AU735205B2 - Vaccine and assay - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: VACCINE AND ASSAY Applicant: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION 000.

0 0. 0 0 0 0 0 The following statement is a full description of this invention, including the best method of performing it known to me: C:\WINWORD\TEMPLATEPO0-11 .DOT VACCINE AND ASSAY The present invention relates to a vaccine for protection of animals against tunicaminyl-uracil toxins, to an assay for detecting and/or isolating tunicaminyluracil toxins and to compounds and compositions used in preparation of the vaccine and assay.

Tunicaminyl-uracil toxins and related compounds are extremely poisonous and are produced by several genera of bacteria including Clavibacter and Streptomyces. One group of tunicaminyl-uracil toxins are the corynetoxins.

These are produced by a bacterium (Clavibacter toxicus) which colonises nematode galls formed in the seedheads of annual ryegrass/wimmera ryegrass (Lolium rigidum), annual beard grass (Polypogon monspeliensis) and blown grass (Agrostis avenacea). They are responsible for annual ryegrass toxicity (ARGT) in Western Australia and South Australia, and floodplain staggers (FPS) in South Australia and new South Wales. ARGT kills between 20,000 to 80,000 sheep annually in Western Australia. During a three month period in 1990/91, 1722 cattle and 2466 sheep died from FPS in New South Wales. The corynetoxins have also caused poisoning of livestock feeding on contaminated hay.

In addition to stock deaths, field evidence suggests lambing losses and productivity losses and an adverse effect on wool growth has been demonstrated.

The number of new farms affected by ARGT is increasing each year.

The tunicaminyl-uracil toxins are cumulative and extremely poisonous.

They exert their effects by inhibiting Uriclinediphospho-Nacetylglocosamine:dolichyl phosphate N-acetylglucosamine-1-phosphate 25 transferase (N-acetylglucosamine phosphate transferase) leading to inhibition of N-glycosylation of glycoproteins and depletion or defective synthesis of essential glycoproteins.

This has raised serious public health and food safety concerns (Edgar 1997). The growing concern with the transfer of the corynetoxins into animal 30 products for human consumption is exacerbated because long-term, low level exposure to tunicaminyl-uracil toxins is likely to cause multi-system adverse SS C:\WNWORD\STACVICDYF\SPECIPO6813.DOC effects.

As well as affecting animal production and the safety of animal products tunicaminyl-uracil toxins such as corynetoxins can also contaminate grain. For example annual ryegrass is a common and increasingly herbicide resistant weed found in many crops (wheat, barley, canola etc.) and annual ryegrass seed is a common contaminant of grain. When the contaminating ryegrass seed is colonized by C. toxicus, the grain can also be contaminated by corynetoxins.

There is a need for analytical methods for monitoring concentrations of tunicaminyl-uracil toxins in grain, animal products and food.

Farm management can reduce stock losses; however, management procedures are time-consuming and costly (Australian Bureau of Statistics, 1989).

The method most commonly used to estimate the toxicity of annual ryegrass paddocks involves counting the number of "bacterial" galls in pasture samples. An immunoassay for the bacterium is also available to detect the presence or absence of bacterium in grass samples. Gall counts are sometimes augmented by estimates of the antimicrobial activity of the galls, but this is at best a semi-quantitative indication of the toxin level. A simple high performance liquid chromatography (HPLC) method for quantifying the corynetoxins in "bacterial" galls is available. Sample preparation for HPLC analysis in samples other than "bacterial" galls is, however, very labour intensive and time consuming. The cost of HPLC analysis is high and the method is insufficiently sensitive for many requirements. Therefore, we have developed a more sensitive, lower cost, accurate and easily applicable immunoassay for measuring the corynetoxins in a variety of samples.

25 The present invention attempts to overcome some of the problems in detecting and immunizing against tunicamine toxins such as tunicaminyl-uracil toxins.

~Accordingly, in one aspect of the present invention there is provided a tunicamine derivative macromolecule conjugate including a tunicamine derivative 30 bonded to a macromolecule. Preferably the tunicamine derivative is a tunicaminyl-uracil unit bonded to a macromolecule.

SS C:IWINWORDSTACV1C DYPXSPECWO6813.DOC 4 We have now found that a tunicamine derivative bonded to a macromolecule may provide an immunogen which can be used in preparation of a vaccine against tunicamine toxins, preferably tunicaminyl-uracil toxins and related poisons. The tunicaminyl-uracil toxin may be a corynetoxin, preferably corynetoxin U17a or H17a.

The tunicamine derivative may also be used in providing an assay for detecting tunicamine toxins. More particularly, it will be used for detecting tunicaminyl-uracil toxins.

The tunicamine derivative will preferably include tunicaminyl uracil or a derivative thereof such as a hydrogenation product or uracil-ring opened product of a hydrogenation product. Accordingly, there is provided a tunicamine derivative-macromolecule conjugate. Preferably, there is provided a tunicaminyluracil macromolecule conjugate.

Tunicamine may be represented by the formula I

OH

(10-amino-2,10-dideoxy-L-galacto-beta-D-allo-undecodialdo-1 ,4-furanose-1 1,7pyranose).

The preferred tunicamine derivatives may be represented by the formula II

OH

SHR 3 H (II) 3 2 R HO OH wherein R is preferably selected from a uracil radical of formula SS C:\WNWORDSTACVICDYFSPECPON6813.OC S or a derivative thereof, preferably the hydrogenated N O derivative such as dihydro uracil, and a ring opened derivative of formula

H

2

N-C-N-CH

2

-CH

2

CO

2

H

II I

O

and R 1 is preferably hydrogen, aliphatic acyl such as straight or branch chain aliphatic substituted carbonyl,

R

2 is an optionally substituted sugar residue preferably selected amino substituted sugar residues wherein the amino is optionally further substituted by hydrogen acyl or alkyl. More preferably R 2 is N-acetyl glucosamine, glucosamine, glucose or galactosamine and most preferably R 2 is of formula:

NHR

3

O

HOH2C

OH

Iwherein R is acetyl or hydrogen.

More preferred R' are hydrogen and fatty acyl groups.

Tunicaminyl-uracil toxins generally contain a constant uracil-tunicamine-Nacetylgucosamine moiety. The fatty acid, linked via an amide bond to the aminogroup C 10 position of the tunicamine unit is the part of the molecule that varies and gives rise to multi-component mixtures of tunicaminyl-uracil toxins produced by different bacterial species. The tunicaminyl-uracil toxins mixture produced by a particular bacterial source is relatively constant and differs from the mixture produced by other bacterial species. The fatty acids incorporated on the amine at Co of the tunicamine produced by different bacteria show different substitution, SS C:\WINWORD\STACVICDYF\SPECIPO5813.DOC branching and chain lengths. For example, the 16 corynetoxins produced by C.

toxicus incorporate fatty acids with chain lengths from C15 to C19. The fatty acids found in corynetoxins are straight chain or have iso or antiiso methyl branching and they are saturated, ap-unsaturated and p-hydroxyl acids (Edgar et a.,1982; Cockrum and Edgar 1983; Frahn et al., 1984). The tunicaminyl-uracil toxins referred to as tunicamycins, produced by Streptomyces lysosuperificus, incorporate fatty acids with chain lengths from C14 to C17. They are either straight-chain, iso or antiiso branched. They include saturated and apunsaturated acids but there are no p-hydroxy acids in the tunicamycin mixture (Cockrum and Edgar 1983). Other tunicaminyl-uracil toxin mixtures, e.g. the streptovirudens produced by Streptomyces griseofulvis, have different but constant mixtures of fatty acids (Cockrum and Edgar, 1983; Eckardt 1983). Some streptovirudens also have dihydro-uricil in place of uracil.

The macromolecule component is preferably bound via one or more hydroxy, amino or carboxyl groups of the tunicamine derivatives. Most preferably the macromolecule component is bound through the amino group substituted on Clo position of the tunicamine group that is C1o of the 2,10-dideoxy-L-galactobeta-D-allo-undecodialdo-1,4-furanose- 11,7-pyranosyl unit or an amino group when present in the group R 2 such as an amino at C2 of 2-deoxy-alpha-Dglucopyranosyl unit. Most preferably the macromolecule is linked via the amino at Co of the tunicamine group. Preferably the macromolecule is a protein such as bovine foetal calf serum protein, bovine serum albumen or the like; high molecular weight plant proteins and synthetic compounds such as polymers. The most preferred macromolecule protein is bovine foetal calf serum. It is also preferable 25 that the macromolecule does not in itself, cause a measurable immune response so that if the conjugate is administered, a predominant immune response is measurable against the tunicamine derivative.

The macromolecule may be bound to or conjugated with the tunicamine derivative at any of a number of sites available for substitution. For example if the 30 macromolecule is protein, then conjugation may occur at one or more of the hydroxy, amino or carboxyl groups of the tunicamine derivative.

