Treatment of immune disease by mucosal delivery of antigens using genetically modified lactobacillus

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DESCRIPTION

Treatment of immune disease by administration through the antigen mucosa using genetically modified Lactobacillus

Description

The present disclosure relates to the treatment of autoimmune and allergic diseases by the administration through the mucosa by microorganisms, in particular Lactococcus lactis, of secreted immunodominant antigens.

Field of the Invention

The immune system has the task of distinguishing between the own and the non-self. The mucous immune system, present along the respiratory, gastrointestinal and genitourinary pathways, has the additional burden of coexisting with an abundance of bacteria and harmless antigens such as food, airborne antigens or commensally bacterial flora. A key feature of the mucous immune system is its ability to remain tolerant to these antigens while effectively retaining the ability to repel pathogens. The introduction of systemically antigen, both by injection and injury, leads to local infiltration of inflammatory cells and production of specific immunoglobulins. In contrast, antigens introduced into mucosal surfaces, such as the gastrointestinal and genitourinary pathways, cause active inhibition of the immune response to those systemically antigens. The specific induction of these responses regulated by the administration of antigen through the gastrointestinal tract is known as oral tolerance. Oral administration of antigen can lead to lack of systemic reactivity and is an attractive alternative to immunosuppressive medical inventions that have no desirable side effects (such as steroids). The invention is based in particular on the field of low dose tolerance, obtained by continued exposure to low dose of antigen. Mucosal tolerance inductions have been proposed as a treatment strategy against autoimmune, allergic and inflammatory diseases.

State of matter

The following discussion of the background of the invention is provided simply to assist the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

Autoimmune, allergic and inflammatory diseases impose a tremendous burden on the patient and society, resulting in decreased quality of life and huge costs. In addition, there is no adequate treatment without acceptable side effects or that is socially appropriate. Current treatments for autoimmune disease are largely palliative, usually immunosuppressive, or anti-inflammatory. Nonimmune therapies, such as hormone replacement in Hashimoto's thyroiditis or type 1 DM, treat outcomes of the autoaggressive response. Treatment with steroids or NSAIDs limits the inflammatory symptoms of many diseases. IVIG is used for CIDP and GBS. It has been shown that more specific immunomodulatory therapies, such as TNF etanercept antagonists, are useful in the treatment of RA. However, these immunotherapies may be associated with a high risk of adverse effects, such as high susceptibility to infection. Celiac disease, which can be characterized by a small chronic intestinal inflammation, can only be treated effectively by a socially restrictive diet that requires lifelong withdrawal of foods containing wheat, rye or barley. Although a strict gluten-free diet can lead to healing of the intestine, gluten intolerance is permanent.

Celiac disease, also known as celiac disease or gluten-sensitive enteropathy, is a chronic inflammatory disease that develops from an immune response to specific dietary grains that contain gluten. The diagnosis can be made based on the classic presentation of diarrhea, fatty stools, meteorism and abdominal pain, weight loss, metabolic bone diseases, anemia, in addition to the presence of serum antibodies with specificity for gliadin and tissue transglutaminase (tTG) (also called antiendomisiales). The mucosal lesion is located in the proximal part of the small intestine, and is characterized by villus atrophy, cryptic cell hyperplasia and lymphocytic infiltration of the epithelium and the lamina propria, which release proinflammatory cytokines such as IL-2 and IFN- , in response to gliadina. Celiac disease can be considered the most common food-sensitive enteropathy in humans, and it can appear at any time in a person's life. The prevalence is in the range of 1: 100 to 1: 300 in western, Arab and Indian populations. Apart from gluten, the disease can be triggered for the first time after surgery, viral infection, severe emotional stress, pregnancy or childbirth. WO 2007/063075 describes the induction of tolerance to -gliadin after co-administration of Lactococcus lactis that secretes -gliadin with IL-10.

Therefore, induction of antigen-specific oral tolerance would be an unattractive therapeutic approach. Although oral tolerance was first described in 1911, it was not until after the 1970s that researchers began addressing the mechanisms involved (Mayer and Shao, 2004a). Several mechanisms for the development of oral tolerance have been proposed, ranging from the deletion of antispecific T lymphocytes, through deviation

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immune and induction of anergy, to suppression by Treg (Mucida et al., 2005). Most researchers agree that there are two different ways of obtaining oral tolerance, high dose tolerance, obtained after a single high dose of antigen, which is based on anergy and / or deletion (Friedman and Weiner, 1994), and low-dose tolerance, obtained by repeated exposure to low-dose antigen, mediated by active suppression of immune responses by CD4 + T lymphocytes, which include regulatory T lymphocytes producing Foxp3 +, IL-10 and / or TGF-. And, more importantly, it has been shown that regulatory T lymphocytes induced by mucosal tolerance mediate circumstantial suppression, a process by which specific regulatory cells for one protein suppress the response of effector cells near another protein. Circumstantial suppression is another important characteristic of antigen-induced suppression because the set of antigens that induces organ-specific autoimmunity is very unknown, and overcomes the phenomenon of epitope dissemination. The spread of epitopes is a complication of autoimmune and allergic diseases, so the onset of the immune response is prolonged over time to include responses to other antigens.

Targeted and more efficient administration of molecules for therapeutic and prophylactic applications is a priority for the pharmaceutical industry. Effective strategies should reduce the required dose, increase safety and improve efficacy by focusing the molecules at the desired site of action. The mucous pathways of drug and vaccine administration offer several logistical and biological advantages compared to injection. Oral administration is particularly attractive as a result of ease of administration. However, gastrointestinal degradation and low levels of absorption generally make this route of administration of peptide and protein drugs ineffective. Alternative mucosal pathways such as nasal, rectal, pulmonary and ocular pathways are also being investigated.

Thus, there is still a problem in the matter of effectively inducing antigen tolerance.

Summary of the Invention

Surprisingly, the present inventors found that an immunodominant antigen that participates in the induction of celiac disease that is released, and preferably is continuously present, in a mucous site of a patient induces a specific immunotolerance for antigen. In particular, when a microorganism such as preferably Lactococcus lactis (LL), which constitutively expresses and secretes an immunodominant antigen that participates in the induction of celiac disease, is released daily at a mucous site, an immune tolerance specific for antigen was induced. The present inventors observed that the administration through the mucosa of such an antigen by a microorganism of L. lactis gives a significantly better suppression of the antigen-specific immune response compared to the single administration through the mucosa of said antigen or said microorganism.

The present inventors demonstrate that the invention can induce oral tolerance much more efficiently than with monotherapy with antigen or control L. lactis alone. In vivo activation of antigen-specific regulatory T lymphocytes was strongly potentiated. Specifically, mucosal administration of a gliadin-derived peptide, which is immunodominant for T-lymphocyte responses mediated by DQ8 by L. Genetically modified lactis induces the suppression of T-cell responses limited by local and systemic DQ8. The treatment resulted in a specific decrease in antigen of the proliferative capacity of splenocytes and inguinal lymph node cells, which was critically dependent on the production of IL-10 and TGF- and was associated with a significant induction of Foxp3 + regulatory T lymphocytes. . Because this approach of bacteria that release antigen has the ability to enhance oral tolerance even in the established hyperreactivity environment, it is applicable to the treatment of celiac disease. The efficacy of the invention was demonstrated in mouse models of autoimmune and allergic disease, as well as in the context of immune inactivation of therapeutic agents.

In a first aspect, the invention relates to a microorganism constitutively secreting an antigen that participates in the induction of celiac disease for use in administration through the mucosa to treat celiac disease, with the proviso that when the The microorganism constitutively secretes said antigen is Lactococcus lactis MG1363 that secretes the alpha-gliadin protein, gliadin peptide HLA-DQ8 or gliadin peptide HLA-DQ8 in deamidated form, then said microorganism is not to be administered orally in combination with Lactococcus lactis MG1363 that produces murine IL-10.

In one embodiment, said microorganism is:

<dl><dt>-</dt><dd>lactic acid or yeast bacteria, more preferably it is Lactococcus lactis (LL); </dd></dl>

<dl><dt>-</dt><dd>it is administered daily; </dd></dl>

<dl><dt>-</dt><dd>it is administered for at least 1 week, more preferably for at least 1 month or 1 year; </dd></dl>

<dl><dt>-</dt><dd>it is administered at least once a day, preferably twice a day; </dd></dl>

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<dl><dt>-</dt><dd>it is given at a dose of at least 10 femtograms at 100 mg per day, such as 100 fg to 10 mg per day, or 1 pg to 1 mg per day, or 10 pg to 100 µg per day, or 100 pg to 10 µg per day, or 1 ng to 1 µg per day, or 10 ng to 100 mg per day; me</dd></dl>

<dl><dt>-</dt><dd>It is administered by spray, capsule, spray, lozenges, bolus, tablet, sachets, liquid, suspension, emulsion or troches. </dd></dl>

In another embodiment, said administration through the mucosa is chosen from the group consisting of rectal administration, oral administration, pulmonary administration, ocular administration, nasal administration, vaginal administration and oral administration.

In another embodiment, said microorganism or composition is formulated as a medicament, medical food or nutritional product.

In another aspect, the invention relates to a pharmaceutical composition consisting of a microorganism and a pharmaceutically acceptable carrier, wherein said microorganism constitutively secretes an antigen, which participates in the induction of celiac disease.

In one embodiment, said microorganism is lactic acid or yeast bacteria, more preferably it is Lactococcus lactis (LL).

In another embodiment, said microorganism is present in a dose of at least 10 femtograms at 100 mg, such as 100 fg to 10 mg, or 1 pg to 1 mg, or 10 pg to 100 µg, or 100 pg to 10 µg, or 1 ng to 1 µg, or 10 ng to 100 mg.

In another embodiment, said pharmaceutical composition is formulated in sprays, capsules, aerosols, lozenges, bolus, tablets, sachets, liquids, suspensions, emulsions or troches.

In yet another embodiment, the invention relates to the pharmaceutical composition as described herein for use in administration through the mucosa to treat celiac disease.

In another aspect, the invention relates to a medicament, nutritional product or medical food for treating, preventing and / or alleviating celiac disease, which comprises at least one microorganism, wherein said microorganism constitutively secretes an antigen involved in induction. of celiac disease, with the proviso that when the microorganism constitutively secreting said antigen is Lactococcus lactis MG1363 that secretes the alphagliadin protein, HLA-DQ8 gliadin peptide or HLA-DQ8 gliadin peptide in deamidated form, then said microorganism is not to be administered orally in combination with Lactococcus lactis MG1363 that produces murine IL10.

In one embodiment, said microorganism is lactic acid or yeast bacteria, more preferably it is Lactococcus lactis (LL).

Detailed description of the invention

Throughout this disclosure, various publications, patents and descriptive reports of published patents are cited by an identifying quotation. The disclosures of these publications, patents and patent specification published are incorporated herein by reference in the present disclosure to more fully describe the state of the art to which the present invention relates.

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, molecular biology (including recombinant techniques), cell biology, biochemistry and immunology, which are within the skill of the subject. Such techniques are fully explained in the literature, such as, "Molecular Cloning: A Laboratory Manual" second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis"

<dl><dt>(M. </dt><dd>J. Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); the series “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (DM Weir &amp; CC Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (JM Miller &amp; MP Calos, eds., 1987); "Current Protocols in Molecular Biology" (FM Ausubel et al., Eds., 1987, and bulletins), "Polymerase Chain Reaction" (Mullis et al., Eds., 1994); and "Current Protocols in Immunology" (J.</dd></dl>

<dl><dt>AND.</dt><dd> Coligan et al., Eds., 1991). </dd></dl>

Definitions

As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular forms "a", "a", "the" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of

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cells, which include mixtures thereof. Similarly, the use of "a compound" for the treatment or preparation of medicaments as described herein contemplates using one or more compounds of the present invention for such treatment or preparation, unless the context clearly dictates otherwise.

As used herein, the term "comprising" is intended to mean that the compositions and procedures include the elements cited, but do not exclude others. "Which consists essentially of" when used to define compositions and procedures should mean that it excludes other elements of any essential significance for the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification process and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting of" should mean that it excludes more than trace elements of other components and substantial procedural steps to administer the compositions of the present invention. The embodiments defined by each of these transition terms are within the scope of the present invention.

Invention

The present inventors demonstrate that administration through the mucosa of an immune dominant antigen that participates in the induction of celiac disease secreted by a microorganism such as preferably L. lactis induces suppression of local and systemic T lymphocyte responses. The treatment produced a specific antigen decrease in the proliferative capacity of splenocytes and inguinal lymph node cells, which was critically dependent on the production of IL-10 and TGF- and was associated with a significant induction of Foxp3 + regulatory lymphocytes. This approach of bacteria that release antigen has the ability to enhance oral tolerance even in the established hyperreactivity environment. Thus, it is applicable for the treatment of celiac disease. The efficacy of the invention was demonstrated in mouse models of autoimmune and allergic disease, as well as in the context of immune inactivation of therapeutic agents.

In this document a method of inducing immune tolerance to an antigen is described, which comprises administration through the mucosa of said antigen by a microorganism.

The invention relates to the use of a microorganism, preferably a non-pathogenic microorganism, more preferably lactic acid or yeast bacteria, even more preferably Lactococcus lactis constitutively secreting an antigen that participates in the induction of celiac disease, for the preparation of a medication, medical food or nutritional product for administration through the mucosa to treat celiac disease in a patient, with the proviso that when the microorganism constitutively secreting said antigen is Lactococcus lactis MG1363 that secretes the alpha-gliadin protein, HLA-DQ8 gliadin peptide or HLA-DQ8 gliadin peptide in deamidated form, then said microorganism is not for administration by orally in combination with Lactococcus lactis MG1363 that produces murine IL-10, wherein said antigen is preferably continuously present in said patient.

Preferably, said antigen is released by a microorganism that expresses antigen. Preferably, said antigen is released by a microorganism that secretes antigen. Also described herein is an antigen that is expressed on the surface of said microorganism that expresses antigen, or said antigen is released after digestion.

Preferably, the present invention relates to the use of a microorganism that expresses antigen for the preparation of a medicament for administration through the mucosa to induce immune tolerance.

Preferably, said immune tolerance is induced in a patient. Said patient is preferably an animal. Said animal is preferably a mammal, and is preferably chosen from the group consisting of mouse, rat, pig, cow, sheep, horses and human being. Preferably, said mammal is a human being. Preferably, said immune tolerance is mucosal tolerance.

Mucous membrane

Mucosa as used herein can be any mucosa such as oral mucosa, rectal mucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa, pulmonary mucosa and nasal mucosa. Administration through the mucosa as used throughout the application encompasses the administration to the mucosa. Administration through the oral mucosa includes oral, sublingual and gingival routes of administration. Accordingly, the present invention relates to a method in which said administration through the mucosa is chosen from the group consisting of rectal administration, oral administration, pulmonary administration, ocular administration, nasal administration, vaginal administration and oral administration. Preferably, said administration through the mucosa is oral administration and said tolerance is oral tolerance.

Mucosal tolerance as used herein throughout the application is the inhibition of specific immune reactivity to an antigen in an animal (including humans), after said animal has been exposed to said antigen by the mucosal route. Preferably, said mucosal tolerance is systemic tolerance. Subsequent exposure

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of the antigen can be each exposure known to the person skilled in the art, such as exposure by parenteral injection, by administration through the mucosa or by endogenous production such as in the case of auto-antigens. Oral tolerance is the inhibition of specific immune reactivity to an antigen in an animal (including humans), after said animal has been exposed to said antigen by the oral route. Oral tolerance at low dose is oral tolerance induced by low doses of antigens, and is characterized by active immune suppression, mediated by regulatory T lymphocytes sensitive to cyclophosphamide that can transfer tolerance to guests without prior treatment. Oral tolerance at high dose is the oral tolerance induced by high doses of antigens, is insensitive to treatment with cyclophosphamide and develops at induction of hyporeactivity of T lymphocytes by anergy and / or deletion of antigen-specific T lymphocytes. The difference in cyclophosphamide sensitivity can be used to distinguish between low dose and high dose tolerance (Strobel et al., 1983). Preferably, said oral tolerance is low dose oral tolerance as described by Mayer and Shao (2004b).

Thus, the present invention relates to a use as described herein, wherein said induction of immune tolerance is at least 1.5, preferably 2, more preferably 3 times or more with respect to before said induction. Alternatively, said antigen is tolerated at least 1.5, 2, 3 times or more with respect to before said induction. The induction of immune tolerance can be measured by methods known in the art. Preferably, said induction of immune tolerance can be measured by modulating a level of cytokines in said animal. As such, the modulation may be an increase in a cytokine level, for example, said increase in a cytokine level is at least 1.5, 2, 3 times or more with respect to before said induction, for example, IL -10 or TGF-. Alternatively, said modulation is a decrease in the level of a particular cytokine level, for example, said decrease in the cytokine level is at least 1.5, 2, 3 times or more with respect to before said induction, for example, IL -12, IL-17 and IFN-. The cytokines that are modulated can be chosen from any relevant cytokine, preferably said cytokines are chosen from the group consisting of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IL-23, TNF-, IFN-, IFN-, MCP-1, TGF-, RANK-L and Flt3L.

Constructions, administration and integration

In the present invention, the microorganism releases the antigen at the intended site, that is, the mucosa. The microorganism expresses said antigen, after which the antigen is constitutively secreted. Therefore, in a preferred embodiment, the microorganism, such as L. lactis, comprises an expression vector that can express the heterologous antigen, for example, the antigen used to induce immune tolerance, secreted under conditions present in the intended mucosa, for example, such as in the gastrointestinal tract. The microorganism, for example, L. lactis, may comprise expression vectors that can express the heterologous antigen secreted to a degree sufficient to induce immune tolerance. A degree of expression is expected as high as possible without damaging the viability of the cell or host to be treated. With greater expression, less frequent and lower doses may be required for tolerance purposes. Naturally, the dosage schedule will not only depend on the amount of antigen, but also on the type of antigen and the presence or absence of other immunogenicity stimulating or suppressing factors in the composition.

