Immunol. Cell Biol. (1990) 68, 377-385
Attempts to induce resistance in mice to Schistosoma japonicum and Schistosoma mansoni by exposure to crude schistosome antigens plus cloned glutathione-S-transferases G. F. Mitchell, K. M. Davern, S. M. Wood, M. D. Wright, V. P. Argyropoulos, K. S. McLeod, W. U. Tiu* and E. G. Garcia' The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia and 'Department of Parasitology, College of Public Health, University of the Philippines, Manila, Philippines (Submitted
22 June 1990. Accepted for publication
9 November
1990).
Summary Several attempts have been made to induce resistance in mice to Schistosoma japonicum (Philippines) or Schistosoma mansoni by exposure to living male and/or female adult worms, their antigens or irradiated cercariae. No resistance was demonstrated in the following cases: re-exposure of mice to cercariae following praziquantel (PZQ) treatment of existing infection; re-exposure of mice following cyclosporin A (CsA) treatment at the time of first cerearial exposure; subcutaneous or intraperitoneal deposition of living male or female worms; repeated intranasal administration of crude worm homogenates plus Bordetella pertussis vaccine (BPV) as adjuvant. Homologous ^^Co-irradiated cercariae were very effective at inducing resistance to infection with 5. mansoni but not to infection with S. japonicum (Philippines) in a limited series of experiments. A regime of infection, immunization with homologous Escherichia co//-derived glutathione-S-transferases (GST), then PZQ treatment followed by homologous re-exposure did not result in significant resistance in either the 5. mansoni or the S. japonicum (Philippines) systems. Mice given irradiated cercariae plus GST were not more resistant to subsequent 5. mansoni infection than mice given irradiated cercariae alone. The results generally confirm and extend those reported by others with the conclusion that resistance to schistosomes in mice is difficult to achieve by exposure to adult worm antigens alone. Moreover, additional immunization with the GST available to date as cloned gene products, and injected in Freund's complete adjuvant, does not influence the outcome of exposure to crude worm antigens including any additive effects of protective irradiated cercariae.
INTRODUCTION A useful though not obligatory starting point in the development of a prophylactic molecular vaccine against infection with human schistosomes is a clear demonstration of induced resistance in a relevant model system (e.g. 1-4). As
Correspondence: Dr Graham F. Mitchell, Immunoparasitology LInit, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia. Abbreviations used in this paper: BPV, Bordetella pertussis vaccine; CsA, cyclosporin A; c/m, cercariae per mouse; FCA, Freund's complete adjuvant; GSH, glutathione; GST, glutathione-S-transferase; IgE immunoglobulin E; i.n., intranasally; i.p., intraperitoneally; KLH, keyhole limpet haemocyanin; PBS, phosphate buffered saline pH 7 3; PZQ, praziquantel; s.c, subcutaneously; SPF, specific pathogen free.
part of a larger programme of vaccine development against schistosomiasis japonica (Philippines) and schistosomiasis mansoni, attempts have been made to induce resistance to first infection, or to reinfection, in mice by a variety of manipulations. In some instances we have sought to extend to Schistosoma japonicum (Philippines) results that have been reported by others for S. japonicum (Chinese) and S. mansoni in the mouse model. The overall objective, however, has been to induce a 'baseline' level of resistance in the highly permissive mouse host on which to build, using more defined molecular vaccines such as the glutathione-S-transferases (GST) (5). It is possible that this combination of partial resistance plus vaccination with defined antigen may more closely resemble the situation in human populations exposed to infection and where schistosome vaccines may be tested in the future.
G. F. MITCHELL £T^Z,.
378
MATERIALS AND METHODS Mice Male and female mice ofthe inbred strain CBA/H, BALB/c.H-2'', A/J and the Fi hybrids C57BL/ 6 X 129/J, BALB/c X 129/J, CBA X C57BL/6 that are all entirely permissive to schistosome infection were derived from a specific pathogen free facility but maintained conventionally from about 6 weeks of age. They were generally used when 8-14 weeks of age. Parasites The maintenance of the lifecycles of S. Japonicum (Philippines) (Sorsogon isolate) in Oncomelania hupensis quadrasi and S. mansoni (Puerto Rico) in Biomphalaria glabrata have been described in detail (6). For routine exposure, anaesthetized mice in dorsal recumbency were given 20 cercariae per mouse (dm) of 5. japonicum or 100 c/m of S. mansoni applied to the shaved abdomen. Worm burdens were determined 40-60 days after challenge using standard perfusion procedures (6) and expressed as the arithmetic mean ± standard error of the mean. Cercariae in water, shed from B. glabraia or following crushing of O. h. quadrasi, were irradiated in an Eldorado 6 teletherapy unit (Atomic Energy of Canada, Commercial Products) charged with 5000 Ci of ^^Co at a dose rate of about 8 Gy/min for a total dose of 300 Gy (i.e. 30 krads). They were applied to the shaved abdomen of anaesthetized mice on at least two occasions. Cercarial numbers indicated are the estimated total number applied to the skin. Antigen preparations Living adult worms were rinsed in cold mouse tonicity phosphate buffered saline pH 7 3 (PBS) and either impanted intraperitoneally (i.p.) after laparotomy or injected through an 18 G needle i.p. or subcutaneously (s.c.) over the dorsum ofthe mouse. Adult worm homogenates (either male or female) were prepared in 2 mL PBS using an homogenizer with up to approximately 100 iiL packed worms, and 0-5 mL Bordeteila pertussis vaccine (BPV) (Commonwealth Serum Laboratories [CSL], Melbourne, Australia) added. Mice were anaesthetized by inhalation with a 2:1 mixture of ether: chloroform (7) and 50 to 100 \iL crude antigen plus adjuvant applied to the external nares (i.e., i.n.). The same mice were given 100150|a.L s.c. Mice were boosted i.n. and s.c. at least once. For injection in Freund's complete adjuvant (Difco Laboratories, Michigan, USA) (FCA), equal volumes of antigen in PBS and FCA were emulsified and 100-250 nL injected i.p. and s.c. Antigens were the Mr 26 000 and Mr 28 000 glutathione-S-transferases (GST) (Sj26, Sm26, Sj28 and Sm28) produced as native or near-native molecules in Escherichia coli and affinity-purified on glutathione-agarose columns (8). Recombinant Sj26 (rSj26) is a full length molecule produced by the plasmid pSj-5; rSm26 lacks an estimated 7 A'-terminal amino acids and is produced in the pEV vector (9, MD Wright, unpublished); rSj28 lacks an estimated 5 A^-terminal amino acids and is produced
in pBluescript (Strategene, La JoUa, California, USA); rSm28 lacks 7 JV-terminal amino acids (compared with a published sequence) and is produced in pBluescript (8). Amounts injected were 25-70 ng in FCA and 2050 |xg for aqueous boosts. Sj26 and Sj28 were coupled at a weight ratio of 1:1 using glutaraldehyde to keyhole limpet haemocyanin (KLH) (Calbiochem, La Jolla, USA). Drugs Praziquantel tablets (Droncit, Bayer) (PZQ) were crushed, suspended in 2 5% cremaphor and sonicated briefly. Mice were dosed orally on three occasions 1 or 2 days apart with 5-6 mg (i.e. around 200-250 mg/kg body weight) in a volume of 0 2 mL. Cyclosporin A (CsA) was injected 5 X s.c. daily at a dose of 1 mg per mouse per dose in olive oil (01-0 2 mL). The pure crystalline form of CsA was a generous gift from Sandoz, of Basel, Switzerland. It was dissolved in ethanol immediately prior to use and then diluted 1:9 v/v with olive oil BP to give a 5-10 mg/mL CsA solution.
