Hypoallergenic variants of Phl p 5, the major allergen from Phleum pratense

Abstract

Disclosed are hypoallergenic variants of Phl p 5, the major allergen from Phleum pratense, and the uses thereof in the treatment of allergic diseases.

Claims

1. A hybrid protein containing a hypoallergenic sequence variant of the Phl p 5 major allergen from Phleum pratense pollen consisting of SEQ ID NO:2 and a hypoallergenic sequence variant of the major allergen Phl p 1 from Phleum pratense pollen consisting of SEQ ID NO: 6.

2. The hybrid protein according to claim 1, wherein said Phl p 1 and Phl p 5 hypoallergenic sequence variants are located at either amino or carboxyl ends with a head-to-tail orientation.

3. The hybrid protein according to claim 2, wherein said Phl p 1 and Phl p 5 are located at the amino- and carboxyl-end, respectively.

4. The hybrid protein of claim 1, wherein said sequence variant of the Phl p 5 major allergen and the hypoallergenic variant of the major allergen Phl p 1 are separated by a linker.

5. The hybrid protein of claim 4, wherein said linker consists of EF dipeptide.

6. The hybrid protein according to claim 4, wherein said linker consists of an amino acid sequence containing from 1 to 8 amino acids.

7. The hybrid protein according to claim 6, consisting of SEQ ID NO:4.

8. A pharmaceutical composition containing an effective amount of a hybrid protein according to claim 1, and pharmaceutically acceptable carriers, excipients or adjuvants.

9. The composition according to claim 8, which is in a form suitable for sublingual, intranasal, subcutaneous or transdermal administration.

10. A method of treating patients allergic to Phleum pratense pollen comprising: administering to said patients in need thereof an effective amount of a hybrid protein according to claim 1 or a pharmaceutical composition thereof.

11. The method of claim 10, wherein said patients have bronchial asthma, allergic rhinitis, allergic conjunctivitis, or allergic oral syndrome.

12. A nucleic acid molecule coding for a hybrid protein according to claim 1.

13. A nucleic acid molecule according to claim 12, whose sequence is selected from SEQ ID NO:8 and 10.

14. An expression vector containing the nucleic acid molecule according to claim 12.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: ELISA analysis of IgE reactivity to Phl p 5 allergen and its hypoallergenic variant SEQ ID NO: 2

(2) FIG. 2: Inhibition of IgE binding to Phl p 5 allergen

(3) FIG. 3: Murine IgG response to respective immunogenic proteins

(4) FIG. 4: IgG response in mice immunised with SEQ ID NO:1

(5) FIG. 5: Murine IgG response to Phl p pollen extract.

(6) FIG. 6: Inhibition of IgE binding to SEQ ID NO: 1 by murine IgG antibodies

(7) FIG. 7: Schematic presentation of hybrid protein SEQ ID NO: 3 and SEQ ID NO: 4

(8) FIG. 8: ELISA analysis of IgE reactivity to hybrid variant SEQ ID NO: 4

(9) FIG. 9: Inhibition of IgE binding to Phl p pollen extract

(10) FIG. 10: Inhibition of IgE binding to (mix wt)

(11) FIG. 11: Murine IgG response to Phl p

(12) FIG. 12: Murine IgG response to SEQ ID NO: 1

(13) FIG. 13: Murine IgG response to Phl p 1

(14) FIG. 14: Inhibition of IgE binding to Phl p by murine IgG antibodies

EXAMPLES

(15) Unless otherwise indicated, the methods used in the following examples are described in Sambrook, Fritsch E T Maniatis “Molecular Cloning: A Laboratory Manual” II Ed. Vol. 1-2-3 CSH Lab Press 1989.

