susceptible proportion of WEHI 129/J and other permissive mouse strains revealed four antigens that were differentially .... Searle Corp., Sydney, Australia), as previously described (4,. 15). ..... McLaren, and Knopf, personal communication), data that .... This work was supported by the Australian National Health and.
Vol. 56, No. 11
INFECTION AND IMMUNITY, Nov. 1988, P. 2948-2952
0019-9567/88/112948-05$02.00/0 Copyright C 1988, American Society for Microbiology
Schistosoma mansoni Antigens Differentially Recognized by Resistant WEHI 129/J Mice MARK D. WRIGHT,* MARK V. ROGERS, KATHY M. DAVERN, AND GRAHAM F. MITCHELL The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia Received 5 May 1988/Accepted 29 July 1988 Mice of the strain WEHI 129/J are genetically resistant to chronic Schistosoma mansoni infection. Resistance is expressed in at least 50% of mice, with the remaining mice showing normal susceptibility to infection. The serum antibody specificities in the resistant proportion of WEHI 129/J were analyzed at various times after exposure to cercariae by using both Western blotting and immunoprecipitation. Comparisons with the susceptible proportion of WEHI 129/J and other permissive mouse strains revealed four antigens that were differentially recognized by resistant mice at various times of infection: Sm25, an Mr 25,000 integral membrane protein of adult worms that was better recognized by resistant mice 40 to 50 days after exposure; Sm67, an Mr 67,000 water-soluble antigen of adult worms that was better recognized by resistant mice at days 30 to 40; Sm120, an Mr 120,000 antigen expressed by cercariae and adult worms that was differentially recognized, although inconsistently, at days 20 to 40 postexposure; and Sm26, an Mr 26,000 glutathione S-transferase that was uniquely recognized by resistant mice at day 20 in two of three experiments. Analysis of antibody specificities in (BALB/c x WEHI 129/J)Fl x WEHI 129/J backcross mice indicated that high responsiveness to Sm25 at days 40 to 50 correlated with resistance. The candidacy of these four molecules as vaccines for schistosomiasis mansoni is discussed.
factors is suggested by the failure to detect any increased or accelerated anti-Sj26 immunoglobulin G antibody response in the resistant proportion of WEHI 129/J mice in the S. japonicum system (8). Here we present a detailed analysis of the protein Abinding serum immunoglobulin antibody response of the S. mansoni-resistant proportion of WEHI 129/J to S. mansoni antigens at various times of infection. Comparison with the antibody response of the susceptible proportion of WEHI 129/J and other permissive mouse strains has revealed four antigens that are better recognized by resistant mice at some stage of infection. Further, we attempt to correlate the inheritance of high responsiveness to these four antigens with the inheritance of resistance in crosses between WEHI 129/J and susceptible BALB/c mice.
In the search for a vaccine against schistosomes, one approach used to identify candidate vaccine antigens is to compare the immune responses of hosts differing in their susceptibility to infection (14). Although mice are generally susceptible to schistosomiasis (20, 21), 129/J mice bred at this institute (hereafter designated WEHI 129/J mice) are genetically resistant to chronic infection with either Schistosoma japonicum (10; M. D. Wright, W. U. Tiu, S. M. Wood, J. C. Walker, E. G. Garcia, and G. F. Mitchell, J. Parasitol., in press) or S. mansoni (25; Wright et al., in press). Resistance is manifest in at least 50% of WEHI 129/ J mice, the remaining proportion showing susceptibility to infection that is characteristic of other mouse strains. The mechanism(s) of resistance that operates in WEHI 129/J mice is not understood. Genetic analysis indicates that resistance is inherited recessively and is probably under the control of one gene (Wright et al., in press). Analysis of the antibody response in mice exposed to S. japonicum reveals a 26-kilodalton (kDa) antigen (Sj26), recognized well by WEHI 129/J mice but poorly by mice of the permissive strain BALB/c (15). Nucleotide sequence analysis of cDNA encoding Sj26 has shown this antigen to be a glutathione Stransferase (GST) (18). Both S. mansoni and S. japonicum have at least two GST isoenzymes of molecular weight 26,000 and 28,000 (Sj26, Sj28, Sm26, and Sm28) (8; W. U. Tiu, K. M. Davern, M. D. Wright, P. G. Board, and G. F. Mitchell, Parasite Immunol., in press). Vaccination with Sj26 can induce significant protection to S. japonicum infection in mice, although inconsistently (18; G. F. Mitchell, E. G. Garcia, K. M. Davem, W. U. Tiu, and D. B. Smith, Trans. R. Soc. Trop. Med. Hyg., in press). Similarly, Sm28 appears to be a host-protective antigen of S. mansoni (1, 2, 24). However, the relevance of the anti-GST immune response in the expression of resistance of WEHI 129/J mice has not been fully elucidated. The involvement of additional *