Normally, the expression system will comprise a genetic construct comprising at least one nucleotide sequence encoding the desired antigen, preferably operably linked to a promoter that can direct the expression of the sequence in the host microorganism. Suitably, the antigen to be expressed can be encoded by a nucleic acid sequence that is adapted to the preferred codon use of the host. The construct may additionally contain (all) other suitable element (s), including enhancers, transcription initiation sequences, signal sequences, indicator genes, transcription termination sequences, etc. ., operable in the selected host, as is known to the person skilled in the art. The construction is preferably in a form suitable for the transformation of the host and / or in a form that can be stably maintained in the host, such as a vector, plasmid or mini-chromosome. Suitable vectors comprising nucleic acid may be chosen or constructed for introduction into microorganisms, for example, bacteria, which contain appropriate regulatory sequences that include promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral, for example, phage, or phagemid, as appropriate. For more details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for nucleic acid manipulation, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Short Protocols in Molecular Biology, second edition, Ausubel et al. eds., John Wiley &amp; Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. They are incorporated herein by reference. In a preferred embodiment, the coding sequences for the biologically active polypeptide and the antigen are contained in an operon, that is, a nucleic acid construct for multi-cistronic expression. In an operon, the transcription of a promoter produces an mRNA comprising more than one coding sequence, each with its own ribosome binding site properly positioned in the 5 'direction. Thus, more than one polypeptide can be translated from a single mRNA. The use of an operon allows the expression of the biologically active polypeptide and the antigen to be coordinated. More preferably, a food grade construction is used.

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In one embodiment, the present invention relates to stably transfected microorganisms, that is, microorganisms in which the gene encoding the antigen has been integrated into the host genome. Techniques for establishing stably transfected microorganisms are known in the art. For example, the gene of interest can be cloned into the host genome by homologous recombination. Preferably, an essential host gene is affected by the homologous recombination event, such as deletion of the gene, one or more amino acid substitutions that lead to an inactive form of the protein encoded by the essential gene, or a frame shift mutation. which produces a truncated form of the protein encoded by the essential gene. In one embodiment, the essential gene is the thyA gene. A preferred technique is described in WO02 / 090551, which is specifically incorporated herein in its entirety. The transforming plasmid can be any plasmid, as long as it cannot complement the essential gene affected, for example, the thyA gene. The plasmid can be self-replicating, preferably carrying one or more genes of interest and one or more resistance markers, or the plasmid is an integrating plasmid. In the latter case, the integrating plasmid itself can be used to affect the essential gene, producing integration at the site of the essential gene, for example, the thyA site, because the function of the essential gene is affected, for example, the thyA gene. Preferably, the essential gene, such as the thyA gene, is substituted by double homologous recombination with a cassette comprising the gene or genes of interest, flanked by sequences that choose target that target insertion in the essential gene, such as the target. target thyA site. It will be appreciated that these sequences that choose target are sufficiently long and sufficiently homologous to allow integration of the gene of interest into the target site.

The genetic construct encoding the antigen of the invention can thus be present in the extrachromosomal host cell, preferably autonomously replicating using its own origin of replication, or it can be integrated into the microbial genomic DNA, for example, bacterial or yeast chromosome, for example, Lactococcus chromosome. In the latter case, a single copy or multiple copies of said nucleic acid can be integrated; integration may occur at a random site of the chromosome or, as described above, at a predetermined site thereof, preferably at a predetermined site, such as in a preferred non-limiting example, at the thyA site of Lactococcus, for example Lactococcus lactis.

Therefore, in one embodiment, the genetic construct encoding the antigen of the invention may further comprise sequences configured to effect insertion of said genetic construct into the genome, for example, a chromosome, of a host cell.

In one example, the insertion of the genetic construct at particular sites within a genome, for example chromosome, of a host cell can be facilitated by homologous recombination. For example, the genetic construction of the invention may comprise one or more regions of homology to said site of integration within the genome, for example, a chromosome, of the host cell. The sequence at said genome site, for example chromosome, can be natural, that is, it occurs in nature, or it can be an exogenous sequence introduced by prior genetic engineering.

For example, said homology region (regions) may (n) be at least 50 bp, preferably at least 100 bp, for example, at least 200 bp, more preferably at least 300 bp, for example at least 400 bp, even more preferably at least 500 bp, for example, at least 600 bp or at least 700 bp, still more preferably at least 800 bp, for example, at least 900 bp, or at least 1000 bp or more.

In a preferred example, two regions of homology may be included, one flanking each side of the relevant expression units present in the genetic construct of the invention. Such a configuration can advantageously insert the relevant sequences, that is, at least those that encode and effect the expression of the antigen of interest, in host cells. Ways of performing homologous recombination, especially in bacterial hosts, and selecting recombinants, are generally known in the art.

Microorganism transformation procedures are known to those skilled in the art, such as, for example, transformation and electroporation of protoplasts.

A high degree of expression can be achieved using homologous expression and / or secretion signals in the expression vectors present in the microorganism, for example, L. lactis. Signals that adequately regulate the expression as they are present in the constructs in the examples are useful. Other expression signals will be apparent to the person skilled in the art. The expression vector can be optimized for expression depending on the microorganism in which it is incorporated, for example, L. lactis For example, specific expression vectors are known that give sufficient levels of expression in Lactococcus, Lactobacillus lactis, casei and plantarum. In addition, systems that have been developed for the expression of heterologous antigens in the non-pathogenic, non-colonizing, non-invasive food grade bacterium Lactococcus lactis are known (see RU patent GB2278358B, which is incorporated herein by reference). A particularly preferred construction according to the invention comprises the multiple copy expression vector described in PCT / NL95 / 00135 (WO-A-96/32487), in which the nucleotide sequence encoding the antigen has been incorporated. Such a construction is particularly suitable for the expression of a

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desired antigen in a lactic acid bacterium, in particular in Lactobacillus, at a high level of expression, and can also be used advantageously to direct the expressed product to the surface of the bacterial cell. The constructs (for example, of PCT / NL95 / 00135) can be characterized in that the nucleic acid sequence encoding the antigen is preceded by a 5 'untranslated nucleic acid sequence comprising at least the minimum sequence required for recognition of ribosomes and RNA stabilization. This can be followed by a translation initiation codon that can go (immediately) followed by a fragment of at least 5 codons from the 5 'end portion of the translated nucleic acid sequence of a lactic acid bacterium gene or a structural or functional equivalent of the fragment. The fragment can also be controlled by a promoter. The contents of document PCT / NL95 / 00135, which includes the different embodiments disclosed therein, and all other documents mentioned in this specification, are incorporated herein by reference. One aspect of the present invention provides a method that allows high level regulated expression of heterologous genes in the host and expression coupling to secretion. In another preferred embodiment, the bacteriophage T7 RNA polymerase and its related promoter are used to develop a powerful expression system according to WO93 / 17117, which is incorporated herein by reference. Preferably, the expression plasmid is derived from pT1 NX.

A promoter employed according to the present invention is preferably constitutively expressed in the bacteria. The inventors observed that constitutive expression of the antigen produced high immune tolerance as opposed to inducible expression. In addition, the use of a constitutive promoter avoids the need to supply an inductor or other regulatory signal for the expression to take place. Preferably, a promoter directs expression at a level at which the bacterial host cell remains viable, that is, retains some metabolic activity, although growth is not maintained. Advantageously then, such an expression can be at a low level. For example, if the expression product accumulates intracellularly, the level of expression can lead to accumulation of the expression product in less than about 10% of the cellular protein, preferably about or less than about 5%, for example, about 1-3%. A promoter can be homologous to the bacteria used, that is, one found in that bacterium in nature. For example, a lactococcal promoter can be used in a lactococcus. A preferred promoter for use in Lactococcus lactis (or other lactococci) is "P1" derived from the Lactococcus lactis chromosome (Waterfield, NR, Lepage, RWF, Wilson, PW et al. (1995)). The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis. Gene 165 (1), 9-15). Another preferred promoter is the usp45 promoter.

The nucleic acid construct or constructs may comprise a secretory signal sequence. Thus, in a preferred embodiment, the nucleic acid encoding an antigen can provide the secretion of said antigen (appropriately coupling a nucleic acid sequence encoding a single sequence to the nucleic acid sequence encoding the antigen). The ability of a bacterium that houses the nucleic acid to secrete the antigen can be tested in vitro under culture conditions that maintain the viability of the organism. Preferred secretory signal sequences include any of those with activity in Gram-positive organisms such as Bacillus, Clostridium and Lactobacillus. Such sequences may include the Bacillus amyloliquefaciens -amylase secretion driver or the staphylokinase enzyme secretion driver secreted by some strains of Staphylococcus, which is known to function in both Gram-positive and Gram-negative hosts (see "Gene Expression Using Bacillus", Rapoport (1990) Current Opinion in Biotechnology1: 21-27), or conductive sequences of numerous other Bacillus enzymes or S-layer proteins (see p. 341-344 by Harwood and Cutting, "Molecular Biological Methods for Bacillus", John Wiley &amp; Co. 1990). Preferably, said secretion signal is derived from usp45 (Van Asseldonk et al., 1993 Mol. Gen. Genet. 240: 428-434). Preferably, said antigen is constitutively secreted.

In an alternative embodiment, the coding sequences for the biologically active polypeptide and the antigen are part of the same nucleic acid vector, or separate vectors, and are individually under the regulatory control of separate promoters. The promoters may be the same or different. A nucleic acid or vector construct comprising a coding sequence for a biologically active polypeptide and a coding sequence for an antigen in which each coding sequence is under the control of a promoter for expression in a non-invasive host, for example, Lactococcus , whether it is an operon or not, is provided by another aspect of the present invention.

Antigens

The sequence encoding the antigen that participates in the induction of celiac disease can be obtained from any natural source and / or can be prepared synthetically using well-known DNA synthesis techniques. The sequence encoding the antigen can then (for example) be incorporated into a suitable expression vector to provide a genetic construct of the invention, which is then used to transform or transfect the intended host. The recombinant thus obtained can then be cultured, after which the collected cells can be used to formulate the composition, optionally after further purification and / or processing steps, such as lyophilization to form a powder.

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An antigen can be any antigen that participates in the induction of celiac disease known to the person skilled in the art. An antigen as used herein throughout the application is preferably any substance that elicits an immune response when introduced into the body of an animal, wherein said immune response may be a T-lymphocyte-mediated and / or B-lymphocyte-mediated response. The antigen may comprise a T-cell epitope and / or a B-cell epitope. The length of the antigen is not particularly limiting, provided that said antigen can be expressed in the microorganism of the invention. The antigen can be a protein or a part thereof, such as a polypeptide or a peptide. The antigens according to the invention include linear and / or conformational epitopes. The responses mediated by T lymphocytes cover Th1, Th2 and / or Th17 responses. Antigens such as, but not limited to, allergens (including food allergens), alo-antigens, self antigens, self-antigens and therapeutic molecules or antigens that induce an immune response are also described herein. Also described herein, said antigen participates in the induction of diseases related to the immune response. Also described herein, said antigen participates in the induction of allergic asthma, multiple sclerosis, type I diabetes, autoimmune uveitis, autoimmune thyroiditis, severe autoimmune myasthenia, rheumatoid arthritis, food allergy or graft versus host disease.

The inventors observed that the secreted immunodominant antigens of the invention suppress systemic inflammatory responses of T lymphocytes, and that these antigens are necessary and sufficient for the induction of a significant tolerogenic effect.

Regulatory T lymphocytes (Treg) play a critical role in the induction and maintenance of oral tolerance. The induction of Treg is an important objective for immunotherapy for various autoimmune, allergic and inflammatory diseases. Current strategies for therapeutic induction of antigen-specific suppressor cells face significant obstacles, and usually require arduous techniques to isolate, manipulate and transfer adequate numbers of regulatory cells. The microorganism antigen release system, for example, L. lactis, of the present invention avoids these problems and effectively induces Treg specific for antigen. In the present invention it was demonstrated that induction of Treg can be achieved by exposing the mucous immune system to low doses of antigen. Exposure to low doses of antigen is preferably continued exposure. Therefore, the present invention relates to antigens that participate in the induction of celiac disease by inducing and / or expanding Treg cells, preferably CD4 + CD25 +, CD4 + CD25- and CD8 + Treg cells.

It was further demonstrated in the present invention that the Treg cells that were induced and / or expanded by the antigens according to the invention function through a mechanism dependent on TGF- and / or IL-10. Previous tests have provided that TGF- plays a critical role in oral tolerance, in addition to the development of peripherally induced Treg.

Accordingly, the present disclosure provides immunodominant antigens that participate in the induction of celiac disease that stimulate endogenous expression of TGF- and / or IL-10.

In addition, TGF--producing Th3 cells for antigen were shown to trigger differentiation of Foxp3 + regulatory cells specific for peripheral antigen. In addition, TGF--dependent conversion of CD4 + CD25-peripheral T lymphocytes into CD25 +, CD45RB- / low suppressor cells has been reported. It was shown that oral tolerance induced by Ag conjugated with CTB is associated with the increase in TGF- by the generation of both Foxp3 + CD25 + and regulatory T lymphocytes Foxi3 + and Foxp3-CD25-CD4 +. These data suggest a key role of 'adaptive' Treg Foxp3 + in the induction and maintenance of oral tolerance. The present inventors also show a significant induction of Foxp3 from the mucosa. In addition, the regulatory T cell induced by the 'mucosa' tends to be antigen specific since L. lactis is only unable to induce this regulation by increasing Foxp3 within TGLAI. Accordingly, the present invention preferably relates to Treg Foxp3 + cells.

The present invention further demonstrated that Treg cells that were induced and / or expanded by the antigens according to the invention decreased inflammation, particularly in spleen and inguinal lymph node cells. In addition, the production of IFN- and IL-12 decreased. Accordingly, the present invention provides immunodominant antigens that participated in the induction of celiac disease that decrease endogenous production of IFN- and / or IL-12 and / or stimulate endogenous expression of TGF- and / or IL-10 . In addition, the present invention relates to antigens that participated in the induction of celiac disease that reduce the proliferation of spleen cells and / or inguinal lymph nodes. It will be appreciated that the present invention also relates to antigens that participate in the induction of celiac disease by suppressing the inflammatory response of antigen-specific T lymphocytes.

It will be appreciated that certain isoforms of HLA-DQ are more commonly associated with a certain autoimmune disease. For example, the small chronic intestinal inflammation that defines celiac disease is characterized by a loss of tolerance to ingested gluten peptides and is strongly associated with a T-lymphocyte response limited to HLA-DQ2 or HLA-DQ8. The expression of HLA-DQ2 or HLA-DQ8 is necessary for the expression of celiac disease, and confer up to 40% of the genetic risk in western populations. One of the most important aspects in the pathogenesis of celiac disease

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it is the activation of an immune response of helper T lymphocytes 1, which occurs when antigen presenting cells that express HLA-DQ2 / DQ8 molecules present gluten peptides to CD4 + T lymphocytes.

DQ8 stands out due to its strong association with not only celiac disease, but also juvenile diabetes. It is also linked to HLA-DR alleles that participate in RA and may increase the risk. HLA-DQ does not spread evenly and certain populations are at high risk; however, then the risk is frequently dependent on the environment (gluten consumption) and the high prevalence of some diseases may be the result of the movement of individuals from low-risk environments to higher-risk environments.

HLA-DQ8 according to the invention is the serotypic representation of a DQA1: DQB1 haplotype. DQ8 represents the DQA1 * 0301 haplotypes: DQB1 * 0302, DQA1 * 0302.DQB1 * # 0302, or DQA1 * 0303 haplotypes: DQB1 * 0302. These haplotypes are associated with some of the most common autoimmune disease known. DQA1 * 0301: DQB1 * 0302 is the most frequent of these 3 haplotypes and represents approximately 80% of global DQ8. Thus, the present invention relates to antigens recognized by haplotypes DQA1 * 0301: DQB1 * 0302, DQA1 * 0302: DQB1 * 0302 and / or DQA1 * 0303: DQB1 * 0302, called "DQ8 epitope".

HLA-DQ2 is expressed in more than 90% of people with celiac disease. HLA DR3-DQ2 is the serotypic representation of an HLA-DRB1: DQA1: DQB1 haplotype. DR3-DQ2 mainly represents the haplotype DRB1 * 0301: DQA1 * 0501: DQB1 * 0201. It is relatively abundant in the western hemisphere. DQ2 is encoded by DQB1 * 02 alleles in combination with other alpha alleles. The two most common Q chains of DQ2 are very similar. Thus, the present invention relates to antigens that participate in the induction of celiac disease recognized by haplotypes DQB1 * 0201, DQB1 * 0202 and / or DQB1 * 0203, called "DQ2 epitope".

The present invention preferably relates to antigens that participate in the induction of celiac disease that are derived from glycoproteins. Preferably, said antigens are derived from gliadin, preferably -gliadin and / or hordein. Gliadins, which can be subdivided into -, -and -gliadins, and hordein are well known in the art, and their sequences are easily recoverable through public domain libraries, such as NCBI. Preferably, said -gliadin is derived from Triticum, such as T. aestivum or T. turgidum.

The present invention demonstrates that CD4 + T lymphocytes recognize native gluten peptides in the context of DQ2 or DQ8.

In one embodiment, the present invention relates to the DQ8 epitope:

QYPSGQGSFQPSQQNPQA, corresponding to residues 203-220 of the sequence recoverable through UniProtKB / TrEMBL entry Q9M4L6 (SEQ ID NO: 4).

Said native DQ8 epitope is preferably encoded by the nucleotide sequence 5'-caa tac cca tca ggt caa ggt tca ttc caa cca tca caa caa aac cca caa gct-3 '(SEQ ID NO: 3)

In one embodiment, the present invention relates to the epitope DQ2:

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, corresponding to residues 57-89 of the recoverable sequence using UniProtKB / TrEMBL entry Q9M4L6 (SEQ ID NO: 8)

Said DQ2 epitope is preferably 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)

Antigens are commonly deamidated in the intestines by, for example, endogenous tissue transglutaminase. Deamidated antigens are more immunoreactive and easily recognized than antigens that are not deamidated. The presence of endogenous tissue transglutaminase is indifferent in case the antigens are deamidated by other means. In one embodiment, the present invention relates to deamidated antigens that participate in the induction of celiac disease, encoded by nucleotide sequences in which the codons for glutamine residues in epitopes are preferably substituted by codons for glutamic acid residues.

In particular, the present invention relates to the deamidated DQ8 epitope

QYPSGEGSFQPSQENPQA (SEQ ID NO: 2).

