PREVENTION OF ALLERGY

Abstract

A preparation comprising peptides from natural allergens for use in the prevention of development of allergy.

Claims

1. A preparation comprising peptides of a natural allergen wherein the peptides are hydrolyzed allergen peptides for use in the prevention of development of allergy, wherein the preparation is administered prior to the development of any allergy against the allergen used for the preparation.

2. The preparation for use according to claim 1 wherein the preparation is administered 2 to 10 times.

3. The preparation for use according to claim 2 wherein the preparation is administered at intervals of 2 to 10 days.

4. The preparation for use according to claim 2 wherein the preparation is administered in constant amounts.

5. The preparation for use according to claim 1 wherein the preparation is free of immune stimulating adjuvants.

6. The preparation for use according to claim 1 wherein said hydrolyzed allergen peptides are obtainable by a) extracting a natural source of allergens comprising allergenic proteins to form an extract, b) purifying of said extract to remove non-protein components to form a purified extract, c) denaturing said purified extract to form a purified denatured extract, d) hydrolysing the purified denatured extract to form hydrolysed allergen peptides.

7. The preparation for use according to claim 1 wherein said hydrolyzed allergen peptides are obtainable by a) extracting a source of allergens comprising allergenic proteins to form an extract, b) purifying the extract to remove non-protein components to form a purified extract, c) denaturing the purified extract with a first denaturing agent to form a purified denatured extract, d) refining the purified denatured extract to remove impurities to form a refined denatured extract, e) denaturing the refined denatured extract with a second denaturing agent to form denatured allergen mixture, and f) hydrolyzing the denatured allergen mixture to form the hydrolyzed allergen peptides.

8. The preparation for use according to claim 1 wherein the allergens are selected among pollen allergens, milk allergens, venom allergens, egg allergens, weed allergens, grass allergens, tree allergens, shrub allergens, flower allergens, vegetable allergens, grain allergens, fungi allergens, fruit allergens, berry allergens, nut allergens, seed allergens, bean allergens, fish allergens, shellfish allergens, seafood allergens, meat allergens, spices allergens, insect allergens, mite allergens, mould allergens, animal allergens, pigeon tick allergens, worm allergens, soft coral allergens, animal dander allergens, nematode allergens, allergens of Hevea brasiliensis.

9. The preparation for use according to claim 1, wherein denaturing is performed with a denaturing agent selected from the group consisting of chaotropic agents, reducing agents and mixtures thereof.

10. The preparation for use according to claim 1, wherein the hydrolysis is performed with an enzyme.

11. The preparation for use according to claim 6, further comprising purifying the hydrolyzed allergens to remove peptides with molecular weights above 10.000 Da and below 1.000 Da, wherein 70% of the peptides are between 10.000 Da and 1.000 Da.

12. The preparation for use according to claim 1 for use as an allergy vaccine.

13. A method of preventing the development of allergy comprising administering a preparation comprising peptides from natural allergens to a patient at risk of developing an allergy to the natural allergens.

14. The preparation for use of claim 9 wherein the denaturing agent is selected from the group consisting of urea, guanidinium chloride, dithiotreitol, thioglycerol, -mercaptoethanol, TCEP (tris (2-carboxyethyl) phosphine) and mixtures thereof.

15. The preparation for use of claim 10, wherein the hydrolysis is performed in the presence of a chaotropic agent and a reducing reagent.

Description

EXAMPLE 1PEANUT

1. Aim of the Study

[0054] Evaluation of the induction of a possible memory effect in Balb/c mice treated twice per week during 3 weeks with 400 g of peanuts peptides (ARA/PEP_SOL UBT13J03) in mannitol-trehalose environment (the injections were performed sub-cutaneously without any adjuvant) and challenged by a unique intraperitoneal (ip) injection of 100 g of peanut native proteins without any adjuvant.

2. Summary of the Protocol

[0055]

TABLE-US-00001 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Number of animals 10 10 10 10 10 10 Treatment: ARA/ ARA/ ARA/ ARA/ ARA/ PLACEBO Antigens in M/T PEP_SOL PEP_SOL PEP_SOL PEP_SOL PEP_SOL 13J03 13J03 13J03 13J03 13J03 Injected dose 400 g 400 g 400 g 400 g 400 g 400 g (treatment) Way of SC SC SC SC SC SC administration (treatment) Injected volume 300 l 300 l 300 l 300 l 300 l 300 l (treatment) Injections J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- calendar 11-14 11-14 11-14 11-14 11-14 11-14 (treatment) et 18 et 18 et 18 et 18 et 18 et 18 Challenge ARA/ ARA/ ARA/L ARA/ ARA/ ARA/ ALL_SOL ALL_SOL ALL_SO ALL_SOL ALL_SOL ALL_SOL NATIVES NATIVES NATIVES NATIVES NATIVES NATIVES 13I12 13I12 13I12 13I12 13I12 13I12 Injected dose 100 g 100 g 100 g 100 g 100 g 100 g (challenge) Way of IP IP IP IP IP IP administration (challenge) Injected volume 200 l 200 l 200 l 200 l 200 l 200 l (challenge) Injection J28 J35 J42 J49 J56 J28 calendar (challenge)

