Rabbits were vaccinated once, by subcutaneous and intradermal injection, with sonicates of oncospheres (TpO) or conditioned media from in vitro maintained mature metacestodes (TpMcES) of Taenia pisiformis. Extracts were either incorporated into or mixed with unilammelar liposomes (reverse phase evaporative vesicles) or emulsified in Freund's Incomplete Adjuvant (FIA). Control groups received liposomes or FIA without antigen, or antigen preparation without adjuvant. Rabbits were challenged orally two weeks after vaccination with approximately 1500 eggs of T. pisiformis and necropsied eight weeks after challenge. A mean of 155 cysts was recovered from seven control rabbits. A 67% reduction in peritoneal cyst numbers was obtained in TpO-IFA vaccinated rabbits compared to 75% for the TpO-liposome entrapped group. The highest level of protection (86%) was obtained when TpO was mixed with but not entrapped in liposomes. Only 32% and 39% reduction in peritoneal cyst numbers was obtained after immunizing with the TpMcES preparation in liposomes or IFA respectively, however >85% of peritoneal metacestodes were dead (necrotic or calcified) and suggests a different immune response than occurs after vaccination with oncosphere extracts. Specific anti-oncospheral or anti-metacestode ES antibody (IgG) responses at two weeks post vaccination were similar in rabbits immunized with liposome or IFA associated extract

Craig, P. S.

Zumbuehl, O.

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Journal of Helminthology (1988) 62, 58-62Immunization against experimental rabbit cysticercosisusing liposome-associated antigen preparationsP. S. CRAIG1 and O. ZUMBUEHL:'Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place,Liverpool L3 5QA, England'' Physikalische Pharmazie-ETH, Sonneggstrasse 5, CH-8092 Zurich, SwitzerlandABSTRACTRabbits were vaccinated once, by subcutaneous and intradermal injection, with sonicates of onco-spheres (TpO) or conditioned media from in vitro maintained mature metacestodes (TpMcES) ofTaenia pisiformis. Extracts were either incorporated into or mixed with unilammelar liposomes (reversephase evaporative vesicles) or emulsified in Freund's Incomplete Adjuvant (FIA). Control groupsreceived liposomes or FIA without antigen, or antigen preparation without adjuvant. Rabbits werechallenged orally two weeks after vaccination with approximately 1500 eggs of T. pisiformis and necro-psied eight weeks after challenge. A mean of 155 cysts was recovered from seven control rabbits.A 67% reduction in peritoneal cyst numbers was obtained in TpO-IFA vaccinated rabbits compared to75% for the TpO-liposome entrapped group. The highest level of protection (86%) was obtained whenTpO was mixed with but not entrapped in liposomes. Only 32% and 39% reduction in peritoneal cystnumbers was obtained after immunizing with the TpMcES preparation in liposomes or IFA respectively,however >85% of peritoneal metacestodes were dead (necrotic or calcified) and suggests a differentimmune response than occurs after vaccination with oncosphere extracts. Specific anti-oncospheral oranti-metacestode ES antibody (IgG) responses at two weeks post vaccination were similar in rabbitsimmunized with liposome or IFA associated extracts.KEY WORDS: Taenia pisiformis, immunization, oncosphere, metacestode, liposomes, rabbitsINTRODUCTIONThe ability to immunize animals against taeniid larval cestode infection using livingoncospheres or their extracts (sonicates or in vitro culture 'ES' preparations) hasbeen convincingly demonstrated to varying degrees in mice, rabbits, sheep andcattle (see reviews, FLISSER et ah, 1979; RICKARD, 1982; RICKARD & WILLIAMS,1982). However, in almost all cases where oncospheral antigen preparations wereused as experimental vaccines, they were associated with an adjuvant, notablyFreund's Complete (FCA) or Incomplete (FIA) adjuvants. Unfortunately Freund'sadjuvant is only acceptable for use in experimental animals and is definitely notacceptable for human use due largely to problems of granuloma formation, plasmacell tumours (in mice) and mineral oil persistence (refs cited by SIDDIQUI et ai,1981).There is therefore a need to investigate alternative ways of presentingimmunizing antigens to the host, especially if practical vaccination against humancysticercosis {Taenia solium) or human