Said deamidated DQ8 epitope is preferably encoded by the nucleotide sequence 5'-caa tac cca tca ggt gaa ggt tca ttc caa cca tca caagaa aac cca caa gct-3 '(SEQ ID NO: 1)

In particular, the present invention relates to the deamidated DQ8 epitope

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LQL QPF PQP ELP YPQ PQL PYP QPE LPY PQP QPF (SEQ ID NO: 6)

Said deamidated DQ8 epitope is preferably 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)

It was further demonstrated that the presence of additional sequences, such as a label, for epitope sequences did not influence the immune response. Accordingly, in other embodiments, said epitope may additionally comprise amino acids, such as, for example, 50 amino acids, 43, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 , 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid (s). Therefore, the present invention relates to DQ8 epitopes comprising a maximum of 50 additional amino acids. In another embodiment, the present invention relates to the amino acid sequence GAPVPYPDPLEPRQYPSGEGSFQPSQENPQA (SEQ ID NO: 16) comprising a DQ8 epitope and an e tag (GAPVPYPDPLEPR (SEQ ID NO: 31)).

Immune response

A disease related to the immune response as used herein is a disease caused by an unwanted immune response of the body against an antigen, whereby said antigen can be both a heterologous antigen and a self-antigen. Diseases related to the immune response include, but are not limited to, allergic reaction that includes food allergy, celiac disease, allergic asthma, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. Diseases related to the immune response also include unwanted immune reactions such as graft versus host disease or immune activation of the medication such as the production of antibodies against non-endogenous factor VIII. According to the invention, the disease is celiac disease. Also described herein are diseases related to the immune response that include, but are not limited to, allergic reaction that includes food allergy, allergic asthma, autoimmune uveitis, autoimmune thyroiditis, autoimmune myasthenia gravis, rheumatoid arthritis, type 1 diabetes and sclerosis multiple. Diseases related to the immune response also include unwanted immune reactions such as graft versus host disease or immuno-activation of the medication such as the production of antibodies against non-endogenous factor VIII.

According to the present invention, the term "immunodominant" refers to the principle antigens that induce an immune response.

In view of the foregoing, it will thus be appreciated that the present invention relates to the use as described herein, wherein said use is therapeutic and / or prophylactic.

Another aspect of the disclosure relates to a method for inducing immune tolerance to an antigen that participates in the induction of celiac disease, which comprises administration through the mucosa of said antigen by a microorganism in combination with administration through the mucosa. of a microorganism that produces the immunomodulatory compound. The immunomodulatory compound and the antigen can be released by the same microorganism, or they can be a different microorganism.

Medication and administration

Compound means any chemical or biological or complex compound, which includes simple or complex organic and inorganic molecules, peptides, peptidomimetics, proteins, protein complexes, antibodies, carbohydrates, nucleic acids or derivatives thereof. An immunomodulatory compound is a compound that modifies the function of the immune system. An immunomodulatory compound as used herein is a compound that induces tolerance; tolerance induction can be obtained, as a non-limiting example, directly by inducing regulatory T lymphocytes such as Treg, Tr1 or Th3, or by shifting the balance of Th1 / Th2 towards Th1 or Th2, or by inhibiting Th17, or in a way indirectly, by activation of immature dendritic cells to tolerate dendritic cells and / or inhibit the Th2 immune response that induces the expression of "co-stimulation" factors on mature dendritic cells. Immunomodulatory and immunosuppressive compounds are known to the person skilled in the art and include, but are not limited to, bacterial metabolites such as spergualin, fungal and Streptomyces metabolites such as tacrolimus, rapamycin or cyclosporine, immunosuppressive cytokines such as IL-4, IL -10, IFN-, TGF- (as a selective adjuvant for regulatory T lymphocytes), Flt3L, TSLP, CTB and Rank-L (as selective tolerant and / or antagonist DC inducing antibodies such as anti-CD40L, anti-CD25, anti-CD20, anti-IgE, anti-CD3, anti-IL-6 (or IL6R) and proteins , peptides or fusion proteins such as the CTL-4-Ig or CTLA-4-agonist fusion protein.

Thus, the immunomodulatory compound can be any immunomodulatory compound known to the person skilled in the art. Preferably, said immunomodulatory compound is an immunosuppressive compound, even more preferably said compound is an immunosuppressive cytokine or antibody. Preferably said cytokine

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Immunosuppressant is a cytokine or antibody that boosts tolerance. Immunosuppressive cytokines are known to those skilled in the art and include, but are not limited to, IL-4, IL-10, IFN- and TGF-, as a selective adjuvant for regulatory T lymphocytes; and Flt3L, TSLP, CTB and Rank-L, as selective tolerogenic DC inducers. Preferably, said immunosuppressive cytokine is selected from the group consisting of IL-4, IL-10, IFN- and Flt3L. It will be appreciated by one skilled in the art that the present invention also relates to functional homologs thereof. A functional counterpart connotes a molecule that has essentially the same or similar function, at least for the intended purposes, but can be structurally differentiated. Most preferably, said cytokine that enhances immunosuppressive tolerance is IL-10, or a functional homologue thereof. Preferably, said immunosuppressive antibody is chosen from the group consisting of anti-IL-2, anti-IL12, anti-IL6, anti-IFN-.

Administration as used herein means any administration procedure known to the person skilled in the art and includes, but is not limited to, coated or uncoated pharmaceutical formulations of the compound for administering capsules, liposomes, spherosomes, polymer particles comprising or carrying. the compound to administer or microorganisms that secrete, show or accumulate the compound to be administered, optionally in the presence of compounds that can enhance administration through the mucosa and / or mucosal uptake.

The compounds or compositions described herein may be administered in pure form, combined with other active ingredients or combined with pharmaceutically acceptable excipients or carriers. The oral compositions will generally include an inert diluent carrier or an edible carrier. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, enema and the like may contain any of the following components, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate; a slide such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate or orange flavoring. If the unit dosage form is a capsule, it may contain, in addition to the material of the above type, a liquid carrier such as fatty oil. In addition, unit dosage forms may contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, Shellac or enteric agents. In addition, the syrup may contain, in addition to the active compounds, sucrose as a sweetener and certain preservatives, inks, dyes and flavorings. It will be appreciated that the form and character of the pharmaceutically acceptable vehicle are imposed by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The vehicle (s) must be "acceptable (s)" in the sense of being compatible with the other formulation components and not harmful to the recipient thereof.

Alternative preparations for administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are dimethyl sulfoxide, alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous vehicles include mixtures of alcohols and water, buffered media and saline. Intravenous vehicles include fluid and nutritive boosters, electrolyte boosters such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelators, inert gases and the like may also be present. Various liquid formulations are possible for these administration procedures, including saline, alcohol, DMSO and water based solutions.

Preferably, said antigen and / or said immunosuppressive cytokine are expressed in low amounts, preferably 0.1 µg or less per dose of bacteria administered in an experimental mouse environment, such amounts that will result in an environment of human disease.

The terms "treatment", "treat" and the like, as used herein, include improvement or elimination of celiac disease developed once it has been established or relief of the characteristic symptoms of such disease. As used herein, these terms also include, depending on the patient's condition, preventing the onset of celiac disease or symptoms associated with celiac disease, which include reducing the severity of celiac disease or symptoms associated with same before the affliction with said disease. Such prevention or reduction before affliction refers to administration of the compound or composition of the invention to a patient who is not at the time of administration afflicted with the disease. "Preventing" also includes preventing the recurrence or prevention of relapse of celiac disease or associated symptoms, for example, after a period of improvement. It should be evident that mental conditions may be responsible for physical symptoms. In this regard, the term "treat" also includes the prevention of celiac disease or improvement or elimination of developed celiac disease once established or relief of the characteristic symptoms of such conditions.

As used herein, the term "medicament" also encompasses the terms "drug," "therapeutic," "potion," or other terms used in the medical field to indicate a preparation with therapeutic or prophylactic effect.

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It will be appreciated that the present compound, that is, the antigen that participates in the induction of celiac disease, is released or expressed in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" is indicated to refer to an amount of a compound or composition of the present invention that will cause a desired therapeutic or prophylactic effect or response when administered according to the desired treatment regimen. . It is observed that when the immunodominant antigen that participates in the induction of celiac disease is continuously present, the inflammatory response of antigen-specific cells is reduced even further. This reduction is significantly greater compared to the administration of the antigen as such, the microorganism as such, or the non-continuous presence of the antigen. The term "continuously present" or "continued presence" according to the invention refers to the constant or uninterrupted presence of an antigen according to the invention at the intended mucous site, for example, the site of inflammation. The presence of the antigen can be measured by techniques well known in the art, such as PCR, ELISA or immunoprecipitation techniques, such as detailed, for example, in the examples section and above. In addition, the presence of L. Lactis can be a measure of the presence of the antigen. Thus, the effects produced by the antigen may be a measure of the presence of the antigen, such as, for example, the presence or increase of endogenous TGF- or IL-10 levels, or a decrease in IFN- levels.  or IL-12, or the presence of Treg cells, as described herein, or a decrease in the proliferative capacity of splenocytes and draining lymph node cells. Thus it will be appreciated that antigen levels may vary, while the antigen is still considered to be continuously present.

Preferably, the compound or composition is provided in a unit dosage form, for example, a tablet, capsule, enema or measured aerosol dose, so that a single dose is administered to the subject, for example, a patient.

The active substances can be administered 1 to 6 times a day, enough to present the desired activity. These daily doses can be administered as a single dose once a day, or they can be administered as two or more smaller doses at the same times of the day or different that in total administer the specified daily dose. Preferably, the active ingredient is administered once or twice a day. For example, one dose could be taken in the morning and one later in the day.

In all aspects of the invention, the daily maintenance dose can be administered for a clinically desirable period in the patient, for example, from 1 day to several years (for example, throughout the entire mammal's lifetime); for example about (2 or 3 or 5 days, 1 or 2 weeks, or 1 month) up and / or, for example, up to about (5 years, 1 year, 6 months, 1 month, 1 week, or 3 or 5 days). Administration of the daily maintenance dose for about 3 to about 5 days or for about 1 week to about 1 year is typical. Other constituents of liquid formulations may include preservatives, inorganic salts, acids, bases, buffers, nutrients, vitamins, or other pharmaceutical products.

The microorganism that releases the antigen can be administered in a dose of at least 104 colony forming units (cfu) at 1012 cfu per day, preferably between 106 cfu to 1012 cfu per day, most preferably between 109 cfu and 1012 cfu per day. According to the procedure as described in Steidler et al. (Science 2000), the antigen and possibly the immunomodulatory compound of, for example, 109 cfu is secreted at least 1 ng to 100 ng. By ELISA, known to a person skilled in the art, the person skilled in the art can calculate the range of antigen secretion in relation to any other dose of cfu.

The antigen that participates in the induction of celiac disease can be administered in a dose that induces a low dose response. Preferably, said antigen is released in a dose of at least 10 fg at 500 µg per day, preferably between 1pg and 250 µg per day, more preferably between 100 pg and 200 µg per day, or preferably 1 ng and 150 µg, or more preferably 10 ng and 125 µg per day, even more preferably 100 ng and 100 µg per day, even more preferably 1 µg and 90 µg per day and most preferably between 10 µg and 75 µg per day, such as, for example, 25 µg, 30 µg, 40 µg, 50 µg, 60 µg or 70 µg per day.

Preferably, the compounds or composition are provided in a unit dosage form, for example, a tablet, solution, capsule or measured aerosol dose, such that a single dose is administered to the subject, for example, a patient.

Depending on the mode of administration, for example oral, or any of those described above, the person skilled in the art knows how to define or calculate the actual dose to be administered to a patient. The person skilled in the art will be able to adjust the doses depending on the patient, microorganism, vector, etc.

The compounds of the present invention can also take the form of a pharmacologically acceptable salt, hydrate, solvate or metabolite. Pharmacologically acceptable salts include basic salts of inorganic and organic acids, which include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, naphthalene sulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid,

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Salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. When the compounds of the invention include an acidic function, such as a carboxy group, then pharmaceutically acceptable cationic pairs suitable for the carboxy group are well known to those skilled in the art and include alkali, alkaline earth, ammonium, quaternary ammonium and similar cations. .

Microorganism

The microorganism according to the invention can be any microorganism, which includes bacteria, yeasts or fungi, suitable for administration through the mucosa. Preferably, said microorganism is a non-pathogenic microorganism, even more preferably said microorganism is a probiotic microorganism. Probiotic organisms are known to the person skilled in the art. Probiotic organisms include, but are not limited to, bacteria such as Lactobacillus sp., Lactococcus sp. and yeasts such as Saccharomyces cerevisiae subspecies boulardii. Preferably, said bacterium is a lactic acid bacterium; even more preferably said lactic acid bacterium is chosen from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus and Weissella. In a further preferred embodiment, said microorganism is Lactococcus lactis. In another preferred embodiment, said lactic acid bacterium is Lactobacillus sp. In another preferred embodiment, said microorganism is Saccharomyces cerevisiae, even more preferably said yeast is Saccharomyces cerevisiae subsp. boulardii.

Most preferably, said probiotic microorganism is a lactic acid bacterium, since the administration of heterologous proteins (i.e., non-lactic acid bacterial proteins) by lactic acid bacteria in the mucosa, which include both oral and vaginal administration, is has described (Steidler and Rottiers, 2006; Liu et al., 2006), which makes these lactic acid bacteria extremely suitable for the administration of said antigen and possibly said immunosuppressive compound. L. lactis is a non-pathogenic, non-invasive, non-colonizing Gram-positive bacterium. A variety of genetically modified L. lactis strains is generated for synthesis and local administration of immunomodulatory proteins to the intestinal mucosa. In addition, a biological containment system is established that makes the clinical application of genetically engineered L. lactis a feasible strategy.

In a preferred embodiment, said microorganism is a thycto mutant of Lactococcus lactis. An especially preferred embodiment uses a thycto mutant of Lactococcus lactis, in which the gene encoding the antigen has been used to affect the thyA gene.

Nutritional products and medical foods

It will be appreciated that the present compounds and compositions can be used as nutritional products, functional or medical food, or as additives in said nutritional products, functional or medical food. Another embodiment provides a food or beverage, preferably suitable for human consumption, comprising a nutritional product and a flavoring, in which the nutritional product comprises an extract of an agricultural product.

The nutritional products, both in the form of a liquid extract and a dry composition, are edible and can be eaten directly by humans, but are preferably provided to human beings in the form of nutritional additives or supplements, for example, in the form of tablets. of the type sold in health food stores or as components in edible solids, more preferably processed food products such as cereals, breads, tofu, cookies, ice cream, biscuits, potatoes, pretzels, cheese, etc., and in drinking liquids, for example, beverages such as milk, sodas, isotonic drinks, fruit juices. Thus, in one embodiment a method is provided to enhance the nutritional value of a food or beverage by mixing the food or beverage with a nutritional product in an amount that is effective to enhance the nutritional value of the food or beverage.

The disclosure further provides a method for enhancing the nutritional value of a food or beverage comprising mixing a food or beverage with a nutritional product to produce a nutritionally potentiated food or beverage, in which the nutritional product is mixed in an amount effective to enhance the nutritional value of the food or drink, wherein the nutritional product comprises an extract from a culture comprising the antigens of the present invention and in which the nutritionally potentiated food or beverage may further comprise a flavoring agent. Preferred flavors include sweeteners such as sugar, corn syrup, fructose, dextrose, maltodextrose, cyclamates, saccharin, phenyl alanine, xylitol, sorbitol, maltitol and herbal sweeteners, for example, stevia.

The nutritional products described herein are intended for human consumption and thus the procedures for obtaining them are preferably performed according to Good Manufacturing Practices (GMP) and any applicable government regulations governing such procedures. Especially preferred procedures use only naturally derived solvents. The nutritional products described herein preferably contain relatively high levels of substances beneficial to health. Nutritional products can be mixed together to increase their beneficial health effects.

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Unlike nutritional products, so-called "medical foods" are not intended for use by the general public and are not available in stores or supermarkets. Medical foods are not those foods included in a healthy diet to reduce the risk of disease, such as reduced fat foods or low sodium foods, nor are they weight loss products. A doctor prescribes a medical food when a patient has special nutrient needs in order to treat a disease or health condition, and the patient is under the continuous care of the doctor. The label must clearly state that the product is intended to be used to treat a specific medical condition or disorder. An example of a medical food is a nutritionally diverse medical food designed to provide targeted nutritional support for patients with chronic inflammatory conditions. The active compounds of this product are, for example, one or more of the compounds described herein. Functional foods can encompass those foods included in a healthy diet to reduce the risk of disease, such as reduced fat foods or low sodium foods, or weight loss products. Therefore, the present invention contemplates a food or beverage comprising a nutritional product according to the invention.

Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention. It is intended, therefore, that the appended claims cover all such equivalent variations as they are within the scope of the invention.

In addition, all terms used in the description of compounds of the present disclosure have their meaning as is well known in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: LL-OVA oral feeding significantly reduces HTR responses. Balb / c mice were sensitized by sc injection of OVA / CFA on days 0 and received a booster immunization of OVA / IFA on day 21. Mice were treated orally with BM9, LLpTREX1, LL-OVA and 1 µg of OVA on days 7-11, 14-18, 21-25 and 28-31. On day 31, the mice were exposed to 10 µg of OVA in 10 µl of saline solution in the ear pinnacle. HTR responses were expressed as the difference in ear thickness before and after exposure to OVA for both ears 24 h after exposure.

Figure 2: The oral feeding of LL-OVA significantly reduces the specific proliferation for OVA (A) and the production of IFN- (B), IL-6 (C) and IL-10 (D) of splenocytes in mass . Balb / c mice were sensitized by sc injection of OVA / CFA on days 0 and received a booster immunization of OVA / IFA on day 21. Mice were treated orally with BM9, LLpTREX1 and LL-OVA on days. 21-25 and 28-31. On day 31, mass splenocytes were isolated and tested for OVA-specific proliferation, which is expressed as the mean cpm ± SEM at different concentrations of OVA and for the production of IFN-, IL-6 and IL- 10 after 72 hours of ex vivo stimulation with 100 µg / ml of OVA.