MATERIALS AND METHODS Mice. Male and female WEHI 129/J and (BALB/c x WEHI 129/J)F1 were produced in a specific-pathogen-free facility but maintained conventionally from 6 weeks of age. Additional 129/J mice (JAX 129/J) were purchased from Jackson Laboratory, Bar Harbor, Maine, and were maintained and bred under conventional conditions as were the backcross mice (BALB/c x WEHI 129/J)F1 x WEHI 129/J. Mice were 8 to 17 weeks old when first used for infection. Parasites and infection. The maintenance of the S. mansoni life cycle has been described in detail elsewhere (Wright et al., in press). Briefly, S. mansoni (Puerto Rican strain) was maintained in albino Biomphalaria glabrata and in mice. In experimental infections, 100 S. mansoni cercariae were applied to the shaved abdominal surface of anesthetized mice by a standard cover slip method (22). Worm burdens were determined at 50 to 60 days after exposure by portal perfusion and are expressed as arithmetic means ± standard errors of the means. Exposed mice were bled from the retroorbital plexus every 10 days, and the sera from individual mice were stored
Corresponding author. 2948
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S. MANSONI ANTIGENS RECOGNIZED BY RESISTANT MICE
at -70°C. Care was taken when pooling sera to ensure that similar worm burdens existed in the serum donors. Antigens. S. mansoni adult worm extract (AWE) was prepared by homogenizing S. mansoni worms on ice in T-NET buffer (15), centrifuging at 13,000 x g for 10 min, and retaining the supernatant. When AWE was prepared for use in Western blotting (immunoblotting), worms were homogenized instead in sample buffer (Tris hydrochloride buffer [pH 6.8] containing 3% [wt/vol] sodium dodecyl sulfate). S. mansoni GST was prepared from AWE by affinity chromatography on glutathione-conjugated agarose as previously described (18, 19). S. mansoni integral membrane proteins and aqueousphase proteins were partitioned using Triton X-114 phase separation as described elsewhere (5, 17). S. mansoni cercarial extract used for Western blotting was prepared by homogenizing pelleted cercariae in sample buffer, centrifuging at 13,000 x g for 10 min, and retaining the supernatant. Electrophoresis and autoradiography. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out under reducing conditions as described by Laemmli and Favre (13). Samples were suspended in sample buffer and electrophoresed on 7.5 or 13% acrylamide slab gels. For autoradiography of '25I-labeled materials, Agfa-Gevaert Curix RP2 X-ray film was used in combination with intensifier screens (Cronex Lightning-Plus, Du Pont Co.,
Wilmington, Del.). Western blotting. A 300-,u antigen preparation was electrophoresed on 13 or 7.5% polyacrylamide gels, and the separated proteins were transferred electrophoretically to nitrocellulose filters (6). Nitrocellulose filters were then blocked for at least 1 h in 5% skimmed milk powder (BLOTTO) in phosphate-buffered saline (pH 7.4). Strips were then cut and incubated overnight in 1/200 dilutions of infected mouse serum. Nitrocellulose strips were extensively washed in BLOTTO for at least 1 h with two further washes in phosphate-buffered saline before being probed with 125I-labeled protein A in BLOTTO for 1 h. Finally, strips were washed extensively in BLOTTO and phosphatebuffered saline before being prepared for autoradiography. Immunoprecipitation. Parasite antigen preparations were iodinated using the Bolton-Hunter reagent, (N-succinimidyl-3,4-hydroxy,5-[125I]iodophenyl)propionate (Amersham Searle Corp., Sydney, Australia), as previously described (4, 15). Radiolabeled parasite proteins were then precleared by incubation with protein A-bearing Staphylococcus aureus Cowan 1 strain; Commonwealth Serum Laboratories, Parkville, Australia) at 4°C to remove proteins binding nonspecifically. When AWE was used, samples containing 106 cpm in 100 RIl were incubated with 5 ,ul of mouse serum at 4°C for at least 1 h. Antigen-antibody complexes were precipitated by the addition of 100 pul of 10% protein A-bearing S. aureus. These were then washed three times in T-NET buffer before electrophoretic analysis. When radiolabeled GST was immunoprecipitated, only 105 cpm per serum sample was used. In this case, immunoprecipitates were washed in T-NET buffer containing 0.1% SDS.
