WO2002072142A2

WO2002072142A2 - Modulation of a balance between the gut mucosal immune system and the intestinal microflora

Info

Publication number: WO2002072142A2
Application number: PCT/NL2002/000165
Authority: WO
Inventors: Nicolaas Adrianus Bos; Franciscus Gerardus Maria Kroese
Original assignee: Rijksuniversiteit Groningen
Priority date: 2001-03-12
Filing date: 2002-03-12
Publication date: 2002-09-19
Also published as: WO2002072142A3; AU2002239159A1

Abstract

The invention relates to the field of biotechnology, especially to the modulation of a balance between the gut mucosal immune system and the intestinal microflora of said gut. The invention provides a nucleic acid library comprising a repertoire of genes or functional fragments thereof said genes or fragments essentially encoding a repertoire of proteinaceous substances capable of binding with a repertoire of antigenic fragments derivable from one or more micro-organisms.

Description

Title Modulation of a balance between the gut mucosal immune system and the intestinal microflora

The invention relates to the field of biotechnology, especially to the modulation of intestinal dysbactenosis in the balance between the gut mucosal immune system and the intestinal microflora of said gut.

The intestinal tract harbors a dynamic, complex bacterial ecosystem The presence and composition of the commensal gut flora is known to be of great importance for resistance to pathogenic microbes (e g via competition for space and nutritional elements) By interaction with the intestinal immune system this normal ecosystem also contributes to a well-balanced homeostasis and colonization resistance to potential pathogens The composition of the gut flora influences the development of the mucosal immune system and the immune system itself modulates the composition of the gut flora

IgA is known to play a crucial role, as it is the most important effector molecule of the humoral immune system In the lamina propria underlying the gut epithelium there is an abundance of plasma cells producing mostly IgA IgA occurs in various polymeric forms including monomers, dimer and even higher multimers In addition to heavy and light chains the polymeric forms of IgA usually contain J chains, allowing secretion of the IgA molecules through the mucosal epithelium The polymeric forms of IgA can bind to the poly-Ig receptor (plgR) that is expressed on the basolateral surface of the mucosal epithehal cells The IgA plgR complex is transcytosed to the apical surface of the mucosal epithelium During transit, a disulfide band is formed between the IgA and the plgR At the apical surface the IgA molecule is released by proteolytic cleavage of the polylgR This cleavage result in a fragment, called secretory component (SC) being retained on the IgA molecule This secretory IgA (slgA) has been shown to be very resistant to proteolytic degradation (review in [1])

The binding capacities of the diverse repertoire of slgA molecules that are produced and secreted at a rate of 2,5 g/day are largely unknown In mice, we have shown that in the small intestine practically all intestinal bacteria are coated with IgA, while in the cecum, large intestine and feces about 10-30% of the commensal bacteria is coated with IgA [2] The effects of slgA on the commensal intestinal flora are not well characterised Described effector functions within the gut lumen involve primarily prevention of attachment ("immune exclusion"), but also neutralisation of viruses or bacterial toxins has been described The efficiency of IgA molecules to exclude bacteria from the gut seems to be different for commensal bacteria and pathogenic bacteria The commensal bacterial population is able to maintain itself in a very constant composition, despite the IgA coating, while pathogens are very efficiently completely excluded from the gut This difference in "immune exclusion" might be related to the overall affinity and specificity of the involved IgA molecules [3]

Evidence for differences in IgA molecules comes from experiments in the mouse It has been shown in mice that two distinct subpopulations of B cells contribute to the production of this IgA [4]

Conventional B2-cells that develop in the bone marrow throughout live are activated by antigen-contact in the Peyer 's patches along the small intestine and then differentiate - during migration to the lamina propπa - into plasma cell They produce IgA that can be highly specific for antigens of (pathogenic) bacteria and can normally be of high affinity (further referred to as 'specific IgA') In contrast, Bl cells originate throughout live mostly from the peritoneal cavity of mice, switch to IgA expression and migrate to the gut LP where they accumulate There they are thought to produce IgA antibodies with high cross- reactivity but rather low affinity for any microbes present in the gut (referred to as "natural" IgA antibodies) [5]

The presence of natural IgA antibodies has also been shown in human secretions There is a strong relationship between colonization of the intestinal tract with bacteria that belong to the commensal flora and the development of IgA production at the site The produced IgA appears to influence the colonization of the intestinal mucosa by microbes in two different ways a) IgA coating of some microbes results in an effective and rapid clearing of this species from the gut and b) IgA coating of other bacterial species results in their maintenance in a stable ecosystem and consequently in the becoming part of the commensal flora