Figure 3: Oral feeding of LL-OVA significantly reduces OVA-specific proliferation of CD4 + splenic T lymphocytes. Balb / c mice were sensitized by sc injection of OVA / CFA on days 0 and received a booster immunization of OVA / IFA on day 21. Mice were treated orally with BM9 (A), LLpTREX1 (B) and LL-OVA (C) on days 21-25 and 28-31. On day 31, mass splenocytes were isolated and OVA-specific proliferation of CD4 + splenic T lymphocytes by CFSE and labeled with CD4-APC and flow cytometry analysis after 90 h of ex vivo restimulation with 100 µg / ml of OVA.

Figure 4: CD4 + T lymphocytes from mice treated with LL-OVA transfer tolerance to recipients without prior treatment. Balb / c mice were sensitized by sc injection of OVA / CFA on days 0 and received a booster immunization of OVA / IFA on day 21. Mice were treated orally with BM9, LLpTREX1 and LL-OVA on days. 21-25 and 28-31. On day 31, CD4 + splenic T lymphocytes were isolated and tested for tolerance transfer capacity. The transfer of tolerance by splenic CD4 + T lymphocytes from mice treated with LL-OVA and LL-pTREX to recipients without prior treatment was evaluated by sensitizing and exposing the latter to an HTR response, which is expressed as the difference in the thickness of the ear before and after exposure to OVA for both ears 24 hours after exposure.

Figure 5: NOD AB ° DQ8 transgenic mice were immunized by sc injection of 100 µg of eDQ8d in CFA on the day

1. Mice were treated orally with LL-eDQ8d or LL-pT1 NX on days 1-10. Control mice received BM9. On day 10, the mice were exposed to 10 µg of eDQ8d in 10 µl of saline in the ear pinnacle. HTR responses are expressed as the average increase 24 hours after injection, after subtraction of ear thickness before exposure to eDQ8d. The results summarize data from 3 independent experiments that include 6 mice per group.

Figure 6: After HTR measurements, the spleens (A) and inguinal lymph nodes (B) of the BM9 (control), LL-pT1NX and LL-eDQ8d groups were isolated and restimulated ex vivo with 50 µg of peptide eDQ8d. The proliferative response specific for eDQ8d of splenocyte mass (p = 0.048) and inguinal lymph node cells

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(p = 0.0022) were expressed as mean cpm.

Figure 7: Cytokine measurements in the spleen supernatant (A) and inguinal lymph node cells (B) were performed 24 hours after restimulation. The results are averages of cytokine secretion in pg / ml representative of at least two individual experiments.

Figure 8: The specific proliferation of decreased splenic eDQ8d depends on IL-10 and TGF-.

EXAMPLES

EXAMPLE A: Induction of specific tolerance for OVA by genetically modified Lactococcus lactis that releases OVA for natural mice sensitized to OVA

Introduction

To this end, the present inventors manipulated by genetic engineering LL that secretes OVA (LL-OVA) and evaluated the induction of systemic tolerance in a therapeutic model for autoimmunity / allergy, specifically the model of immunization with OVA.

Materials and procedures

Bacteria and media: The Lactococcus lactis strain MG1363 (LL) was genetically modified and used throughout this study. The bacteria were grown in GM17E medium consisting of M17 broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose and 5 µg / ml erythromycin (Abbott). The stock suspensions of LL strains were stored at 20 ° C in 50% glycerol in GM17E medium. The mother suspensions were diluted 500 times in GM17E medium and incubated at 30 ° C overnight. Within 16 hours they reached a saturation density of 2x109 colony forming units (CFU) per ml. Bacteria were collected by centrifugation and resuspended in BM9 medium at 2 x 1010 bacteria / ml. Each mouse received 100 µl of this suspension daily through an intragastric catheter.

Plasmids: The mRNA sequence encoding Gallus gallus egg albumin was recovered from Genbank (accession number AY223553) and published data. Total chicken uterus RNA was isolated and cDNA was synthesized using 2 µg total RNA, 2 µM oligo dT primers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mM DTT (Sigma-Aldrich, Zwijndrecht, The Countries Low), 0.5 mM dNTP (Invitrogen, Merelbeke, Belgium), 20 U of RNAsin (Promega Incorporation Benelux) and 100 U of Superscript II reverse transcriptase (Invitrogen) in a volume of 25 µl. An OVA cDNA fragment was amplified by polymerase chain reaction (PCR) using the following primers: direct 5'-GGCTCCATCGGTGCAGCAAGCATGGAATT-3 '(SEQ ID No.: 9) and reverse 5'-ACTAGTTAAGGGGAAACACATCTGCCAAAGAAGAGQAQ-3' Nº: 10). The reaction conditions were 94 ° C for 2 min 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 with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the Lactococcal P1 promoter 17. MG1363 strains transformed with plasmids carrying OVA cDNAs were designated OVA secreting L. lactis (LL-OVA). L. lactis-pTREX1, which is MG1363 containing the empty vector pTREX1, served as a control (LL-pTREX).

Mice: Seven week old female BALB / c mice were obtained from Charles River Laboratories (Calco, Italy) and housed in a conventional animal shelter under conditions without specific pathogens. Animal studies were approved by the Ethical Committee of the Department of Molecular Biomedical Research at the University of Ghent (file nº 07/029).

Antigen: V quality OVA protein without intact LPS was used as antigen in all experiments (Sigma Aldrich).

Immunization of mice and induction of oral tolerance: Balb / c mice were immunized by sc injection of 100 µg of OVA in 100 µl of a 1: 1 mixture of CFA (Difco, BD Bioscience, Erembodegem, Belgium) and saline solution at the base from the tail the first day. LL-OVA, LL-pTREX1 or 1 µg of purified OVA dissolved in 100 µl of BM9 were administered daily on days 7-11, 14-18, 21-25 and 28-31 (guideline 1) and on days 21- 25 and 28-31 (guideline 2). Control mice received only BM9. Antigen or bacterial suspensions were introduced into the stomach using a 18 gauge stainless animal feed needle. On day 21, a booster immunization was administered by sc injection of 100 µg of OVA into 100 µl of a mixture 1: 1 of IFA (Sigma-Aldrich). Tolerance induction was assessed by HTR responses, cytokine measurement and OVA specific proliferation, and adoptive transfer experiments.

Delayed type hyperreactivity responses: Antigen-specific HTR responses were evaluated by injection of OVA on day 31. Twenty-four hours later HTR measurements were made. For the measurement of antigen-specific HTR responses, the mice were exposed to 10 µg of OVA in 10 µl of saline in the ear pinnacle. Ear swelling, defined as the increase in ear thickness due to exposure, was measured blinded 24 hours after exposure using a digital micrometer (Conrad, Belgium).

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HTR responses were expressed as the difference in ear thickness before and after exposure to OVA for both ears.

Specific proliferation for OVA and cytokine assays: On day 39, spleens were collected and splenocytes were evaluated for specific proliferation for OVA and cytokine production. Individual cell suspensions were prepared from spleens by passing the cells through 70 µm cell sieves (Becton / Dickinson Labware). Erythrocytes in cell suspensions were lysed by incubation with red blood cell lysis buffer. CD4 + T lymphocytes were enriched using the CD4 + T lymphocyte isolation kit and midiMACS columns (Miltenyi Biotec, Germany).

To test the proliferation of total splenocyte populations, 2 x 105 cells were cultured in 96-well U-bottom plates in a total volume of 200 µl of complete medium [ie, RPMI-1640 containing 10% fetal bovine serum (SBF), 10 U / ml penicillin, 10 µg / ml streptomycin, 2 mM L-glutamax, 0.4 mM sodium pyruvate] both alone and with OVA, added at concentrations ranging from 1.2 to 100 µg / ml Proliferation was further evaluated by labeling with 5,6-CFSE (Invitrogen, Merelbeke, Belgium). Splenocytes were resuspended in PBS at 107 / ml and incubated in a final concentration of 10 µM CFSE for 12 min at 37 ° C. The labeled cells were washed twice with complete cold medium on ice before culturing 2 x 105 cells in 96-well U-bottom plates in a total volume of 200 µl of complete medium with 100 µg / ml of OVA. After 90 h of culture at 37 ° C and 5% CO2 in a humidified incubator, the cells were collected and the cells stained with allophycocyanin-labeled anti-CD4 (BD, Biosciences) and proliferation was determined using cytometry of flow (FACSCanto, BD Biosciences).

To test the proliferation of CD4 + T lymphocytes, 2 x 105 CD4 + T lymphocyte cells were cultured in 96-well U-bottom plates with OVA-loaded splenocytes treated with mitomycin C, 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 µl of complete medium. The cells were grown for 90 h at 37 ° C and 5% CO2 in a humidified incubation oven. For proliferation assays, 1 µCi / well of [3 H] -thymidine was added during the last 18 h of culture, DNA was collected on fiberglass filter mats (Perkin Elmer, Boston, USA) and the DNA bound radioactivity was measured in a scintillation counter (Perkin Elmer). For cytokine measurements, the cell culture supernatants used in the different proliferation assays were collected after 72 h of culture and frozen at -20 ° C. Cytokine production was quantified using Mouse Flex Set Cytometric Bead Array (BD Biosciences, Mountain View, CA, USA).

Adoptive transfer experiments: On day 39, spleens were collected from the treatment groups. Individual cell suspensions were obtained by slicing the spleens and filtering them through 70 µm cell sieves (Becton / Dickinson Labware). The cell suspensions were enriched for CD4 + T lymphocytes, as described above. Cells enriched in CD4 + were transferred adoptively to BALB / c accepting mice without prior treatment by iv injection of 1 x 106 CD4 + T lymphocytes. One day after the adoptive transfer, all mice were sensitized by injecting 100 µg of OVA / 25 µl of saline / 25 µl of IFA (Sigma-Aldrich) sc at the base of the tail, and 5 days from here, mice were exposed according to the HTR protocol described above.

Statistical analysis: The significance of differences between groups in ear thicknesses and cytokine measurements were tested using unilateral ANOVA. Statistical significance is indicated as * (p &lt;0.05) or ** (p &lt;0.01).

Results

LL-OVA significantly enhances tolerance inducing capacity in the OVA immunization model compared to free OVA

To study oral tolerance induction, mice were fed orally as described above. The administration of LL-OVA to BALB / c mice sensitized to OVA led to a significant decrease in HTR response compared to sensitized control mice (BM9 group) and mice treated with LLpTREX1 or 1 µg of purified OVA (Figure 1).

These data were accompanied by a significantly decreased proliferative capacity and production of IFN-, IL-10 and IL6 (Figure 2) of the splenocytes in mass of mice treated with LL-OVA with respect to groups treated with BM9 or LLpTREX1.

LL-OVA enhances oral tolerance by CD4 + T lymphocytes

To assess whether CD4 T lymphocytes mediate or not in the induction of oral tolerance, the response of proliferative CD4 T lymphocytes specific to OVA in splenocytes was studied. Flow cytometry showed that only 0.8% of CD4 + splenic T lymphocytes proliferate after OVA re-stimulation in the LL-OVA group compared with 4.5% and 11.6% in the BM9 and LL-pTREX1 (Figure 3). In addition, the adoptive transfer of splenic CD4 + T cells from the LL-OVA treated group to BALB / c mice without prior treatment demonstrated that these cells

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they could transfer tolerance, since these cells were able to reduce the HTR response after immunizing and exposing the acceptor mice to OVA (Figure 4).

conclusion

Here, the present inventors demonstrated that intragastric administration of L. lactis that secretes OVA suppresses OVA-specific T lymphocyte responses by inducing regulatory CD4 +. The present inventors demonstrated that this induction of immune tolerance is more potent than the free OVA protein and that it could be established in a therapeutic environment.

EXAMPLE B: Oral-specific induction of antigen tolerance by genetically modified Lactococcus lactis by administering immunodominant epitopes of DQ8 specific to gluten-sensitized class II transgenic mice

Introduction

Celiac disease, also known as celiac disease or gluten-sensitive enteropathy, is a chronic inflammatory disease that develops from an immune response to specific dietary grains that contain gluten. Celiac disease is a complex multigenic disorder that is strongly associated with genes that encode the variants of human leukocyte antigen HLA-DQ2 or HLA-DQ8. One of the most important aspects in the pathogenesis of celiac disease is the activation of an immune response of cooperating T lymphocytes 1. This occurs when antigen presenting cells expressing HLA-DQ2 / DQ8 molecules present gluten peptides to T lymphocytes. CD4 (+). Both kinds of gluten proteins, gliadins and glutenins, contain peptides that bind DQ2 and DQ8. It is generally accepted that the immune response, such as IFN-producción production of gluten-specific T lymphocytes, triggers the destruction of the mucosa in the small intestine of patients with celiac disease. Therefore, the activation of a harmful T lymphocyte immune response in the intestine of patients with celiac disease seems to be a key to the onset and progression of the disease.

Antigen-specific immune suppression is an attractive therapeutic goal for the treatment of celiac disease. The active administration of gluten proteins / recombinant peptides to the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for tolerance induction. To this end, the present inventors manipulated by genetic engineering LL that secretes the deamidated DQ8 (LL-eDQ8d) and evaluated the local and systemic immune response in class II NOD AB ° DQ8 transgenic mice sensitized to gluten after oral supplementation.

Thus, the present inventors demonstrate that oral administration of L. lactis that produces the gliadin peptide suppresses specific immune responses for gliadin by inducing antigen-specific CD4 + regulatory T lymphocytes.

Materials and procedures

Bacteria and media: The Lactococcus lactis strain MG1363 (LL) was genetically modified and used throughout this study. The bacteria were grown in GM17E medium, which was M17 broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose and 5 µg / ml erythromycin (Abbott). The stock suspensions of LL strains were stored at -20 ° C in 50% glycerol in GM17E medium. The mother suspensions were diluted 200 times in GM17E medium and incubated at 30 ° C overnight. A saturation density of 2 x 109 colony-forming units (CFU) per ml was reached within 16 hours of cultivation. Bacteria were collected by centrifugation and concentrated 10 times in BM9 inoculation buffer at 2 x 109 bacteria / 100 µl. For treatment, each mouse received 100 µl of this suspension daily by intragastric catheter.

Plasmids: The sequence encoding the deamidated DQ8 epitope (encoding DQ8d: caa tac cca tca ggt gaa ggt tca ttc caa cca tca caa gaa aac cca caa gct (SEQ ID NO: 1)) was retrieved from published data. In summary, two glutamine residues within the alpha-gliadin peptide were changed to glutamic acids to stimulate the deamidated immunodominant alphagliadin response for patients with celiac disease carrying DQ8, and this epitope is recognized by T lymphocytes of these mice. The DQ8d cDNA fragment was synthetically constructed (Operon, The Netherlands) and amplified by polymerase chain reaction (PCR) using the following direct and reverse 5'-caatacccatcaggtgaaggttc-3 'primers (SEQ ID NO: 11) and 5'-cgactagttaagcttgtgggttttcttgtgat-3 '(SEQ ID NO: 12). For the purposes of detection, an e (e) label was attached to the fragment, which consists of the following sequence ggt gct 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 the DQ8d gene, the PCR product that was produced in step 1 (DQ8d) was used as a template in a PCR with 5'ggtgctccagttccatacccagatccacttgaaccacgtcaatacccatca-3' oligonucleotides (SEQ ID NO: 14) and 5'-cgactagttaagcttgtgggttttcttgtgat-3 '(SEQ ID NO: 15). The amplified fragment was fused with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the lactococcal P1 promoter. MG1363 strains transformed with plasmids carrying eDQ8d cDNA were designated eDQ8d secreting Lactococcus lactis (LL-eDQ8d). LL-pT1NX, which is MG1363 that contains the vector

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empty pT1NX, served as control.

Functional analysis of secreted epitopes: For the functional analysis of the secreted eDQ8d epitope, a proliferation assay was performed with clones of human T lymphocytes derived from the intestines of patients with celiac disease (CD). The bacteria were grown overnight as described above, diluted 1:50 and grown for another 4 or 6 hours, respectively. Clones of gluten-specific T lymphocytes were generated from a small intestinal biopsy taken from patient S, a patient with adult Dutch CD who had taken a gluten-free diet for several years. The patient gave informed consent to the study, which was approved by the hospital's ethical committee. The patient was serologically typed to be HLA-DR3 / 4, DQ2 / 8, thus carrying both DQ dimers associated with CD. The T lymphocyte clone II29 was found to respond to an alpha-gliadin-derived peptide with a core of at least 9 amino acids QGSFQPSQQ, when bound to HLA-DQ8. It was found that the deamidation of the glutamine residue P1 and / or P9

(Q) in glutamic acid (E) by the activity of tissue transglutaminase substantially enhanced the stimulating capacity of the T lymphocytes of this gluten peptide. Proliferation assays were performed in duplicate or triplicate in 150 µl of culture medium (Iscove) in 96-well U-bottom plates (Falcon) using 104 T lymphocytes stimulated with 105 peripheral blood mononuclear cells of the same HLA-DQ e irradiated at 3000 RAD in the absence or presence of supernatant at various concentrations. After 48 hours, the cultures were pulsed with 0.5 uCi of 3H-thymidine, collected 18 hours from here, after which the incorporation of 3H-thymidine was determined as a measure of proliferation.

Mice: Transgenic mice expressing HLA-DQ8 in a context deficient in endogenous MHC II (AB ° DQ8 +) were backcrossed with NOD mice for 10 generations and crosslinked to produce congenital AB ° DQ8 + NOD mice. Mice from seven to sixteen weeks of age were used for the experiments. The mice were weaned and kept in a conventional animal farm until 8-12 weeks of age.

Antigen and antibodies: Deamidated DQ8 epitopes with e-tag (GAPVPYPDPLEPRQYPSGEGSFQPSQENPQA (SEQ ID No. 16)) and without (QYPSGEGSFQPSQENPQA (SEQ ID No. 2)) were synthesized. For T lymphocyte phenotyping, antibodies to CD4 and CD25 were purchased from BD-Biosciences (San Jose, CA), and anti-Foxp3 APC staining kits were purchased from eBiosciences (San Diego, USA), respectively. Anti-IL-10 neutralizing monoclonal antibody (1 µg / ml, clone JES052A5), neutralizing monoclonal antibody for TGF- (1 µg / ml, clone 1D11) and neutralizing antibodies for LAP (1 µg / ml, clone 27235) were obtained from R&amp;D Systems (Minneapolis, MN).