hydatidosis (Echinococcus granulosus andE. multilocularis) ever becomes a feasible proposition. The use of liposomes couldprovide a useful and acceptable alternative to mineral oil based adjuvants.Liposomes, originally described by BANGHAM et al. (1965) are microvesiclesartificially composed of phospholipid membranes enclosing an aqueous compart-ment, which have remarkable similarities to biological membranes and exhibitimmune adjuvant properties (TOM, 1980). Liposomes are readily taken up bymacrophages and thus the liver is an important target organ. Most applications todate of liposomes in parasitology have involved targetting chemotherapy ofprotozoal infections (CROFT, 1986).of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X00011226Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 17:22:23, subject to the Cambridge Core termsImmunization against rabbit cysticercosis using liposome-associated antigens 59Here we report the use of liposomes to immunize rabbits against oral infectionwith T. pisiformis, a natural taeniid of rabbits. The results were compared with theuse of Freund's Incomplete Adjuvant.MATERIALS AND METHODSRabbits and vaccination protocolThirty-two, 16 week old female NZ white rabbits were divided into vaccinationand control groups (see Table I). Rabbits were vaccinated intradermally andsubcutaneously 0-5 ml total volume (approximately 300 ug protein) on Day 0,challenged orally two weeks later with approximately 1500 eggs per rabbit of T.pisiformis and necropsied eight weeks after challenge. The numbers of cysts in theperitoneal cavity (including liver surface) were counted and their viability notedmacroscopically. Control groups were injected with FIA or liposomes withoutantigen or with immunizing antigen alone. Rabbits were ear bled at variousintervals and sera stored at —30°C until used.Immunizing extracts of T. pisiformisOncospheres were artificially hatched from eggs of T. pisiformis and purified onPercoll (Pharmacia) to remove shell components and unhatched eggs (RAJASEKAR-IAH et ai, 1980), and sonicated in 0-15 M phosphate buffered saline pH 7-2 (PBS)as described by CRAIG & RICKARD (1981). The oncospheral sonicate wascentrifuged at 500 g for 30 mins at 4°C and the supernatant used as the immunogen(TpO). Each rabbit was vaccinated with the product of approximately 75 000oncospheres.An excretory-secretory (ES) antigen extract was prepared from the maturemetacestode stage maintained in vitro, essentially as described elsewhere (CRAIG,1984). Briefly, 'conditioned' serum-free media (DMEM + 20 mM hepes + 1%glucose) was harvested every third day from 20 mestacestodes in a 10*ml culture,pooled and concentrated through a 10 000 Mwt filter (Amicon Corp) anddesignated TpMcES.LiposomesUnilammelar reverse phase evaporative vesicles (REVs) were preparedessentially as described by SZOKA & PAPHADJOPOULOS (1978). 20 mg sphingo-TABLE I. Mean numbers of cysts of Taenia pisiformis present in peritoneal cavity of vaccinated orcontrol rabbitsVaccination groupControl (lip)(IFA)TpO aloneTpO+lipTpO+IFATpO+lip ("free")TpMcES aloneTpMcES±lipTpMcES ± IFANo. rabbits74444333No.Range64-31049-8022-8034-7312-4374, 95, 16560, 105, 15053, 88, 140cystsMean±S.D.155±9062±1439±18a52±1922±14b111+48105±4594±44% cysts necroticmean8305080108595100% protection60756786283239Students t-test: b vs a—p<005. lip=liposomes. IFA=Incomplete Freund's Adjuvant. TpO = T. pisiformisoncosphere extract. TpMcES=T. pisiformis metacestode "ES" extract.of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X00011226Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 17:22:23, subject to the Cambridge Core terms60 P. S. CRAIG and O. ZUMBUEHLmyelin (Sigma) and 8 mg cholesterol (recrystallized) were dissolved in 3 mlchloroform in a stoppered glass test tube suspended in a 37°C water bath. To thiswas added 3 ml of ether and then 1 ml of antigen and the mixture bath sonicated (at37°C). Solvent was removed by rotary evaporation at 37°C for 30 mins. Aftervortex mixing REVs (liposomes) were produced.In addition, liposomes were prepared exactly as above but without antigen('empty') and used as controls. Also a batch of 'empty' liposomes were incubated inantigen solution at room temperature for 1 hr (i.e. antigen not entrapped inliposomes).Freund's AdjuvantThe TpO and TpMcES antigen preparations were emulsified in