Example 1

Site-Specific Mutagenesis of the cDNA Coding for Phl p 5 Allergen

(16) Site-specific mutagenesis of the cDNA coding for wt Phl p 5 allergen (SEQ ID NO: 7, preceded at 5′ by a sequence coding six histidines) was carried out by cDNA cloning in a prokaryotic vector (pBluescript, GenBank acc. n. X52327) followed by PCR amplification. The oligonucleotides used as primers in the PCR reaction (Table 1) carried the appropriate base substitutions. For each mutagenesis, a complementary oligonucleotide binding to a corresponding region of the DNA strand was used (19). After amplification, the unaltered original template was selectively degraded with enzymatic digestion catalyzed by the restriction enzyme Dpn I. Escherichia coli cells were then transformed with the mutagenized molecules. Clones obtained from single bacterial colonies were sequenced according to Sanger to determine the correct base modification and the absence of non-specific mutations in the cDNA.

(17) TABLE-US-00001 TABLE 1 Sequences of the oligonucleotides used as primers  in site-specific mutagenesis. The mutated bases are in bold character. Oligonucleotide Sequence Phl p5 P158L Caa ggt tat cct tgc cgg cga gct g  (SEQ ID NO: 13)

Example 2

Construction of a Plasmid Coding for Wild-Type Phl p 1-Phl p 5 Hybrid Molecule (wtHybrid)

(18) The hybrid molecule containing the genetic information for wild-type Phl p 1-Phl p 5 hybrid was obtained by fusion of cDNAs coding for the single allergens.

(19) The cDNAs encoding mature Phl p 1 and Phl p 5 proteins were obtained separately by PCR using Php1 DIM Kpn FW (Cgc ggt acc atc ccc aag gtt ccc ccg gg—SEQ ID NO: 14) and Php1 DIM Eco RV (Gc gaa ttc ctt gga cga gta get ggt—SEQ ID NO: 15) oligonucleotide primers for Phl p 1 and Php5 DIM Eco FW (cgc gaa ttc gcc gat cta ggt tac ggc cc—SEQ ID NO: 16) and Php5 DIM Bam RV (Gcg gga tcc tca gac ttt gta gcc acc—SEQ ID NO: 17) oligonucleotide primers for Phl p 5. Phl p 1 SEQ ID NO: 11 and Phl p 5 SEQ ID NO: 7 cDNAs were used as templates.

(20) The amplification product obtained from Phl p 1 was re-amplified by replacing Php1 DIM Kpn FW con Phl p 1 DIM His FW (gcg ggt acc cat atg cat cac cat cac cat cac atc ccc aag gtt ccc ccg SEQ ID NO:18), whereby a sequence coding for six histidines upstream of Phl p 1 sequence was inserted. A Kpn I site and a Nde I site (containing the ATG) were inserted at 5′ of Phl p 1 amplification product, and a Eco R I site was inserted at its 3′ in place of the stop codon. An Eco R I site was inserted in place of the ATG at 5′ of Phl p 5, and a Bam H I site was inserted at its 3′ after the stop codon. The amplified products were purified and digested with Kpn I and Eco R I restriction enzymes (Phl p 1), or Eco R I and Bam H I (Phl p 5) (restriction sites are underlined in the primers), and subsequently inserted into Kpn I/Bam H I sites of pEt 3c vector (Stratagene, La Jolla, Calif.) to obtain a construct capable of expressing a Phl p 1-Phl p 5 fusion protein preceded by a sequence of six histidines. Introduction of Eco R I restriction site, that is necessary for cloning of fragments, allowed for insertion of two amino acids (glutamic acid and phenylalanine) at the junction of the two proteins without altering the reading frame (FIG. 8).

(21) Clones obtained from single bacterial colonies were sequenced by the Sanger method to verify that base change was correct, and the absence of unspecific mutations in the cDNA.

Example 3

Construction of a Plasmid Coding for Mutant Phl p 1-Phl p 5 Hybrid Molecule (MutHybrid)

(22) The hybrid molecule coding for mutant Phl p 1-Phl p 5 hybrid was obtained following the method described in EXAMPLE 2 for the wild-type hybrid variant.

(23) The oligonucleotide pairs used in the PCR reaction were identical, while the cDNAs used as templates encoded for two hypoallergenic variants whose sequences are herein identified as SEQ ID NO: 12 (for Phl p 1 mutant) and SEQ ID NO: 8 (Phl p 5 mutant).

