VARIANTS OF GROUP 5 ALLERGENS OF THE TRUE GRASSES HAVING REDUCED ALLERGENEITY DUE TO MUTAGENESIS OF PROLINE RESIDUES

Abstract

The present invention relates to the preparation and use of recombinant variants of group 5 allergens of the Poaceae (true grasses), which are characterised by reduced IgE reactivity compared with known wild-type allergens and at the same time substantially retained reactivity with T-lymphocytes.

Claims

1. A hypoallergenic variant of a group 5 allergen of the true grass family (Poaceae) wherein the prolines which correspond in an alignment to the prolines in positions 57, 58, 117, 146, 155, 180, 211 in the amino acid sequence of wild-type Phl p 5.0109 have been mutated singly or in combinations.

2. The hypoallergenic variant of claim 1, wherein the proline which corresponds in an alignment to proline 211 of wild-type Phl p 5.0109 is not deleted, but instead is replaced by another amino acid.

3. The hypoallergenic variant of claim 1, wherein the prolines which correspond in an alignment to the prolines in positions 146 and 155 in the amino acid sequence of wild-type Phl p 5.0109 are not mutated.

4. The hypoallergenic variant of claim 1, further wherein the amino acids which correspond in an alignment to lysine in position 61 and glutamic acid in position 205 in the amino acid sequence of wild-type Phl p 5.0109 have been mutated singly or in combinations.

5. The hypoallergenic variant of claim 1, wherein the group 5 allergen is a group 5 allergen of the Pooideae sub-family.

6. The hypoallergenic variant of claim 1, wherein the group 5 allergen is selected from the group consisting of Phl p 5, Lol p 5, Poa p 5, Hol l 5, Dac g 5, Pha a 5, Ant o 5, Fes p 5, Hor v 5, Sec c 5 and Tri a 5.

7. The hypoallergenic variant of claim 1, wherein it is a fragment or a variant of a hypoallergenic variant of claim 1 or a multimer of one or more hypoallergenic variants of claim 1 or wherein one or more hypoallergenic variants of claim 1 or fragments, variants or multimers thereof are a constituent of a recombinant fusion protein.

8. A DNA molecule which encodes for a hypoallergenic variant of claim 1.

9. A recombinant expression vector containing a DNA molecule of claim 8 functionally connected to an expression control sequence.

10. A non-human host organism transformed with a DNA molecule of claim 8.

11. A process for the preparation of a hypoallergenic variant by cultivation of a non-human host organism of claim 10 and isolation of the corresponding allergen variant from the culture.

12. The hypoallergenic variant of claim 1, formulated as medicament.

13. The DNA molecule of claim 8, formulated as medicament.

14. A recombinant expression vector of claim 9 as medicament.

15. A method for the prevention and/or therapeutic treatment of type 1 allergies in the triggering of which group 5 allergens of the true grasses are causally involved, comprising administering a hypoallergenic variant of claim 1.

16. A method for immunotherapeutic DNA vaccination comprising administering to a patient at least one DNA molecule of claim 8.

17. Pharmaceutical preparation comprising at least one hypoallergenic variant of claim 1 and optionally further active compounds and/or assistants for the prevention and/or therapeutic treatment of type 1 allergies.

18. A pharmaceutical preparation of claim 17, wherein the further active compounds are allergens of the true grasses or variants thereof.

19. A pharmaceutical preparation comprising at least one hypoallergenic variant of claim 8 and optionally further active compounds and/or assistants for the prevention and/or therapeutic treatment of type 1 allergies.

20. A pharmaceutical preparation comprising at least one hypoallergenic variant of claim 9 and optionally further active compounds and/or assistants for the prevention and/or therapeutic treatment of type 1 allergies.

Description

EXPLANATIONS OF THE FIGURES

[0131] FIG. 1a: Alignment of deduced amino acid sequences of the group 5 allergens of the Poaceae: Alignment of ripe group 5 sequences of various species. The boxes show the position of the α-helices of Phl p 6 (PDB entry 1 NLX) and the C-terminal half of a Phl p 5.02 fragment (PDB entry 1 L3P). The amino acid positions of the proline residues are labelled in accordance with their position in ripe Phl p 5.0109.

[0132] Sequence references: Phl p 5.0101 (SEQ ID NO: 8) (Phleum pratense IUIS sequence, UniProtKB Q40960), Phl p 5.0104 (SEQ ID NO: 9) (Phleum pratense IUIS sequence, UniProtKB P93467), Phl p 5.0109 (SEQ ID NO: 2) (Phleum pratense IUIS sequence, UniProtKB Q84UI2), Phl p 5.0201 (SEQ ID NO: 10) (Phleum pratense IUIS sequence, UniProtKB Q40963), Phl p 6.0101 (SEQ ID NO: 11) (Phleum pratense IUIS sequence, UniProtKB P43215), Lol p 5.0101 (SEQ ID NO: 12) (Lolium perenne IUIS sequence, UniProtKB Q40237), Pha a 5.0101 (SEQ ID NO: 13) (Phalaris aquatica IUIS sequence, UniProtKB P56164), Dac g 5 (SEQ ID NO: 14) (Dactylis glomerata, UniProtKB Q93XD9), Hol 15.0101 (SEQ ID NO: 15) (Holcus lanatus IUIS sequence, UniProtKB 023972), Hol 15.0201 (SEQ ID NO: 16) (Holcus lanatus IUIS sequence, UniProtKB 23971), Poa p 5.0101 (SEQ ID NO: 17) (Poa pratensis IUIS sequence, UniProtKB Q9FPR0), Tri a 5 (SEQ ID NO: 18) (Triticum aestivum, UniProtKB Q70JP9), Hor v 5 (SEQ ID NO: 19) (Hordeum vulgare, EST TC190653).

[0133] FIG. 1b: Preservation of the proline residues

[0134] FIG. 2: Working model of the position of the proline residues in the 3D structure of Phl p 5a

[0135] 3D homology model of the N-terminal (amino acids 31-139 of Phl p 5.0109; model molecule: Phl p 6, PDB entry 1 NLX; depicted on the left-hand side) and C-terminal 4-helix bundle (amino acids 155-255; model molecule: Phl p 5b fragment, PDB entry 1 L3P; right).

[0136] a. Highly simplified model of the two 4-helix bundles. H1-H8: helices 1-8. Prolines are shown with their position designation and atomic structure. Proline residues P146 and P256 are in regions which it has not been possible to show owing to the lack of structural data in the 3D models of the model molecules (dotted lines). The loop depicted between helix 2 and 3 is speculative, since the model molecule Phl p 6 has no region which is homologous to this Phl p 5a region.

[0137] b. Surface model. All proline residues which can be depicted are exposed at the surface (coloured black).

[0138] FIG. 3: rPhl p 5a wt (IUIS entry Phl p 5.0109); cDNA sequence (GenBank entry: AJ555152; 855 bp), SEQ ID NO:1

[0139] FIG. 4: rPhl p 5a wt (IUIS entry Phl p 5.0109); deduced amino acid sequence (UniProtKB entry: Q84U12; 284 aa), SEQ ID NO:2

[0140] FIG. 5: N-terminal histidine fusion component; DNA sequence (57 bp), SEQ ID NO:3

[0141] FIG. 6: N-terminal histidine fusion component; amino acid sequence (19 aa) SEQ ID:4

[0142] FIG. 7: Results of the IgE inhibition tests with Phl p 5a variants with proline deletions in individual loops on use of a serum pool (“6His” disclosed as SEQ ID NO: 6)

[0143] (a)+(b): Data from one individual experiment in each case with double determination. The symbols represent the means of the duplicates on measurement of eight inhibitor concentrations in each case. The horizontal lines of the error bars show the individual values of the double determination.

[0144] (c): Summary of the results of a number of individual experiments.

[0145] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0146] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0147] The bar height shows the mean obtained from a number (n) of individual experiments. The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0148] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0149] FIG. 8: Comparison of the IgE inhibition of rPhl p 5a d[P117, 180]+6His with the single-position proline mutants rPhl p 5a d[P117]+6His and d[P180]+6His (“6His” disclosed as SEQ ID NO: 6)

[0150] Summary of the Results of a Number of Individual Experiments:

[0151] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0152] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0153] The bar height shows the mean obtained from a number (n) of individual experiments. The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0154] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0155] FIG. 9: Comparison of the IgE inhibition of MPV.3+6His with the single-position proline mutants rPhl p 5a d[P57, 58]+6His and d[P229]+6His (“6His” disclosed as SEQ ID NO: 6)

[0156] Summary of the Results of a Number of Individual Experiments:

[0157] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0158] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0159] The bar height shows the mean obtained from a number (n) of individual experiments.

