TREATMENT OF IMMUNE DISEASE BY MUCOSAL DELIVERY OF ANTIGENS USING GENETICALLY MODIFIED LACTOCOCCUS
20190076511 ยท 2019-03-14
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K39/4611
HUMAN NECESSITIES
A61K39/001
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K39/46433
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K39/0008
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to the treatment of autoimmune and allergic diseases by mucosal delivery by micro-organism, in particular Lactococcus lactic, of secreted immunodominant antigens.
Claims
1-44. (canceled)
45. A method for treating type 1 diabetes in a subject in need thereof, the method comprising orally administering to the subject a composition comprising lactic acid bacteria comprising a nucleic acid sequence integrated into a chromosome of the lactic acid bacteria, wherein said nucleic acid sequence encodes a beta cell autoantigen, wherein said beta cell autoantigen is constitutively expressed and secreted by said lactic acid bacteria, and wherein said subject has established autoimmunity to said beta cell autoantigen.
46. The method of claim 45, wherein said beta cell autoantigen is insulin, proinsulin, or an insulin fragment.
47. The method of claim 46, wherein said beta cell autoantigen is human proinsulin B24-C36, insulin, or InsB.sub.9-23.
48. The method of claim 45, wherein said lactic acid bacteria is Lactococcus lactis.
49. The method of claim 45, wherein said oral administration a. induces regulatory T-cells (Treg); b. reduces proliferation of spleen and inguinal lymph node cells; and/or c. suppresses an inflammatory antigen specific T cell response.
50. The method of claim 49, wherein said Treg cells are Foxp3.sup.+ Treg cells.
51. The method of claim 45, wherein said lactic acid bacteria is: (i) administered daily; (ii) administered for at least 1 week; (iii) administered at least once a day; (iv) administered in a dose of at least 10 femtogram to 100 mg per day; and/or (v) administered in a formulation selected from spray, capsule, aerosol, lozenges, bolus, tablet, sachets, liquid, suspension, emulsion, and troches.
52. The method of claim 45, wherein said lactic acid bacteria is administered to said subject daily.
53. The method of claim 45, wherein said composition is formulated as a medicament, medical food, nutraceutical, or beverage.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
Examples
Example A: Induction of OVA-Specific Tolerance by Genetically Modified Lactococcus lactis Delivering OVA to OVA-Sensitized Wild-Type Mice
Introduction
[0100] For this purpose we genetically engineered OVA secreting LL (LL-OVA) and evaluated the induction of systemic tolerance in a therapeutic model for autoimmunity/allergy, namely the OVA immunization model.
Materials and Methods
[0101] Bacteria and media: The Lactococcus lactis MG1363 (LL) strain was genetically modified and used throughout this study. Bacteria were cultured in GM17E medium consisting of M17broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose and 5 ?g/ml erythromycin (Abbott). Stock suspensions of LL strains were stored at ?20? C. in 50% glycerol in GM17E medium. Stock suspensions were diluted 500-fold in GM17E medium and incubated at 30? C. overnight. Within 16 hours they reached a saturation density of 2?10.sup.9 colony forming units (CFU) per ml. Bacteria were harvested by centrifugation and resuspended in BM9 medium at 2?10.sup.10 bacteria/ml. Each mouse received 100 ?l of this suspension daily through an intragastric catheter.
[0102] Plasmids: The mRNA sequence encoding Gallus gallus Ovalbumin was retrieved from Genbank (accession number AY223553) and from published data. Total RNA was isolated from chicken uterus and cDNA was synthesized using 2 ?g total RNA, 2 ?M oligo dT primers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mM DTT (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mM dNTP (Invitrogen, Merelbeke, Belgium), 20 U Rnasin (Promega Incorporation Benelux) and 100 U superscript II reverse transcriptase (Invitrogen) in a volume of 25 ?l. An OVA cDNA fragment was amplified by Polymerase Chain Reaction (PCR) using the following primers: forward 5-GGCTCCATCGGTGCAGCAAGCATGGAATT-3 (SEQ ID NO:9) and reverse 5-ACTAGTTAAGGGGAAACACATCTGCCAAAGAAGAGAA-3 (SEQ ID NO:10). Reaction conditions were 94? C. for 2 minutes followed by 30 cycles at 94? C. for 45 seconds, 62? C. for 30 seconds and 72? C. for 90 seconds. The amplified fragment was fused to the Usp45 secretion signal of the erythromycin resistant pT1NX vector, downstream of the lactococcal P1 promotor17. MG1363 strains transformed with plasmids carrying OVA cDNA were designated L. lactis secreting OVA (LL-OVA). The L. lactis-pTREX1, which is MG1363 containing the empty vector pTREX1, served as control (LL-pTREX).
[0103] Mice: Seven-week old female Balb/c mice were obtained from Charles River Laboratories (Calco, Italy) and were housed in a conventional animal facility under specific pathogen-free conditions. The animal studies were approved by the Ethics Committee of the Department for Molecular Biomedical Research at Ghent University (file No. 07/029).
[0104] Antigen: Intact, LPS-free OVA grade V protein was used as antigen in all experiments (Sigma Aldrich).
[0105] Immunization of mice and induction of oral tolerance: Balb/c mice were immunized by s.c. injection of 100 ?g OVA in 100 ?l of a 1:1 mixture of CFA (Difco, BD Bioscience, Erembodegem, Belgium) and saline solution at the base of the tail on the first day. LL-OVA, LL-pTREX1 or 1 ?g purified OVA dissolved in 100 ?l BM9 were administered daily on days 7-11, 14-18, 21-25 and 28-31 (regime 1), and on days 21-25 and 28-31 (regime 2). Control mice received only BM9. Antigen or bacterial suspensions were introduced into the stomach using an 18-gauge stainless animal feeding needle. On day 21, a boost immunization was given by s.c. injection of 100 ?g OVA in 100 ?l of a 1:1 mixture of IFA (Sigma-Aldrich). Tolerance induction was assessed by DTH responses, measurement of cytokines and OVA-specific proliferation, and adoptive transfer experiments.
[0106] Delayed-type Hypersensitivity responses: Antigen-specific DTH responses were assessed by injection of OVA on day 31. Twenty-four hours later DTH measurements were performed. For measurement of antigen-specific DTH responses, mice were challenged with 10 ?g OVA in 10 ?l saline in the auricle of the ear. Ear swelling, defined as the increase in ear thickness due to challenge, was measured in a blinded fashion 24 hours after challenge using a digital micrometer (Conrad, Belgium). The DTH responses were expressed as the difference in ear thickness before and after the OVA challenge for both ears.
[0107] OVA-specific proliferation and cytokine assays: On day 39, the spleens were harvested and the splenocytes were assessed for OVA-specific proliferation and cytokine production. Single cell suspensions of spleens were prepared by passing the cells through 70-?m cell strainers (Becton/Dickinson Labware). Erythrocytes in the cell suspensions were lysed by incubation with red cell lysis buffer. CD4.sup.+ T cells were enriched using CD4.sup.+ T cell isolation kit and midiMACS columns (Miltenyi Biotec, Germany).
[0108] To assay proliferation of total splenocyte populations, 2?10.sup.5 cells were cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium (i.e., RPMI-1640 containing 10% fetal calf serum (FCS), 10 U/ml penicillin, 10 ?g/ml streptomycin, 2 mM L-glutamax, 0.4 mM sodium pyruvate) either alone or with OVA, added at concentrations ranging from 1.2 to 100 ?g/ml. The proliferation was further assessed by 5,6-CFSE labeling (Invitrogen, Merelbeke, Belgium). The splenocytes were resuspended in PBS at 10.sup.7/ml and incubated in a final concentration of 10 ?M CFSE for 12 minutes at 37? C. Labeled cells were washed twice with ice-cold complete medium before being cultured at 2?10.sup.5 cells in 96-well U-bottom plates in a total volume of 200 ?l complete medium with 100 ?g/ml OVA. After 90 hours of culture at 37? C. and 5% CO.sub.2 in a humidified incubator, the cells were harvested and the cells were stained with allophycocyanin-labeled anti-CD4 (BD, Biosciences) and proliferation was determined using flow cytometry (FACSCanto, BD Biosciences).
[0109] To assay proliferation of CD4.sup.+ cells, 2?10.sup.5 cells CD4.sup.+ T cells were cultured in 96-well U-bottom plates with mitomycin C treated-OVA loaded splenocytes, acting as antigen presenting cells, at ratios 1/1, 1/0.3, 1/0.1, 1/0.03 and 1/0 in a total volume of 200 ?l complete medium. Cells were cultured for 90 hours at 37? C. and 5% CO.sub.2 in a humidified incubator. For proliferation assays, 1 ?Ci/well [3H]-thymidine was added for the last 18 hours of culture, DNA was harvested on glass fiber filter mats (Perkin Elmer, Boston, USA), and DNA-bound radioactivity was measured on a scintillation counter (Perkin Elmer). For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays were collected after 72 hours of culture and frozen at ?20? C. Cytokine production was quantified using the Mouse Flex Set Cytometric Bead Array (BD Biosciences, Mountain View, Calif., USA).
[0110] Adoptive transfer experiments: On day 39, the spleens were collected from the treatment groups. Single cell suspensions were obtained by mincing the spleens and straining them through 70-?m cell strainers (Becton/Dickinson Labware). The cell suspensions were enriched for CD4.sup.+ T cells, as described above. CD4.sup.+-enriched cells were adoptively transferred to na?ve BALB/c acceptor mice by the i.v injection of 1?10.sup.6 CD4.sup.+ T cells. One day after adoptive transfer, all mice were sensitized by injection 100 ?g OVA/25 ?l saline/25 ?l IFA (Sigma-Aldrich) s.c. at the tail base, and 5 days thereafter, mice were challenged according to the DTH protocol described above.
[0111] Statistical analysis: Significance of differences between groups in ear-thicknesses and cytokine measurements were tested using one-way ANOVA. Statistical significance is indicated as *(p<0.05) or **(p<0.01).
Results
LL-OVA Significantly Enhance the Tolerance-Inducing Capacity in OVA Immunization Model Compared to Free OVA
[0112] To study the induction of oral tolerance, mice were orally fed as described above. Administration of LL-OVA to OVA-sensitized BALB/c mice led to a significant decrease in DTH response compared to the sensitized control mice (BM9 group) and mice treated with LL-pTREX1 or 1 ?g purified OVA (
[0113] These data were accompanied by a significant decreased proliferative capacity and IFN-?, IL-10 and IL-6 production (
LL-OVA Enhances Oral Tolerance Via CD4.SUP.+ T Cells
[0114] To assess whether CD4 T cells mediate the induction of oral tolerance, the OVA-specific proliferative CD4 T cell response in the splenocytes was studied. Flow cytometry demonstrated that only 0.8% of the CD4.sup.+ splenic T cells proliferate after OVA restimulation in the LL-OVA group compared to 4.5% and 11.6% in the BM9 and LL-pTREX1 groups (
Conclusion
[0115] Here, we demonstrated that intragastric administration of OVA-secreting L. lactis suppresses OVA-specific T cell responses via the induction of CD4.sup.+ regulatory. We demonstrated that this immune tolerance induction is more potent than free OVA protein, and that this could be established in a therapeutic setting.