SS C:%WINVRDSTACV1CIDYF\SPECI PO8813.DOC The macromolecule may be bound directly to the tunicamine derivative or it may be bound via a linking group. To achieve conjugation, available tunicaminyluracil containing substances may require chemical modification to generate a suitable linking group. In each case a variety of attachment procedures have been employed involving, for example, carbodiimide reagents, Nhydroxysuccinimide, mixed anhydride, succine anhydride, iodoacetic acids, carbonyldiimidazole, glutaraldehyde etc. to form amide, ester, ether or other covalent links with the carrier macromolecules. The tunicaminyl-uracil toxins or their chemical modified variant compounds can be attached directly to the carrier or through a linking structure. Preferably a linking group will be present.

0 0 II II Examples of linking groups include diradial groups of formula -C-A-Cwhere A is a hydrocarbon diradial or derivate thereof. Preferred linking groups O O II II have the formula C-(CH 2 wherein n is an integer, preferably n is an integer in the range of from 1 to 10. The linking group may be derived from an acylating agent which is preferably selected from diacids, internal anhydrides or imides of a diacid, acid chlorides; glutaraldehyde, carbodiimide and heterobifunctional cross linkers.

Succinic anhydride is a preferred reagent for use in preparing a linking group.

In another aspect of the present invention there is provided a method of :o preparing a tunicamine derivative macromolecule conjugate said method including: providing a tunicamine derivative as represented by the formula II

SOH

H.H H

H

Ro o (Il) 0

R

2 HO OH 25 wherein R is selected from a uracil radical of formula 25 wherein R is selected from a uracil radical of formula SS C:%WIORD\STACVICDYF\SPECPO6M13.OOC 8 0

NH

or a derivative thereof, preferably the hydrogenated derivative such as dihydrouracil and a ring opened derivative of formula

H

2

N-C-N-CH

2

-CH

2

CO

2

H

0 and R 1 is selected from the group including hydrogen, aliphatic acyl including straight or branch chain aliphatic substituted carbonyl;

R

2 is an optionally substituted sugar residue preferably selected amino substituted sugar residues wherein the amino is optionally further substituted by hydrogen acyl or alkyl. More preferably R 2 is N-acetyl glucosamine, glucosamine, glucose or glactosamine and most preferably

R

2 is of formula:

NHR

3 HOH2C OH 2 OH 15 wherein R is acetyl or hydrogen, and reacting the tunicamine derivative with a macromolecule such that the macromolecule binds to one or more hydroxy, amino or carboxyl groups of the tunicamine derivative; and reacting the tunicamine with a macromolecule such that the 20 macromolecule binds to one or more hydroxy, amino or carboxyl groups of the tunicamine.

In a further aspect of the present invention, there is provided a method of producing antibodies to a tunicamine derivative including administering to a SS C:\WNWORD\STACVIC1DYF\SPECI\PO6813.DOC 9 subject an effective amount of a tunicamine derivative macromolecule conjugate; and recovering antibodies from the subject.

Preferably the tunicamine derivative is tunicaminyl-uracil.

Administration of the tunicamine derivative macromolecule conjugate to the subject is intended to immunize the subject so that antibodies are produced which are reactive against a tunicamine. The macromolecule is intended to enhance the immune response to the tunicamine. Adjuvants such as oils may be used to stimulate antibody production. Antibodies to the tunicamine moiety will be reactive to the molecule from which it is derived. Therefore if tunicaminyl-uracil is the derivative then antibodies produced will be reactive to tunicaminyl-uracil.

The antibodies produced may be monoclonal or polyclonal and may be specific to one or more tunicamine moieties of a tunicamine derivative.

Genetic and cellular material involved in the production of antibodies to the tunicamine such as DNA, RNA, mRNA, amino acid and polypeptide sequences and cells (antibody producing cells such as B-cells) may be identified and recovered from the subject producing the antibodies. These antibody genetic materials are indicative of antibodies to the tunicamine derivatives.

All known substances incorporating the tunicamine moiety or more preferably the tunicaminyl-uracil moiety have low molecular weights and are not antigenic. Polyclonal or monoclonal antibodies are therefore produced after attaching (conjugating) suitable derivatives to carrier macromolecules (such as hemocyanin, ovalbumin, bovine serum albumin, foetal calf serum etc.). Other suitable carriers include high molecular weight plant proteins and synthetic compounds, such as polymers.

Immunization, using a tunicamine derivative-macromolecule or tunicaminyluracil-macromolecules conjugates, can be performed by a variety of different protocols. In general the conjugates are administered to experimental animals by a primary injection of an aqueous emulsion with oil adjuvant, and/or other adjuvants such as alum precipitates, DEAE-dextran or Quil A etc. followed by one or more boosting injections. During and after the immunization procedure, sera taken from the animals may be screened for production of specific antibodies to SS C:\WNWORD\STACVICDYFSPECIPO6813.DOC the tunicamine or tunicaminyl-uracil grouping using plates coated with suitable tunicamine or tunicaminyl-uracil derivatives and tunicamine or tunicaminyl-uracil labelled reagents as in competitive assays or anti-species IgG labelled reagent as in "sandwich" assays.

The polyclonal anti-tunicaminyl-uracil antibodies needed for isolating and assaying tunicaminyl-uracil toxins can be conveniently produced in large quantities using sheep. The anti-tunicamine or anti-tunicaminyl-uracil antisera can be used in the assay procedures and isolation methods either before or after purification.

Monoclonal anti-tunicaminyl-uracil toxin antibodies may be prepared by methods which are well known in the art, again using tunicamine derivative macromolecule or tunicaminyl-uracil macromolecule carrier conjugates as the primary immunization agent.

In another aspect of the present invention there is provided a vaccine against a tunicamine derivative, said vaccine including an immunogenically effective amount of a tunicamine derivative macromolecule conjugate, said conjugate being capable of effecting an immunological response to said tunicamine derivative.

Preferably in the vaccine, the derivative is tunicaminyl-uracil. Accordingly, there is provided a vaccine against a tunicaminyl-uracil wherein the tunicaminyluracil macromolecule conjugate is an active immunogen. The tunicaminyl-uracil vaccine will be effective for stimulating protection in animals against tunicaminyluracil toxins. Such a vaccine would allow protected animals to feed with impunity on tunicaminyl-uracil toxin contaminated feed and reduce production losses resulting from toxicity and under-utilization of pasture due to fear of the tunicaminyl-uracil toxicity. The vaccine is expected to lead to an increased clearance rate for the toxins resulting in safer animal products.

Accordingly, in a further aspect of the present invention, there is provided a method of preventing and/or treating an animal against tunicaminyl-uracil poisoning, said method including administering an effective amount of a tunicamine derivative macromolecule conjugate to said animals.

SS C:\WINWORDSTACVICYFSPECIPO6813.DOC 11 Preferably the tunicamine derivative is a tunicaminyl-uracil so that the conjugate is a tunicaminyl-uracil macromolecule conjugate.

Preferably the tunicaminyl-uracil poisoning is caused by a tunicaminyluracil toxin such as a corynetoxin. The corynetoxin may be corynetoxin U17a or H17a. The tunicaminyl-uracil poisoning (toxicity) may result in annual ryegrass toxicity (ARGT) or flood plain staggers (FPS).

The administration of the tunicamine derivative macromolecule conjugate or tunicaminyl-uracil macromolecule conjugate may be a single dose or more preferably repeated smaller doses. Administration of the tunicaminyl derivative macromolecule conjugate or tunicaminyl-uracil macromolecule conjugate may be via any route which can induce an immunological reaction including oral, intravenous, intramuscular, subcutaneous, intranasal, intradermal or suppository routes.

In yet another aspect of the present invention, there is provided a method of detecting the presence of a tunicamine derivative in a sample said method including contacting said sample with an antibody to a tunicamine derivative and detecting conjugates of the antibody and tunicamine derivative.

The tunicamine derivative may be a tunicaminyl-uracil and the antibodies may be reactive to a tunicaminyl-uracil. The antibodies may be prepared as described herein using a tunicaminyl-uracil macromolecule conjugate. Preferably the method is to detect the presence of tunicaminyl-uracil and indicate the potential for tunicaminyl-uracil toxin poisoning. More preferably the toxin is a corynetoxin such as corynetoxin U17a and H17a.

The method of detecting tunicamine derivatives can be applied to immunoassays. The immunoassays of this invention include competitive binding assays and "sandwich" assays and other established formats of immunoassays.

They provide rapid, highly sensitive, specific methods of detecting and quantifying substances incorporating a tunicaminyl-uracil moiety and anti-tunicaminyl-uracil antibodies. For instance, a solid-phase competitive enzyme linked immunosorbent assay (ELISA) may be performed in one and a half to four hours, depending on the accuracy required, and is capable of detecting and quantitating SS C:\WNWORD\STACVIC\DY\SPECI\PO613.DOC 12 levels of tunicaminyl-uracil toxins as low as 0.25-1 ng/ml. The sandwich assay can also be performed as a rapid means of screening hybridomas for antitunicaminyl-uracil antibodies and measuring tunicaminyl-uracil substances in various sample materials.