The invention provides a nucleic acid library comprising a repertoire of genes or functional fragments thereof said genes or fragments essentially encoding a repertoire of proteinaceous substances capable of binding with a repertoire of antigenic fragments derivable from one or more micro-organisms, in particular wherein said micro-organisms are intestinal With such a library according to the invention, preferably wherein said repertoire of proteinaceous substances capable of binding with a micro-organism are derivable from a repertoire of immunoglobuhns or functional fragments thereof, a method of treating patients with a disturbed balance between the gut mucosal immune system and the intestinal microflora by oral administration of commensal bacteria or yeast cells that are producing a polyclonal mixture of slgA molecules is provided The slgA molecules preferably represent the normal repertoire of IgA molecules that are produced in the lamina propria of the human gut Genes encoding such IgA molecules are cloned and inserted according to standard recombinant DNA technology The IgA molecules are preferably expressed in a gram-positive host commensal bacterium or in a commensal yeast cell , if so desired in combination with J chain and the extra cellulair part of the poly-Ig receptor such that said immunoglobuhns commonly allow for continuous intestinal secretion of slgA The poly-Ig receptor can also be expressed as membrane bound protein holding the IgA complex on the outer membrane In this way proteotypic enzymes produced by epithetial cells release the slgA molecules from the bacteria into the lumen Of course, use of other types of immunoglobuhns such as IgM or IgG instead of IgA, or functional fragments thereof, is also provided

For this purpose the invention provides for example for the production of a such a nucleic acid library of 'natural' IgA encoding genes from human mucosal IgA plasma cells In the lamina propria of the small and large intestinal wall a great number of IgA producing plasma cells can be found The produced IgA reflects the immunological history of the encounters of that person with intestinal bacteria evoking specific and natural IgA production, which in healthy individuals maintains a balance with the gut microflora RNA is for example isolated from healthy gut tissue samples obtained by elective intestine surgery from patients with no known intestinal problems Samples can be either obtained from small or large intestine By RT-PCR with primers specific for lmmunoglobulin variable regions and the constant IgA genes in combination with primers for the kappa and lambda light chains, IgA encoding immunoglobulin genes or fragments thereof is specifically amplified. The obtained PCR products are cloned into bacterial or yeast expression vectors. 2. Also is provided a nucleic acid derived from a library according to the invention, and in particular a set of vectors comprising a library or a nucleic acid according to the invention, in particular wherein said vectors comprise a virus or a plasmid, such as a bacteriophage, a transposon, cosmid an, artificial chromosome constructs YAC/BAC, and the like. Alternatively, the invention provides a set of vectors wherein said virus comprises an enteric virus such as a rota- or coronavirus. Also is provided a set of host cells comprising a set of vectors according to the invention. Cloned IgA heavy and light immunoglobulin genes are preferably transformed into gut commensal bacteria, in combination with vectors expressing the J chain and the poly-Ig-receptor gene. After expression of the IgA molecules they polymerize through the J chain, which results in binding to the poly-Ig receptor protein. After secretion of this product by the bacteria, locally present peptidases cut off part of the poly-Ig receptor, resulting in the secretory component attached to the polymeric IgA molecules. In this way, a source of locally produced polyclonal secretory IgA, is provided which is well suited for the gut micro- environment. Candidate bacterial species are gram-positive bacteria such as lactobacilli or streptococci that both have been used for expression of cloned products and are known commensal bacteria, which are already used as probiotics in humans. Lactobacilli until now have been used for induction of mucosal immune responses by oral vaccination. Preferably, there is no induction of mucosal immune responses, but on the contrary mucosal immune responses are suppressed by local production of 'natural' IgA. Candidate yeast cells are Saccharomyces cerevisiae or S. boulardie, which has been described to be able to express immunoglobuhns [7;8] and are known to be used as probiotics [9; 10].