Example B
[0116] Induction of Antigen-Specific Oral Tolerance by Genetically Modified Lactococcus lactis Delivering DQ8-Specific Immunodominant Gliadin Epitopes to Gluten-Sensitized Class II Transgenic Mice
Introduction
[0117] Celiac disease, also known as celiac sprue or gluten-sensitive enteropathy, is a chronic inflammatory disease that develops from an immune response to specific dietary grains that contain gluten. Celiac is a complex multigenic disorder that is strongly associated with the genes that encode the human leukocyte antigen variants HLA-DQ2 or HLA-DQ8. One of the most important aspects in the pathogenesis of Celiac is the activation of a T-helper 1 immune response. This arises when antigen-presenting cells that express HLA-DQ2/DQ8 molecules present the toxic gluten peptides to CD4(.sup.+) T cells. Both classes of gluten proteins, gliadins and glutenins, contain peptides that bind DQ2 and DQ8. It is generally accepted that the immune response, such as the production of IFN-? from gluten-specific T cells, triggers destruction of the mucosa in the small intestine of celiac disease patients. Hence, the activation of a detrimental immune T cell response in the intestine of celiac disease patients appears to be key in the initiation and progression of the disease.
[0118] Antigen-specific immune suppression is an attractive therapeutic goal for the treatment of celiac disease. Active delivery of recombinant gluten proteins/peptides at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. For this purpose we genetically engineered deamidated DQ8 epitope secreting LL (LL-eDQ8d) and evaluated the local and systemic immune response in gluten-sensitized NOD AB? DQ8 class II transgenic mice after oral supplementation.
[0119] Here, we demonstrate that oral delivery of gliadin peptide producing L. lactis suppresses gliadin-specific immune responses via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Materials and Methods
[0120] Bacteria and media: The Lactococcus lactis MG1363 (LL) strain was genetically modified and used throughout this study. Bacteria were cultured in GM17E medium, being M17 broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose and 5 ?g/ml erythromycin (Abbott). Stock suspensions of LL strains were stored at ?20? C. in 50% glycerol in GM17E medium. Stock suspensions were diluted 200-fold in GM17E medium and incubated at 30? C. overnight. Within 16 hours of culture, a saturation density of 2?10.sup.9 colony forming units (CFU) per ml was reached. Bacteria were harvested by centrifugation and 10-fold concentrated in BM9 inoculation buffer at 2?10.sup.9 bacteria/100 ?l. For treatment, each mouse received 100 ?l of this suspension daily by intragastric catheter.
[0121] Plasmids: The sequence encoding the deamidated DQ8 epitope, (encoding DQ8d: caa, tac cca tca ggt gaa ggt tea ttc caa cca tea caa gaa aac cca caa get (SEQ ID NO:1)), was retrieved from published data. In summary, two glutamine residues within the alpha-gliadin peptide were changed into glutamic acids to stimulate the deamidated immunodominant alpha-gliadin response for the DQ8 carrying celiac disease patients, and this epitope is recognized by T cells of these mice. The DQ8d cDNA fragment was synthetically constructed (Operon, The Netherlands) and amplified by Polymerase Chain Reaction (PCR) using the following forward and reverse primers 5-caatacccatcaggtgaaggttc-3 (SEQ ID NO:11) and 5-cgactagttaagcttgtgggttttcttgtgat-3 (SEQ ID NO:12). For detection purposes an e-tag (e) was attached to the fragment, consisting of the following sequence ggt get cca gtt cca tac cca gat cca ctt gaa cca cgt (SEQ ID NO:13). To add the e-tag to the 5 end of DQ8d gene, the PCR product that was produced in step 1 (DQ8d) was used as template in a PCR with oligonucleotides 5-ggtgctccagttccatacccagatccacttgaaccacgtcaatacccatca-3 (SEQ ID NO:14) and 5-cgactagttaagcttgtgggttttcttgtgat-3 (SEQ ID NO:15). The amplified fragment was fused to the Usp45 secretion signal of the erythromycin resistant pT1 NX vector, downstream of the lactococcal P1 promoter. MG1363 strains transformed with plasmids carrying eDQ8d cDNA were designated Lactococcus lactis secreting eDQ8d (LL-eDQ8d). The LL-pT1 NX, which is MG1363 containing the empty vector, pT1 NX, served as control.
[0122] Functional analysis secreted epitopes: For functional analysis of the secreted eDQ8d epitope a proliferation assay with human T cell clones derived from the intestines of celiac disease (CD) patients was performed. Bacteria were grown overnight as described before, deluded 1:50 and grown for another 4 or 6 hours respectively. T cell clones specific for gluten were generated from a small intestinal biopsy taken from patient S, an adult Dutch CD patient that had been on a gluten-free diet for several years. The patient gave informed consent to the study, which was approved by the hospital ethics committee. The patient was typed serologically to be HLA-DR3/4, DQ2/8, thus carrying both CD-associated DQ dimers. T cell clone 1129 was found to respond to an alpha-gliadin derived peptide with a minimal 9 amino acid core QGSFQPSQQ (SEQ ID NO:4), when bound to HLA-DQ8. Deamidation of the P1 and/or P9 glutamine residue (Q) into glutamic acid (E) by the activity of tissue transglutaminase was found to substantially enhance the T cell stimulatory capacity of this gluten peptide. Proliferation assays were performed in duplicate or triplicate in 150 ?l culture medium (Iscoves) in 96-well flat-bottomed plates (Falcon) using 10.sup.4 T cells stimulated with 10.sup.5 HLA-DQ-matched and 3000 RAD irradiated Peripheral blood mononuclear cells in the absence or presence of supernatant at several concentrations. After 48 hours, cultures were pulsed with 0.5 uCi of .sup.3H-thymidine, harvested 18 hours thereafter upon which .sup.3H-thymidine incorporation was determined as a measure for proliferation.
[0123] Mice: Transgenic mice that express HLA-DQ8 in an endogenous MHC II-deficient background (AB? DQ8.sup.+) were backcrossed to NOD mice for 10 generations and intercrossed to produce congenic NOD AB? DQ8.sup.+ mice. Seven to sixteen week old mice were used for the experiments. Mice were weaned and maintained in a conventional animal facility until 8-12 weeks of age.
[0124] Antigen and Antibodies: Deamidated DQ8 epitopes with (GAPVPYPDPLEPRQYPSGEGSFQPSQENPQA (SEQ ID NO:16)) and without (QYPSGEGSFQPSQENPQA (SEQ ID NO:2)) e-tag were synthesized. For T-cell phenotyping, CD4 and CD25 antibodies were purchased from BD-Biosciences (San Jose, Calif.), and APC anti-Foxp3 staining kits were purchased from eBiosciences (San Diego, USA) respectively. Anti-IL-10 neutralizing monoclonal antibody (1 ?g/ml, clone JES052A5), TGF-? neutralizing monoclonal antibody (1 ?g/ml, clone 1D11) and LAP neutralizing antibodies (1 ?g/ml, clone 27235) were obtained from R&D Systems (Minneapolis, Minn.).
[0125] Oral feeding and DTH (Delayed-type hypersensitivity) reaction: NOD AB? DQ8 mice on a gluten free chow were sensitized by subcutaneous injection of 100 ?g deamidated eDQ8 peptides in 100 ?l of a 1:1 CFA (purchased from Difco of Becton, Dickinson and Company, San Jose, Calif.) saline solution in the tail base at day 1. The peptide used for the sensitization had the same sequence as the secreted epitope. Mice were fed BM9 as a negative control, LL-pT1NX or LL-eDQ8d [all at days 1-10 dissolved in 100 ?l BM9]. Feedings were performed by intragastric administrations of antigen or bacterial suspensions using an 18-gauge stainless gavage needle. Ten days after immunization, antigen-specific DTH responses were assessed. Twenty-four hours thereafter DTH measurements were performed. For measurement of antigen-specific DTH responses, mice were challenged with 10 ?g eDQ8d in 10 ?l saline in the auricle of the ear. The increase in ear thickness was measured in a blinded fashion using an engineer's micrometer (Mitutoyo, Tokyo, Japan) at 24 hours after challenge. DTH responses were expressed as the difference in increase 24 hours after eDQ8d injection, following subtraction of ear-thickness before challenge. Subsequently mice were sacrificed, spleen and lymph nodes were harvested and cells were assessed for DQ8d-specific proliferation and cytokine production. For e-tag interference NOD AB? DQ8 mice were immunized with 100 ?g deamidated DQ8 peptides with (eDQ8d) or without E-tag (DQ8d) in 100 ?l of a 1:1 Complete Freund's Adjuvant (CFA, Difco, BD) saline solution in the tail base at day 1. At day 7 mouse DTH measurements were performed as described above with 10 ?g DQ8d with or without e-tag, corresponding to the peptide used for the immunization.
[0126] Cell cultures, proliferation and cytokine production assays: Cell suspensions of spleen and lymph nodes were prepared at day 11 of the experiment by homogenizing the tissue with a tissue grinder in 1?PBS. Erythrocytes were removed from the spleen cell suspensions by incubation with ACK (Ammonium Chloride/Potassium (lysing buffer)). Cells were incubated in 96-well microtiter plates at 5?10.sup.5 cells/well in 0.2-ml volumes at 37? C. in RPMI 1640 (1.5% Hepes, 1% Penstrep and 10% FBS) with supplements containing either medium alone, 10 ?g Con A, or 50 ?g eDQ8d epitope. In a separate experiment IL-10, TGF-?, IL10 & TGF-? or LAP neutralizing antibodies were added to splenocytes of LL-eDQ8d treated mice. After 24 hours, proliferation was assessed by addition of 1 ?Ci/well [.sup.3H]-thymidine for the last 24 hours of culture. DNA-bound radioactivity was harvested onto glass fiber filter mats and thymidine-incorporation measured on a scintillation counter (Perkin Elmer). Results were expressed as mean cpm of triplicate wells. For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays, described above, were collected after 24 hours of culture and frozen at ?20? C. until cytokine analysis was performed. Cytokine production was quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences).
[0127] Flow cytometric analysis: Spleens and gut-associated lymph node tissue (GALT) of BM9, LL-pT1 NX or LL-eDQ8d treated mice were isolated, prepared as described above and stained for CD4, CD25 and Foxp3. Intracellular staining was performed for Foxp3 according to the manufacturer's instructions (eBiosciences, San Diego, Calif.) and subsequently measured using flow cytometry on a Becton Dickinson FACSCaliburs. For analysis cells were gated on CD4.sup.+CD25.sup.+ and CD4.sup.+CD25.sup.? subpopulations and within these populations Foxp3 histograms were used to determine Mean Fluorescence Intensity (MFI).
[0128] Statistical analysis: Results from cytokine measurements are expressed as means?SEM. eDQ8d-specific proliferation, ear-thickness and cytokine measurements were tested for significance using one-way ANOVA followed by the student's t-test comparison: two samples assuming equal variance, to determine the differences between individual groups. For all tests a p value <0.05:*, <0.01:** was used to indicate statistical significance for both tests.