RESULTS Lack of resistance to reinfection after PZQ treatment of existing infection It has been demonstrated repeatedly in both S. mansoni/mouse and S. japonicum (Chinese)/ mouse systems that PZQ treatment of infection does not lead subsequently to resistance to cercarial challenge. Results of two such experiments in S. mansoni and S. japonicum (Philippines) are presented in Table 1. PZQ given at a time during infection and at high doses to effect substantial reductions in worm burdens (rather than complete elimination of worms) does not result in any resistance to reinfection. In an attempt to more closely simulate 'natural infections' with schistosomes, mice were exposed over 50 days to trickle infections with 7 X 2-4 c/m and dosed some weeks later with high doses of PZQ. Mice were either killed to examine worm burdens or, after several weeks, re-exposed to 20 c/m (ideally, of course, a trickle challenge would be more appropriate than the pulse challenge used in this protocol). It is apparent from Table 2 that no evidence of resistance to reinfection was evident in PZQ-treated infected mice. Thus the immune response induced by trickle infection has not obviously altered the state of immunity that might exist after drug treatment of one-time infected mice. From Table 2 it can also be seen that PZQ at the high standard doses used in mice (i.e. 3 X approximately 250 mg/kg body weight) (10,11) was still not able to eliminate all worms following trickle exposure just as such high doses
IMMUNIZATION AGAINST SCHISTOSOMES
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Table 1. Lack of resistance to reinfection in schistosome-infected mice treated with PZQ. Mouse strain First (infection) infection (c/m) (C57BL/6X129/J)Fi 20 5. japonicum
PZQ'
Second infection (c/m) t
20 20
(BALB/cX 129/J)Fi 5. mansoni
20 20
100 100
No. of mice • ~ 8 6 11 8
Worm burdens (Day 42 or 46 after challenge) ~ 5-0 ±0-7 0-8 ±0-3 (males only) 8-2±l-l 6-4 ±1-5
7 0-7 ± 0-6 (males only) 8 17-6 ± 2 - 4 * 6 31-2±9-9* • Praziquantel given orally (250 mg/kg) on days 50, 51 and 52 (or S. japonicum infection and on days 80, 82 and 84 for S. mansoni infection. t Second infection on Day 125 or 111 in the two experiments. * Not significantly different, the apparent difference in the means only reflecting a very high worm burden (80) in one mouse in the control group. Two other experiments have given similar results with no evidence of protection against reinfection. 100 100
Table 2. Lack of resistance to reinfection with 5. japonicum in (C57BL/6 X 129/J)Fi mice exposed to trickle infections and treated with PZQ. Exposure to cercariae (Day 0 to 50)
PZQ (Day 74, 75 ,76)
2, 3 or 4 cercariae on 7 occasions over 50 days
—
Nil
— • Mice killed on Day 90. t Mice killed on Day 76. * Mice killed on Day 40 after challenge.
are unable to remove all worms following onetime exposure to cercariae (Table 1). Male worms dominate in these residual worm burdens. [Unpublished experiments have suggested that PZQ may be relatively more effective wben worm burdens in chronically infected mice are low.] Lack of resistance to reinfection after CsA treatment of short-term infection A surprisingly persistent effect of CsA, injected s.c. in oil, has been reported in the S. mansoni/ mouse system (12,13). That is, mice given CsA are resistant to inflection with S. mansoni when exposed to cercariae several weeks or even months later. To determine whether this was the case for S. japonicum (Philippines) (and indeed, to determine whether CsA had any effect on this parasite), one group of A/J mice was exposed to
Challenge (Day 118)
No. of mice
20 c/m
7 8 8
1-7 ± 0 - 7 ' 5-8 ± 0-61 13-8 ± 1 - 8 *
20 c/m
6
10-5 ± 2-0*
Worm burdens
20 c/m and injections of CsA or diluent commenced. Another group received CsA or diluent but a delayed exposure to cercariae. It can be seen from Table 3 that CsA, as reported by at least six groups, is a powerful anti-schistosome reagent wben administered at about the time of exposure to S. japonicum cercariae, or 3-4 weeks prior to cercarial challenge. The persistent effect of CsA renders the protocol invalid as a means of detecting resistance to reinfection in drug-cured infected mice (Group 4 versus Group 5 in Table 3). To address the question of resistance to reinfection, tbe protocol was altered. CBA/H mice were infected with 100 cercariae per mouse of S. mansoni at about the time of CsA injection and reehallenged around 6 weeks later. Mice were killed at a time that allowed differentiation between the worms derived from first (mature
G. F. MITCHELL ETAL.
380
Table 3. Persistence of anti-schistosome effect of cyclosporin A in A/J mice exposed to S. japonicum (Philippines). Injection/Infection Group
Day 1-5
1 2 3 4 5
Diluent CsA Diluent CsA CsA
Day 1 20 c/m + +
Day 28 20 c/m
No. of mice
Worm burdens
+ + +
8 8 7 8 8
9-0±l 7* 0-3 ± 0-2 • 6-6±l 3 + 0-9 ±0-4 + l-3±0-4 +
+
* Mice killed on Day 40. + Mice killed on Day 88. Table 4. Lack of resistance to reinfection with S. mansoni in infected CBA/H mice treated with cyclosporin A. Worm burdens (Day 70-73)
Injection/Infection Type of mouse
Day 1-5
Intact
Zero Diluent CsA CsA Zero Diluent CsA
Nude
Diluent CsA CsA
Day 2 (100 c/m)
Day 42 (100 c/m)
+
—
+
—
No. of mice
Mature worms
8 6 7 6 8 7 8
7-4 ±0-8* 7-7+1-7 0 3 + 02
4 3 3
12-8 ±2-4* 2-0 ± 1-2* l-7± 1-2
Immature worms
9-7± 15 13-8±1 4 15-0+ 2-2 7-5±M
23-7 ±4-9
* Some smaller (stunted) worms recorded in these groups.
worms) and second exposure (immature worms). It can be seen from Table 4 that (a) CsA has had a dramatic eflfect on worm numbers and that the diluent alone (olive oil) has had no effect [some preliminary experiments with other oily vehicles indicated some effect of the vehicle or diluent alone; see reference 14]; (b) the CsA effect on schistosomes may stiil be present, though weakly, when the drug is given 42 days prior to cercarial challenge; and (c) no evidence for resistance to reinfection was demonstrable in this experiment. Additional limited data in Table 4 reinforce those reported by others — namely, that CsA has an effect on schistosomes in the hypothymic nude mouse. That is, an intact immune system is not required for expression of the schistosomulocidal effects of CsA (12,15). Lack of resistance to reinfection after injection of living adult worms It is possible that exposure to antigens of living adult worms other than via the portal system
and liver, as occurs in natural infection, may lead to an entirely different type of sensitization. Mice were implanted or injected on several occasions with living adult male or female schistosomes either i.p. or s.c. over the dorsum ofthe mouse and subsequently challenged percutaneously with cercariae. Data in Table 5 indicate that no clear cut or consistent resistance could be demonstrated using these regimes of antigen exposure. Lack of resistance to infection after intranasal administration of worm antigens Evidence exists that the lung is an important site of schistosomule elimination in resistant mice (16-18). To sensitize the lungs of mice, crude adult worm homogenates of either adult male or female schistosomes were administered i.n. (and also s.c.) together with BPV. With other antigens, this manipulation in appropriately anaesthetized mice results virtually in a quantitative deposition of antigens into the lungs (at least transiently) and induction of immunogiobulin E
IMMUNIZATION AGAINST SCHISTOSOMES
381
Table 5. Lack of resistance in mice exposed to living adult worms subcutaneously and/or intraperitoneally. Expt. no. (mouse strain and sex) 1 (C57BL/6X129/J)Fi (M)
2 (C57BL/6X129/J)Fi (M)
3 (CBAXC57BL/6)Fi (M-i-F)
Injection/ Implantation 2X105'. japonicum male worms (Day 0 and 50) i.p. + s.c. Control 2X 12 5". mansoni male worms (Day 0 and 61) i.p. Control 50 5. japonicum male worms over 60 days (4 injections) s.c. 30 S. japonicum female worms over 60 days (4 injections) s.c. Control
4 (C57BL/6X129/J)F| (M)
~200 S. mansoni male worms over 64 days(3 injections) s.c. ~ 120 5. mansoni female worms over 64 days(3 injections) s.c. Control
(IgE) antibodies (7). It can be seen from Table 6 that no resistance to infection was demonstrated in mice repeatedly exposed intranasally to crude antigens and BPV. Lack of resistance to reinfection in schistosome-infected mice immunized with GST and treated with PZQ Genes encoding four GST isoenzymes of Mr 26 000 and Mr 28 000 in S. japonicum (Philippines) and S. mansoni (i.e. Sj26, Sj28, Sm26 and Sm28) have been cloned and native or near native proteins expressed in E. coli (8). Inconsistent (if any) vaccinating effects have been demonstrated in these laboratories with these GST (see the Discussion section). Mice infected with 5. mansoni or S. japonicum (Philippines) were immunized and boosted with homologous E. co//-derived GST then treated with PZQ. Sev-
Challenge
No. of mice
Worm burdens (Day 40-47 after challenge)
(Day 82) 20 c/m S. japonicum
6
5-5± 13
20 c/m
5
3-2 ±0-6
(Day 77) 100 c/m S. mansoni
5
12 8 ± 4 2
10
[P=005] 23-1 ±3-0
(Day 103) 20 c/m 5'. japonicum
6
5-8 ± 1-0
20 c/m
5
7 8 ± 1-4
20 c/m
4
7-0± 1-8
(Day 95) 100 c/m 5. mansoni
4
16 5 ±6-2
100 c/m
4
23 3 ±6-3
100 c/m
5
20-8±2 4
100 c/m
eral weeks later they were challenged with cercariae ofthe homologous schistosome and worm burdens determined in the usual manner. Data in Table 7 indicate that no resistance was demonstrated in infected mice immunized with GST then treated with PZQ before challenge. In a related protocol, S. japonicum-infected (BALB/c X 129/J)Fi mice were injected twice with Sj26 and Sj28 conjugated to KLH, then treated with PZQ before boosting with GSTKLH. No resistance to reinfection was apparent despite high antibody titres to GST (unpubl. data). Resistance to homologous reinfection after exposure to irradiated cercariae of S. mansoni but not S. japonicum (Philippines) Cercariae of S. mansoni or S. japonicum were exposed to ^"Co irradiation and applied on at