Tunicaminyl-uracil toxins and other tunicaminyl-uracil-containing substances can be extracted from samples with, for example methanol/water or in alkaline aqueous solution (such as pH 8-10). The extracts diluted in assay buffer are suitable for analysis.

In a competitive assay, tunicaminyl-uracil toxin standards such as purified corynetoxins or tunicamycins and diluted sample extract aliquots in assay buffer may be incubated with a tunicaminyl-uracil-enzyme conjugate in assay recepticles such as tubes or wells of a microtitre tray coated with the anti-tunicaminyl-uracil antiserum. After washing, the recepticles are incubated with a substrate that is converted by the enzyme into a coloured product. After a predetermined time to allow the colour to develop, a standard curve is constructed by plotting the optical density of the colour in the wells against the amount of standard tunicaminyl-uracil toxins added. This curve is then used to determine the amount of tunicaminyluracil toxins in the unknown samples.

A variant of the competitive ELISA employs an enzyme labelled, secondary antibody, specific for IgG of the animal species in which the primary antibody is elicited. Tunicaminyl-uracil standards and diluted extract aliquots in assay buffer are incubated with anti-tunicaminyl-uracil antibodies in the wells of microtitre trays coated with suitable tunicaminyl-uracil derivatives or conjugates, after washing, incubated with anti-species IgG-enzyme conjugate. The recepticle is washed again and incubated with a substrate to allow the colour to develop as in a S*competitive assay.

We have found that tunicaminyl-uracil toxins exhibit a surprising propensity to bond to a solid phase surface of an immuno assay so that antibody detection of tunicaminyl-uracil toxins on the surface of a solid phase can be used to quantitatively measure the presence of the toxins in a sample Accordingly we Sprovide a method of analysis of a sample for tunicaminyl-uracil toxin including SS C:\WINWORD\STACVICDYF\SPECI\PO813.DOC 13 treating a solid phase with the sample to be analysed and treating the solid phase with an antibody to said toxin to detect the presence of said toxin on the solid phase. The solid phase is preferably also either treated with a glutaraldehyde (another reagent known to improve surface performance) or a buffer containing a protein such as BSA. Glutaraldehyde is preferably used as a pretreatment however the protein may be used prior to or in conjunction with the sample. We have found that the sensitivity of the assay is enhanced if the amount of protein such as BSA in the buffer is reduced from concentrations about 0.5 generally used in ELISA to a concentration of less than 0.3% by weight and more preferably from 0.05 to 2.5% by weight.

Radioimmunoassay can be developed by using 14, 1251, or 3H labelled tunicaminyl-uracil toxins. Tunicaminyl-uracil labelled reagents are produced using an enzyme, a radioactive material 14 C, 251, 3 an optical label such as fluorescent material or with some other easily detected entity biotin) as a tracer). Radiolabelled tunicaminyl-uracil standards and diluted sample extract aliquots in assay buffer are incubated with anti-tunicaminyl-uracil antibodies in a tube or in a 96 well plate. After an incubation period the tunicaminyl-uracil moiety becomes bound to the antibodies and the unbound materials are separated using one of the procedures such as ammonium sulphate precipitation, charcoal absorption, double antibody precipitation or filtration through a filter. Bound and unbound labelled tunicaminyl-uracil toxins can be counted and a standard curve constructed by plotting the bound or unbound counts against the amount of o standard tunicaminyl-uracil toxins added. This curve is then used to determine the amount of tunicaminyl-uracil toxins in the unknown samples.

25 The immunoassays of this invention can be used to detect and quantify tunicaminyl-uracil toxins in a liquid sample. As well as extracts of foodstuffs, the liquid samples may include essentially all biological fluids such as serum and !.urine, lymph, extracts of tissues, seed heads of plants and plant materials etc.

The liquid samples may also be extracts or supernatants of microbial cultures including both laboratory and naturally occurring microbial cultures. rumen fluid).

SS C:\WINWORDSTACV1C\DYF\SPECIPPO613.DOC 14 In another aspect of the present invention there is provided a method of isolating and/or purifying a tunicamine derivative from a sample, said method including: contacting said sample with immobilized anti-tunicamine derivative antibody to form an anti-tunicamine derivative antibody tunicamine derivative complex; and extracting the tunicamine derivative from the anti-tunicamine derivative antibody tunicamine derivative complex.

Preferably the tunicamine derivative is tunicaminyl-uracil N-acetyl glucosamine. The method is particularly useful for isolating tunicaminyl-uracil substances from samples. In particular, the method is for isolating tunicaminyluracil substances such as tunicaminyl-uracil toxins such as corynetoxins including corynetoxin U17a or H17a. The method may be used in solid phase assays and affinity purification including immunopurification. The anti-tunicamine derivatives and more preferably the anti-tunicaminyl-uracil antibodies are prepared according to the present invention and described herein by using the tunicaminyl-uracil macromolecule described herein.

In the solid phase assays and affinity purification methods of this invention, the anti- tunicaminyl-uracil antibodies are preferably immobilized by affixing them to a variety of solid phases. Well-known solid phases include ELISA trays and beads formed from glass, polystyrene, polypropylene, dextran, magnetic bead, gold, silver and other solid materials: tubes formed from or coated with such •materials etc. The antibodies can be either covalently or non-covalently bound to the solid phase, by techniques such as covalent bonding via an amide or ester 25 linkage or adsorption. Those skilled in the art will know many other suitable solidphases and methods of immobilizing antibodies thereon, or will be able to ascertain such using no more than routine experimentation.

Immunopurification of tunicaminyl-uracil substances may be performed according to the following procedure. Antibodies that bind the tunicaminyl-uracil 30 toxins are immobilized by affixing them to a solid phase (see above) to form an immunoadsorbent that specifically adsorbs the tunicaminyl-uracil moiety. A liquid C:NORDTACC PECP .DOC SS C:WINWORDSTACVICWDY'FSPECIO68 1 sample from which the tunicaminyl-uracil materials are to be isolated is contacted with the immunoadsorbent under conditions that allow the tunicaminyl-uracilcontaining substances in the liquid to be adsorbed by the immunoadsorbent. The immunoadsorbent and the adsorbed tunicaminyl-uracil-containing substances are then separated. Usually, the immunoabsorbent is washed, and then the tunicaminyl-uracil-containing materials are recovered from the immunoabsorbent.

In conventional affinity chromatography, the immunoadsorbent usually comprises antibody-conjugated particles that are packed into a column. This is a convenient embodiment for tunicaminyl-uracil affinity purification. Antitunicaminyl-uracil antibody-conjugated particles, preferably beads, are packed into a column and the tunicaminyl-uracil containing liquid is passed through the column. The tunicaminyl-uracil materials are retained because of the binding affinity of the immunoadsorbent for the tunicaminyl-uracil moiety. After washing, the tunicaminyl-uracil materials are recovered, most often by elution with an eluant that causes the bound tunicaminyl-uracils to dissociate from the immunoadsorbent.

The tunicaminyl-uracil-specific antibodies of this invention may be useful in purifying the tunicaminyl-uracil toxins and related substances as fine chemicals for use in research aimed at achieving a greater understanding of the production and physiological and biochemical mechanism of tunicaminyl-uracil toxins and associated phenomena. Other benefits from this aspect of the invention will be a means of supplying tunicaminyl-uracil toxins for use as authentic standards in *assays and a means of concentrating low levels of tunicaminyl-uracil toxins from samples to allow their unequivocal identification by physicochemical methods 25 such as HPLC and HPLC-MS. The other reagents and methods of this invention will also aid such research by providing means of detecting and quantitating tunicaminyl-uracil-containing substances and identifying tunicaminyl-uracil binding S" proteins and receptors in vivo and in vitro.

In addition, tunicaminyl-uracil-specific antibodies of this invention may be 30 used in the preparation of affinity-purified tunicaminyl-uracil containing SS C:\WINWORD\STACVC\DYFSPECI\PO6813.DOC substances for use in the production of tunicaminyl-uracil macromolecule carrier conjugates as disclosed herein.

As broadly described above, the present invention pertains to chemical synthesis and immunochemical production of reagents useful in detecting, isolating and quantitating substances incorporating a tunicaminyl-uracil moiety and in protectively immunizing animals against their toxic effects. The assays and detection methods employ tunicaminyl-uracil labelled reagents and antitunicaminyl-uracil antibodies that react specifically with substances incorporating a tunicaminyl-uracil moiety. The latter are also employed in affinity purification of substances incorporating a tunicaminyl-uracil moiety while protective immunization involves vaccination with tunicaminyl-uracil macromolecule conjugates.

