Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Reexamination Certificate
2000-09-09
2004-12-21
Priebe, Scott D. (Department: 1635)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
C424S093200, C424S093400, C435S471000, C435S488000, C435S243000, C435S252100, C435S252300, C435S252330, C435S252800
Reexamination Certificate
active
06833130
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to recombinant microorganisms (i.e., bacteria, yeast, and fungi) that display an oligosaccharide-comprising binding moiety that can compete with a ligand for binding to a receptor for the ligand, and use of the microorganisms to deliver the oligosaccharide to a human or other animal. The microoganism can be used for adsorbing toxins or pathogenic microorganisms from a particular environment.
BACKGROUND OF THE INVENTION
Surfaces of all cells express a complexity of oligosaccharides that provide a number of functions. A primary one of these functions is determining on their own or together with other molecules, interactions with other cells or molecules. The nature, linkage and conformation of sugar residues of the oligosaccharides, and in particular residues at or close to the non-reducing terminus of an oligosaccharide, determines whether the oligosaccharide will or will not participate in a particular receptor-ligand interaction.
The susceptibility of an animal to infection and the eventual physiological site affected by an infection is to a large extent determined by the expression on the cell surface of such oligosaccharide receptors. In the case of enteric infections, one primary prerequisite for pathogenesis is that the microorganism persists in the intestinal lumen of the host. Generally this requires some form of adherence to the lumeinal epithelium (forming the mucosal surface of the gut), otherwise the micro-organism is flushed from the gut. Additionally for a toxigenic organism, the toxin also needs to bind to the luminal epithelium and, for some toxins, needs to be absorbed systemically to be effective, otherwise, it too would be flushed from the gut.
Certain surface structures of pathogenic and other bacteria known as adhesins mediate adherence to luminal epithelial cells. A number of adhesins are known and organisms without adhesins are generally of low virulence. Adhesins are proteinaceous factors which promote the adherence of bacteria and viruses to cells of their hosts. Adhesins can be either fimbrial or filamentous in structure or they may be afimbrial. Adhesins associated with fimbriae may be associated with accessory proteins such as tip proteins at an extreme end of the fimbrial structure and such tip proteins can be regarded as lectins. The receptor on the host cell has in some cases been determined and shown to be a carbohydrate such as an oligosaccharide associated with a glycolipid or a glycoprotein.
The best characterised system from a molecular and biological viewpoint, is the P-fimbriae (also called pili) produced by uropathogenic
Escherichia coli.
This tip adhesin binds the glycolipid Gb
3
(globotriaosyl ceramide—see below) and the fimbrial subunits can be purified by affinity chromatography using a Gb
3
mimic. Another well characterised group of adhesins are those associated with enterotoxigenic
E. coli
(ETEC) strains which infect pigs to cause scours. These are termed the K88 type and a number of variants are known, being K88ab, K88ac and K88ad. The adhesins associated with these fimbriae have been shown to have different receptor requirements, which includes the presence of both glycolipid and protein receptors. The carbohydrate requirement has been characterised in at least some of these, for example K88ad uses carbohydrates of the lactoneotetraose series of glycolipids.
A number of toxins have been identified and include, Shiga toxins (also referred to as Shiga-like toxins and verotoxins), toxins produced by various species of Clostridia, including tetanus toxin, botulinum toxin, and
C. diffcile
toxins A and B, Staphylococcal enterotoxins,
Escherichia coil
heat labile and heat stable enterotoxins and cholera toxin. Without toxin activity the majority of otherwise enterotoxigenic bacteria would be less capable of causing disease.
The receptors for the majority of adhesins and toxins identified to date are carbohydrate in nature. For example, the glycolipid globotriaosyl ceramide (Gb
3
) which has the structure Gal&agr;[1→4]Gal&bgr;[1→4]Glc-ceramide, is the preferred receptor for most members of the Shiga toxin family. Similarly, the ganglioside G
M1
is the receptor for cholera toxin and
E. coli
heat labile enterotoxin type I.
C. difficile
toxin A binds to several host receptors, all of which have in common a Gal&bgr;[1→4]GlcNAc moiety. The neurotoxin produced by
C. botulinum
is also believed to be specific for a sialic acid containing glycoprotein or glycolipid present on neurons. The terminal Gal&agr;[1→4]Gal&bgr; moiety present on Gb
3
is the receptor for P pili, the major adhesin of uropathogenic
E. coli
strains. Similarly, asialo-G
M1
is the receptor for adhesive pili (CFAs) of some enterotoxigenic
E. coli
strains. The sialated gangliosides NeuGc-GM
3
and NeuNAc-GM
3
have also been identified as the target cell receptors for porcine rotavirus strains, and it is presumed that rotavirus strains causing disease in humans also bind specific oligosaccharide moieties present on cell surface glycolipids.
The elucidation of the nature of oligosaccharides acting as receptors for particular toxins and pathogenic microorganisms has opened up a promising avenue in the diagnosis and potential treatment or prevention of diseases caused by these agents. The use of the sugar residues forming receptors for toxins or adhesins has been proposed as a means of specifically identifying the toxins or bacteria involved in an infection. For example, the ganglioside receptor G
M1
is used as a specific capturing agent in ELISA assays for the presence of cholera toxin.
It has also been proposed to use synthetically prepared oligosaccharides as a means of adsorbing toxins or the like from samples. Examples of proposed uses of receptors for adsorbing toxins or pathogenic organisms out of a sample include Krivan et al in U.S. Pat. No. 5,696,000 which discusses the pharmaceutical use of certain tetra- and tri- saccharide receptors coupled to a carrier such as a liposome to inhibit the adherence of micro-organisms to susceptible cells. A similar use for toxins such as Shiga toxin, can be seen in U.S. Pat. No. 5,849,714 to Rafter et al which discloses the use of a synthetic construct of sugar residues making up the globotriose receptor, coupled by a linker to an inert support for use in treatment of bacterial dysentery.
A problem arises, however, in the synthesis and delivery of these compounds, because oligosaccharides are difficult or expensive to synthesise chemically, the conformation may not be appropriate, and the oligosaccharide may preferably need to be presented in an immobilized (non-diffusible) form. Thus, there is a need to provide a mechanism for delivery and presentation of the oligosaccharide moiety in an appropriate conformation in the environment where the toxin or pathogenic organism is to be adsorbed (for example the gastrointestinal tract).
SUMMARY OF THE INVENTION
The invention provides, in a first embodiment, a recombinant microorganism that displays on its surface a binding moiety that, when administered to an animal, competes with a ligand for binding to a receptor for the ligand. The binding moiety includes an oligosaccharide that is composed of at least one sugar residue that is attached to an acceptor moiety by a glycosyltransferase that is encoded by an exogenous nucleic acid which is present in the microorganism. The oligosaccharide can further include at least a second sugar residue that is attached to the acceptor moiety by at least a second glycosyltransferase. One or more of the additional glycosyltransferases can also be encoded by one or more exogenous nucleic acids that are present in the microorganism.
The receptor is typically present on a surface of a cell. Cells of interest include, for example epithelial or endothelial cells, in particular those that are present in an animal mucosal membrane.
The binding moiety is, in some embodiments, a mimic of a receptor for a toxin or adhesin of a pathogenic organis
Morona Renato
Paton Adrienne W.
Paton James C.
Priebe Scott D.
Whiteman Brian
Women's and Children's Hospital
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