[0160] The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0161] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0162] FIG. 10: Comparison of the IgE inhibition of MPV.3+6His with the single-position proline mutants rPhl p 5a d[P211]+6His, P211L+6His, K61E+6His and E205K+6His (“6His” disclosed as SEQ ID NO: 6)

[0163] Summary of the Results of a Number of Individual Experiments:

[0164] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0165] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0166] The bar height shows the mean obtained from a number (n) of individual experiments.

[0167] The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0168] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0169] FIG. 11: Test for functional allergeneity of MPV.3+6His (“6His” disclosed as SEQ ID NO: 6)

[0170] Evidence of the reduced functional allergeneity of MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) by means of basophil activation test with basophils of four clinically defined grass pollen allergy sufferers (P). Horizontal line: level of stimulation by a negative control.

[0171] FIG. 12: IgE inhibition test with MPV.4+6His (“6His” disclosed as SEQ ID NO: 6) and MPV.4

[0172] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0173] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0174] The bar height shows the mean obtained from a number (n) of individual experiments.

[0175] The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0176] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0177] FIG. 13: Determination of the molecular weight of MPV.5

[0178] Chromatogram of an analytical SEC with online molecular weight determination.

[0179] The figure shows the relative UV signal at 280 nm (right-hand Y axis) and the molecular weight (left-hand Y axis; measurement point line in the peak region), plotted against the elution time (X axis).

[0180] For online determination of the protein concentration, the OptilabrEX refractive index detector (Wyatt, Santa Barbara, USA) was employed. The light scattering by the particles was determined using the MiniDAWN Treos multiangle detector (Wyatt). The particle weight was calculated using the ASTRA 5.3.2.17 software (Wyatt) via Debeye formalism with a refractive-index increment of 0.180 ml/g.

[0181] Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t.sub.0) is at 20.45 min (corresponds to ˜670 kD).

[0182] Eluent: 20 mM Tris 8.0, 150 mM NaCl

[0183] FIG. 14: Determination of the molecular weight of MPV.7 Chromatogram of an analytical SEC with online molecular weight determination.

[0184] The figure shows the relative UV signal at 280 nm (right-hand Y axis) and the molecular weight (left-hand Y axis; measurement point line in the peak region), plotted against the elution time (X axis).

[0185] For online determination of the protein concentration, the OptilabrEX refractive index detector (Wyatt, Santa Barbara, USA) was employed. The light scattering by the particles was determined using the MiniDAWN multiangle detector Treos (Wyatt). The particle weight was calculated using ASTRA 5.3.2.17 software (Wyatt) via Debeye formalism with a refractive-index increment of 0.180 ml/g.

[0186] Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t.sub.0) is at 20.45 min (corresponds to ˜670 kD).

[0187] Eluent: 20 mM Tris 8.0, 150 mM NaCl

[0188] FIG. 15: Determination of the molecular weight of MPV.4

[0189] Chromatogram of an analytical SEC with online molecular weight determination.

[0190] The figure shows the relative UV signal at 280 nm (right-hand Y axis) and the molecular weight (left-hand Y axis; measurement point line in the peak region), plotted against the elution time (X axis).

[0191] For online determination of the protein concentration, the OptilabrEX refractive index detector (Wyatt, Santa Barbara, USA) was employed. The light scattering by the particles was determined using the MiniDAWN Treos multiangle detector (Wyatt). The particle weight was calculated using ASTRA 5.3.2.17 software (Wyatt) via Debeye formalism with a refractive-index increment of 0.180 ml/g.

[0192] Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t.sub.0) is at 20.45 min (corresponds to ˜670 kD).

[0193] Eluent: 20 mM Tris 8.0, 150 mM NaCl

[0194] FIG. 16: Determination of the molecular weight of MPV.6

[0195] Chromatogram of an analytical SEC with online molecular weight determination.

[0196] The figure shows the relative UV signal at 280 nm (right-hand Y axis) and the molecular weight (left-hand Y axis; measurement point line in the peak region), plotted against the elution time (X axis).

[0197] For online determination of the protein concentration, the OptilabrEX refractive index detector (Wyatt, Santa Barbara, USA) was employed. The light scattering by the particles was determined using the MiniDAWN Treos multiangle detector (Wyatt). The particle weight was calculated using ASTRA 5.3.2.17 software (Wyatt) via Debeye formalism with a refractive-index increment of 0.180 ml/g.

[0198] Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t.sub.0) is at 20.45 min (corresponds to ˜670 kD).

[0199] Eluent: 20 mM Tris 8.0, 150 mM NaCl

[0200] FIG. 17: IgE binding to immobilised MPV.4, MPV.5, MPV.6 and MPV.7

[0201] Test for binding of IgE to Phl p 5 proteins immobilised on nitrocellulose membrane strips.

[0202] rPhl p 5b wt: recombinant Phl p 5b isoform

[0203] rPhl p 5a wt: recombinant Phl p 5a isoform

[0204] nPhl p 5a/b: Phl p 5 allergen of natural origin, consisting of protein of the a and b isoform (Allergopharma)

[0205] MPV.4: d[P57, P58, P117, P146, P155, P180, P229] K61E E205K P211L

[0206] MPV.5: d[P57, P58, P117, P180, P229] K61E E205K P211L

[0207] MPV.6: d[P57, P58, P117, P146, P155, P180, P229] P211L

[0208] MPV.7: d[P57, P58, P117, P180, P229] P211L

[0209] HSA: albumin from human serum (negative control).

[0210] Total: control for uniform charging of the strips. Staining with reagent DB71 (Sigma, USA)

[0211] SP: serum pool Bor18/100 (Allergopharma)

[0212] FIG. 18: IgE inhibition test with MPV.5 and MPV.4

[0213] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0214] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0215] The bar height shows the mean obtained from a number (n) of individual experiments.

[0216] The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0217] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0218] FIG. 19: IgE inhibition test with MPV.6 and MPV.7

[0219] Left-hand bar (dark grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 5 μg/ml.

[0220] Right-hand bar (pale grey): IgE inhibition relative to rPhl p 5a wt at an inhibitor concentration of 1.25 μg/ml.

[0221] The bar height shows the mean obtained from a number (n) of individual experiments.

[0222] The error bars show maximum (horizontal line of the upper error bar) and minimum (horizontal line of the lower error bar) values obtained in a number (n) of evaluated individual experiments.

[0223] Solid phase: rPhl p 5a wt. Serum pool: Bor 18/100, Allergopharma.

[0224] FIG. 20: Test for functional allergeneity of MPV.4

[0225] Evidence of the reduced functional allergeneity of MPV.4 by means of basophil activation test with basophils of four clinically defined grass pollen allergy sufferers (P).

[0226] Horizontal line: level of stimulation by a negative control.

[0227] FIG. 21: Test for functional allergeneity of MPV.5

[0228] Evidence of the reduced functional allergeneity of MPV.5 by means of basophil activation test with basophils of four clinically defined grass pollen allergy sufferers (P).

[0229] Horizontal line: level of stimulation by a negative control.

[0230] FIG. 22: Test for functional allergeneity of MPV.6

[0231] Evidence of the reduced functional allergeneity of MPV.6 by means of basophil activation test with basophils of clinically defined grass pollen allergy sufferers (P).

[0232] Horizontal line: level of stimulation by a negative control.

[0233] FIG. 23: Test for functional allergeneity; MPV.7

[0234] Evidence of the reduced functional allergeneity of MPV.7 by means of basophil activation test with basophils of four clinically defined grass pollen allergy sufferers (P).

[0235] Horizontal line: level of stimulation by a negative control.

[0236] FIG. 24: rPhl p 5b wt precursor (IUIS entry Phl p 5.0201); deduced amino acid sequence 284 aa (Swiss prot: 040963.2), SEQ ID NO:5

[0237] Even without further embodiments, it is assumed that a person skilled in the art will be able to utilise the above description in the broadest scope. The preferred embodiments should therefore merely be regarded as descriptive disclosure, which is, however, in no way limiting in any way.

[0238] The following examples are thus intended to explain the invention without limiting it. Unless indicated otherwise, percent data mean percent by weight. All temperatures are indicated in degrees Celsius. “Conventional work-up”: water is added if necessary, the pH is adjusted, if necessary, to values between 2 and 10, depending on the constitution of the end product.

[0239] The following hypoallergenic variants according to the invention were prepared by biotechnological methods and characterised. However, the preparation and characterisation of the substances can also be carried out by other methods for the person skilled in the art. For example, the hypoallergenic variants according to the invention can also be synthesised chemically. The invention likewise relates to the hypoallergenic variants according to the invention described below.

