanus toxoid (TT, Statens Seruminstitut, Copenhagen, Denmark), 1 µg/ml tuberculin-purified protein derivate (PPD, Statens Seruminstitut), or 1 µg/ml phytohemeagglutinin (PHA, Sigma-Aldrich Inc.; 3 days stimulation) with [3H]thymidine (1 µCi/well, Amersham Biosciences) present during the last 18 h of culture. Stimulation index (SI) was calculated as mean (of triplicates) cpm for antigen-stimulated PBMC divided by mean cpm for nonstimulated PBMC cultured in medium alone. Presented are the highest SI values obtained, regardless of the stimulating antigen concentration used.

Cytokine Analysis—Stimulation of cytokine production was analyzed in long term PBMC cultures essentially as earlier described (41). The cells were stimulated with 10 µg/ml rLep d 2.01, rLep d 2.02, shufflant L4 (cytoplasmic and periplasmic fraction), R2, TT, PPD (1 µg/ml), or left unstimulated in cRPMI only. After 5 and 8 days of culture, interleukin 2 (IL-2, 20 units/ml; Proleukin, Chiron, Emeryville, CA) was added and at day 12, cell supernatants were collected for cytokine measurements by ELISA (Diaclone, Besançon, France). Interferon {gamma} (IFN-{gamma}), IL-5, and IL-13 were measured, and the detection limits for the assays were 12.5, 7.8, and 3.2 pg/ml, respectively.

Immunization of Mice—Female AJ mice were immunized in groups of 8–9, with either 10 µg of the wild-type rLep d 2.01, or 10 µg of one of the two shuffled allergens L4 (periplasmic) or R2 (adsorbed to 1 mg of aluminum hydroxide hydrate, Sigma-Aldrich). Control mice were immunized with PBS and aluminum hydroxide hydrate only. Three injections were administered intraperitoneally every 2 weeks. One week after the last immunization (day 35), mice were sacrificed, and blood was drawn by cardiac puncture. Sera were collected and stored at -20 °C until analysis.

Detection of Allergen-specific Mouse Antibodies—Immunoglobulin G1(IgG1) and IgG2a antibodies to rLep d 2.01 or rGly d 2 was analyzed in the mouse serum samples by ELISA (42). In brief, 96-well plates (Nunc, Roskilde, Denmark) were coated with 5 µg/ml rLep d 2.01 or rGly d 2 in 0.1 M carbonate buffer pH 9.6 at 4 °C overnight. To reduce nonspecific binding, plates were blocked with 1% bovine serum albumin in PBS. The mouse sera were diluted 1:5000 for IgG1- and 1:1000 for IgG2a-detection and added to plates for 1 h. IgG1-levels were detected with a rat anti-mouse IgG1 (BD Biosciences) diluted 1:1000, and a biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) diluted 1:2000 followed by alkaline phosphatase-conjugated streptavidin (DakoCytomation, Glostrup, Denmark) diluted 1:3000. IgG2a-levels were detected with an alkaline phosphatase-conjugated goat anti-mouse IgG2a (Southern Biotech, Birmingham, AL) diluted 1:4000. Phosphatase substrate (Sigma) was used as substrate, and the color reaction was monitored at 405 nm.

Blocking Capacity of Mouse Sera—The blocking capacity of the sera from immunized mice was investigated using inhibition ELISA. Plates were coated as for the direct ELISA described above. The mouse sera were diluted 1:100, 1:1000, and 1:10000 and added to rLep d 2.01 or rGly d 2-coated plates for 1 h. A pool of human sera from eight patients allergic to L. destructor or serum from a single patient having high levels of IgE to L. destructor (27.8 kUA/liter) was used to test the blocking capacity of the mouse sera. The human sera were diluted 1:2 and allowed to incubate in mouse serum preincubated and washed plates for 1 h. Human IgE bound to the parental allergen was detected using an HRP-conjugated polyclonal rabbit anti-human IgE (DakoCytomation), diluted 1:4000. Color development was performed and monitored as previously described (43). The blocking capacity is expressed as percentage signal of control wells (i.e. wells with no mouse serum added).

The ability of sera from immunized mice to block mediator release from a rat mast cell line transfected with the {alpha}-chain of human Fc{epsilon}RI (44) and sensitized with patient IgE was assessed in a degranulation assay. Transfectants were maintained in Dulbecco's modified Eagle's-Glutamax medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 international units/ml), streptomycin (100 µg/ml), and G418 (1 mg/ml) (all from Invitrogen). For functional assays, transfectants were seeded in 96-well plates at 1 x 104 in 100 µl of complete medium (without G418) containing serum (1:10 dilution) from a patient sensitized to L. destructor (11 kilounits/liter). After 48 h, IgE-sensitized cells were washed and stimulated in 100 µl of Tyrodes buffer (10 mM HEPES pH 7.3, 135 mM NaCl, 5 mM KCl, 5.6 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2, and 5% bovine serum albumin) prepared in 40% D2O and containing 50 ng/ml rLep d 2.01 that had been absorbed with serum from mice immunized with recombinant allergens. Four serum pools from groups of mice immunized with rLep d 2.01, L4, R2, and from the control group of nonimmunized (PBS) mice were used for absorbtion. Each mouse serum pool (diluted 1:2 in PBS) was incubated with 5 µg/ml rLep d 2.01 for 60 min at room temperature. Absorbed rLep d 2.01 was then added at 1:100 dilution in stimulation assays, and samples were run in duplicates. Degranulation was measured by determining release of the granular enzyme beta-hexosaminidase, expressed as percentage of total beta-hexosaminidase in cells after correction for spontaneous release in nonstimulated cultures (net release). Stimulation in the presence of 1 µg/ml rabbit anti-human IgE was used as control to determine maximal degranulation of IgE-sensitized cells (44).

