Immunological studies with liposome-bound corynetoxins

Veterinary Immunology and Immunopathology, 19 (1988) 21-30 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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Immunological Studies with Liposome-Bound Corynetoxins A.S. McWILLIAM ~and P. VOGEL

Animal Health Division, Western Australian Department of Agriculture, South Perth, W.A. 6151 (Australia) 1Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX 1 3RE (Great Britain); author to whom correspondence should be addressed. (Accepted 13 November 1987)

ABSTRACT McWilliam, A.S. and Vogel, P., 1988. Immunological studies with liposome-bound corynetoxins. Vet. Immunol. Immunopathol., 19: 21-30. Attempts were made to raise antibodies against corynetoxins, a family of toxins responsible for annual ryegrass toxicity. The glycolipid nature of corynetoxins made them ideally suited for incorporation into the structure of small unilamellar liposomes. Sheep were injected with corynetoxin liposomes with and without adjuvants such as lipid A and muramyl dipeptide, and the sera tested for anti-corynetoxin antibody. Similarly, rabbits were injected with hydrolysed corynetoxin coupled to human IgG and keyhole limpet haemocyanin and with corynetoxin coupled to bovine serum albumin. These preparations were administered with complete Freund's adjuvant. The failure of any of these preparations to elicit an anti-corynetoxin antibody response in either sheep or rabbits is discussed.

INTRODUCTION

Annual ryegrass toxicity (ARGT) is a rapidly spreading neurological and hepatoxic disease of livestock which occurs in southern and western Australia and South Africa. The disease is characterized by ataxia, convulsions, a pale enlarged liver and haemorrhage in various organs (Berry and Wise, 1975). Histopathologically, changes in the central nervous system (Berry et al., 1980) and liver (Berry et al., 1982) are seen. ARGT results when animals ingest annual ryegrass (Lolium rigidum) containing galls induced by a nematode (Anguina agrostis ) (Stynes and Bird, 1980 ) and subsequently colonized by Corynebacteriurn rathayi. The bacteria produce the causal agent of ARGT, a family of highly toxic glycolipids known as corynetoxin (CT) (Fig. 1 ) which is similar to tunicamycin (TM) in both structure (Frahn et al., 1984) and biological activity (Vogel et al., 1982; Jago et al., 1983 ). 0165-2427/88/$03.50

© 1988 Elsevier Science Publishers B.V.

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OH o

.o

~O

I

.

.

R~HN~~jC@OH

'/ /~/H

CORYNETOXINS acid

$-hydroxy

(a)

C17 , C19

Iso

(1)

C16 -

normal

(n)

antelso

C18

series

RCO

Q,B -

-

unsat,

saturated

C17, C19

C15, C17, C19

C16

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C16, C18

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Fig. l. Structureoftheglycolipidcorynetoxin ~mily.(A~erFrahnetaL, 1984). As stock losses continue there is a growing demand by farmers for either prophylactic and/or therapeutic treatment of affected animals. Richards et al. (1979) found that chlordiazepoxide treatment of ARGT-affected sheep was effective in a pen situation. However, this treatment was ineffective and impractical in the field (Norris et al., 1981). This paper describes our attempts to raise antibodies to CT for possible vaccine production and for research and diagnostic purposes. In particular, a method is described for the incorporation of CT into the structure of small unilamellar vesicles (SUV) and the use of these for inoculation of sheep and rabbits. MATERIALS AND METHODS

Preparation of corynetoxin Corynetoxin was isolated from bacterial galls by the method of Vogel et al. (1984). By dissolving CT in methanol-HC1 (pH 3) and incubating for 1 h at room temperature, a form of CT was obtained which had a fourfold reduction

23 in LD~o in 2-week-old rats. This modified CT (MCT) was indistinguishable from CT by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Nuclear magnetic resonance (NMR) studies on HPLC-purified MCT confirmed its similarity to CT.