382
G. F, MITCHELL ETAL.
Table 6. Lack of resistance in mice given crude antigen + BPV intranasally and subcutaneously. Mouse strain (sex)
A/J (F)
BALB/c.H-2'' (F)
Antigen
Challenge
(Day 0 + 36) 5. mansoni male worm extract + BPV i.n. + s.c. S. mansoni female worm extract + BPV i.n. + s.c.. BPV alone i.n. + s.c. (Day 0, 22 + 56) S. japonicum male worm extract + BPV i.n. + s.c. S. japonicum female worm extract + BPV i.n. + s.c. BPV alone i.n. + s.c.
(Day 43) 100 c/m S. mansoni
30-5 ±4-1*
100 c/m
30-1 ±7-8
100 c/m
46 4 ±5-6
No. of mice
Worm burdens (Day 56 and 41 after challenge)
(Day 57) 20 c/m S. japonicum
6 8± 16
20 c/m
!-4± 1-1
20 c/m 6 0±l 3
* Majority of mice in this group were unusual in having more female than male worms.
Table 7. Lack of resistance to reinfection in (C57BL/6 X 129/J)Fi mice following immunization of infected mice with GST prior to PZQ treatment. Injection/Infection First infection (Day 0) 20 c/m S. japonicum 20 c/m S. japonicum Nil Nil 100 c/m S. mansoni 100 c/m S. mansoni Nil Nil
GST + FCA (Day 14) 33 Hg rSj26* +33 Hg rSj28 in FCA FCA alone GST in FCA FCA alone
GST (Day 36) 25 Hg rSj26 +25 Hg rSj28 PBS
PZQ
Worm burdens No. of mice
(Day 40 and 42 after challenge)
5
l l - 4 ± 1-7
+
5
9 6 ± 14
+ +
Challenge
(Days 42, 43, 44) (Day 65) 20 c/m + S. japonicum +
6 5
8-7±M 10-6 ±2-4
(Day 20) 30 Hg rSm26 +70 Hg rSm28 in FCA FCA alone
GST + PBS + (Day 37) (Days 47, 48, 49) (Day 68) 20 Hg rSm26 + 100 c/m +30 Hg S. mansoni rSm28 PBS + +
6
13-3±1 8
7
13-6±2-5
GST in FCAFCA alone
GST PBS
7 6
12 9 ±2-3 13-3+1-6
+ +
• rSj26, rSj28, rSm26, rSm28 are the Mr 26 000 and Mr 28 000 GST of 5. japonicum and S. mansoni produced in, and purified from, E. coli.
IMMUNIZATION AGAINST SCHISTOSOMES
least two occasions to the skin of mice that were subsequently challenged. As demonstrated by numerous other investigators and reviewed by Taylor and Bickle (1), clear evidence of resistance to homologous (but not heterologous) challenge was demonstrated in mice previously given irradiated cercariae of 5". mansoni. In contrast, no convincing homologous resistance was demonstrated in mice given S. japonicum (Philippines) cercariae exposed to ^^Co irradiation (Table 8). Groups of mice were exposed to ^^Coirradiated cercariae of S. mansoni percutaneously and later immunized with Sm26 and Sm28 GST (results not shown). Both these sensitizing manipulations were repeated and mice challenged with 100 c/m S. mansoni. GST immunization did not change the level of resistance expected from exposure to irradiated cercariae alone (i.e. approximately 75% resistance). Thus the mean worm burden in 11 X (C57BL/6X 129/J)Fi hybrids given a total of 120 (ig GST and approximately 2000 irradiated cercariae on 2 X 2 occasions over 35 days was 86% that in controls.
383
DISCUSSION The experiments reported in this paper were prompted by the generally disappointing vaccinating effects of E. co/z-derived schistosome GST in naive mice. The Mr 26 000 and Mr 28 000 GST isoenzymes of 5. Japonicum (i.e. Sj26 and Sj28) and S. mansoni (i.e. Sm26 and Sm28) have been expressed as full-length or near full-length molecules in E. coli and affinity purified on glutathione (GSH) columns. Numerous vaccination studies in mice using individual isoenzymes or combinations have been conducted (19,20, and Mitchell G. F., Wright M. D., Davem K. M., Tiu W. U., Garcia E. G., unpublished). No consistent high level protection has been demonstrated in any mouse strain, the (C57BL/6X129/J)Fi mouse and FCA being used for most experiments. Attempts to increase anti-GST responses or induce antibodies that cross-react between isoenzymes by the use of pGEX fusion proteins of Sj26 (21-23) or conjugation of GST to powerful antigens such as sheep erythrocytes have been generally unsuccessful, with no protection being demonstrated. Far more impressive vaccination data have been
Table 8. Resistance in mice exposed to ^^Co-irradiated cercariae of 6". mansoni but not 5. japonicum (Philippines). Mouse strain (sex) (CBA/H X C57BL/6)Fi (M + F)
Irradiated cercariae
Challenge
(Day 0 + 30) ~ 1200 S. mansoni
(Day 51) 100 c/m S. mansoni
Nil
ll-5±3-2 61% resistance (P< 0-001)
~340 S. japonicum 8 0 Nil 8 8-8 ± 1-3* * One male worm found in the seven mice. t As evidenced by the worm burdens after 100 c/m, the infectivity ofthe cercariae used in this experiment was obviously low. * This difference is insignificant; however, the number of mice with < 6 worms in the experimental group was 4/8 whereas this number of mice in the control group was 1/8.