EXAMPLE 1: VARIANTS OF PHL P 5A WITH ONE OR TWO ADJACENT PROLINE DELETIONS

[0240] The preparation of variants rPhl p 5a d[P57, P58]+6His, rPhl p 5a d[P85]+6His, rPhl p 5a d[P117]+6His, rPhl p 5a d[P146, P155]+6His, rPhl p 5a d[P180]+6His, rPhl p 5a d[P211]+6His, rPhl p 5a d[P229]+6His and rPhl p 5a d[P256]+6His and immunological characterisation thereof is described below (“6His” disclosed as SEQ ID NO: 6). The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0241] Construction by Genetic Engineering:

[0242] The DNA of the variants are synthesised by the bonding of long overlapping DNA oligonucleotides and amplification of the DNA by a PCR standard method. The codons are selected so that the deduced amino acid sequence is based on that of Phl p 5.0109 (FIG. 3, 4). The mutations for the proline deletions are introduced using specific oligonucleotides which lack the corresponding codons for proline in the PCR reactions. The oligonucleotides are selected so that the deduced protein carries a hexahistidine (SEQ ID NO: 6) fusion component at the 5′ end (FIG. 5, 6). The cDNAs are ligated into expression vector pTrcHis2 Topo (Invitrogen, Carlsbad, USA). The correctness of the DNA is confirmed by sequencing.

[0243] Expression, Purification and Biochemical Analysis:

[0244] The expression is carried out in Escherichia coli (Top10 strain; Invitrogen). rPhl p 5a wt and the variants are purified by specific binding of the N-terminal histidine residues to an Ni2+ chelate matrix (immobilised metal ion affinity chromatography, IMAC; material: HiTrap, GE Healthcare, Uppsala, Sweden). The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy.

[0245] Evidence of Reduced IgE Binding:

[0246] The investigation of the IgE binding ability of the test substances is carried out using an EAST-IgE inhibition test (enzyme allergosorbent test). In this method, the allergen/IgE interaction can be investigated in solution, enabling interfering masking of epitopes of the test substance to be excluded, for example by immobilisation on a membrane.

[0247] The EAST inhibition test is carried out as follows. Microtitre plates are coated with the allergens, here recombinant wild-type Phl p 5.0109 (rPhl p 5a wt).

[0248] After removal of the unbound allergen molecules by washing, the plate is blocked with bovine serum albumin in order to prevent later non-specific binding. IgE antibodies of allergy sufferers in the form of a representative pool of individual allergy sufferer sera (serum pool) in suitable dilution are incubated with the allergen-coated microtitre plates. The amount of allergen-bound IgE antibodies is quantified photometrically via an anti-human-IgE/alkaline phosphatase conjugate by the reaction of a substrate to give a coloured end product.

[0249] The binding of the IgE antibodies is inhibited substance-specifically by a soluble allergen or the substance to be tested (recombinant modified allergen) as a function of the concentration. The results depicted in FIG. 7. of IgE inhibition tests with the recombinant allergen variants of Phl p 5a show that reduced IgE binding ability of Phl p 5a is caused by deletion of proline residues P57 and P58; P117; P146 and P155; P180; P211 or P229, but not by deletion of P85 or P256. A reduced inhibitory action indicates a loss of IgE epitopes.

EXAMPLE 2: VARIANTS OF PHL P 5A WITH COMBINATIONS OF PROLINE DELETIONS

[0250] The preparation and immunological characterisation of variant rPhl p 5a d[P117, P180]+6His (“6His” disclosed as SEQ ID NO: 6) is described below by way of example for hypoallergenic variants according to the invention of group 5 allergens of the Poaceae with combinations of a plurality of proline deletions corresponding to amino acid positions of 57 and 58, 117, 146, and 155, 180, 211 and 229, based on Phl p 5.0109. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0251] Construction by Genetic Engineering:

[0252] The variants are synthesised by the bonding of long overlapping DNA oligonucleotides and amplification of the DNA by PCR. The codons are selected so that the deduced amino acid sequence is based on that of Phl p 5.0109. The mutations for the proline deletions are introduced using specific oligonucleotides which lack the corresponding codons for proline in the PCR reactions. The oligonucleotides are selected so that the deduced protein carries a hexahistidine (SEQ ID NO: 6) fusion component at the 5′ end. The cDNAs are transformed into expression vector pTrcHis2 Topo (Invitrogen) and in Escherichia coli. The correctness of the DNA is confirmed by sequencing.

[0253] Expression, Purification and Biochemical Analysis:

[0254] The expression is carried out in Escherichia coli (Top10 strain; Invitrogen). The variants are purified by IMAC. The purity of the eluted proteins is checked by SDS-PAGE, and the absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy.

[0255] Evidence of Reduced IgE Binding:

[0256] The investigation of the IgE binding ability of the test substances is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which were employed in the form of a representative serum pool. The results depicted in FIG. 8 show that IgE inhibition of variant rPhl p 5a d[P117, P180]+6His is significantly lower than that of the variants with only one of proline deletions rPhl p 5a d[P117]+6His and rPhl p 5a d[P180]+6His (“6His” disclosed as SEQ ID NO: 6). This result shows that the combination of individual proline deletions can result in increased reduction of the IgE binding ability of Phl p 5a.

EXAMPLE 3: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P85, P117, P146, P155, P180, P211, P229, P256] (MPV.1+6HIS (“6HIS” DISCLOSED AS SEQ ID NO: 6))

[0257] Allergen variant MPV.1+6His (“6His” disclosed as SEQ ID NO: 6) has a combination of the deletions of all Phl 5a proline residues investigated. The aim of the preparation is IgE binding ability reduced to the maximum with acceptable protein solubility.

[0258] Construction by Genetic Engineering:

[0259] The variants are synthesised by the bonding of long overlapping DNA oligonucleotides and amplification of the DNA by PCR. The codons are selected so that the deduced amino acid sequence is based on that of Phl p 5.0109. The mutations for the proline deletions are introduced using specific oligonucleotides which lack the corresponding codons for proline in the PCR reactions.

[0260] The oligonucleotides are selected so that the deduced protein carries a hexahistidine (SEQ ID NO: 6) fusion component at the 5′ end. The cDNA are transformed into expression vector pTrcHis2 Topo (Invitrogen) and in Escherichia coli. The correctness of the DNA is confirmed by sequencing.

[0261] Expression:

[0262] The expression is carried out in Escherichia coli (Top10 strain; Invitrogen). The proteins are deposited by the host cell as insoluble inclusion bodies.

[0263] Test of Solubility:

[0264] The protein aggregates are isolated in a purity of about 80% after cell digestion using a standard method and solubilised in a denaturing manner by treatment with a 6 molar solution of guanidinium hydrochloride.

[0265] The denatured proteins are diluted 1:50 in a volume of 25 ml for this testing at 4° C. and kept at 4° C. overnight. On the following day, any visible precipitate formation is checked organoleptically. After partial concentration, the samples are centrifuged, and the clear supernatants are investigated for the presence of insoluble microaggregates by means of UV-Vis spectroscopy.

[0266] The systematic investigation of the solubility behaviour of variant MPV.1+6His (“6His” disclosed as SEQ ID NO: 6) shows that subsequent conversion of the proteins into a guanidinium hydrochloride-free formulation is always accompanied by the formation of protein aggregates and is thus not possible (Table 1)

EXAMPLE 4: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P117, P146, P155, P180, P211, P229] (MPV.2+6HIS (“6HIS” DISCLOSED AS SEQ ID NO: 6))

[0267] The preparation and immunological characterisation of variant MPV.2+6His (“6His” disclosed as SEQ ID NO: 6) is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of protein deletions corresponding to amino acid positions 57, 58, 117, 146, 155, 180, 211 and 229, based on Phl p 5.0109. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0268] Construction by Genetic Engineering:

[0269] The variants are synthesised by the bonding of long overlapping DNA oligonucleotides and amplification of the DNA by PCR. The codons are selected so that the deduced amino acid sequence is based on that of Phl p 5.0109.

[0270] The mutations for the proline deletions are introduced using specific oligonucleotides which lack the corresponding codons for proline in the PCR reactions.

[0271] The oligonucleotides are selected so that the deduced protein carries a hexahistidine (SEQ ID NO: 6) fusion component at the 5′ end. The cDNA are ligated into expression vector pTrcHis2 Topo (Invitrogen) and transformed in Escherichia coli. The correctness of the DNA is confirmed by sequencing.