Example 4

Production of Phl p 5 Proteins, Respective Mutants, wtHybrid and MutHybrid

(24) Wild-type Phl p 1 (SEQ ID NO:11) and Phl p 5 (SEQ ID NO:7) cDNAs, mutagenized cDNAs (SEQ ID NO:12 and SEQ ID NO:8), and engineered wt and Mut Hybrid cDNAs (SEQ ID NO: 9 e 10), preceded by the sequence coding for six histidines, were cloned in an expression vector and expressed in Escherichia coli cells according to standard protocols. Cells were collected by centrifugation and resuspended in 100 mM NaH.sub.2PO.sub.4 buffer, pH 8 and lysed by sonication. The recombinant proteins were separated by centrifugation. Soluble Phl p 5 protein (SEQ ID NO: 1) was purified from the supernatant by affinity chromatography using Ni-NTA agarose columns bound to nitrilotriacetic acid (Qiagen, Milan, Italy) which chelates nickel ions interacting with the six-histidine portion fused to the allergen. Mutagenized Phl p 5 (SEQ ID NO: 2) and wt and mutant Hybrid (SEQ ID NO: 3 and 4), separated as aggregate in the pellet, were resuspended in denaturing buffer 8 M urea, 100 mM NaH.sub.2PO.sub.4, 10 mM Tris pH 8 and stirred for 60 min at 20° C. The solubilized recombinant proteins were separated from insoluble debris by centrifugation and purified from the supernatant under denaturing conditions by affinity chromatography using Ni-NTA agarose columns. Purified proteins were refolded by dialysis in a 5 mM (NH.sub.4)HCO.sub.3 solution for 18 hours at 4° C.

Example 5

Characteristics of Sera from Allergic Subjects

(25) Sera were collected from subjects with a clinical history of seasonal allergy to Phleum pratense pollen, and high EAST reactivity specific for Phl p 1 and Phl p 5 allergens and used in single or pooled form. A pool of sera from non-allergic subjects was used as a negative control.

Example 6

ELISA Analysis of Phl p 5 Variants Reactivity to IgEs from a Serum Pool

(26) Equivalent amounts (25 pmol) in 50 mM carbonate/bicarbonate buffer, pH 9.6 of wt allergen (SEQ ID NO: 1), mutagenized variant (SEQ ID NO:2) and unrelated antigen (BSA) were adsorbed on wells of polystirene plates for ELISA assay by incubation at 4° C. for 16 hours. The wells were washed with washing solution (60 mM phosphate buffer, pH 6.5, containing 0.05% Tween-20), and blocked with dilution solution (2% BSA in PBS 1×). 100 μl aliquots, diluted 1:5 in dilution buffer, of a pool of eleven human sera from EAST positive or non-allergic subjects (data not shown) were added to each sample and incubated at 25° C. for 2 hours. After three washes, peroxidase-conjugated anti human-IgE serum (1:4000 in diluting buffer) was added, followed by incubation at 25° C. for 1.5 hours. After three washes, the colorimetric reaction was developed by adding 100 μl of TMB reagent (BioFX Laboratories, Owings Mills, Md.) and incubating for 15 minutes at 25° C. The reaction was stopped by adding 100 μl of 1 N HCl and read at 450 nm using a microplate reader spectrophotometer. Results were confirmed by three independent experiments.

(27) The same protocol was applied with some modifications to test IgE reactivity of the engineered wt and Mut hybrids. Serial dilution of the hybrid allergens (SEQ ID NO:3 and 4) were prepared in a 1:2 ratio, starting from 0.4 nmol/ml and adsorbed on wells of polystirene plates. A pool of sera positive to Phleum pratense pollen was diluted 1:8 in diluting solution.