384
G. F. MITCHELL £7" ^L.
reported with full length recombinant or native Sm28 against S. mansoni infection (24,25). An hypothesis we have developed (5) is that aggressive immune attack at the schistosomule or schistosome surface releases GST and that antibodies to GST, through neutralization of the repair function of these enzymes, increase the damage inflicted by the initial immune insult. It follows from this that anti-GST immunity alone would not be host-protective and that other immune responses are required to inflict initial damage. With this in mind we have attempted to sensitize mice through exposure to infection or exposure to crude antigen preparations and to test the effects of GST immunization superimposed on a 'baseline' level of resistance or at least a degree of sensitization. Exposure of mice to infection followed by drug treatment does not lead to resistance reinfection. This statement applies to mice exposed by trickle infections of cercariae. Moloney et al. (26) in the S. japonicum (Chinese) system demonstrated clearly that resistance to reinfection in mice was only demonstrated early after PZQ administration, an effect readily ascribed to 'pathology-related resistance' (16). Exposure of mice to living adult worms in ectopic sites also does not lead to resistance nor does repeated administration of crude antigen plus adjuvant into the airways to sensitize the lungs. Abbreviation of infection with CsA also does not leave the host resistant to reinfection (allowing for a remarkably prolonged effect of CsA injected in an oil vehicle). Irradiated cercariae in the S. mansoni system resulted in good levels of homologous resistance as expected. No such resistance to homologous infection was demonstrated in mice given relatively large numbers of ^"Co-irradiated cercariae of S. japonicum (Philippines). Good levels of resistance to S. japonicum (Chinese) using ultraviolet (UV)-irradiated cercariae have been
reported by Moloney et al. (27). Interestingly, no resistance to S. japonicum (Philippines) was demonstrated in mice exposed to irradiated S. japonicum (Chinese) cercariae (28). In a recent review we have emphasized differences in transit times in lungs for cercariae of S. japonicum versus S. mansoni, the latter persisting in the lungs for much longer than S. japonicum (29). It is possible that persistence of S. mansoni cercariae in lungs increases either the sensitization for and/or expression of resistance to challenge. It becomes important to determine whether there are differences between S. japonicum (Chinese) and S. japonicum (Philippines) in this regard and also between UV-irradiated versus ^"Coirradiated cercariae of S. japonicum in lung persistence and therefore 'sensitization' of this organ. Disappointingly, E. co//-derived GST in FCA did not change the lack of resistance to reinfection after PZQ treatment of existing infection in mice. Also, the GST (of 5. mansoni expressed in E. coli) did not appear to increase the levels of resistance induced by exposure to irradiated cercariae. Three out of the four GST presently available (8) are not full length proteins and lack A'-terminal amino acids. Whether this is important in the lack of any consistent vaccinating effect of GST (using the one adjuvant, FCA) obtained in this laboratory remains to be determined. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Edna McConnell Clark Foundation, the Australian International Development Assistance Bureau (AIDAB) and the United Nations Development Programme (UNDP)/World Bank/World Health Organization (WHO) Special Programme for Research and Training in Tropical DiseasesRockefeller Foundation Health Sciences for the Tropics-Partnerships in Research Programme.
REFERENCES 1. Taylor, M. G. and Bickle, Q. D. 1986. Irradiated schistosome vaccines. Parasitol Today 2: 132134. 2. Capron, A., Dessaint, J. P., Capron, M., Ouma, J. H. and Butterworth, A. E. 1987. Immunity to schistosomes: Progress toward vaccine. Science 238: 1065-1072. 3. Dean, D. A. 1983. Schistosoma and related genera: Acquired resistance in mice. Exp. Parasitol. 55: 1-104.
4. Sher, A., James, S. L., Correa-Oliveira, R., Hieny, S. and Pearce, E. 1989. Schistosome vaccines: Current progress and future prospects. Parasilotogy 98: S61-S6S. 5. Mitchell, G. F. 1989. Glutathione S-transferases: Potential components of anti-schistosome vaccines? Parasitol. Today 5: 34-37. 6. Wright, M. D., Tiu, W. U., Wood, S. M., Walker, J. C, Garcia, E. G. and Mitchell, G. F. 1988. Schistosoma mansoni and S. japonicum worm
IMMUNIZATION AGAINST SCHISTOSOMES numbers in 129/J mice of two types and dominance of susceptibility in FI hybrids. / Parasitol. 74:618-622. 7. O'Donnell, I. J. and Mitchell, G. F. 1978. An investigation of the allergens of Ascaris lumbricoidcs using a radio-allergosorbent test (RAST) and sera of naturally infected humans: Comparison with an allergen for mice identified by a passive cutaneous anaphylaxis test. Aust. J. Biol. Sci. 31: 459-487. 8. Henkle, K. J., Davern, K. M., Wright, M. D., Ramos, A. J. and Mitchell, G. F. 1990. Comparison of the cloned genes of the 26- and 28-kilodalton glutathione-S-transferases of Schistosoma japonicum and Schistosoma mansoni. Mol. Biochem. Parasitol. 40: 23-34. 9. Crowl, R., Seamans, C , Lomedico, P. and McAndrew, S. 1985. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene 3S, 31-38. 10. Doenhoff, M. J., Sabah, A. A. A., Fletcher, C , Webbe, G. and Bain, J. 1987. Evidence for an immune-dependent action of praziquantel on Schistosoma mansoni in mice. Trans. Roy. Soc. Trop. Med. Hyg. 81: 247-254. 11. Brindley, P. J., Strand, M., Norden, A. P. and Sher, A. 1989. Role of host antibody in the chemotherapeutic action of praziquantel against Schislosoma mansoni: Identification of target antigens. Mot. Biochem. Parasitol. 34: 99-108. 12. Bout, D., Deslee, D. and Capron, A. 1986. Antischistosomal effects of cyclosporin A: Cure and prevention of mouse and rat schistosomiasis mansoni. Infect. Immun. 52: 823-827. 13. Pons, H.A.,Adams, S. andStadecker, M.J. 1988. Schistosoma mansoni: The basis for the antischistosomal effect of cyclosporin A. Exp. Parasitol. 67: 190-198. 14. Salafsky, B., Fusco, A. C, Li, L. H., Mueller, J. and Ellenberger, B. 1989. Schistosoma mansoni: Experimental chemoprophylaxis in mice using oral antipenetration agents. Exp. Parasitol. 69: 263-271. 15. Brannon, L. R., Chappel, L. H., Woo, J. and Thomson, A. W. 1989. Antischistosomal activity of cyclosporin A: Studies on murine spleen cells and the influence of a cyclosporin antagonist on resistance to infection. Immunology 67: 382387. 16. Wilson, R. A., Coulson, P. S. 1989. When and how does immune elimination of Schistosoma mansoni occur? Parasitol. Today 5: 274-278. 17. McLaren, D.J. 1989. Will the real target of immunity to schistosomiasis please stand up? Parasitol. Today 5: 279-282. 18. Dean, D. A., Mangold, B. L., Georgi, J. R. and Jacobbson, R. H. 1984. Comparison of Schistosoma mansoni migration patterns in normal and irradiated cercaria-immunized mice by means of
385
autoradiographicanalysis./)w. / Trop. Med. Hyg. 33: 89-96. 19. Smith, D. B., Davern, K. M., Board, P. G., Tiu, W. U., Garcia, E. G. and Mitchell, G. F. 1986. Mr 26 000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione-S-transferase. Proc. Natl. Acad. Sci. USA 83: 8793-8707. 20. Mitchell, G. F., Garcia, E. G., Davern, K. M., Tiu, W. U. and Smith, D. B. 1988. Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum. Trans. Roy. Soc. Trop. Med. Hyg. 82: 885-889. 21. Smith, D. B., Rubira, M. R., Simpson, R. J., Davern, K. M., Tiu, W. U., Board, P. G. and Mitchell, G. F. 1988. Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione-S-transferase. Molec. Biochem. Parasitol. 27: 249-256. 22. Smith, D. B., and Johnson, K. S. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 61: 31-40. 23. Davern, K. M., Tiu, W. U., Samaras, N., Gearing, D. P., Hall, B. E., Garcia, E. G. and Mitchell, G. F. 1990. Schistosoma japonicum: monoclonal antibodies to the Mr 26 000 schistosome glutathioneS-transferase (Sj26) in an assay for circulating antigen in infected individuals. Exp. Parasitol. 70: 293-304. 24. Bailout, J-M., Grzych, J-M., Pierce, R. J. and Capron, A. 1987. A purified 28 000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. /. Immunol. 138: 3448-3453. 25. Balloul, J-M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J-M., Pierce, R. J., Cavallo, D., Lecocq, J. P. and Capron, A. 1987. Molecular cloning of a protective antigen of schistosomes. Nature 326: 149-153. 26. Moloney, N. A., Hinchcliffe, P. and Webbe, G. 1987. Loss of resistance to reinfection with Schistosoma japonicum in mice after treatment with praziquantel. Trans. Roy. Soc. Trop. Med. Hyg. 81: 247-254. 27. Moloney, N. A., Hinchcliffe, P. and Webbe, G. 1987. Passive transfer of resistance to mice with sera from rabbits, rats or mice vaccinated with ultra violet attenuated cercariae of Schistosoma japonicum. Parasitology 94: 497-508. 28. Moloney, N. A., Garcia, E. G. ahd Webbe, G. 1985. The strain specificity of vaccination with ultra violet attenuated cercariae of the Chinese strain of Schistosoma japonicum. Trans. Roy. Soc. Trop. Med. Hyg. 79: 245-247. 29. Mitchell, G.F., Tiu, W.U. and Garcia, E.G. 1991. Infection characteristics of Schistosoma japonicum in mice and relevance to assessment of schistosome vaccines. Adv. Parasitol. (in press).
