Immunization of mice with recombinant Sjc26GST induces a pronounced anti-fecundity effect after experimental infection with Chinese Schistosoma japonicum

Vaccine, Vol. 13, No. 6, pp. 603407, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264-410x/95 $10.00+0.00

Immunization of mice with recombinant Sjc26GST induces a pronounced antifecundity eaffect after experimental infection with Chinese Schistosoma japonicum Liu Shuxian’, Song Guangchen*, Donald P. McManus+

Xu Yuxian*,

Wen Yang+ and

We report the cloning, by polymerase chain reaction (PCR), of a cDNA encoding a japonicum ( Chinese) 26 kDa glutathione-S-tranferase (GST) (Sjc26GST), expression of the cDNA, affinity purtjication of the recombinant GST and its vaccine efficacy in o,utbred NIH mice using Freund’s as adjuvant. The most striking feature of the vaccination experiments was the pronounced reduction in the number of eggs in the livers and spleens of immunized mice. A relatively low but significant level of protection in terms of reduced worm viability against challenge infection was also observed. Further, the level of anti-Sjc26GST antibody in immunized mice was signtficantly higher than in control mice at week 6 post-challenge infection. These results closely mirror the protection conferred by immunization of animals with the 28 kDa GST of S. mansoni (Sm28) where a reduction in worm viability, worm fecundity and egghatching ability have been reported following challenge with S. mansoni. In terms of developing a vaccine ag
Schistosoma

Keywords: Schistosoma

japonicum;

Chinese strain; recombinant

26 kDa

The glutathione-S-transferases (GSTs) are a family of cytosolic or membrane-bound enzymes present in all organisms with their main function being the detoxification of electrophilic compounds’. The schistosome GSTs have been shown to exist as 26 and 28 kDa molecules, each consisting of several isoforms2-6; full-length or near full-length genes for the 26 kDa (Sj26, Sm26) and 28 kDa (Sj28, Sm28) GSTs of Philippine S. japonicum and S. mansoni have been cloned and expressed in Escherichia coli’-“. Much recent interest has focused on GSTs as potential components of anti-schis,tosome vaccines’ ’ . Impressive vaccination data. in terms of reduction in worm burden, have been reported with full-length recombinant or native Sm28 against S. mansoni in rodents and primates9m’4, although less encouraging protection has *Department of Immunology, Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine, Shanghai 200025, Peoples Republic of China. ‘Molecular Parasitology Unit, Tropical Health Program, The Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia. (Received 9 March 1994; revised 2 August 1994; accepted 9 August 1994)

GST:

Sjc26GST;

anti-fecundity

immunity

been demonstrated in mice with near full-length recombinant Sm28 and Sj28 proteins lacking N-terminal amino acids”. 16. Similarly, attempts to reduce parasite load in mice immunized with Philippine S. japonicum recombinant Sj2615 or mice exposed to crude schistosome antigens plus recombinant 26 and 28 GSTS’~ have, generally, been disappointing. Significantly, in addition to impeding worm viability, Sm28 has been shown to decrease schistosome fecundity in baboons and rodents as well as the hatching capacity of eggs14. “, with potentially important consequences for disease transmission and granuloma-derived pathology. We have isolated and purified, by affinity chromatography on a glutathione agarose matrix, a fraction containing native 26 and 28 kDa GSTs from adult worms of Chinese S. japonicum”. A comparative study of the antigenicity, immunogenicity and protective efficacy of this fraction and Philippine recombinant Sj26 carried out subsequently”. ” yielded similar results; 26.2-32.5% protection (in terms of reduced worm burden) was stimulated in mice immunized with the two antigen sources and challenged with cercariae of Chinese S. japonicum. In this paper, we have carried out

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Anti-fecundity immunity induced by S. japonicum 26 kDa GST: S. Liu et al. a vaccination trial on mice using purified recombinant Sj26 (Chinese) (reSjc26GST) on Chinese S. juponicum, determining the effect of the molecule on worm burden and, for the first time with Sj26GST. on worm fecundity.

