Compositions for antigen-specific induction of tolerance

Abstract

The invention relates to an immunogenic composition for sublingual, perlingual or oral administration, comprising at least an antigen and at least an adjuvant that is a bacterium selected from a Bifidobacterium and a lactic acid bacterium, and/or a combination of a corticosteroid with vitamin D3 or any metabolite or analog of the latter, in a pharmaceutically acceptable carrier that is suitable for sublingual, perlingual, or oral administration. Such compositions allow to elicit antigen-specific immune tolerance.

Claims

1. A method for inducing an antigen-specific tolerance in an individual in need thereof, comprising the sublingual or perlingual administration to the individual of a therapeutically effective amount of an immunogenic composition comprising a mixture of: an adjuvant that is a naturally occurring bacteria that is a Bifidobacterium or a lactic acid bacterium; and at least one antigen against which antigen-specific tolerance is to be induced, said antigen being in free form.

2. The method of claim 1, wherein the composition elicits or improves antigen-specific regulatory T cell responses.

3. The method of claim 1, wherein the composition induces production of IL10 by T cells.

4. The method of claim 1, wherein said adjuvant is a naturally occurring bacterium of the genus Bifidobacterium.

5. The method of claim 1, wherein said adjuvant is a naturally occurring bacterium of a genus selected from the group consisting of Lactobacillus, Lactococcus and Streptococcus.

6. The method of claim 1, wherein said adjuvant is Lactobacillus plantarum.

7. The method of claim 1, wherein said antigen is an allergen.

8. The method of claim 1, wherein said antigen is a protein allergen selected from the group consisting of a protein allergen of the genus Dermatophagoides; a protein allergen of the genus Felis; a protein allergen of the genus Ambrosia; a protein allergen of the genus Lolium; a protein allergen of the genus Cryptomeria; a protein allergen of the genus Alternaria; a protein allergen of the genus Alder; a protein allergen of the genus Betula; a protein allergen of the genus Blomia; a protein allergen of the genus Quercus; a protein allergen of the genus Olea; a protein allergen of the genus Artemisia; a protein allergen of the genus Plantago; a protein allergen of the genus Parietaria; a protein allergen of the genus Canine; a protein allergen of the genus Blattella; a protein allergen of the genus Apis; a protein allergen of the genus Cupressus; a protein allergen of the genus Juniperus; a protein allergen of the genus Thuya; a protein allergen of the genus Chamaecyparis; a protein allergen of the genus Periplaneta; a protein allergen of the genus Agropyron; a protein allergen of the genus Secale; a protein allergen of the genus Triticum; a protein allergen of the genus Cynorhodon; a protein allergen of the genus Dactylis; a protein allergen of the genus Festuca; a protein allergen of the genus Poa; a protein allergen of the genus Avena; a protein allergen of the genus Holcus; a protein allergen of the genus Anthoxanthum; a protein allergen of the genus Arrhenatherum; a protein allergen of the genus Agrostis; a protein allergen of the genus Phleum; a protein allergen of the genus Phalaris; a protein allergen of the genus Paspalum; and a protein allergen of the genus Sorghum.

9. The method of claim 1, wherein said antigen is a protein allergen selected from the group consisting of Bet v I; Bet v II; Blo t I; Blo t III; Blo t V; Blo t XII; Cyn d I; Der p I; Der p II; Der p III; Der p VII; Der f I; Der f II; Der f III; Der f VII; Fel d I; Amb a I.1; Amb a I.2; Amb a I.3: Amb a I.4; Amb a II; Lol p I; Lot p II; Lol p III; Lot p IV; Lol p IX; Cry j I; Cry j II; Can f I; Can f II; Jun s I; Jun v I; Jun a I; Jun a II; Dac g I; Dac g V; Poa p I; Phl p I; Phl p V; Phl p VI and Sor h I.

10. The method of claim 1, wherein said antigen is a protein involved in an autoimmune disease or graft rejection.

11. The method of claim 1, which comprises sublingual administration of the immunogenic composition to the individual.

12. The method of claim 1, which comprises perlingual administration of the immunogenic composition to the individual.

13. The method of claim 1, wherein the adjuvant further includes a combination of a corticosteroid with vitamin D3 or any metabolite or analog of the latter.

14. The method of claim 13, wherein the corticosteroid is dexamethasone or fluticasone.

15. The method of claim 14, wherein the adjuvant is said bacteria and a combination of (i) dexamethasone and (ii) vitamin D3 or 1,25-dihydroxyvitamin D3.