In a further embodiment, the invention provides an immunohistology assay method for detecting tunicaminyl-uracil toxin in the tissue animals suspected of having injested tunicaminyl-uracil toxins the assay method including treating the tissue with antibody raised from a tunicaminyl-uracil macromolecule conjugate according to any one of claims 1 to 6 and optionally further treating the tissue with a labelled antibody selective for said antibody to provide a measure of the pressure of said tunicaminyl-uracil toxin in said tissue.

The present invention will now be more fully described with reference to the following examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on •the generality of the invention described above.

EXAMPLE 1 25 PREPARATION AND CHARACTERIZATIONS OF TUNICAMINYL-URACIL PROTEIN CONJUGATES.

This example demonstrates methods of production of immunogens for inducing antibodies against tunicaminyl-uracil toxins (TMU).

Production of immunogen involving conjugation via amino groups.

30 Creating amino groups and conjugation via the amino groups of tunicaminyl-uracil toxins to carboxyl groups of carrier proteins yield more effective S* SS C:\WINWORDSTACVIC\DYFSPECIPO6813.DOC immunogens and produced antibodies with greater affinity for free toxins in circulation.

A preferred method of attaching tunicaminyl-uracil toxins (TMU) and TMU derivatives to proteins is depicted in Scheme 1. The fatty acid and/or acetyl group of a TMU is removed to generate free amino groups which is then coupled, to a carboxyl group or groups on proteins. [Condensation with aldehyde groups generated on the protein, and, if required, subsequent reduction of the imine generated to give a C-N bond, is another approach which can be employed.] Cleavage of the fatty acid and acetyl groups of TMUs can be achieved by one of a number of means. A preferred way of doing this is to use a reagent such as trifluoroacetic acid trifluoroacetic anhydride. This replaces the amide fatty acids and acetyl groups with trifluoroacetyl (TFA) groups. As well as the amino group on the C11 tunicamine moiety and the amino group of the glucosamine being converted to TFA derivatives in this process, all of the hydroxyl groups associated with the TMUs are also converted to their TFA derivatives by this reagent. The TFA groups is removed by one of a number of means. One preferred way of removing the TFA groups is to use an ammonia-methanol mixture. Once the TFA groups have been removed the free primary amino groups are available for coupling to carboxyl groups on proteins using condensation catalysts such as carbodiimides and a range of commercially available products designed for this purpose.

For example, to generate free amino groups for conjugation, 20 mg of tunicamycins or corynetoxins were treated with 20 ml trifluroacetic anhydride and 0.4 ml of trifluroacetic acid at 105 0 C for 48 hours (Scheme After drying under 25 nitrogen, trifluoroacetamide groups were cleaved, by treating with 80 ml of methanolic ammonia at room temperature for 7 days (Scheme The progress S* of the reaction was followed using cellulose thin layer chromatography (E.Merck 5567-7, acetonitrile 50:water 50: ammonium hydroxide 0.04). The reaction products were detected with a ninhydrin spray and identified by mass 30 spectrometry. The amino tunicaminyl-uracil product (mainly the diamino compound shown in Scheme 1) generated in this way (subsequently referred to SS C:\WINWORD\STACVIC\DYFSPECI\PO6813.DOC 18 as amino tunicaminyl-uracil derivative was then attached to carrier proteins by established conjugation methods, using carbodiimide, glutaraldehyde, heterobifunctional and homobifunctional cross linkers.

eeS 19 OH0

CH

3 0 O

H-N

CH

3 -~CH(CH 2 )nCHCH CNH 0 c 0 N NHA~ HO O 0

OH

HOH

2

COH

I 00 C TFAAITFA

F

3 COC H H OCOCF 3

HCOCF

3

FCCOOF

03

F

3 C3COH 2

OCCCC

3 HOCF33 Methanol/NH 3 OH 0 HH H OH O 0

HOH

2

OH

O H SS C:WNODSA~CY\PCPS1.O Scheme 1. The hydroxyl and amide groups of ARGT toxins were transformed to trifluroacetamide groups, which were then cleaved with methanolic ammonia to form amino groups. These amino groups were used to link to carrier proteins using various reagents such as glutaraldehyde, carbodiimide and heterobifunctional and homobifunctional cross-linkers.

(ii) Production of immunogen after creating a carboxyl group on the tunicaminyl-uracil toxin.

Tunicaminyl-uracil toxins were chemically modified to give free carboxyl groups suitable for linking the modified toxins to carrier proteins using carbodiimide reagents, N-hydroxysuccinimide, mixed anhydride etc. to form covalent links with the carrier macromolecules. The tunicaminyl-uracil toxins or their modified synthetic variant compounds can be attached directly to the carrier or through a linking structure.

Succinylation This example demonstrates a method of producing an immunogen for individual antibodies against tunicaminyl-uracil toxins (TMU). The alcohol groups on the carbohydrate moieties of TMU are used for coupling to proteins. Succinic anhydride, is used to acylate the hydroxyls and, in the process, generate free carboxyl groups which are used to form esters and amides with hydroxyl and 20 amino groups on proteins. The primary hydroxyl group on the Nacetylglucosamine moiety was succinylated, using succinic anhydride, to give a linker with a terminal carboxyl group as follows: In 10 ml reaction vessel, 20 mg tunicamycins in 2.5 ml dry pyridine (as 8 mg/ml were reacted with 24 mg of succinic anhydride (10x molar) at 70 0

C

25 overnight. Pyridine was evaporated and unreacted succinic acid was washed with 15 ml diethyl ether and succinylated tunicamycins was dried using a rotary evaporator. Later, succinylated tunicamycins were redissolved in water as 2 mg/ml or in dimethyl formamide (DMF) as 3-4 mg/ml for conjugation to carrier proteins as previously described in the first approach. When DMF is used, the SS C:\WINWORD\STACVICDYF\SPECIP O6 8 1 3.DOC 21 DMF concentration should not be more than 10 of the total water volume in which carrier protein was dissolved. Succinylated tunicamycins in DMF should also be added into protein solution dropwise while stirring to avoid the precipitation of the carrier protein. This is shown in Scheme 2.

OH Tunicamycin /TM

OH

Tunicamycin 0 0 Succinic anhydride TM 0 -C-(CH 2 2

-C-OH

Succinylated tunicamycin

EDC

H

2 N-Prote M -C-(CH2)2-C N Succinvlated tunicamvcin- protein conjugate SS C:\WNWORD\STACVIC\DYFSPECIPO6813.DOC 22 Scheme 2. Changing one or more hydroxyl groups of the carbohydrate moiety to produce a free carboxyl group using succinic anhydride for linking to carrier protein.

Hydrogenation This example demonstrates yet another method of producing immunogens for inducing antibodies against tunicaminyl-uracil toxins. The uracil in the TMU is converted by hydrogenation, e.g. using a rhodium catalyst, to a dihydrouracil.

The latter is unstable in dilute alkali and the ring opens to give a carboxyl group suitable for linking to proteins. This is illustrated in Scheme 3. However, a specific example is given below: mg tunicamycins (Sigma cat T7765) in 6.7 ml methanol (as 3 mg/ml) were added into a 10 ml reaction vessel together with 20 mg of rhodium (5 on alumina). After 2 hours of hydrogenation, reduced tunicamycins were filtered through a 0.2 i solvent resistant filter. The reaction vessel was washed 4x with 3 ml methanol for complete recovery. The combined solvent was evaporated using a rotary evaporator. Reduced tunicamycin was redissolved in 0.1M sodium hydroxide as 2.5 mg/ml. After 2 hours of reaction period, the solution was neutralised with 0.1M hydrochloric acid and ring-opened and reduced tunicamycin concentration was adjusted to 1 mg/ml with water. At that stage, the solution was ready for conjugation to a carrier protein. Ring-opened tunicamycins 20 ml (of 1 mg /ml) were added slowly to the mixture of 33 mg of 10 mg/ml proteins (such as fetuin, cationised bovine serum albumin or hemocyanin) and 100 mg of 1-ethyl-3 (3dimethylaminopropyl) carbodiimide-HCL while stirring. The reaction mixture was then stirred at room temperature for 24 hours in the dark. After the reaction period, 25 ring-opened tunicamycins unattached to protein were separated from the protein conjugate by ultrafiltration using a 10,000 MW cutoff membrane. The retentate was washed 4 times with 20 ml of 40 ethanol and redissolved in 33 ml water as 1 mg protein/ml and stored at -25 0 C in small aliquots. During vaccination, the proteintoxin conjugate can be injected as 0.25-0.5 mg carrier protein/sheep.

SS C:NWORDSTACCDYFSPECPO6813DO SS C:\WlNWORDSTACVCDYFSPECIPO6813.DOC HO OH

H

2 I Rhodium HO OH a.

a a.

a.

a.

a a. a a.

a a a a a 0.1 m NaOH J HO OH SS C:WNOD.TCnOFSEIP81.O 24 Scheme 3. Reduction and ring-opening of the uracil ring to produce a free carboxyl group for linking to the carrier protein.

