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"Composition and Method for the Treatment and Prevention of Enteric Bacterial Infections" in Patent Application Approval Process

May 13, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Robins-Browne, Roy Michael (Templestowe, AU); Rawlin, Grant Thomas (Kilmore East, AU); Lichti, Gottfried (Essendon, AU), filed on December 23, 2013, was made available online on May 1, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Biotechnology Companies).

This patent application is assigned to Immuron Limited.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention may be used in the treatment and prophylaxis of gastrointestinal diseases caused by a range of organisms including E. coli, Salmonella, Campylobacter, Helicobacter, Vibrio, Shigella, Yersinia and Aeromonas bacteria. For the purposes of exposition, however, the invention will be explained in its application to enterotoxigenic E. coli (ETEC) in humans. It will be understood however that the broadest part of the invention is not limited by this exemplary application.

"Diarrhoea caused by enterotoxigenic E. coli (ETEC) causes significant discomfort in adults and can lead to death through dehydration of young and older people. A significant fraction of the diarrhoea suffered by travellers to destinations such as Mexico, Africa and South-East Asia is caused by ETEC.

"One treatment currently used for travellers' diarrhoea is prophylactic antibiotic therapy, for example with Amoxycillin. However antibiotic resistance has reduced the effectiveness of antibiotic therapy and side-effects such as constipation or diarrhoea are common.

"Symptomatic therapy is used for vomiting and diarrhoea, for example with loperamide hydrochloride, atropine sulphate and diphenoxylate hydrochloride. However the inappropriate use of Loperamide and Atropine leads to severe constipation and the inappropriate use of diphenoxylate may lead to dependence. These agents are also unsuitable for administration to children.

"A further treatment includes fluid replacement therapy using isotonic drinks. However fluid replacement therapy is merely palliative, and does not decrease clinical diarrhoea

"Furthermore symptomatic relief and fluid therapy will treat the symptoms but will not remove the cause.

"Milk and egg products have been shown to have potential therapeutic and preventative roles in the relief of symptoms of gastrointestinal disorders

"Peterson and Campbell in U.S. Pat. No. 3,376,198 teach the immunisation of milk-producing ungulates to produce antibodies or 'protective principles' against bacteria and viruses.

"Carlander et al in BioDrugs 2002; 16(6):433-7 teach the use of antibodies derived from egg yolk to decrease Pasteurella bacteria in the opening of the gastrointestinal tract (the mouth).

"Shimamoto et al in Hepatogastroenterology 2002 49(45): 709-714 teaches the use of specific antibodies against Helicobacter pylori raised in eggs to decrease the number of H. pylori bacteria in the stomach of patients.

"Linggood et al in U.S. Pat. No. 4,971,794 teach the use of hyperimmune bovine colostrum as a source of antibodies to E. coli. The cows were vaccinated using preparations of pili from a mixture of strains. The patent teaches that the vaccine must comprise antigens of a plurality of strains of E. coli expressing Type 1 pili, CFA 1 pili, CFA 2 pili and K88 pili. K88 is associated with porcine ETEC.

"Hastings in U.S. Pat. No. 5,017,372 teaches the use of hyperimmune colostrum from ungulates as a source of antibodies to E. coli. The ungulate vaccine was made using the following method: Various E. coli bacteria were grown under conditions so that CFA 1 or CFA 2 or both were produced. The bacteria were then lysed by ultrasound to release these antigens and heat labile toxins.

"Freedman et al in The Journal of Infectious Diseases 1998, 177:662-7 teaches the use of hyperimmune colostrum as a source of antibodies against E. coli. A vaccine was made by growing bacteria strains in a broth designed to optimise CFA expression, then purifying the CFA using precipitation followed by size-exclusion or ion exchange chromatography.