16. A method for inducing an antigen-specific tolerance in an individual in need thereof, comprising the sublingual or perlingual co-administration to the individual of a therapeutically effective amount of: a first composition comprising an adjuvant that is a naturally occurring bacteria that is a Bifidobacterium or a lactic acid bacterium, and a separate second composition comprising at least one antigen against which antigen-specific tolerance is to be induced, wherein the antigen is in free form.

17. The method of claim 16, wherein said first composition further includes a combination of a corticosteroid with vitamin D3 or any metabolite or analog of the latter.

18. A method for treating an allergy in an individual in need thereof, comprising administering by sublingual or perlingual route to the individual a therapeutically effective amount of an immunogenic composition comprising a mixture of: an adjuvant that is a naturally occurring bacteria that is a Bifidobacterium or a lactic acid bacterium; and at least an allergen to which the patient has developed or is likely to develop a sensitivity, wherein the allergen is in free form.

19. The method of claim 18, wherein said adjuvant is a naturally occurring bacterium of the genus Bifidobacterium.

20. The method of claim 18, wherein said adjuvant is a naturally occurring bacterium of a genus selected from the group consisting of Lactobacillus, Lactococcus and Streptococcus.

21. The method of claim 18, wherein said adjuvant is Lactobacillus plantarum.

22. The method of claim 18, wherein said antigen is a protein allergen selected from the group consisting of a protein allergen of the genus Dermatophagoides; a protein allergen of the genus Felis; a protein allergen of the genus Ambrosia; a protein allergen of the genus Lolium; a protein allergen of the genus Cryptomeria; a protein allergen of the genus Alternaria; a protein allergen of the genus Alder; a protein allergen of the genus Betula; a protein allergen of the genus Blomia; a protein allergen of the genus Quercus; a protein allergen of the genus Olea; a protein allergen of the genus Artemisia; a protein allergen of the genus Plantago; a protein allergen of the genus Parietaria; a protein allergen of the genus Canine; a protein allergen of the genus Blattella; a protein allergen of the genus Apis; a protein allergen of the genus Cupressus; a protein allergen of the genus Juniperus; a protein allergen of the genus Thuya; a protein allergen of the genus Chamaecyparis; a protein allergen of the genus Periplaneta; a protein allergen of the genus Agropyron; a protein allergen of the genus Secale; a protein allergen of the genus Triticum; a protein allergen of the genus Cynorhodon; a protein allergen of the genus Dactylis; a protein allergen of the genus Festuca; a protein allergen of the genus Poa; a protein allergen of the genus Avena; a protein allergen of the genus Holcus; a protein allergen of the genus Anthoxanthum; a protein allergen of the genus Arrhenatherum; a protein allergen of the genus Agrostis; a protein allergen of the genus Phleum; a protein allergen of the genus Phalaris; a protein allergen of the genus Paspalum; and a protein allergen of the genus Sorghum.

23. The method of claim 18, wherein said antigen is a protein allergen selected from the group consisting of Bet v I; Bet v II; Blo t I; Blo t III; Blo t V; Blo t XII; Cyn d I; Der p I; Der p II; Der p III; Der p VII; Der f I; Der f II; Der f III; Der f VII; Fel d I; Amb a I.1; Amb a 1.2; Amb a 1.3; Amb a 1.4; Amb a II; Lol p I; Lot p II; Lol p III; Lot p IV; Lol p IX; Cry j I; Cry j II; Can f I; Can f II; Jun s I; Jun v I; Jun a I; Jun a II; Dac g I; Dac g V; Poa p I; Phl p I; Phl p V; Phl p VI and Sor h I.

24. The method of claim 18, which comprises sublingual administration of the immunogenic composition to the individual.

25. The method of claim 18, which comprises perlingual administration of the immunogenic composition to the individual.

26. The method of claim 18, wherein said adjuvant further includes a combination of a corticosteroid with vitamin D3 or any metabolite or analog of the latter.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1: Experimental design

(2) A. Sensitization was performed by two intraperitoneal (i.p.) injections in 14 days interval with 10 g ovalbumin (OVA) or phosphate buffer saline (PBS) (control) adsorbed to 2 mg Al(OH).sub.3. The sensitization was followed by a 20 min aerosol challenge with 1% w/v OVA on 5 consecutive days. Blood samples (bleeding) were taken in order to perform the anti-OVA antibody titration. Measurements of airway responsiveness were performed by whole body plethysmography. After sacrifice of mice, spleen cells and lung were isolated for cytokine analysis and inflammation detection respectively.