[0272] Expression:

[0273] The expression is carried out in Escherichia coli (Top10 strain; Invitrogen). The proteins are deposited by the host cell as insoluble inclusion bodies.

[0274] Test of Solubility:

[0275] MPV.2+6His (“6His” disclosed as SEQ ID NO: 6) exhibits poor solubility throughout the pH range of 4.5-9.0 investigated. The formation of visible precipitates can only be prevented with a solutions comprising Tween 80 and L-arginine monohydrochloride, but microaggregates are also detected in this batch below (Table 2).

EXAMPLE 5: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P229] K61E, E205K, P211L (MPV.3+6HIS (“6HIS” DISCLOSED AS SEQ ID NO: 6))

[0276] The preparation and immunological characterisation of variant MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of deletions of proline residues 57, 58, 117, 146, 155, 180 or 229 corresponding to the amino acid positions in Phl p 5 5.0109, in which proline residue 211 is mutated into any desired other amino acid or in which lysine 61 is additionally converted into glutamate or glutamate 205 is converted into lysine. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0277] The effect of mutations K61E, E205K and P211L present in the amino acid sequence of MPV.3+6His on the IgE binding ability is investigated by preparing variants rPhl p 5a K61E+6His, rPhl p 5a E205K+6His and rPhl p 5a P211L+6His and characterising them immunologically (“6His” disclosed as SEQ ID NO: 6).

[0278] Construction by Genetic Engineering:

[0279] The nucleotide sequence encoding for MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) is surprisingly generated in this example by a number of polymerase errors in a cDNA synthesis. The DNA is ligated into expression vector pTrcHis2 Topo (Invitrogen). The DNA encoding for variants rPhl p 5a K61E+6His, rPhl p 5a E205K+6His and rPhl p 5a P211L+6His are prepared specifically and ligated into expression vector pTrcHis2 Topo (“6His” disclosed as SEQ ID NO: 6). The correctness of the generated cDNA sequences is checked by DNA sequencing.

[0280] Expression:

[0281] The expression is carried out in Escherichia coli (Top10 strain; Invitrogen). The proteins are deposited by the host cell as insoluble inclusion bodies.

[0282] Test of the Solubility of MPV.3+6his (“6his” Disclosed as SEQ ID NO: 6):

[0283] The protein aggregates are isolated in a purity of about 80% after cell digestion and solubilised in a denaturing manner by treatment with a 6 molar solution of guanidinium hydrochloride. The series testing of various solutions in order to check the solubility properties of MPV.3+6His in a non-denaturing environment is carried out analogously to the experiments with variants MPV.1+6His and MPV.2+6His (“6His” disclosed as SEQ ID NO: 6).

[0284] Surprisingly, it is observed that variant MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) has high solubility at slightly basic pH during the dilution process (Table 3). This behaviour is in contrast to variants MPV.1+6His and MPV.2+6His investigated above and represents a crucial advantage for the preparation of the protein in a non-denaturing environment (“6His” disclosed as SEQ ID NO: 6).

[0285] Purification of MPV.3+6his, and Mutants rPhl p 5a K61E+6his, rPhl p 5a E205K+6his and rPhl p 5a P211L+6his (“6his” Disclosed as SEQ ID NO: 6):

[0286] The denatured proteins are diluted 1:50 in 20 mM Tris, 150 mM NaCl, pH 9.0, and kept at 8° C. overnight. The chromatographic purification is carried out by IMAC (HiTrap material, GE Healthcare) and SEC (Superdex 75 material, GE Healthcare). The proteins are finally in stable and soluble form in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5.

[0287] Biochemical Analysis:

[0288] The purity is checked by SDS-PAGE. The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy.

[0289] Evidence of the Reduced IgE Binding of MPV.3+6his (“6his” Disclosed as SEQ ID NO: 6):

[0290] The investigation of the IgE binding ability of MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which are employed in the form of a representative serum pool. The results depicted in FIG. 9 show that the IgE inhibition of MPV.3+6His is significantly lower than that of variants rPhl p 5a d[P57, P58]+6His and rPhl p 5a d[P229]+6His (“6His” disclosed as SEQ ID NO: 6).

[0291] For further characterisation of mutations K61E, E205K and P211L of variant MPV.3+6His, the three variants rPhl p 5a K61E+6His, rPhl p 5a E205K+6His and rPhl p 5a P211L+6His are investigated by EAST (“6His” disclosed as SEQ ID NO: 6).

[0292] Variant rPhl p 5a P211L+6His exhibits a comparably low inhibition behaviour to the point mutant rPhl p 5a d[P211]+6His tested previously (“6His” disclosed as SEQ ID NO: 6). Variants rPhl p 5a K61E+6His and rPhl p 5a E205K+6His exhibit slightly reduced IgE binding (FIG. 10) (“6His” disclosed as SEQ ID NO: 6).

[0293] It can be concluded that the three point mutations K61E, E205K and P211L contribute to the significantly reduced IgE binding of MPV.3+6His (“6His” disclosed as SEQ ID NO: 6).

[0294] Evidence of the Reduction of the Functional Allergeneity of MPV.3+6his (“6his” Disclosed as SEQ ID NO: 6):

[0295] The functional action of MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) in the crosslinking of membrane-bound IgE of the effector cells and activation thereof is investigated in vitro.

[0296] For the basophil activation test, heparinised whole blood of grass pollen allergy sufferers is incubated with various concentrations of the test substances. Allergenic substances are able to bind specific IgE antibodies which are associated with the high-affinity IgE receptors of the basophilic granulocytes. The crosslinking of the IgE/receptor complexes triggered by the allergen molecules results in signal transduction, which results in degranulation of the effector cells and thus the triggering of the allergic reactions in vivo.

[0297] The allergen-induced activation of basophilic immunocytes can be determined in vitro by quantification of the expression of a surface protein (CD203c) coupled to the signal transduction of IgE-receptor crosslinking (Kahlert et al., Clinical Immunology and Allergy in Medicine Proceedings of the EAACI 2002 (2003) Naples, Italy 739-744). The number of surface proteins expressed on a cell and the percentage value of the activated cells of a cell pool is measured highly sensitively via the binding of a fluorescence-labelled monoclonal antibody to the surface protein and subsequent analysis by fluorescence-activated flow cytometry.

[0298] MPV.3+6His (“6His” disclosed as SEQ ID NO: 6) exhibits reduced activation of basophilic granulocytes of grass pollen allergy sufferers here compared with rPhl p 5a wt and thus functionally reduced allergeneity (FIG. 11)

EXAMPLE 6: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, 117, 146, 155, 180, P229] K61E, E205K, P211L (MPV.4)

[0299] The preparation and immunological characterisation of variant MPV.4 is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of deletions of proline residues 57, 58, 117, 146, 155, 180 or 229 corresponding to the amino acid positions in Phl p 5.0109), in which proline residue 211 is mutated into any desired other amino acid or in which lysine 61 is additionally converted into glutamate or glutamate 205 is converted into lysine. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0300] Construction of MPV.4 (+6his (“6his” Disclosed as SEQ ID NO: 6)) by Genetic Engineering:

[0301] In order to prepare the cDNA of the histidine fusion protein of MPV.4 (MPV.4+6His), a fragment of the already cloned cDNA of MPV.1+6His is re-cloned into the plasmid MPV.3+6His/pTrcHis2 Topo which is already present (“6His” disclosed as SEQ ID NO: 6).

[0302] In order to prepare the fusion component-free protein (MPV.4), the DNA is ligated into vector pTMP (Allergopharma, Reinbek) without the section encoding for the histidine fusion component. The correctness of the sequence is checked by DNA sequencing.

[0303] Expression of MPV.4 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0304] The expression is carried out either as histidine fusion protein (expression vector pTrcHis2Topo; Invitrogen) in Escherichia coli (Top10 strain; Invitrogen) or without fusion component (expression vector pTMP) in Escherichia coli (BL21 strain; Merck, Darmstadt). In both cases, the recombinant proteins are deposited as inclusion bodies. The proteins are solubilised using a 6 molar solution of guanidinium hydrochloride.

[0305] Test of the Solubility of MPV.4+6his (“6his” Disclosed as SEQ ID NO: 6):

[0306] The series testing of various solutions in order to check the solubility properties of MPV.4+6His in a non-denaturing environment is carried out analogously to the experiments with variant MPV.1+6His (“6His” disclosed as SEQ ID NO: 6). It is observed that variant MPV.4+6His (“6His” disclosed as SEQ ID NO: 6) has solubility at slightly basic pH (Table 4)

[0307] Purification of MPV.4+6his (“6his” Disclosed as SEQ ID NO: 6):

[0308] The fusion protein is purified on a preparative scale by IMAC (HiTrap material, GE Healthcare) and SEC (Superdex 75 material, GE Healthcare) and is finally present in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5.