"Tackett et al in The New England Journal of MedicineMay 12, 1988 pp 1240-1243 describe the pooling of multiple inactivated (formaldehyde or glutaraldehyde treated) bacterial whole cell suspension in a vaccine administered to cattle. The whole-cell suspensions comprise E. coli of O serogroups: O6, O8, O15, O20, O25, O27, O63, O78, O114, O115, O128, O148, O153 and O159 as well as heat-labile enterotoxins, cholera toxin, CFA 1 and E. coli surface antigen 3. O antigens of E. coli such as O6, O8, O15, O20, O25, O27, O63, O78, O114, O115, O128, O148, O153 and O159 are heat stable antigens located on the bacterial cell wall and not on protruding structures such as pili (fimbriae) or flagella. These O antigens are composed of polysaccharide moieties linked to a core lipooligosaccharide complex common to the wall material of most Gram negative bacteria. Because of the close association between the O antigens and the cell wall, O antigen based vaccines have been made from cell walls or whole inactivated bacteria. The lipopolysaccharide endotoxins are a normal part of the outer cell wall of the bacteria and their toxic regions are embedded in the cell wall (see 'Endotoxins in Health and Disease', 1999 Chapter 12 and other chapters). A further reason for the fact that normal veterinary practice uses whole cell bacterial antigens rather than individual O antigen moieties is that O antigens are endotoxins--the use of high concentrations of endotoxins in pregnant cattle is considered problematic in prior art in terms of animal welfare and productivity.

"The use of milk and egg products in the prior art for the prevention of symptoms of gastrointestinal disorders is associated with a number of problems.

"Cholera toxin (see Tackett et al., 1988) are likely to be difficult to register because of their high toxicity. Further, heat labile toxins, although highly immunogenic are not likely to be protective.

"The extent of protection obtained from antibodies produced as above is unsatisfactory. One of the attendant problems is that large quantities of immune concentrate are needed to produce a satisfactory prophylactic outcome. When whole cell antigens are used, an enormous variety of antibody responses arise and assays for antibody titre are difficult to interpret. A key issue is whether a particular detected antibody is protective or not. Whole bacterial cells have many antigens that are unlikely to be related to protection. In addition, vaccines comprising Gram negative bacteria are more likely to produce adverse vaccine reactions and therefore present regulatory problems due to adverse animal ethics reports and therapeutic goods adverse reaction reports. Adverse vaccine reactions are likely to stop dairy farmers from participating in any production venture for milk antibodies.

"The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "We have made the surprising discovery that enhanced treatment or prophylaxis of enteric disease caused by Gram negative bacteria in animals and humans can be achieved by administering a vaccine or by administering hyperimmune material raised against said vaccine, wherein the vaccine is characterised in that it comprises one or more cell wall antigens reactive in a manner characteristic of O group serotypes or reactive in a manner characteristic of lipopolysaccharide-associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments.

"Accordingly in one aspect the invention provides a method of treatment or prophylaxis of enteric disease caused by Gram negative bacteria, the method including the step of administering a vaccine or a hyperimmune material raised against said vaccine to an individual, wherein the vaccine comprises one or more cell wall antigens reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments.

"In another aspect the invention provides a composition for use in the treatment or prophylaxis of enteric disease caused by Gram negative bacteria, the composition comprising hyperimmune material prepared by immunizing a host animal with a vaccine comprising one or more cell wall antigens reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments.

"In yet another aspect the invention provides a method of preparing hyperimmune material for treatment or prophylaxis of gastrointestinal dysfunction caused by enterotoxigenic Gram negative bacteria such as ETEC, said hyperimmune materials being raised against a vaccine comprising one or more cell wall antigens reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments.

"In a further aspect the invention provides the use of cell wall antigens in the manufacture of hyperimmune material for treatment or prophylaxis of gastrointestinal disease caused by Gram negative bacteria such as E. coli (ETEC) wherein the cell wall antigens are reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments.

"In yet another aspect the invention provides a process for the preparation of immunoglobulins for treatment or prophylaxis of gastrointestinal disease in animals comprising immunising a host animal with a vaccine comprising one or more cell wall antigens reactive in a manner characteristic of O group serotypes, or reactive in a manner characteristic of lipopolysaccharide associated antigens, at least some of said antigens being separated from bacterial cell walls or wall fragments to thereby induce the host animal to produce hyperimmune material comprising the immunoglobulins.