(3) For tolerance induction in a therapeutic model, sensitized mice were sublingually treated with PBS (control), OVA or OVA plus adjuvant every week, twice a week, during 2 months at a concentration of 500 g. Blood samples (bleeding) were taken for humoral response analysis. Measurements of airway responsiveness were performed by whole body plethysmography. After sacrifice of mice, spleen and lung were removed for in vitro cytokine assays and inflammation analysis respectively.

(4) B. For tolerance induction in a prophylactic model, mice were sublingually treated with PBS, OVA or OVA plus adjuvant every week, twice a week, during 1 month at a concentration of 500 g. Sensitization was performed by two intraperitoneal (i.p.) injections in 14 days interval with 10 g ovalbumin (OVA) or phosphate buffer saline (PBS) (control) adsorbed to 2 mg Al(OH)3. The sensitization was followed by a 20 min aerosol challenge with 1% w/v OVA on 5 consecutive days. Blood samples (bleeding) were taken for humoral response analysis. Measurements of airway responsiveness were performed by whole body plethysmography. After sacrifice of mice, spleen and lung were removed for in vitro cytokine assays and inflammation analysis respectively.

(5) FIG. 2: Humoral, cellular, respiratory responses, and inflammation after sensitization

(6) A. OVA-specific IgG1 and IgG2a (dilution 1/100) antibody levels were measured in sera from 4 mice sensitized with OVA.

(7) B. OVA-specific IgE (dilution 1/10) antibody level was measured in sera from 3 mice sensitized with OVA.

(8) C. IFN-, IL-5 and IL10 levels in spleen cell of mice sensitized with OVA were measured by Elispot.

(9) D. The airway responsiveness was determined for sensitized mice with PBS or OVA by measurement of Penh index in response to methacholine (50 mg/ml). Eight mice per group were displayed (.box-tangle-solidup.=mean).

(10) E. Representative lung sections obtained 48 h after the last challenge. Paraffin tissue sections were stained with HES (Hematoxylin, eosin, safran). One arrow (top) indicates bronchial mucus secretion and the other arrow (bottom) indicates specific cellular infiltration.

(11) FIG. 3: Airway responsiveness after tolerization

(12) The airway responsiveness was determined for healthy mice, mice sensitized with OVA and tolerized with PBS, mice sensitized with OVA and tolerized with OVA and mice sensitized with OVA and tolerized with OVA+dexamethasone (Dex)+1,25-dihydroxyvitamin D3 (vitD3) (panel A) or OVA+L. plantarum (panel B) by measurement of Penh value in response to methacholine (50 mg/ml). Four to eight mice per group were displayed (horizontal bar median).

(13) FIG. 4: TGF- production after tolerization

(14) TGF- production was determined for mice sensitized with OVA and tolerized with PBS (n=2), mice sensitized with OVA and tolerized with OVA (n=3) and mice sensitized with OVA and tolerized with OVA+VitD3+Dex (n=4 (panel A) or OVA+L. plantarum (n=3) (panel B) by Elipsot.

EXAMPLE

Sublingual Administration of Ovalbumin with (i) Lactobacillus plantarum or (ii) 1,25-dihydroxyvitamin D3+dexamethasone

(15) Summary:

(16) Adjuvants inducing IL10 gene expression by human T cells in vitro (Lactobacillus plantarum or 1,25-dihydroxyvitamin D3+dexamethasone) were mixed with ovalbumin and administered sublingually to Balb/c mice sensitized to ovalbumin. When compared with ovalbumin alone or untreated animals, each of those adjuvants could enhance the clinical efficacy of the vaccine (based on improvement of the respiratory function). Clinical improvement was correlated with an increase in ovalbumin-specific TGF producing T lymphocytes in the spleen (i.e. T cells with a regulatory profile, subtype Tr1 or Th3).

(17) Materials and Methods:

(18) 1. Animals, Culture Medium, Reagents and Antigens

(19) Six-week-old female BALB/c mice were purchased from Charles River (L'Arbresle, France) and maintained on an ovalbumin-free diet. Each experimental group consisted of two to eight mice. The international levels of ethical standards for the use of animals were applied.