Oral feeding and HTR reaction (delayed type hyperreactivity): NOD AB ° DQ8 mice with a gluten-free feed were sensitized by subcutaneous injection of 100 µg deamidated eDQ8 peptides in 100 µl of 1: 1 CFA (purchased from Difco de Becton, Dickinson and Company, San Jose, CA) and saline solution in the base tail on day 1. The peptide used for sensitization had the same sequence as the secreted epitope. Mice were fed with BM9 as a negative control, LL-pT1NX or LL-eDQ8d [all on days 1-10 dissolved in 100 µl of BM9]. Feeds were made by intragastric administrations of antigen or bacterial suspensions using a 18 gauge stainless nasogastric tube needle. Ten days after immunization, the specific HTR responses for antigen were evaluated. Twenty-four hours from here HTR measurements were made. For the measurement of antigen-specific HTR responses, mice were exposed to 10 µg of eDQ8d in 10 µl of saline in the ear pinnacle. The increase in ear thickness was measured in a blinded manner using an engineer micrometer (Mitutoyo, Tokyo, Japan) 24 h after exposure. HTR responses were expressed as the difference in increase 24 hours after eDQ8d injection, after subtraction of ear thickness before exposure. Subsequently, the mice were sacrificed, the spleen and lymph nodes were collected and the cells were evaluated for specific proliferation for DQ8d and cytokine production. For interference of the e-label, NOD AB ° DQ8 mice were immunized with 100 µg of deamidated DQ8 peptides with (eDQ8d) or without e-mark (DQ8d) in 100 µl of 1: 1 Freund's complete adjuvant (CFA, Difco, BD) and saline solution in the base tail on day 1. On day 7, mouse HTR measurements were performed as described above with 10 µg of DQ8d with or without e-tag, corresponding to the peptide used for immunization .

Cell cultures, proliferation and cytokine production assays: Suspensions of spleen cells and lymph nodes were prepared on day 11 of the experiment by homogenizing the tissue with a tissue mill in 1 X PBS. Erythrocytes were removed from spleen cell suspensions by incubation with ACK (ammonium / potassium chloride (lysis buffer)). Cells were incubated in 96-well microtiter plates at 5 x 105 cells / well in 0.2 ml volumes at 37 ° C in RPMI 1640 (1.5% Hepes, 1% Penstrep and 10% SBF) with supplements containing both medium alone, 10 µg of Con A and 50 µg of epitope eDQ8d. In a separate experiment, neutralizing antibodies to IL-10, TGF-, IL10 and TGF- or LAP were added to splenocytes from mice treated with LL-eDQ8d. After 24 h, proliferation was evaluated by the addition of 1 µCi / well of [3 H] -thymidine during the last 24 h of culture. DNA-bound radioactivity was collected on fiberglass filter mats and thymidine incorporation was measured in a scintillation counter (Perkin Elmer). The results were expressed as mean cpm of triplicate wells. For cytokine measurements, the cell culture supernatants used in the different proliferation assays, described above, were collected after 24 h of culture and frozen at -20 ° C until they were

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He performed the cytokine analysis. Cytokine production was quantified using the mouse inflammation cytometric pearl assay (BD Biosciences).

Flow cytometry analysis: Spleens and intestine-associated lymph node tissue (TGLAI) from mice treated with BM9, LL-pT1NX or LL-eDQ8d 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, CA) and subsequently measured using flow cytometry in a Becton Dickinson FACSCaliburs. For analysis, cells were regulated on subpopulations of CD4 + CD25 + and CD4 + CD25- and within these populations Foxp3 histograms were used to determine the average fluorescence intensity (MFI).

Statistical analysis: The results of cytokine measurements are expressed as means ± SEM. Proliferation specific to eDQ8d, ear thickness and cytokine measurements were tested for significance using unilateral ANOVA followed by the comparison of the Student t test: two samples that assume equal variance, to determine differences between individual groups . For all tests a value of p &lt;0.05: *, &lt;0.01: ** was used to indicate statistical significance for both tests.

Results

Mucosal administration of eDQ8d epitopes by L. lactis significantly decreases the HTR response induced by DQ8d and the proliferative capacity of spleen cells and mass inguinal lymph nodes.

Daily intragastric administration of LL-eDQ8d in class II NOD AB ° transgenic mice immunized with DQ8eDQ8d led to a significant decrease in HTR response compared to sensitized control negative mice (Figure 5). Control mice (fed with BM9) were clearly immunized with eDQ8d, but daily intragastric administration of LL-eDQ8d significantly reduced HTR (13.1 x 10-2 mm versus 5.1 x 102 mm, p = 0, 0031). Ear swelling was also slightly reduced in mice treated with LL-pT1NX compared to controls (9.3 x 10-2 mm versus 13.1 x 10-2 mm, p = 0.0343), but at a degree much lower than in mice treated with LL-eDQ8d. Non-ABQ transgenic NOD AB mice showed only a minor increase in ear thickness (3.2 x 10-2 mm). These data indicate that orally administered LL-eDQ8d suppresses systemic inflammatory T lymphocyte responses in immunized NOD AB ° DQ8 transgenic mice and that the secreted antigen is necessary for the induction of a significant tolerogenic effect. These data were accompanied by a significantly decreased proliferative capacity of splenocytes and inguinal lymph node cells (Figure 6). The reduced proliferative response was accompanied by a significant regulation by increase of IL-10 and a regulation by decrease in the production of IL-12 after stimulation by eDQ8d ex vivo of splenocytes (Figure 7). In addition, LL-eDQ8d significantly reduced the production of IFN- induced by eDQ8d in the inguinal lymph nodes compared to mice treated with BM9 and LL-pT1NX. Together, these data indicate that LL-eDQ8d treatment suppresses T lymphocyte activation after eDQ8d stimulation and suggests that DC activation can also be modulated.

The reduced splenic proliferation of eDQ8d depends on IL-10 and TGF-, and the treatment of LL-DQ8d significantly increases the splenic and Foxp3 expression of TGLAI

The functional importance of TGF-, IL-10 and LAP (membrane-associated TGF-sobre) on the splenic proliferative response specific for eDQ8d was analyzed using neutralizing antibodies. Neutralizing antibodies to IL-10, TGF- or LAP did not significantly interfere with the decrease in splenic proliferative response of mice treated with LL-eDQ8d, but the addition of a combination of neutralizing monoclonal antibodies to TGF- and IL10 abolished the decrease in the specific proliferative capacity for eDQ8d of splenocytes from mice treated with LL-eDQ8d (Figure 8). These data strongly suggest that treatment with LL-eDQ8d can suppress T lymphocyte activation in transgenic mice class II NOD AB ° DQ8 immunized with eDQ8d and that this suppression depends on both IL-10 and TGF-. In addition, a regulation by significant increase of Foxp3 within splenic CD4 + CD25 + was observed, in addition to the population of CD4 + CD25-cells of mice treated with LL-eDQ8d compared to the control (BM9) (IMF 171 vs. 61 and 35 versus 6, respectively). Surprisingly, Foxp3 was also regulated by an increase in the population of CD4 + CD25-in gut-associated lymph node tissue (TGLAI) of mice treated with LL-eDQ8d compared to those treated with BM9 (IMF 73 vs. 30) , but not in the CD4 + CD25 + population of TGLAI. The feeding of LL-pT1NX also induced some regulation by an increase in Foxp3, but exclusively in the population of CD4 + CD25-splenic T lymphocytes and to a lesser degree than LL-eDQ8d (IMF 15 vs. 35, respectively).

conclusion

The data of the present inventors demonstrated that administration through the mucosa of a gliadin-derived immunodominant peptide for DQ8-mediated T lymphocyte responses by genetically modified L. lactis induces the suppression of local and systemic DQ8-limited T lymphocyte responses. in transgenic mice class II NOD AB ° DQ8. The treatment produced a specific decrease for antigen of the proliferative capacity of

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Splenocytes and inguinal lymph node cells, which was critically dependent on the production of IL-10 and TGF- and was associated with a significant induction of Foxp3 + regulatory T lymphocytes. Because this approach of bacteria that release antigen has the ability to enhance oral tolerance even in the established hyperreactivity environment, it can be applied for the treatment of celiac disease and possibly other autoimmune and / or allergic diseases.

Native DQ8 epitope

The previous experiments are repeated with the native -gliadin epitope, that is, QYPSGQGSFQPSQQNPQA (SEQ ID NO: 4), corresponding to residues 203-220 of the sequence recoverable by UniProtKB / TrEMBL entry Q9M4L6. Said native DQ8 epitope is encoded by the nucleotide sequence 5'-caa tac cca tca ggt caa ggt tca ttc caa cca tca caa caa aac cca caa gct-3 '(SEQ ID NO: 3).

The results with the native -gliadin DQ8 epitope are essentially identical to the results described above for the deamidated Q-gliadin DQ8 epitope.

Trial in celiac patients using the DQ8 epitope

In a preliminary study, L. lactis engineered according to the invention is used as a therapeutic agent in a trial in patients with celiac disease. The inventors' findings provide promises that this approach is effective in a specific way for antigen.

Celiac disease is a particularly attractive target for this approach, due to the ability of LL to administer the antigen at the site of the primary response to achieve both direct and circumstantial tolerance.

Trial in celiac patients using the DQ2 epitope

There are no transgenic mice that express HLA-DQ2 in an endogenous MHC II deficient environment comparable to HLA-DQ8 mice as used above. Therefore, the experiments described above for DQ8 epitopes were not possible in an appropriate mouse model. Therefore, the present inventors carry out some preliminary experiments in patients with celiac disease, using both the native -gliadin DQ2 epitope, also deamidated.

Specifically, the above experiments are repeated using:

Deamidated DQ8 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 cca caca cca caca 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 cca ca cca caa cca ttc (SEQ ID NO: 7)

The results with the native and deamidated -gliadin DQ2 epitope are essentially indicated in the results described above for the Q-gliadin DQ8 epitopes.

EXAMPLE C: Induction of tolerance to factor VIII and coagulation factor IX after oral administration of

L. lactis that secretes said factors

Introduction

Several therapeutic (recombinant) proteins, such as interferon, factor VIII / IX and antibodies (Remicade), are administered at high doses for prolonged treatment periods. However, a complication associated with its use is the development of specific immune responses for proteins such as antibodies. These antibodies (Ab), also called inhibitors, make therapeutic proteins less effective. Examples include the formation of inhibitors for factor VIII / IX in hemophilia, erythropoietin (Epo) in patients receiving therapy for chronic renal failure and IFN-in patients receiving treatment for multiple sclerosis. Here, the present inventors demonstrate that oral administration of factor VIII (and factor IX) by L. lactis suppresses inhibitor formation to said factor by inducing antigen-specific CD4 + regulatory T lymphocytes.

Material and procedures

Bacteria and plasmids: L. lactis strain MG1363 is used throughout this study. Bacteria are grown in GM17 medium, that is, M17 (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at -20 ° C in 50% glycerol in GM17. For intragastric inoculations, the mother suspensions are diluted 200 times in fresh GM17 and incubated at 30 ° C. They reach a saturation density of

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two x 109 colony forming units (CFU) per ml within 16 hours. Mixed bacterial suspensions are used throughout this study. Therefore, the bacteria that are mixed are collected by centrifugation and the sediments of both bacterial cultures are concentrated 10 times in BM9 medium (Schotte, Steidler et al., 2000). For treatment, each mouse receives 100 µl of this suspension per intragastric catheter.

CDNA or human FIXII and FIX cDNA fragments, which represent epitopes of CD4 + T lymphocytes specific for FVIII and FIX, are amplified fused with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the lactococcal P1 promoter.

The 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-pT1NX, which is MG1363 containing the empty vector pT1NX, serve as a control.

Quantification of FVIII and FIX: FVIII or FIX of LL-FVIII and LL-IX, respectively, are determined using adsorption enzyme immunoassay (ELISA) specific for human FVIII and FIX, which have been previously described (Chuah et al., 2003). Recombinant proteins are also analyzed by Western blot analysis and COATests and aPTT assays, as described (Chuah et al., 2003; Van den Driessche et al., 1999). The NH2 end of this protein is determined by automated Edman degradation. Since FVIII and FIX are normally expressed in the liver in which they undergo extensive post-translational modifications, coagulation factors produced from engine-manipulated L. lactis can be biologically inactive. However, these post-translational differences will probably have no impact on the capacity of these recombinant proteins produced by L. lactis to induce immune tolerance. In fact, most inhibitors that have been characterized in detail to date normally recognize amino acid residues (Villard et al., 2003), rather than glycosylated moieties.

Animals: Mice with hemophilia A or B obtained by inactivation of murine FVIII or FIX genes using homologous recombination in ES cells as described by (Bi et al. (1995) and Wang et al. (1997)) are raised in the laboratory. These recipient mice generate neutralizing antibodies when exposed to purified recombinant FVIII or FIX antigen in the presence of CFA (Mingozzi et al., 2003). The inhibitory state can be monitored over time using specific Bethesda or ELISA assays for anti-FVIII / anti-FIX. Receptor mice exposed to FVIII or FIX (+ CFA) normally develop inhibitors 2-3 weeks after antigen exposure.

Experimental environment: Mice 4-6 weeks old receive FVIII, FIX, LL-FVIII, LL-FIX or LL-pT1NX or LL-OVA (an irrelevant antigen) (1 or 10 µg). As a positive control for tolerance induction, the present inventors inject mice with adeno-associated viral vectors (AAV) that express FIX of a hepatocyte-specific promoter. Recipient mice develop specific immune tolerance for FIX that prevents the induction of anti-FIX antibodies after subsequent exposure to FIX + CFA.

In a prophylactic environment, FVIII, FIX, LL-FVIII, LL-FIX alone are administered orally to mice with hemophilia A or B using a gastric catheter, using different intervals and treatment doses. These recipient mice are subsequently exposed to 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 collected by retro-orbital bleeding. The development of antibodies directed against FVIII or FIX is evaluated using Bethesda assays (Kasper et al., 1975) or using a modified ELISA specific for anti-FVIII or anti-FIX (Van den Driessche et al., 1999) at different intervals of time.

In a therapeutic environment, mice with hemophilia A or B are first immunized with FVIII or FIX, as described (Mingozzi et al., 2003). Inhibitor status is monitored over time using Bethesda or ELISA assays specific for anti-FVIII / anti-FIX. Mice with low or high inhibitor titers are subsequently treated with FVIII, FIX, LL-FVIII, LL-FIX only using different intervals and treatment doses and the inhibitor titers are determined over time. The specificity of the possible immune tolerance is evaluated by exposing the mice that receive FVIII, FIX, LL-FVIII, LL-FIX only with an irrelevant antigen (tetanus toxoid or OVA).

Cell cultures, proliferation and cytokine assay: Individual spleen and lymph node cell suspensions are prepared by passing the cells through 70 µm filter cell sieves (Becton / Dickinson Labware). The erythrocytes are removed from the spleen cell suspensions by incubation with red blood cell lysis buffer.

Proliferation assays of populations of total splenocytes, 2 x 105 cells are grown in 96-well U-bottom plates in a total volume of 200 µl of complete medium both alone and with purified FVIII or FIX, and with and without monoclonal antibodies anti-IL-10 or anti-TGF- neutralizers. FVIII and FIX are added at concentrations ranging from 1 to 100 µg / ml. Neutralizing antibodies are added at 1, 0.1 and 0.01 µg / ml. For CD4 + T cell proliferation assays and CD4 + CD25- T cell populations, 0.2 x 105 CD4 + T cell cells or CD4 + CD25 T cells are grown on 96-well U-bottom plates with 1 x 105 cells CD4-irradiated, which serve as antigen presenting cells, and FVIII or FIX (0 or 100 µg / ml) in a total volume of 200 µl of complete medium both with and without neutralizing antibodies. After 72 h at 37 ° C in a humidified incubator with 5% CO2,

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Proliferation is assessed by the addition of 1 µCi / well of [3 H] -thymidine. DNA-bound radioactivity is collected 16-18 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer).

For cytokine measurements, the cell culture supernatants used in the different proliferation assays are collected after 24, 48 and 72 h of culture and frozen at -20 ° C until the cytokine analysis is performed. Cytokine production is quantified using the mouse inflammation cytometric pearl assay (BD Biosciences, Mountain View, CA, USA).

In vivo T regulatory activity assay: In order to test for active suppression of antibody formation in mice, splenocytes, purified CD4 + T lymphocytes, CD4 + CD25- or CD4 + CD25 + T lymphocytes isolated from different groups of L Treated experimental lactis were adopted adoptively to C3H / HeJ mice without prior treatment. Untreated mice are used as control. The number of transfected cells is 107 for complete spleen cells, spleen cells depleted in subpopulations or positively selected CD4 + cells and CD4 + CD25- and CD4 + CD25 + T cells. Receptor mice (n = 4-5 per experimental cohort) were injected subcutaneously with 5 µg of hF.IX in CFA 36 hours after the adoptive transfer. Plasma anti-hF.IX IgG titers were measured 2.5 weeks after immunization.

Results

LL-FVIII and LL-IX significantly enhance tolerance-inducing ability in mice with hemophilia A or B compared to free FVIII or FIX

To study oral tolerance induction, mice are fed orally as described above (experimental environment). The addition of LL-FVIII or LL-FIX significantly enhances the induction of tolerance towards FVIII and FIX since the proliferative response specific for splenocyte factor is significantly reduced in this group compared to the control and free FVIII and FIX groups.

LL-FIIIV and LL-FIX enhance oral tolerance in association with specific titers for reduced FVIII and FIX and production of IFN- and more IL-10 and TGF- in response to that factor

To study oral tolerance induction, mice are fed orally as described above (experimental environment). Antibodies specific for FVIII and FIX and the production of cytokines in response to said factor in splenocytes and lymph nodes are quantified as described above. Inhibitor formation and production of proinflammatory cytokine IFN- is strongly reduced and immunosuppressive cytokines IL-10 and TGF- significantly increase in the LL-FVIII / FIX group compared to control and FVIII / IX groups free.

LL-FVIII / FIX enhances oral tolerance using CD4 + T lymphocytes

To assess whether CD4 + T lymphocytes mediate or not induce oral tolerance, the factor-specific proliferative CD4 + T lymphocyte response is studied in splenocytes and lymph nodes. Thus, mice are fed orally as described above (experimental environment) and the proliferation of factor-specific CD4 + T cells is determined as described in Cell cultures, proliferation and cytokine assay. The response of CD4 specific T lymphocytes for factor in the LL-FVIII / FIX group is significantly reduced compared to control and free FVIII / IX groups.