Liposome preparation Small unilamellar vesicles (SUV) were prepared using a modification of the method of Batzri and Korn (1973) in which L-v~-dipalmitoyl phosphatidylcholine (DPPC) (8 mg), cholesterol (4 mg) and either CT or MCT (2.5 mg) were dissolved in methanol (4 ml) and injected below the surface of 10 ml of rapidly stirred TRIS/HC1 (pH 8.4) at 55 °C. Mixing was continued without heating for 30 rain and the SUV concentrated by centrifugation at 150 000 x g for 3 h at 22°C. The SUV were washed twice with 10 ml T R I S / H C I (pH 8.4) and resuspended in 5 ml of buffer.

Lipid A Lipid A was prepared from lipopolysaccharide (LPS) of Salmonella abortusequi (Sigma Chemical Company, U.S.A. ), by a modification of the method of Dancey et al. (1977). LPS was dissolved in 1% (v/v) acetic acid at 3 rag/ ml. The solution was heated in a boiling water bath until a precipitate formed (approximately 2 h ). The precipitate was filtered, washed twice with distilled water and once with acetone, and air-dried.

Electron microscopy Liposome structure was confirmed by negative staining of the SUV preparation on formvar-carbon-coated grids using phosphotungstic acid ( 2 %, w~v ). Grids were examined in a Siemens 102 transmission electron microscope at 80 kV.

Corynebacterium rathayi Corynebacterium rathayi was isolated from bacterial galls and grown on Kado and Heskett medium (Kado and Heskett, 1970) for 7 days at 25°C. Bacteria were suspended in TRIS/HC1 (pH 8.4) at I g wet weight/10 ml. Preparation of inoculum (a) MCT-liposomes with muramyl dipeptide (MDP) A suspension of SUV containing M C T (3 ml) was mixed with 0.3 ml of Nacetyl-muramyl-l-alanyl-d-isoglutamine (MDP, Sigma Chemical Company, St.

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Louis, MO, U.S.A. ) in TRIS/HC1 (pH 8.4) at 1 mg/ml and left overnight at 4°C.

(b) MCT-liposomes with lipid A Lipid A was dissolved at 1 mg/ml in chloroform/methanol (1:1, v/v). This solution was incorporated with the methanolic solution of MCT, DPPC and cholesterol. The SUV were then prepared as described. However, before centrifugation the chloroform was removed by evaporation under reduced pressure at 37 ° C.

(c) MCT-liposomes and methylated bovine serum albumin (MeBSA) One hundred and fifty microliters of a 2 mg/ml solution of MeBSA (Sigma Chemical Co.) were mixed with 3 ml of MCT-liposome preparation and left overnight at 4 ° C.

(d) Corynebacterium and complete Freund's adjuvant (CFA) Equal volumes of CFA (C.S.L., Australia.) and C. rathayi suspension, prepared as described, were emulsified. Sheep were injected intramuscularly with 5 ml of the emulsion, containing 0.25 g wet weight of Corynebacterium rathayi, into several sites in the flank mucles.

(e) MCT with MeBSA and CFA MCT (1 mg) was dissolved in I ml TRIS/HC1 (pH 8.4) containing 500 ~g MeBSA. The solution was left overnight at 4 ° C and then made up to 3 ml with TRIS/HC1 buffer. Equal volumes of this solution and CFA were emulsified and sheep injected intramuscularly into multiple sites with 1.2 ml of emulsion which contained 200 ~g MCT.