3. Denomination of the Groups

[0056]

TABLE-US-00002 Group 1 13J03-DP/Challenge J28 Group 2 13J03-DP/Challenge J35 Group 3 13J03-DP/Challenge J42 Group 4 13J03-DP/Challenge J49 Group 5 13J03-DP/Challenge J56 Group 6 PLACEBO (M/T)/Challenge J28

4. Correspondence Between Days Post-Beginning of the Treatment and Days Post-Challenge

[0057] All the groups have the same calendar for the treatment phase till the Day 28

TABLE-US-00003 Groups Days post-beginning of the treatment Days Post-Challenge 1 and 6 28-35-42-49-56-63-70 0-7-14-21-28-35-42 2 35-42-49-56-63-70 0-7-14-21-28-35 3 42-49-56-63-70 0-7-14-21-28 4 49-56-63-70 0-7-14-21 5 56-63-70 0-7-14

[0058] Caution:

[0059] ARA/PEP_SOL is commonly used for peanut peptides.

[0060] ARA/ALL_SOL is commonly used for peanut proteins

5. Results

[0061] Here are the results obtained by ELISA for the production of specific IgG anti-ARA proteins (nativenative proteins coating)

[0062] a. Evolution of IgG Production Before any Challenge (from Day 0 to Day 28):

[0063] FIGS. 1 and 2 show that the treatment of the mice with peanut peptides as drug product leads to a very weak production of specific IgG anti-ARA PROTEINS till the Day 28. The mice treated with PLACEBO show no specific IgG anti-ARA PROTEINS production.

[0064] b. Evolution of IgG Production after the Challenge (from the Challenge Day to the Day 42 Post-Challenge): Caution: Not all Groups Reach this Day 42 Post-Challenge!!

[0065] FIGS. 3 and 4 show the production of specific IgG anti-ARA PROTEINS in the sera of mice belonging to the ARA-peptides treated groups and to the Placebo-group. This production increases with different speed according to the moment when the challenge occurred. When the challenge is performed at Day 28, 35 and 56 post-beginning of the treatment, we can observe that the specific IgG anti-ARA PROTEINS production take off from Placebo-s one 2 weeks after the challenge. For the groups with the challenge occurred at Day 42 and 49 post-beginning of the treatment, no difference in the IgG production is observed to the Placebo's one till Day 21 post-challenge (and even to Day 28 post-Challenge for the D42-Challenge Group=Group 3).

[0066] For the group 1, when the Challenge is performed at Day 28 post-beginning of the treatment, a maximum in the specific IgG anti-ARA PROTEINS production seems to be reached at Day 28 post-Challenge after the going down to a plate state at D35 (and later) post-treatment.

[0067] For the group 2, when the Challenge is performed at Day 35 post-beginning of the treatment, a maximum is reached at Day 35 post-challenge but we don't have the late time-points so, we may not conclude if this point is well a maximum or if the production will increase again at late time-points.

[0068] The higher titers values of each group are the following ones:

TABLE-US-00004 Specific IgG anti-ARA PROTEINS Days Post- MEAN/MEDIAN TITERS Challenge Maximum value Titers Group 1 D 28 = 10.892/11.653 Group 2 D 35 = 8.900/8.894 Group 3 D 28 = 2.103/1.022 Group 4 D 21 = 1.849/947 Group 5 D 14 = 8.171/6.543 Group 6 D 42 = 990/990