equal volumesof Freund's Incomplete Adjuvant (Sigma)Enzyme linked immunosorbent assay (ELISA)The method of ELISA for detecting specific IgG antibodies to the TpMcES andTpO antigen preparations was essentially as described previously (CRAIG &RICKARD, 1981; CRAIG, 1984). Antigens were used to coat plates at ~ 5 ug prot/mland all incubations carried out at 4°C. Rabbit sera were used at a dilution of 1:150.RESULTSThe mean numbers of cystic metacestodes obtained from the peritoneal cavityof rabbits in vaccinated and control groups is shown in Table I. A mean of 155 cystsper rabbit (range 64-310) were counted in the control group (FIA or liposomeswithout antigen) and approximately 92% appeared viable. Vaccination of rabbitswith oncospheral sonicate TpO entrapped inside liposomes resulted in a 75%reduction in cyst numbers. This was comparable to the 67% reduction in cystnumbers obtained when TpO-FIA was used to immunize rabbits. However thegreatest degree of protection, 86% was obtained in the group vaccinated withliposomes which had only been passively incubated in the TpO preparation, i.e. theantigen was not entrapped within the liposomes. Vaccination with TpO aloneresulted in 60% protection.In contrast, the level of protection obtained when rabbits were vaccinated withthe TpMcES preparation, either in liposomes or FIA or alone was much lower, i.e.32%, 39% and 28% respectively. However, a greater proportion of the cysts(85-100%) observed in the TpMcES vaccinated rabbits were dead, necrotic and/orpartially calcifying, compared to the TpO vaccinated groups (Table I).Measurement of specific IgG antibody responses using TpO or TpMcES antigenpreparations in the ELISA revealed very similar responses in rabbits for bothliposome or FIA associated vaccine groups (Fig. 1). There was perhaps someindication that anti-TpO antibody responses were maintained for a longer durationin the TpO-FIA group compared to the TpO-liposome group.DISCUSSIONA significant degree of protection (75% and 86%) against oral challenge with T.pisiformis eggs was obtained in rabbits immunized with a crude low speed sonicateof T. pisiformis oncospheres entrapped in, or passively coated onto liposomes(REVs). This level of protection was at least as good as that obtained when theoncospheral sonicate (TpO) was injected after being emulsified in Freund'sIncomplete Adjuvant (i.e. 67%). RAJASEKARIAH et al. (1985) used similar TpOof use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X00011226Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 17:22:23, subject to the Cambridge Core termsImmunization against rabbit cysticercosis using liposome-associated antigensFIG. 1.One. group61QOMcES groupo oVac 2wpv 2wpcchall.8wpcFIG. 1. Serum antibody responses (IgG-ELISA) against a) TpO or b) TpMcES extracts in groups ofrabbits vaccinated by a) oncospheral or b) metacestode (McES) extracts in liposomes or FIA, or PBS'controls' in liposomes. wpv=weeks post vaccination; wpc=weeks post challenge.sonicates to vaccinate rabbits, and obtained 90% protection, after two intramuscularinjections two weeks apart in Freund's Complete Adjuvant followed by FIArespectively. In the present study the greatest level of protection (86%) wasachieved after a "single" injection with TpO coated onto the surface of liposomes.This level of protection was greater than the 60% level obtained after vaccinationof rabbits with TpO alone or the 76% level obtained when the TpO preparationwas entrapped inside liposomes. This suggests that functional antigen in TpO maybe better presented to the immune effector cells, e.g. macrophage on the liposomesurface. Interestingly VAN ROOIJEN & VAN NIEUWMEGEN (1980) observed a similarresult when they measured the immune response in rabbits against human serumalbumin (HSA) that had been coated on 'empty' liposomes or entrapped in thevesicles. The greatest responses were associated with liposome surface coatedHSA, and these authors suggested that adjuvant effects of liposomes are mediatedby antigen exposed on the liposome surface.In contrast to the immunity obtained by TpO vaccination against invadingoncospheres from the oral egg challenge, which is probably effective in rabbits 3—5days after infection (RAJASEKARIAH et al., 1985), it appears from the present studyof use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X00011226Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 