MATERIALS

AND METHODS

Mice

Sixty-eight male outbred NIH mice (bred in a specific pathogen-free facility at the Institute of Parasitic Diseases, Shanghai), 18-25 g in weight and 6-8 weeks old were used in vaccination experiments.

intravenously 7 days later with 10 pg reSjc26GST in 0.85% (w/v) saline. An adjuvant control group, comprising 11 mice, was subjected to the same immunization schedule as the immune-challenge group but the reSjc26GST was replaced by PBS. Twenty-six non-immunized mice were used as a challenge control group. Five days after final boosting, the immune-challenge and adjuvant control and the challenge control mice were groups, anaesthetised with 0.01 ml g-’ body weight of 5.0 mg ml-’ sodium pentobarbitol (Nembutal) (Sigma) and infected percutaneously with 40 cercariae by the cover glass method. Worm recovery and tissue sampling

Parasites

The lifecycle of a Chinese population of S. juponicum is maintained in the Shanghai laboratory. The parasite was originally collected in Oncomelania hupensis hupensis snails from Guichi County, Anhui Province. Adult worms were recovered from infected mice or rabbits by perfusion and washed thoroughly free of contaminating host components.

Vaccinated and control mice were sacrificed at the same time with perfusion being undertaken 6 weeks after challenge infection. The worm reduction rate (% protection) was calculated according to the formula: % Protection i

Recombinant

Sjc26GST

Total RNA was isolated from adult Chinese S. japonicunz using guanidinium thiocyanate-caesium chloride gradient centrifugation”. A cDNA fragment encoding the 26 kDa GST of Chinese S. japonicum was obtained by in vitro reverse transcription of the total RNA using AMV reverse transcriptase (Promega) and amplification by a standard PCR protocol with 2 primers and Taq DNA polymerase (Perkin Elmer primers Cetus). The (Primer I: two STAGAATTCGCATGTCCCCTATACTA3’; Primer II: SGAGATTCCGACATTTATATTTAG3’) used were synthesized by a DNA autosynthesisor (Model 281 A DNA synthesiser) according to the published nucleotide sequence of the cDNA encoding the Sj26 GST of Philippine S. juponicum’“. The PCR-amplified Sjc26 cDNA was subcloned into M13mp18 (New England Biolabs) and sequenced using standard procedure?‘. The cDNA was also ligated to the expression vector PBV220 and cloned and expressed in the host bacterium DH5a’“. PBV220 and DHSa were kindly supplied by the Institute of Virology, Chinese Academy of Preventive Medicine, Beijing. Positive clones were selected by restriction mapping and by assaying the expressed products for GST enzyme activity23. Purified reSjc26GST was obtained by loading a 25 g lysate of DH5a cells expressing the Sjc26GST cDNA on an affinity column with oxidised and reduced glutathione agarose (Sigma) matrix; reSjc26GST was eluted from the column with high salt buffer and PBS containing glutathione (Sigma)‘.

Mean number

I-

Mean number

schedule

An immune-challenge group of 31 mice was immunized with the reSjc26GST. Each mouse was immunized subcutaneously (s.c.) with 50 pg of purified recombinant reSjc26GST emulsified in an equal volume of complete Freund’s adjuvant (FCA) (Difco). The mice were boosted S.C. with 50 pg reSjc26GST emulsified in an equal volume of incomplete Freund’s adjuvant (IFCA) (Difco) after 14 days and further boosted