Results
[0129] Mucosal Delivery of eDQ8d Epitopes by L. lactic Significantly Decreases the DQ8d-Induced DTH Response and Proliferative Capacity of Bulk Spleen and Inguinal Lymph Node Cells.
[0130] Daily intragastric administration of LL-eDQ8d in eDQ8d-immunized NOD AB? DQ8 class II transgenic mice led to a significant decrease in DTH response compared to the sensitized negative control mice (
Decreased Splenic eDQ8d-Specific Proliferation Depends on IL-10 and TGF-?, and LL-DQ8d Treatment Significantly Increases Splenic and GALT Foxp3 Expression
[0131] The functional importance of TGF-?, IL-10, and LAP (membrane-associated TGF-?) on the eDQ8d-specific splenic proliferative response was analyzed using neutralizing antibodies. IL-10-, TGF-?- or LAP-neutralizing antibodies did not significantly interfere with the decreased splenic proliferative response of LL-eDQ8d treated mice, but adding a combination of TGF-? and IL-10 neutralizing monoclonal antibodies completely abolished the decreased eDQ8d-specific proliferative capacity of splenocytes of LL-eDQ8d treated mice (
Conclusion
[0132] Our data demonstrated that mucosal delivery of a gliadin derived peptide immunodominant for DQ8-mediated T-cell responses by genetically modified L. lactis, induces suppression of local and systemic DQ8 restricted T-cell responses in NOD AB? DQ8 class II transgenic mice. Treatment resulted in an antigen-specific decrease of the proliferative capacity of the splenocytes and inguinal lymph node cells, which was critically dependent on the production of IL-10 and TGF-? and associated with a significant induction of Foxp3.sup.+ regulatory T cells. Because this approach of antigen-delivering bacteria has the capacity for potentiating oral tolerance even in the setting of established hypersensitivity, it may be applicable for the treatment of celiac disease and possibly other autoimmune and/or allergic diseases.
Native DQ8 Epitope
[0133] The above experiments are repeated with the native ?-gliadin epitope, i.e., QYPSGQGSFQPSQQNPQA (SEQ ID NO:4), corresponding to residues 203-220 of the sequence retrievable via UniProtKB/TrEMBL entry Q9M4L6. The native DQ8 epitope is encoded by the nucleotide sequence 5-caa tac cca tea ggt caa ggt tea ttc caa cca tea caa caa aac cca caa get-3 (SEQ ID NO:3).
[0134] The results with the native ?-gliadin DQ8 epitope are essentially identical to the results described above for the deamidated ?-gliadin DQ8 epitope.
Trial in Celiac Patients Using DQ8 Epitope
[0135] In a preliminary study, engineered L. lactis according to the invention are used as a therapeutic in a trial in patients with Celiac disease. Our findings provide promise that this approach is effective in an antigen-specific manner.
[0136] Celiac disease is an especially attractive target for this approach, due to the ability of the LL to deliver the antigen at the site of the primary response to achieve both direct and bystander tolerance.
Trial in Celiac Patients Using DQ2 Epitope
[0137] No transgenic mice exist expressing HLA-DQ2 in an endogenous MHC II-deficient background, comparable to HLA-DQ8 mice as used above. Accordingly, the experiments described above for DQ8 epitopes were not possible in an appropriate mouse model. We therefore conduct some preliminary experiments in patients with celiac disease, using both native as well as deamidated ?-gliadin DQ2 epitope.
[0138] Specifically, the above experiments are repeated using: deamidated DQ2 epitope LQLQPFPQPELPYPQPQLPYPQPELPYPQPQPF (SEQ ID NO:6), encoded by the nucleotide sequence 5-tta caa tta caa cca ttc cca caa cca gaa tta cca tac cca tta cca tac cca caa cca gaa tta cca tac cca caa cca caa cca ttc (SEQ ID NO:5) and the native DQ2 epitope: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:8), encoded by the nucleotide sequence 5-tta caa tta caa cca ttc cca caa cca caa tta cca tac cca tta cca tac cca caa cca caa tta cca tac cca caa cca caa cca ttc (SEQ ID NO:7)
[0139] The results with the native and deamidated ?-gliadin DQ2 epitope are essentially identical to the results described above for the ?-gliadin DQ8 epitopes.
Example C
[0140] Induction of Tolerance to Clotting Factor VIII and Factor IX Following Oral Administration of L. lactis Secreting the Factors
Introduction
[0141] Several therapeutic (recombinant) proteins, such as interferon's, factor VIII/IX and antibodies (Remicade) are administered at high doses over prolonged treatment periods. However, a complication associated with their use is the development of protein-specific immune responses, such as antibodies. These antibodies (Abs), also called inhibitors, render the therapeutic proteins less effective. Examples include the formation of inhibitors for factor VIII/IX in hemophilia, erythropoietin (Epo) in patients undergoing therapy for chronic renal failure, and IFN- in patients undergoing treatment for multiple sclerosis. Here, we demonstrate that oral delivery of the Factor VIII (and Factor IX) by L. lactis suppresses inhibitor formation to the factor via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Material and Methods
[0142] Bacteria and plasmids: The L. lactis strain MG1363 is used throughout this study. Bacteria are cultured in GM17 medium, i.e., M17 (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at ?20? C. in 50% glycerol in GM17. For intragastric inoculations, stock suspensions are diluted 200-fold in fresh GM17 and are incubated at 30? C. They reach a saturation density of 2?10.sup.9 colony-forming units (CFU) per ml within 16 hours. Throughout this study, mixed bacterial suspensions are used. Therefore, the bacteria that are mixed are harvested by centrifugation and pellets of both bacterial cultures are concentrated 10-fold in BM9 medium (Schotte, Steidler et al., 2000). For treatment, each mouse receives 100 ?l of this suspension by intragastric catheter.
[0143] Human FVIII and FIX cDNA or cDNA-fragments, representing FVIII- and FIX-specific CD4.sup.+ T-cell epitopes, are amplified fused to the Usp45 secretion signal of the erythromycin-resistant pT1 NX vector, downstream of the lactococcal P1 promoter.
[0144] MG1363 strains transformed with plasmids carrying human FVIII (and/or epitope fragment), FIX (and/or epitope fragment), were designated L. lactis secreting LL-FVIII, LL-FIX. LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serve as control.
[0145] Quantification of FVIII and FIX: FVIII or FIX from LL-FVIII and LL-IX, respectively are determined using human FVIII and FIX-specific enzyme-linked immunosorbent assay (ELISA), that have been described previously (Chuah et al., 2003). The recombinant proteins are also analyzed by Western blot analysis and COATests and aPTT assays, as described (Chuah et al., 2003; VandenDriessche et al., 1999). The NH2-terminus of this protein is determined by automated Edman degradation. Since FVIII and FIX are normally expressed in the liver where they undergo extensive post-translational modifications, the clotting factors produced from the engineered L. lactis may be biologically inactive. However, these post-translational differences will likely have no repercussions on the ability of these L. lactis-produced recombinant proteins to induce immune tolerance. Indeed, most inhibitors that have been characterized in detail to date typically recognize amino acid residues (Villard et al., 2003), rather than glycosylated moieties.
[0146] Animals: Hemophilia A or B mice obtained by knocking out the murine FVIII or FIX genes using homologous recombination in ES cells as described by (Bi et al., (1995) and Wang et al., (1997), are bred in the laboratory. These recipient mice generate neutralizing antibodies when challenged with purified recombinant FVIII or FIX antigen in the presence of CFA (Mingozzi et al., 2003). The inhibitor status can be monitored over time using Bethesda assays or anti-FVIII/anti-FIX-specific ELISAs. Recipient mice challenged with FVIII or FIX (+CFA) typically develop inhibitors 2-3 weeks after antigenic challenge.
[0147] Experimental setting: Four- to six-week-old mice receive FVIII, FIX, LL-FVIII, LL-FIX, or LL-pT1NX or LL-OVA (an irrelevant antigen) (1 or 10 ?g). As a positive control for tolerance induction, we inject mice with adeno-associated viral vectors (AAV) expressing FIX from a hepatocyte-specific promoter. Recipient animals develop FIX-specific immune tolerance that prevents induction of anti-FIX antibodies upon subsequent challenge with FIX+CFA.
[0148] In a prophylactic setting, FVIII, FIX, LL-FVIII, LL-FIX alone are administered orally to hemophilia A or B mice using a gastric catheter, using different treatment intervals and doses. These recipient mice are subsequently challenged with purified recombinant FVIII or FIX antigen, in the presence of CFA (Mingozzi et al., 2003). Control animals are exposed to LL-pT1NX and LL-OVA. Plasma is harvested by retro-orbital bleeding. The development of antibodies directed against FVIII or FIX is assessed using Bethesda assays (Kasper et al., 1975) or using a modified anti-FVIII or anti-FIX specific ELISA (VandenDriessche et al., 1999) at different time intervals.
[0149] In a therapeutic setting, hemophilia A or B mice are first immunized with FVIII or FIX, as described (Mingozzi et al., 2003). The inhibitor status is monitored over time using Bethesda assays or anti-FVIII/anti-FIX specific ELISAs. Mice with low or high inhibitor titers are subsequently treated with FVIII, FIX, LL-FVIII, LL-FIX alone using different treatment intervals and doses and inhibitor titers are determined over time. The specificity of the possible immune tolerance is assessed by challenging the mice that receive FVIII, FIX, LL-FVIII, LL-FIX alone with an irrelevant antigen (tetanus toxoid or Ova).
[0150] Cell cultures, proliferation and cytokine assay: Single cell suspensions of spleen and lymph nodes are prepared by passing the cells through 70 ?m filter cell strainers (Becton/Dickinson Labware). Erythrocytes are removed from the spleen cell suspensions by incubation with red cell lysis buffer.
[0151] Proliferation assays of total splenocyte populations, 2?10.sup.5 cells are cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium either alone or with purified FVIII or FIX, and either with or without anti-IL-10 or anti-TGF-? neutralizing monoclonal antibodies. FVIII and FIX is added at concentrations ranging from 1 to 100 ?g/ml. The neutralizing antibodies are added at 1, 0.1 and 0.01 ?g/ml. For proliferation assays of CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cell populations, 0.2?10.sup.5 cells CD4.sup.+ T cells or CD4.sup.+CD25.sup.? T cells are cultured in 96-well U-bottom plates with 1?10.sup.5 irradiated CD4.sup.? cells, acting as antigen presenting cells, and FVIII or FIX (0 or 100 ?g/ml) in a total volume of 200 ?l complete medium either with or without neutralizing antibodies. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 16-18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on a scintillation counter (Perkin Elmer).
[0152] For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays are collected after 24, 48 and 72 hours of culture and frozen at ?20? C. until cytokine analysis is performed. Cytokine production is quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).