Carboxylation In this example, free carboxyl groups were introduced for conjugation by using various dicarboxylic acids (COOH-(CH 2 )n-COOH) such as adipic acid, pimelic acid, suberic acid, sebacic acid and 1,10 decanedicarboxylic acid etc (Scheme 4) as follows: Tunicamycins (20 mg) was reacted with 6x molar equivalent of dicarboxylic acid in the presence of 3x molar equivalent of dicyclohexylcarbodimide (DCC) and 0.2x molar equivalent of 4 dimethylaminopyridine (DMAP) in 2.5 ml dry DMF for 20 hours at room temperature. The solvent was evaporated and unreacted dicarboxylic acids and DMAP were removed by washing 5x with 15 ml of diethyl ether. The ether insoluble tunicaminyl-uracil derivatives with free carboxyl groups can be conjugated to carrier proteins as previously described.

The conjugates produced by these approaches were complementary, and generate immunogens that expose different aspects of the toxins and were successful in inducing antibodies against the tunicaminyl-uracil toxins in sheep.

SS C:\W1NWORD\STACVIC\DYF\SPECI\POS813.DOC Tunicamycin ii-

OH

Tunicamycin HO OC-C H -C 2 H OOC-C Hr- 2 Adipic acid

DCC

DMAP

/TO-C-(C

H 2 4 -C-O H

EDC

H2N en .09.

to too..

C

Scheme 4. Introduction of free carboxyl groups for conjugation, by using various dicarboxylic acids (COOH-(CH 2 The proteins used in preparing immunogens in accordance with Example 1 include bovine foetal calf serum protein, haemocyamines, ovalbumin and BSA.

SS C:WNGDSA~CDF.PCVO1.O 26 EXAMPLE 2 Tunicaminyl-uracil labelled reagents tunicaminyl-uracil enzyme conjugates, etc.) Tunicaminyl-uracil toxins were chemically modified to give free carboxyl groups or amino groups as above and linked to enzymes using carbodiimide reagents, N-hydroxysuccinimide, mixed anhydride etc. to form covalent links with the enzyme molecules. The tunicaminyl-uracil toxins or their modified forms can be attached directly to the enzymes or through a heterobifuntional or homobifunctional linking structure.

EXAMPLE 3 Antibody Production The tunicamine derivative of the immunogen may comprise a tunicamycin antibiotic or other tunicaminyl-uracil toxins, differing in regard to the series of fatty acids linked to the amino group of the central tunicamine unit. Such toxins are described by Eckhardt (1983). These toxins are of low molecular weight (800- 900) and do not naturally induce an immune response in animals. We have chemically modified the commercially available tunicamycins (Sigma Cat. T7765), and linked them as haptens to proteins to give immunogenic conjugates. Sheep were injected with the conjugates in an oil adjuvant.

Figure 1 shows the structure of corynetoxin U17a.

Anti-tunicaminyl-uracil antisera were prepared in sheep by subcutaneous injection of emulsions (250-500 jg/dose/sheep) made from tunicaminyl-uracil protein conjugates in an oil adjuvant. Booster injections were given at four-week intervals. Ten ml of blood were taken two weeks after the injection to monitor the 25 specific anti-tunicaminyl-uracil antibodies. Radio-immunoassay (RIA) and ,enzyme-linked immunosorbent assay (ELISA) were developed for assessing the production of antibodies in vaccinated sheep.

EXAMPLE 3A- RADIOIMMUNOASSAY A radio-immunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for assessing the production of antibodies in vaccinated sheep. Sera (0.1 ml) were incubated with 3000 counts per minute (cpm) of

**G

C

SS C:\WINWORD\STACVIC\DYRSPECI\PO6813.DOC 27 tritium-labelled tunicamycins, specific activity of 3000 cpm/8.9 ng, with or without additional unlabelled tunicamycins, at 4°C overnight. Tunicamycins bound to antibodies and unbound toxins were separated by adding an equal volume of saturated ammonium sulphate to precipitate the antibody fraction. Increased binding of tunicamycins to serum antibodies can be detected in sheep after vaccination. (Figure 2).

Moreover, tritium-labelled tunicamycins bound to the antibody fraction can be displaced by adding unlabelled corynetoxins and tunicamycins, demonstrating competition between labelled and unlabelled tunicamycins for anti-toxin antibodies in vaccinated sheep sera (Figure 3).

Figure 2 is a bar graph showing antibody bound fraction of tunicamycins in the serum of the sheep before and after the vaccination.

Figure 3 is a graph showing antibody bound fraction of the tritium-labelled toxins in the serum of vaccinated sheep can be displaced by adding additional unlabelled toxins showing competition between the labelled and unlabelled toxins to bind anti-toxins antibodies.

EXAMPLE 3(B) ENZYME LINKED IMMUNOSORBENT ASSAY After vaccination, increased binding of sheep serum IgG to microtitre plates, coated with modified tunicamycin attached to bovine serum albumin (BSA) 20 or coated directly using the amino derivatives of the tunicaminyl-uracil toxins, can be detected using anti-sheep IgG conjugated to peroxidase (Figures 4 Figure 4 is a graph showing binding of sheep serum IgG to amino tunicaminyl-uracil-glucosamine-BSA conjugate- coated plates before and after vaccination with a tunicaminyl-uracil-glucosamine-BSA conjugate. Binding 25 detected using anti-sheep IgG peroxidase enzyme. Microtitre trays were coated with 25 ng of amino tunicaminyl-uracil-glucosamine-BSA conjugate/well in a carbonate buffer (pH 9.6).

Figure 5 is a graph showing binding of sheep serum IgG to the wells of a microtitre plate coated with 4 ng amino tunicaminyl-uracil glucosamine derivative/well before and after vaccination. Binding detected using anti-sheep IgG peroxidase enzyme. Microtitre trays were coated with amino tunicaminyl- SS C:\WINWORDSTACV1CDYFSPECIP0681.DOC 28 uracil derivative in carbonate buffer (pH 9.6).

EXAMPLE 4 Competitive ELISA for Toxin Detection Serum IgG from vaccinated sheep was purified using a protein G sephrose 4 fast flow affinity column (Pharmacia). After dialysis against phosphate buffered saline, purified IgG was aliquoted and stored at -20 0 C. The tunicamycin standard solution was prepared by weighing crystalline tunicamycins and dissolving them in a known volume of methanol. The concentrations of Corynetoxin U17a and H17a standards, purified by preparative HPLC, were estimated by multiplication of optical density at 260 nm by their respective molecular weights (858 and 876) and dividing by 9650 (eMax at 260 nm).

(ii) Covalent linkage of the amino tunicaminyl-uracil-glucosamine derivatives to microtitre plates.

To analyse the cross reactions of purified serum IgG with different 15 tunicaminyl-uracil toxins, microtitre plates pre-treated with glutaraldehyde were coated with 4 ng of amino tunicaminyl-uracil-glucosamine derivative per well. The plates were washed and different amounts of free corynetoxin U17a, corynetoxin 'H17a, and tunicamycin standards were added to the wells in 100 .l volumes along with the optimum dilution of purified IgG in 100 1. After a two-hour period 20 of competition for antibody binding between the solid and liquid phase toxins, the unbound reagents were washed out. The antibodies bound to the wells were detected by the addition of anti-sheep IgG conjugated to peroxidase. After two hours incubation, the plates were washed and 3, 5, tetramethylbenzidine (Sigma Cat. T2885) substrate was added and incubated for a further 20 minutes 25 before stopping solution was added. A comparison of the optical densities of the wells at 450nm for different amounts of corynetoxin U17a, corynetoxin H17a, and tunicamycins is shown in Figure 6.

Figure 6 is a graph comparing the optical densities of the wells with different amounts of free corynetoxin U17a, corynetoxin H17a, and tunicamycin standards, showing competition for binding with purified vaccinated sheep serum IgG in microtitre plates coated with chemically modified tunicamycin. Bars SS C:%WNMRDTACNAC\DYF\SECUPM13.DOC 29 indicate standard errors of the means of four replicates.

Cross-reactivity of different tunicaminyl-uracil toxins and compounds similar to or forming a part of the chemical structure of tunicaminyl-uracil toxins were compared at 80 and 50 inhibition of the binding of purified IgG to plates coated with 4 ng of chemically modified tunicamycin (Table 1).

Table 1. Cross-reactivity of different tunicaminyl-uracil toxins and compounds similar to or forming a part of the chemical structure of tunicaminyl-uracil toxins at and 50% inhibition of the binding of purified IgG to 4 ng of chemically modified tunicamycin coated plate.