Statistical Analysis—The immunoglobulin levels induced in immunized mice were compared with PBS-immunized mice using the non-parametric Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
DNA Shuffling—DNA shuffling yielded a library of chimeric allergen sequences. DNA sequencing revealed that numerous clones with different patterns of shuffled sequences and point mutations were present in the library. As an example, schematic representations of sequences from nine clones that were later selected in the screening procedure are shown in Fig. 1. Eight of these nine shuffled allergen genes had mixed sequences, originating either from all three, or from two of the parental genes and additional point mutations leading to amino acid substitutions; one of the selected variants had only one amino acid substitution compared with Lep d 2.02 (L4 in Fig. 1).


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 1.
Schematic representation of nine shuffled allergen sequences. The diagram shows the parental gene origin of amino acid sequence segments of nine shuffled clones from the library of chimeric allergen sequences obtained by DNA shuffling. Amino acid sequences derived from Gly d 2 (open), Lep d 2.01 (shaded), and Lep d 2.02 (filled) are indicated as bars. Amino acid changes that are not derived from any parental gene are indicated by boxes on top of each bar. Four low IgE-binding clones (L1–L4) and five reduced IgE-binding clones (R1–R5) are shown.

 
Excluding additional diversity from TaqDNA polymerase errors, the theoretical size of the library constructed by DNA shuffling of the starting genes Lep d 2.02, Gly d 2, and the hybrid gene GlyLep was over 100 million. Following ligation of DNA assemblies into expression vector and transformation into E. coli, two libraries of about 1 million colonies were generated from two different fragment sizes of the starting genes. Western blot analysis for recombinant protein expression from 20 randomly picked colonies indicated that the frequency of clones not expressing full-length protein was less than 20% (data not shown). One thousand colonies from each library were grown for recombinant protein production and screened simultaneously for IgE binding and protein expression levels. Seventy-four clones with apparent high protein expression and reduced IgE binding were selected for re-growth and further analysis by IgE binding, protein expression, anti-His6 Western blot, and DNA sequencing. About 49% of the selected clones were shown to express high levels of recombinant protein (as high or higher than wild-type Lep d 2.02 in the same expression vector), while about 32 and 19% expressed moderate and low levels of protein, respectively.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 2.
Screening of high expressing, full-length shuffled allergens with reduced binding capacity to patient IgE. Expressed allergen clones in E. coli soluble extracts were screened for low capacity to compete with wild-type rLep d 2.02 for binding to patient IgE in a bead-based IgE binding competition assay. Protein concentration was determined using a bead-based competition ELISA based on expression of C-terminal His6 tags. Expression levels (abscissa) and IgE binding intensities (ordinate) are shown for each of 80 shuffled clones from one 96-well plate. Raw values in relative luminescence units (RLU) for each assay were plotted together. The encircled area represents shuffled clones (filled circles) with high recombinant protein expression levels and low IgE binding. Parental control proteins (rLep d 2.02) are indicated with open circles and negative control extracts are indicated with filled triangles.

 
Expression and Screening of Shuffled Allergens—Variant genes from the shuffled library were cloned into expression vectors encoding a His6 tag in-frame at the 3'-end of the cloned genes. Proteins recovered from the periplasm of expressing bacteria were then subjected to screening to enrich shuffled allergens with hypoallergenic properties. The C-terminal His6 tags enabled quantification and selection by a screening procedure based on protein quantification of His6 tag-bearing proteins. Thus, truncated proteins not binding to an anti-His6 antibody and low expressing clones giving low signal in the quantification assay were eliminated in this screening step. In a bead-based IgE binding competition assay, proteins with low capacity to compete with labeled wild-type rLep d 2.02 for binding to patient IgE were selected. Combination of the two screening principles resulted in scattergrams where high expressing, full-length shuffled allergens with reduced or low IgE binding capacity could be identified (Fig. 2).

Among the clones exhibiting high expression and reduced or low IgE binding capacity, nine shuffled allergens were chosen for further characterization. Western blot analysis of the nine selected shuffled allergens confirmed that they were all expressed as full-length proteins, because they could be detected with an anti-His6 antibody (data not shown). The sequence chimerism of the selected shuffled allergens is shown in Fig. 1.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3.
IgE binding analysis of nine shuffled allergens. Nine expressed and purified shuffled proteins were assayed for their respective abilities to compete with wild-type rLep d 2.02 for binding to IgE in a pool of sera from nine patients allergic to L. destructor. The patient IgE binding intensity of each shuffled protein (L1–L4 and R1–R5) and two independent dilution series of wild-type rLep d 2.02 (wt A and wt B) were measured by competition to biotinylated rLep d 2.02. The IgE binding in wells with increasing concentrations of competing wild-type or shuffled allergens (abscissa) is expressed as a fraction of maximal IgE signal in the IgE competition ELISA (ordinate). Expressed clones with little or no binding to patient IgE (L1–L4) did not compete with biotinylated rLep d 2.02, whereas clones with reduced binding (R1–R5) competed for IgE binding with wild-type rLep d 2.02 only at >80 times higher concentrations.