Example 7

ELISA Inhibition Assay—Monomeric Variant SEQ ID NO: 2 Ability to Inhibit Binding of Phl p 5 to IgEs in Serum

(28) Equal amounts (1.25 pmol) of wild type Phl p 5 (SEQ ID NO: 1), in 50 mM carbonate/bicarbonate buffer, pH 9.6, were adsorbed onto wells of polystyrene plates for ELISA assay by incubating at 4° C. for 16 hours. Wells were then washed with washing solution (60 mM phosphate buffer, pH 6,5, containing 0.05% Tween-20), and free sites were blocked with diluting solution (2% BSA, PBS 1×). Aliquots (100 μl) of a 1:10 dilution of pooled human sera positive to Phl p 5 were pre-incubated with two-fold serial dilutions of wt or mutagenized allergens starting from 25 pmol/ml at 25° C. for 2 hours. The mixes were then added to each well, and incubated at 4° C. for 16 hours. After three washes, anti-human IgE peroxidase-conjugated serum diluted 1:4000 in diluting buffer was added, and incubated at 25° C. for 1.5 hours. Colorimetric reaction development was obtained by adding 100 μl TMB reagent and incubating for 15 minutes at 25° C. The reaction was stopped by adding 100 μl 1 N HCl, and evaluated by reading at 450 nm with a spectrophotometer. Inhibition percentage was calculated by using the following formula: 100×[(A−B)/A], where A is absorbance at 450 nm in the absence of inhibitor, and B is absorbance in the presence of inhibitor. Data are representative of three independent experiments.

Example 8

ELISA Inhibition Assay—Ability of SEQ ID NO: 3 and SEQ ID NO: 4 to Inhibit Binding of Human Serum IgEs to Phl p Pollen Extract or Phl p 1 and Phl p 5 Mix

(29) Equal amounts (0.05 μg) of Phleum pratense, in 50 mM carbonate/bicarbonate buffer, pH 9.6, were adsorbed onto wells of polystyrene plates for ELISA assay by incubating at 4° C. for 16 hours. Wells were then washed with washing solution (60 mM phosphate buffer, pH 6,5, containing 0.05% Tween-20), and free sites were blocked with diluting solution (2% BSA in PBS 1×). Aliquots (70 μl) of a 1:10 dilution of a pool of human sera positive to Phleum pratense pollen were pre-incubated with equimolar amounts (150 nmol/ml) of wild type allergen, mutagenized or engineered variants (hybrids) or 0.5 μg/ml of Phleum pratense pollen extract at 25° C. for 2 hours. The mixes were then added to each well, and incubated at 4° C. for 16 hours. After three washes, anti-human IgE peroxidase-conjugated serum diluted 1:4000 in diluting buffer was added, and incubated at 25° C. for 1.5 hours. After three washes, colorimetric reaction development was obtained by adding 100 μl TMB reagent and incubating for 15 minutes at 25° C. The reaction was stopped by adding 100 μl 1 N HCl, and evaluated by reading at 450 nm with a spectrophotometer. Inhibition percentage was calculated by using the following formula: 100×[(A−B)/A], where A is absorbance at 450 nm in the absence of inhibitor, and B is absorbance in the presence of inhibitor.

(30) To test the ability of wt and mutagenised hybrid proteins, SEQ ID NO: 3 and 4, to inhibit Phl p 1 and Phl p 5 mix, the same protocol was applied with the following modifications: the amount of protein adsorbed on wells was 12.5 pmol of Phl p 1 and Phl p 5, the inhibitor concentration started from 0.1 nmol/ml (two-fold serial dilution), the dilution of the pool of sera positive to Phleum pratense was 1:10.

(31) TABLE-US-00002 TABLE 2 Hybrid wild-type- and mutagenized-molecules inhibit IgE binding to Phleum pratense pollen extract Phleum wt Mut SEQ ID SEQ ID serum pratense Mix Mix NO: 3 NO: 4 1 74.7 57.6 53 67.7 49.9 2 74.5 13.7 6.3 16.9 13.2 3 82.2 43.3 25 50.9 41.2 4 78.8 16.7 10 18.9 12.5 5 79.1 59.2 56.6 68.1 54.5 6 87.5 81.1 60.4 87.9 75.8 7 91.3 52.4 30.3 53.4 39 8 78.9 67.8 53.8 69.6 63.7 9 28 62.9 39.6 60.4 51.6 10 41.6 54.9 16.3 49.3 43.2 Mean 71.7 51.0 35.1 54.3 44.5 % inhibition Standard 20.4 21.3 20.4 22.3 19.9 deviation