Attempts to induce resistance in mice to Schistosoma japonicum and Schistosoma mansoni by exposure to crude schistosome antigens plus cloned glutathione-S-transferases G. F. Mitchell, K. M. Davern, S. M. Wood, M. D. Wright, V. P. Argyropoulos, K. S. McLeod, W. U. Tiu* and E. G. Garcia' The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia and 'Department of Parasitology, College of Public Health, University of the Philippines, Manila, Philippines (Submitted
22 June 1990. Accepted for publication
9 November
1990).
Summary Several attempts have been made to induce resistance in mice to Schistosoma japonicum (Philippines) or Schistosoma mansoni by exposure to living male and/or female adult worms, their antigens or irradiated cercariae. No resistance was demonstrated in the following cases: re-exposure of mice to cercariae following praziquantel (PZQ) treatment of existing infection; re-exposure of mice following cyclosporin A (CsA) treatment at the time of first cerearial exposure; subcutaneous or intraperitoneal deposition of living male or female worms; repeated intranasal administration of crude worm homogenates plus Bordetella pertussis vaccine (BPV) as adjuvant. Homologous ^^Co-irradiated cercariae were very effective at inducing resistance to infection with 5. mansoni but not to infection with S. japonicum (Philippines) in a limited series of experiments. A regime of infection, immunization with homologous Escherichia co//-derived glutathione-S-transferases (GST), then PZQ treatment followed by homologous re-exposure did not result in significant resistance in either the 5. mansoni or the S. japonicum (Philippines) systems. Mice given irradiated cercariae plus GST were not more resistant to subsequent 5. mansoni infection than mice given irradiated cercariae alone. The results generally confirm and extend those reported by others with the conclusion that resistance to schistosomes in mice is difficult to achieve by exposure to adult worm antigens alone. Moreover, additional immunization with the GST available to date as cloned gene products, and injected in Freund's complete adjuvant, does not influence the outcome of exposure to crude worm antigens including any additive effects of protective irradiated cercariae.
INTRODUCTION A useful though not obligatory starting point in the development of a prophylactic molecular vaccine against infection with human schistosomes is a clear demonstration of induced resistance in a relevant model system (e.g. 1-4). As
Correspondence: Dr Graham F. Mitchell, Immunoparasitology LInit, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia. Abbreviations used in this paper: BPV, Bordetella pertussis vaccine; CsA, cyclosporin A; c/m, cercariae per mouse; FCA, Freund's complete adjuvant; GSH, glutathione; GST, glutathione-S-transferase; IgE immunoglobulin E; i.n., intranasally; i.p., intraperitoneally; KLH, keyhole limpet haemocyanin; PBS, phosphate buffered saline pH 7 3; PZQ, praziquantel; s.c, subcutaneously; SPF, specific pathogen free.
part of a larger programme of vaccine development against schistosomiasis japonica (Philippines) and schistosomiasis mansoni, attempts have been made to induce resistance to first infection, or to reinfection, in mice by a variety of manipulations. In some instances we have sought to extend to Schistosoma japonicum (Philippines) results that have been reported by others for S. japonicum (Chinese) and S. mansoni in the mouse model. The overall objective, however, has been to induce a 'baseline' level of resistance in the highly permissive mouse host on which to build, using more defined molecular vaccines such as the glutathione-S-transferases (GST) (5). It is possible that this combination of partial resistance plus vaccination with defined antigen may more closely resemble the situation in human populations exposed to infection and where schistosome vaccines may be tested in the future.
G. F. MITCHELL £T^Z,.
378
MATERIALS AND METHODS Mice Male and female mice ofthe inbred strain CBA/H, BALB/c.H-2'', A/J and the Fi hybrids C57BL/ 6 X 129/J, BALB/c X 129/J, CBA X C57BL/6 that are all entirely permissive to schistosome infection were derived from a specific pathogen free facility but maintained conventionally from about 6 weeks of age. They were generally used when 8-14 weeks of age. Parasites The maintenance of the lifecycles of S. Japonicum (Philippines) (Sorsogon isolate) in Oncomelania hupensis quadrasi and S. mansoni (Puerto Rico) in Biomphalaria glabrata have been described in detail (6). For routine exposure, anaesthetized mice in dorsal recumbency were given 20 cercariae per mouse (dm) of 5. japonicum or 100 c/m of S. mansoni applied to the shaved abdomen. Worm burdens were determined 40-60 days after challenge using standard perfusion procedures (6) and expressed as the arithmetic mean ± standard error of the mean. Cercariae in water, shed from B. glabraia or following crushing of O. h. quadrasi, were irradiated in an Eldorado 6 teletherapy unit (Atomic Energy of Canada, Commercial Products) charged with 5000 Ci of ^^Co at a dose rate of about 8 Gy/min for a total dose of 300 Gy (i.e. 30 krads). They were applied to the shaved abdomen of anaesthetized mice on at least two occasions. Cercarial numbers indicated are the estimated total number applied to the skin. Antigen preparations Living adult worms were rinsed in cold mouse tonicity phosphate buffered saline pH 7 3 (PBS) and either impanted intraperitoneally (i.p.) after laparotomy or injected through an 18 G needle i.p. or subcutaneously (s.c.) over the dorsum ofthe mouse. Adult worm homogenates (either male or female) were prepared in 2 mL PBS using an homogenizer with up to approximately 100 iiL packed worms, and 0-5 mL Bordeteila pertussis vaccine (BPV) (Commonwealth Serum Laboratories [CSL], Melbourne, Australia) added. Mice were anaesthetized by inhalation with a 2:1 mixture of ether: chloroform (7) and 50 to 100 \iL crude antigen plus adjuvant applied to the external nares (i.e., i.n.). The same mice were given 100150|a.L s.c. Mice were boosted i.n. and s.c. at least once. For injection in Freund's complete adjuvant (Difco Laboratories, Michigan, USA) (FCA), equal volumes of antigen in PBS and FCA were emulsified and 100-250 nL injected i.p. and s.c. Antigens were the Mr 26 000 and Mr 28 000 glutathione-S-transferases (GST) (Sj26, Sm26, Sj28 and Sm28) produced as native or near-native molecules in Escherichia coli and affinity-purified on glutathione-agarose columns (8). Recombinant Sj26 (rSj26) is a full length molecule produced by the plasmid pSj-5; rSm26 lacks an estimated 7 A'-terminal amino acids and is produced in the pEV vector (9, MD Wright, unpublished); rSj28 lacks an estimated 5 A^-terminal amino acids and is produced
in pBluescript (Strategene, La JoUa, California, USA); rSm28 lacks 7 JV-terminal amino acids (compared with a published sequence) and is produced in pBluescript (8). Amounts injected were 25-70 ng in FCA and 2050 |xg for aqueous boosts. Sj26 and Sj28 were coupled at a weight ratio of 1:1 using glutaraldehyde to keyhole limpet haemocyanin (KLH) (Calbiochem, La Jolla, USA). Drugs Praziquantel tablets (Droncit, Bayer) (PZQ) were crushed, suspended in 2 5% cremaphor and sonicated briefly. Mice were dosed orally on three occasions 1 or 2 days apart with 5-6 mg (i.e. around 200-250 mg/kg body weight) in a volume of 0 2 mL. Cyclosporin A (CsA) was injected 5 X s.c. daily at a dose of 1 mg per mouse per dose in olive oil (01-0 2 mL). The pure crystalline form of CsA was a generous gift from Sandoz, of Basel, Switzerland. It was dissolved in ethanol immediately prior to use and then diluted 1:9 v/v with olive oil BP to give a 5-10 mg/mL CsA solution.
