Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

EXPERIMENTALPARASITOLOGY57, 279-286 (1984)

Schistosoma mansoni: Interactive Effects of Irradiation and Cryopreservation on Parasite Maturation and Immunization of Mice E. R. JAMES AND A. R. DOBINSON London

School

of Hygiene

and Tropical Medicine, St. Albans, Hertfordshire

Winches Farm Field Station, AL4 OXQ, England, U.K.

(Accepted for publication

395 Hatfield

Road,

8 February 1984)

JAMES, E. R., AND DOBINSON, A. R. 1984. Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice. Experimental Parasitology 57,279-286. Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of mCo irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CD. Development into adult worms was prevented with 4 krad for IC schistosomula, but for Cl schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed. INDEX DESCRIPTORS.Schistosoma mansoni; Trematode; Blood fluke; Schistosomula; 60Co-irradiation; Attenuation; Cryopreservation; Cryoprotectant; Ethanediol; Rapid cooling; Vaccination; Storage.

The effectiveness of irradiated cercariae or schistosomula in protecting against a challenge infection has been demonstrated for several species, Schistosoma mansoni (Smithers 1962; Smithers and Terry 1965; Erickson and Caldwell 1965; Eveland and Morse 1978; Minard et al. 1978a, b; Bickle et al. 1979b), S. haematobium (Webbe et al. 1982), S. juponicum (Hsu et al. 1965, 1969), and for S. mattheei and S. bovis (Taylor et al. 1976a; Bushara et al. 1978; Bickle et al. 1979~). Irradiated schistosomula have also been used with success in limited field trials against S. bovis (Majid et al. 1980) and S. juponicum (Hsu et al. 1983). The doses of irradiation used in these different studies have varied between the minimum dose required to sterilize the adult

worms and which will still allow a small population of stunted worms to develop (approximately 2.3 krad for S. munsoni and 2.7 krad for S. mattheei; Bickle et al. 1979a, c; Minard et al. 1978b), to high doses (48 to 144 krad) which will prevent the cercariae or schistosomula from migrating away from the site of exposure/injection (Hsu et al. 1963; Minard et al. 1978a, b). The level of resistance stimulated by either percutaneously applied cercariae (Bickle 1978; Minard et al. 1978a) or intramuscularly injected, artificially transformed schistosomula (Bickle et al. 1979b) depends on the level of irradiation used. The optimum level of protection occurs when intermediate irradiation doses between those producing sterilization or which inhibit migration (Bickle et al. 1979b; Minard et al. 1978a) are used. 279

OWl-4894184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

280

JAMES AND DOBINSON

A crucial step in the potential utilization of a live radiation-attenuated vaccine has been the development of an effective cryopreservation technique for the schistosomula (James and Farrant 1977; James 1981), facilitating indefinite storage. The capacity of cryopreserved, radiation-attenuated schistosomula to immunize mice has been demonstrated by Bickle and James (1978) and Murrell e? al. (1979). The process of cryopreservationdamages some schistosomula and may itself produce some degree of attenuation. Here we set out to investigate the underlying trends in the interaction between cryopreservation and different levels of 6oCo irradiation on the capacity of the schistosomula to survive and develop into adult worms, and on their ability to protect mice against a challenge infection.