Example 9

Immunization of Balb/c Mice

(32) Seven groups of mice each composed of five Balb/c strain female animals (Charles River) were subcutaneously immunized with 150 pmol of wt, mutagenized or engineered (hybrid) allergen or 10 μg of Phleum pratense pollen extract mixed with 2 mg of Al(OH.sub.3) in 200 μl saline. Other two boosts were performed after 21 and 42 days. As a control, five mice received the same treatment with an unrelated antigen. Two, five and seven weeks following first immunization, blood collection was performed from jugular vein of mice, and antibody response to the respective immunogen, wt molecules and Phleum pratense pollen extract was checked by ELISA. Sera from mice were tested singularly or pooled based on immunogenic type and time elapsed from first immunization.

Example 10

ELISA Analysis of Specific Murine IgG Response

(33) Equal amounts of Phleum pratense pollen extract (20 μg/ml), wt Phl p 5 or Phl p 1, SEQ ID NO: 2 and SEQ ID NO: 4 variants (2 μg/ml), in 50 mM carbonate/bicarbonate buffer, pH 9.6, were adsorbed onto wells of polystyrene plates for ELISA assay by incubating at 4° C. for 16 hours. Wells were then washed with washing solution (60 mM phosphate buffer, pH 6,5, containing 0.05% Tween-20), and free sites were blocked with diluting solution (2% BSA in PBS 1×). Equal aliquots (100 μl) of each mouse serum or pooled sera were added to each well at a 1:1000 (FIG. 5) or 1:5000 dilution in diluting buffer, and incubated at 25° C. for 2 hours. After three washes, anti-total mouse IgG peroxidase-conjugated serum diluted 1:8000 in diluting buffer was added and incubated at 25° C. for 1.5 hours. After three washes, colorimetric reaction development was obtained by adding 100 μl TMB reagent and incubating for 20 minutes at 25° C. The reaction was stopped by adding 100 μl 1 N HCl and read at 450 nm with a spectrophotometer. Data show the mean reactivity obtained by analysis of the sera from 5 mice for each group.

Example 11

ELISA Inhibition Assay. IgGs Against SEQ ID NO: 2 and SEQ ID NO: 4 Inhibit Binding Between Phleum pratense Pollen Extract and IgEs in the Sera of Allergic Patients Positive to Phleum pratense

(34) Equal amounts of Phleum pratense pollen extract (1 μg) in 50 mM carbonate/bicarbonate buffer, pH 9.6, were adsorbed onto wells of polystyrene plates for ELISA assay by incubating at 4° C. for 16 hours. Wells were then washed with washing solution (60 mM phosphate buffer, pH 6,5, containing 0.05% Tween-20), and free sites were blocked with diluting solution (BSA 2%, PBS 1×). Aliquots (100 μl) of 1:15 diluted pools from mouse sera collected after seven weeks from first immunization were incubated at 4° C. for 16 hours. After three washes, seven 1:10 diluted human sera positive to Phleum pratense were added at 4° C. for 16 hours. After three washes, anti-human IgE peroxidase-conjugated serum diluted 1:4000 in diluting buffer was added, and incubated at 25° C. for 1.5 hours. After three washes, colorimetric reaction development was obtained by adding 100 μl TMB reagent (BioFX Laboratories, Owings Mills, Md.), and incubating for 20 minutes at 25° C. The reaction was stopped by adding 100 μl 1 N HCl and read at 450 nm with a spectrophotometer. Inhibition percentage was calculated by using the following formula: 100×[(A−B)/A], where A is absorbance at 450 nm when antigen is pre-incubated with serum from not-immunized mice, and B is absorbance in the presence of serum from immunized mice.

Example 12

Statistical Analysis

(35) In the figures, results are expressed as mean values plus corresponding standard deviations.

(36) UNISTAT 5.5 Light for Excel software was used for statistical analyses. Data were analyzed by paired t-Test.

BIBLIOGRAPHY

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