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of worms

of worms

in immune-challenge

in adjuvant

or challenge

mice control

mice

i

At the end of perfusion, the livers and spleens of each mouse from the three groups were weighed. The livers or spleens were homogenized, passed through nylon screen (size 260 holes/inch), the homogenates subjected to digestion with 20% (w/v) NaOH and the number of eggs present determined by light microscopic examination. Total egg counts were expressed for each group of mice as the mean number of eggs per gram of mouse liver or spleen, and the mean number of eggs per pair of adult schistosomes. Assay for anti-reSjC26GST

antibodies in sera

Blood was taken from mice in each of the 3 groups just prior to perfusion. Anti reSjc26GST antibody levels in sera (diluted 1:10 in PBS) were determined by ELISA. Plates were coated with 1 pg reSjcGST26 per well. Foetal calf serum (FCS) (3%) (Sino-American Biotechnology Co.) was used for blocking at 37°C for 1 h. Horse radish peroxidase labelled goat anti-mouse IgG antibody (Promega) was used at 1:lOOO dilution. Ophenylenediamine (Sigma) was used as substrate at a concentration of 0.4 mg ml-‘. Plates were also coated with a 1:500 dilution of 1% soluble egg antigen (SEA) for assay of anti-SEA antibodies. Statistical

analysis

Student’s t-test was used for comparisons of data. RESULTS Purification

Immunization

=

of reSjc26GST

The PCR-amplified cDNA fragment, putatively expressing Sjc26GST, subcloned into M 13mpl8 and sequenced (540 nucleotides from both ends of the cDNA), showed 99.6 and 100% identity, respectively, in nucleotide and amino acid sequence with the published Philippine Sj26 cDNA sequence”. Such close identity between the 26GSTs of Chinese and Philippine S, japonicum has been confirmed in another studyz5. Affinity chromatography on glutathione-agarose

xl00

Anti-fecundity immunity induced

yielded purified reSjc26GST with a protein concentration of 1.16 mg ml-’ and with GST enzyme activity of 55.1 IU mg-‘. A single band, at approximately 26 kDa, was evident when the reSjc26GST fraction was subjected to SDS-PAGE (data not shown). Anti-Sjc26GST reSjc26GST

antibody in mice immunized

with

For the determination, by ELISA, of specific antibodies in mice infected with S. japonicum, preliminary experiments ((data not shown) indicated that more consistent results were obtained when the reSjc26GST was diluted for coating on ELBA plates relative to its protein concentration rather than its GST enzyme activity. O.D. values (mean&SD.) of murine anti-Sjc26GST antibod:y in the immune-challenge, challenge control and adjuvant control groups by ELISA were 0.78?0.41., 0.41kO.23 and 0.40+0.24, respectively; reference positive (a pool of infected mouse sera) and negative (a pool of normal mice sera) sera gave values of 0.62kO.14 and 0.16?0.02, respectively. The mean O.D. value for anti-Sjc26GST antibody levels in immune-challenge mice was significantly higher than the challenge and adjuvant controls (PC 0.01). Sera from mice immunized with reSjc26GST and all the control groups were also tested in ELISA with SEA. O.D. values for the immune-challenge, challenge control and adjuvant control groups were 0.65t0.22, 0.72kO.22 and 0.73kO.20, respectively; the reference positive and negative sera gave values of 0.65f0.21 and 0.19+0.02, respectively. There were no significant differences in O.D. levels between the immune-challenge and challenge and adjuvant control groups 0, > 0.05). Protective immunity induced by reSjc26GST

in mice

There were no statistically significant differences @ > 0.05) in the weights of livers and spleens from mice in the immune-challenge and the two control groups (Table 1).

The number of worms recovered for each group after perfusion is shown in Table 1. Worm reduction rates of 23.7% (immune-challenge vs adjuvant control) 0, c 0.05) and 26.4% (immune-ch,allenge vs challenge control) (p < 0.01) were obtained. Vaccination experiments with mice immunized with native Chinese Sj26-28 GSTs or Table 1

Results of vaccination

of NIH mice against Schistosoma Immune-challenge

Wt of livers (g) Wt of spleens (g) Worm burden Eggs/mouse liver Eggs/g liver Eggs/liver/worm pair Eggs/mouse spleen Eggs/g spleen Eggs/spleen/worm pair