[0309] Purification of the Non-Fusion Protein MPV.4:

[0310] The solution of the denatured non-fusion protein are firstly diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0, analogously to the purification scheme of the fusion protein and kept at 4° overnight. In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC).

[0311] At high ion strength, proteins bind to an HIC column with specific strength, depending on their surface hydrophobicity, enabling them to be eluted selectively by gradual reduction of the salt concentration.

[0312] The diluted protein solution is firstly diluted further 1:2 with 20 mM Tris, 2 M ammonium sulfate, 150 mM NaCl, pH 9.0, in order to achieve a final ammonium sulfate concentration of 1 M. This protein solution is then passed through a HiTrap butyl-S FF HIC column (GE Healthcare, Uppsala, Sweden) and, after binding of the target protein, eluted gradually with 20 mM Tris, 150 mM NaCl, pH 9.0.

[0313] As preparation for the second purification step, the HIC eluates are re-buffered in 20 mM Tris, 50 mM NaCl, pH 8.0, via a Sephadex G25 column (GE Healthcare).

[0314] As the second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare), and the material passing through the column is collected. The target protein is located in the material passing through the column, while foreign proteins bind to the column and are effectively removed.

[0315] In the AIEX process, proteins having a negative surface charge are bound to the column material at low ion strength, while uncharged or positively charged proteins do not bind.

[0316] In the final step, the AIEX output is subsequently purified via an SEC column (Superdex 75, GE Healthcare), so that the target protein is finally present in 20 mM Tris, 150 mM NaCl, pH 8.0.

[0317] Biochemical analysis of MPV.4 (+6His (“6His” disclosed as SEQ ID NO: 6)): The purity of the proteins is checked by SDS-PAGE. The identity of the fusion component-free MPV.4 is confirmed by determination of the molecular weight by means of mass spectroscopy (MALDI-TOF) and sequencing of the N-terminal amino acids (sequence: A-D-L-G-Y-G-P-A-T (SEQ ID NO: 7)) (Table 6). The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy.

[0318] Molecular weight analysis of MPV.4 by means of SEC/MALS/RI surprisingly shows that the mass of the eluted proteins is between 28.2 and 49.3 kD (average mass 36.9 kD). Since the theoretical mass of the monomer is 27.6 kD, it can be assumed that a mixture of monomers and dimers is present (FIG. 15, Table 6). Qualitatively the same result is obtained with the fusion protein.

[0319] Evidence of the Reduced IgE Binding of MPV.4 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0320] The investigation of the IgE binding ability is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which are employed in the form of a representative serum pool.

[0321] The results depicted in FIG. 12 show that MPV.4 has IgE binding reduced to the same extent both with and without fusion component. It is thus shown that the reduced IgE reactivity is not dependent on the presence of the histidine tag.

[0322] A simple test method for the determination of the IgE reactivity of specific IgE from allergy sufferer sera on membrane-bound test proteins is the strip test. For this purpose, the test substances in the same concentration and amount are bound alongside one another to a strip of nitrocellulose membrane under non-denaturing conditions. A series of such membrane strips can be incubated in parallel with different allergy sufferer sera. After a washing step, the specifically bound IgE antibodies are rendered visible on the membrane by a colour reaction, promoted by an anti-human IgE/alkaline phosphatase conjugate.

[0323] The results of variant MPV.4 using individual grass pollen allergy sufferer sera are depicted in FIG. 17. Sera of allergy sufferers with antibodies against natural Phl p 5 (nPhl p 5a/b, mixture of Phl p 5a and b isoform) are used. The IgE antibodies likewise react with the recombinant rPhl p 5a wt and the likewise investigated recombinant wild-type b isoform (rPhl p 5b wt).

[0324] It is clear that the Phl p 5-specific IgE antibodies of all allergy sufferer sera bind variant MPV.4 to a greatly reduced extent, while recombinant wild-type proteins rPhl p 5a wt and rPhl p 5b wt are bound just as strongly as nPhl p 5a/b.

[0325] Evidence of the reduction of the functional allergeneity of MPV.4: The functional action of MPV.4 in the crosslinking of membrane-bound IgE of the effector cells and activation thereof is investigated both with fusion proteins and with non-fusion proteins in vitro. Both the fusion protein and the fusion component-free MPV.4 exhibit greatly reduced activation of basophilic granulocytes of grass pollen allergy sufferers here compared with rPhl p 5a wt and thus highly functionally reduced allergeneity (FIG. 20).

EXAMPLE 7: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P117, P180, P229] K61E, E205K, P211L (MPV.5)

[0326] The preparation and immunological characterisation of variant MPV.5 is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of deletions of proline residues 57, 58, 117, 180 or 229 corresponding to the amino acid positions in Phl p 5.0109, in which proline residue 211 is mutated into any desired other amino acid or in which lysine 61 is additionally converted into glutamate or glutamate 205 is converted into lysine. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0327] Construction of MPV.5 (+6his (“6his” Disclosed as SEQ ID NO: 6)) by Genetic Engineering:

[0328] In order to prepare the cDNA of the histidine fusion protein (MPV.5+6His), a fragment of the already cloned cDNA of rPhl p 5a d[P117, P180]+6His is re-cloned into the plasmid MPV.3+6His/pTrcHis2 Topo which is already present (“6His” disclosed as SEQ ID NO: 6).

[0329] In order to prepare the fusion component-free protein (MPV.5), the DNA is ligated into vector pTMP (Allergopharma, Reinbek) without the section encoding for the histidine fusion component. The correctness of the sequence is checked by DNA sequencing.

[0330] Expression of MPV.5 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0331] The expression is carried out either as histidine fusion protein (expression vector pTrcHis2Topo; Invitrogen) in Escherichia coli (Top10 strain; Invitrogen) or without fusion component (expression vector pTMP; Allergopharma) in Escherichia coli (BL21 strain; Merck, Darmstadt).

[0332] In both cases, the recombinant proteins are deposited as inclusion bodies. The proteins are solubilised by means of a 6 molar solution of guanidinium hydrochloride.

[0333] Purification of MPV.5+6his (“6his” Disclosed as SEQ ID NO: 6):

[0334] The fusion protein is purified on a preparative scale by IMAC (HiTrap material, GE Healthcare) and SEC (Superdex 75 material, GE Healthcare) and is finally present in 25 mM sodium phosphate buffer with 150 mM NaCl, pH 7.5.

[0335] Purification of the Non-Fusion Protein:

[0336] The IB solution of the denatured non-fusion protein are firstly diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0, analogously to the purification scheme of the fusion protein and kept at 4° overnight.

[0337] In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC). The dilute protein solution is firstly diluted further 1:2 with 20 mM Tris, 2 M ammonium sulfate, 150 mM NaCl, pH 9.0, in order to achieve a final ammonium sulfate concentration of 1 M. This protein solution is then passed through a HiTrap butyl-S FF HIC column (GE Healthcare, Uppsala, Sweden) and, after binding of the target protein, eluted gradually with 20 mM Tris, 150 mM NaCl, pH 9.0.

[0338] As preparation for the second purification step, the HIC eluates are re-buffered in 20 mM Tris, 50 mM NaCl, pH 8.0, via a Sephadex-G25 column (GE Healthcare). As the second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare), and the material passing through the column, which comprises the target protein, is collected.

[0339] In the final step, the AIEX output is subsequently purified via an SEC column (Superdex 75, GE Healthcare), so that the target protein is finally present in 20 mM Tris, 150 mM NaCl, pH 8.0.

[0340] Biochemical Analysis of MPV.5 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0341] The purity of the proteins is checked by SDS-PAGE. The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. The identity of the fusion component-free MPV.5 is confirmed by determination of the molecular weight by means of mass spectroscopy (MALDI-TOF) and sequencing of the N-terminal amino acid sequence (sequence: A-D-L-G-Y-G-P-A-T (SEQ ID NO: 7)) (Table 6).

[0342] Analysis of MPV.5 by SEC/MALS/RI shows that the protein is exclusively in the form of the monomer (FIG. 13, Table 6). A comparable result is obtained with the fusion protein. The high tendency towards dimerisation which is observed in the case of variant MPV.4 is thus dependent on the presence of mutation d[P146, P155].