[0153] In vivo T regulatory activity assay: In order to test for active suppression of antibody formation in mice, splenocytes, bead-purified CD4.sup.+ T cells, CD4.sup.+CD25.sup.? or CD4.sup.+CD25.sup.+ T cells isolated from the different experimental L. Lactis-treated groups are adoptively transferred to na?ve C3H/HeJ mice. Untreated mice are used as control. The number of transferred cells is 10.sup.7 for whole spleen cells, subpopulation-depleted spleen cells, or positively selected CD4.sup.+ cells and CD4.sup.+CD25.sup.? and CD4.sup.+CD25.sup.+ T cells. Recipient mice (n=4-5 per experimental cohort) were subcutaneously injected with 5 ?g hF.IX in cFA 36 hours after adoptive transfer. Anti-hF.IX IgG titers in plasma were measured 2.5 weeks after immunization.
Results
LL-FVIII and LL-IX Significantly Enhances the Tolerance-Inducing Capacity of in Hemophilia A or B Mice Compared to Free FVIII or FIX
[0154] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). Addition of LL-FVIII or LL-FIX significantly enhances the tolerance induction towards FVIII and FIX as the factor-specific proliferative response of splenocytes is significantly reduced in this group in comparison to the control and free FVIII and FIX groups.
LL-FIIIV and LL-FIX Potentiate Oral Tolerance in Association with Reduced FVIII- and FIX-Specific Titers and IFN-? and More IL-10 and TGF-? Production in Response to the Factor
[0155] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). FVIII and FIX-specific antibodies and cytokine production in response to the factor in splenocytes and lymph nodes are quantified as described above. The inhibitor formation and production of the proinflammatory cytokine, IFN-? is strongly reduced and the immunosuppressive cytokines IL-10 and TGF-? is significantly increased in the LL-FVIII/FIX group in comparison to the control and free FVIII/IX groups.
LL-FVIII/FIX Enhances Oral Tolerance Via CD4.SUP.+ T Cells
[0156] To assess whether CD4.sup.+ T cells mediate the induction of oral tolerance, the factor-specific proliferative CD4.sup.+ T-cell response is studied in the splenocytes and lymph nodes. Therefore, mice are orally fed as described above (experimental setting) and the factor-specific CD4.sup.+ T cell proliferation is determined as described in Cell cultures, proliferation and cytokine assay. The factor-specific CD4 T-cell response in the LL-FVIII/FIX group is significantly reduced in comparison to the control and free FVIII/IX groups.
Antigen-Induced T Regulatory Cells Following LL-FVIII/FIX Therapy can Transfer Protection from Inhibitor Formation In Vivo
[0157] In order to test for active suppression of antibody formation in mice treated with the oral tolerance protocol, we adoptively transfer splenocytes from the different treated groups as described above (In vivo T regulatory activity assay). Compared with controls and free FVIII/IX groups, antifactor IgG formation is significantly reduced in the LL-FVIII/FIX group, indicating activation of regulatory CD4.sup.+ T cells in our combination oral tolerance protocol.
Conclusion
[0158] Our data demonstrate that mucosal delivery of recombinant FVIII- or FIX secreting L. lactis are more potent than free FVIII or FIX in suppressing the formation of FVIII- and FIX-specific inhibitors in Hemophilia A and B mice respectively.
Example D
[0159] Induction of Tolerance to an Allergen, Der p 1 Following Oral Administration of L. lactis Secreting the Allergen
Introduction
[0160] Allergic asthma is a chronic inflammatory disorder of the airways. It is characterized by reversible airway obstruction, elevated serum levels of allergen-specific, immunoglobulin E, mucus hypersecretion and airway hyperresponsiveness (AHR) to ronchospasmogenic stimuli. Its symptoms are made worse by exposure to an allergen (e.g., tree, grass and weed pollen, dust and dust mites, mold, animal dander) to which the patient has been sensitized. Type 2 T-helper (Th2) lymphocytes play a crucial role in the initiation, progression and persistence of the disease. Current data suggest that Th2 responses to allergens are normally suppressed by regulatory T cells. Furthermore, suppression by this subset is decreased in allergic individuals. Here, we demonstrate that oral delivery of allergen by L. lactis suppresses asthma-like responses via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Material and Methods
[0161] Two Mouse models of allergic asthma that mimics human disease are the Ova allergen model and the humanized SCID model.
[0162] The Ova allergen model: OVA-sensitized mice are inhalationally challenged with OVA aerosol that leads to Th2 cytokine-dependent eosinophilic airway inflammation, bronchial hyperreactivity, and IgE production, findings highly characteristic of human allergic asthma (Brusselle, 1994, Clin. Exp. Allergy 24:73; Kips et al., 1996, Am. J. Respir. Crit. Care Med. 153:535; Brusselle et al., 1995, Am. J. Respir. Cell Mol. Biol. 12:254).
[0163] Bacteria: The L. lactis strain MG1363 is used throughout this study. Bacteria are cultured in GM17 medium, i.e., M17 (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at ?20? C. in 50% glycerol in GM17. For intragastric inoculations, stock suspensions are diluted 500-fold in fresh GM17 and incubated at 30? C. They reached a saturation density of 2?10.sup.9 colony-forming units (CFU) per mL within 16 hours. Bacteria are harvested by centrifugation and concentrated 10-fold in BM9 medium. For treatment, each mouse receives 100 ?L of this suspension daily by intragastric catheter.
[0164] Plasmids: The mRNA sequence encoding Gallus gallas Ovalbumin is retrieved from Genbank (accession number AY223553). Total RNA is isolated from chicken uterus and cDNA is synthesized using 2 ?g total RNA, 2 ?M oligo dT primers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mM DTT (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.5 mM dNTP (Invitrogen, Merelbeke, Belgium), 20 U Rnasin (Promega Incorporation Benelux) and 100 U superscript II reverse transcriptase (Invitrogen) in a volume of 25 ?l OVA cDNA fragment is amplified by Polymerase Chain Reaction (PCR) using the following conditions: 94? C. for 2 minutes followed by 30 cycles at 94? C. for 45 seconds, 62? C. for 30 seconds and 72? C. for 90 seconds, with the following forward and reverse primers 5-GGCTCCATCGGTGCAGCAAGCATGGAATT-3 (SEQ ID NO:17) and 5-ACTAGTTAAGGGGAAAC-ACATCTGCCAAAGAAGAGAA-3 (SEQ ID NO:18).
[0165] The amplified fragment is fused to the Usp45 secretion signal of the erythromycin resistant pT1 NX vector, downstream of the lactococcal P1 promoter.
[0166] MG1363 strains transformed with plasmids carrying OVA cDNA are designated LL-OVA. LL-pTREX1, which is MG1363 containing the empty vector, serve as control.
[0167] Quantification of OVA: OVA from LL-OVA are determined using an in house developed OVA-specific enzyme-linked immunosorbent assay (ELISA). Production of the recombinant proteins is also assessed by Western blot analysis.
[0168] Mice: BALB/c mice (6 to 8 weeks of age) are purchased from Charles River Laboratories (Calco, Italy). The mice are maintained under specific pathogen-free conditions.
[0169] Immunization of mice: Mice are immunized i.p. with 10 ?g of OVA (grade V; Sigma-Aldrich) in 1 mg of aluminum hydroxide (alum). This immunization is repeated after 21 days (on days 0 and 21). Control mice receive a saline injection instead of the OVA/alum solution. 26 days after the immunization, sensitized mice inhale an aerosolized solution of 1% OVA dissolved in PBS for 10 minutes. OVA inhalation is conducted for 3 days in a row (days 47, 48, and 49). Control mice inhale PBS alone under the same conditions as used for the experimental group.
[0170] Induction of oral tolerance: Mice receive LL-OVA, LL-pTREX1, 1 ?g OVA or BM9 on days 0-4, 7-11, 14-18 and 21-25. As positive control for oral tolerance induction mice are fed 30 mg OVA by intragastric catheter that reduce bronchial eosinophilia and airway hyperresponsiveness, with high dose feeding being more effective than low-dose feeding.
[0171] Measurement of airway hyperresponsiveness (AHR): 24 hours after the final inhalation (day 50), airway hyperresponsiveness is assessed by methacholine-induced airflow obstruction. The mice are exposed for 2.5 minutes to nebulized physiologic saline (Otsuka Pharmaceutical), followed by incremental doses (1-30 mg/ml) of nebulized methacholine. These mice are placed in a whole-body plethysmograph for 2.5 minutes following nebulization, and enhanced pause (Penh) is measured using Biosystem XA WBP system (Buxco Electronics). Penh represents pulmonary airflow obstruction and is calculated using the formula: Penh=((Te?Tr)/(Tr?PEF/PIF)), where Penh=enhanced pause (dimensionless), Te=expiratory time (seconds), Tr=relaxation time (seconds), PEF=peak expiratory flow (milliliters per second), and PIF=peak inspiratory flow (milliliters per second). Penh is measured and averaged approximately every 5 s, and the cumulative values are averaged as the Penh value for each time point. Airway hyperresponsiveness is expressed as PC200Mch (200% provocative concentration of methacholine), which is the concentration of methacholine that doubled the baseline Penh value.
[0172] Analysis of bronchoalveolar lavage fluid (BALF): After the measurement of airway hyperresponsiveness, bronchoalveolar lavage samples are obtained. The mice are euthanized by i.p. injection of overdose ketamin and xylazin, and then the lungs are lavaged with 0.5 ml of saline four times. The lavage fluid is centrifuged and the cells are resuspended in 1 ml of saline with 1% BSA. Total cell numbers are counted using a hemocytometer. Cytospin samples are prepared by centrifuging the suspensions at 300 rpm for 5 minutes. To clearly distinguish the eosinophils from the neutrophils, three different stains are applied: Diff-Quick, May-Grunwald-Giemsa, and Hansel (eosin) stains. At least 300 leukocytes are differentiated by light microscopy based on the standard morphologic criteria. The level of IL-13, IL-4 and IL-5 in BALF is detected by Cytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA) following the manufacturer's instructions.
[0173] Measurement of serum total IgE and OVA-specific Ig: On day 50, blood samples are obtained from retro-orbital sinus under anesthesia. After the samples had fully coagulated, they are centrifuged, and the sera is collected and stored at ?80? C. until use. Total IgE is assayed by ELISA using paired Abs (BD Pharmingen) according to the manufacturer's instructions. To measure OVA-specific IgE, IgG1, and IgG2a in sera, microtiter plates (Maxisorp, Nunc, VWR International, Haasrode, Belgium) are coated with 2 ?g/ml OVA. Subsequently, the wells are blocked with 0.1% casein in PBS, after which the plates are incubated with mouse serum samples diluted 1:10 to 1:20480 in PBS containing 0.1% casein and 0.05% TWEEN? 20 (PBS-CT), with goat anti-mouse IgG2a-HRP (Southern Biotechnology Associates (SBA), Imtec ITK Diagnostics, Antwerpen, Belgium, dilution 1:5000), goat anti-mouse IgG1-HRP or goat anti-mouse IgE-HRP (SBA, dilution 1:5000). After washing, substrate (3,3,5,5 tetramethylbenzidine (TMB) substrate reagent, Pharmingen, Becton Dickinson, Erembodegem, Belgium) is added to each well. Finally, reactions are stopped by adding 1M H.sub.2SO.sub.4 to the wells. The absorbances are read at 450 nm. ELISA scores are expressed as titers, which are the inverse of the highest dilution that still had on (OD.sub.450 higher than the calculated cutoff value. The cutoff is calculated as the mean OD.sub.450 of five non-immunized mice increased with three times the SD.