4.04 0.80 14.07 i !690.3 666.5 382.6 45.8 57.3 6.51

f * + f + f f f +

1.05 0.32 4.15 13.9 3.5 2.0 3.7 4.6 0.52

group (31”)

by S. japonicum 26 /da

GST S. Liu et al.

recombinant Philippine Sj26 and then challenged with cercariae of Chinese S. japonicum have provided similar levels of protection (26.2-32.5%)20. Effect on egg counts in liver and spleen

There was a substantial reduction in the number of eggs present in the liver and spleens of the immunechallenge group mice when compared with the challenge and, especially, adjuvant-controls (Table 1) (p < O.OOl0.05). The number of eggs in the liver was reduced by 55.5 and 70.8% (eggs g-’ liver) and 30.6 and 59.2% (eggs/ liver/schistosome pair), respectively; egg numbers were reduced in the spleen by 72.8 and 82.7% (eggs g-’ spleen) and 66.9 and 78.8% (eggslspleenfschistosome pair), respectively. We cannot explain why the adjuvant control had significantly (p < 0.01-0.05) more eggs/ schistosome worm pair than the challenge control. DISCUSSION

An advantage of utilising enzymes such as the GSTs as vaccine candidates for schistosomiasis is their ability to bind specific substrates which can be readily incorporated for purification of gene expression products by affinity chromatographyZ6. Furthermore, the GSTs provide an important function as detoxifying agents and antibody-mediated neutralization of this detoxification function could render the schistosome vulnerable to toxic products generated by immune attack at the tegumental surface or even the gut. GSTs may also be involved in the solubilization of nutrients in the schistosome gut such as haematin and this function could also be inhibited by antibody-mediated mechanisms’. ’ ’ . It has been hypothesised”’ I6 that aggressive immune attack at the schistosomule or adult 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 assault. As a consequence of this hypothesis, anti-GST immunity alone would not be host-protective and that other immune responses are required to inflict initial damage. The gene encoding a 26 kDa GST (reSjp26GST) from S. japonicum (Philippine strain) has been cloned and expressed’.’ but vaccination of mice with reSjp26GST and native Sjp26GST is not sufficient to induce consistent protective immunity in terms of decreased worm burdens.

japonicumusing reSjc26GST Challenge 3.51 0.89 19.12 5271.8 1498.0 551.6 187.7 210.4 19.65

control group (26)

+ 0.78 f 0.03 ZII3.63 i 90.0 * 25.6 f 9.4 f 6.4 + 7.1 * 0.73

Adjuvant 3.78 0.86 18.45 8642.4 2285.3 936.6 283.6 330.2 30.74

control group (11) f f i + + i f i +

1.01 0.34 3.04 447.2 126.2 51.7 16.1 18.7 1.71

“Numbers of mice (male, 6-8 weeks old) per group are shown in brackets. All results are presented as means f SD. The immune-challenge group were immunized with 50 pg reSjc26GST + FCA (s.c.), followed by boosts of 50 pg reSjc26GST + IFCA (s.c.) after 14 days and 10 pg reSjc26GST + saline (i.v.) 7 days later. The adjuvant control group was subjected to the same schedule except that the reSjC26GST was replaced by PBS. The challenge control mice and, 5 days after final boosting, the adjuvant control and immune-challenge groups were infected with 40 S. japonicum cercarLae. Six weeks after challenge, all mice were sacrificed concurrently; adult worm burdens and egg numbers in spleens and livers were compared in the three groups

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Anti-fecundity

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S. japonicum 26 kDa GST S. Liu et al.