COMPOUND CROSS-REACTION 1 Corynetoxin U17a 2 Corynetoxin H17a 3 Tunicamycins 4 N-acetylglucosamine 5 D-glucose 6 Uracil 7 Uridine 8 Uridine 5'-diphosphate 9 Uridine 5'-diphosphate-Nacetylglucosamine 100 94.9 204 No cross-reactivity No cross-reactivity 0.011 0.045 0.003 0.008 100 91.1 212 No cross-reactivity No cross-reactivity 0.009 0.036 0.004 0.006 10 Sheep injected with modified tunicamycin conjugated to proteins were found to produce antibodies against tunicaminyl-uracil toxins (corynetoxin U17a, corynetoxin H17a and tunicamycins). Cross-reactions were very strong with all three of the tunicaminyl-uracil toxins tested; there was very little or no crossreaction with compounds similar to or forming a part of the structure of tunicaminyl-uracil toxins (Table 1).

Surprisingly one sheep (No. 237), vaccinated with amino tunicaminyluracil derivative conjugated to foetal bovine serum (FBS) using a heterobifunctional (Succinimidyl 4-[N-maleimido-methyl]cyclohexane-1-carboxylate) linker, gave a SS C:\WINWORDSTACVIC\DYFSPECI\PO6813.DOC particularly novel type of standard curve not previously reported in the literature.

When various amount of free tunicaminyluracil toxins were added to wells treated with 0.2 glutaraldehyde and coated with amino tunicaminyluracil-glucosamine derivative-bovine serum albumin (BSA) conjugate in carbonate buffer, pH 9.6., increasing rather than decreasing enzymic activity (increasing optical density) was observed following the addition of anti-sheep IgG horse radish peroxidase (Figure The same novel phenomenon is produced when the wells are left uncoated, provided 0.125 to 0.25 BSA is included in the assay buffer (0.125 to 0.25 BSA and 0.05 tween 20 in phosphate buffered saline, pH These results indicate that tunicaminyluracil toxin added to the wells becomes attached, probably associated with BSA, to the surface of the wells and antibodies in the serum from sheep 237 bind quantitatively to the newly attached toxin. Anti-sheep IgG-HRP then binds to the sheep antibodies in proportion to the added toxin.

The cross-reactivity of the antiserum from sheep no. 237 with corynetoxin 15 U17a, corynetoxin H17a and a tunicamycin mixture is shown in Figure 12. The enzymic activity (increasing optical density) is double the blank value (no toxin in well) when as little as 12.5 pg of free corynetoxin U17a is added into the well.

Moreover, the antiserum showed no cross-reactivity to a range of compounds similar to or forming a part of the chemical structure of tunicaminyluracil toxins.

20 For example, no significant cross reactivity was detected when as much as 16 pg of N-acetylglucosamine, D-glucose, uracil, uridine, uridine 5'-diphosphate, or uridine 5'-diphosphate-N-acetylglucosamine were each added to the wells. That is up to 1.3 x 10 6 times higher than the addition of 12.5 pg of corynetoxin U17a referred to above.

25 Figure 7. Standard curve produced by adding tunicaminyluracil toxins to microtitre tray in the presence of antibodies from sheep no. 237 (1/8000 dilution).

The data was generated using 0.2 glutaraldehyde-treated microtitre trays, coated with 1 ng amino tunicarinyluracil-glucosamine derivative-BSA in carbonate buffer, pH 9.6. The graph shows increasing enzymic activity (increasing optical density) indicating increasing attachment of anti-sheep IgG HRP in wells corresponding to increasing amounts of added tunicaminyluracil SS C:\WINWORDISTACVICDYF\SPECIPO6813.DOC 31 toxins. Values are mean SE of 4 determinations.

A number of approaches have been developed for detecting corynetoxins in field and laboratory samples. A bioassay using nursling rats (Stynes et al., 1979), takes 5 days and is costly due to the requirement for laboratory animals.

Methods currently used to estimate toxicity, such as counting the number of "bacterial" galls in pasture samples and hay (Riley, 1992), immunoassay for the presence of bacterium (Riley and Mckay, 1991) and estimates of the antimicrobial activity of the galls (Riley and Ophel, 1992), have provided a useful guide to predict the level of the risk of toxicity; however, they do not measure the level of corynetoxins, directly. Moreover, the number of "bacterial" galls or the level of bacterium does not always directly correlate with the production of corynetoxins.

Production of corynetoxins increases dramatically as the grass matures (Stynes and Bird, 1983) and the toxin production appears to be correlated to infection of the bacterium with a bacteriophage (Ophel et al., 1993). Weather condition also 15 appears to influence the likelihood of animals being poisoned.

Corynetoxins can be quantified by HPLC (Cockrum and Edgar, 1985).

However, sample preparation for HPLC analysis can be very labour intensive, time consuming, costly and the method has relatively low sensitivity. HPLC analysis of corynetoxins involves detection by UV absorption. UV-absorbing 20 impurities, with similar retention times to those of corynetoxins in the samples, can interfere with the HPLC assay, reducing both specificity and sensitivity of the method.

The ELISA method, we have developed, for measuring corynetoxins and other tunicaminyl-uracil toxins overcomes previous limitations and offers several 25 advantages over other screening methods. Sensitivity and specificity are high, no clean up of sample is required, sample throughput is rapid, the cost per assay and equipment costs are lower than other methods. As multiple samples can be tested simultaneously, the ELISA is suitable for routine monitoring of corynetoxins and other tunicaminyl-uracil toxins in large numbers of samples.

SS C:WINWORD\STACVIC\DYF\SPECI\PO6813.DOC 32 EXAMPLE COATING OF MICROTITRE TRAYS USED IN IMMUNOASSAYS TO DETECT AND MEASURE TMUS.

The diamino TMU according to Example 1 is particularly useful in coating microtitre trays used in immunoassays for corynetoxins. In one application microtitre trays coated with this TMU derivative have been used in a competitive enzyme-linked-immuno-sorbent-assay (ELISA) in which free toxin in samples and the surface-coated diamino TMU derivative compete for binding to TMU antisera in solution. This provides the basis of an ELISA for detecting and quantifying corynetoxins. It has been found that the stability of coating by the diamino derivative of TMUs can be improved by pre-treating the microtitre tray wells with glutaraldehyde.

EXAMPLE 6 VACCINE AGAINST TUNICAMINYL-URACIL TOXINS 15 Challenge With Toxins Commercially available tunicamycins were chemically modified and linked as haptens to proteins as in Example 1. Sheep were injected with the conjugates in an oil adjuvant. Using RIA and ELISA as in Example 2 and 3, it can be demonstrated that the binding of the radiolabelled and unlabelled tunicamycins 20 and corynetoxins increases in the sera of vaccinated sheep.

S Induction of an immune response against tunicaminyl-uracil toxins does not imply that the vaccinated animal will be protected from poisoning. Resistance to poisoning must be assessed and correlated with the immune responsiveness of the animal to the vaccine. To determine if vaccinated sheep were protected, we 25 conducted two pen trials, one simulating a chronic challenge and the other an acute challenge.

a) Chronic Challenge Ten vaccinated and ten unvaccinated sheep were individually housed.

Mean body weight of the vaccinated and unvaccinated sheep were 30.6±1.3 (SE) and 30.7±1.3 kg before vaccination and 32.6±1.5 and 33.6±0.9 kg at the beginning of the challenge. No significant differences in body weights nor any SS C:%WINWORDSTACAC\YFSPECIP6813.DOC 33 adverse affects were observed in sheep due to vaccination. All sheep were injected subcutaneously with 1 pg tunicamycins/kg/day, five days per week. Food intakes were monitored daily. Blood samples were collected and body weight changes were recorded weekly.

After 7 weeks, sheep had received an accumulated dose of 35 lg toxins/kg, which approximates an LD 50 dose (Jago and Culvenor, 1987). By that time, five of the unvaccinated group had shown convulsions, starting as early as 19pjg toxins/kg, compared to only one vaccinated sheep which first convulsed after receiving 30jg toxins/kg. (Figure 8).

Figure 8 is a graph comparing the occurrence of convulsions in vaccinated sheep and unvaccinated sheep.

Four of the unvaccinated group had died (at 20, 21, 23 and 25 [ig toxins/ kg), compared to no death in the vaccinated group. (Figure 9).

15 Figure 9 is a bar chart comparing the number of deaths observed in vaccinated sheep with unvaccinated sheep.

Figure 10 shows the group liveweight changes recorded during *subcutaneous injection of tunicamycin at a rate of 1 [g/day, five days a week b) Acute Challenge 20 Ten vaccinated and ten unvaccinated sheep were used in the trial. Mean body weights of the vaccinated and unvaccinated sheep were 28.6+0.5 and 28.7+0.4 kg respectively before vaccination and 36.0+0.6 and 34.9+1.0 kg before the beginning of oral dosing. No significant differences in body weights nor any adverse affects were observed in sheep due to vaccination.

Figure 11 shows the cumulative score of sheep displaying convulsive episodes.

Figure 12 is a bar chart showing the cumulative score of sheep deaths.