[0174] Histological examination of lung tissue: After bronchoalveolar lavage samples are obtained, the lungs are perfused with physiologic saline and are resected from the mice. The lungs are fixed with neutralized buffered formalin and embedded in paraffin. Sections (3-?m thick) are stained with H&E or periodic acid-Schiff (PAS). The intensity of histological changes in the lungs is evaluated with four grading scores (0, no inflammation; 1, slight/mild; 2, moderate; and 3, severe), according to the distribution and intensity of the following findings: 1) epithelial shedding or undulation of the nuclei of bronchial epithelial cells, 2) increase in the number of goblet cells, 3) infiltration of inflammatory cells from vessels into the mucosal and submucosal area of the bronchus and peribronchial interstitium, and 4) hypertrophy and thickening of the smooth-muscle cell layer.
[0175] RT-PCR for analysis of cytokine and chemokine gene expression in the lung: The lungs are removed after perfusion with physiologic saline, and total RNA is extracted using ISOGEN (Nippon Gene) according to the manufacturer's instructions. Total RNA (10 ?g) is reverse-transcribed using oligo(dT)15 primer (Promega) and Superscript II RNase H-reverse transcriptase (Invitrogen Life Technologies) at 42? C. for 2 hours. To ensure that each sample contained the same amount of cDNA, the ?-actin cDNA concentration of each sample is first determined using ?-actin-specific primers. These samples are amplified for the appropriate number of cycles, such that the amount of PCR product remained on the linear part of the amplification curve. The PCR products are electrophoresed in a 2% agarose gel and were visualized by ethidium bromide staining. The levels of IL-13, eotaxin, IL-10, IFN-?, and TGF-? are determined using the following specific primer sets.
TABLE-US-00001 Thesenseprimerfor?actin (SEQIDNO:19) 5-ACGACATGGAGAAGATCTGG-3, andtheantisenseprimer (SEQIDNO:20) 5-TCGTAGATGGGCACAGTGTG-3. ThesenseprimerforIL-13 (SEQIDNO:21) 5-TCTTGCTTGCCTTGGTGGTCTCGC-3, andtheantisense (SEQIDNO:22) 5-GATGGCATTGCAATTGGAGATGTTG-3. Thesenseprimerforeotaxin (SEQIDNO:23) 5-GGGCAGTAACTTCCATCTGTCTCC-3, andtheantisenseprimer (SEQIDNO:24) 5-CACTTCTTCTTGGGGTCAGC-3. ThesenseprimerforIL-10 (SEQIDNO:25) 5-TACCTGGTAGGAGTGATGCC-3, andtheantisense (SEQIDNO:26). 5-GCATAGAAGCATACATGATG-3. ThesenseprimerforIFN-? (SEQIDNO:27) 5-CATAGATGTGGAAGAAAAGA-3, andtheantisense (SEQIDNO:28) 5-TTGCTGAAGAAGGTAGTAAT-3. ThesenseprimerforTGF-? (SEQIDNO:29) 5-CTTTAGGAAGGACCTGGGTT-3 andtheantisense (SEQIDNO:30) 5-CAGGAGCGCACAATCATG33-3.
[0176] Cell cultures, proliferation and cytokine assay: One day after the final inhalation (day 50) single cell suspensions of spleen and mediastinal lymph nodes are prepared by passing the cells through 70 ?m filter cell strainers (Becton/Dickinson Labware). Erythrocytes are removed from the spleen cell suspensions by incubation with red cell lysis buffer. CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cells are enriched using CD4.sup.+ T cell isolation kit (Miltenyi Biotec, Germany) or CD4.sup.+CD25.sup.+ regulatory T cell isolation kit (Miltenyi Biotec, Germany), respectively and MACS columns (midiMACS; Miltenyi Biotec).
[0177] Proliferation assays of bulk splenocyte and LN populations, 2?10.sup.3 cells are cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium either alone or with purified OVA. OVA is added at concentrations ranging from 1 to 100 ?g/ml. For proliferation assays of CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cell populations, 2?10.sup.5 cells CD4.sup.+ T cells or CD4.sup.+CD25.sup.? T cells are cultured in 96-well U-bottom plates with mitomycin treated splenocytes that are loaded with 1 mg/ml OVA for 16 hours, acting as antigen presenting cells, at ratio's CD4.sup.+ T cell or CD4.sup.+CD25.sup.? T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 ?l complete medium. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on a scintillation counter (Perkin Elmer).
[0178] For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays is collected after 24, 48 and 72 hours of culture and frozen at ?80? C. until cytokine analysis is performed. Cytokine production is quantified using the Mouse Inflammation Cytometric Bead Array (BD Biosciences, Mountain View, Calif., USA).
[0179] In vivo T regulatory activity assay: One day after the final inhalation (day 21), spleens of the treated mice are digested with 0.1% collagenase (Sigma-Aldrich) at 37? C. for 20 minutes. In some experiments, single-cell suspensions of whole spleen cells are prepared and cultured with Con A (2 ?g/ml; Sigma-Aldrich) for 48 hours. Cells are collected, and 10.sup.7 cells are adoptively transferred i.v. into na?ve BALB/c mice. For negative selection, CD4.sup.+, CD8.sup.+, CD11c.sup.+, CD19.sup.+, or CD11b.sup.+ cells are depleted from the whole spleen cells using magnetic beads (MACS; Miltenyi Biotec) with biotinylated anti-mouse CD4, CD8, CD11c, CD19, and CD11b mAb (BD Pharmingen), according to the manufacturer's instructions. The efficiency of depletion is examined by flow cytometry (>99%). CD4.sup.+, CD4.sup.+CD25.sup.? cells are purified using CD4.sup.+ T cell isolation kit. Regulatory T cell isolation kit following the manufacturer's instructions. The purity of positively selected cells is checked using flow cytometry. For cell transfer experiments, cells are transferred into BALB/c mice from the tail veins just before their first immunization or just after their second immunization with OVA/alum. The number of transferred cells is 10.sup.7 for whole spleen cells, subpopulation-depleted spleen cells, or positively selected CD4.sup.+ cells and CD4.sup.+CD25.sup.? cells. In the Humanized SCID (hu-SCID) Model (as Described by Duez et al., 2000; Hammad et al., 2000).
[0180] In this model, the allergic immune response to the house dust mite (HDM) allergen Der p 1 can be studied. Such hu-SCID mice reconstituted i.p. with PBMC from HDM-allergic patients and subsequently exposed to aerosols of HDM produce human IgE, develop a pulmonary infiltrate composed of activated T cells and DCs, and exhibit AHR in response to bronchoconstrictor agents (Pestel et al., 1994, J. Immunol., 153:3804; Duez et al., Am. J. Respir. Crit. Care Med., vol. 161, pp. 200-206, 2000).
Bacteria
[0181] The L. lactis strain MG1363 is used throughout this study. Bacteria are cultured in GM17 medium, i.e., M17 (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at ?20? C. in 50% glycerol in GM17. For intragastric inoculations, stock suspensions are diluted 200-fold in fresh GM17 and incubated at 30? C. They reached a saturation density of 2?10.sup.9 colony-forming units (CPU) per mL within 16 hours. Bacteria are harvested by centrifugation and concentrated ten-fold in BM9 medium. For treatment, each mouse receives 100 ?L of this suspension daily by intragastric catheter.
Plasmids
[0182] Der p 1, a 222 amino-acid residue globular glycoprotein, is one of the major allergens from Dermatophagoides pteronyssinus (Dpt) mites. DNA sequence with optimal L. lactis codon usage encoding the Der p 1 protein is synthesized, amplified and fused to the Usp45 secretion signal of the erythromycin resistant pT1 NX vector downstream of the lactococcal P1 promoter. MG1363 strains transformed with plasmids carrying murine Der p 1, Der p 1 aa52-71 and Der p 1 aa117-133 cDNA are designated LL-Derp1, LL-Derp1aa52-71 and LL-Derp1aa117-133. LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serve as control.
Quantification of Der p 1
[0183] Der p 1 from LL-Derp1 is determined using an in house developed Der p 1-specific enzyme-linked immunosorbent assay (ELISA). Production of the recombinant proteins is also assessed by Western blot analysis.
Patients
[0184] Blood is collected from donors sensitive or not sensitive to house dust mites. Allergic patients present the usual features of house dust mite sensitization. Skin prick tests toward Dermatophagoides pteronyssinus (Dpt) allergen (Stallergenes, Fresnes, France) (diameter ?10 mm) are positive, and all patients have serum-specific IgE antibodies. Total IgE concentrations are greater than 150 IU/ml (150-1600 IU/ml). Healthy donors are tested as negative controls (total IgE levels are less than 150 IU/ml, and they have negative skin prick tests toward commonly inhaled allergens).
Human Peripheral Blood Mononuclear Cell Preparation
[0185] Platelet rich plasma is obtained after centrifugation (120?g, 15 minutes) and discarded. Blood cells are then diluted in RPMI 1640 (Life Technologies, Paisley, Scotland) (vol/vol) and layered over a Ficoll gradient (Pharmacia, Uppsala, Sweden). After centrifugation (400?g, 30 minutes), PBMCs are harvested at the interface and washed three times in sterile RPMI medium before transfer.
Mice
[0186] C.B.-17 SCID mice (6-8 weeks old) are maintained in isolators with sterilized bedding in a specific animal facility. The SCID colony is regularly checked for absence of mouse serum immunoglobulins by ELISA.
Peripheral Blood Mononuclear Cells Transfer in SCID Mice: PBMC Hu-SCID Mice
[0187] SCID mice are between 6 and 8 weeks old at the time of cell transfer. The mice are reconstituted by intraperitoneal injection of 10?10.sup.6 mononuclear cells from allergic patients or healthy donors in 400 ?l of RPMI via a 23-gauge needle. On the same day, they receive intraperitoneally 2 index reactivity [IR] units Dpt. Four days after the cell reconstitution, SCID mice are exposed to daily allergen aerosols containing 100 IR units of Dpt (100 IR units are equivalent to approximately 200 ?g of protein contained in the Dpt extract) for 4 successive days (day 0 to day 4). The control group is not exposed to Dpt. One day before airway responsiveness measurement (day 35 and day 60), hu-SCID mice are exposed to another aerosol of 100 IR units of Dpt solution.
Experimental Setting
[0188] Mice receive L. lactis engineered to express Der p 1 or an irrelevant antigen (OVA) as negative control.
[0189] The engineered L. lactis bacteria are administered orally to SCID mice using a gastric catheter, using different treatment intervals and doses starting one day after PBMC reconstitution. Induction of oral tolerance is assessed by measuring human serum IgE antibodies, analysis of pulmonary infiltration, measurement of AHR and analysis of cell populations and cytokine production in the BALF. Furthermore, induction of tolerance is assessed by analysis of the proliferative T cell response against Der p 1.
Assessment of Airway Responsiveness (AHR)
[0190] Airway responsiveness (expressed as provocative dose of carbachol causing a 50% increase in lung resistance) is measured on day 35 or day 60 as described by Duez et al., 2000.