6. Conclusions

[0069] From all these results, we can conclude the following points: [0070] The treatment with ARA-peptides as described in this protocol induces a very weak production of specific IgG anti-ARA PROTEINS till Day 28 (post-beginning of the treatment) [0071] No specific IgG production is observed in Placebo groups during the treatment phase till Day 28 (post-beginning of the treatment) [0072] The challenge with a unique dose of 100 g of ARA native proteins potentializes the effect of the treatment allowing an induction of the production of specific IgG anti-ARA PROTEINS reaching a maximum 4 weeks after the challenge for the group challenged at Day 28 post-beginning of the treatment. This effect is not observed in the Placebo-groups that received also the same challenge. So, we can conclude that the challenge is not sufficient per se to induce the IgG production 4 weeks after the challenge (as observed in Placebo-groups) but allows the expression of the ARA-peptides treatment. Obviously, the ARA-peptides treatment can prime the immune system allowing a memory effect after a challenge with native proteins. [0073] The amplitude of the IgG response depends on the moment separating the end of the treatment and the challenge. The response is higher groups challenged at Day 28, 35 and 56 post-beginning of the treatment. Surprisingly, the groups challenged at Day 42 and 49 post-beginning of the treatment seem not to show a significative difference than the placebo-group. Maybe the production is delayed in these groups . . . . We could answer that question when we will have tested the late time-points. [0074] All these results are related to ARA native proteins that are the closest proteins to natural allergens.

EXAMPLE 2HOUSE DUST MITE

[0075] Evaluation of the induction of a possible memory effect in Balb/c mice treated twice per week during 3 weeks with 400 g of house dust mite peptides (HDM/PEP_SOL UBT15H20) in mannitol-trehalose environment (the injections were performed sub-cutaneously without any adjuvant) and challenged by a unique ip injection of 100 g of house dust mite native proteins without any adjuvant.

7. Summary of the Protocol

[0076]

TABLE-US-00005 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Number of animals 15 15 15 15 10 10 10 10 Treatment: HDM/ HDM/ HDM/PE HDM/PE PLACEBO PLACEBO PLACEBO PLACEBO Antigens in M/T PEP_SOL PEP_SOL P_SOL P_SOL 15H20 15H20 15H20 15H20 Injected dose 400 g 400 g 400 g 400 g 400 g 400 g 400 g 400 g (treatment) Way of administration SC SC SC SC SC SC SC SC (treatment) Injected volume 300 l 300 l 300 l 300 l 300 l 300 l 300 l 300 l (treatment) Injections calendar J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7-11- J0-4-7- (treatment) 11-14 11-14 11-14 11-14 11-14 11-14 14 11-14 et 18 et 18 et 18 et 18 et 18 et 18 et 18 et 18 Challenge HDM/ HDM/ HDM/ HDM/ HDM/ HDM/ HDM/ HDM/ ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL NATIVES NATIVES NATIVES NATIVES NATIVES NATIVES NATIVES NATIVES 13G04nat 13G04nat 13G04nat 13G04nat 13G04nat 13G04nat 13G04nat 13G04nat Injected dose 100 g 100 g 100 g 100 g 100 g 100 g 100 g 100 g (challenge) Way of administration IP IP IP IP IP IP IP IP (challenge) Injected volume 200 l 200 l 200 l 200 l 200 l 200 l 200 l 200 l (challenge) Injection calendar J28 J35 J42 J49 J28 J35 J42 J49 (challenge)

8. Denomination of the Groups

[0077]

TABLE-US-00006 Group 1 15H20-DP/Challenge J28 Group 2 15H20-DP/Challenge J35 Group 3 15H20-DP/Challenge J42 Group 4 15H20-DP/Challenge J49 Group 5 PLACEBO (M/T)/Challenge J28 Group 6 PLACEBO (M/T)/Challenge J35 Group 7 PLACEBO (M/T)/Challenge J42 Group 8 PLACEBO (M/T)/Challenge J49

9. Correspondence Between Days Post-Beginning of the Treatment and Days Post-Challenge

[0078] All the groups have the same calendar for the treatment phase till the Day 28

TABLE-US-00007 Groups Days post-beginning of the treatment Days Post-Challenge 1 and 5 28-35-42-49-56-63-70-77-84-91 0-7-14-21-28-35-42-49- 56-63 2 and 6 35-42-49-56-63-70-77-84-91-98 0-7-14-21-28-35-42-49- 56-63 3 and 7 42-49-56-63-70-77-84-91-98-105 0-7-14-21-28-35-42-49- 56-63 4 and 8 49-56-63-70-77-84-91-98-105-112 0-7-14-21-28-35-42-49- 56-63

10. Results

[0079] Here are the results obtained by ELISA for the production of specific IgG anti-House Dust Mite proteins (nativenative proteins coating)

[0080] a. Evolution of IgG Production Before any Challenge (from Day 0 to Day 28):

[0081] FIGS. 5 and 6 show that the treatment of the mice with house dust mite peptides as drug product leads to a weak production of specific IgG anti-HDM PROTEINS till the Day 28. The mice treated with PLACEBO show no specific IgG anti-HDM PROTEINS production (Groups 5 to 8 are at the same level)