The invention provides that that administration of such a set of vectors or host cells capable of expressing IgA (or a functionally related binding molecule) restores the balance between the gut commensal bacteria and the mucosal immune system and in this way prevents unwanted immunological reactions towards these gut bacteria. Passive immunotherapy with IgA purified from serum, plasma and milk has been suggested in patents US5833984, US5371196 and JP406605A. Oral administration of enteric film-coated IgA has been proposed to get efficient transfer to the gut lumen (US4335099). Also usage of a mixture of active Ig concentrate, mixed with probiotic bacteria was suggested for beneficial effects on the gastrointestinal health (US5531988) Production and usage of slgA by transport of dimeric IgA through a cultured epithelial cell expressing the poly-Ig receptor and harvesting at the opposite site has been described (WO9116061) Also the production of either synthetic poly-Ig receptor or secretory component and the formation of poly-Ig-receptor- J-IgA complexes has been described [6] and suggested in WO09857993 and WO9830577 Furthermore the usage of monoclonal IgA antibodies specific for several different pathogens, produced by hybridoma technology or recombinant DNA technology is proposed for immunotreatment (US5670626)

However, the invention provided the insight that local production of slgA is provided by oral administration of (host cells) e g commensal bacteria or yeast cells, provided with a set of vectors according to the invention Secondly, the recombinant IgA molecules that will be produced by these host cells (preferably these are of intestinal origin, these are generally recognised as safe (G R A S ) represents at least a subpopulation of the large number of different IgA molecules naturally produced along the intestinal tract This results in restoration of the normal dynamic balance between the gut flora and the humoral mucosal immune system Thirdly, the slgA product, if so desired, can be anchored to the outside of the plasma membrane of the host cells, such as the transformed commensal bacterial or yeast cells, with the poly-Ig receptor similarly to the normal physiological passage of IgA to the intestinal lumen, after which naturally occurring enzymes cut the polylg receptor resulting in the release of slgA into the intestinal lumen

Furthermore, the invention provides use of a library or a nucleic acid or a set of vectors host cells according to the invention in the preparation of a pharmaceutical or nutraceutical composition In one embodiment, the invention provides application of a mixture of the recombinant gut commensal bacteria or yeast cells to patients with a disturbed balance between the microflora and the mucosal immune system Oral administration of a mixture of such live recombinant gut bacteria or yeast cells will result in a local production of a polyclonal mixture of IgA antibodies which should reflect the natural IgA production that is normally present to maintain the balance with the normal gut flora The duration of the colonization will depend on locally present niches for the utilized bacterial or yeast strains Permanent colonization might be possible and might be beneficial for maintaining the balance with the gut flora for extended time periods If temporary colonization is obtained, this can be prolonged as long as wanted by repetitive administration of the recombinant bacterial or yeast cells

A method of treating patients with a disturbed balance between the gut microflora and the mucosal immune system by oral administration of slgA- producing commensal bacterial or yeast cells, in particular wherein said preparation is produced from a large library of different human IgA-encoding heavy and light chain genes A particularly beneficial embodiment is wherein an individual commensal bacterial or yeast species is capable of expressing a human IgA heavy chain, a human kappa or lambda light chain, a J-chain and the poly-Ig receptor, especially wherein the produced polymeric IgA-J-Poly-Ig complex is expressed on the outer side of the plasma membrane, whereafter the slgA can be cleaved from the membrane by enzymes naturally present in the intestinal tract resulting in local release of slgA

Preferably, such a commensal bacterium as provided by the invention comprises of a member of the group of gram -positive bacteria, such as those that belong to the group of intestinal bacterial cells that are used as probiotics As said, also transformed yeast cells are provided, especially those which comprise a member of the group of saccharamyces, such as those that belong to the group of intestinal yeast cells that are used as probiotics

The invention for example provides a method wherein patients are treated which have an acute or a chronic disturbance of their intestinal flora, such as those which have a (if only temporarily) impaired mucosal immune system because of cytostatic or immunosuppressive treatments for diseases unrelated to intestinal problems Other patients likely to benefit from a method as provided by the invention are those which have a dysregulated mucosal immune system, because of an autoimmune disease, or those which have an impairment in their ability to produce or transport slgA to the intestinal lumen

Potential applications of a pharmaceutical or nutraceutical composition as provided herewith are for example patients with a disturbed balance between the commensal bacterial flora and the mucosal immune system, with patients with chronic gut inflammations, such as IBD, with patients with a disturbed (mucosal) immune system, such as transplantation patients (in particular liver transplantation), oncological patients undergoing chemotherapy or irradiation, patients with a selective IgA deficiency, patients with autoimmune diseases, with patients with a disturbed gut microflora, such as patients undergoing antibiotic treatment, or with patients with an acute gastrointestinal infection All these can benefit from immunotreatment as provided herewith, be it that the balance is disturbed because of alteration in the gut flora itself or due to (temporal) impairment of the immune system because of treatments for other diseases, 1 e in general for the treatment of an intestinal dysbacteriosis or for the prevention of the occurrence thereof The invention also provides a food or feed comprising a composition according to the invention and the use of such a food or feed in modulating intestinal dysbacteriosis

References

1. Brandtzaeg P, Farstad IN, Johansen FE, Morton HC, Norderhaug IN, Yamanaka T (1999) The B cell system of human mucosae and exocrine glands. Immunol.Rev. 171:45-87.