Ground and homogenized annual ryegrass seedheads, infected with bacterium Clavibacter toxicum, were orally administered at 170 g/day/sheep, for six days during the first week and for 5 days during the second week. Corynetoxin concentration in the ground seed was estimated by HPLC to be 83 ig/gm. After SS C:\WNWORD\STACVICDYF\SPECI\PO6813.DOC 12 days, all the surviving sheep had received a total of 155 mg corynetoxins or 3.44 mg corynetoxins per kg (in 11 doses). Feed intakes were monitored daily, blood sampling and body weight changes were recorded weekly. The sheep were monitored for a further 3 weeks after dosing ceased.

Nine of the unvaccinated group (90 had shown convulsions, starting as early as day 6, compared to only one vaccinated sheep 11 days after dosing started (Fig. 11). Nine unvaccinated sheep died, starting at day 7, compared to one death in the vaccinated group on day 13, one day after dosing ceased (Fig.

12). Group liveweight changes are shown in Figure 13.

Figure 13 shows group liveweight changes recorded during oral challenge with corynetoxins.

All remaining vaccinated sheep and the one surviving control animal showed no clinical signs of poisoning during the experiment and for 3 weeks of subsequent monitoring.

15 Following subcutaneous administration of a total of 35p.g pure tunicamycins/kg 7 weeks, at a rate of 1 pg/kg/day, 5 days/week, 50% of the unvaccinated sheep had displayed convulsions and 40% had died. This result accords with the quantitative toxicity measurements of Jago and Culvenor (1987) and reinforces their demonstration of a cumulative effect. Following the same 20 level of exposure, only one sheep had convulsed in the vaccinated group and none had died.

When a total dose of 155mg of corynetoxins (3.44 mg/kg) was administered orally to unvaccinated sheep over 11 days, 90% displayed convulsions and 90% died. This result compares with Jago and Culvenor's 25 estimate of an oral lethal dose in sheep of between 3.2 and 5.6mg/kg. By contrast, vaccinated sheep displayed only 10% convulsion and 10% deaths when exposed to the same oral dose of corynetoxins.

In both the acute and chronic trials the two vaccinated sheep which showed clinical signs of poisoning were found to have not responded as well as the other sheep to the vaccine and had low antibody titres (data not shown) thus indicating that protection is correlated to antibody titer.

SS C:%WNWORDSTACVICDYFSPECIIPOl3.DOC A significant degree of protection was achieved in sheep vaccinated with a synthetic immunogen against ARGT/FPS. Protection appeared to be correlated with antibody titer.

References Australian Bureau of Statistics (1989) Ryegrass toxicity in Western Australia.

Catalogue No. 7421.5.

Berry, P.H. and Stynes, B.A. (1980) Annual ryegrass toxicity, revised by Vogel, P.

and Brown, A.G.P. 1986. Australian Standard Diagnostic Techniques for Animal Diseases NO. 39, CSIRO Australia 1987.

Bryden, Trengove, Davis, Giesecke, P.R. and Curran., G.C.

(1994) Corynetoxicosis of livestock: a nematode-bacterium disease complex associated with different grasses. In: Colegate, S.M. and Dorling, P.R. (eds), Plant-Associated Toxins: Agricultural, Phytochemical and Ecological Aspects, 15 CAB International, United Kingdom, pp 410-415.

Cockrum, P.A. and Edgar, J.A. (1983) High-performance liquid chromatographic comparison of the tunicaminyl-uracil-based antibiotics corynetoxin, .tunicamycin, streptovirudin and MM19290. J. Chromatography 268:245-254.

Cockrum, P.A. and Edgar, J.A. (1985) Rapid estimation of corynetoxins in 20 bacterial galls from annual ryegrass (Lolium rigidum Gaudin) by highperformance liquid chromatography. Australian Journal of Agricultural Research 36, 35-41.

Cockrum, Culvenor, Edgar, Jago, Payne, A.L. and Bourke, C.A. (1987) Toxic tunicaminyl-uracil antibiotics identified in water- 25 damaged wheat responsible for the death of pigs. Australian Journal of *"Agricultural Research, 38, 245-253.

Davies, White, Williams, Allen, J.G. and Croker, K.P. (1996) Sublethal exposure to corynetoxins affects production of grazing sheep.

Australian Journal of experimental Agriculture 36, 649-655.

Davies, Williams, White, C.L. and Hocking Edwards, J.E. (1997) Tunicamycin reduces wool growth by slowing the mitotic activity of wool SS C:\WNWORD\STACVIC\DYF\SPECIPO813.DOC 36 follicles, Australian Journal of Agricultural Research 48, 331-336.

Eckardt, K. (1983). Tunicamycins, streptovirudins and corynetoxins, a special subclass of nucleoside antibiotics. J Nat Prod (Lloydia) 46: 544-550.

Edgar, J.A. (1997). Corynetoxins: a food safety risk? Microbiology Australia 18: 11-12.

Edgar, Frahn, Cockrum, Anderton, Jago, Culvenor, C.C.J, Jones, Murray, K. and Shaw, K.J. (1982) Corynetoxins, causative agents of annual ryegrass toxicity; their identification as tunicamycin group antibiotics. Journal of The Chemical Society Chemical Communications, 222- 224.

Edgar, Cockrum, Stewart, Anderton, N.A. and Payne, A.L. (1994) Identification of corynetoxins as the cause of poisoning associated with annual beard grass (Polypogon monspeliensis Desf.) and blown grass (Agrostis avenacea C. Gemelin). In: Colegate, S.M. and Dorling, P.R. (eds), Plant- 15 Associated Toxins: Agricultural, Phytochemical and Ecological Aspects, CAB International, United Kingdom, pp 393-398.

Frahn, Edgar, Jones, Cockrum, Anderton, N. and Culvenor, C.C.J (1984) Structure of corynetoxins, metabolites of Corynebacterium rathayi responsible for toxicity of annual ryegrass (Lolium rigidum) pastures.

20 Australian Journal of Chemistry 37,165-182.

Jago M.V. (1985). Causative action of corynetoxins in annual ryegrass toxicity. In "Plant Toxicology" (eds AA Seawright, MP Hegarty, LF James and RF Keeler) pp 569-577 The Queensland Poisonous Plants Committee, Yeerongpilly.

Jago, M.V. and Culvenor, C.C.J. (1987) Tunicamycin and corynetoxin poisoning 25 in sheep. Australian Veterinary Joumal 64, 232-235.

Mckay A.C. and Ophel K.M. (1993). Toxigenic Clavibacter/Anguina associations infecting grass seedheads. Ann Rev Phytopath 31: 151-167.

Ophel, Bird, A.F. and Kerr, A. (1993) Association of bacteriophage particles with toxin production by Clavibactor toxicus, the causal agent of annual ryegrass toxicity. Phytopathology 83, 676-681.

Peterson JE Jago MV, Stewart PL (1996). Permanent testicular damage SS C:\WINWORDLSTACVICDYF\SPECInPO613.DOC -I 4.

37 induced in rats by a single dose of tunicamycin Reprod Toxicol 10: 61-69.

Riley, I.T. and Mckay, A.C. (1991) Inoculation of Lolium rigidum with Clavibactor sp., the toxigenic bacteria associated with annual ryegrass toxicity. Joural of Applied bacteriology 71, 302-306.

Riley, I. (1992) Paddock sampling for management of annual ryegrass toxicity.

WA. Journal of Agriculture 33, 51-56.

Riley, I.T. and Ophel, K.M. (1992) Rapid detection of corynetoxins produced by Clavibactor toxicum. Letters in Applied Microbiology 14, 96-99.

Roberts, W.D. and Bucat, J. (1992) The surveillance of annual ryegrass toxicity in Western Australia. Miscellaneous publication No. 39/92 Department of Agriculture Western Australia.

Stewart P.L., May, C. and Jago, M.V. (1998). Reduction and recovery of N-acetylglucosamine- 1-phosphate transferase activity in sheep and rat liver following a single 15 subcutaneous dose of tunicamycin. Aust Vet J 76:20-21 Stynes, Petterson, Lloyd, Payne, A.L. and. Lanigan, G.W. (1979) The production of toxin in annual ryegrass, Lolium rigidum, infected with a nematode, Anguina sp. and Corynebacterium rathayi. Australian Joumal of Agricultural Research 30, 201-209.

20 Stynes, A. and Bird, A.F. (1983) Development of annual ryegrass toxicity.

Australian Journal of Agricultural Research 34, 653-660.

Vogel, Petterson, Berry, Frahn, Anderton, Cockrum, P.A., Edgar, Jago, Lanigan, Payne, A.L. and Culvenor, C.C.J.

(1981) Isolation of a group of glycolipid toxins from seedheads of annual 25 ryegrass (Lolium rigidum Gaud.) infected by Corynebacterium rathayi.

Australian Journal of Experimental Biology and Medical Science 59, 455-467.