Human IgE Measurements
[0191] Several days after transplantation with human cells, mice are bled from the retro-orbital sinus under anesthesia. Total human IgE is investigated by a two-site immuno-radiometric method with the use of two different mouse mAbs specific for the E-chain (Immunotech International, Luminy, France). At least 20 ?l of serum is used in a duplicate test. The sensitivity of the method permits the detection of 0.1 IU/ml (0.24 ng/ml).
[0192] Specific IgE Ab against Dpt allergen is quantified by ELISA. Briefly, plastic tubes (Maxisorb Startube, Nunc, Denmark) are coated overnight with Dpt allergen in 0.1 M carbonate/bicarbonate buffer (pH 9.6) at 4? C. and saturated with 1% BSA in 0.1 M PBS (pH 7.4) for 2 hours at room temperature. After washing, the tubes are incubated for 2 hours at room temperature and overnight at 4? C. with Hu-SCID mice serum diluted in PBS containing BSA (1%) and TWEEN? (0.01%). After extensive washings, a HRP-labeled anti-human IgE Ab is added. After washing, substrate (3,3%5,5 tetramethylbenzidine (TMB) substrate reagent, Pharmingen, Becton Dickinson, Erembodegem, Belgium) is added to each well. Finally, reactions are stopped by adding 1M H.sub.2SO.sub.4 to the wells. The absorbances are read at 450 nm.
Histological Examination of the Lung
[0193] Lungs are excised at day 35 and fixed in paraformaldehyde and processed from paraffin embedding. Paraffin tissue sections are stained for the detection of human CD45.sup.+ cells after which human cells on the murine lung sections were quantified by histological scoring as described by Duez et al., 2000.
Analysis of Bronchoalveolar Lavage Fluid (BALF)
[0194] BALF is analyzed as described in the OVA allergen model.
[0195] Cell Cultures, Proliferation and Cytokine Assay: Single cell suspensions of spleen are prepared by passing the cells through 70 ?m filter cell strainers (Becton/Dickinson Labware). Erythrocytes are removed from the spleen cell suspensions by incubation with red cell lysis buffer. CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cells are enriched using human CD4.sup.+ T cell isolation kit (Miltenyi Biotec, Germany) or human CD4.sup.+CD25.sup.+ Regulatory T cell isolation kit (Miltenyi Biotec, Germany), respectively and MACS columns (midiMACS; Miltenyi Biotec).
[0196] Proliferation assays of bulk splenocyte, 2?10.sup.5 cells are cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium either alone or with purified Der p 1, and either with or without anti-IL-10 or anti-TGF-? neutralizing monoclonal antibodies. Der p 1 is added at concentrations ranging from 1 to 100 ?g/ml. The neutralizing antibodies are added at 1, 0.1 and 0.01 ?g/ml. For proliferation assays of human CD4.sup.? T cells and human CD4.sup.+CD25.sup.? T cell populations, 2?10.sup.5 cells CD4.sup.+ T cells or CD4.sup.+CD25.sup.? T cells are cultured in 96-well U-bottom plates with mitomycin treated human PBMC that are loaded with 1 mg/ml Der p 1 for 16 hours, acting as antigen presenting cells, at ratio's CD4.sup.+ T cell or CD4.sup.+CD25.sup.? T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 ?l complete medium either with or without neutralizing antibodies. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on a scintillation counter (Perkin Elmer).
[0197] For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays is collected after 24, 48 and 72 hours of culture and frozen at ?80? C. until cytokine analysis is performed. Cytokine production is quantified using the Human Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).
Results
[0198] LL-OVA and LL-Der p 1 Significantly Enhances the Tolerance-Inducing Capacity in OVA- and huSCID Mice Model For Asthma, Respectively.
[0199] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). Addition of LL-OVA/Derp1 significantly enhances the tolerance induction towards OVA/Derp1 as the allergen-specific proliferative response of the splenocytes is significantly reduced in the LL-OVA/Derp1 group in comparison to the control and free OVA/Derp1 groups.
LL-OVA/Derp1 Potentiates Oral Tolerance in Association with Reduced AHR, Eosinophilic Infiltration, Serum IgE Levels, and Lowered IL-13, IL-4 and IL-5 Cytokine Production in Response to the Allergen.
[0200] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). AHR, eosinophilic BALF infiltration, IgE titer as well as cytokine production in response to the antigens is determined as described above. AHR, eosinophilic BALF infiltration, IgE titer is strongly reduced, and IL-13, IL-4 and IL-5 significantly lowered in the LL-OVA/Derp1 group in comparison to the control and free OVA/Derp1 groups.
LL-OVA/Derp1 Enhances Oral Tolerance Via CD4.SUP.+ T Cells
[0201] To assess whether CD4 T cells mediate the induction of oral tolerance, the allergen-specific proliferative CD4 T-cell response is studied in the splenocytes and lymph nodes. Therefore, mice are orally fed as described above (experimental setting) and the allergen-specific CD4.sup.+ T cell proliferation is determined as described in Cell cultures, proliferation and cytokine assay. The allergen-specific CD4 T cell response in the LL-OVA/Derp1 group is significantly reduced in comparison to the control and free-OVA/Derp1 groups.
Antigen-Induced T Regulatory Cells Following LL-OVA Therapy can Transfer Protection from Asthma-Like Responses In Vivo
[0202] In order to test for active suppression of asthma-like responses in mice treated with the oral tolerance protocol, we adoptively transfer splenocytes from the different treated groups as described above (in vivo T regulatory activity assay). Compared with controls and free OVA groups, asthma-like responses are significantly reduced in the LL-OVA group, indicating activation of regulatory CD4.sup.+ T cells in our combination oral tolerance protocol.
Conclusion
[0203] Our data demonstrate that mucosal delivery of allergen secreting L. lactis is more potent than free allergen to induce allergen-specific immune tolerance via the induction of antigen-specific CD4.sup.+ regulatory T cells, even in the setting of established hypersensitivity.
Example E
[0204] Induction of Tolerance to BLG Food Allergen Following Oral Administration of L. lactis Secreting the Allergen
Introduction
[0205] Food allergy is a disease affecting approximately 2% to 5% of the population. In human beings, elevated IgE antibodies as well as the presence of IL-4-producing, antigen-specific T lymphocytes suggest a Th2-skewed mechanism. Here, we demonstrate that oral delivery of a food allergen by L. lactis suppresses allergen-specific immune responses via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Material and Methods to the Examples
Bacteria and Plasmids
[0206] The L. lactis strain MG1363 is used throughout this study. Bacteria are cultured in GM17 medium, i.e., M17 (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at ?20? C. in 50% glycerol in GM17. For intragastric inoculations, stock suspensions are diluted 200-fold in fresh GM17 and incubated at 30? C. They reach a saturation density of 2?10.sup.9 colony-forming units (CFU) per mL within 16 hours. Bacteria are harvested by centrifugation and concentrated ten-fold in BM9 medium. For treatment, each mouse receives 100 ?L of this suspension daily by intragastric catheter. Bovine ?-lactoglobulin cDNA is amplified and fused to the Usp45 secretion signal of the erythromycin resistant pT1 NX vector, downstream of the lactococcal P1 promoter. MG1363 strains transformed with plasmids carrying murine BLG is designated LL-BLG. LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serve as control.
Quantification of Bovine ?-Lactoglobulin (BLG)
[0207] BLG from LL-BLG is determined using an in house developed BLG-specific enzyme-linked immunosorbent assay (ELISA) and Western blot analysis.
Experimental Setting
[0208] The murine model of food allergy used to explore the protective effect of L. lactis is a mouse model of food-induced IgE-type response as described by Frossard et al. (J. Allergy Clin. Immunol. 113:958-964, 2004). Mice receive LL-BLG or an irrelevant antigen (OVA) as negative control. As a positive control for tolerance induction, mice receive a high dose of BLG in the drinking water that prevents the mice from anaphylaxis upon oral challenge with BLG.
[0209] In a prophylactic setting, the engineered L. lactis bacteria that produce BLG are administered orally to the mice using a gastric catheter, using different treatment intervals and doses. Subsequently, these recipient mice are orally challenged with purified BLG antigen, in the presence of cholera toxin. Control animals are exposed to L. lactis engineered with a control vector that does not express BLG (but OVA instead). Induction of tolerance is assessed by analysis of anaphylaxis after intragastric antigen challenge, by measuring BLG-specific IgG1, IgG2a and IgE titers in serum and feces, by determining the number of antibody secreting cells in spleen and PP, by analysis of the T cell proliferation and cytokine production in MLN, PP and spleen.
[0210] To evaluate whether the induction of immune tolerance towards BLG could be enhanced by L. lactis, mice are administered with LL-BLG or with 1 ?g free BLG.
Oral Sensitization to BLG
[0211] Four- to 5-week-old female C3H/HeOuJ mice (Charles River) are immunized at days 0, 7, 14, and 21 by intragastric gavage with 20 mg of BLG (Sigma) and 10 ?g of CTX, purchased from List Biological Laboratories in 0.2 moUL NaHCO.sub.3. The positive control group (tolerized mice) receive 0.8 mg/mL BLG in their drinking water ad libitum for 4 weeks. The total amount of protein given (22.4 mg) is similar to the total amount of BLG given to the sensitized mice. To demonstrate that the tolerization procedure also enduringly activate the peripheral and not only the mucosal immune system, a group of tolerized mice is injected twice with 80 ?g ip BLG adsorbed to 1 mg alum at days 28 and 42.
Antigen Challenge
[0212] On day 28, all mice are challenged by intragastric gavage with 100 mg BLG in 0.4 mL 0.2 mol NaHCO.sub.3. Anaphylaxis is observed and graded by using a reaction score (0, no reaction, to 3, severe reaction or death) described in detail elsewhere (Frosssard et al., 2001). The core body temperature is measured by infrared at the ear before challenge and 30 minutes after gavage. The animals are killed, and blood is collected by cardiac puncture into EDTA-containing tubes, and plasma is obtained for histamine measurement by commercial ELISA kit (Immunotech, Marseille, France).
Cell Cultures, Proliferation and Cytokine Assay
[0213] Single cell suspensions of spleen, mesenteric lymph nodes and PP are prepared as described by Frossard et al. (2004). CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cells are enriched using CD4.sup.+ T cell isolation kit (Miltenyi Biotec, Germany) or CD4.sup.+CD25.sup.+ Regulatory T cell isolation kit (Miltenyi Biotec, Germany), respectively and MACS columns (midiMACS; Miltenyi Biotec).
[0214] Proliferation assays of bulk splenocyte and LN populations, 2?10.sup.5 cells are cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium either alone or with purified BLG, and either with or without anti-IL-10 or anti-TGF-? neutralising monoclonal antibodies. BLG is added at concentrations ranging from 1 to 100 ?g/ml. The neutralizing antibodies are added at 1, 0.1 and 0.01 ?g/ml. For proliferation assays of CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cell populations, 2?10.sup.5 cells CD4.sup.+ T cells or CD4.sup.+CD25.sup.? T cells are cultured in 96-well U-bottom plates with mitomycin treated splenocytes that are loaded with 1 mg/ml BLG for 16 hours, acting as antigen presenting cells, at ratio's CD4.sup.+ T cell or CD4.sup.+CD25.sup.? T cell/APCs 1/1, 1/0.3, 1/0.1, 1/0.03, 1/0 in a total volume of 200 ?L complete medium either with or without neutralizing antibodies. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on a scintillation counter (Perkin Elmer).