974
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43
Sera from WEHI 129/J, (BALB/c x WEHI 129/J)F1, and JAX 129/J mice were collected at various times after exposure to 100 cercariae per mouse, pooled, and subjected to immunoblot analysis. WEHI 129/J sera were divided into
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two pools: resistant mice (with no worms detected by portal perfusion at day 50) and susceptible mice (with detectable worms). Previous studies have shown F1 and JAX 129/J mice to be entirely permissive to chronic S. mansoni infection (Wright et al., in press). There were two major antigens clearly recognized well by the resistant proportion of WEHI 129/J, a 25-kDa species (Sm25) and a 67-kDa species (Sm67) (Fig. 1). Sm25 was first recognized by all strains at day 30; by day 40, the resistant WEHI 129/J mice recognized this antigen with greater intensity, and this better recognition was accentuated by day 50. Sm67 was well recognized at day 30 only by the resistant proportion of WEHI 129/J mice. At day 40, Sm67 was also seen by the susceptible WEHI 129/J mice and to a lesser extent by the JAX 129/J and F1 mice. Analysis of high-molecular-weight antigens by immunoblotting (Fig. 2) revealed an additional antigen recognized by resistant WEHI 129/J of 120 kDa (Sm120). Sm120 was seen solely by resistant mice at day 20, and this reactivity appeared to decrease between days 30 and 50 after exposure to cercariae. B
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FIG. 2. Western blot analysis of sera from mice exposed to S. mansoni against AWE separated on SDS-PAGE (7.5% polyacrylamide gel) 10 (A), 20 (B), 30 (C), 40 (D), and 50 (E) days postexposure. Serum sources (lanes): 1, resistant WEHI 129/J mice; 2, susceptible WEHI 129/J mice; 3, (BALB/c x WEHI 129/J)F1 mice; 4, JAX 129/J mice.
2950
WRIGHT ET AL. A
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FIG. 3. Western blot analysis of sera from mice exposed to S. mansoni against S. mansoni Triton X-114-soluble (TX-114) and water-soluble (Aq) proteins 10 (A), 20 (B), 30 (C), 40 (D), and 50 (E) days postexposure. Serum sources (lanes): 1, resistant WEHI 129/J mice; 2, susceptible WEHI 129/J mice; 3, (BALB/c x WEHI 129/ J)F1 mice; 4, JAX 129/J mice.