 
IgE Binding of Shuffled Allergens— The IgE binding properties of the nine shuffled allergens selected from the scattergram analysis were further analyzed in IgE binding competition experiments. The ability to compete with wild-type rLep d 2.02 for binding to IgE in the bead-based IgE binding assay was determined for all nine hypoallergen candidates, and based on the results they were named L1–4 for low IgE binding and R1–5 for reduced IgE binding. Fig. 3 shows that much higher concentrations were needed for the shuffled allergens than for wild-type rLep d 2.02 to obtain 50% inhibition of the binding of rLep d 2.02 to IgE, ranging from ~2.3 µg/ml for R2 and R5 to no inhibition at the highest concentration tested (100 µg/ml) for L1, L2, and L3, and only slight inhibition at 100 µg/ml for L4. In contrast, 50% homologous inhibition of the IgE signal with rLep d 2.02 was obtained at only 0.03 µg/ml protein concentration. Thus, compared with wild-type rLep d 2, all nine shuffled allergens selected from the screening exhibited strongly reduced binding to mite allergic patient IgE, corresponding to about 80-fold reduction to almost complete loss of binding.

T-cell Reactivity of Shuffled Allergens—Next, we wanted to study whether the nine shuffled allergens with strongly reduced IgE binding capacity had retained their ability to activate T cells from mite allergic patients. For this purpose, the nine hypoallergen candidates, L1–4 and R1–5, were expressed, purified, and analyzed by SDS-PAGE (Fig. 4). The endotoxin content in the wild-type allergens and the nine shuffled allergens ranged between 0.37 and 8.5 ng/mg protein, median 1.16 ng/mg. The amount of endotoxin did not correlate with proliferation when the allergens were analyzed in the lymphoproliferation assay.


Figure 4
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 4.
Gel analysis of purified shuffled proteins. Recombinant allergens and shuffled proteins purified from cytoplasmic E. coli expression (L1–L3, R1–R5, Lep d 2-Gly d 2 chimera (Gly-Lep), Gly d 2, Lep d 2.02c, and L4c) or periplasmic E. coli expression (L4p, Lep d 2.02p) were analyzed by SDS-PAGE and stained with Coomassie Blue. Sizes of the SeeBlue molecular weight markers (M) are indicated.

 
Purified shuffled allergens and parental allergens (rLep d 2.01, rLep d 2.02, rGly d 2, and the Gly d 2 (1–21)-Lep d 2.01 (22–125) chimeric allergen) were tested for stimulation of T-cell proliferation and cytokine production in PBMC cultures obtained from seven patients allergic to L. destructor. First, the ability to stimulate T-cell proliferation was measured. The proliferative responses, expressed as stimulation indices (SI), for all shufflants, wild-type allergens, and positive controls are shown for each patient in Table 1. All patients except for one (patient no. 6, Table 1) displayed positive stimulation indices, i.e. SI ≥ 3, to at least one of the wild-type allergens rLep d 2.01, rLep d 2.02, or rGly d 2. The magnitude of the proliferative responses to the different shufflants varied between patients, and the patients also showed individual patterns of reactivity. No patient displayed T-cell reactivity to the Lep d 2-Gly d 2 chimeric construct, which was used together with the wild-type allergen genes as a template for DNA shuffling. The two shuffled allergens stimulating most frequent positive T-cell responses were L4 (positive SI values for 6/7 patients) and R2 (positive for 4/7 patients).


View this table:
[in this window]
[in a new window]

  		TABLE 1
Lymphoproliferative responses to wild-type and shuffled allergens T cell proliferation in PBMC cultures from seven mite allergic patients, number 1–7, stimulated with wild-type allergens (Lep d 2.01, Lep d 2.02 (expressed as cytoplasmic, c, and periplasmic, p, protein) and Gly d 2), Lep d 2-Gly d 2 chimera (Gly-Lep), nine shuffled allergens (L1–L4 and R1–R5; L4 expressed as cytoplasmic, c, and periplasmic, p, protein) and the positive controls tetanus toxoid, TT, and tuberculin-purified protein derivate, PPD. The proliferation is expressed as SI, i.e. mean cpm-value of triplicate in stimulated cultures divided by mean cpm value of triplicate in nonstimulated cultures. SI-values ≥ 3 are considered positive. Positive SI values are shown in bold.

 
The ability of L4 and R2 to stimulate cytokine production was further investigated. Long term PBMC cultures were established for four patients (patient no. 1, 2, 3, and 6) to evaluate the cytokine profiles of the response stimulated by rLep d 2.01, rLep d 2.02, L4, and R2. The concentrations of IL-5, IL-13, and IFN-{gamma} were measured in the cell culture supernatants (Fig. 5). As shown in Fig. 5a, rLep d 2.01 generally stimulated higher concentrations of IFN-{gamma} than the shuffled allergens and rLep d 2.02. L4 and R2 stimulated similar levels of IL-5 and IL-13 concentrations as rLep d 2.01 and rLep d 2.02 (Fig. 5, b and c) and IFN-{gamma} levels similar to rLep d 2.02 (Fig. 5a). In conclusion, two of the shuffled allergens, L4 and R2, stimulated T-cell proliferation of a similar magnitude as wild-type rLep d 2. These two shuffled allergens also stimulated cytokine secretion, but no consistent difference could be observed regarding the Th1/Th2 profile of cytokines (here represented by IFN-{gamma} for Th1 and IL-5 and IL-13 for Th2) stimulated by L4 or R2 compared with the wild-type allergens.


Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 5.
The shuffled allergens L4 and R2 induce cytokine levels of similar magnitude as wild-type rLep d2. Long term PBMC cultures from four patients were stimulated with rLep d 2.01, rLep d 2.02, shufflant L4 or R2 and the concentrations of IFN-{gamma} (a), IL-5 (b), and IL-13 (c) were measured by ELISA in the cell culture supernatants. The cytokine concentrations are expressed as ng/ml, and mean concentrations stimulated by each protein are indicated.

 


Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 6.
The shuffled allergens L4 and R2 induce allergen-specific antibodies with blocking capacity. Groups of mice (n = 8–9/group) were immunized with shuffled allergens L4, R2, or wild-type rLep d 2.01. Sera were analyzed by direct ELISA for rLep d 2.01-(a and c) and rGly d 2-specific (b and d) IgG1 (a and c), and IgG2a (b and d) antibodies. The capacity of the mouse sera to block the binding of human sera from an L. destructor-sensitized patient to rLep d 2.01 (e) and rGlyd2(f) was analyzed in an inhibition ELISA. ***, p < 0.001 using Mann-Whitney non-parametric test, comparing immunized mice with control mice.

 
Immunogenicity of Shuffled Allergens in Vivo—To investigate whether L4 and R2 had immunogenic properties, mice were immunized with either rLep d 2.01 or one of the shuffled allergens L4 or R2. As shown in Fig. 6, both L4 and R2 induced significantly elevated levels of IgG1 and IgG2a antibodies to both rLep d 2 (Fig. 6, a and c) and rGly d 2 (Fig. 6, b and d), indicating that the shuffled allergens are immunogenic and induce a cross-reactive immune response against the wild-type allergens.

To examine if the mouse sera could block the binding of human IgE to rLep d 2.01 and rGly d 2, an inhibition ELISA was performed using serum from one single L. destructor-sensitized patient (27.8 kilounits/liter) or with a serum pool from eight L. destructor-sensitized patients. Serum from L4-immunized mice blocked human IgE binding to rLep d 2.01 equally well as serum from rLep d 2.01-immunized mice, both when serum was from one patient (Fig. 6e), or the serum pool was used in the competition assay (data not shown). Sera from R2-immunized mice were able to block human IgE binding to rLep d 2.01, but at higher serum concentrations compared with sera from L4-immunized mice (Fig. 6e). Similar results were obtained when assaying for blocking of human IgE binding to rGly d 2 (Fig. 6f).

Finally, we tested whether the antibodies induced by immunization of mice with L4 and R2 were able to block rLep d 2-stimulated mediator release from effector cells carrying IgE from a patient sensitized to L. destructor. Recombinant Lep d 2.01 was preincubated with serum pools from mice immunized with the shuffled hypoallergen candidates L4 and R2, with wild-type rLep d 2 or with PBS. The pretreated rLep d 2 preparations were then analyzed in a degranulation assay measuring release of beta-hexosaminidase. As shown in Fig. 7, preincubation with serum from mice immunized with wild-type rLep d 2 resulted in almost 3-fold reduction of rLep d 2-stimulated beta-hexosaminidase release compared with control serum from non-immunized mice, whereas preincubation with the L4 and R2 serum pools resulted in 1.7- and 1.2-fold reduction of mediator release, respectively. Comparable data were obtained when cells had been sensitized with patient plasma instead of serum (not shown).

Taken together, both shuffled hypoallergen candidates, L4 more than R2, are capable of inducing blocking IgG antibodies recognizing the wild-type allergens Lep d 2.01 and Gly d 2, despite the fact that they both possess a strongly reduced ability to bind patient IgE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mites are a common cause of allergic disease world-wide (45, 46), and group 2 allergens are known to be major allergens in several mite species including Dermatophagoides spp, L. destructor (47) and G. domesticus (37). Allergen-specific immunotherapy to mite allergy is routinely carried out using mite extracts, but there have been several recent attempts to develop improved and standardized allergen preparations based on recombinant allergens. Hypoallergens with reduced IgE binding capacity but retained immunogenicity have been obtained by disrupting one or more of the disulfide bonds in the group 2 mite allergens Der p 2 (18), Der f 2 (19), and Lep d 2 (48). The disruption of disulfide bonds likely results in a loss of correct protein folding and conformational B-cell epitopes. In this study we tried a new approach and generated chimeric Group 2 mite allergen genes with the use of directed molecular evolution by multigene DNA shuffling and screening. Expressed mutant allergens were screened for reduced IgE reactivity but preserved T-cell epitopes and two hypoallergen candidates meeting these criteria were identified. Thus, as a proof of concept, we here show that DNA shuffling and screening can be used to create hypoallergenic allergens for potential use in allergen-specific immunotherapy without the use of structural data on the wild-type allergens.


Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 7.
L4 induces antibodies with blocking capacity in an effector cell degranulation assay. rLep d 2.01 was preincubated with pools of sera from mice immunized with wild-type rLep d 2.01 (Lepd2), L4, R2, or non-immunized mice (PBS, control sera). Then the capacity to stimulate mediator release (beta-hexosaminidase) from Fc{epsilon}RI-humanized mast cells sensitized with serum from a patient allergic to L. destructor was measured and expressed as % released beta-hexosaminidase of maximum release. Results of stimulation with rLep d 2.01 preabsorbed with the denoted serum pools are shown to the left and represented by filled columns. Mean of duplicates with S.D. is shown. Anti-IgE stimulation was used as a positive control measuring maximum IgE-mediated release (open column, mean of eight measurements with S.D.).

 
DNA shuffling and screening methods have previously been used to improve various properties of cytokines, antibodies, co-stimulatory molecules, and viral antigens (30–35). In the previous applications of these methods, desired variants were identified using positive screening or selection procedures based on exploiting improvements or altered characteristics in shufflants compared with parental proteins, such as increased binding to a receptor, ligand, or antibody. For identification of hypoallergens with reduced binding to patient IgE, the screening procedures present additional challenges. Variants that carry frame-shifts, truncations, misassembled regions, or other mutations that give rise to poor or no expression, protein fragments, or grossly mutated proteins may turn up frequently in the screen as appearing to be low IgE binding clones of interest. In addition to eliminating undesired mutants from the screen, information on the amount of expressed protein to be used when evaluating changes in apparent IgE binding is essential. To perform high throughput screens for hypoallergenic shufflants with reduced binding to patient IgE, an additional parallel screen to identify clones expressing acceptable levels of full-length protein and to quantify expression levels was required. For that purpose we exploited the C-terminal His6 purification tag to develop a method for indirect confirmation of full-length protein and quantification in the crude E. coli extracts. Using screening techniques involving parallel IgE binding and protein quantification assays, numerous full-length clones were identified with reduced binding to patient IgE. Additional characterization, including SDS-PAGE, Western blot, and DNA sequencing was still needed to eliminate clones with small, internal in-frame deletions. The majority of shuffled variants making it through these tests were confirmed to have reduced IgE binding when the protein was purified.

Most of the hypoallergen candidates selected after the screening procedure were shown to comprise recombined DNA sequences originating from two or more of the different parental genes. Interestingly, one of the candidates, L4, had an intact Lep d 2.02 sequence with only one single amino acid substitution, a change of a positively charged (lysine) to a negatively charged (glutamic acid) amino acid residue. The expressed L4 recombinant protein exhibited strongly reduced IgE binding. A possible explanation for the decreased IgE binding capacity is that the substituted amino acid residue is part of a dominant B-cell epitope. Such dominant B-cell epitopes have not been identified for Lep d 2, but it has been shown that the IgE binding epitopes of Lep d 2 are conformational because synthetic peptides spanning the Lep d 2 sequence were not able to bind IgE in patient sera (49). Thus, another plausible explanation for the decreased IgE reactivity is that the single amino acid substituted in L4 is important for the overall allergen structure and that the substitution leads to disruption of one or more conformational epitopes. Among the nine shuffled allergen sequences presented in Fig. 1, L4 is the only sequence with this specific substitution. As expected, L4 exhibited T-cell reactivity comparable to that of wild-type rLep d 2. In addition, the levels of induced IgG1 and IgG2a in L4-immunized mice were comparable to those of rLep d 2-immunized mice. Most of the other eight shuffled allergens tested for T-cell reactivity induced decreased lymphoproliferative responses when compared with wild-type Lep d 2. Because all eight sequences include both point mutations and sequences of mixed origin, it is not surprising that they exhibit both reduced B-cell and T-cell reactivity. However, all of the allergen shufflants were shown to induce T-cell proliferation in PBMC cultures from at least one of the tested L. destructor allergic patients. The allergen shufflants L4 and R2 exhibited the most frequent proliferative responses, and R2 was thus considered to be the most promising hypoallergen candidate apart from L4. We have earlier shown that the most dominant T-cell epitope of Lep d 2.01 is situated between residues 60 and 75 and the second most dominant between residues 10 and 25 (41). The reduced T-cell reactivity of the eight shufflants may partly be caused by the fact that all except R5 contain Gly d 2 sequences in at least one of the two most dominant T-cell epitope regions. Shufflant R5 only comprises Lep d 2.01 and Lep d 2.02 sequences but still exhibits comparably low T-cell reactivity. However, in contrast to L4, the sequence of R5 is chimeric and includes several point mutations which could possibly reduce the reactivity since several parts of the sequence in addition to those harboring the two dominant epitopes can stimulate T-cell proliferation (41).

Several mechanisms for how successful allergen-specific immunotherapy leads to allergen non-responsiveness and symptom relief have been described (3, 5). These include changes in the T-cell response to allergens, either through immune deviation from a Th2-dominated response to a more Th1-skewed response or by induction of a regulatory T-cell response. Allergen-specific immunotherapy also induces allergen-specific antibodies with the capacity to bind allergen and block their interaction with IgE on mast cells, thereby preventing immediate type reactions. In a recent study where hypoallergenic derivatives of the major birch pollen allergen Bet v 1 were used to treat birch pollen allergic patients, the Bet v 1 hypoallergen treatment led to high levels of IgG antibodies recognizing wild-type Bet v 1 (10, 50). The two mite Group 2 hypoallergen candidates, L4 and R2, were tested for their ability to induce an antibody response in mice. Immunization with both candidates generated IgG antibodies (IgG1 and IgG2a) that recognized Lep d 2 and Gly d 2. When sera from immunized mice were used in inhibition ELISA experiments, both L4 and R2 antisera were shown to block binding of human IgE to Lep d 2 as well as to Gly d 2. Furthermore, serum from mice immunized with L4 displayed blocking capacity in a functional assay measuring Lep d 2-stimulated mediator release from Fc{epsilon}RI-humanized mast cells sensitized with serum from a patient allergic to L. destructor. Antiserum from mice immunized with R2 only had a limited ability to block mediator release in this assay. Thus, antibodies induced by L4 were more potent than R2 both in the ELISA inhibition assay and in the mediator release assay to block binding of patient IgE to Lep d 2. These results may be explained by the fact that L4 differs from Lep d 2.02 by only one amino acid substitution. However, it is notable that L4, which exhibited the most reduced IgE binding capacity of the two candidates, still induces IgG with high capacity to block IgE binding.