[0082] b. Evolution of IgG Production after the Challenge (from the Challenge Day to the Day 63 Post-Challenge):

[0083] FIGS. 7 and 8 show the production of specific IgG anti-HDM PROTEINS in the sera of mice belonging to the HDM-peptides treated groups. This production increases to reach a maximum at Day 14 post-Challenge before going down to reach a relative plate state at Day 28 (for Groups 1, 2 and 4) or at Day 35 (for Group 3) post-Challenge. This production (the levels of IgG titers) seems to be linked to the moment when the Challenge occurred: the IgG productions are higher in Groups 3 and 4 (respectively challenged at Day 42 and 49 post-beginning of the treatment) than in Groups 1 and 2 (respectively challenged at Day 28 and 35 post-beginning of the treatment).

[0084] The titers values at these specific time-points are the following ones:

TABLE-US-00008 Days Specific IgG anti-HDM PROTEINS MEAN/MEDIAN TITERS Post-Challenge Maximum value Titers Plate State value Titers Group 1 D 14 = 9.074/7.528 D 28 = 7.257/6.976 Group 2 D 14 = 19.203/15.715 D 28 = 11.813/11.040 Group 3 D 14 = 52.547/38.826 D 35 = 29.552/27.798 Group 4 D 14 = 41.817/28.886 D 28 = 28.356/24.059 Group 5 D 63 = 458/458 / Group 6 D 56 = 500/500 / Group 7 D 63 = 748/748 / Group 8 D 56 = 462/462 /

11. Conclusions

[0085] From all these results, we can conclude the following points: [0086] The treatment with HDM-peptides as described in this protocol induces a weak production of specific IgG anti-HDM PROTEINS till Day 28 (post-beginning of the treatment) [0087] No specific IgG production is observed in Placebo-groups during the treatment phase till Day 28 (post-beginning of the treatment) [0088] The challenge with a unique dose of 100 g of HDM native proteins potentializes the effect of the treatment allowing an induction of the production of specific IgG anti-HDM PROTEINS reaching a maximum 2 weeks after the challenge. This effect is not observed in the Placebo-groups that received also the same challenge. So, we can conclude that the challenge is not sufficient alone to induce the IgG production (as observed in Placebo-groups) but allows the expression of the HDM-peptides treatment. Obviously, the HDM-peptides treatment can prime the immune system allowing a memory effect after a challenge with native proteins. [0089] The amplitude of the IgG response depends on the moment separating the end of the treatment and the challenge. The response is higher for late-challenged groups. [0090] All these results are related to HDM native proteins that are the closest proteins to natural allergens.

EXAMPLE 3RAGWEED

[0091] Evaluation of the induction of a possible memory effect in Balb/c mice treated twice per week during 3 weeks with 100 g or 400 g of ragweed peptides (RAG/PEP_SOL UBT16E03) in mannitol-trehalose environment (the injections were performed sub-cutaneously without any adjuvant) and challenged by a unique ip injection of 100 g of ragweed native proteins without any adjuvant.

Summary of the Protocol

[0092]

TABLE-US-00009 Groupe 1 Groupe 2 Groupe 3 Groupe 4 Groupe 5 Groupe 6 Groupe 7 Number of animals 10 10 10 10 10 10 10 Treatment: RAG/ RAG/ RAG/ RAG/ RAG/ RAG/ PLACEBO Antigens in M/T PEP_SOL PEP_SOL PEP_SOL PEP_SOL PEP_SOL PEP_SOL 16E03 16E03 16E03 16E03 16E03 16E03 Injected dose 100 g 100 g 100 g 400 g 400 g 400 g 100 g (treatment) Way of SC SC SC SC SC SC SC administration (treatment) Injected volume 200 l 200 l 200 l 200 l 200 l 200 l 200 l (treatment) Injections calendar J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- J0-4-7- (treatment) 11-14 11-14 11-14 11-14 11-14 11-14 11-14 and 18 and 18 and 18 and 18 and 18 and 18 and 18 Challenge RAG/ RAG/ RAG/ RAG/ RAG/ RAG/ RAG/ ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL ALL_SOL NAT 16E17 NAT 16E17 NAT 16E17 NAT 16E17 NAT 16E17 NAT 16E17 NAT 16E17 Injected dose 100 g 100 g 100 g 100 g 100 g 100 g 100 g (challenge) Way of IP IP IP IP IP IP IP administration (challenge) Injected volume 200 l 200 l 200 l 200 l 200 l 200 l 200 l (challenge) Injection calendar J28 J42 J56 J28 J42 J56 J28 (challenge)