To further characterize these antigens, the same serum pools were used to probe blotted Triton X-114 and aqueousphase S. mansoni antigen fractions (Fig. 3). Sm25 is a putative integral membrane protein, because it clearly partitioned into the hydrophobic detergent fraction. By contrast, Sm67 is an aqueous-phase (hydrophilic) antigen. The minor antigen Sml20 was not identified in either of these fractions and may therefore be a structural antigen located in the detergent-insoluble material. Sml20 is recognized by resistant mice relatively early in infection (day 20), which suggests that this antigen may be expressed in younger schistosomes. This was confirmed by immunoblot analysis of cercarial extract probed with day 10 and 20 infected-mouse sera, which showed a 120-kDa antigen clearly recognized at day 20 by resistant mice (data not shown). Immunoprecipitation analysis in the S. japonicum-WEHI 129/J system has previously implicated the schistosome GSTs as antigens whose recognition may have relevance in the resistance of this mouse strain (15, 18). However, the immunoblot analyses in the S. mansoni system showed no reactivity of any infected-mouse serum against molecules with Mrs corresponding to that of GST, i.e., 26,000 or 28,000 (Fig. 1, 2, and 3). One explanation for the failure to detect anti-GST reactivity is that the antigenicity of these molecules is destroyed or reduced by the blotting procedure. To examine whether any anti-GST activity could be detected by alternative approaches, iodinated AWE was analyzed by immunoprecipitation with mouse sera. The better recognition of Sm25 and Sm67 by sera from resistant WEHI 129/J
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FIG. 4. Immunoprecipitation and SDS-PAGE analysis (13% polyacrylamide gel) of Bolton-Hunter reagent-labeled GST reacted with sera from mice exposed to S. mansoni 20 (A), 30 (B), 40 (C), and 50 (D) days postexposure. Serum sources (lanes): 1, resistant WEHI 129/J mice; 2, susceptible WEHI 129/J mice; 3, (BALB/c x WEHI 129/J)Fl mice; 4, JAX 129/J mice; 5, unexposed WEHI 129/ J mice.
mice on day 30, 40, or 50, although not as striking as that in immunoblots, was confirmed by immunoprecipitation. However, in contrast to the immunoblotting data, the sera precipitated bands at 26 and 28 kDa, presumably GST. To confirm that these 26- and 28-kDa antigens were indeed GST, radiolabeled GST purified from adult worms was immunoprecipitated (Fig. 4). Clearly, there was anti-GST antibody activity in serum, and Sm26 was recognized solely by resistant mice at day 20. At day 30, JAX 129/J and susceptible WEHI 129/J mice also recognized Sm26, whereas the anti-Sm26 response in F1 mice was delayed until day 40. No significant anti-Sm28 activity could be detected until day 40, when F1 mice recognized this antigen. At day 50, JAX 129/J mice had also become high responders to Sm28, whereas both WEHI 129/J serum pools remained relatively low in anti-Sm28 antibody levels. Other studies have indicated that chronically infected WEHI 129/J mice can recognize Sm28 well, so the low anti-Sm28 response demonstrated in these particular WEHI 129/J mice is not consistent (Tiu et al., in press). Further studies examining the responsiveness of mice to Sm28 have shown inconsistency in the response of infected individuals to this antigen. Only two of eight JAX 129/J mice, two of four WEHI 129/J mice, and one of four BALB/c mice had significant antiSm28 responses at day 50 after exposure (data not shown). In contrast, the response to Sm26 was far more consistent with the exception of BALB/c mice, which had previously been shown to be low responders to Sm26 (Tiu et al., in press). One potentially powerful approach to identifying antigens whose recognition is relevant in the expression of resistance is to correlate the inheritance of resistance with the inheritance of high responsiveness to these antigens (9). Previous work has established that approximately one-quarter of (BALB/c x WEHI 129/J)F1 x WEHI 129/J backcross mice inherit the resistant phenotype with respect to S. mansoni
S. MANSONI ANTIGENS RECOGNIZED BY RESISTANT MICE
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FIG. 5. Western blot analysis of sera from S. mansoni-exposed mice against AWE separated by SDS-PAGE (13% polyacrylamide gel) 30 (A), 40 (B), and 50 (C) days postexposure. Serum sources (lanes): 1, resistant WEHI 129/J mice; 2, susceptible WEHI 129/J mice; 3, (BALB/c x WEHI 129/J)F1 mice; 4, resistant (BALB/c x WEHI 129/J)F1 x WEHI 129/J backcross mice (i.e., these mice had no worms when perfused at day 50); 5, partially resistant backcross mice (4.4 0.5 worms per mouse); 6, susceptible backcross mice (20.5 1.4 worms per mouse). ±
±