In conclusion, we demonstrate that directed molecular evolution represents a powerful approach for generating allergens with hypoallergenic properties. The two group 2 mite hypoallergen candidates that were characterized exhibited strongly reduced IgE reactivity, preserved T-cell reactivity and were able to induce wild-type allergen-specific blocking antibodies. These results suggest that DNA shuffling and screening are promising methods by which to generate novel hypoallergen candidates for potential use in allergen-specific immunotherapy.


   FOOTNOTES
 
* This work was supported in part by grants from the Swedish Foundation for Health Care Sciences and Allergy Research, the Swedish Research Council, Swedish Asthma and Allergy Association's Research Foundation, the Swedish Cancer and Allergy Foundation, Hesselman's Foundation, Magnus Bergvall's Foundation, Konsul Th. C. Bergh's Foundation, Åke Wiberg's Foundation, the Stockholm County Council, and the Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Karolinska Institutet, Dept. of Medicine, Clinical Immunology and Allergy Unit, Karolinska University Hospital, 171 76 Stockholm, Sweden. Tel.: 46-8-51776441; Fax: 46-9-335724; E-mail: guro.gafvelin{at}ki.se.

3 The abbreviations used are: ASIT, allergen-specific immunotherapy; HTP, high-throughput; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PPD, tuberculin-purified protein derivate; SI, stimulation index; TT, tetanus toxoid; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; IFN, interferon; IL, interleukin. Back