Antigen-induced regulatory T lymphocytes following LL-FVIII / FIX therapy may transfer protection from inhibitor formation in vivo

In order to test the active suppression of antibody formation in mice treated with the oral tolerance protocol, the present inventors adopt adoptively splenocytes from the different groups treated as described above (T in vivo regulatory activity test). Compared with controls and groups of free FVIII / IX, the formation of anti-IgG factor is significantly reduced in the LL-FVIII / FIX group, which indicates activation of regulatory CD4 + T lymphocytes in the combination oral tolerance tolerance protocol. The present inventors.

conclusion

The data of the present inventors demonstrate that administration through the mucosa of L. lactis that secretes recombinant FVIII or FIX is more potent than FVIII or FIX in suppressing the formation of specific inhibitors for FVIII and FIX in mice with hemophilia A and B respectively.

EXAMPLE D: Induction of tolerance to an allergen, Der p 1, after oral administration of L. lactis secreting said allergen

Introduction

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Allergic asthma is a chronic inflammatory disorder of the respiratory tract. It is characterized by reversible airway obstruction, elevated serum levels of allergen-specific immunoglobulin E, mucosal hypersecretion and airway hyperreactivity (HVR) to bronchospasmogenic stimuli. Its symptoms are worsened by exposure to an allergen (for example, tree, grass and grass pollen, dust and dust mites, mold, animal dander) to which the patient has been sensitized. Cooperative T type 2 (Th2) lymphocytes play a crucial role in the onset, progression and persistence of the disease. Current data suggest that Th2 responses to allergens are normally suppressed by regulatory T lymphocytes. In addition, suppression by this subset decreases in allergic individuals. Here, the present inventors demonstrate that oral administration of allergen by L. lactis suppresses asthma-like responses by inducing antigen-specific CD4 + regulatory T lymphocytes.

Material and procedures

Two mouse models of allergic asthma that mimic human disease are the OVA allergen model and the humanized SCID model.

Ova allergen model: OVA-sensitized mice are exposed by aerosol inhalation of OVA leading to eosinophilic airway inflammation dependent on Th2 cytokines, bronchial hyperreactivity and IgE production, highly characteristic findings 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).

Bacteria: Strain MG1363 of L. lactis is used throughout this study. Bacteria are grown in GM17 medium, that is, M17 (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at -20 ° C in 50% glycerol in GM17. For intragastric inoculations, the mother suspensions are diluted 500 times in fresh GM17 and incubated at 30 ° C. A saturation density of 2 x 109 colony forming units (CFU) per ml was reached within 16 hours. Bacteria are collected by centrifugation and concentrated 10 times in BM9 medium. For treatment, each mouse receives 100 µl of this suspension daily by intragastric catheter.

Plasmids: The mRNA sequence encoding Gallus gallus egg albumin is retrieved from Genbank (accession number AY223553). Total chicken uterus RNA is isolated and cDNA is synthesized using 2 µg total RNA, 2 µM oligo dT primers (Promega Corporation Benelux, Leiden, The Netherlands), 0.01 mM DTT (Sigma-Aldrich, Zwijndrecht, The Countries Low), 0.5 mM dNTP (Invitrogen, Merelbeke, Belgium), 20 U of RNAsin (Promega Incorporation Benelux) and 100 U of Superscript II reverse transcriptase (Invitrogen) in a volume of 25 µl. The OVA cDNA fragment is amplified by polymerase chain reaction (PCR) using the following conditions: 94 ° C for 2 min followed by 30 cycles at 94 ° C for 45 seconds, 62 ° C for 30 seconds and 72 ° C for 90 seconds , with the following direct and inverse primers 5'-GGCTCCATCGGTGCAGCAAGCATGGAATT-3 '(SEQ ID NO: 17) and 5'-ACTAGTTAAGGGGAAAC-ACATCTGCCAAAGAAGAGAA-3' (SEQ ID NO: 18).

The amplified fragment is fused with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the lactococcal P1 promoter.

MG1363 strains transformed with plasmids carrying OVA cDNAs are designated LL-OVA. LL-pTREX1, which is MG1363 that contains the empty vector, serves as a control.

Quantification of OVA: LL-OVA OVA is determined using an adsorption enzyme immunoassay (ELISA) specific for internally developed OVA. The production of recombinant proteins is also evaluated by Western blot analysis.

Mice: BALB / c mice (6 to 8 weeks old) are purchased from Charles River Laboratories (Calco, Italy). Mice are kept under conditions without specific pathogens.

Immunization of mice: Mice are immunized ip with 10 µg of OVA (quality 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 an injection of saline instead of the OVA / alum solution. 26 days after immunization, the sensitized mice inhale an aerosolized solution of 1% OVA dissolved in PBS for 10 min. OVA inhalation is performed for 3 days in a row (days 47, 48 and 49). Control mice inhale PBS only under the same conditions that are used for the experimental group.

Oral tolerance induction: Mice receive LL-OVA, LL-pTREX1, 1 µg of OVA or BM9 on days 0-4, 7-11, 14-18 and 21-25. As a positive control for oral tolerance induction, mice are fed 30 mg of OVA per intragastric catheter that reduces bronchial eosinophilia and airway hyperreactivity, with high-dose feeding more effective than low-dose feeding.

Measurement of airway hyperreactivity (HVR): 24 h after final inhalation (day 50), airway hyperreactivity is assessed by obstruction of methacholine-induced airflow. The rats

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they are exposed for 2.5 min to nebulized physiological saline (Otsuka Pharmaceutical), followed by increasing dose (1-30 mg / ml) of nebulized methacholine. These mice are placed on a full-body plethysmograph for 2.5 min after nebulization, and the enhanced pause (Penh) is measured using the Biosystem XA WBP (Buxco Electronics) system. "Penh" represents the obstruction of the pulmonary air flow and is calculated using the formula: Penh = ((Te -Tr) / (Tr x PEF / PIF)), in which Penh = enhanced (dimensionless) pause, Te = time expiratory (seconds), Tr = relaxation time (seconds), PEF = peak expiratory flow (millimeters per second) and PIF = peak inspiratory flow (millimeters per second). Penh is measured and averaged approximately every 5 s, and the accumulated values ​​are averaged as the Penh value for each moment of time. Hyperreactivity of the airways is expressed as PC200Mch (200% stimulating concentration of methacholine), which is the concentration of methacholine that doubles the initial Penh value.

Bronchoalveolar lavage fluid (BALF) analysis: After measuring the airway hyperreactivity, bronchoalveolar lavage samples are obtained. Mice are sacrificed by ip injection of ketamine and xylazine overdose and then the lungs are washed with 0.5 ml of saline four times. The wash fluid is centrifuged and the cells are resuspended in 1 ml of saline with 1% BSA. Total cell numbers are counted using a hemocytometer. Cytocentrifugation samples are prepared by centrifuging the suspensions at 300 rpm for 5 min. To clearly distinguish eosinophils from neutrophils, three different stains are applied: Diff-Quick, May-Grünwald-Giemsa and Hansel (eosin) stains. At least 300 leukocytes are differentiated by optical microscopy based on conventional morphological criteria. The level of IL-13, IL-4 and IL-5 in BALF is detected by cytometric pearl assay (BD Biosciences, Mountain View, CA, USA) following the manufacturer's instructions.

Measurement of total serum IgE and OVA specific Ig: On day 50, retroorbital sinus blood samples are obtained under anesthesia. After the samples have completely coagulated, they are centrifuged, and the serum is collected and stored at -80 ° C until use. Total IgE is tested by ELISA using paired Ab (BD Pharmingen) according to the manufacturer's instructions. To measure IgE, IgG1 and IgG2a specific for OVA in sera, microtiter plates (Maxisorp, Nunc, VWR International, Haasrode, Belgium) are coated with 2 µg / ml of OVA. Subsequently, the wells are blocked with 0.1% casein in PBS, after which the plates are incubated with samples of mouse serum diluted from 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, Antwerp, Belgium, 1: 5000 dilution], goat anti IgG1-HRP - goat anti-mouse mouse or IgE-HRP (SBA, dilution 1: 5000). After washing, the substrate [3,3 ', 5,5'-tetramethylbenzidine (TMB), Pharmingen, Becton Dickinson, Erembodegem, Belgium] substrate is added to each well. Finally, the reactions are stopped by adding 1M H2SO4 to the wells. Absorbances are read at 450 nm. ELISA scores are expressed as titers, which are the inverse of the highest dilution that still had an OD450 greater than the calculated cut-off value. The cut-off point is calculated as the average OD450 of 5 non-immunized mice increased by three times the ED.

Histological examination of lung tissue: After obtaining bronchoalveolar lavage samples, the lungs are perfused with physiological saline and removed from the mice. The lungs are fixed with neutralized buffered formalin and incorporated in paraffin. Sections (3 µm thick) are stained with H&amp;E or periodic acid-Schiff (PAS). The intensity of histological changes in the lungs is evaluated with four classification scores (0, no inflammation; 1, mild / mild; 2, moderate; and 3, serious) according to the distribution and intensity of the following findings: 1) epithelial dissemination or undulation of the nuclei of bronchial epithelial cells, 2) increase in the number of goblet cells, 3) infiltration of inflammatory cells from the vessels in the mucosal and submucosal area of ​​the bronchus and peribronchial interstitium and 4) hypertrophy and thickening of the smooth muscle cell layer.

RT-PCR for the analysis of the cytokine and chemokine gene expression in the lung: The lungs are excised after perfusion with physiological saline and the total RNA is extracted using ISOGEN (Nippon Gene) according to the manufacturer's instructions. Total RNA (10 µg) is reverse transcribed using oligo primer (dT) 15 (Promega) and RNase H-Superscript II-reverse transcriptase (Invitrogen Technologies) at 42 ° C for 2 h. To ensure that each sample contained the same amount of cDNA, the concentration of -actin cDNA of each sample is first determined using primers specific for -actin. These samples are amplified during the appropriate number of cycles, so that the amount of PCR product remains in the linear part of the amplification curve. The PCR products are electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The levels of IL-13, eotaxin, IL-10, IFN- and TGF- are determined using the following sets of specific primers.

The direct sense primer for -actin 5'-ACGACATGGAGAAGATCTGG-3 '(SEQ ID NO: 19), and

5'-TCGTAGATGGGCACAGTGTG-3 'counter sense primer (SEQ ID NO: 20).

The direct sense primer for IL-13 5'-TCTTGCTTGCCTTGGTGGTCTCGC-3 '(SEQ ID NO: 21) and opposite direction 5'-GATGGCATTGCAATTGGAGATGTTG-3' (SEQ ID NO: 22).

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The direct sense primer for eotaxin 5'-GGGCAGTAACTTCCATCTGTCTCC-3 '(SEQ ID NO: 23) and the opposite direction primer 5'-CACTTCTTCTTGGGGTCAGC-3' (SEQ ID NO: 24).

The direct sense primer for IL-10 5'-TACCTGGTAGGAGTGATGCC-3 '(SEQ ID NO: 25), and

the opposite direction 5'-GCATAGAAGCATACATGATG-3 '(SEQ ID NO: 26).

The direct sense primer for IFN- 5'-CATAGATGTGGAAGAAAAGA-3 '(SEQ ID NO: 27), and

the opposite direction 5'-TTGCTGAAGAAGGTAGTAAT-3 '(SEQ ID NO: 28).

The direct sense primer for TGF- 5'-CTTTAGGAAGGACCTGGGTT-3 '(SEQ ID NO: 29), and

the opposite direction 5'-CAGGAGCGCACAATCATGTT-3 '(SEQ ID NO: 30).

Cell cultures, proliferation and cytokine assay: One day after final inhalation (day 21), individual spleen cell suspensions and mediastinal lymph nodes are prepared by passing the cells through 70 µm filter cell sieves (Becton / Dickinson Labware) Erythrocytes are removed from spleen cell suspensions by incubation with red cell lysis buffer. CD4 + T lymphocytes and CD4 + CD25-T lymphocytes are enriched using a CD4 + T lymphocyte isolation kit (Miltenyi Biotec, Germany) or a CD4 + CD25 + regulatory T lymphocyte isolation kit (Miltenyi Biotec, Germany), respectively, and MACS columns (midiMACS; Miltenyi Biotec).

Proliferation assays of splenocyte and LN populations in bulk, 2 x 105 cells are cultured in 96-well U-bottom plates in a total volume of 200 µl of complete medium both alone and with purified OVA. OVA is added at concentrations ranging from 1 to 100 µg / ml. For proliferation assays of CD4 + T lymphocytes and populations of CD4 + CD25- T lymphocytes, 2 x 105 CD4 + T lymphocytes or CD4 + CD25-T lymphocytes are cultured in 96-well U-bottom plates with spleomycytes treated with mitomycin that are loaded with 1 mg / ml of OVA for 16 h, acting as antigen presenting cells, at ratios of CD4 + T lymphocytes or CD4 + CD25- / APC T lymphocytes of 1/1, 1 / 0.3, 1/0, 1, 1 / 0.03, 1/0 in a total volume of 200 µl of complete medium. After 72 h at 37 ° C in a humidified incubator with 5% CO2, the proliferation is evaluated by the addition of 1 µCi / well of [3 H] -thymidine. DNA-bound radioactivity is collected 18 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer).

For cytokine measurements, the cell culture supernatants used in the different proliferation assays are collected after 24, 48 and 72 h of culture and frozen at -80 ° C until cytokine analysis is performed. Cytokine production is quantified using the mouse inflammation cytometric pearl assay (BD Biosciences, Mountain View, CA, USA).

Test of the regulatory activity of T in vivo: One day after the final inhalation (day 21), the spleens of the treated mice are digested with 0.1% collagenase (Sigma-Aldrich) at 37 ° C for 20 min. In some experiments, individual cell suspensions of whole spleen cells are prepared and cultured with Con A (2 µg / ml; Sigma-Aldrich) for 48 h. The cells are collected and 107 cells are transferred in an adoptive manner iv to BALB / c mice without prior treatment. For negative selection, CD4 +, CD8 +, CD11c +, CD19 + or CD11b + lymphocytes are depleted of the entire spleen cells using magnetic beads (MACS; Miltenyi Biotec) with anti-CD4 mAb, CD8, CD11c, CD19 and CD11b biotinylated mouse (BD Pharmingen), according to the manufacturer's instructions. The effectiveness of depletion is examined by flow cytometry (&gt; 99%). CD4 +, CD4 + CD25-lymphocytes are purified using a CD4 + T lymphocyte isolation kit and a regulatory T lymphocyte isolation kit according to the manufacturer's instructions. The purity of positively selected cells is checked using flow cytometry. For cell transfer experiments, the cells are transferred to 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 107 for complete spleen cells, spleen cells depleted in a subpopulation or CD4 + lymphocytes and CD4 + CD25-positively selected lymphocytes.

In the humanized SCID model (hu-SCID) (as described by Duez et al., 2000; Hammad et al., 2000)

In this model the allergic immune response to the house dust mite allergen (HDM) Der p 1 can be studied. Such ip-reconstituted hu-SCID mice with CMSP from patients allergic to HDM and subsequently exposed to HDM aerosols produce human IgE, develop a pulmonary infiltrate composed of activated T lymphocytes and DC and have HVR in response to bronchoconstrictor agents (Pestel et al., 1994, J Immunol, 153: 3804; Duez et al., Am J Respir Crit Care Med, vol. 161, p. 200-206, 2000).

Bacteria

L. lactis strain MG1363 is used throughout this study. Bacteria are grown in GM17 medium, that is, M17 (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose. The mother suspensions of all strains are

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Store at -20 ° C in 50% glycerol in GM17. For intragastric inoculations, the mother suspensions are diluted 200 times in fresh GM17 and incubated at 30 ° C. A saturation density of 2 x 109 colony forming units (CFU) per ml was reached within 16 hours. Bacteria are collected by centrifugation and concentrated 10 times in BM9 medium. For treatment, each mouse receives 100 µl of this suspension daily by intragastric catheter.

Plasmids

Der p 1, a globular glycoprotein of 222 amino acid residues, is one of the main allergens of Dermatophagoides pteronyssinus (Dpt) mites. The DNA sequence is synthesized using codons of optimal L. lactis encoding the Der p 1 protein, amplified and fused with the Usp45 secretion signal of the erythromycin resistant pT1NX vector downstream of the lactococcal P1 promoter. The MG1363 strains transformed with plasmids carrying murine Der p 1 cDNA, Der p 1 aa 52-71 and Der p 1 aa 117-133 are designated LL-Derp1, LL-Derplaa52-71 and LL-Derp1aa117-133. LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serves as a control.

Quantification of Der p1

Der p 1 of LL-Derp1 is determined using an adsorption enzyme immunoassay (ELISA) specific for internally developed Der p 1. The production of recombinant proteins is also evaluated by Western blot analysis.

Patients

Blood is collected from donors sensitive or not sensitive to house dust mites. Allergic patients have the usual sensitization characteristics to house dust mites. Dermatophagoides pteronyssinus (Dpt) allergen skin puncture tests (Stallergènes, 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 below 150 IU / ml and have negative skin puncture tests towards commonly inhaled allergens).

Preparation of human peripheral blood mononuclear cells

Platelet rich plasma is obtained after centrifugation (120 xg, 15 minutes) and discarded. Then, the blood cells are diluted in RPMI 1640 (Life Technologies, Paisley, Scotland) (vol / vol) and arranged in layers on a Ficoll gradient (Pharmacia, Uppsala, Sweden). After centrifugation (400 xg, 30 minutes), the CMSPs are collected at the interface and washed three times in sterile RPMI medium before transfer.

Mice

CB-17 SCID mice (6-8 weeks of age) are kept in insulators with sterilized beds in a specific animal farm. The SCID colony is checked regularly to determine the absence of mouse serum immunoglobulins by ELISA.