infection (Wright et al., in press). It is therefore of interest to determine whether the resistant proportion of backcross mice has inherited any increased or accelerated response against the four differentially recognized antigens identified above. Sera from WEHI 129/J, F1, and backcross mice were collected at various times after exposure and subjected to immunoblot analysis (Fig. 5). Backcross mouse sera were divided into three pools: resistant mice (no worms), partially resistant mice (4.4 + 0.5 worms per mouse), and susceptible 1.4 worms per mouse). WEHI 129/J mouse mice (20.5 serum was pooled into resistant and susceptible groups as described previously. High responsiveness to Sm25 at days 40 and 50 correlated strongly with resistance, as shown by the relative reactivities of the resistant mice (Fig. 5, lanes 1, 4, and 5) compared with the susceptible mice (Fig. 5, lanes 2, 3, and 6). No such correlation exists for Sm67, since resistant and susceptible mice reacted equally with this antigen in this experiment. Similarly, analysis of day 20 sera showed no correlation between resistance and responsiveness to either Sm26 or Sml20 (data not shown). ±
DISCUSSION The results presented in this paper identify four S. mansoni antigens which at various times of infection are better recognized by resistant WEHI 129/J mouse sera: Sm25, Sm26, Sm67, and Sml20. In assessing the likely relevance of immune responses against these antigens to the expression of resistance in WEHI 129/J mice, it is essential to consider at what time in infection WEHI 129/J mice eliminate their parasites. Preliminary work indicates that S. mansoni worms are killed at around day 20 after exposure (El Saghier, McLaren, and Knopf, personal communication), data that are compatible with previous work in the S. japonicumWEHI 129/J system suggesting that attrition occurs in the postlung, pre-egg-laying stage (8, 10). Therefore, immune responses examined at day 20 are likely to have the greatest relevance to the expression of resistance in WEHI 129/J mice. Antigens that are differentially recognized by resistant mice at later time points may represent internal antigens released by dead and dying worms that are not normally
2951
accessible to the immune system of permissive mice. Alternatively, low sensitivity of assays at early time points versus later times in the course of infection may not allow relevant specificities to be detected early. Moreover, early relevant T-cell reactivities may express as antibody specificities later. There are two antigens that are better recognized by resistant mice at day 20: Sm26, a GST; and Sml20, an antigen expressed both by adilt worms and cercariae (Fig. 2 and 4). The unique appearance of anti-Sm26 antibodies in resistant WEHI 129/J mice at day 20 (two different pools being used in this analysis) is of particular interest. However, we could find no accelerated response to Sm26 in the resistant proportion of backcross mice; in this particular experiment, resistant WEHI 129/J serum at day 20 also did not recognize Sm26 (data not shown). Sj26, the homologous molecule in S. japonicum, is also differentially recognized by S. japonicum-exposed WEHI 129/J mice (15) as well as giving variable but nontheless significant protection on occasions in vaccinated mice (18; Mitchell et al., in press). However, in contrast to the data presented here, previous studies have failed to demonstrate any increased or accelerated response to Sj26 in the resistant proportion of WEHI 129/J mice (8). Vaccination with the other S. mansoni GST isoenzyme, Sm28, has been shown to be protective in a variety of hosts (1, 2, 24). However, protein A-binding antibodies to Sm28 appear to have no relevance to the expression of resistance in the WEHI 129/J model because responsiveness to Sm28 at the time points examined is inconsistent and does not correlate with resistance in the WEHI 129/J mouse (Fig. 4). Furthermore, anti-Sm28 antibodies are not detectable until day 40, some 20 days after presumed parasite attrition (Fig. 4). The possible relevance of responsiveness to Sml20 in the expression of resistance is supported by the fact that resistant and not susceptible mice recognize this antigen on immunoblots at day 20, the proposed time of parasite attrition (Fig. 2). However, this antigen has not been differentially recognized in all immunoblot experiments performed, nor is it differentially recognized by immunoprecipitation
analysis. There are two antigens differentially recognized by resistant mice at time points later than the presumed time of parasite attrition: Sm25 and Sm67, which are both major immunogens. Sm25 is not differentially recognized by resistant WEHI 129/J mice until day 40 (Fig. 1 and 2). High responsiveness to Sm25 at day 40 or 50 strongly correlates with the resistant phenotype in backcross analysis (Fig. 5).