[0215] For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays is collected after 24, 48 and 72 hours of culture and frozen at ?80? C. until cytokine analysis will be performed. Cytokine production is quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA).
In Vivo T Regulatory Activity Assay
[0216] In order to test for active suppression of antibody formation in mice, splenocytes, bead-purified CD4.sup.+ T cells, CD4.sup.+CD25.sup.? or CD4.sup.+CD25.sup.+ T cells isolated from the different experimental L. Lactis-treated groups are adoptively transferred to na?ve C3H/HeOuJ mice. Untreated mice are used as control. The number of transferred cells is 10.sup.7 for whole spleen cells, subpopulation-depleted spleen cells, or positively selected CD4.sup.+ cells and CD4.sup.+CD25 and CD4.sup.+CD25.sup.+ T cells. If Tregs are implicated, subsequent challenge of these mice with BLG antigen should prevent induction of humoral immune responses against BLG and anaphylaxis.
Enzyme-Linked Immunoassays for BLG-Specific Serum and Feces Antibodies
[0217] Sera are obtained from tail bleedings at day 0, 7, 14, 21 and 28. Feces are obtained at the same times and resuspended in PBS plus 1% FCS (Life technologies) supplemented with pepstatin 1:1000 (Fluka) at 0.1 mg/mL. The samples are mechanically disaggregated and vortexed for 2 minutes, followed by two centrifugations at 4? C. for 20 minutes at 14,000 rpm.
[0218] Sera and feces are assayed for BLG-specific IgE, IgG1, IgG2a and/or IgA antibody levels by a method adapted from Adel-Patient et al. (2000, J. Immunol. Methods). In brief, MaxiSorp microtiter plates (Nunc) are coated for 18 hours at room temperature with 250 ng/well streptavidin (Fluka), followed by 300 ?L of a solution of polyvinylpyroliddon K25 (Fluka) overnight. One microgram of biotinylated BLG is incubated for 3 hours, and diluted sera (1:6666 and 1:2222 for IgG1, 1:666 and 1:222 for IgG2a, 1:66 and 1:22 for IgE) or feces (1:3, 1:10, and 1:33) in PBS plus 10% horse serum is added in duplicates in presence of 0.5 ?g/mL goat anti-mouse IgA, rat anti-mouse IgG1 or anti-mouse IgG2a peroxidase-labeled antibodies (Southern Biotechnologies) for 2 hours. For IgE measurement, a monoclonal rat anti-mouse IgE Ab (clone R35-72, BD Pharmingen) followed by peroxidase-coupled anti-rat Ab (Caltag) is added. Optical density is measured at 490 nm. Results are expressed as arbitrary units, with pooled sera from BLG plus alum-immunized mice used as a reference serum.
Antigen-Specific Antibody Production is Measured by Means of ELISPOT
[0219] Peyer's patches are excised mechanically from the gut and incubated for 30 minutes in HBSS medium supplemented with 5 mmol EDTA (Life Technologies). Similarly, Peyer patches and mesenteric lymph nodes are gently crushed and filtered through a 70-m nylon filter. Spleen cells are preincubated for 5 minutes in Tris-buffered NH.sub.4Cl to remove red blood cells. Lymphoblasts are isolate on a Percoll 60%/66% gradient (Amersham).
[0220] For the measurement of BLG-specific IgG1, IgG2a and IgA antibodies, ELISPOT plates (Millipore) are coated with streptavidin overnight at 37? C., followed by addition of 1 g of biotinylated BLG for 3 hours. Lymphoblasts isolated on a Percoll 60%166% gradient from are resuspended at two different concentrations, 1 and 2?10.sup.6 in Iscove's modified Dulbecco's medium supplemented with penicillin, streptomycin, L-glutamine, gentamicin, polymixin B, and 5% FCS for 24 hours at 37? C., followed by overnight incubation at 4? C. with anti-IgA, anti-IgG1 and anti-IgG2a antibodies (Southern Biotechnology). Amino-ethyl-carbazole, 100 ?L/well, is added for 10 minutes, and the spots are automatically counted by using the KS ELISPOT 4.2.1 Software (Zeiss) and expressed as cell-forming units per 10.sup.6 cells (CFU).
LL-BLG Significantly Enhances the Tolerance-Inducing Capacity of BLG in Murine Model of Food Allergy
[0221] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). Addition of LL-BLG significantly enhances the tolerance induction towards BLG as the allergen-specific proliferative response of the splenocytes is significantly reduced in the LL-BLG group in comparison to the control and free-BLG groups.
[0222] LL-BLG potentiates oral tolerance in association with reduced BLG-specific antibody response and lowered IL-4 cytokine production in response to the allergen.
[0223] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). BLG-specific antibody response and cytokine production in response to the factor is determined as described above. BLG-specific antibodies levels and IL-4 are significantly lowered in the LL-BLG group in comparison to the control and free-BLG groups.
Results
LL-BLG Enhances Oral Tolerance Via CD4.SUP.+ T Cells
[0224] To assess whether CD4 T cells mediate the induction of oral tolerance, the allergen-specific proliferative CD4 T-cell response is studied in the splenocytes and lymph nodes. Therefore, mice are orally fed as described above (experimental setting) and the allergen-specific CD4.sup.+ T cell proliferation is determined as described in cell cultures, proliferation and cytokine assay. The allergen-specific CD4 T-cell response in the LL-BLG group is significantly reduced in comparison to the control and free-BLG groups.
Antigen-Induced T Regulatory Cells Following LL-BLG Therapy can Transfer Protection from Allergic-Like Responses In Vivo
[0225] In order to test for active suppression of allergic-like responses in mice treated with the oral tolerance protocol, we adoptively transfer splenocytes from the different treated groups as described above (In vivo T regulatory activity assay). Compared with controls and free-BLG groups, allergic-like responses are significantly reduced in the LL-BLG group, indicating activation of regulatory CD4.sup.+ T cells in our combination oral tolerance protocol.
Conclusion
[0226] Our data demonstrate that mucosal delivery of allergen secreting L. lactis is more potent than free allergen to induce allergen-specific immune tolerance via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Example F
[0227] Induction of Tolerance to Insulin Following Oral Administration of L. lactis Secreting the Auto-Antigen
Introduction
[0228] Autoimmunity is characterized by spontaneous inflammatory tissue damage and by impaired physiological function resulting from loss of tolerance to self-antigen. It is associated with a partially overactive immune system, which is characterized by an excess of T helper (Th) cells. Predisposing factors, such as susceptibility genes and environmental factors are difficult to influence, therefore recent efforts to develop immunotherapies are focused on re-establishing the functional balance between pathogenic effector cells and immunoregulatory T cells by depleting the former and/or enhancing the latter. Autoimmune destruction of pancreatic islet beta cells is the major cause of Type 1 diabetes mellitus (T1D). This destruction is associated with cellular and humoral immune responses to several beta cell auto-antigens, both of which can precede the clinical onset of disease.
[0229] Here, we demonstrate that oral delivery of an auto-antigen delivering L. lactis suppresses diabetic-specific immune responses via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Material and Methods
Bacteria and Plasmids
[0230] The L. lactis strain MG1363 is used throughout this study. Bacteria are cultured in GM17 medium, i.e., M17 (Difco Laboratories, Detroit, Mich.) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at ?20? C. in 50% glycerol in GM17. For intragastric inoculations, stock suspensions are diluted 200-fold in fresh GM17 and incubated at 30? C. They reach a saturation density of 2?10.sup.9 colony-forming units (CFU) per mL within 16 hours. Bacteria are harvested by centrifugation and concentrated 10-fold in BM9 medium. For treatment, each mouse receives 100 ?L of this suspension daily by intragastric catheter. DNA sequence with optimal L. lactis codon usage encoding the human proinsulin II B24-C36 peptide (hpllp), porcine insulin and immunodominant-peptide InsB.sub.9-23 (B9-23 is essentially the same across many species human, rat and mouse) are synthesized, amplified and fused to the Usp45 secretion signal of the erythromycin resistant pT1NX vector, downstream of the lactococcal P1 promoter.
[0231] MG1363 strains transformed with plasmids carrying murine hpllp, Insulin, InsB.sub.9-23 are designated LL-hpllp, LL-insulin, LL-InsB.sub.9-23. LL-pT1NX, which is MG1363 containing the empty vector pT1NX, served as control. Expression of these proteins is determined using antigen-specific ELISA and Western blot analysis.
Mice
[0232] Non-obese female and male diabetic (NOD) mice and NOD-severe combined immunodeficient (SCID) (Balb/c background) mice are purchased from the Jackson laboratory. Balb/c wild-type (WT) mice are purchased from Charles River Italy. Mice are maintained in a specific pathogen-free central animal facility. Mice are treated and used in agreement with the institutional guidelines.
Experimental Setting
[0233] In a prophylactic setting, the LL-hpllp, LL-insulin, LL-InsB.sub.9-23 are administered orally to NOD mice starting from day 21 of age (weaning), and using the optimal feeding regime or until 100 days of age (when most mice develop diabetes). In addition, LL-pT1NX is administered orally as a negative control. For the positive (tolerizing) control group, 3-week-old NOD mice are treated orally with 0.8 mg human insulin/hpllp/InsB.sub.9-23 for 3 times a week for 2 or 4 weeks. Development of diabetes is determined by continuous monitoring of urine glucose levels three times a week and in case of glucosuria monitoring of blood glucose levels. Pancreases are collected at 12-23 weeks and at the end of experiment (35 weeks), and serial sections are stained with hematoxylin/eosin to score mononuclear cell infiltration or by immunohistochemistry to analyze T cell infiltration.
[0234] In a therapeutic setting the LL-hpllp, LL-insulin, LL-InsB.sub.9-23 are administered orally to diabetic NOD females showing stable glycosuria and hyperglycemia (12-23 weeks). In addition, LL-pT1 NX is administered orally as a negative control. For the positive (tolerizing) control group, diabetic NOD mice are treated as described in Bresson et al., 2006. Complete remission is defined as the disappearance of glycosuria and a return to normal glycemia.
[0235] In a syngeneic islet transplantation setting, female NOD mice with recent-onset diabetes are treated orally for 3 weeks with LL-hpllp, LL-insulin, LL-InsB.sub.9-23, or with LL-pT1 NX as a negative control. After 3 weeks, 500 freshly isolated pancreatic islets from non-diabetic NOD mice are transplanted to diabetic NOD mice. Blood glucose is then monitored 3 times weekly until diabetes recurrence or until 15 weeks after grafting. Animals with 2 consecutive glucose levels 250 mg/dL are considered diabetic and will be subsequently killed for serum collection and histological analysis of the graft.
[0236] The precise mechanisms of tolerance induction are analyzed in vitro, in vivo after re-challenging the NOD mice with specific auto-antigens and by adoptive T-cell transfer into NOD-SCID mice.