Transfer of peripheral blood mononuclear cells in SCID mice: Hu-SCID mice with CMSP

SCID mice are between 6 and 8 weeks old at the time of cell transfer. Mice are reconstituted by intraperitoneal injection of 10 x 106 mononuclear cells from allergic patients or healthy donors in 400 µl of RPMI using a 23 gauge needle. On the same day they receive 2 units of Dpt reactivity index [IR] intraperitoneally. Four days after cell reconstitution, SCID mice are exposed to allergen aerosols daily containing 100 units of IR of Dpt (100 units of IR are equivalent to approximately 200 µg of protein contained in Dpt extract) for 4 successive days (day 0 to day 4). The control group is not exposed to Dpt. One day before the measurement of airway reactivity (day 35 and day 60), the hu-SCID mice are exposed to another aerosol of 100 IR units of Dpt solution.

Experimental environment

Mice receive genetically engineered L. lactis to express Der p 1 or an irrelevant antigen (OVA) as a negative control.

Genetically engineered L. lactis bacteria are administered orally to SCID mice using a gastric catheter, using different intervals and treatment doses starting one day after reconstitution with CMSP. Oral tolerance induction is evaluated by measuring human serum IgE antibodies, pulmonary infiltration analysis, HVR measurement and analysis of cell populations and cytokine production in the BALF. In addition, tolerance induction is assessed by analysis of the proliferative response of T lymphocytes against Der p 1.

Airway reactivity assessment (HVR)

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The reactivity of the airways (expressed as a stimulant dose of carbacol that causes a 50% increase in lung resistance) is measured on day 35 or day 60 as described by Duez et al., 2000.

Human IgE measurements

Several days after transplantation with human cells, blood is drawn from the mice from the retroorbital sinus under anesthesia. Total human IgE is investigated by a two-site immunoradiometric procedure with the use of two different mouse mAbs specific for the  chain (Immunotech International, Luminy, France). At least 20 µl of serum is used in a duplicate test. The sensitivity of the procedure allows the detection of 0.1 IU / ml (0.24 ng / ml).

The specific IgE Ab against the 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 PBS 0 , 1 M (pH 7.4) for 2 h at room temperature. After washing, the tubes are incubated for 2 h at room temperature and overnight at 4 ° C with serum of Hu-SCID mice diluted in PBS containing BSA (1%) and Tween (0.01%). After extensive washing, an HRP-labeled human anti-IgE Ab is added. After washing, substrate [3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate, Pharmingen, Becton Dickinson, Erembodegem, Belgium] is added to each well. Finally, the reactions are stopped by adding 1M H2SO4 to the wells. Absorbances are read at 450 nm.

Histological examination of the lung

The lungs are removed on day 35 and fixed in paraformaldehyde and processed for incorporation into paraffin. The paraffin tissue sections are stained for the detection of human CD45 + cells, after which the human cells were quantified in the murine lung sections by histological scoring as described by Duez et al., 2000.

Analysis of bronchoalveolar lavage fluid (BALF)

BALF is analyzed as described in the OVA allergen model.

Cell cultures, proliferation and cytokine assay:

Individual spleen cell suspensions are prepared by passing the cells through 70 µm filter cell sieves (Becton / Dickinson Labware). The erythrocytes are removed from the spleen cell suspensions by incubation with red blood cell lysis buffer. CD4 + T lymphocytes and CD4 + CD25-T lymphocytes are enriched using a human CD4 + T lymphocyte isolation kit (Miltenyi Biotec, Germany) or a human CD4 + CD25 + regulatory T lymphocyte isolation kit (Miltenyi Biotec, Germany), respectively , and MACS columns (midiMACS; Miltenyi Biotec).

Mass splenocyte proliferation assays, 2 x 105 cells are cultured in 96-well U-bottom plates in a total volume of 200 µl of complete medium both alone and with purified Der p1, and both with and without anti-neutralizing monoclonal antibodies -IL-10 or anti-TGF-. Der p1 is added at concentrations ranging from 1 to 100 µg / ml. Neutralizing antibodies are added at 1, 0.1 and 0.01 µg / ml. For proliferation assays of human CD4 + T lymphocytes and populations of CD4 + CD25-human T lymphocytes, 2 x 105 CD4 + T lymphocytes or CD4 + CD25-T lymphocytes are cultured in 96-well U-bottom plates with human CMSP treated with mitomycin loaded with 1 mg / ml of Der p1 for 16 h, acting as antigen presenting cells, at ratios of CD4 + T lymphocytes or CD4 + CD25- / APC T lymphocytes of 1/1, 1 / 0.3, 1 / 0.1, 1 / 0.03, 1/0 in a total volume of 200 µl of complete medium both with and without neutralizing antibodies. After 72 h at 37 ° C in a humidified incubator with 5% CO2, proliferation is evaluated by the addition of 1 µCi / well of [3 H] -thymidine. DNA-bound radioactivity is collected 18 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer).

For cytokine measurements, the cell culture supernatants used in the different proliferation assays are collected after 24, 48 and 72 h of culture and frozen at -80 ° C until cytokine analysis is performed. Cytokine production is quantified using the mouse inflammation cytometric pearl assay (BD Biosciences, Mountain View, CA, USA).

Results

LL-OVA and LL-Der p 1 significantly enhances the tolerance-inducing capacity in the OVA model and in huSCID mice for asthma, respectively

To study oral tolerance induction, mice are fed orally as described above (experimental environment). The addition of LL-OVA / Derp1 significantly enhances the induction of tolerance towards OVA / Derp1 since the allergen-specific proliferative response of splenocytes is significantly reduced in

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the LL-OVA / Derp1 group compared to the control and free OVA / Derp1 groups.

LL-OVA / Derp1 enhances oral tolerance in association with reduced HVR, eosinophilic infiltration, serum IgE levels and reduced production of cytokines IL-13, IL-4 and reduced IL-5 in response to said allergen

To study oral tolerance induction, mice are fed orally as described above (experimental environment). HVR, infiltration of eosinophilic BALF, IgE titre, in addition to cytokine production in response to said antigens, is determined as described above. HVR, infiltration of eosinophilic BALF, IgE titre are strongly reduced, and IL-13, IL-4 and IL-5 were significantly reduced in the LL-OVA / Derp1 group compared to the control and OVA / Derp1 groups free.

LL-OVA / Derp1 enhances oral tolerance using CD4 + T lymphocytes

To assess whether CD4 T lymphocytes mediate or not induce oral tolerance, the allergen-specific proliferative CD4 T lymphocyte response is studied in splenocytes and lymph nodes. Therefore, mice are fed orally as described above (experimental environment) and proliferation of allergen-specific CD4 + T cells is determined as described in Cell cultures, proliferation and cytokine assay. The allergen-specific CD4 T lymphocyte response in the LL-OVA / Derp1 group is significantly reduced compared to the control and free OVA / Derp1 groups.

Antigen-induced regulatory T lymphocytes following LL-OVA therapy can transfer protection from asthma-like responses in vivo

In order to test the active suppression of asthma-like responses in mice treated with the oral tolerance protocol, the present inventors adopt adoptively splenocytes from the different groups treated as described above (T in vivo regulatory activity test). Compared to controls and free OVA groups, asthma-like responses are significantly reduced in the LL-OVA group, which indicates activation of regulatory CD4 + T cells in the combination oral tolerance protocol of the present inventors.

conclusion

The data of the present inventors demonstrate that administration through the L. lactis mucosa that secretes allergen is more potent than the free allergen to induce allergen specific immune tolerance by inducing antigen-specific CD4 + regulatory T lymphocytes, even in the hyperreactivity environment established.

EXAMPLE E: Induction of tolerance to BLG food allergen after oral administration of L. lactis secreting said allergen

Introduction

Food allergy is a disease that affects approximately 2% to 5% of the population. In humans, elevated IgE antibodies, in addition to the presence of IL-4-producing antigen-specific T lymphocytes, suggest a mechanism deviated towards Th2. Here, the present inventors demonstrate that oral administration of a food allergen by L. lactis suppresses allergen-specific immune responses by inducing antigen-specific CD4 + regulatory T lymphocytes.

Material and procedures for the examples

Bacteria and plasmids

L. lactis strain MG1363 is used throughout this study. Bacteria are grown in GM17 medium, that is, M17 (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at -20 ° C in 50% glycerol in GM17. For intragastric inoculations, the mother suspensions are diluted 200 times in fresh GM17 and incubated at 30 ° C. They reach a saturation density of 2 x 109 colony forming units (CFU) per ml within 16 hours. Bacteria are collected by centrifugation and concentrated 10 times in BM9 medium. For treatment, each mouse receives 100 µl of this suspension daily by intragastric catheter. Bovine -lactoglobulin cDNA is amplified and fused with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the lactococcal P1 promoter.

MG1363 strains transformed with plasmids carrying murine BLG are designated LL-BLG. LL-pT1 NX, which is MG1363 containing the empty vector pT1 NX, serves as a control.

Quantification of bovine -lactoglobulin (BLG)

BLG of LL-BLG is determined using an adsorption enzyme immunoassay (ELISA) specific to BLG developed

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internally and Western blot analysis.

Experimental environment

The murine food allergy model used to explore the protective effect of L. lactis is a food-induced IgE-type mouse response model as described by Frossard et al. (J Allergy Clin Immunol 113: 958-964, 2004). Mice receive LL-BLG or an irrelevant antigen (OVA) as a negative control. As a positive control for tolerance induction, mice receive a high dose of BLG in drinking water that prevents anaphylaxis in mice after oral exposure to BLG.

In a prophylactic environment, genetically engineered L. lactis bacteria that produce BLG are administered orally to mice using a gastric catheter, using different intervals and treatment doses. Subsequently, these recipient mice are orally exposed to purified BLG antigen, in the presence of cholera toxin. Control animals are exposed to L. Genetically engineered lactis with a control vector that does not express BLG (but OVA instead). Tolerance induction is evaluated by anaphylaxis analysis after intragastric exposure to antigen, measuring IgG1, IgG2a and IgE titers specific for serum BLG and feces, determining the number of cells that secrete antibodies in spleen and PP, by analysis of T lymphocyte proliferation and cytokine production in MLN, PP and spleen.

To assess whether induction of immune tolerance to BLG could be enhanced or not by L. lactis, mice are administered with LL-BLG or with 1 µg of free BLG.

Oral sensitization to BLG

Four to 5 week old female C3H / HeOuJ mice (Charles River) are immunized on days 0, 7, 14 and 21 by intragastric nasogastric tube with 20 mg of BLG (Sigma) and 10 µg of CTX, purchased from List Biological Laboratories in 0.2 mol / l NaHCO3. The positive control group (tolerated mice) receives 0.8 mg / ml of BLG in their drinking water at will for 4 weeks. The total amount of protein administered (22.4 mg) is similar to the total amount of BLG administered to sensitized mice. To demonstrate that the tolerization procedure also permanently activates the peripheral immune system and not just the mucous, a group of tolerated mice is injected twice with 80 µg of BLG ip adsorbed at 1 alum on days 28 and 42.

Antigen exposure

On day 28, all mice are exposed by intragastric nasogastric tube to 100 mg of BLG in 0.4 ml of 0.2 moles of NaHCO3. Anaphylaxis is observed and classified using a reaction score (0, no reaction, up to 3, severe reaction or death) described in detail elsewhere (Frosssard et al., 2001). The core body temperature is measured by infrared in the ear before exposure and 30 minutes after the nasogastric tube. Animals are sacrificed and blood is collected by cardiac puncture in tubes containing EDTA, and plasma is obtained for histamine measurement by a commercial ELISA kit (Immunotech, Marseille, France).

Cell cultures, proliferation and cytokine assay

Suspensions of individual spleen cells, mesenteric lymph nodes and PP are prepared as described by Frossard et al. (2004). CD4 + T lymphocytes and CD4 + CD25-T lymphocytes are enriched using the CD4 + T lymphocyte isolation kit (Miltenyi Biotec, Germany) or the CD4 + CD25 + regulatory T lymphocyte isolation kit (Miltenyi Biotec, Germany), respectively, and MACS columns (midiMACS; Miltenyi Biotec).

Proliferation assays of mass splenocyte and LN populations, 2 x 105 cells are grown in 96-well U-bottom plates in a total volume of 200 µl of complete medium both alone and with purified BLG, and with and without anti-IL-10 or anti-TGF- neutralizing monoclonal antibodies. BLG is added at concentrations ranging from 1 to 100 µg / ml. Neutralizing antibodies are added at 1, 0.1 and 0.01 µg / ml. For CD4 + T cell proliferation assays and CD4 + CD25- T cell populations, 2 x 105 CD4 + T cell cells or CD4 + CD25 T cells are grown in 96-well U-bottom plates with spleomycytes treated with mitomycin that are loaded with 1 mg / ml of BLG for 16 h, acting as antigen presenting cells, at ratios of CD4 + T lymphocyte or CD4 + CD25- / APC T 1/1, 1 / 0.3, 1 / 0.1 , 1 / 0.03, 1/0 in a total volume of 200 µl of complete medium both with and without neutralizing antibodies. After 72 h at 37 ° C in a humidified incubator with 5% CO2, proliferation is evaluated by adding 1 µCi / well of [3 H] -thymidine. The DNA-bound radioactivity is collected 18 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer).

For cytokine measurements, the cell culture supernatants used in the different proliferation assays are collected after 24, 48 and 72 h of culture and frozen at -80 ° C until the cytokine analysis is performed. Cytokine production is quantified using the mouse inflammation cytometric pearl assay (BD Biosciences,

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Mountain View, CA, USA).

Test of regulatory activity of T in vivo

In order to test the active suppression of antibody formation in mice, splenocytes, CD4 + T lymphocytes, purified CD4 + CD25- or CD4 + CD25 + T lymphocytes in beads isolated from the different experimental groups treated with

L. lactis are adopted adoptively to C3H / HeOuJ mice without prior treatment. Untreated mice are used as control. The number of transfected cells is 107 for complete spleen cells, spleen cells depleted in subpopulations or positively selected CD4 + cells and CD4 + CD25- and CD4 + CD25 + T cells. If Treg is involved, after exposure of these mice to the BLG antigen, they should prevent the induction of humoral immune responses against BLG and anaphylaxis.

Adsorption enzyme immunoassay for serum antibodies and feces specific for BLG

Serums from tail hemorrhages are obtained on day 0, 7, 14, 21 and 28. Feces are obtained at the same time and resuspended in PBS plus 1% of SBF (Life Technologies) supplemented with 1: 1000 pepstatin (Fluka ) at 0.1 mg / ml. The samples are mechanically disintegrated and vortexed for 2 minutes, followed by two centrifugations at 4 ° C for 20 minutes at 14,000 rpm.

Serums and feces are tested for levels of IgE, IgG1, IgG2a and / or IgA antibody specific for BLG by a method adapted from Adel-Patient et al. (2000, J. Immunol Methods). In summary, MaxiSorp microtiter plates (Nunc) are coated for 18 hours at room temperature with 250 ng / well of streptavidin (Fluka), followed by 300 µl of a K25 polyvinylpyrrolidone solution (Fluka) overnight. One microgram of biotinylated BLG is incubated for 3 hours and 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 in duplicate in the presence of 0.5 µg / ml of goat anti-mouse IgA antibodies , of rat anti-mouse IgG1 or anti-mouse IgG2a labeled with peroxidase (Southern Biotechnologies) for 2 hours. For IgE measurement, a mouse anti-mouse IgE monoclonal rat Ab (clone R35-72, BD Pharmingen) is added followed by peroxidase-coupled anti-rat Ab (Caltag). The optical density is measured at 490 nm. The results are expressed as arbitrary units, using pooled sera from mice immunized with BLG plus alum as reference serum.

Production of antibodies specific for antigen measured by ELISPOT

Peyer plates are mechanically removed from the intestine and incubated for 30 minutes in HBSS medium supplemented with 5 mmol of EDTA (Life Technologies). Similarly, Peyer plates and mesenteric lymph nodes are gently crushed and filtered through a 70 µm nylon filter. Spleen cells are previously incubated for 5 minutes in Tris buffered NH4Cl to remove red blood cells. Lymphoblasts are isolated on a gradient of 60% / 66% Percoll (Amersham).

For the measurement of IgG1, IgG2a and IgA antibodies specific for BLG, ELISPOT plates (Millipore) are coated with streptavidin overnight at 37 ° C, followed by the addition of 1 g of biotinylated BLG for 3 hours. Lymphoblasts are isolated on a gradient of 60% / 66% Percoll and resuspended at two different concentrations, 1 and 2 x 106 in Dulbecco's medium modified by lscove supplemented with penicillin, streptomycin, L-glutamine, gentamicin, polymyxin B and 5% SBF for 24 hours at 37 ° C, followed by overnight incubation at 4 ° C with antilgA, anti-IgG1 and anti-IgG2a (Southern Biotechnology) antibodies. Amino-ethyl-carbazole, 100 µl / well, is added over 10 minutes and the spots are automatically counted using the KS ELISPOT 4.2.1 software (Zeiss) and expressed as cell-forming units per 106 cells (CFU).

LL-BLG significantly enhances the tolerance inducing capacity of BLG in the murine food allergy model

To study oral tolerance induction, mice are fed orally as described above (experimental environment). The addition of LL-BLG significantly enhances the induction of tolerance towards BLG since the allergen-specific proliferative response of splenocytes is significantly reduced in the LL-BLG group compared to the control and free BLG groups.

LL-BLG enhances oral tolerance in association with reduced response of BLG-specific antibodies and decreased production of cytokine IL-4 in response to said allergen.

To study oral tolerance induction, mice are fed orally as described above (experimental environment). The BLG-specific antibody response and cytokine production in response to that factor are determined as described above. The specific antibody levels for BLG and IL-4 decrease significantly in the LL-BLG group compared to the control and free BLG groups.

Results

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LL-BLG enhances oral tolerance through CD4 + T lymphocytes

To assess whether CD4 T lymphocytes mediate or not induce oral tolerance, the allergen-specific proliferative CD4 T lymphocyte response is studied in splenocytes and lymph nodes. Therefore, mice are fed orally as described above (experimental environment) and the proliferation of allergen-specific CD4 + T cells is determined as described in Cell cultures, proliferation and cytokine assay. The allergen-specific CD4 T lymphocyte response in the LL-BLG group is significantly reduced compared to the control and free BLG groups.