[0343] Evidence of the Reduced IgE Binding of MPV.5 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0344] The investigation of the IgE binding ability is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which are employed in the form of a representative serum pool. MPV.5 exhibits IgE binding ability which is reduced to an equal extent both with and without fusion component. The IgE binding ability is somewhat higher than that of variant MPV.4, which may be due to better accessibility of IgE epitopes in the case of MPV.5, which is exclusively in monomeric form (FIG. 18).

[0345] The results of variant MPV.5 in the strip test using individual grass pollen allergy sufferer sera are depicted in FIG. 17. It is clear that the Phl p 5-specific IgE antibodies of all allergy sufferer sera bind variant MPV.5 to a greatly reduced extent.

[0346] Evidence of the Reduction of the Functional Allergeneity of MPV.5 (+6his (“6his” Disclosed as SEQ ID NO: 6)):

[0347] The functional action of MPV.5 in the crosslinking of membrane-bound IgE of the effector cells and activation thereof is investigated both with fusion proteins and with non-fusion proteins in vitro. MPV.5 exhibits greatly reduced activation of basophilic granulocytes of grass pollen allergy sufferers here compared with rPhl p 5a wt and thus highly functionally reduced allergeneity (FIG. 21).

EXAMPLE 8: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P117, P146, P155, P180, P229] P211L (MPV.6)

[0348] The preparation and immunological characterisation of variant MPV.6 is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of deletions of proline residues 57, 58, 117, 146, 155, 180 or 229 corresponding to the amino acid positions in Phl p 5.0109, in which proline residue 211 is mutated into any desired other amino acid. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0349] Construction by Genetic Engineering:

[0350] For the preparation of the DNA, a DNA fragment of MPV.2+6His (“6His” disclosed as SEQ ID NO: 6) is ligated into vector rPhl p 5a d[57, 58, 117, 180, 229] P211L/pTMP (Allergopharma) present at this time. The correctness of the sequence is checked by DNA sequencing.

[0351] Expression:

[0352] The expression is carried out exclusively as fusion component-free protein in expression vector pTMP (Allergopharma) in Escherichia coli (strain BL21; Merck, Darmstadt). The recombinant proteins are deposited as inclusion bodies (IB).

[0353] Purification:

[0354] The IB solution of the denatured non-fusion protein is firstly diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0, and kept at 4° overnight.

[0355] In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC).

[0356] The dilute protein solution is firstly diluted further 1:2 with 20 mM Tris, 2 M ammonium sulfate, 150 mM NaCl, pH 9.0, in order to achieve a final ammonium sulfate concentration of 1 M. This protein solution is then passed through a HiTrap butyl-S FF HIC column (GE Healthcare, Uppsala, Sweden) and, after binding of the target protein, eluted gradually with 20 mM Tris, 150 mM NaCl, pH 9.0.

[0357] As preparation for the second purification step, the HIC eluates are re-buffered in 20 mM Tris, 50 mM NaCl, pH 8.0, via a Sephadex G25 column (GE Healthcare). As the second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare), and the material passing through the column, which comprises the target protein, is collected. In the final step, the AIEX output is subsequently purified via an SEC column (Superdex 75, GE Healthcare), so that the target protein is finally present in 20 mM Tris, 150 mM NaCl, pH 8.0.

[0358] Biochemical Analysis:

[0359] The purity of the proteins is checked by SDS-PAGE. The identity of the fusion component-free MPV.6 is confirmed by determination of the molecular weight by means of mass spectroscopy (MALDI-TOF) and sequencing of the N-terminal amino acid sequence (sequence: A-D-L-G-Y-G-P-A-T (SEQ ID NO: 7)) (Table 6).

[0360] The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. In the analysis of MPV.6 by means of SEC/MALS/RI, the purified proteins elute in two peaks. Peak 1 represents the monomeric form and peak 2 the dimeric form (FIG. 16; Table 6).

[0361] The dimerisation ability is, as also shown with reference to variants MPV.4 and MPV.5, attributable to the presence of mutation d[P146, 155].

[0362] Evidence of Reduced IgE Binding:

[0363] The investigation of the IgE binding is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which are employed in the form of a representative serum pool. The results depicted in FIG. 19 show that MPV.6 has greatly reduced IgE binding. The results in the strip test using individual grass pollen allergy sufferer sera are depicted in FIG. 17. It is clear that the Phl p 5-specific IgE antibodies of all allergy sufferer sera bind variant MPV.6 to a greatly reduced extent.

[0364] Evidence of the Reduction of Functional Allergeneity:

[0365] The functional action of MPV.6 in the crosslinking of membrane-bound IgE and the activation of basophilic granulocytes is greatly reduced compared with rPhl p 5a wt, as shown by the results with whole blood of two grass-pollen allergy sufferers (FIG. 22).

EXAMPLE 9: HYPOALLERGENIC VARIANT RPHL P 5A D[P57, P58, P117, P180, P229] P211L (MPV.7)

[0366] The preparation and immunological characterisation of variant MPV.7 is described below by way of example for hypoallergenic variants of group 5 allergens of the Poaceae with combinations of deletions of proline residues 57, 58, 117, 180 or 229 corresponding to the amino acid positions in Phl p 5a wild-type or Phl p 5.0109, in which proline residue 211 is mutated into any desired other amino acid. The recombinant unmodified allergen (rPhl p 5 wt+6His (“6His” disclosed as SEQ ID NO: 6)) is prepared and investigated analogously, and the hypoallergenic variants of the other group 5 allergens according to the invention of the true grasses and their wild-type proteins, in particular Lol p 5 and Poa p 5, can also be prepared and investigated analogously.

[0367] Construction by Genetic Engineering:

[0368] The DNA is prepared by preparation of a DNA fragment by means of specific oligonucleotides by PCR processes and then re-cloning into vector rPhl p 5a d[P57, P58, P117, P180, P211, P229]/pTMP (Allergopharma) already present at this time. The correctness of the sequence is checked by DNA sequencing.

[0369] Expression:

[0370] The expression is carried out as fusion component-free protein in expression vector pTMP (Allergopharma) in Escherichia coli (strain BL21; Merck, Darmstadt). The recombinant proteins are deposited as inclusion bodies (IB).

[0371] Test of Solubility:

[0372] The series testing in order to check the solubility properties of the recombinant protein in a non-denaturing environment shows high solubility of the protein in the slightly basic pH range (Table 5).

[0373] Purification:

[0374] The IB solution of the denatured non-fusion protein is firstly diluted 1:50 with 20 mM Tris, 150 mM NaCl, pH 9.0, and kept at 4° overnight. In a first purification step, the target protein is enriched by hydrophobic interaction chromatography (HIC). The dilute protein solution is firstly diluted further 1:2 with 20 mM Tris, 2 M ammonium sulfate, 150 mM NaCl, pH 9.0, in order to achieve a final ammonium sulfate concentration of 1 M. This protein solution is then passed through a HiTrap butyl-S FF HIC column (GE Healthcare, Uppsala, Sweden) and, after binding of the target protein, eluted gradually with 20 mM Tris, 150 mM NaCl, pH 9.0.

[0375] As preparation for the second purification step, the HIC eluates are re-buffered in 20 mM Tris, 50 mM NaCl, pH 8.0, via a Sephadex G25 column (GE Healthcare). As the second purification step, the eluates are applied to an anion exchange chromatography column (HiTrap Q HP, GE Healthcare), and the material passing through the column, which comprises the target protein, is collected. In the final step, the AIEX output is subsequently purified via an SEC column (Superdex 75, GE Healthcare), so that the target protein is finally present in 20 mM Tris, 150 mM NaCl, pH 8.0.

[0376] Biochemical Analysis:

[0377] The purity of the proteins is checked by SDS-PAGE. The identity of the fusion component-free MPV.7 is confirmed by determination of the molecular weight by means of mass spectroscopy (MALDI-TOF) and sequencing of the N-terminal amino acid sequence (sequence: A-D-L-G-Y-G-P-A-T (SEQ ID NO: 7)) (Table 6). The absence of insoluble protein aggregates is confirmed by UV-Vis spectroscopy. Analysis by SEC/MALS/RI shows that the eluted proteins are exclusively in the form of monomers (FIG. 14; Table 6). This is attributable to the absence of mutation d[P146, P155].

[0378] Evidence of Reduced IgE Binding:

[0379] The investigation of the IgE binding is carried out using an EAST inhibition test with IgE antibodies of allergy sufferers which are employed in the form of a representative serum pool. The results depicted in FIG. 19 show that MPV.7 has greatly reduced IgE binding. The results in the strip test using individual grass pollen allergy sufferer sera are depicted in FIG. 17. It is clear that the Phl p 5-specific IgE antibodies of all allergy sufferer sera bind variant MPV.7 to a greatly reduced extent.