(D Hydrolysed CT (HCT) coupled to human immunoglobulin G (HIgG) and keyhole limpet haemocyanin (KLH) CT (20 mg) was hydrolysed in 3 M hydrochloric acid (3 ml) for 3.5 h at 100 ° C. The hydrolysate was then neutralized by passage through a 10-ml column of Amberlite TR-45 ( O H - ) resin (BDH, U.K.) and eluted with distilled water. The sample was lyophilized and washed with dry ether. Eighteen railligrams of HCT and 10 mg of either KLH (Sigma Chemical Co.) or HIgG (provided by Dr G. Stewart, Clinical Immunology, Princess Margaret Hospital, Perth, Western Australia) were dissolved in 0.5 ml of distilled water, and 200 mg of 1-ethyl-3- (3-dimethyl-aminopropyl) -carbodiimide hydrochloride was added. The solution was made up to 1.75 ml with distilled water and agitated overnight at room temperature. The solution was dialysed against 0.1 M phosphate-buffered saline (PBS) (pH 8.0) and emulsified with an equal volume of CFA. Rabbits were injected intramuscularly with the emulsion such that each received 5 mg HCT. Booster injections were given 14 days later using incom-

25 plete Freund's adjuvant (IFA). A coupling ratio of approximately 1200 molecules of HCT per KLH molecule was found.

(g) CT coupled to BSA CT was coupled to BSA using sodium periodate according to the method of Smith et al. (1970) at molar concentrations of toxin:periodate of 1:10 and 1:1. The toxin:protein ration was 25:1.

Determination of MCT incorporation into liposomes The degree of incorporation of MCT into SUV was determined by dissolving 1 ml of lyophilized MCT-SVU preparation in 5 ml of methanol and submitting the solution to reverse phase HPLC analysis on a C18 ~t-Bondapak column (3.9 mm X 30 cm, Waters Associates). The column was run in methanol-water (3:1, v/v) at 2.0 ml/min and the effluent monitored at 254 nm. Pure CT was used to generate a standard curve of peak height versus amount of CT.

Removal of serum albumin As the binding of CT to serum albumin produced false positive results in the antibody assays, albumin was removed prior to testing of the sera. Blue Sepharose CL-6B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) (5 ml) was washed with 0.1 M phosphate buffer (pH 7.0) and then incubated for 1 h at room temperature with 0.5 ml antiserum diluted 1:4 with phosphate buffer. After centrifugation the supernatant was removed, lyophilized and reconstituted to 1/4 volume with distilled water.

Inoculation Sheep Five groups of three sheep (1-year-old Merino ewes, 29-35 kg) received inoculations with preparations a-e {from Materials and Methods). All SUV preparations were administered intraperitoneally (IP) as 1 ml of solution containing 80 pg MCT. Booster injections were given on days 7, 21, 30, 37 and 43. A control group of three animals received the same dose of pure unmodified CT in TRIS/HC1 (pH 8.4) IP on the same days.

Rabbits Groups of two rabbits (6 months old) received inoculations of preparations f and g, administered intramuscularly (IM) such that each received 3 mg CTcarrier. Booster injections were given on days 7, 14 and 21.

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Antibody assays Agglutination assay Anti-CT antibodies were assayed using an agglutination assay in which CT was hydrophobically bound to Phenyl-Sepharose CL-4B. Phenyl-Sepharose CL-4B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) was washed with TRIS/HC1 (pH 8.4) and 0.1 ml of packed gel was incubated with 1 mg CT in TRIS/HC1 in i ml total volume for 1 h at 37 ° C. The gel was washed twice with TRIS/HC1 buffer and resuspended in 2 ml of buffer. This suspension was used in a standard agglutination assay with anti-CT antiserum.

Immunodiffusion Immunodiffusion analyses were performed according to the method of Ouchterlony (1958).

Complement fixation test (CFT) In a CFT the MCT was used at a concentration of I mg/ml in TRIS/HC1 pH 8.4, guinea pig complement at a dilution of 1:25 and rabbit haemolytic serum at 1:500.