17:22:23, subject to the Cambridge Core terms62 P. S. CRAIG and O. ZUMBUEHLthat a different immune response is active in the TpMcES vaccinated rabbits and isdirected at the later metacestode stages. This was manifested by the presence oflarge numbers of dead or necrotic larvae in the peritoneal cavity. Theseobservations fit in with the concept of post-encystment immunity, where theimmune response affects the mature or established parasite rather than the earlyoncosphere stage (see reviews by GEMMELL & SOULSBY, 1968; RICKARD &WILLIAMS, 1982). However from our own experiments we cannot say at what timeafter infection metacestodes become susceptible to the immune response, but it islikely to be after emergence from the liver (i.e. 2-3 weeks post infection).In summary, liposome entrapped low speed supernatants of TpO sonicates gaveas good protection against the development of rabbit cysticercosis as TpOemulsified in FIA. Functional oncospheral antigen(s) in T. pisiformis may be moreeffectively presented to the host immune system on the liposomes surface.Vaccination with the TpMcES from the mature metacestode (in liposomes or FIA)did not prevent infection, but appeared to be effective in destroying the moremature metacestodes.ACKNOWLEDGEMENTSThis work was supported by the Wellcome Trust and the EEC (STD).REFERENCESBANGHAM, A. D., STANDISH, M. M. & WATKINS, J. C. (1965) Diffusion of univalent ions acrossthe lamellae of swollen phospholipids. Journal of Molecular Biology, 13, 238-252.CRAIG, P. S. (1984) Circulating antigens, antibodies and immune complexes in experimental Taeniapisiformis infections of rabbits. Parasitology, 89, 121-131.CRAIG, P. S. & RICKARD, M. D. (1981) Anti-oncospheral antibodies in the serum of lambsexperimentally infected with either Taenia ovis or Taenia hydatigena. Zeilschrift furParasitenkunde, 64, 169-177.CROFT, S. L. (1986) Liposomes in the treatment of parasitic diseases. Pharmacy International, 7,229-233.FLISSER, A., PEREZ-MONTFORT, R. & LARRALDE, C. (1979) The immunology of human andanimal cysticercosis: a review. Bulletin of the World Health Organization, 57, 839-856.GEMMELL, M. A. & SOULSBY, E. J. L. (1968) The development of acquired immunity totapeworms and progress towards active immunization, with special reference to Echinococcusspp. Bulletin of the World Health Organization, 39, 45-55.RAJASEKARIAH, G. R., RICKARD, M. D. & MITCHELL, G. F. (1980) Density gradientseparation of Taenia pisiformis oncospheres. Journal of Parasitology, 66, 355-356.RAJASEKARIAH, G. R., RICKARD, M. D. & O'DONNELL, I. J. (1985) Taenia pisiformis:protective immunization of rabbits with solubilized oncospheral antigen. ExperimentalParasitology, 59, 321-327.RICKARD, M. D. (1982) Immunization against infection with larval taeniid cestodes using oncospheralantigens. In: Cysticercosis: Present State of Knowledge and Perspectives (editors A. Flisser, K.Willms, J. P. Laclette, C. Larralde, C. Ridaura & F. Beltran): Academic Press: London, pp.633-646.RICKARD, M. D. & WILLIAMS, J. F. (1982) Hydatidosis/cysticercosis: immune mechanisms andimmunization against infection. Advances in Parasitology, 21, 229-296.SIDDIQUI, W. A., KAN, S. C , KRAMER, K., CASE, S. & PALMER, K. (1981) Use of a syntheticadjuvant in an effective vaccination of monkeys against malaria. Nature, 289, 64—66.SZOKA, F. & PAPAHADJOPOULOS, D. (1978) Procedure for preparation of liposomes with largeinternal aqueous space and high capture by reverse phase evaporation. Proceedings of theNational Academy of Science, USA, 75, 4194-4198.TOM, B. H. (1980) An overview: liposomes and immunobiology—macrophages, liposomes andtailored immunity. In: Liposomes and Immunobiology (editors B. H. Tom & H. R. Six):Elsevier North Holland: Amsterdam, pp. 3-22.VAN ROOIJEN, N. & VAN NIEUWMEGEN, R. (1980) Liposomes in immunology: evidence thattheir adjuvant effect results from surface exposition of the antigens. Cellular Immunology, 49,402-407.Accepted 13th May 1987.of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X00011226Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 17:22:23, subject to the Cambridge Core terms

Immunization against experimental rabbit cysticercosis using liposome-associated antigen preparations

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