[0380] Evidence of the Reduction of Functional Allergeneity:

[0381] The functional action of MPV.7 in the crosslinking of membrane-bound IgE and the activation of basophilic granulocytes of grass pollen allergy sufferers is greatly reduced compared with rPhl p 5a wt (FIG. 23).

[0382] T-Cell Reactivity of the Hypoallergenic Phl p 5a Variants:

[0383] In order to investigate the T-cell reactivity, oligoclonal T-cell lines of grass pollen allergy sufferers are established by conventional methods with stimulation with natural nPhl p 5a or rPhl p 5a wt molecules. In a proliferation test, the different T-cell lines were stimulated with the reference allergen rPhl p 5a wt and the modified recombinant allergen variants. The proliferation rate was determined by conventional methods by the incorporation of [3H]-thymidine.

[0384] The results of the proliferation tests of MPV.4 and MPV.7 with T-cell lines of 12 grass pollen allergy sufferers are depicted here by way of example for the modified allergen variants described.

[0385] The T-cell reactivity of variant MPV.4, which carries the most mutations of all molecules investigated, is not reduced, in spite of the modifications of the amino acid sequence compared with unmodified rPhl p 5a wt, which demonstrates the retention of crucial T-cell epitopes (Table 7).

[0386] Mutant MPV.7 contains the smallest number of modified amino acid positions of all multiproline mutants investigated. As expected, the molecule stimulates human T-lymphocytes comparably well to the unmodified allergen rPhl p 5a (Table 8).

TABLE-US-00001 TABLE 1 Solubility behaviour of MPV.1 + 6His (“6His” disclosed as SEQ ID NO: 6) K61E, d[P57, 58] d[P85] d[P117] d[146, 155] d[P180] d[P211] P211L d[P229] d[P256] E205K x x x x x x x x Evalua- Soln. pH NaCl Buffer substance Additive OC.sup.1 A.sub.280/A.sub.330.sup.2 tion.sup.3 1 4.5 0.15M 0.02M Na acetate none − n.p. − 2 5.5 0.15M 0.02M Na citrate none − n.p. − 3 6.5 0.15M 0.02M Ka phosph. none − n.p. − 4 7.5 0.15M 0.02M Na phosph. none − n.p. − 5 8.0 0.15M 0.02M Tris none − n.p. − 6 9.0 0.15M 0.02M Tris none − n.p. − 7 8.0  0.075M 0.02M Tris 0.5M L-arginine HCl; + 12.5 (−) − 0.005% (w/v) Tween 80 8 8.0 none 0.02M Tris 0.005% (w/v) Tween 80 + 10.2 (−) − 9 8.0 none 0.02M Tris 0.5M L-arginine HCl − n.p. − 10 7.5 none 0.02M Na phosph. 10% (w/v) glycerine − n.p. − .sup.1The proteins isolated from inclusion bodies were firstly denatured using 6M guanidinium hydrochloride, subsequently diluted 1:50 in a non-denaturing solution (soln. 1-10) and kept at 4° C. overnight. On the following day, an organoleptic check (OC) with respect to turbidity caused by visible macroaggregates or precipitates was carried out. (−) turbidity; (+) clear solution. .sup.2UV/Vis spectral analysis for the detection of insoluble microaggregates in clear solutions by determination of the ratio of the absorption at 280 and 330 nm. Test of the batches after centrifugation. A.sub.280/A.sub.330 ≦ 20: precipitation (−); A.sub.280/A.sub.330 ≦ 30: precipitation tendency (∘); A.sub.280/A.sub.330 > 30: no precipitation (+). (n.p.) not performed. .sup.3Evaluation of the solubility behaviour based on organoleptic and spectrophotometric analysis. (−) tending towards insoluble; (+) soluble.

TABLE-US-00002 TABLE 2 Solubility behaviour of MPV.2 + 6His (“6His” disclosed as SEQ ID NO: 6) K61E, d[P57, 58] d[P85] d[P117] d[146, 155] d[P180] d[P211] P211L d[P229] d[P256] E205K x x x x x x Evalua- Soln. pH NaCl Buffer substance Additive OC.sup.1 A.sub.280/A.sub.330.sup.2 tion.sup.3 1 4.5 0.15M 0.02M Na acetate none − n.p. − 2 5.5 0.15M 0.02M Na citrate none − n.p. − 3 6.5 0.15M 0.02M Ka phosph. none − n.p. − 4 7.5 0.15M 0.02M Na phosph. none − n.p. − 5 8.0 0.15M 0.02M Tris none − n.p. − 6 9.0 0.15M 0.02M Tris none − n.p. − 7 8.0  0.075M 0.02M Tris 0.5M L-arginine HCl; + 11.5 (−) − 0.005% (w/v) Tween 80 8 8.0 none 0.02M Tris 0.005% (w/v) Tween 80 − n.p. − 9 8.0 none 0.02M Tris 0.5M L-arginine HCl − n.p. − 10 7.5 none 0.02M Na phosph. 10% (w/v) glycerine − n.p. − .sup.1The proteins isolated from inclusion bodies were firstly denatured using 6M guanidinium hydrochloride, subsequently diluted 1:50 in a non-denaturing solution (soln. 1-10) and kept at 4° C. overnight. On the following day, an organoleptic check (OC) with respect to turbidity caused by visible macroaggregates or precipitates was carried out. (−) turbidity; (+) clear solution. .sup.2UV/Vis spectral analysis for the detection of insoluble microaggregates in clear solutions by determination of the ratio of the absorption at 280 and 330 nm. Test of the batches after centrifugation. A.sub.280/A.sub.330 ≦ 20: precipitation (−); A.sub.280/A.sub.330 ≦ 30: precipitation tendency (∘); A.sub.280/A.sub.330 > 30: no precipitation (+). (n.p.) not performed. .sup.3Evaluation of the solubility behaviour based on organoleptic and spectrophotometric analysis. (−) tending towards insoluble; (+) soluble.

TABLE-US-00003 TABLE 3 Solubility behaviour of MPV.3 + 6His (“6His” disclosed as SEQ ID NO: 6) K61E, d[P57, 58] d[P85] d[P117] d[146, 155] d[P180] d[P211] P211L d[P229] d[P256] E205K x x x x Evalua- Soln. pH NaCl Buffer substance Additive OC.sup.1 A.sub.280/A.sub.330.sup.2 tion.sup.3 1 4.5 0.15M 0.02M Na acetate none − n.p. − 2 5.5 0.15M 0.02M Na citrate none − n.p. − 3 6.5 0.15M 0.02M Ka phosph. none − n.p. − 4 7.5 0.15M 0.02M Na phosph. none − n.p. − 5 8.0 0.15M 0.02M Tris none + 36.0 (+) + 6 9.0 0.15M 0.02M Tris none + 42.6 (+) + 7 8.0  0.075M 0.02M Tris 0.5M L-arginine HCl; + 37.5 (+) + 0.005% (w/v) Tween 80 8 8.0 none 0.02M Tris 0.005% (w/v) Tween 80 + 26.0 (∘) − 9 8.0 none 0.02M Tris 0.5M L-arginine HCl − n.p. − 10 7.5 none 0.02M Na phosph. 10% (w/v) glycerine − n.p. − .sup.1The proteins isolated from inclusion bodies were firstly denatured using 6M guanidinium hydrochloride, subsequently diluted 1:50 in a non-denaturing solution (soln. 1-10) and kept at 4° C. overnight. On the following day, an organoleptic check (OC) with respect to turbidity caused by visible macroaggregates or precipitates was carried out. (−) turbidity; (+) clear solution. .sup.2UV/Vis spectral analysis for the detection of insoluble microaggregates in clear solutions by determination of the ratio of the absorption at 280 and 330 nm. Test of the batches after centrifugation. A.sub.280/A.sub.330 ≦ 20: precipitation (−); A.sub.280/A.sub.330 ≦ 30: precipitation tendency (∘); A.sub.280/A.sub.330 > 30: no precipitation (+). (n.p.) not performed. .sup.3Evaluation of the solubility behaviour based on organoleptic and spectrophotometric analysis. (−) tending towards insoluble; (+) soluble.