(20) Culture medium for spleen cell consisted of RPMI 1640 containing 10% fetal calf serum and 1% L-glutamine (all from Invitrogen, France).

(21) Ovalbumin (OVA, Garde V) was purchased from Sigma Chemicals. Phosphate-buffer saline (PBS) and alum were purchased from Invitrogen (France) and Pierce (Rockford, Ill.), respectively.

(22) 2. Adjuvants

(23) A strain of lactic acid bacteria (LAB), Lactobacillus plantarum, was used for in vivo applications in BALB/c mice (Microbiologics, Inc., Saint Cloud) at 2.10.sup.8 colony forming units (CFU)/ml in sterile PBS.

(24) 1,25-dihydroxyvitamin D3 (1 mg/kg) and dexamethasone (0.03 g/kg) in sterile PBS were also used.

(25) 3. Mice Sensitization and Tolerization (FIG. 1)

(26) Sensitization was performed by two intraperitoneal (i.p.) injections in 14 days interval with 10 g OVA adsorbed to 2 mg Al(OH).sub.3 in a total volume of 100 l. The sensitization was followed by a 20 min aerosol challenge with 1% w/v OVA on 5 consecutive days with an aerosol delivery system (Buxco Europe; Ltd, UK). Control animals were sham treated with sterile PBS.

(27) For tolerance induction in a therapeutic model, sensitized mice were sublingually treated with OVA or OVA plus adjuvant every week, twice a week, during 2 months at a concentration of 500 g (20 l). Control mice were sensitized with OVA followed by sham tolerization with PBS.

(28) For tolerance induction in a prophylactic model, mice were sublingualy treated with PBS, OVA or OVA plus adjuvant every week, twice a week, during 1 month at a concentration of 500 g. Sensitization was performed by two intraperitoneal. (i.p.) injections in 14 days interval with 10 g ovalbumin (OVA) or phosphate buffer saline (PBS) (control) adsorbed to 2 mg Al(OH).sub.3. The sensitization was followed by a 20 min aerosol challenge with 1% w/v OVA on 5 consecutive days

(29) 4. Determination of Airway Responsiveness

(30) Measurements of airway responsiveness were performed by whole body plethysmography (Buxco Europe; Ltd, UK). Airflow signals were recorded in response to aerosolized methacholine (50 mg/ml), 1 minute, four times. The higher value of Penh (enhanced pause) was taken into account for the calculation of the Penh index (Penh index=Penh measured/Penh PBS).

(31) 5. Determination of Serum Allergen-Specific Antibody Levels

(32) Blood samples were collected after the sensitization in order to perform the anti-OVA antibody titration by ELISA. For detection of IgG1 and IgG2a antibodies, the mouse anti-serum was incubated with purified OVA (0.1 g) coated onto ELISA plates (Nunc, Denmark). Biotinylated-rat anti-mouse IgG1 or IgG2a antibodies (dilution 1/100, BD Pharmingen) were applied and streptavidine-peroxidase-conjugated rat anti-mouse IgG antibodies (dilution 1/400, BD Pharmigen) were used for detection. An o-phenylene diamine and hydrogen peroxyde substrate (Sigma) was added, the reaction stopped and the optic density (OD) read using an ELISA reader at 492 nm.

(33) For detection of IgE antibody; mouse IgE antibodies (Bethyl laboratories) were coated onto Elisa plates. The mouse anti-serum was applied and OVA-digoxigenin (dilution 1/20, Roche) plus HRPO-conjugated rabbit anti-digoxigenin (dilution 1/1000, Roche) were used for detection. An ABTS substrate was added (Roche), the reaction stopped and the OD read using an ELISA reader at 405 nm.

(34) 6. Dosage of TGF-

(35) Dosage of TGF- from supernatants of spleen cell cultures was performed with an ELISA kit (R&D systems).

(36) 7. Spleen Cell Cultures for Cytokine Analysis by Elispot

(37) Mice were killed by cervical dislocation. Spleen cells were isolated and plated in triplicate in 96-well flat-bottomed ELISPOT plates (Millipore, Mass.) at densities of 210.sup.5 cells per well. Different conditions were used: medium alone, OVA (100 g/ml) and PMA (phorbol myristyl acetate) (50 ng/ml, Sigma)/ionomycine (500 ng/ml, Sigma) which was used as positive control. Spot forming cells (SFCs) were monitored for IFN-, IL-5 and IL10. Elispot kits were from R&D systems Europe.