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

SS C:\WNWORD\STACVICIDYFSPECIPO6813.DOC

Claims (43)

1. A tunicamine derivative macromolecule conjugate including a tunicamine derivative bound to a macromolecule wherein said conjugate is capable of effecting an immune response.

2. A tunicamine derivative macromolecule conjugate according to claim 1 wherein said tunicamine derivative is tunicaminyl-uracil or a derivative thereof including a hydrogenation product or uracil-ring opened product of a hydrogenation product.

3. A tunicamine derivative macromolecule conjugate according to claim 1 or 2 wherein said macromolecule is linked to the tunicamine derivative via one or more hydroxyl, amino or carboxyl groups of the tunicamine derivative, said molecule selected from the group including proteins and synthetic compounds including polymers.

4. A tunicamine derivative macromolecule conjugate according to any one of claims 1 to 3 wherein said macromolecule is selected from the group consisting of bovine foetal calf serum protein, bovine serum albumin or the like, and high molecular weight plant proteins.

A tunicamine macromlecule conjugate according to any oneof caims 1 to 4 wherein said tunicamine derivative protein of the conjugate is derived from a compound of formula II OH H H H QH R NH 0 R R HO OH wherein R is selected from a uracil radial of formula 0 NH N O SS C:\WINWORDLSTACVICDYFSPECtPO6813.DOC 39 or a derivative thereof, such as the hydrogenated derivative and a ring opended derivative of formula H 2 N-C-N-CH 2 -CH 2 CO 2 H O and R 1 is selected from the group consisting of hydrogen, aliphatic acyl including straight or branch chain aliphatic substituted carbonyl; and R 2 is an optionally substituted sugar residue and wherein said conjugate is substituted, optionally via a linking group, at one or more hydroxyl or amino group of said tunicamine derivative.

6. A tunicamine derivative according to claim 5 wherein R 2 is of formula NHR 3 OH2C OH OH wherein R 3 is hydrogen or acetyl.

7. A tunicamine derivative macromolecule conjugate according to claim 3 or claim 5 wherein the macromolecule is linked to said tunicamine derivative by a linking group selected from the group consisting of diradial groups of formula 0 0 II II where A is a hydrocarbon diradial or derivate thereof.

8. A tunicamine derivative macromolecule conjugate according to claim 7 O O II II -C-(CH -C- wherein the linking group has the formula (CH 2 n wherein n is an integer, preferably n is an integer in the range of from 1 to

9. A method of preparing a tunicamine derivative macromolecule conjugate said method including: providing a tunicamine derivative according to formula II ss C:%WNVVwRDSTACVIC DYFTscpEIpoe13.DOC H H OH RI NH II) R 2 1 R HO OH wherein R is selected from a uracil radical of formula 0 INH |1 or a derivative thereof, such as the hydrogenated N 0 derivative and a ring opened derivative of formula H 2 N-C-N-CH 2 -CH 2 CO 2 H 0 and R 1 is selected from the group consisting of hydrogen, aliphatic acyl including straight or branch chain aliphatic substituted carbonyl; and reacting the tunicamine derivative with a macromolecule such that the macromolecule binds to one or more hydroxy, amino or carboxyl groups of the tunicamine derivative; and R 2 is an optionally substituted sugar residue and wherein said conjugate is substituted, optionally via a linking group, at one or more hydroxyl or amino group of said tunicamine derivative, said method including reacting the tunicamine with a macromolecule such that the macromolecule binds via one or more hydroxy, amino or carboxyl groups of the tunicamine.

10. A method according to claim 9 further including linking the macromolecule to the tunicamine or tunicamine derivative via a linking group to form amide, ester, SS C:AWNORDWSTACVCfYPSPECIPO88l3.DOC ether or other covalent links.

11. A method according to claim 8 wherein the linking group is selected from 0 O II II the group including diradial groups of formula where A is a hydrocarbon diradial or derivate thereof.

12. A method according to claim 9 wherein the linking groups have the formula O O II II -C(CH 2 wherein n is an integer in the range of from 1 to 12.

13. A tunicamine derivative macromolecule conjugate prepared according to any one of claims 8 to 12.

14. A method of producing antibodies and antibody related genetic materials to a tunicamine derivative said method including administering to a subject an effective immunological amount of a tunicamine derivative macromolecule conjugate; and recovering antibodies from the subject.

A method according to claim 14 wherein said tunicamine derivative macromolecule conjugate is according to any one of claims 1 to 5 or claim 13.

16. A method according to claim 14 or 15 wherein administration of the tunicamine derivative macromolecule conjugate further includes an adjuvant.

17. An antibody to a tunicamine derivative macromolecule conjugate said antibody being capable of immunologically reacting with a tunicamine derivative.

18. An antibody to a tunicamine derivative prepared by the method according to any one of claims 14 to 16.

19. An antibody according to claim 17 or 18 which is polyclonal or monoclonal.

A vaccine against a tunicamine derivative said vaccine including an immunologically effective amount of a tunicamine derivative macromolecule conjugate said conjugate being capable of effecting an immunological response to said tunicamine derivative.

21. A vaccine according to claim 20 wherein said tunicamine derivative macromolecule is according to any one of claims 1 to 5 or claim 13.

22. A method of prevention or treatment of tunicaminyl-uracil poisoning in SS C:\WINWORDSTACVC\DYNSPECIPOs613.DOC 42 animals, said method including administering an effective amount of a tunicamine derivative macromolecule conjugate to said animal.

23. A method according to claim 22 wherein said tunicamine derivative macromolecule conjugate is according to any one of claims 1 to 5 or claim 13.

24. A method according to claim 22 wherein the animal is suffering tunicaminyl poisoning caused by the tunicaminyl-uracil toxin from bacteria including Streptomyces spp and Clavibactor toxicus.

A method according to claim 24 wherein the tunicaminyl-uracil toxin is a corynetoxin.

26. A method of detecting the presence of a tunicamine derivative in a sample said method including contacting said sample with an antibody to a tunicamine derivative and detecting complexes of the antibody and tunicamine derivative.

27. A method according to claim 26 wherein said antibody is according to any one of claims 17 to 19.

28. A method according to claim 26 or 27 which is an ELISA wherein the contacting of the sample with an antibody to a tunicamine derivative is conducted on a solid support coated with the tunicamine derivative.

29. A method according to claim 28 which is a competitive ELISA, wherein said solid support is coated with a diamino tunicamine derivative and free tunicamine derivative and coated tunicamine derivative complete for binding to an antibody to a tunicamine derivative.

A method according to claim 28 or 29 wherein the solid support is a surface of a microtitre tray.

31. A method according to any one of claims 26 to 30 wherein the detection of a tunicamine derivative indicates a tunicaminyl-uracil toxin.

32. A method according to claim 31 wherein the tunicaminyl-uracil toxin is from a bacteria including Streptomyces spp or Clavibactor toxicus.

33. A method according to claim 32 wherein the tunicaminyl-uracil toxin is a corynetoxin.

34. A method of isolating and/or purifying a tunicamine derivative from a sample, said method including: SS C:\WlNWORDOSTACVICDYFLSPECIrPO6813.DOC 43 contacting said sample with immobilized anti-tunicamine derivative antibody to form an anti-tunicamine derivative antibody tunicamine derivative complex; and extracting the tunicamine derivative from the anti-tunicamine derivative antibody tunicamine derivative complex.

A method according to claim 34 wherein said antibody is according to any one of claims 17 to 19.

36. A method according to claim 34 or 35 wherein the tunicamine derivative is a tunicaminyl-uracil toxin.

37. A method according to claim 36 wherein the tunicaminyl-uracil toxin is a corynetoxin.

38. An immunohistology assay method for detecting tunicaminyl-uracil toxin in the tissue of animals suspected of having injested tunicaminyl-uracil toxins the assay method including treating the tissue with antibody raised from a tunicaminyl-uracil macromolecule conjugate according to any one of claims 1 to 6 and optionally further treating the tissue with a labelled antibody selective for said antibody to provide a measure of the presence of said tunicaminyl-uracil toxin in said tissue.

39. A method of analysis of a sample for tunicaminyl-uracil toxin including treating a solid phase with the sample to be analysed and treating the solid phase with an antibody to said toxin to detect the presence of said toxin on the solid phase.

A method according to claim 39 wherein the antibody is prepared according to claim 14.

41. A method according to claim 40 wherein the solid phase is contacted with a protein buffer.

42. A method according to claim 41 wherein the protein is present in an amount of no more than 0.5 by weight. SS C:vWNVVRDSTAcVtCODY1SPECIC883.DOC 44

43. A conjugate according to any one of claims 1 to 6 substantially as herein described with reference to any one of the examples. DATED: 15 May, 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys for: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION *se ase sees 06 Goes*: 0 0 SS C:%WWORDSTACVICZDYFSPECIPO6813.DOC