TABLE-US-00004 TABLE 4 Solubility behaviour of MPV.4 + 6His (“6His” disclosed as SEQ ID NO: 6) K61E, d[P57, 58] d[P85] d[P117] d[146, 155] d[P180] d[P211] P211L d[P229] d[P256] E205K x x x x x x x Evalua- Soln. pH NaCl Buffer substance Additive OC.sup.1 A.sub.280/A.sub.330.sup.2 tion.sup.3 1 4.5 0.15M 0.02M Na acetate none − n.p. − 2 5.5 0.15M 0.02M Na citrate none − n.p. − 3 6.5 0.15M 0.02M Ka phosph. none − n.p. − 4 7.5 0.15M 0.02M Na phosph. none + 33.6 (+) + 5 8.0 0.15M 0.02M Tris none + 34.3 (+) + 6 9.0 0.15M 0.02M Tris none + 32.0 (+) + 7 8.0  0.075M 0.02M Tris 0.5M L-arginine HCl; + 34.8 (+) + 0.005% (w/v) Tween 80 8 8.0 none 0.02M Tris 0.005% (w/v) Tween 80 + 23.9 (∘) − 9 8.0 none 0.02M Tris 0.5M L-arginine HCl − n.p. − 10 7.5 none 0.02M Na phosph. 10% (w/v) glycerine − n.p. − .sup.1The proteins isolated from inclusion bodies were firstly denatured using 6M guanidinium hydrochloride, subsequently diluted 1:50 in a non-denaturing solution (soln. 1-10) and kept at 4° C. overnight. On the following day, an organoleptic check (OC) with respect to turbidity caused by visible macroaggregates or precipitates was carried out. (−) turbidity; (+) clear solution. .sup.2UV/Vis spectral analysis for the detection of insoluble microaggregates in clear solutions by determination of the ratio of the absorption at 280 and 330 nm. Test of the batches after centrifugation. A.sub.280/A.sub.330 ≦ 20: precipitation (−); A.sub.280/A.sub.330 ≦ 30: precipitation tendency (∘); A.sub.280/A.sub.330 > 30: no precipitation (+). (n.p.) not performed. .sup.3Evaluation of the solubility behaviour based on organoleptic and spectrophotometric analysis. (−) tending towards insoluble; (+) soluble.

TABLE-US-00005 TABLE 5 Solubility behaviour of MPV.7 K61E, d[P57, 58] d[P85] d[P117] d[146, 155] d[P180] d[P211] P211L d[P229] d[P256] E205K x x x x x Evalua- Soln. pH NaCl Buffer substance Additive OC.sup.1 A.sub.280/A.sub.330 .sup.2 tion.sup.3 1 4.5 0.15M 0.02M Na acetate none − n.p. − 2 5.5 0.15M 0.02M Na citrate none − n.p. − 3 6.5 0.15M 0.02M Ka phosph. none − n.p. − 4 7.5 0.15M 0.02M Na phosph. none − n.p. − 5 8.0 0.15M 0.02M Tris none + 32.5 (+) + 6 9.0 0.15M 0.02M Tris none +  72.5 (+). + 7 8.0  0.075M 0.02M Tris 0.5M L-arginine HCl; + 37.6 (+) + 0.005% (w/v) Tween 80 8 8.0 none 0.02M Tris 0.005% (w/v) Tween 80 − n.p. − 9 8.0 none 0.02M Tris 0.5M L-arginine HCl + 30.4 (+) + 10 7.5 none 0.02M Na phosph. 10% (w/v) glycerine + 43.0 (+) + .sup.1The proteins isolated from inclusion bodies were firstly denatured using 6M guanidinium hydrochloride, subsequently diluted 1:50 in a non-denaturing solution (soln. 1-10) and kept at 4° C. overnight. On the following day, an organoleptic check (OC) with respect to turbidity caused by visible macroaggregates or precipitates was carried out. (−) turbidity; (+) clear solution. .sup.2 UV/Vis spectral analysis for the detection of insoluble microaggregates in clear solutions by determination of the ratio of the absorption at 280 and 330 nm. Test of the batches after centrifugation. A.sub.280/A.sub.330 ≦ 20: precipitation (−); A.sub.280/A.sub.330 ≦ 30: precipitation tendency (∘); A.sub.280/A.sub.330 > 30: no precipitation (+). (n.p.) not performed. .sup.3Evaluation of the solubility behaviour based on organoleptic and spectrophotometric analysis. (−) tending towards insoluble; (+) soluble.

TABLE-US-00006 TABLE 6 Results of the molecular weight analysis of MPV.4, MPV.5, MPV.6 and MPV.7 compared with rPhl p 5a wt SEC-MALS- Phl p 5a MW.sub.calc. RI.sup.2 variant [kD] .sup.1 Peak MW [kD] Assessment Wild type 28.3 1 27.3 Only monomers detectable MPV.4 27.6 1 28.2-49.3* Monomer and dimers detectable in mixed peak MPV.5 27.8 1 27.2 Only monomers detectable MPV.6 27.6 1 53.0 Dimer peak Monomers and 2 29.8 Monomer peak dimers detect- able MPV.7 27.8 1 27.2 Only monomers detectable .sup.1 Calculated molecular weight (MW.sub.calc.) without starting methionine on the basis of the amino acid sequence (Software: DNA-Star, Lasergene, USA) .sup.2Determination of the particle mass by SEC-MALS. The figure quoted is the average mass of the eluted protein particles in the peak window set with the exception of the measurement of MPV.4 (*), in which the scattering range of the masses in the peak as a whole is indicated. For online determination of the protein concentration, the OptilabrEX refractive index detector (RI) (Wyatt, Santa Barbara, USA) was employed. The light scattering by the particles was determined using the MiniDAWN Treos multiangle detector (Wyatt). The particle mass was calculated using ASTRA 5.3.2.17 software (Wyatt) via Debeye formalism with an assumed refractive-index increment of 0.180 ml/g. Column: Superdex 200 GL 10/300 (GE Healthcare, Uppsala, Sweden). The size exclusion (t.sub.0) is at 20.45 min (corresponds to ~670 kD). Eluent: 20 mM Tris 8.0, 150 mM NaCl

TABLE-US-00007 TABLE 7 Evidence of the T-cell reactivity of MPV.4 Stimulation index.sup.1 Reactivity of MPV.4 Donor.sup.2 T-cell line rPhl p 5a wt MPV.4 relative to rPhl p 5a wt.sup.3 3 3.10 3.0 4.0 1.33 8 8.2 13 12.7 0.98 8 8.3 2.9 4.0 1.38 19 19.1 7.2 9.9 1.38 19 19.2 3.5 4.9 1.40 21 21.210 4.5 5.8 1.29 23 23.22 4.3 2.2 0.51 55 55.184 26.7 28.5 1.07 55 55.193 49.9 46.2 0.93 59 59.57 4.1 4.4 1.07 59 59.91 7.7 9.0 1.17 60 60.162 4.9 2.6 0.53 65 65.115 6.7 9.1 1.36 116 116.34 9.4 2.8 0.30 128 128.40 8.8 8.9 1.01 137 137.41 2.8 6.5 2.32 137 137.43 2.6 2.5 0.96 1.12 average 0.45 SD .sup.1Stimulation index (SI), calculated from [.sup.3H] measurement values of the proliferation test. cpm measurement values of allergen-stimulated cell cultures/cpm measurement values of unstimulated cell cultures. .sup.2Donor: clinically defined grass pollen allergy sufferer. .sup.3Calculated using SI (MPV.4)/SI (rPhl p 5a wt). SD: standard deviation.

TABLE-US-00008 TABLE 8 Evidence of the T-cell reactivity of MPV.7 Reactivity of Stimulation index.sup.1 MPV.7 relative to Donor.sup.2 T-cell line rPhl p 5a wt MPV.7 rPhl p 5a wt.sup.3 3 3.10 2.0 2.2 1.10 8 8.2 27.7 23.6 0.85 8 8.3 5.2 8.2 1.58 11 11.2 2.1 2.7 1.29 11 11.3 2.4 2.4 1.00 19 19.1 4.8 6.5 1.35 21 21.2 3.7 3.4 0.92 21 21.210 3.9 5.4 1.38 55 55.181 12.4 14.8 1.19 55 55.184 123.6 149.5 1.21 59 59.91 4.5 5.0 1.11 60 60.162 2.9 2.6 0.90 65 65.115 4.6 4.7 1.02 70 70.126 6.5 6.5 1.00 116 116.34 8.5 2.5 0.29 128 128.40 4.8 3.5 0.73 1.06 average 0.30 SD .sup.1Stimulation index (SI), calculated from [.sup.3H] measurement values of the proliferation test. cpm measurement values of allergen-stimulated cell cultures/cpm measurement values of unstimulated cell cultures. .sup.2Donor: clinically defined grass pollen allergy sufferer. .sup.3Calculated using SI (MPV.7)/SI (rPhl p 5a wt). SD: standard deviation.