Inhibition of bacterial inhibition assay The bacterial inhibition assay (BIA) for CT (Stynes and Vogel, 1983) was modified to allow detection of anti-CT antibodies. It was hoped that as well as detecting antibodies, this assay would measure the CT neutralizing capacity of the antisera. Test sera were incubated with CT at 37 ° C for 1 h and a BIA performed. The BIA relies on inhibition of the growth of Corynebacterium tritici by concentrations of CT > 125 ng/ml. The presence of anti-CT antibodies would result in binding of CT and hence a reduction in CT-induced inhibition of bacterial growth. A positive control consisted of a solution of bovine serum albumin to which CT binds strongly.

Enzyme immunoassay An enzyme immunoassay (Engvall et al., 1971) was modified to allow detection of sheep anti-CT antibody. As the binding of CT to polystyrene ELISA plates could not be confirmed, CT bound to Phenyl-Sepharose 4B was used as the solid phase. Washed and centrifuged Phenyl-Sepharose 4B (0.43 g) was mixed with 6 mg CT and made up to 3.5 ml with TRIS/HC1. After incubation for 90 min at room temperature, the gel was washed with TRIS/HC1 (pH 8.4) and incubated with 1.5 mg ferritin in 1 ml TRIS buffer for 1 h at room temperature. The gel was washed twice with TRIS buffer and resuspended in 200 pl PBS. Twenty-five microlitres of gel suspension were placed in glass tubes and incubated at room temperature for 1 h with 200 #l of test sera. The gel was then washed with PBS, centrifuged and 200 #l of a 1:400 dilution of alkaline

27 phosphatase conjugated to rabbit anti-sheep IgG (Kirkegaard and Perry, Maryland, U.S.A.) added. After 1 h at room temperature, the gel was washed twice with PBS. Two hundred microlitres of a 1 m g / m l solution of Sigma 104 alkaline phosphatase substrate in diethanolamine buffer (pH 9.8) were then added. After 1 h the gel was centrifuged and the optical density of the supernatant measured at 405 nm. RESULTS

Liposome incorporationof MCT Using the HPLC assay for MCT it was determined that 75% of the added MCT was incorporated into SUV. This figure is too high for CT trapped in the aqueous phase of the SUV, hence it may be assumed that most of the MCT was incorporated as part of the phospholipid structure of the SUV. Electron microscopy of negatively stained liposome preparations confirmed that SUV were present and had a diameter of approximately 30 nm.

Antibody production No antibodies to CT were detected in the sera of any of the inoculated animals using the assays described; however, antibodies were detected against the carrier molecules, HIgG, KLH and BSA by immunodiffusion.

Challenge experiments Following inoculation with the preparations a-e (Materials and Methods) all fifteen sheep received increasing intravenous (IV) doses of pure CT (1, 2, 5, 7.5, 10, 12/Lg/kg body weight) weekly over a period of 6 weeks, to determine whether they had developed any resistance to CT. Compared with a group of three uninoculated control animals, inoculated animals showed no significant difference in the amount of toxin necessary to induce visual signs of toxicity such as ataxia, tremors, convulsions or death. DISCUSSION While glycolipids are not usually antigenic, antibodies against them have been produced by using cell membranes, micellar suspensions of purified lipid and heterologous protein, emulsions of glycolipid with MeBSA and CFS or by incorporating the glycolipid into liposomes (Alving, 1977). We incorporated the glycolipid corynetoxins into the structure of liposomes as a means of rendering them immunogenic. Phosphatidylcholine liposomes were chosen as they are biodegradable, non-immunogenic and act as an adju-