Interestingly, studies of S. mansoni-infected humans showed a correlation between resistance and responsiveness to a 24.5-kDa antigen (7). As previously shown, Sm25 partitions into the Triton X-114-soluble phase and thus is a putative integral membrane protein (17) (Fig. 3). Integral membrane proteins interact directly with the hydrophobic core of a lipid bilayer (23), so there is a possibility that epitopes of this molecule are exposed on the schistosome surface and vulnerable to immune attack. There is evidence that a 25-kDa antigen is indeed present in the tegumental membrane of both 5-day-old lung worms and adult worms (16). Certainly Sm25 is worthy of further investigation with regard to its vaccine potential. Sm67 partitions into the aqueous phase after Trixon X-114 phase separation and thus is a hydrophilic protein. It is first recognized by resistant mice at day 30; however, by day 50 there is no apparent difference in responsiveness of resistant and susceptible mice to this molecule (Fig. 1 and 2). It is possible that this molecule corresponds to the S. mansoni
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WRIGHT ET AL.
heat shock protein of 70 kDa as characterized and cloned by Hedstrom et al. (11), since both molecules are major immunogens. The relationship of Sm67 to a recently described 68-kDa protective antigen (12) remains unknown. Further work will attempt to isolate these differentially recognized antigens from a parasite cDNA library by using sera eluted from appropriate regions of immunoblots (3). We have, as yet, no proof that any of these four antigens is responsible for the expression of resistance in WEHI 129/J mice. The only definitive test is whether these antigens can protect appropriately vaccinated mice from chronic S. mansoni infection. ACKNOWLEDGMENTS This work was supported by the Australian National Health and Medical Research Council and the Rockefeller Foundation Great Neglected Diseases Network. We thank Anthea Baker, Susan Wood, and Karen McLeod for superb technical assistance. LITERATURE CITED 1. Balloul, J. M., J. M. Grzych, R. J. Pierce, and A. Capron. 1987. A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis. J. Immunol. 138:3448-3453. 2. Balloul, J. M., P. Sondermeyer, D. Dreyer, M. Capron, J. M. Grzych, R. J. Pierce, D. Carvallo, J. P. Lecocq, and A. Capron. 1987. Molecular cloning of a protective antigen of schistosomes. Nature (London) 326:149-153. 3. Beall, J. A., and G. F. Mitchell. 1986. Identification of a particular antigen from a parasite cDNA library using antibodies affinity purified from selected portions of Western blots. J. Immunol. Methods. 86:217-223. 4. Bolton, A. E., and W. M. Hunter. 1973. The labelling of proteins to high specific radioactivities by conjugation to a 125I containing acylating agent. Application to the radioimmunoassay. Biochem. J. 133:529-539. 5. Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256:1604-1607. 6. Burnette, W. N. 1981. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and protein A. Anal. Biochem. 112:195-205. 7. Butterworth, A. E., M. Capron, J. S. Cordingley, P. R. Dalton, D. W. Dunne, H. C. Kariuki, G. Kimani, D. Koech, M. Mugambi, J. H. Ouma, M. A. Prentice, B. A. Richardson, T. K. Arap Siongok, R. F. Sturrock, and D. W. Taylor. 1985. Immunity after treatment of human schistosomiasis mansoni. II. Identification of resistant individuals and analysis of their immune responses. Trans. R. Soc. Trop. Med. Hyg. 79:393-408. 8. Davern, K. M., W. U. Tiu, G. Morahan, M. D. Wright, E. G. Garcia, and G. F. Mitchell. 1987. Responses in mice to Sj26, a glutathione S-transferase of Schistosoma japonicum worms. Immunol. Cell Biol. 65:473-482. 9. Erlich, J. H., R. F. Anders, I. C. Roberts-Thomson, J. W. Schrader, and G. F. Mitchell. 1983. An examination of differences in serum antibody specificities and hypersensitivity reactions as contributing factors to chronic infection with the intestinal protozoan parasite, Giardia muris, in mice. Aust. J. Exp. Biol. Med. Sci. 61:599-615.