(38) 8. Lung Histology

(39) Mice were killed by cervical dislocation. Lung were isolated and embedded in paraffin. Micrometer paraffin sections were stained with HES (hematoxylin, eosin, safran).

(40) 9. Gene Expression by Human T Cells

(41) Dendritic cells (DC) were treated with candidate adjuvants for 24 h, washed and cultured with purified allogenic nave CD4+ T cells. mRNAs were extracted 24 h to 5 days later and gene expression was assessed by quantitative PCR with primers specific for the tested genes. Only data above 2 fold increase were considered as significant.

(42) Results

(43) A. Selection of IL-10-inducing Adjuvants in Human In Vitro Model

(44) Human in vitro model consisting in coculture of human preated DC with allogenic T cells have been used to select IL10-inducing adjuvants. Briefly, human DC were treated with adjuvants. After 24 h, DC were washed and cultured with allogenic nave CD4+ T cells. After 24 h to 5 days, gene expression in human T cells was assessed by quantitative PCR.

(45) Among several putative adjuvants tested, Lactobacillus plantarum and 1,25-dihydroxyvitamin D3 in combination with dexamethasone (Vit/Dex) have been shown liable to induce IL10 gene expression by human T cells in vitro (Table 1).

(46) TABLE-US-00001 TABLE 1 Summary of screening data for IL10 inducing adjuvants Gene expression by T cells Adjuvants Tbet IFN GATA3 Foxp3 TGF IL10 Vit/Dex + + + +++ L. plantarum +++ ++ R848 + LTA ++ Pam3CSK4 LPS pg Imiquimod + + ssPolyU

(47) B. Immunotherapy Experiments

(48) 1. Murine Models of Sensitization with OVA

(49) Mice, immunized i.p. with OVA absorbed to Al(OH).sub.3 and subsequently exposed to an OVA solution via aerosol, were analysed for their humoral, cellular, respiratory responses and inflammation. The sensitization procedure has been shown to induce OVA-specific IgG1 and IgE, but little IgG2a antibodies in serum (FIGS. 2A and 2B). In accordance with the production of allergen-specific IgE antibodies, spleen cells from the OVA-sensitization mice displayed strong Th2 profile in response to OVA (FIG. 2C). OVA-sensitized mice present airway hyperresponsiveness to methacholine when compared to PBS-treated mice (FIG. 2D). Finally, the sensitization protocol has been shown to induce specific cellular infiltration of the lung and bronchial mucus secretion (FIG. 2E).

(50) 2. Sublingual Tolerization in a Therapeutic Model of Type I Allergy

(51) After having sensitized the mice, the inventors have tested different protocol of tolerization and particularly the capacity of candidate adjuvants to upregulate antigen-specific regulatory T cell responses and accelerate the tolerance induction by sublingual way. Lactobacillus plantarum and 1,25-dihydroxyvitamin D3 in combination with dexamethasone have been tested as adjuvants for upregulation of antigen-specific regulatory T cell responses.

(52) Sublingual treatment with OVA administered together with L. plantarum (FIG. 3A) or 1,25-dihydroxyvitamin D3 plus dexamethasone (FIG. 3B) led to a reduction of airway responsiveness as compared to PBS-treated mice or animals treated with OVA alone.

(53) Finally, TGF- production was enhanced in supernatant of spleen cell cultures of mice treated with OVA and L. plantarum (FIG. 4A) or 1,25-dihydroxyvitamin D3 plus dexamethasone (FIG. 4B) compared to PBS-treated mice or animals treated with OVA alone.

(54) 3. Sublingual Tolerization in a Prophylactic Model of Type I Allergy

(55) Sublingual desensitization is also tested in a murine prophylactic model of asthma. Briefly, Balb/c mice are sublingually administered with an adjuvant to be tested (1,25-dihydroxyvitamin D3+dexamethasone) in association with an antigen (OVA) (500 g) 2 times per week during 4 weeks prior to being sensitized and challenged by OVA.

(56) Results indicate that pre-treatment of mice with OVA+adjuvant according to the invention is liable to induce a reduction of airway responsiveness following subsequent sensitization.

(57) 4. Murine Models of Sensitization with Birch Pollen and House Dust Mite

(58) The adjuvants according to the invention are tested in murine model of sensitization either to birch pollen or house dust mite and also seem promising for preventing or treating allergies with these allergens.