Detection of Diabetes
[0237] Glucose monitoring: urine glucose is measured by using Diastix (Miles) and is confirmed by blood glucose measurements with the blood glucose monitoring system OneTouch Ultra (LifeScan Inc.). Diabetes is defined as two consecutive blood glucose values superior to 250 mg/dl.
[0238] Insulitis: Mice are killed by CO.sub.2 asphyxiation and the pancreas is fixed in 10% formalin overnight, embedded in paraffin, and serial 5 ?m sections are stained with hematoxylin and eosin. The insulitis score (mean?SD) is determined by microscopically grading the degree of cellular infiltration in 10-15 islets/mouse as follows: 0, no visible sign of islet infiltration; 1, peri-islet infiltration; 2, <50% infiltration; 3, >50% infiltration.
[0239] Islet isolation and transplantation: Islets of insulitis- and diabetes-free 14- to 21-day old donor NOD mice are isolated after asceptic removal by digesting the pancreatic glands with collagenase in Hanks' balanced salt solution during vigorous shaking. Islet isolation is carried out by direct hand-picking under a stereo-microscope. Diabetic recipient NOD mice were anaesthetized by intraperitoneal injection of avertin (0.02 ml/g BWT), the left kidney was exposed via lumbar incision and 500 freshly isolated islets were given under the renal capsule.
Immunohistochemistry
[0240] To detect insulin, CD4 and CD8 expression in pancreatic R cells, primary Abs (guinea pig anti-swine insulin from Dako (dilution 1:300), anti-CD4 RM4.5 and anti-CD8a IHC from BD Biosciences (dilution 1:50)) are applied to frozen tissue sections as described in Christen et al., 2004.
In Vitro Proliferation Assay
[0241] Single cell suspensions of spleen, mesenteric LN (MLNs) and PLNs are prepared. Proliferation assays of total splenocyte populations, 2?10.sup.5 cells are cultured in 96-well U-bottom plates in a total volume of 200 ?l complete medium either alone or with graded concentrations (1-100 ?g/ml) of purified human insulin or peptides specific for CD4 T cells (InsB.sub.9-23, H-2.sup.d or g restricted) or for CD8 T cells (InsB.sub.15-23, K.sup.d restricted) (Sigma), and either with or without anti-IL-10 or anti-TGF-? neutralizing monoclonal antibodies. The neutralizing antibodies are added at 1, 0.1 and 0.01 ?g/ml. For proliferation assays of total CD3.sup.+ T cells, CD8.sup.+ T cells, CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cell populations, 0.2?10.sup.5 cells T cells are cultured in 96-well U-bottom plates with 1?10.sup.5 irradiated splenocytes from WT Balb/c mice loaded with insulin or GAD65 or peptides specific for CD4.sup.+ or CD8.sup.+ T cells, in a total volume of 200 ?l complete medium either with or without neutralizing antibodies. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 16-18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation is measured on a scintillation counter (Perkin Elmer). T cells are purified from PLNs or spleens by negative selection through magnetic bead separation using CD3.sup.+, CD4.sup.+ or CD8.sup.+ isolation kit (MACS; Milteny Biotec, Auburn, Calif.). CD4.sup.+ T cells are used as total cells or further separated into CD25.sup.+ and CD25.sup.? by MACS using CD25.sup.+ isolation kit (Milteny Biotec). The purity (>90%) of the cell populations is determined by flow cytometric analysis.
[0242] For cytokine measurements, supernatants of the cell cultures used in the different proliferation assays (antigen-specific stimulation), described above, are collected after 72 hours of culture and frozen at ?80? C. until cytokine analysis is performed. Cytokine production is quantified using the Mouse Inflammation Cytometric Bead Assay (BD Biosciences, Mountain View, Calif., USA). Purified CD3.sup.+ T cells, CD4.sup.+ T or CD8.sup.+ T cells are cultured and stimulated in vitro non-specifically with an anti-CD3/anti-CD28 mixture (1 ?g/ml each) for 24 hours or they remain unstimulated as control. The supernatants are harvested, and analyzed for IL-10, IL-4, IL-5 and IFN? production using BD? Cytometric Bead Array flex set on a BD FACSArray Bioanalyzer using the FCAP array software (BD Biosciences). Capture ELISA experiments are used to determine TGF-?1 using the Quantikine kit (R&D Systems).
In Vitro T Cell Proliferation Inhibition Assay
[0243] 2?10.sup.4 purified total splenic CD4.sup.+CD25.sup.? T cells isolated from recently diabetic female NOD (8-12 weeks) are co-cultured with varying numbers of CD8.sup.+ T cells, CD4.sup.+ T cells and CD4.sup.+CD25.sup.? T cell populations isolated from the spleen, MLN or PLNs from the different experimental groups in the presence of 2?10.sup.4 T-cell-depleted irradiated insuline- or peptides-loaded splenocytes from WT Balb/c mice. After 72 hours at 37? C. in a 5% CO.sub.2 humidified incubator, proliferation is assessed by addition of 1 ?Ci/well [.sup.3H]-thymidin. DNA-bound radioactivity is harvested 16-18 hours later onto glass fiber filter mats (Perkin Elmer, Boston, USA) and thymidine-incorporation measured on a scintillation counter (Perkin Elmer).
In Vitro Cytotoxicity Assay
[0244] Lymphoblast targets used are Con A-activated splenocytes from BALB/c mice. A total of 10.sup.6 target cells are labeled with 100 ?Ci of .sup.5lCr (Amersham International, Buckinghamshire, U.K) for 90 minutes at 37? C., washed three times and then incubated with 1 ?g/ml peptide (InsB.sub.15-23 or an irrelevant peptide) at 37? C. for 1 hour. Target cells are washed two times and seeded at 10.sup.4 cells per well. CD8.sup.+ T cells, isolated from spleen, MLNs and PLNs are added to each well, in triplicate, at various effector:target (E:T) ratios. The plates are centrifuged at 500 rpm for 2 minutes, and incubated at 37? C. for 4 hours. After incubation, supernatants are collected for determination of .sup.5lCr release (% lysis=100?(test cpm-spontaneous cpm)/(total cpm-spontaneous cpm)). For the indirect killing assay, CD8.sup.+ T cells are incubated with 5 ?g/ml anti-CD3 antibody (clone 145-2C11, Pharmingen) prior to incubation with effectors.
Adoptive Transfer of Diabetes
[0245] NOD-SCID mice at 8-10 wk are injected i.v. with 2?10.sup.7 or i.p. with 5?10.sup.6 splenocytes isolated from diabetic female NOD mice (6 weeks, 12 weeks and 18 weeks) combined with or without graded numbers of bead-purified CD3.sup.+ T cells, CD8.sup.+ T cells, CD4.sup.+ T cells, CD4.sup.+CD25.sup.? or CD4.sup.+CD25.sup.+ T cells isolated from the different experimental L. Lactis-treated groups. Untreated mice are used as control. Development of diabetes is determined by continuous monitoring of blood glucose levels three times a week.
Results
[0246] LL-Hpllp, LL-Insulin, LL-InsB.sub.9-23 Delays Diabetes Recurrence after Syngeneic Islet Transplantation
[0247] To assess whether LL-hpllp, LL-Insulin and LL-InsB(9-23) induce oral tolerance, diabetes recurrence after syngeneic islet transplantation is studied. Therefore, mice are orally fed as described above (experimental setting) and pancreatic islets are transplanted as described (Islet isolation and transplantation). Diabetes recurrence is delayed in the LL-hpllp/insulin/InsB.sub.9-23 group in comparison to the control.
LL-Hpllp, LL-Insulin, or LL-InsB.sub.9-23 Significantly Enhances the Tolerance-Inducing Capacity of Freehpllp, Insulin, or InsB.sub.9-23 in the Non-Obese Diabetic Mouse
[0248] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). Addition of LL-hpllp, LL-insulin, LL-InsB.sub.9-23 significantly enhances the tolerance induction towards auto-antigen as the auto-antigen-specific proliferative response of the splenocytes is significantly reduced in the LL-hpllp/insulin/InsB.sub.9-23 group in comparison to the control and free hpllp/insulin/InsB.sub.9-23 groups.
LL-Hpllp, LL-Insulin, or LL-InsB.sub.9-23 Potentiates Oral Tolerance in Association with Reduced Insulitis, Deceased Rate of Beta Cell Destruction, and Increased IL-10 Production by Splenocytes
[0249] To study the induction of oral tolerance, mice are orally fed as described above (experimental setting). The presence of insulitis, the rate of beta-cell destruction and cytokine production in response to the auto-antigen is determined as described above. Histological analysis shows a significant lower degree of insulitis and beta cell destruction and increased IL-10 production in the LL-hpllp/insulin/InsB.sub.9-23 group in comparison to the control and free-hpllp/insulin/InsB.sub.9-23 groups.
LL-hpllp, LL-Insulin, LL-InsB.sub.9-23 Enhances Oral Tolerance Via CD4.sup.+ T Cells
[0250] To assess whether CD4 T cells mediate the induction of oral tolerance, the auto-antigen-specific proliferative CD4 T-cell response is studied in the splenocytes and lymph nodes. Therefore, mice are orally fed as described above (experimental setting) and the auto-antigen-specific CD4.sup.+ T cell proliferation is determined as described (in vitro proliferation assay). The auto-antigen-specific CD4 T cell response in the LL-hpllp/insulin/InsB.sub.9-23 group in comparison to the control and free-hpllp/insulin/InsB.sub.9-23 groups.
Example F5
[0251] Autoaggressive CD8.sup.+ Responses are Suppressed in NOD Mice Following LL-InsB.sub.9-23 Therapy
[0252] To examine whether our combination approach induce suppressive CD4.sup.+ T cells that are capable of modulating diabetes by bystander suppressive mechanisms, we analyze the effect on CD8.sup.+ autoaggresive T cells. The percentage and/or activity of antigen-specific autoaggressive CD8.sup.+ cells is strongly reduced after LL-InsB.sub.9-23 therapy.
Antigen-Induced T Regulatory Cells Following LL-InsB.sub.9-23 Therapy can Transfer Protection from Autoimmune-Like Responses In Vivo
[0253] In order to test for active suppression of diabetic-like responses in mice treated with the oral tolerance protocol, we adoptively transfer splenocytes from the different treated groups as described above (adoptive transfer of diabetes). Compared with controls and free-InsB.sub.9-23 group, diabetic-like responses are significantly reduced in the LL-InsB.sub.9-23 group, indicating activation of regulatory CD4.sup.+ T cells in our combination oral tolerance protocol.
Conclusion
[0254] We demonstrate that oral delivery of an auto-antigen delivering L. lactis suppresses diabetic-specific immune responses via the induction of antigen-specific CD4.sup.+ regulatory T cells.
Discussion
[0255] On the whole, the above presented data indicates that oral supplementation of a genetically modified L. lactis secreting antigens can decrease systemic inflammation induced by that antigen, even in a sensitized subject. Advantageously, the Lactococcus-mediated suppression often appears more potent than after mucosal administration of free antigen. Potentially, the suppression may be mediated by the induction of Foxp3.sup.+ regulatory T cells,
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