   ACKNOWLEDGMENTS
 
We thank Liana Sheppard, Sasha Lazetic, and Neda Bigdeli for excellent technical assistance. We also thank the staff at the blood bank at the regional hospital in Visby, Sweden for assistance in blood sample collection and the mite allergic patients for their contribution.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Spiegelberg, H. L., Horner, A. A., Takabayashi, K., and Raz, E. (2002) Curr. Opin. Allergy Clin. Immunol. 2, 547-551[CrossRef][Medline] [Order article via Infotrieve]
  2. Robinson, D. S. (2003) Pediatr. Pulmonol. 36, 369-375[CrossRef][Medline] [Order article via Infotrieve]
  3. Till, S. J., Francis, J. N., Nouri-Aria, K., and Durham, S. R. (2004) J. Allergy Clin. Immunol. 113, 1025-1034[CrossRef][Medline] [Order article via Infotrieve]
  4. Zhu, D., Kepley, C. L., Zhang, K., Terada, T., Yamada, T., and Saxon, A. (2005) Nat. Med. 11, 446-449[CrossRef][Medline] [Order article via Infotrieve]
  5. Larche, M., Akdis, C. A., and Valenta, R. (2006) Nat. Rev. Immunol. 6, 761-771[CrossRef][Medline] [Order article via Infotrieve]
  6. Ball, T., Sperr, W. R., Valent, P., Lidholm, J., Spitzauer, S., Ebner, C., Kraft, D., and Valenta, R. (1999) Eur. J. Immunol. 29, 2026-2036[CrossRef][Medline] [Order article via Infotrieve]
  7. Moverare, R., Elfman, L., Vesterinen, E., Metso, T., and Haahtela, T. (2002) Allergy 57, 423-430[CrossRef][Medline] [Order article via Infotrieve]
  8. Akdis, C. A., and Blaser, K. (2001) Trends Immunol 22, 175-178[CrossRef][Medline] [Order article via Infotrieve]
  9. Valenta, R., Vrtala, S., Focke-Tejkl, M., Bugajska, S., Ball, T., Twardosz, A., Spitzauer, S., Gronlund, H., and Kraft, D. (1999) Biol. Chem. 380, 815-824[CrossRef][Medline] [Order article via Infotrieve]
  10. Niederberger, V., Horak, F., Vrtala, S., Spitzauer, S., Krauth, M. T., Valent, P., Reisinger, J., Pelzmann, M., Hayek, B., Kronqvist, M., Gafvelin, G., Gronlund, H., Purohit, A., Suck, R., Fiebig, H., Cromwell, O., Pauli, G., van Hage-Hamsten, M., and Valenta, R. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, Suppl. 2, 14677-14682[Abstract/Free Full Text]
  11. Beezhold, D. H., Hickey, V. L., and Sussman, G. L. (2001) J. Allergy Clin. Immunol. 107, 1069-1076[CrossRef][Medline] [Order article via Infotrieve]
  12. Swoboda, I., De Weerd, N., Bhalla, P. L., Niederberger, V., Sperr, W. R., Valent, P., Kahlert, H., Fiebig, H., Verdino, P., Keller, W., Ebner, C., Spitzauer, S., Valenta, R., and Singh, M. B. (2002) Eur. J. Immunol. 32, 270-280[CrossRef][Medline] [Order article via Infotrieve]
  13. Rabjohn, P., West, C. M., Connaughton, C., Sampson, H. A., Helm, R. M., Burks, A. W., and Bannon, G. A. (2002) Int. Arch. Allergy Immunol. 128, 15-23[CrossRef][Medline] [Order article via Infotrieve]
  14. Holm, J., Gajhede, M., Ferreras, M., Henriksen, A., Ipsen, H., Larsen, J. N., Lund, L., Jacobi, H., Millner, A., Wurtzen, P. A., and Spangfort, M. D. (2004) J. Immunol. 173, 5258-5267[Abstract/Free Full Text]
  15. Karisola, P., Mikkola, J., Kalkkinen, N., Airenne, K. J., Laitinen, O. H., Repo, S., Pentikainen, O. T., Reunala, T., Turjanmaa, K., Johnson, M. S., Palosuo, T., Kulomaa, M. S., and Alenius, H. (2004) J. Immunol. 172, 2621-2628[Abstract/Free Full Text]
  16. Neudecker, P., Lehmann, K., Nerkamp, J., Haase, T., Wangorsch, A., Fotisch, K., Hoffmann, S., Rosch, P., Vieths, S., and Scheurer, S. (2003) Biochem. J. 376, 97-107[CrossRef][Medline] [Order article via Infotrieve]
  17. Spangfort, M. D., Mirza, O., Ipsen, H., Van Neerven, R. J., Gajhede, M., and Larsen, J. N. (2003) J. Immunol. 171, 3084-3090[Abstract/Free Full Text]
  18. Smith, A. M., and Chapman, M. D. (1996) Mol. Immunol. 33, 399-405[CrossRef][Medline] [Order article via Infotrieve]
  19. Takai, T., Yokota, T., Yasue, M., Nishiyama, C., Yuuki, T., Mori, A., Okudaira, H., and Okumura, Y. (1997) Nat. Biotechnol. 15, 754-758[CrossRef][Medline] [Order article via Infotrieve]
  20. Drew, A. C., Eusebius, N. P., Kenins, L., de Silva, H. D., Suphioglu, C., Rolland, J. M., and O'Hehir R, E. (2004) J. Immunol. 173, 5872-5879[Abstract/Free Full Text]
  21. Vrtala, S., Hirtenlehner, K., Susani, M., Akdis, M., Kussebi, F., Akdis, C. A., Blaser, K., Hufnagl, P., Binder, B. R., Politou, A., Pastore, A., Vangelista, L., Sperr, W. R., Semper, H., Valent, P., Ebner, C., Kraft, D., and Valenta, R. (2001) Faseb J. 15, 2045-2047[Abstract/Free Full Text]
  22. Karamloo, F., Schmid-Grendelmeier, P., Kussebi, F., Akdis, M., Salagianni, M., von Beust, B. R., Reimers, A., Zumkehr, J., Soldatova, L., Housley-Markovic, Z., Muller, U., Kundig, T., Kemeny, D. M., Spangfort, M. D., Blaser, K., and Akdis, C. A. (2005) Eur. J. Immunol. 35, 3268-3276[CrossRef][Medline] [Order article via Infotrieve]
  23. Saarne, T., Kaiser, L., Gronlund, H., Rasool, O., Gafvelin, G., and van Hage-Hamsten, M. (2005) Clin. Exp. Allergy 35, 657-663[CrossRef][Medline] [Order article via Infotrieve]
  24. Vrtala, S., Hirtenlehner, K., Vangelista, L., Pastore, A., Eichler, H. G., Sperr, W. R., Valent, P., Ebner, C., Kraft, D., and Valenta, R. (1997) J. Clin. Investig. 99, 1673-1681[Medline] [Order article via Infotrieve]