MATERIALS

AND METHODS

Schistosoma mansoni (Puerto Rican strain) cercariae were obtained from laboratory-reared albino Biomphalaria glabrata (also of Puerto Rican origin), and concentrated to approximately lOOO/ml on a Millipore filtration apparatus (XX15 047 00) with 8-pm filters. Cercariae were resuspended in lactalbumin hydrolysate with Earle’s salts buffered with sodium bicarbonate (Elac medium, 041 1250, Gibco Biocult), concentrated to approximately lO,OOO/ml, transferred to a “universal” tube (Sterilin), and artificially transformed into schistosomula by the “syringe” technique (James and Taylor 1976), based on that of Colley and Wikel(l974) using 10 passages through a 21-G needle to disrupt the cercarial bodies and tails. The universal tube was then completely filled with Elac medium, and the schistosomular suspension was incubated for 3 hr at 37 C, with the tube lying horizontally in a water bath to prevent the schistosomula from forming a pellet in the tube. Tails were removed by agitating the suspension and allowing it to settle under gravity for 10 min; the tailrich supematant was discarded and the pellet of schistosomula was resuspended in 1 ml of Elac prior to cryopreservation. The schistosomula were cryopreserved by the two-step addition of ethanediol technique of James (1981) using incubations in 10% v/v ethanediol (AnalaR grade, BDH Chemicals Ltd) for 10 min at 37 C and 5 min at 0 C, followed by 35% ethanediol for 10 min at 0 C. The suspension of schistosomula was then deposited in 20-)11 aliquots onto glass slivers

(40 x 5 mm) supported on an aluminum block resting on crushed ice, which were then immediately plunged into liquid nitrogen (cooling rate approximately 5100 C mini over the range 0 C to -60 C). Frozen schistosomula were stored either for 2 hr or overnight in liquid nitrogen prior to thawing. Samples were warmed, with simultaneous dilution of the cryoprotectant by dropping each 20-(11 sample on its glass sliver into 2 ml of Elac medium prewarmed to 42 C and agitating rapidly. The 6-ml glass tube containing the thawed parasite suspension was then placed into another water bath at 37 C, and the schistosomula were allowed to sediment for 10 min. The schistosomula were irradiated in a ‘Gammabeam 60’ 12-point cobalt source at a dose rate of 4.3 krad/min. Fresh (I) schistosomula and those to be cryopreserved subsequently (IC) were irradiated in Elac medium in a polystyrene “universal” tube. Schistosomula already frozen were irradiated on their glass slivers within a 3.6-ml polypropylene “cryotube” (Nunc, Gibco Biocult) immersed in 400 ml liquid nitrogen in a circular, expanded-polystyrene container (wall thickness 15 mm and id. 90 mm). Schistosomula suspended in 35% ethanediol (EI) were irradiated in polypropylene tubes in ice in the polystyrene container used above for CI schistosomula. The amount of radiation absorbed by the polystyrene container and a thickness of 45 mm of liquid nitrogen or crushed ice was determined using a Farmer Dosimeter Mk.2 (Baldwin Instrument Co, Ltd, Dartford, UK), and was found to be 12.5%. Thus, the levels of irradiation indicated by the Gammabeam 60 have been adjusted downwards for the CI and EI groups by this amount. Cryopreserved samples on slivers were thawed individually in 2 ml Elac and sedimented, the schistosomula from at least 10 slivers were pooled for each treatment, counts of three replicate aliquots were made to estimate the total numbers of organisms present, and the volumes were adjusted to give a concentration of approximately 100 schistosomula/O. 1 ml. A further five replicate counts were made to estimate more precisely the number of organisms being injected. The viability of thawed schistosomula was determined by microscopic observation as described previously (James 1981); the number of organisms which appeared normal both morphologically and in their motility was expressed as a percentage of the total number of organisms present in the sample. This method is useful for quick viability assessment, but is obviously fairly subjective and does not necessarily correlate with infectivity. The mice used were 6- to g-week-old male outbred Swiss T.O. (Theiler’s Original) (Tuck and Sons Ltd.) for maturation or CBA/CA (Batttin and Kingman Ltd.) for vaccination studies. Infection with the radiationattenuated schistosomula was by intramuscular (i.m.) injection into the hamstring muscle of the left hindlimb. Challenge infections of cercariae were given per-

Schistosoma

mansoni: ATTENUATION

BY IRRADIATION

cutaneously on the belly by the ring method of Smithers and Terry (1965), as adapted by Doenhoff et al. (1978). Perfusion of the adult worms at 40 days postinfection was as described by Doenhoff et al. (1978). The mean percentage development to adult worms was derived from