8. Denomination of the Groups

[0093]

TABLE-US-00010 Group 1 16E03-DP 100 g/Challenge D 28 Group 2 16E03-DP 100 g/Challenge D 42 Group 3 16E03-DP 100 g/Challenge D 56 Group 4 16E03-DP 400 g/Challenge D 28 Group 5 16E03-DP 400 g/Challenge D 42 Group 6 16E03-DP 400 g/Challenge D 56 Group 7 PLACEBO (M/T)/Challenge J28

9. Correspondence Between Days Post-Beginning of the Treatment and Days Post-Challenge

[0094] All the groups have the same calendar for the treatment phase till the Day 28

TABLE-US-00011 Groups Days post-beginning of the treatment Days Post-Challenge 1, 4 28-35-42-49-56-63-70-77-84-91 0-7-14-21-28-35-42-49- and 7 56-63 2 and 5 42-49-56-63-70-77-84-91-98-105 0-7-14-21-28-35-42-49- 56-63 3 and 6 56-63-70-77-84-91-98-105-112-119 0-7-14-21-28-35-42-49- 56-63

10. Results

[0095] Here are the results obtained by ELISA for the production of specific IgG anti-Ragweed proteins (nativenative proteins coating)

[0096] a. Evolution of IgG Production Before any Challenge (from Day 0 to Day 28):

[0097] FIGS. 9 and 10 show that the treatment of mice with both concentration of ragweed peptides as drug product leads to a weak production of specific IgG anti-RAG PROTEINS until Day 28 (Group 1-6). The mice treated with PLACEBO show no production of specific IgG anti-RAG PROTEINS (Group 7)

[0098] b. Evolution of IgG Production after the Challenge (from the Challenge Day to Day 56 Post-Challenge):

[0099] FIGS. 11 and 12 show the production of specific IgG anti-RAG PROTEINS in the sera of mice belonging to the RAG-peptides treated groups. This production increases to reach a maximum at Day 14-21 post-Challenge before going down to reach a relative plate state at Day 28-42. This production (the levels of IgG titers) seems to be linked to the moment when the Challenge occurred: the IgG productions are higher in Groups 3 and 6 (respectively challenged at Day 56 post-beginning of the treatment) than in Groups 1; 2; 4 and 5 (respectively challenged at Day 28 and Day 42 post-beginning of the treatment).

[0100] The titers values at these specific time-points are the following ones:

TABLE-US-00012 Specific IgG anti-RAG PROTEINS Days Post- MEAN/MEDIAN TITERS Challenge Maximum value Titers Plate State value Titers Group 1 D 49 = 2.604/2.319 D 56 = 799/722 Group 2 D 21 = 11.569/9.041 D 42 = 2.137/1.720 Group 3 D 14 = 28.382/15.552 D 28 = 6.931/4.226 Group 4 D 7 = 2.655/2.341 D 56 = 443/351 Group 5 D 14 = 10.744/6.699 D 42 = 2.620/1.361 Group 6 D 21 = 29.673/27.507 D 28 = 7.352/7.042 Group 7 D 63 = 99/99 D 28 = 99/99

11. Conclusions

[0101] From all these results, we can conclude the following points: [0102] The treatment with RAG-peptides as described in this protocol induces a weak basal production of specific IgG anti-RAG PROTEINS till Day 28 (post-beginning of the treatment) [0103] No specific IgG production is observed in Placebo-groups during all the study (post-treatment and post-challenge) [0104] The challenge with a unique dose of 100 g of RAG native proteins potentializes the effect of the treatment allowing an induction of the production of specific IgG anti-RAG PROTEINS reaching a maximum 2 weeks after the challenge. This effect is not observed in the Placebo-groups that received also the same challenge. So, we can conclude that the challenge is not sufficient alone to induce the IgG production (as observed in Placebo-groups) but allows the expression of the RAG-peptides treatment effect. In conclusion, the RAG-peptides treatment can prime the immune system allowing a memory effect after a challenge with native proteins. [0105] The amplitude of the IgG response depends on the delay between the end of the treatment and the challenge. The response is higher for late-challenged groups. [0106] The amplitude of the IgG response after challenge is dependent of the dose of the treatment with a higher response at 400 g than at 100 g when the challenge is performed at Day 28 and Day 42. When the challenge is performed later (Day 56) the IgG responses are similar both dosages, probably due to the amplitude of the response. [0107] All these results are related to RAG native proteins that are the closest proteins to natural allergens.