Here we have reported the cloning of a cDNA encoding Sjc26GST, expression of the cDNA, affinity purification of the resulting recombinant GST and its vaccine efficacy in mice using Freund’s as adjuvant. A relatively low but significant level of protection in terms of reduced worm viability against challenge infection was obtained. The level of anti-Sjc26GST antibody in immunized mice was also significantly higher than in control mice at 6 weeks post-challenge infection. However, the most striking feature of the vaccination experiments was the pronounced reduction in the number of eggs in the livers and spleens of immunized mice. These results mirror closely the protection conferred by immunization of animals with Sm2814 and Sm28 peptide fragments” where a reduction of worm viability, worm fecundity and egg-hatching ability has been reported. It therefore appears that in terms of developing a vaccine strategy against schistosomiasis japonica, the results of our study indicate that immunisation with reSjc26GST can provide two complementary goals in human or animal populations-some reduction in worm burden following exposure to infection or reinfection and reduction of pathology by a decrease in worm fecundity with this direct effect also affecting the transmission of S. juponiczcm. The mechanism of antifecundity immunity requires elucidation and we do not know whether Sjc26GST can affect egg hatching ability as occurs with Sm28 or whether it can induce antiembryonation immunity (see Ref. 27). In our experiments, the anti-Sjc26GST antibody level of immune-challenge mice was significantly higher than in the challenge-control and adjuvant control mice but there was no significant difference between the three groups in anti-SEA antibody levels. Previous experiments’” have shown that sera from mice immunised with recombinant Philippine Sj26 contained antibodies which reacted with SEA of Chinese S. although the titre was relatively low. jqwnicuin, Antibodies in sera from these mice and from mice immunized with native 26-28 kDa GSTs of Chinese S. juponicum were shown in immunoblots to specifically bind to SEA components with molecular sizes of 97-120 kDa”‘. Whether these components are targets of antiembryonation or anti-miracidial immunity remains to be established. Vaccination trials on domestic animals are currently in progress to determine whether the same anti-viability and anti-fecundity effects observed in mice can be induced using reSjc26GST. ACKNOWLEDGEMENTS We are grateful to Dr W. Tiu (College of Public Health, Manila, The Philippines) for kindly providing E. coli containing a plasmid (pSj5) that directs synthesis of native Sj26. This research was supported by the National “863” Bio-Tek programme. This investigation also received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