2. Kroese FG, de-Waard R, Bos NA (1996) B-l cells and their reactivity with the murine intestinal microflora. Semin. Immunol. 8: 11-18.

3. Bos NA, Cebra JJ, Kroese FGM (2000) B-l cells and the intestinal microflora. Curr. Topics in Microbiol. Immunol. 252:211-220

4. Kroese FGM, Cebra JJ, van der Cammen MJF, Kantor AB, Bos NA (1995) Contribution of Bl cells to intestinal IgA production in the mouse. Methods: A companion to Methods in Enzymology 8:37-43.

5. Kroese FGM, Bos NA (1999) Peritoneal Bl cells switch in vivo to IgA and these IgA antibodies can bind to bacteria of the normal intestinal microflora. Curr. Topics in Microbiol.Immunol. 246:343-349.

6. Chintalacharuvu KR, Morrison SL (1999) Production and characterization of recombinant IgA. Immunotechnology. 4:165-174.

7. Horwitz AH, Chang CP, Better M, Hellstrom KE, Robinson RR (1988) Secretion of functional antibody and Fab fragment from yeast cells. Proc.Natl.Acad.Sci.U.S.A. 85:8678-8682.

8. Wood CR, Boss MA, Kenten JH, Calvert JE, Roberts NA, Emtage JS (1985) The synthesis and in vivo assembly of functional antibodies in yeast. Nature 314:446-449.

9. Lewis SJ, Freedman AR (1998) Review article: the use of biotherapeutic agents in the prevention and treatment of gastrointestinal disease. Aliment.Pharmacol.Ther. 12:807-822.

10. Chaucheyras F, Fonty G, Bertin G, Gouet P (1995) In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in association with an archaea methanogen is stimulated by a probiotic strain of Saccharomyces cerevisiae. Appl.Environ. Microbiol. 61:3466-3467.

Claims

I . A nucleic acid library comprising a repertoire of genes or functional fragments thereof said genes or fragments essentially encoding a repertoire of proteinaceous substances capable of binding with a repertoire of antigenic fragments derivable from one or more micro-organisms.

2 . A library according to claim 1 wherein said micro-organisms are intestinal.

3 . A library according to claim 1 or 2 wherein said repertoire of proteinaceous substances capable of binding with a micro-organism are derivable from a repertoire of immunoglobulins or functional fragments thereof.

4 . A library according to claim 3 wherein said immunoglobulins commonly allow for intestinal secretion.

5 . A library according to claim 3 or 4 wherein said immunoglobulins comprises IgA.

6 . A nucleic acid derived from a library according to anyone of claims 1 to 5.

7 . A set of vectors comprising a library according to anyone of claims 1 to 5 or a nucleic acid according to claim 6.

8 . A set according to claim 7 wherein said vectors comprise a virus or a plasmid.

9 . A set according to claim 8 wherein said virus comprises an enteric virus such as a rota- or coronavirus.

10 . A set of host cells comprising a set of vectors according to claim 7 or 8.

I I . A set according to claim 10 wherein said cells comprise a micro-organism such as a bacterium or yeast.

12 . A set according to claim 11 wherein said micro-organism is an enteric micro-organism.

13 . A set according to claim 11 or 12 wherein said micro-organism is considered a G.R.A.S. micro-organism.

14 . Use of a library according to anyone of claims 1 to 5 or a nucleic acid according to claim 6 or a set of vectors according to anyone of claims 7 to 9 or a set of host cells according to anyone of claims 10 to 13 in the preparation of a pharmaceutical or nutraceutical composition.

15 . Use according to claim 14 wherein said composition is for the treatment of an intestinal dysbacteriosis or for the prevention of the occurrence thereof.

16 . A composition comprising a library according to anyone of claims 1 to 5 or a nucleic acid according to claim 6 or a set of vectors according to anyone of claims 7 to 9 or a set of host cells according to anyone of claims 10 to 13.

17 . A method of pharmaceutical or nutraceutical treatment comprising administering a composition according to claim 16 or 17 to an individual.

18 . A method according to claim 17 wherein said treatment comprises treatment of an intestinal dysbacteriosis.

19 . A food or feed comprising a composition according to claim 16.

20 . Use of a food or feed according to claim 19 in modulating intestinal dysbacteriosis.