28 vant for incorporated or attached haptens (Van Rooijen and Van Nieuwmegen, 1980a). As antibodies to glycolipids are directed primarily against the carbohydrate moiety (Rapport and Graf, 1969) and the adjuvant effect of liposomes is thought to be due to the surface exposition of antigen (Van Rooijen and Van Nieuwmegen, 1980b ), small unilamellar vesicles (SUV) were chosen to optimize the surface exposition of CT and hence the chances of producing anti-CT antibodies. In addition, the glycolipid nature of CT makes it ideally suited for incorporation into the structure of these liposomes. Although liposomes have been used to raise anti-mannosyl, anti-galactosyl and anti-glycolipid antibodies (Das et al., 1982a, b; Lingwood et al., 1980), the administration of CT incorporated into SUV failed to induce any detectable anti-CT antibody response. This failure may be due to several factors arising from the nature and toxicity of CT. Firstly, low molecular weight glycolipid molecules, such as CT (MW =800 daltons), are classically very poor immunogens and may require extended immunization regimes for antibody production. Secondly, the extreme toxicity of CT (LDso, IP, 2-week-old rats = 100 #g/ kg) means that only small amounts can be administered. There is also evidence that CT has a cumulative effect when administered IP to sheep (unpublished results). In addition, the inability to administer more than minute amounts of CT is contradictory to the requirement that larger amounts of glycolipid than protein are necessary for raising antibodies (Radunz and Berzborn, 1970). Thirdly, Wood and Kabat (1981) found that carbohydrate antigens which protrude furthest from the liposome surface were able to induce the best antibody response. Thus CT may not protrude sufficiently from the surface of the SUV. Finally, CT is essentially identical to tunicamycin, an antibiotic which blocks the formation of lipid-linked oligosaccharides, thereby preventing glycosylation of proteins (Tkacz and Lampen, 1975). Tunicamycin has been shown to inhibit the in vitro secretion of immunoglobulin from plasma cells by amounts ranging from complete inhibition of IgE, 85% of IgA, 81% of IgM and 28% of IgG (Hickman et al., 1977; Hickman and Kornfeld, 1978). It is therefore possible that CT may impair or prevent production and secretion of anti-CT antibodies by cells of the immune system; however, the production of antibodies to the carrier portion of CT conjugates suggests that there is no general immunosuppression produced by CT. Attempts were made to augment the adjuvant effect of the liposomes by incorporation of the B cell mitogen, lipid A or muramyl dipeptide (MDP) in the liposome inoculant. Both MDP (Jolivet et al., 1981; Audibert and Chedid, 1980) and lipid A (Van Rooijen and Van Nieuwmegen, 1980c; Dancey et al., 1977 ) have been found to increase the adjuvant effect of liposomes containing protein and lipid antigens. However, no anti-CT antibodies were detected in sera of animals inoculated with these preparations. Further attempts to produce antibodies involved inoculation with conjugates of CT to BSA, HCT to KLH and HIgG and inoculation with CT-pro-

29 ducing c u l t u r e s of Corynebacterium rathayi. A g a i n no a n t i - C T a n t i b o d i e s were d e t e c t e d in a n y s e r a w i t h a n y of t h e s e schedules. T h e n o n - a n t i g e n i c i t y o f cory n e t o x i n is s i m i l a r to t h a t f o u n d w i t h t h e a n t i b i o t i c l i n c o m y c i n ( W h i t e , 1966). I n conclusion, we h a v e d e s c r i b e d a m e t h o d for t h e i n c o r p o r a t i o n of large a m o u n t s of C T into t h e s t r u c t u r e of S U V a n d h a v e u s e d t h e s e for t h e inocul a t i o n o f sheep. D e s p i t e t h e fact t h a t a n u m b e r of d i f f e r e n t i n o c u l a n t s a n d a d j u v a n t s were used, no a n t i - c o r y n e t o x i n a n t i b o d i e s were detected. ACKNOWLEDGEMENTS T h i s r e s e a r c h was s u p p o r t e d b y a g r a n t f r o m t h e A u s t r a l i a n W o o l C o r p o r a tion. T h e a u t h o r s w i s h to e x p r e s s t h e i r g r a t i t u d e to P r o f e s s o r W . J . P e n h a l e , School of V e t e r i n a r y Studies, M u r d o c h U n i v e r s i t y , W e s t e r n A u s t r a l i a for critically r e v i e w i n g t h e m a n u s c r i p t .

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