Antigen-induced regulatory T lymphocytes following LL-BLG therapy may transfer protection from allergic-like responses in vivo

In order to test the active suppression of allergic-like responses in mice treated with the oral tolerance protocol, the present inventors adopt adoptively splenocytes from the different treated groups as described above (T in vivo regulatory activity test). Compared to controls and groups of free BLG, allergic-like responses are significantly reduced in the LL-BLG group, which indicates activation of CD4 + regulatory T lymphocytes in the combination oral tolerance protocol of the present inventors.

conclusion

The data of the present inventors demonstrate that administration through the L. lactis mucosa that secretes allergen is more potent than the free allergen in inducing allergen specific immune tolerance by inducing antigen-specific CD4 + regulatory T lymphocytes.

EXAMPLE F: Induction of insulin tolerance after oral administration of L. lactis that secretes said autoantigen

Introduction

Autoimmunity is characterized by spontaneous inflammatory tissue injury and altered physiological function resulting from the loss of tolerance to an antigen itself. It is associated with a partially hyperactive immune system, which is characterized by an excess of T helper lymphocytes (Th). It is difficult to influence predisposing factors, such as susceptibility genes and environmental factors, so recent efforts to develop immunotherapies focus on restoring the functional balance between pathogenic effector cells and immunoregulatory T lymphocytes reducing the former and / or enhancing the latter . Autoimmune destruction of beta cells from pancreatic islets is the leading cause of type 1 diabetes mellitus (T1D). This destruction is associated with cellular and humoral immune responses to several beta cell autoantigens, both of which may precede the clinical onset of the disease.

Here, the present inventors demonstrate that oral administration of an autoantigen that releases L. lactis suppresses specific immune responses for diabetics by inducing antigen-specific CD4 + regulatory T lymphocytes.

Material and procedures

Bacteria and plasmids

L. lactis strain MG1363 is used throughout this study. Bacteria are grown in GM17 medium, that is, M17 (Difco Laboratories, Detroit, MI) supplemented with 0.5% glucose. Stock suspensions of all strains are stored at -20 ° C in 50% glycerol in GM17. For intragastric inoculations, the mother suspensions are diluted 200 times in fresh GM17 and incubated at 30 ° C. They reach a saturation density of 2 x 109 colony forming units (CFU) per ml within 16 hours. Bacteria are collected by centrifugation and concentrated 10 times in BM9 medium. For treatment, each mouse receives 100 µl of this suspension daily by intragastric catheter.

DNA sequence is synthesized using codons of L. lactis optimum encoding human proinsulin II B24-C36 peptide (hpllp), porcine insulin and InsB9-23 immunodominant peptide (B9-23 is essentially the same throughout many species human, rat and mouse), are amplified and fused with the Usp45 secretion signal of the erythromycin-resistant pT1NX vector, downstream of the lactococcal P1 promoter.

MG1363 strains transformed with plasmids carrying murine hpllp, insulin, InsB9-23 are designated LL-hpllp, LLinsulina, LL-InsB9-23. LL-pT1NX, which is MG1363 containing the empty vector pT1NX, served as a control. The expression of these proteins is determined using antigen specific ELISA and Western blot analysis.

Mice

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Non-obese male and female diabetic mice (NOD) and NOD mice with severe combined immunodeficiency (SCID) (Balb / c background) are purchased from Jackson laboratory. Natural mice (WT) Balb / c are purchased from Charles River Italy. Mice are kept in a central animal without specific pathogens. Mice are treated and used according to institutional guidelines.

Experimental environment

In a prophylactic environment, LL-hpllp, LL-insulin, LL-InsB9-23 are administered orally to NOD mice starting from day 21 of age (weaning) and using the optimal feeding schedule or up to 100 days of age (when most mice develop diabetes). In addition, LL-pT1NX is administered orally as a negative control. For the positive control group (tolerant), 3-week-old NOD mice are treated orally with 0.8 mg of human insulin / hpllp / InsB9-23 for 3 times a week for 2 or 4 weeks. The development of diabetes is determined by continuous monitoring of urine glucose levels three times a week and in the case of glycosuria by monitoring blood glucose levels. The pancreas are collected at 12-23 weeks and at the end of the experiment (35 weeks) and the serial sections are stained with hematoxylin / eosin to score mononuclear cell infiltration or by immunohistochemistry to analyze T lymphocyte infiltration.

In a therapeutic environment, LL-hpllp, LL-insulin, LL-InsB9-23 are administered orally to diabetic NOD females showing glucosuria and stable hyperglycemia (12-23 weeks). In addition, LL-pT1NX is administered orally as a negative control. For the positive control group (tolerant), diabetic NOD mice are treated as described in Bresson et al., 2006. Complete remission is defined as the disappearance of glycosuria and the return to normal blood glucose.

In a syngeneic islet transplant environment, female NOD mice with newly occurring diabetes are treated orally for 3 weeks with LL-hpllp, LL-insulin, LL-InsB9-23 or with LL-pT1 NX as a negative control. After 3 weeks, 500 recently isolated pancreatic islets from non-diabetic NOD mice are transplanted into diabetic NOD mice. Then, blood glucose is monitored 3 times weekly until the recurrence of diabetes or up to 15 weeks after grafting. Animals with 2 consecutive glucose levels ≥ 250 mg / dl are considered diabetic and will subsequently be sacrificed for serum collection and graft histological analysis.

Precise tolerance induction mechanisms are analyzed in vitro, in vivo after reexposing NOD mice to specific autoantigens and by adoptive transfer of T lymphocytes to NOD-SCID mice.

Diabetes screening:

Glucose monitoring: urine glucose is measured using Diastix (Miles) and confirmed by blood glucose measurements with the OneTouch Ultra blood glucose monitoring system (LifeScan Inc.). Diabetes is defined as 2 consecutive blood glucose values ​​greater than 250 mg / dl.

Insulitis: Mice are sacrificed by asphyxiation with CO2 and the pancreas is fixed in 10% formalin overnight, incorporated into paraffin and 5 µm sections in series are stained with hematoxylin and eosin. The insulitis score (mean ± SD) is determined by microscopically classifying the degree of cell infiltration into 10-15 islets / mouse as follows: 0, with no visible signs of islet infiltration; 1, peri-islet infiltration; 2, &lt;50% infiltration; 3,&gt; 50% infiltration.

Isolation and transplantation of islets: The islets of donor NOD mice aged 14 to 21 days without insulitis and diabetes are isolated after aseptic elimination by digesting the pancreatic glands with collagenase in saline solution balanced with Hanks during vigorous agitation. Islet isolation is carried out by direct hand selection under a stereomicroscope. NOD diabetic receptor mice were anesthetized by intraperitoneal injection of avertine (0.02 ml / g BWT), the left kidney was exposed by lumbar incision and 500 newly isolated islets were administered under the renal capsule.

Immunohistochemistry

To detect insulin, the expression of CD4 and CD8 in pancreatic β cells, primary Ab (of Dako swine anti-insulin guinea pig [1: 300 dilution], RM4.5 anti-CD4 and IHC anti-CD8a of BD Biosciences [dilution 1 : 50]) apply to frozen tissue sections as described in Christen et al., 2004.

In vitro proliferation assay

Individual spleen cell suspensions, mesenteric LN (MLN) and PLN are prepared. Proliferation assays of populations of total splenocytes, 2 x 105 cells are cultured in 96-well U-bottom plates in a total volume of 200 µl of complete medium both alone and with graded concentrations (1-100 µg / ml) of human insulin purified or specific peptides for CD4 T lymphocytes (InsB9-23, restricted by H-2d og) or for CD8 T T lymphocytes (InsB1523, restricted by Kd) (Sigma), and both with and without anti-IL-10 or anti-TGF- neutralizing monoclonal antibodies. Neutralizing antibodies are added at 1, 0.1 and 0.01 µg / ml. For total CD3 + T cell proliferation assays,

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CD8 + T lymphocytes, CD4 + T lymphocytes and populations of CD4 + CD25- T lymphocytes, 0.2 x 105 T lymphocyte cells are grown in 96-well U-bottom plates with 1 x 105 irradiated splenocytes of loaded Balb / c WT mice with insulin or GAD65 or specific peptides for CD4 + or CD8 + T lymphocytes, in a total volume of 200 µl of complete medium both with and without neutralizing antibodies. After 72 h at 37 ° C in a humidified incubator with 5% CO2, proliferation is evaluated by adding 1 µCi / well of [3 H] -thymidine. The DNA-bound radioactivity is collected 1618 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer). T lymphocytes are purified from PLN or spleens by negative selection through separation of magnetic beads using the CD3 +, CD4 + or CD8 + isolation kit (MACS; Milteny Biotec, Auburn, CA). CD4 + T lymphocytes are used as total cells or are further separated into CD25 + and CD25 by MACS using the CD25 + isolation kit (Milteny Biotec). The purity (&gt; 90%) of cell populations is determined by flow cytometry analysis.

For cytokine measurements, the cell culture supernatants used in the different proliferation assays (antigen specific stimulation), described above, are collected after 72 h of culture and frozen at 80 ° C until the cytokine analysis is performed . Cytokine production is quantified using the mouse inflammation cytometric pearl assay (BD Biosciences, Mountain View, CA, USA). Purified CD3 +, CD4 + T lymphocytes or purified CD8 + T lymphocytes are cultured and stimulated in vitro not specifically with a mixture of anti-CD3 / anti-CD28 (1 µg / ml each) for 24 hours or remain unstimulated as a control. Supernatants are collected and analyzed for the production of IL-10, IL-4, IL-5 and IFN- using BD ™ Cytometric Bead Array flex set in a BD FACSArray bioanalyzer using FCAP array software (BD Biosciences). Capture ELISA experiments are used to determine TGF-usando1 using the Quantikine kit (R&amp;D Systems).

In vitro T cell proliferation inhibition assay

two x 104 purified total CD4 + CD25-splenic T lymphocytes isolated from recently diabetic female NOD mice (8-12 weeks) are co-cultured with varying numbers of CD8 + T lymphocytes, CD4 + T lymphocytes and CD4 + CD25-isolated T lymphocyte populations spleen, MLN or PLN of the different experimental groups in the presence of 2 x 104 splenocytes loaded with insulin or reduced irradiated peptides in T lymphocytes of Balb / c WT mice. After 72 h at 37 ° C in a humidified incubator with 5% CO2, proliferation is evaluated by adding 1 µCi / well of [3 H] -thymidine. The DNA-bound radioactivity is collected 16-18 h later on fiberglass filter mats (Perkin Elmer, Boston, USA) and thymidine incorporation is measured in a scintillation counter (Perkin Elmer).

In vitro cytotoxicity test

The lymphoblast targets used are splenocytes activated with Con A from BALB / c mice. A total of 106 target cells are labeled with 100 µCi of 51 Cr (Amersham International, Buckinghamshire, UK) for 90 min at 37 ° C, washed three times and then incubated with 1 µg / ml peptide (InsB15-23 or a peptide irrelevant) at 37 ° C for 1 h. The target cells are washed twice and seeded at 104 cells per well. CD8 + T lymphocytes, isolated from spleen, MLN and PLN, are added to each well, in triplicate, to various effector ratios: target (E: D). The plates are centrifuged at 500 rpm for 2 min and incubated at 37 ° C for 4 h. After incubation, the supernatants are collected for the determination of the release of 51 Cr [[% lysis = 100 x (test cpm - spontaneous cpm) / (total cpm - spontaneous cpm)]. For the indirect destruction assay, CD8 + T lymphocytes are incubated with 5 µg / ml anti-CD3 antibody (clone 145-2C11, Pharmingen) before incubation with effectors.

Adoptive diabetes transfer

NOD-SCID mice at 8-10 weeks are injected iv with 2 x 107 or ip with 5 x 106 splenocytes isolated from diabetic female NOD mice (6 weeks, 12 weeks and 18 weeks) combined with or without graduated numbers of CD3 + T lymphocytes , CD8 + T lymphocytes, CD4 + T lymphocytes, CD4 + CD25- or CD4 + CD25 + T lymphocytes purified in beads isolated from the different experimental groups treated with L. lactis. Untreated mice are used as control. The development of diabetes is determined by continuous monitoring of blood glucose levels three times a week.

Results

LL-hpllp, LL-insulin, LL-InsB9-23 delays the recurrence of diabetes after transplantation of syngeneic islets

To assess whether LL-hpllp, LL-Insulin and LL-InsB (9-23) induce or not oral tolerance, the recurrence of diabetes after transplantation of syngeneic islets is studied. Therefore, mice are fed orally as described above (experimental environment) and pancreatic islets are transplanted as described (Isolation and transplantation of islets). The recurrence of diabetes is delayed in the LL-hplip / insulin / insB9-23 group compared to the control.

LL-hpllp, LL-insulin or LL-InsB9-23 significantly enhance the tolerance capacity of free hpllp, insulin or InsB9-23 in the non-obese diabetic mouse

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To study oral tolerance induction, mice are fed orally as described above (experimental environment). The addition of LL-hpllp, LL-insulin, LL-InsB9-23 significantly enhances the induction of tolerance towards autoantigen since the splenocyte-specific autoantigen proliferative response is significantly reduced in the LL-hpllp / insulin / InsB9 group -23 compared to the control and hpllp / insulin / lnsB9-23 free groups.

LL-hpllp, LL-insulin or LL-lnsB9-23 enhance oral tolerance in association with reduction of insulitis, decrease in the rate of destruction of beta cells and increase in the production of IL-10 by splenocytes To study tolerance induction orally, mice are fed orally as described above (experimental environment). The presence of insulitis, the rate of destruction of beta cells and the production of cytokines in response to said autoantigen is determined as described above. Histological analysis shows a lower degree of significant insulitis and destruction of beta cells and high IL-10 production in the LL-hpllp / insulin / insB9-23 group compared to the control and hplip / insulin / insB9-23 groups. free.

LL-hpllp, LL-insulin, LL-InsB9-23 enhances oral tolerance by CD4 + T lymphocytes

To assess whether or not CD4 T lymphocytes mediate oral induction tolerance, the response of proliferative CD4 T lymphocytes specific for autoantigen is studied in splenocytes and lymph nodes. Thus, mice are fed orally as described above (experimental environment) and the proliferation of CD4 + T lymphocytes specific for autoantigen is determined as described (in vitro proliferation assay). The response of CD4 T lymphocytes specific for autoantigen in the LL-hpllp / insulin / InsB9-23 group compared to the control and free hpllp / insulin / InsB9-23 groups.

Example F5: Autoaggressive CD8 + responses are suppressed in NOD mice after LL-InsB9-23 therapy

To examine whether this combination approach of the present inventors induces CD4 + T lymphocytes or suppressors that can modulate diabetes by circumstantial suppressor mechanisms, the present inventors analyze the effect on CD8 + autoaggressive T lymphocytes. The percentage and / or activity of antigen-specific CD8 + lymphocytes is strongly reduced after LL-InsB9-23 therapy.

Antigen-induced regulatory T lymphocytes following LL-InsB9-23 therapy can transfer protection from autoimmune-like responses in vivo

In order to test the active suppression of diabetic-like responses in mice treated with the oral tolerance protocol, the present inventors adopt adoptively splenocytes from the different treated groups as described above (adoptive transfer of diabetes). Compared to controls and the free InsB9-23 group, diabetic-like responses are significantly reduced in the LL-InsB9-23 group, which indicates activation of CD4 + regulatory T lymphocytes in the combination oral tolerance protocol of the present inventors. .

conclusion

The present inventors demonstrate that oral administration of L. lactis that releases autoantigen suppresses specific immune responses for diabetics by inducing antigen-specific CD4 + regulatory T lymphocytes.

DISCUSSION

Taken together, the data presented above indicates that oral supplementation of genetically modified L. lactis that secretes antigens can decrease systemic inflammation induced by that antigen, even in a sensitized subject. Advantageously, suppression mediated by Lactococcus often seems more potent than after administration through the free antigen mucosa. Possibly, suppression can be mediated by induction of Foxp3 + regulatory T lymphocytes.

REFERENCES

-Friedman A. and Weiner, HL (1994). Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl Acad. Sci, 91, 6688-6692.

-Gasson, MJ Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154, 1-9 (1983).

-Liu et al. (2006) Engineered vaginal Lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor Cyanovirin-NAAC 50, 3250-3259.

-Marietta, E. et al. A new model for dermatitis herpetiformis that uses HLA-DQ8 transgenic NOD mice. J. Clin.

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<dl><dt>Invest 114, 1090-1097 (2004). </dt><dd /></dl>

<dl><dt>-</dt><dd>Mayer, L. and Shao, L. (2004a). The use of oral tolerance in the therapy of chronic inflammatory / autoimmune diseases. J. Pediatr. Gastroenterol Nutr. 39, S746-S747.</dd></dl>

<dl><dt>5 </dt><dd>- Mazzarella, G. et al. An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients. Gut 52, 57-62 (2003).</dd></dl>

<dl><dt>-</dt><dd>Mucida, D., Kutchukhidze, N., Erazo, A., Russo, M., Lafaille, JJ and Curotto de Lafaille, MA (2005). Oral tolerance in the absence of naturally occurring Tregs. J. Clin. Invest. 115, 1923-1933.</dd></dl>

<dl><dt>10 </dt><dd>- Steidler, L. and Rottiers, P. (2006) Therapeutic drug delivery by genetically modified Lactococcus lactis. Ann NY Acad. Sci. 1072, 176-186.</dd></dl>

<dl><dt>-</dt><dd>Strobel, S., Mowat, AM, Drummond, HE, Pickering, MG and Ferguson, A. (1983) Immunological responses to fed protein antigens in mice. II oral tolerance for CMI is due to activation of cyclophosphamide-sensitive cells by gut-processed antigen. Immunology, 49, 451-456.</dd></dl>

<dl><dt>15 </dt><dd>- Tobagus, IT, Thomas, WR, &amp; Holt, PG Adjuvant costimulation during secondary antigen challenge directs qualitative aspects of oral tolerance induction, particularly during the neonatal period. J. Immunol. 172, 2274-2285 (2004).</dd></dl>

<dl><dt>-</dt><dd>Van Asseldonk, M. et al. Cloning of Usp45, A Gene Encoding A Secreted Protein from Lactococcus-Lactis Subsp Lactis Mg1363. Gene 95, 155-160 (1990).</dd></dl>

<dl><dt>20 </dt><dd>- Waterfield, NR, Lepage, RWF, Wilson, PW, &amp; Wells, JM The Isolation of Lactococcal Promoters and Their Use in Investigating Bacterial Luciferase Synthesis in Lactococcus-Lactis. Gene 165, 9-15 (1995).</dd></dl>