AND CRYOPRESERVATION

281

/ /’

100

i

i i i i i

i

i i _ i

Percentage development = no. of adult worms at perfusion x 100 no. of schistosomula injected

i i

RESULTS

The purpose of this initial experiment was to determine the dose of irradiation which would prevent development of Schistosoma mansoni schistosomula to adult worms. Groups of 5 T.O. mice were injected i.m. either with schistosomula which had been irradiated before cryopreservation (IC schistosomula), schistosomula which had been irradiated when already in the frozen state (CI schistosomula), schistosomula irradiated in the presence of ethanediol (EI), or untreated unfrozen irradiated controls (I). Schistosomula in groups I, EI, and IC were prepared on the same day from the same batch of cercariae; those in the CI group were prepared from a batch of cercariae harvested, transformed, and cryopreserved 2 days prior to the other groups. Groups I and EI were prepared, counted, and injected into their respective groups of mice first, and then the IC schistosomula were cryopreserved. This did mean, however, that, owing to the large number of sequential counts and injections which had to be performed, the holding time in culture was extended for the IC schistosomula, which were almost 5 hr old post-transformation before they were cryopreserved. These factors may have been responsible for the low percentage recoveries in this group. Increased culture time does not affect the viability of the schistosomula per

0

2

4

6 rradiation

6 dcs

x) (bad)

FIG. 1. Effect of irradiation dose on the mean percentage of adult Schistosoma mansoni developing by 40 days postinfection with 100 irradiated and/or cryopreserved schistosomula. Schistosomula irradiated and then cryopreserved (IC) (0) schistosomula irradiated in the frozen state (CI) (A), irradiated unfrozen controls (I) (0) and schistosomula irradiated while suspended in 35% v/v ethanediol (EI) (A). The CI Okrad controls were irradiated by mistake and thus discarded.

se, but, rather, reduces their ability to be cryopreserved (James 1981); schistosomula of S. mansoni cryopreserve optimally when approximately 90- 180 min old. The mean numbers of organisms injected for the four treatment groups were 87, 95, 91, and 61 for the I, IC, EI, and CI groups, respectively. The overall viabilities of the IC and CI schistosomula were 42 and 45%, respectively, following cryopreservation, and of the two control groups (EI and I) were 96%. The percentage recoveries of adult worms at perfusion after 40 days are

282

JAMES

AND

shown in Fig. 1 and indicate that the amount of irradiation required to prevent any schistosomula from developing into adult worms was 4 krad for I and IC schistosomula, 7 krad for EI, and in excess of 8.8 krad for CI schistosomula. There has thus been considerable radioprotection in the EI and CI groups. A second experiment was carried out to determine the dose of irradiation required to produce optimum protection with the different treatments. Groups of 10 mice were vaccinated i.m. with 100 viable IC schistosomula or with 200 viable CI schistosomula; availability of cercariae at the time of the experiment dictated the numbers of schistosomula which were used for vaccination. The mice were challenged percutaneously with 200 cercariae 35 days later, and perfused after a further 40 days. It is unlikely that any of the worms recovered at perfusion could have been derived from the vaccination, since the previous experiment has shown that 4 krad is sufficient to prevent development of IC schistosomula into adult worms, while 8.8 krad only allowed a mean of 1.6 adult worms to survive to 40 days postinfection with CI schistosomula and, in a further group of animals perfused at 63 days, no adult worms were recovered. Optimum protection of mice vaccinated with IC schistosomula was achieved with a dose level of 9.05 krad (44.6% reduction in adult worm numbers). Optimum protection with CI schistosomula was achieved with an irradiation level of 15.8 krad (65% reduction in adult worm numbers). The percentage reductions are shown graphically in Fig. 2 together with the results obtained by Bickle ef al. (1979b), who found that the optimum dose for uncryopreserved (I) schistosomula was 20 krad. The points in Fig. 2 have been joined using curvilinear interpolation. Student’s t test indicated that the mean