RESULTS Lack of resistance to reinfection after PZQ treatment of existing infection It has been demonstrated repeatedly in both S. mansoni/mouse and S. japonicum (Chinese)/ mouse systems that PZQ treatment of infection does not lead subsequently to resistance to cercarial challenge. Results of two such experiments in S. mansoni and S. japonicum (Philippines) are presented in Table 1. PZQ given at a time during infection and at high doses to effect substantial reductions in worm burdens (rather than complete elimination of worms) does not result in any resistance to reinfection. In an attempt to more closely simulate 'natural infections' with schistosomes, mice were exposed over 50 days to trickle infections with 7 X 2-4 c/m and dosed some weeks later with high doses of PZQ. Mice were either killed to examine worm burdens or, after several weeks, re-exposed to 20 c/m (ideally, of course, a trickle challenge would be more appropriate than the pulse challenge used in this protocol). It is apparent from Table 2 that no evidence of resistance to reinfection was evident in PZQ-treated infected mice. Thus the immune response induced by trickle infection has not obviously altered the state of immunity that might exist after drug treatment of one-time infected mice. From Table 2 it can also be seen that PZQ at the high standard doses used in mice (i.e. 3 X approximately 250 mg/kg body weight) (10,11) was still not able to eliminate all worms following trickle exposure just as such high doses
IMMUNIZATION AGAINST SCHISTOSOMES
379
Table 1. Lack of resistance to reinfection in schistosome-infected mice treated with PZQ. Mouse strain First (infection) infection (c/m) (C57BL/6X129/J)Fi 20 5. japonicum
PZQ'
Second infection (c/m) t
20 20
(BALB/cX 129/J)Fi 5. mansoni
20 20
100 100
No. of mice • ~ 8 6 11 8
Worm burdens (Day 42 or 46 after challenge) ~ 5-0 ±0-7 0-8 ±0-3 (males only) 8-2±l-l 6-4 ±1-5
7 0-7 ± 0-6 (males only) 8 17-6 ± 2 - 4 * 6 31-2±9-9* • Praziquantel given orally (250 mg/kg) on days 50, 51 and 52 (or S. japonicum infection and on days 80, 82 and 84 for S. mansoni infection. t Second infection on Day 125 or 111 in the two experiments. * Not significantly different, the apparent difference in the means only reflecting a very high worm burden (80) in one mouse in the control group. Two other experiments have given similar results with no evidence of protection against reinfection. 100 100
Table 2. Lack of resistance to reinfection with 5. japonicum in (C57BL/6 X 129/J)Fi mice exposed to trickle infections and treated with PZQ. Exposure to cercariae (Day 0 to 50)
PZQ (Day 74, 75 ,76)
2, 3 or 4 cercariae on 7 occasions over 50 days
—
Nil
— • Mice killed on Day 90. t Mice killed on Day 76. * Mice killed on Day 40 after challenge.
are unable to remove all worms following onetime exposure to cercariae (Table 1). Male worms dominate in these residual worm burdens. [Unpublished experiments have suggested that PZQ may be relatively more effective wben worm burdens in chronically infected mice are low.] Lack of resistance to reinfection after CsA treatment of short-term infection A surprisingly persistent effect of CsA, injected s.c. in oil, has been reported in the S. mansoni/ mouse system (12,13). That is, mice given CsA are resistant to inflection with S. mansoni when exposed to cercariae several weeks or even months later. To determine whether this was the case for S. japonicum (Philippines) (and indeed, to determine whether CsA had any effect on this parasite), one group of A/J mice was exposed to
Challenge (Day 118)
No. of mice
20 c/m
7 8 8
1-7 ± 0 - 7 ' 5-8 ± 0-61 13-8 ± 1 - 8 *
20 c/m
6
10-5 ± 2-0*
Worm burdens
20 c/m and injections of CsA or diluent commenced. Another group received CsA or diluent but a delayed exposure to cercariae. It can be seen from Table 3 that CsA, as reported by at least six groups, is a powerful anti-schistosome reagent wben administered at about the time of exposure to S. japonicum cercariae, or 3-4 weeks prior to cercarial challenge. The persistent effect of CsA renders the protocol invalid as a means of detecting resistance to reinfection in drug-cured infected mice (Group 4 versus Group 5 in Table 3). To address the question of resistance to reinfection, tbe protocol was altered. CBA/H mice were infected with 100 cercariae per mouse of S. mansoni at about the time of CsA injection and reehallenged around 6 weeks later. Mice were killed at a time that allowed differentiation between the worms derived from first (mature
G. F. MITCHELL ETAL.
380
Table 3. Persistence of anti-schistosome effect of cyclosporin A in A/J mice exposed to S. japonicum (Philippines). Injection/Infection Group
Day 1-5
1 2 3 4 5
Diluent CsA Diluent CsA CsA
Day 1 20 c/m + +
Day 28 20 c/m
No. of mice
Worm burdens
+ + +
8 8 7 8 8
9-0±l 7* 0-3 ± 0-2 • 6-6±l 3 + 0-9 ±0-4 + l-3±0-4 +
+
* Mice killed on Day 40. + Mice killed on Day 88. Table 4. Lack of resistance to reinfection with S. mansoni in infected CBA/H mice treated with cyclosporin A. Worm burdens (Day 70-73)
Injection/Infection Type of mouse
Day 1-5
Intact
Zero Diluent CsA CsA Zero Diluent CsA
Nude
Diluent CsA CsA
Day 2 (100 c/m)
Day 42 (100 c/m)
+
—
+
—
No. of mice
Mature worms
8 6 7 6 8 7 8
7-4 ±0-8* 7-7+1-7 0 3 + 02
4 3 3
12-8 ±2-4* 2-0 ± 1-2* l-7± 1-2
Immature worms
9-7± 15 13-8±1 4 15-0+ 2-2 7-5±M
23-7 ±4-9
* Some smaller (stunted) worms recorded in these groups.
worms) and second exposure (immature worms). It can be seen from Table 4 that (a) CsA has had a dramatic eflfect on worm numbers and that the diluent alone (olive oil) has had no effect [some preliminary experiments with other oily vehicles indicated some effect of the vehicle or diluent alone; see reference 14]; (b) the CsA effect on schistosomes may stiil be present, though weakly, when the drug is given 42 days prior to cercarial challenge; and (c) no evidence for resistance to reinfection was demonstrable in this experiment. Additional limited data in Table 4 reinforce those reported by others — namely, that CsA has an effect on schistosomes in the hypothymic nude mouse. That is, an intact immune system is not required for expression of the schistosomulocidal effects of CsA (12,15). Lack of resistance to reinfection after injection of living adult worms It is possible that exposure to antigens of living adult worms other than via the portal system
and liver, as occurs in natural infection, may lead to an entirely different type of sensitization. Mice were implanted or injected on several occasions with living adult male or female schistosomes either i.p. or s.c. over the dorsum ofthe mouse and subsequently challenged percutaneously with cercariae. Data in Table 5 indicate that no clear cut or consistent resistance could be demonstrated using these regimes of antigen exposure. Lack of resistance to infection after intranasal administration of worm antigens Evidence exists that the lung is an important site of schistosomule elimination in resistant mice (16-18). To sensitize the lungs of mice, crude adult worm homogenates of either adult male or female schistosomes were administered i.n. (and also s.c.) together with BPV. With other antigens, this manipulation in appropriately anaesthetized mice results virtually in a quantitative deposition of antigens into the lungs (at least transiently) and induction of immunogiobulin E
IMMUNIZATION AGAINST SCHISTOSOMES
381
Table 5. Lack of resistance in mice exposed to living adult worms subcutaneously and/or intraperitoneally. Expt. no. (mouse strain and sex) 1 (C57BL/6X129/J)Fi (M)
2 (C57BL/6X129/J)Fi (M)
3 (CBAXC57BL/6)Fi (M-i-F)
Injection/ Implantation 2X105'. japonicum male worms (Day 0 and 50) i.p. + s.c. Control 2X 12 5". mansoni male worms (Day 0 and 61) i.p. Control 50 5. japonicum male worms over 60 days (4 injections) s.c. 30 S. japonicum female worms over 60 days (4 injections) s.c. Control
4 (C57BL/6X129/J)F| (M)
~200 S. mansoni male worms over 64 days(3 injections) s.c. ~ 120 5. mansoni female worms over 64 days(3 injections) s.c. Control
(IgE) antibodies (7). It can be seen from Table 6 that no resistance to infection was demonstrated in mice repeatedly exposed intranasally to crude antigens and BPV. Lack of resistance to reinfection in schistosome-infected mice immunized with GST and treated with PZQ Genes encoding four GST isoenzymes of Mr 26 000 and Mr 28 000 in S. japonicum (Philippines) and S. mansoni (i.e. Sj26, Sj28, Sm26 and Sm28) have been cloned and native or near native proteins expressed in E. coli (8). Inconsistent (if any) vaccinating effects have been demonstrated in these laboratories with these GST (see the Discussion section). Mice infected with 5. mansoni or S. japonicum (Philippines) were immunized and boosted with homologous E. co//-derived GST then treated with PZQ. Sev-
Challenge
No. of mice
Worm burdens (Day 40-47 after challenge)
(Day 82) 20 c/m S. japonicum
6
5-5± 13
20 c/m
5
3-2 ±0-6
(Day 77) 100 c/m S. mansoni
5
12 8 ± 4 2
10
[P=005] 23-1 ±3-0
(Day 103) 20 c/m 5'. japonicum
6
5-8 ± 1-0
20 c/m
5
7 8 ± 1-4
20 c/m
4
7-0± 1-8
(Day 95) 100 c/m 5. mansoni
4
16 5 ±6-2
100 c/m
4
23 3 ±6-3
100 c/m
5
20-8±2 4
100 c/m
eral weeks later they were challenged with cercariae ofthe homologous schistosome and worm burdens determined in the usual manner. Data in Table 7 indicate that no resistance was demonstrated in infected mice immunized with GST then treated with PZQ before challenge. In a related protocol, S. japonicum-infected (BALB/c X 129/J)Fi mice were injected twice with Sj26 and Sj28 conjugated to KLH, then treated with PZQ before boosting with GSTKLH. No resistance to reinfection was apparent despite high antibody titres to GST (unpubl. data). Resistance to homologous reinfection after exposure to irradiated cercariae of S. mansoni but not S. japonicum (Philippines) Cercariae of S. mansoni or S. japonicum were exposed to ^"Co irradiation and applied on at
