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

Abstract

The present invention relates to the treatment of autoimmune and allergic diseases by mucosal delivery by micro-organism, in particular Lactococcus lactis, of secreted immunodominant antigens.

Description

[0001]The present invention relates to the treatment of autoimmune and allergic diseases by mucosal delivery by micro-organisms, in particular Lactococcus lactis, of secreted immunodominant antigens.FIELD OF THE INVENTION[0002]The immune system has the task of distinguishing between self and non-self. The mucosal immune system, present along the respiratory, gastrointestinal and genitourinary tracts, has the additional burden of coexisting with an abundance of bacteria and innocuous antigens, such as food, airborne antigens or the commensally bacterial flora. A key feature of the mucosal immune system is its ability to remain tolerant to these antigens while retaining the capacity to repel pathogens effectively. Introduction of antigen systemically, whether by injection or injury, leads to local infiltration of inflammatory cells and specific immunoglobulin production. By contrast, antigens introduced at mucosal surfaces, such as the gastrointestinal and genitourinary tracts, elicit...

AI Technical Summary

Benefits of technology

[0033]A high degree of expression can be achieved by using homologous expression and / or secretion signals on the expression vectors present in the micro-organism, e.g. L. lactis. Suitably expression regulating signals as 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 optimised for expression depending on the micro-organism, e.g. L. lactis, it is incorporated in. For instance, specific expression vectors that gave sufficient levels of expression in Lactococcus, Lactobacillus lactis, casei and plantarum are known. Moreover, systems are known which have been developed for the expression of heterologous antigens in the non-pathogenic, non-colonising, non-invasive food-grade bacterium Lactococcus lactis (see UK patent GB2278358B, which is incorporated herein by reference). A particularly preferred construct according to the invention comprises the multi-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 construct is particularly suitable for expression of a desired antigen in a lactic acid bacterium, in particular in a Lactobacillus, at a high level of expression, and also can be used advantageously to direct the expressed product to the surface of the bacterial cell. The constructs (e.g. of PCT / NL95 / 00135) may be characterised in that the nucleic acid sequence encoding the antigen is preceded by a 5′ non-translated nucleic acid sequence comprising at least the minimal sequence required for ribosome recognition and RNA stabilisation. This can be followed by a translation initiation codon which may be (immediately) followed by a fragment of at least 5 codons of the 5′ terminal part of the translated nucleic acid sequence of a gene of a lactic acid bacterium or a structural or functional equivalent of the fragment. The fragment may also be controlled by the promoter. The contents of PCT / NL95 / 00135, including the differing 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 which permits the high level regulated expression of heterologous genes in the host and the coupling of expression to secretion. In a further preferred embodiment, the T7 bacteriophage RNA polymerase and its cognate 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.

[0034]A promoter employed in accordance with the present invention is preferably expressed constitutively in the bacterium. The inventors observed that constitutive expression of the antigen resulted in increased immune tolerance in contrast to inducible expression. Furthermore, the use of a constitutive promoter avoids the need to supply an inducer or other regulatory signal for expression to take place. Preferably, the promoter directs expression at a level at which the bacterial host cell remains viable, i.e. retains some metabolic activity, even if growth is not maintained. Advantageously then, such expression may be at a low level. For example, where the expression product accumulates intracellularly, the level of expression may lead to accumulation of the expression product at less than about 10% of cellular protein, preferably about or less than about 5%, for example about 1-3%. The promoter may be homologous to the bacterium employed, i.e. one found in that bacterium in nature. For example, a Lactococcal promoter may be used in a Lactococcus. A preferred promoter for use in Lactococcus lactis (or other Lactococci) is “P1” derived from the chromosome of Lactococcus lactis (Waterfield, N R, Lepage, R W F, Wilson, P W, 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.

[0043]The present invention further demonstrated that the Treg cells which were induced and / or expanded by the antigens according to the invention decreased inflammation, in particular in the spleen and inguinal lymph node cells. Moreover, the IFN-γ and IL-12 production was decreased. Accordingly, the present invention provides immunodominant antigens which decrease endogenous IFN-γ and / or IL-12 production, and / or stimulate endogenous TGF-β and / or IL-10 expression. Moreover, the present invention relates to antigens reducing proliferation of spleen and / or inguinal lymph node cells. It will be appreciated that the present invention relates also to antigens suppressing inflammatory antigen specific T cell response.

[0066]Delivery as used here means any method of delivery known to the person skilled in the art and includes, but is not limited to, coated or non-coated pharmaceutical formulations of the compound to deliver, capsules, liposomes, oil bodies, polymer particles comprising or carrying the compound to deliver or micro-organisms secreting, displaying or accumulating the compound to deliver, optionally in presence of compounds that may enhance mucosal delivery and / or mucosal uptake.

[0085]Nutraceuticals, whether in the form of a liquid extract or dry composition, are edible and may be eaten directly by humans, but are preferably provided to humans in the form of additives or nutritional supplements e.g., in the form of tablets of the kind sold in health food stores, or as ingredients in edible solids, more preferably processed food products such as cereals, breads, tofu, cookies, ice cream, cakes, potato chips, pretzels, cheese, etc., and in drinkable liquids e.g., beverages such as milk, soda, sports drinks, and fruit juices. Thus, in one embodiment a method is provided for enhancing the nutritional value of a food or beverage by intermixing the food or beverage with a nutraceutical in an amount that is effective to enhance the nutritional value of the food or beverage.

[0087]The nutraceuticals described herein are intended for human consumption and thus the processes for obtaining them are preferably conducted in accordance with Good Manufacturing Practices (GMP) and any applicable government regulations governing such processes. Especially preferred processes utilize only naturally derived solvents. The nutraceuticals described herein preferably contain relatively high levels of health-enhancing substances Nutraceuticals may be intermixed with one another to increase their health-enhancing effects.

Problems solved by technology

Autoimmune, allergic and inflammatory diseases place a tremendous burden on the patient and society, resulting in decreased quality of life and huge costs.

Moreover, no adequate treatment exists without acceptable side effects or which is socially appropriate.

Nevertheless, these immunotherapies may be associated with increased risk of adverse effects, such as increased susceptibility to infection.

However, gastrointestinal degradation and low levels of absorption generally render this route of peptide and protein drug delivery ineffective.

Thus, there remains a problem in the art to effectively induce tolerance of antigens.

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