DOBINSON

irradiation

dose (kradd)

2. Effect of irradiation dose on the mean percentage reduction in a challenge infection of 200 percutaneously applied cercariae of Schistosoma mansoni. Vaccinated with 200 schistosomula which were irradiated and then cryopreserved (IC) (O), or cryopreserved and then irradiated while frozen (CI) (A). Results of Bickle et al. (1979b) using irradiated unfrozen schistosomula (I) (0). FIG.

worm burdens obtained from mice vaccinated with IC schistosomula irradiated at 9 and 14 krad were significantly different from the challenge controls (P < 0.05). The worm burdens in all other groups were not significantly different from the challenge controls. The level of protection achieved with IC schistosomula irradiated at 9 krad was not significantly different from those irradiated at 14 or 27 krad, but was different from that achieved in the other groups. The mean worm burdens from mice vaccinated with CI schistosomula irradiated at all dose levels were significantly different from challenge controls. The resistance produced by vaccination with CI schistosomula irradiated at 18 krad was not significantly different from that obtained with an irradiation dose of 27 krad, but was signif-

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DISCUSSION

In this study we have set out to investigate the trends in the interaction between cryopreservation and irradiation and, specifically, the effect on the dose(s) of irradiation required to prevent development of Schistosoma mansoni schistosomula into adult worms and the dose(s) which optimally attenuate the schistosomula for immunization. The maturation of irradiated and cryopreserved parasites is affected by the sequence in which the irradiation and cryopreservation steps are performed. There was little difference between the levels of irradiation required to prevent development of I or IC schistosomula into adult worms (approximately 3.5 and 3 krad, respectively). However, for organisms irradiated in the frozen state (CI), the irradiation dose required to produce the same degree of attenuation as for I and IC schistosomula was approximately 2.6 to 3 times greater. Most of this effect could clearly be ascribed to the radioprotective properties of ethanediol, since those organisms which were irradiated while simultaneously exposed to ethanediol also required an increase in radiation dose by about 2 to 2.3 times (compared to I and IC schistosomula, respectively). Interestingly, at irradiation doses of 0 and 2 krad, schistosomula which were exposed to ethanediol gave very high (>lOO%) percentage recoveries. We can offer no satisfactory explanation for this observation, but have noted before (James 1981) that larger numbers of schistosomula develop into adult worms following exposure to ethanediol compared to untreated controls; the effect appears to have been pronounced in this study. Many compounds, in addition to being cryoprotective, are also radioprotective

IRRADIATION

AND

CRYOPRESERVATION

283

and ethanediol, in particular, has considerable radioprotective properties (Chapman and Reuvers 1977). The mechanisms of radioprotection are complex and various (Chapman et al. 1975), and a compound such as ethanediol may radioprotect in several different ways operating with several different time scales. For instance, the sequelae to enzyme damage which may adversely affect cells during a relatively long time period (seconds or minutes) are thought to be prevented or restricted by the stabilizing action of radioprotectors, while the damaging actions of free-radicals generated during irradiation within fractions of a second can be limited by these compounds. Ethanediol is apparently an effective free-radical scavenger even at relatively low concentrations, and its overall radioprotective effect at a concentration similar to that used in this study (35% v/v or approximately 6.3 M) is considerable, reducing the damaging effects of irradiation on Chinese hamster tissue culture cells by 50% (Chapman and Reuvers 1977). This level of protection is comparable to that observed here with schistosomula. The process of cryopreservation can itself generate free radicals (Swartz 1971), and this may be one of the mechanisms by which ethanediol cryoprotects . It has been shown previously that cryopreserved, radiation-attenuated schistosomula are capable of stimulating a high degree of protection against a challenge infection (Bickle and James 1978), but it is now apparent that the two processes, cryopreservation and @‘Co irradiation, interact in their effects on the immunogenicity of schistosomula. Bickle et al. (1979b) found that the highest level of protective immunity induced by unfrozen irradiated schistosomula occurred with an irradiation dose of 20 krad. On the basis of the effects which irradiation, together with cryopreservation, have on the development of schistosomula into adult worms, it might be expected that