INFECT. IMMUN. 10. Garcia, E. G., W. U. Tiu, and G. F. Mitchell. 1983. Innate resistance to Schistosoma japonicum in a proportion of 129/J mice. J. Parasitol. 69:613-615. 11. Hedstrom, R., J. Culpepper, R. A. Harrison, N. Agabian, and G. Newport. 1987. A major immunogen in Schistosoma mansoni infections is homologous to the heat-shock protein Hsp-70. J. Exp. Med. 165:1430-1435. 12. King, C. H., R. A. Lett, J. Nanduri, S. El-Ibiary, P. A. S. Peters, G. R. Olds, and A. A. F. Mahmoud. 1987. Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni. J. Immunol. 139:4218-4224. 13. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:
575-599. 14. Mitchell, G. F. 1985. Exploitation of genetically-based variation in host and parasite in the development of parasite vaccines, p. 431-439. In E. Skamene (ed.), Progress in leukocyte biology series. Alan R. Liss Inc., New York. 15. Mitchell, G. F., J. Beall, K. M. Cruise, W. U. Tiu, and E. G. Garcia. 1985. Antibody responses to the antigen Sj26 of Schistosoma japonicum worms that is recognized by genetically resistant 129/J mice. Parasite Immunol. 7:165-178. 16. Payares, G., D. J. McLaren, W. H. Evans, and S. R. Smithers. 1985. Antigenicity and immunogenicity of the tegumental outer membrane of adult Schistosoma mansoni. Parasite Immunol. 7: 43-61. 17. Rogers, M. V., K. M. Davern, J. A. Smythe, and G. F. Mitchell. 1988. Immunoblotting analysis of the major integral membrane protein antigens of Schistosoma japonicum. Mol. Biochem. Parasitol. 29:77-88. 18. Smith, D. B., K. M. Davern, P. G. Board, W. U. Tiu, E. G. Garcia, and G. F. Mitchell. 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 83:8703-8707. 19. Smith, D. B., M. R. Rubira, R. J. Simpson, K. M. Davern, W. U. Tiu, P. G. Board, and G. F. Mitchell. 1988. Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase. Mol. Biochem. Parasitol. 27:249-256. 20. Smithers, S. R., and M. J. Doenhoff. 1982. Schistosomiasis, p. 527-607. In S. Cohen and K. S. Warren (ed.), Immunology of parasitic infections. Blackwell Scientific Publications, Oxford. 21. Smithers, S. R., A. J. G. Simpson, X. Yi, P. Omer-Ali, C. Kelly, and D. J. McLaren. 1987. The mouse model of schistosome immunity. Acta Trop. 44(Suppl.):12-30. 22. Smithers, S. R., and R. J. Terry. 1965. Infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55:695-700. 23. Tanford, C., and J. A. Reynolds. 1976. Characterization of membrane proteins in detergent solutions. Biochim. Biophys. Acta 457:133-170. 24. Taylor, J. B., A. Vidal, G. Torpler, D. J. Meyer, C. Roitsch, J. M. Balloul, C. Southaw, P. Sondermeyer, S. Pemble, J. P. Lecocq, A. Capron, and B. Ketterer. 1988. The glutathione transferase activity and tissue distribution of a cloned Mr 28k protective antigen of Schistosoma mansoni. EMBO J. 7:465472. 25. Tiu, W. U., J. Ehl, J. C. Walker, D. B. Smith, and G. F. Mitchell. 1986. Resistance of 129/J mice to Schistosoma mansoni. Aust. J. Exp. Biol. Med. Sci. 64:345-472.