382
G. F, MITCHELL ETAL.
Table 6. Lack of resistance in mice given crude antigen + BPV intranasally and subcutaneously. Mouse strain (sex)
A/J (F)
BALB/c.H-2'' (F)
Antigen
Challenge
(Day 0 + 36) 5. mansoni male worm extract + BPV i.n. + s.c. S. mansoni female worm extract + BPV i.n. + s.c.. BPV alone i.n. + s.c. (Day 0, 22 + 56) S. japonicum male worm extract + BPV i.n. + s.c. S. japonicum female worm extract + BPV i.n. + s.c. BPV alone i.n. + s.c.
(Day 43) 100 c/m S. mansoni
30-5 ±4-1*
100 c/m
30-1 ±7-8
100 c/m
46 4 ±5-6
No. of mice
Worm burdens (Day 56 and 41 after challenge)
(Day 57) 20 c/m S. japonicum
6 8± 16
20 c/m
!-4± 1-1
20 c/m 6 0±l 3
* Majority of mice in this group were unusual in having more female than male worms.
Table 7. Lack of resistance to reinfection in (C57BL/6 X 129/J)Fi mice following immunization of infected mice with GST prior to PZQ treatment. Injection/Infection First infection (Day 0) 20 c/m S. japonicum 20 c/m S. japonicum Nil Nil 100 c/m S. mansoni 100 c/m S. mansoni Nil Nil
GST + FCA (Day 14) 33 Hg rSj26* +33 Hg rSj28 in FCA FCA alone GST in FCA FCA alone
GST (Day 36) 25 Hg rSj26 +25 Hg rSj28 PBS
PZQ
Worm burdens No. of mice
(Day 40 and 42 after challenge)
5
l l - 4 ± 1-7
+
5
9 6 ± 14
+ +
Challenge
(Days 42, 43, 44) (Day 65) 20 c/m + S. japonicum +
6 5
8-7±M 10-6 ±2-4
(Day 20) 30 Hg rSm26 +70 Hg rSm28 in FCA FCA alone
GST + PBS + (Day 37) (Days 47, 48, 49) (Day 68) 20 Hg rSm26 + 100 c/m +30 Hg S. mansoni rSm28 PBS + +
6
13-3±1 8
7
13-6±2-5
GST in FCAFCA alone
GST PBS
7 6
12 9 ±2-3 13-3+1-6
+ +
• rSj26, rSj28, rSm26, rSm28 are the Mr 26 000 and Mr 28 000 GST of 5. japonicum and S. mansoni produced in, and purified from, E. coli.
IMMUNIZATION AGAINST SCHISTOSOMES
least two occasions to the skin of mice that were subsequently challenged. As demonstrated by numerous other investigators and reviewed by Taylor and Bickle (1), clear evidence of resistance to homologous (but not heterologous) challenge was demonstrated in mice previously given irradiated cercariae of 5". mansoni. In contrast, no convincing homologous resistance was demonstrated in mice given S. japonicum (Philippines) cercariae exposed to ^^Co irradiation (Table 8). Groups of mice were exposed to ^^Coirradiated cercariae of S. mansoni percutaneously and later immunized with Sm26 and Sm28 GST (results not shown). Both these sensitizing manipulations were repeated and mice challenged with 100 c/m S. mansoni. GST immunization did not change the level of resistance expected from exposure to irradiated cercariae alone (i.e. approximately 75% resistance). Thus the mean worm burden in 11 X (C57BL/6X 129/J)Fi hybrids given a total of 120 (ig GST and approximately 2000 irradiated cercariae on 2 X 2 occasions over 35 days was 86% that in controls.
383
DISCUSSION The experiments reported in this paper were prompted by the generally disappointing vaccinating effects of E. co/z-derived schistosome GST in naive mice. The Mr 26 000 and Mr 28 000 GST isoenzymes of 5. Japonicum (i.e. Sj26 and Sj28) and S. mansoni (i.e. Sm26 and Sm28) have been expressed as full-length or near full-length molecules in E. coli and affinity purified on glutathione (GSH) columns. Numerous vaccination studies in mice using individual isoenzymes or combinations have been conducted (19,20, and Mitchell G. F., Wright M. D., Davem K. M., Tiu W. U., Garcia E. G., unpublished). No consistent high level protection has been demonstrated in any mouse strain, the (C57BL/6X129/J)Fi mouse and FCA being used for most experiments. Attempts to increase anti-GST responses or induce antibodies that cross-react between isoenzymes by the use of pGEX fusion proteins of Sj26 (21-23) or conjugation of GST to powerful antigens such as sheep erythrocytes have been generally unsuccessful, with no protection being demonstrated. Far more impressive vaccination data have been
Table 8. Resistance in mice exposed to ^^Co-irradiated cercariae of 6". mansoni but not 5. japonicum (Philippines). Mouse strain (sex) (CBA/H X C57BL/6)Fi (M + F)
Irradiated cercariae
Challenge
(Day 0 + 30) ~ 1200 S. mansoni
(Day 51) 100 c/m S. mansoni
Nil
ll-5±3-2 61% resistance (P< 0-001)
~340 S. japonicum 8 0 Nil 8 8-8 ± 1-3* * One male worm found in the seven mice. t As evidenced by the worm burdens after 100 c/m, the infectivity ofthe cercariae used in this experiment was obviously low. * This difference is insignificant; however, the number of mice with < 6 worms in the experimental group was 4/8 whereas this number of mice in the control group was 1/8.
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G. F. MITCHELL £7" ^L.