284

JAMES AND DOBINSON

IC schistosomula would require a similar irradiation dose to the unfrozen controls (I) to achieve optimum protection, while CI schistosomula would require a dose greater by approximately 2.6 to 3 times (i.e., around 56 krad). This is not the case. For IC schistosomula, the optimum irradiation dose is reduced to approximately 9 krad while, for CI schistosomula, the level of irradiation giving optimal protection is around 16 krad. The process of cryopreservation has thus contributed to the attenuation of the schistosomula by acting synergistically with the irradiation although, in the case of CI schistosomula, this effect has been overshadowed by the radioprotective effects of ethanediol and cryopreservation. Stek (personal communication) has also found that the optimal irradiation dose with the NMRI strain of S. mansoni is shifted to a lower level if irradiated schistosomula are subsequently cryopreserved (IC). However, no direct comparison is possible with the NMRI strain since, uncryopreserved, optimal protection is attained with schistosomula irradiated with 56 krad (Minard el al. 1978a). The lower peak level of protection, at 45%, obtained with IC schistosomula may have been due to the smaller number of vaccine organisms used, although Bickle ef al. (1979b) obtained similar levels of protection using 100, 500, or 5000 attenuated schistosomula. The difference between this peak and that obtained with CI schistosomula (65%) would probably not be significant since considerable variation in the degree of protection is often seen between experiments (Minard et al. 1978a, b; Bickle et al. 197913). However, this degree of protection afforded by 16-krad CI schistosomula (65%) was close to the 69% protection obtained previously with cryopreserved, unirradiated (C) organisms (Bickle and James 1978). The prospects for a live, radiation-attenuated vaccine being produced against Schistosomu munsoni are currently fairly

remote, as indicated by studies in baboons (Taylor et al. 1976b; James et al., in preparation). However, good protection has been achieved against S. huemutobium cercariae using radiation-attenuated, homologous schistosomula (Webb et al. 1982), and limited field trials are in progress in cattle against S. juponicum in China (Hsu et al. 1983) and against S. bovis in the Sudan (Majid et al. 1980; Taylor, personal communication). The only feasible method for storing a live, radiation-attenuated vaccine is by cryopreservation. In designing a system for producing such a vaccine, there may well be a logistical advantage to cryopreserving the schistosomula before performing the irradiation step. However, whether cryopreservation preceeds irradiation, or vice versa, the results reported here indicate that these two processes interact significantly, and that the degree of protection achievable with a specific irradiation dose may be modified considerably if the live organisms are cryopreserved. Additionally, the order in which the irradiation and cryopreservation steps are carried out considerably alters the degree of protection attainable at a specific irradiation dose. ACKNOWLEDGMENTS We are grateful to Dr. Quentin Bickle for allowing us to quote, by way of comparison, from a previous publication (Bickle et al. 1979b), and for helpful discussions. We would also like to thank Mr. G. J. M. Durrant and Mr. D. C. Mackay of the MRC irradiation unit, Mill Hill, for technical help; and the Edna McConnell Clark Foundation for financial support. REFERENCES BICKLE, Q. D. 1978. Studies on development of resistance in experimental murine schistosomiasis. PhD Thesis, London University. BICKLE, Q. D., AND JAMES, E. R. 1978. Immunization of mice using radiation-attenuated cryopreserved schistosomula of S. mansoni. Transactions of the Royal

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,!$Chisf0sOmU

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ATTENUATION

BY IRRADIATION

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diated S. rodhaini cercariae. Journal of Helminthology 50, 215-221. WEBBE, G., STURROCK, R. F., JAMES, E. R., AND JAMES, C. 1982. Schistosoma haematobium in the baboon (Papio anubis): Effect of vaccination with irradiated larvae on the subsequent infection with percutaneously applied cercariae, Transaction of the Royal

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