"The hyperimmune material that is produced according to the methods of the invention may be hyperimmune colostrum or hyperimmune colostrum extract or hyperimmune egg yolk or hyperimmune egg yolk extract.

"At least some of the cell wall antigens are considered to be separated from intact bacterial cell walls or cell wall fragments if, after centrifugation to remove whole cells and substantial cell fragments, at least 10% of said antigens can be detected in the supernatant liquor. This centrifugation should be performed under conditions where a) said antigens do not form micelles or other aggregated structures and b) cell wall integrity is not compromised. Preferably at least 20% of said antigens can be detected in the supernatant liquor, more preferably at least 30%. Preferably said antigens are substantially separated from intact bacterial cell walls.

"Another procedure that can be used to determine if cell wall antigens are separated or disassociated from intact bacterial cell walls or cell wall fragments is as follows: 1) Perform an assay on an aqueous liquor comprising O antigens and Gram negative bacterial cell walls or cell wall fragments to determine total O antigen content per ml using vigorous conditions which will degrade cell wall structure and release O antigens; 2) Establish which specific concentration of Gram negative cells, grown in culture, have an O antigen content equivalent to the O antigen content determined in step 1; 3) Measure the cell wall content (quantity) per ml of the cell culture identified in step 2. This cell wall content will naturally contain the level of O antigen measured in step 1. 4) Compare the cell wall content in step 3 with the content of the original liquor. If equivalent, it may be inferred that all O antigens in the original liquor have their usual association with the cell wall. However, if the cell wall content of the original liquor is less than the cell wall content in step 3 then the original liquor contains O antigens in excess of the expected O antigen content of cell walls and therefore some O antigen is dissociated from the cell walls or wall fragments. If the weight of Gram negative cell wall material (per ml) in Step 3 is W, then it is preferred that the weight per ml of Gram negative wall material in the original liquor is less than 0.8 W, preferably less than 0.3 W, more preferably less than 0.1 W.

"The quantity of lipopolysaccharide-associated antigen (O antigen) present in an aqueous liquor can be determined using methods known to the art such as heating, proteinase K digestion and further heating in the presence of phenol (see example 11). Other assays for lipopolysaccharide-associated antigens have been described by Lyngby et al. One convenient method of standardising lipopolysaccharide-associated antigens is to compare with a cell cuture having a specified number of Gram negative organisms per ml, or a specified quantity of Gram negative wall material per ml. It may be advantageous to compare with cell cultures having 10.sup.4, 10.sup.6 or 10.sup.8 organisms per ml, or higher.

"The quantity of Gram negative cell wall material (intact walls and fragments) present in an aqueous liquor can be determined using assays for specific cell wall markers known in the art. For example peptidoglycans are associated with the cell walls of Gram negative bacteria, and methods of analysing peptidoglycans have been described by Li et al.

DESCRIPTION OF THE FIGURES

"FIG. 1 shows a silver-stained SDS-PAGE gel from the analysis of LPS from a liquor preparation. Shown on the SDS-PAGE gel are samples before and after phenolic pretreatment. After the treatment this gel shows a typical LPS ladder. Also shown in left lane as a control is a boiled bacterial lysate."

URL and more information on this patent application, see: Robins-Browne, Roy Michael; Rawlin, Grant Thomas; Lichti, Gottfried. Composition and Method for the Treatment and Prevention of Enteric Bacterial Infections. Filed December 23, 2013 and posted May 1, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2806&p=57&f=G&l=50&d=PG01&S1=20140424.PD.&OS=PD/20140424&RS=PD/20140424

Keywords for this news article include: Antibodies, Therapy, Cell Wall, Endotoxins, Immunology, O Antigens, Blood Proteins, Immunoproteins, Immunoglobulins, Immuron Limited, Serum Globulins, Bacterial Toxins, Biological Factors, Cellular Structures, Lipopolysaccharides, Gram-Negative Bacteria, Biotechnology Companies, Bacterial Polysaccharides.

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Source: Life Science Weekly


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