reported with full length recombinant or native Sm28 against S. mansoni infection (24,25). An hypothesis we have developed (5) is that aggressive immune attack at the schistosomule or schistosome surface releases GST and that antibodies to GST, through neutralization of the repair function of these enzymes, increase the damage inflicted by the initial immune insult. It follows from this that anti-GST immunity alone would not be host-protective and that other immune responses are required to inflict initial damage. With this in mind we have attempted to sensitize mice through exposure to infection or exposure to crude antigen preparations and to test the effects of GST immunization superimposed on a 'baseline' level of resistance or at least a degree of sensitization. Exposure of mice to infection followed by drug treatment does not lead to resistance reinfection. This statement applies to mice exposed by trickle infections of cercariae. Moloney et al. (26) in the S. japonicum (Chinese) system demonstrated clearly that resistance to reinfection in mice was only demonstrated early after PZQ administration, an effect readily ascribed to 'pathology-related resistance' (16). Exposure of mice to living adult worms in ectopic sites also does not lead to resistance nor does repeated administration of crude antigen plus adjuvant into the airways to sensitize the lungs. Abbreviation of infection with CsA also does not leave the host resistant to reinfection (allowing for a remarkably prolonged effect of CsA injected in an oil vehicle). Irradiated cercariae in the S. mansoni system resulted in good levels of homologous resistance as expected. No such resistance to homologous infection was demonstrated in mice given relatively large numbers of ^"Co-irradiated cercariae of S. japonicum (Philippines). Good levels of resistance to S. japonicum (Chinese) using ultraviolet (UV)-irradiated cercariae have been
reported by Moloney et al. (27). Interestingly, no resistance to S. japonicum (Philippines) was demonstrated in mice exposed to irradiated S. japonicum (Chinese) cercariae (28). In a recent review we have emphasized differences in transit times in lungs for cercariae of S. japonicum versus S. mansoni, the latter persisting in the lungs for much longer than S. japonicum (29). It is possible that persistence of S. mansoni cercariae in lungs increases either the sensitization for and/or expression of resistance to challenge. It becomes important to determine whether there are differences between S. japonicum (Chinese) and S. japonicum (Philippines) in this regard and also between UV-irradiated versus ^"Coirradiated cercariae of S. japonicum in lung persistence and therefore 'sensitization' of this organ. Disappointingly, E. co//-derived GST in FCA did not change the lack of resistance to reinfection after PZQ treatment of existing infection in mice. Also, the GST (of 5. mansoni expressed in E. coli) did not appear to increase the levels of resistance induced by exposure to irradiated cercariae. Three out of the four GST presently available (8) are not full length proteins and lack A'-terminal amino acids. Whether this is important in the lack of any consistent vaccinating effect of GST (using the one adjuvant, FCA) obtained in this laboratory remains to be determined. Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, the Edna McConnell Clark Foundation, the Australian International Development Assistance Bureau (AIDAB) and the United Nations Development Programme (UNDP)/World Bank/World Health Organization (WHO) Special Programme for Research and Training in Tropical DiseasesRockefeller Foundation Health Sciences for the Tropics-Partnerships in Research Programme.
REFERENCES 1. Taylor, M. G. and Bickle, Q. D. 1986. Irradiated schistosome vaccines. Parasitol Today 2: 132134. 2. Capron, A., Dessaint, J. P., Capron, M., Ouma, J. H. and Butterworth, A. E. 1987. Immunity to schistosomes: Progress toward vaccine. Science 238: 1065-1072. 3. Dean, D. A. 1983. Schistosoma and related genera: Acquired resistance in mice. Exp. Parasitol. 55: 1-104.
4. Sher, A., James, S. L., Correa-Oliveira, R., Hieny, S. and Pearce, E. 1989. Schistosome vaccines: Current progress and future prospects. Parasilotogy 98: S61-S6S. 5. Mitchell, G. F. 1989. Glutathione S-transferases: Potential components of anti-schistosome vaccines? Parasitol. Today 5: 34-37. 6. Wright, M. D., Tiu, W. U., Wood, S. M., Walker, J. C, Garcia, E. G. and Mitchell, G. F. 1988. Schistosoma mansoni and S. japonicum worm
IMMUNIZATION AGAINST SCHISTOSOMES numbers in 129/J mice of two types and dominance of susceptibility in FI hybrids. / Parasitol. 74:618-622. 7. O'Donnell, I. J. and Mitchell, G. F. 1978. An investigation of the allergens of Ascaris lumbricoidcs using a radio-allergosorbent test (RAST) and sera of naturally infected humans: Comparison with an allergen for mice identified by a passive cutaneous anaphylaxis test. Aust. J. Biol. Sci. 31: 459-487. 8. Henkle, K. J., Davern, K. M., Wright, M. D., Ramos, A. J. and Mitchell, G. F. 1990. Comparison of the cloned genes of the 26- and 28-kilodalton glutathione-S-transferases of Schistosoma japonicum and Schistosoma mansoni. Mol. Biochem. Parasitol. 40: 23-34. 9. Crowl, R., Seamans, C , Lomedico, P. and McAndrew, S. 1985. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene 3S, 31-38. 10. Doenhoff, M. J., Sabah, A. A. A., Fletcher, C , Webbe, G. and Bain, J. 1987. Evidence for an immune-dependent action of praziquantel on Schistosoma mansoni in mice. Trans. Roy. Soc. Trop. Med. Hyg. 81: 247-254. 11. Brindley, P. J., Strand, M., Norden, A. P. and Sher, A. 1989. Role of host antibody in the chemotherapeutic action of praziquantel against Schislosoma mansoni: Identification of target antigens. Mot. Biochem. Parasitol. 34: 99-108. 12. Bout, D., Deslee, D. and Capron, A. 1986. Antischistosomal effects of cyclosporin A: Cure and prevention of mouse and rat schistosomiasis mansoni. Infect. Immun. 52: 823-827. 13. Pons, H.A.,Adams, S. andStadecker, M.J. 1988. Schistosoma mansoni: The basis for the antischistosomal effect of cyclosporin A. Exp. Parasitol. 67: 190-198. 14. Salafsky, B., Fusco, A. C, Li, L. H., Mueller, J. and Ellenberger, B. 1989. Schistosoma mansoni: Experimental chemoprophylaxis in mice using oral antipenetration agents. Exp. Parasitol. 69: 263-271. 15. Brannon, L. R., Chappel, L. H., Woo, J. and Thomson, A. W. 1989. Antischistosomal activity of cyclosporin A: Studies on murine spleen cells and the influence of a cyclosporin antagonist on resistance to infection. Immunology 67: 382387. 16. Wilson, R. A., Coulson, P. S. 1989. When and how does immune elimination of Schistosoma mansoni occur? Parasitol. Today 5: 274-278. 17. McLaren, D.J. 1989. Will the real target of immunity to schistosomiasis please stand up? Parasitol. Today 5: 279-282. 18. Dean, D. A., Mangold, B. L., Georgi, J. R. and Jacobbson, R. H. 1984. Comparison of Schistosoma mansoni migration patterns in normal and irradiated cercaria-immunized mice by means of
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autoradiographicanalysis./)w. / Trop. Med. Hyg. 33: 89-96. 19. Smith, D. B., Davern, K. M., Board, P. G., Tiu, W. U., Garcia, E. G. and Mitchell, G. F. 1986. Mr 26 000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione-S-transferase. Proc. Natl. Acad. Sci. USA 83: 8793-8707. 20. Mitchell, G. F., Garcia, E. G., Davern, K. M., Tiu, W. U. and Smith, D. B. 1988. Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum. Trans. Roy. Soc. Trop. Med. Hyg. 82: 885-889. 21. Smith, D. B., Rubira, M. R., Simpson, R. J., Davern, K. M., Tiu, W. U., Board, P. G. and Mitchell, G. F. 1988. Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione-S-transferase. Molec. Biochem. Parasitol. 27: 249-256. 22. Smith, D. B., and Johnson, K. S. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 61: 31-40. 23. Davern, K. M., Tiu, W. U., Samaras, N., Gearing, D. P., Hall, B. E., Garcia, E. G. and Mitchell, G. F. 1990. Schistosoma japonicum: monoclonal antibodies to the Mr 26 000 schistosome glutathioneS-transferase (Sj26) in an assay for circulating antigen in infected individuals. Exp. Parasitol. 70: 293-304. 24. Bailout, J-M., Grzych, J-M., Pierce, R. J. and Capron, A. 1987. A purified 28 000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. /. Immunol. 138: 3448-3453. 25. Balloul, J-M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J-M., Pierce, R. J., Cavallo, D., Lecocq, J. P. and Capron, A. 1987. Molecular cloning of a protective antigen of schistosomes. Nature 326: 149-153. 26. Moloney, N. A., Hinchcliffe, P. and Webbe, G. 1987. Loss of resistance to reinfection with Schistosoma japonicum in mice after treatment with praziquantel. Trans. Roy. Soc. Trop. Med. Hyg. 81: 247-254. 27. Moloney, N. A., Hinchcliffe, P. and Webbe, G. 1987. Passive transfer of resistance to mice with sera from rabbits, rats or mice vaccinated with ultra violet attenuated cercariae of Schistosoma japonicum. Parasitology 94: 497-508. 28. Moloney, N. A., Garcia, E. G. ahd Webbe, G. 1985. The strain specificity of vaccination with ultra violet attenuated cercariae of the Chinese strain of Schistosoma japonicum. Trans. Roy. Soc. Trop. Med. Hyg. 79: 245-247. 29. Mitchell, G.F., Tiu, W.U. and Garcia, E.G. 1991. Infection characteristics of Schistosoma japonicum in mice and relevance to assessment of schistosome vaccines. Adv. Parasitol. (in press).