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S-transferases of Schis&oma japonicum and Schistosoma mansoni. Parasite Immunol. 1988, 10, 693-706 O’Leary, K.A. and Tracy, J.W. Purification of three cytosolic glutathione S-transferases from adult Schistosoma manscni. Arch. Biochem. Biophys. 1988, 164, l-12 Wright, M.D., Davern, K.M. and Mitchell, G.F. The functional and immunological significance of some schistosome surface molecules. Parasitol. Today 1991, 7, 56-58 O’Leary, K.A., Hathaway, K.M. and Tracy, J.W. Schistosoma mansoni: single-step purification and characterization of glutathione-S-transferase isoenzyme. Exp. Paras&/. 1992, 75, 47-55 Trottein, F., Godin, C., Pierce, R.J., Sellin, B., Taylor, M.G., Gorillot, I. et a/. Inter-species variation of schistosome 28-kDa glutathione Stransferases. MO& B&hem. Parasitol. 1992, 54, 63-72 Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G. and Mitchell, G.F. Mr 26000 antigen of Schis@soma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione S-transferase. Proc. Nat/ Acad. Sci. USA 1986, 83, 8703-8707 Smith, D.B., Rubira, M.R., Simpson, R.J., Davern, K., Tiu, W.U., Board, P.G. and Mitchell, G.F. Expression of an enzymatically active parasite molecule in Escherichia co//~ Schistosoma japonicum glutathione-Stransferase. MO&. B&hem. Parasol. 1988, 27, 249-256 Balloul, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J. et a/. Molecular cloning of a protective antigen of schistosomes. Nature 1987, 326, 149-153 Henkle, K., Davern, K., Wright, M., Ramos, A. and Mitchell, G.F. Comparison of the cloned genes of the 26- and 28-kilodalton glutathione-S-transferases of Schistosoma japonicum and Schistosoma mansoni. Molec. Biochem. Par&to/. 1990, 40, 23-34 Mitchell, G.F. Glutathione S-transferases-potential components of anti-schistosome vaccines. Pat&to/. Today 1989, 5, 34-37 Balloul, J.M., Grzych, J.M., Pierce, R. and Capron, A. A purified 28,000 Dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. J. Immunol. 1987, 136, 3448-3453 Capron, A., Dessaint, J.P., Capron, M., Ouma, J. and Butterworth, A.E. Immunity to schistosomes: progress towards a vaccine. Science 1987, 238, 1065-l 072 Boulanger, D., Reid, G.D.F, Sturrock, R.F., Wolowczuk, I., Balloul, J.M., Grezel, D. et a/. Immunization of mice and baboons with the recombinant Sm28GST affects both worm viability and fecundity after experimental infection with Schistosoma mansoni. Parasit immunol. 1991, 13, 473-490 Mitchell, G.F., Garcia, E., Davern, K., Tiu, W. and Smith, D.B. Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schisfosoma japonicum in mice. Trans. R. Sot. Trop. Med. Hyg. 1988, 82, 885-889 Mitchell, G.F., Davern, K., Wood, S.M., Wright, M.D., Argyroupoulos, V.P., McLeod, KS. ef a/. Attempts to induce resistance in mice to Schistosoma japonicum and Schistosoma mansoni by exposure to crude schistosome antigens plus cloned glutathione-S-transferase. lmmunol. Cell. Biol. 1990, 68, 377-385 Xu, C.-B., Verwaerde, C., Gras-Masse, H., Fontaine, J., Bossus, M., Trottein, F. et a/. Schistosoma mansoni 28-kDa glutathione S-transferase and immunity against parasite fecundity and egg viability. J. Immunol. 1993, 150, 940-949 Liu, S.X., Song, G.C., Ding, L.Y., Xu, Y.X., Yang, W. and McManus, D.P. GST antigen from Chinese strain of Schistosoma japonicum. Chinese J. Parasitol. Par. Dis. 1992, 10, 312-313 Liu, S.X., Ding, L.Y., Song, G.C., Chen, C.Y. and Cai, Z.H. The antigenicity of GST antigen extracted from Chinese strain of Schistosoma japonicum. S. E. Asian J. Trap. Med. Pub. H/th 1993, 24, 61-64 Liu, S.X., Song, G.C., Ding, L.Y., Xu, Y.X., Cai, Z.H. and Wu, G.Z. Comparative study on antigenicity and immunogenicity of 26-28 kDa antigen and recombinant Sj26 (rSj26) of Schistosoma japonicum. S. E. Asian J. Trop. Med Pub. HHh 1993, 24, 65-69 Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. Isolation of biologically active ribonucleic acid from

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sources enriched in ribonuclease. Biochemistry 1979, 18, 5294-5299 Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, 1989 Cai, Z.H., Song, G.C., Xu, Y.X. and Liu, S.X. The temperaturedependent expression of GST of Schistosoma japonicum (Philippine strain). SE. Asian J. Trap. Med. Pub. Hfth 1993, 24, 57-60 Habig, W.H., Past, M.J. and Jacoby, W.B. Glutathione S transferase. J. Biot. Chem. 1974, 249, 7130-7318

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Yang, W., Waine, G.J., Becker, M., Liu, S.X. and McManus, D.P. The 26-kDa glutathione-S-tranferases from Chinese and Philippine Schistosoma japonicum are identical. Acta Trap. 1994, 57, 345-349 Bergquist, R. Prospects of vaccination against schistosomiasis. Stand. J. Infect. Dis. 1990, 78 (suppl.), 60-71 Garcia, E.G., Rivera, P.T., Mitchell, G.F., Evardome, R., Almonte, R.E. and Tiu, W.U. Effects of induction of antiembryonation immunity on liver granulomas, spleen weight and portal pressure in mice infected with Schistosoma japonicum. Acta Trap. 1989, 46, 93-99

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