  25. Westritschnig, K., Focke, M., Verdino, P., Goessler, W., Keller, W., Twardosz, A., Mari, A., Horak, F., Wiedermann, U., Hartl, A., Thalhamer, J., Sperr, W. R., Valent, P., and Valenta, R. (2004) J. Immunol. 172, 5684-5692[Abstract/Free Full Text]
  26. Stemmer, W. P. (1994) Nature 370, 389-391[CrossRef][Medline] [Order article via Infotrieve]
  27. Stemmer, W. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10747-10751[Abstract/Free Full Text]
  28. Crameri, A., Raillard, S. A., Bermudez, E., and Stemmer, W. P. (1998) Nature 391, 288-291[CrossRef][Medline] [Order article via Infotrieve]
  29. Punnonen, J. (2000) Int. Arch. Allergy Immunol. 121, 173-182[CrossRef][Medline] [Order article via Infotrieve]
  30. Chang, C. C., Chen, T. T., Cox, B. W., Dawes, G. N., Stemmer, W. P., Punnonen, J., and Patten, P. A. (1999) Nat. Biotechnol. 17, 793-797[CrossRef][Medline] [Order article via Infotrieve]
  31. Soong, N. W., Nomura, L., Pekrun, K., Reed, M., Sheppard, L., Dawes, G., and Stemmer, W. P. (2000) Nat. Genet. 25, 436-439[CrossRef][Medline] [Order article via Infotrieve]
  32. Lazetic, S., Leong, S. R., Chang, J. C., Ong, R., Dawes, G., and Punnonen, J. (2002) J. Biol. Chem. 277, 38660-38668[Abstract/Free Full Text]
  33. Leong, S. R., Chang, J. C., Ong, R., Dawes, G., Stemmer, W. P., and Punnonen, J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 1163-1168[Abstract/Free Full Text]
  34. Wright, A., Semyonov, A., Dawes, G., Crameri, A., Lyons, R., Stemmer, W. P., Apt, D., and Punnonen, J. (2005) Hum. Gene Ther. 16, 881-892[CrossRef][Medline] [Order article via Infotrieve]
  35. Apt, D., Raviprakash, K., Brinkman, A., Semyonov, A., Yang, S., Skinner, C., Diehl, L., Lyons, R., Porter, K., and Punnonen, J. (2006) Vaccine 24, 335-344[CrossRef][Medline] [Order article via Infotrieve]
  36. Schmidt, M., Olsson, S., van der Ploeg, I., and van Hage-Hamsten, M. (1995) FEBS Lett. 370, 11-14[CrossRef][Medline] [Order article via Infotrieve]
  37. Gafvelin, G., Johansson, E., Lundin, A., Smith, A. M., Chapman, M. D., Benjamin, D. C., Derewenda, U., and van Hage-Hamsten, M. (2001) J. Allergy Clin. Immunol. 107, 511-518[CrossRef][Medline] [Order article via Infotrieve]
  38. Kronqvist, M., Johansson, E., Pershagen, G., Johansson, S. G., and van Hage-Hamsten, M. (1999) Clin. Exp. Allergy 29, 35-41[CrossRef][Medline] [Order article via Infotrieve]
  39. Johansson, E., Eriksson, T. L., Olsson, S., Kronqvist, M., Whitley, P., Johansson, S. G., Gafvelin, G., and van Hage-Hamsten, M. (1999) Int. Arch. Allergy Immunol. 120, 43-49[CrossRef][Medline] [Order article via Infotrieve]
  40. Kronqvist, M., Johansson, E., Magnusson, C. G., Olsson, S., Eriksson, T. L., Gafvelin, G., and van Hage-Hamsten (2000) Clin. Exp. Allergy 30, 670-676[CrossRef][Medline] [Order article via Infotrieve]
  41. Eriksson, T. L., Gafvelin, G., Elfman, L. H., Johansson, C., Van Hage-Hamsten, M., and Olsson, S. (2001) Clin. Exp. Allergy 31, 1881-1890[CrossRef][Medline] [Order article via Infotrieve]
  42. Parvaneh, S., Johansson, E., Elfman, L. H., and van Hage-Hamsten, M. (2002) Clin. Exp. Allergy 32, 80-86[CrossRef][Medline] [Order article via Infotrieve]
  43. Adedoyin, J., Johansson, S. G., Gronlund, H., and van Hage, M. (2006) J. Immunol. Methods 310, 117-125[CrossRef][Medline] [Order article via Infotrieve]
  44. Marchand, F., Mecheri, S., Guilloux, L., Iannascoli, B., Weyer, A., and Blank, U. (2003) Allergy 58, 1037-1043[CrossRef][Medline] [Order article via Infotrieve]
  45. Sporik, R., Chapman, M. D., and Platts-Mills, T. A. (1992) Clin. Exp. Allergy 22, 897-906[CrossRef][Medline] [Order article via Infotrieve]
  46. Platts-Mills, T. A., Vervloet, D., Thomas, W. R., Aalberse, R. C., and Chapman, M. D. (1997) J. Allergy Clin. Immunol. 100, S2-S24[CrossRef][Medline] [Order article via Infotrieve]
  47. Thomas, W. R., Smith, W. A., Hales, B. J., Mills, K. L., and O'Brien, R. M. (2002) Int. Arch. Allergy Immunol. 129, 1-18[CrossRef][Medline] [Order article via Infotrieve]
  48. Olsson, S., van Hage-Hamsten, M., and Whitley, P. (1998) Mol. Immunol. 35, 1017-1023[CrossRef][Medline] [Order article via Infotrieve]
  49. Elfman, L. H., Whitley, P., Schmidt, M., and van Hage-Hamsten, M. (1998) Int. Arch. Allergy Immunol. 117, 167-173[CrossRef][Medline] [Order article via Infotrieve]
  50. Gafvelin, G., Thunberg, S., Kronqvist, M., Gronlund, H., Gronneberg, R., Troye-Blomberg, M., Akdis, M., Fiebig, H., Purohit, A., Horak, F., Reisinger, J., Niederberger, V., Akdis, C. A., Cromwell, O., Pauli, G., Valenta, R., and van Hage, M. (2005) Int. Arch. Allergy Immunol. 138, 59-66[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
N. Mothes-Luksch, S. Stumvoll, B. Linhart, M. Focke, M.-T. Krauth, A. Hauswirth, P. Valent, P. Verdino, T. Pavkov, W. Keller, et al.
Disruption of Allergenic Activity of the Major Grass Pollen Allergen Phl p 2 by Reassembly as a Mosaic Protein
J. Immunol., October 1, 2008; 181(7): 4864 - 4873.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/6/3778    most recent
M607938200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gafvelin, G.
Right arrow Articles by Punnonen, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gafvelin, G.
Right arrow Articles by Punnonen, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement