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Application of cloning techniques to development of a synthetic vaccine against schistosomiasis
Veterinary Parasitology, 10 (1982) 255--259 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
255
APPLICATION OF CLONING TECHNIQUES TO DEVELOPMENT OF A SYNTHETIC VACCINE AGAINST SCHISTOSOMIASIS
PAUL M. KNOPF, BRAHM S. SRIVASTAVA and ROBERT H. BARKER, Jr. Division o f Biology and Medicine, Brown University, Providence, R I 02912 (U.S.A.)
ABSTRACT Knopf, P.M., Srivastava, B.S. and Barker, R.H., Jr., 1982. Application of cloning techniques to development of a synthetic vaccine against schistosomiasis. Vet. Parasitol., 10:255--259 Techniques for identification of protection-inducing and target antigens of Schistosoma mansoni are described. The potential for use of these antigens in developing a synthetic vaccine by application of monoclonal antibody and recombinant DNA technologies is presented.
INTRODUCTION
Analysis of resistance to reinfection in laboratory models of human schistosomiasis has led to identification of stages of the parasite either sensitive to or capable of inducing a protective immune response (Knopf and Cioli, 1980; Mangold and Knopf, 1981). As a next step in vaccine development, large scale production of the pertinent life cycle stage of the parasite for mass immunization, may n o t be feasible. Thus, identification and isolation of the relevant protection-sensitive ("target") antigens or protection-inducing ("protective") antigens (not necessarily identical; possibly produced by different life cycle stages) are required, along with development of immunization protocols using these antigens. With the recent applications of cell hybridization and recombinant DNA technologies for producing specific proteins, there are reasons to be optimistic about the prospects for generating sufficient quantities of critical immunogenic proteins. The following conditions must be satisfied in order to apply these techniques: (1) Passive transfer of resistance with sera from resistant animals, leading to identification of host protective antibody. (2) Identification of target and/or protective antigens using host protective antibody in an immunoassay. (3) Demonstration that these critical antigens are proteins (or glycoproteins). (4) Identification of parasite stage synthesizing these functional proteins. If conditions (1) and (2) are satisfied, it may be possible to produce mono-
0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
256
clonal antibody useful as a specific reagent for antigen isolation and identification. If conditions (3) and (4) are also satisfied, recombinant DNA technology may be applicable. Variations of these strategies are conceivable and are no less important in revealing the mechanisms by which parasitic organisms escape immune elimination or induce their pathogenic reactions. However, this presentation focuses upon applications relevant to vaccine production. MATERIALS AND METHODS
Passive transfer of resistance Reduced yields of parasites at different stages of the life cycle, or reduced pathogenesis, are criteria used to demonstrate the existence or induction of resistance to a challenge infection. Establishing an immunological mechanism to account for these observations is essential to distinguish antigen-specific, from nonspecific, reactions. The classical approach to this problem has been a demonstration of the transfer of protection with serum from resistant animals, ultimately with an immunoglobulin (Ig) fraction of the serum. Passive immunization with serum Ig both establishes the existence of a specific resistance mechanism and provides a m e t h o d o l o g y for antigen identification. Passive transfer of resistance to a cercarial challenge, with serum or the IgG fraction, has been demonstrated most reproducibly in the rat--Schistosoma mansoni model (Phillips et al., 1977; Mangold and Knopf, 1981). There are examples of passive transfer of specific resistance with lymphocytes (cell-mediated immunity) (Phillips et al., 1975). Combinations of cells and serum have also been required to achieve resistance transfer in certain instances (Maddison and Kagan, 1979). Failure to transfer resistance with serum alone has also been attributed to a requirement for combinations of t w o different Ig classes, e.g., IgG (high concentration in serum) and IgE (insufficient concentration in serum) (Phillips and Colley, 1978). Finally the levels of accessory factors in normal recipients of serum from resistant animals may account for a failure to demonstrate an antibody-mediated immune protection reaction, e.g., eosinophils have been implicated (Butterworth, 1977). If protection is mediated b y cellular (or a combination of cellular and humoral) immune mechanisms, the techniques for detecting the relevant antigens are more complex.
Antigen identification Once antibody which reacts with target antigen is available (anti-target Ab), antigen identification is possible. These antigens m a y be present on the surface of the parasite which is the target stage, or they may be released parasite products which serve some functional role in parasite survival (e.g., enzymes, mitogens, anti-coagulation factors, anti-complement factors, etc.).
257 Furthermore, nontarget stages of the parasite may prove to be a better source of these antigens. Adult schistosomes elaborate products which induce resistance to reinfection (Smithers and Terry, 1967; Knopf and Cioli, 1980). The protective antibody (identified as the effector molecules) eliminates immature parasites, but not adult worms ("concomitant" immunity). I have termed these protection-inducing molecules as protective antigens. Antigen identification strategies are dependent upon the particular parasite being studied. One method involves absorption of the protective serum, containing anti-target Ab, with different life cycle stages of the parasite and measuring changes in the protective titer of the serum. Employing this strategy in our studies, we have revealed the existence of two distinguishable activities in protective serum from S. mansoni-resistant rats following its absorption with the schistosomula stage of the parasite. One activity is responsible for protection while the other appears to interfere with the protective activity (B.S. Srivastava, R.H. Barker and P.M. Knopf, manuscript in preparation). Both activities can be attributed to antibodies in the serum. Another strategy employs antigen-labeling methods. Extrinsic radiolabeling of surface macromolecules and biosynthetic radiolabeling techniques have been used (Brink, 1977; Hayunga et al., 1979). In my laboratory, we are using biosynthetic radiolabeling of protein antigens of different schistosome life cycle stages (cercariae, schistosomula, 4-week-old worms). Soluble, radiolabeled proteins released by freeze--thaw or detergent lysis are subjected to immunoprecipitation with either protective or nonprotective rat antischistosome sera. We have identified a subset of protein antigens uniquely precipitated by antibody in protective rat serum and are currently developing procedures for isolating these antigens in sufficient quantities for immunization studies (R.H. Barker, B.S. Srivastava and P.M. Knopf, manuscript in preparation).
Monoclonal antibody technology Even before target or protective antigens have been identified, a collection of monoclonal antibody producing cells may be generated. One method for achieving this is accomplished by hybridization of myeloma cells (in continuous culture) with antibody-producing cells from immunized donor animal (Kohler and Milstein, 1976). The limitation is the development of a highly effective selection technique for identifying continuously growing cells producing the desired antibody. The stability of antibody production by clones derived from the selection procedure appears to be a matter of chance at the present time. Monoclonal anti-schistosome antibodies have been produced in several laboratories, with the exciting claim that certain of these monoclonal antibodies are capable of protecting mice against schistosome infections. Protective anti-sporozoite monoclonal antibody has been documented in rodent malaria (Yoshida et al., 1980).
258
Recombinant DNA technology If the target or protective antigens are proteins, recombinant DNA technology may be applied for generating plasmid-converted bacteria, hopefully capable of producing this parasite protein. These techniques are being applied to solve mass production problems of human hormones (insulin, growth hormone) and human interferon (Denniston and Enquist, 1981). The production of a viral antigen capable of inducing protective immunity has been reported and establishes the validity of this approach to vaccine production (Kupper et al., 1981). The goal of this procedure is to develop ("engineer") a plasmid vector into which has been inserted the double-stranded DNA coding, partially or completely, for amino acid sequences of the target or protective antigen(s). A variety of approaches for accomplishing this goal are available (Denniston and Enquist, 1981). The antigen-coding DNA sequences are inserted in the vicinity of a promoter site for m R N A transcription and the sequences are embedded between the appropriate start/stop signals for translation of the genetic information into polypeptide chains. This recombinant plasmid vector also contains genetic information for initiation of DNA replication and drug resistance genes. Incorporation of this recombinant plasmid into a suitable bacterial host (drug sensitive) is followed by growth of these bacteria in a drug containing medium. The resultant drug resistant bacteria are plated and colonies tested for production of antigen, utilizing monoclonal Ab. If successful, a source of the target or protective antigen is available. If the antigen proves to be a glycoprotein and the polysaccharide is essential for antigenicity, it may be possible to glycosylate the protein in vitro with appropriate enzyme systems. There are other critical problems, e.g., that the coding information of the parasite template is in phase and correctly translated, and that the protein is stable within the bacteria. The protein may only contain a portion of the antigenic determinants of the target or protective antigen, depending upon the composition of the parasite DNA inserted into the plasmid. Some of these technical problems may be solved using recombinant plasmids for transformation of eukaryote cells (e.g., yeast, cultured mammalian cells).
Immunization protocol The assumption that proteins of the parasite will be sufficient for induction of protection is essential to this approach. Some of the experiments required to satisfy this assumption must certainly proceed before the investment in recombinant DNA technology be undertaken. There are several strategies which can be attempted without the availability of large quantities of purified protein. It may prove necessary to immunize with several proteins to achieve sufficient protection. Inclusion of safe adjuvants or incorporation of protein into liposomes may be required to improve immuno-
259
genicity of the proteins. The cellular products of the recombinant DNA plasmid, if not precisely identical to the parasite antigen, would have to be tested to ascertain their efficacy. ACKNOWLEDGEMENT
Supported by a research grant from the Edna McConnell Clark Foundation.
REFERENCES Brink, L.H., 1977. Schistosoma mansoni: Characterization and comparison of the surface of developing schistosomula and adult worms. Ph.D. Thesis, University of London, London School of Hygiene and Tropical Medicine, pp. 148--205. Butterworth, A.E., 1977. The eosinophil and its role in immunity to helminth infection. In: W. Arber, W. Henle, P.H. Hofschneider, J.H. Humphrey, J. Klein, P. Koldovsky, H. Koprowski, D. Maal6e, F. Melchers, R. Rott, H.G. Schweiger, L. Syru~ek and P.K. Vogt (Editors), Current Topics in Microbiology and Immunology. Vol. 77, SpringerVerlag, Berlin/Heidelberg, pp. 127--168. Denniston, K.J. and Enquist, L.W., (Editors) 1981. Recombinant DNA. In: Benchmark Papers in Microbiology. Vol. 15, Academic Press, NY, 400 pp. Hayunga, E.G., Murrell, K.D., Taylor, D.W. and Vannier, W.E., 1979. Isolation and characterization of surface antigens from S c h i s t o s o m a mansoni. Parasitology, 65: 488--506. Knopf, P.M. and Cioli, D., 1980. S c h i s t o s o m a mansoni: resistance to an infection with cercariae induced by the transfer of live adult worms to the rat. Int. J. Parasitol., 10: 13--19. Kohler, G. and Milstein, C., 1976. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol., 6: 511--519. Kupper, H., Keller, W., Kurz, C., Forss, S., Schaller, H., Franze, R., Strohmaier, K., Marquardt, O., Zaslavsky, V.G. and Hofschneider, P.H., 1981. Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli. Nature, 289: 555-559. Maddison, S.E. and Kagan, I.G., 1979. Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse. J. Parasitol., 65: 515--519. Marigold, B.L. and Knopf, P.M., 1981. Host protective immune responses to S c h i s t o s o m a mansoni infections in the rat. I. Kinetics of hyperimmune serum-dependent sensitivity and elimination of schistosomes in a passive transfer assay. Parasitology, 83: 559--574. Phillips, S.M. and Colley, D.G., 1978. Immunological aspects of host responses to schistosomiasis: resistance, immunopathology and eosinophil involvement. Prog. Allergy, 24: 49--182. Phillips, S.M., Reid, W.A., Bruce, J.I., Hedlund, K., Colvin, R.C., Campbell, R., Diggs, C.L. and Sadun, E.H., 1975. The cellular and humoral immune response to S c h i s t o s o m a mansoni infection in inbred rat. I. Mechanism during initial exposure. Cell. Immunol., 19: 99--116. Phillips, S.M., Reid, W.A. and Sadun, E.H., 1977. The cellular and humoral immune response to S c h i s t o s o m a mansoni infections in inbred rats. II. Mechanisms during reexposure. Cell. Immunol., 28: 75--89. Smithers, S.R. and Terry, R.J., 1967. Resistance to experimental infection with S. mansoni in rhesus monkeys induced by the transfer of adult worms. Trans. R. Soc. Trop. Med. Hyg., 61: 517--533. Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V. and Aikawa, M., 1980. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science, 207: 71--73.
255
APPLICATION OF CLONING TECHNIQUES TO DEVELOPMENT OF A SYNTHETIC VACCINE AGAINST SCHISTOSOMIASIS
PAUL M. KNOPF, BRAHM S. SRIVASTAVA and ROBERT H. BARKER, Jr. Division o f Biology and Medicine, Brown University, Providence, R I 02912 (U.S.A.)
ABSTRACT Knopf, P.M., Srivastava, B.S. and Barker, R.H., Jr., 1982. Application of cloning techniques to development of a synthetic vaccine against schistosomiasis. Vet. Parasitol., 10:255--259 Techniques for identification of protection-inducing and target antigens of Schistosoma mansoni are described. The potential for use of these antigens in developing a synthetic vaccine by application of monoclonal antibody and recombinant DNA technologies is presented.
INTRODUCTION
Analysis of resistance to reinfection in laboratory models of human schistosomiasis has led to identification of stages of the parasite either sensitive to or capable of inducing a protective immune response (Knopf and Cioli, 1980; Mangold and Knopf, 1981). As a next step in vaccine development, large scale production of the pertinent life cycle stage of the parasite for mass immunization, may n o t be feasible. Thus, identification and isolation of the relevant protection-sensitive ("target") antigens or protection-inducing ("protective") antigens (not necessarily identical; possibly produced by different life cycle stages) are required, along with development of immunization protocols using these antigens. With the recent applications of cell hybridization and recombinant DNA technologies for producing specific proteins, there are reasons to be optimistic about the prospects for generating sufficient quantities of critical immunogenic proteins. The following conditions must be satisfied in order to apply these techniques: (1) Passive transfer of resistance with sera from resistant animals, leading to identification of host protective antibody. (2) Identification of target and/or protective antigens using host protective antibody in an immunoassay. (3) Demonstration that these critical antigens are proteins (or glycoproteins). (4) Identification of parasite stage synthesizing these functional proteins. If conditions (1) and (2) are satisfied, it may be possible to produce mono-
0304-4017/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
256
clonal antibody useful as a specific reagent for antigen isolation and identification. If conditions (3) and (4) are also satisfied, recombinant DNA technology may be applicable. Variations of these strategies are conceivable and are no less important in revealing the mechanisms by which parasitic organisms escape immune elimination or induce their pathogenic reactions. However, this presentation focuses upon applications relevant to vaccine production. MATERIALS AND METHODS
Passive transfer of resistance Reduced yields of parasites at different stages of the life cycle, or reduced pathogenesis, are criteria used to demonstrate the existence or induction of resistance to a challenge infection. Establishing an immunological mechanism to account for these observations is essential to distinguish antigen-specific, from nonspecific, reactions. The classical approach to this problem has been a demonstration of the transfer of protection with serum from resistant animals, ultimately with an immunoglobulin (Ig) fraction of the serum. Passive immunization with serum Ig both establishes the existence of a specific resistance mechanism and provides a m e t h o d o l o g y for antigen identification. Passive transfer of resistance to a cercarial challenge, with serum or the IgG fraction, has been demonstrated most reproducibly in the rat--Schistosoma mansoni model (Phillips et al., 1977; Mangold and Knopf, 1981). There are examples of passive transfer of specific resistance with lymphocytes (cell-mediated immunity) (Phillips et al., 1975). Combinations of cells and serum have also been required to achieve resistance transfer in certain instances (Maddison and Kagan, 1979). Failure to transfer resistance with serum alone has also been attributed to a requirement for combinations of t w o different Ig classes, e.g., IgG (high concentration in serum) and IgE (insufficient concentration in serum) (Phillips and Colley, 1978). Finally the levels of accessory factors in normal recipients of serum from resistant animals may account for a failure to demonstrate an antibody-mediated immune protection reaction, e.g., eosinophils have been implicated (Butterworth, 1977). If protection is mediated b y cellular (or a combination of cellular and humoral) immune mechanisms, the techniques for detecting the relevant antigens are more complex.
Antigen identification Once antibody which reacts with target antigen is available (anti-target Ab), antigen identification is possible. These antigens m a y be present on the surface of the parasite which is the target stage, or they may be released parasite products which serve some functional role in parasite survival (e.g., enzymes, mitogens, anti-coagulation factors, anti-complement factors, etc.).
257 Furthermore, nontarget stages of the parasite may prove to be a better source of these antigens. Adult schistosomes elaborate products which induce resistance to reinfection (Smithers and Terry, 1967; Knopf and Cioli, 1980). The protective antibody (identified as the effector molecules) eliminates immature parasites, but not adult worms ("concomitant" immunity). I have termed these protection-inducing molecules as protective antigens. Antigen identification strategies are dependent upon the particular parasite being studied. One method involves absorption of the protective serum, containing anti-target Ab, with different life cycle stages of the parasite and measuring changes in the protective titer of the serum. Employing this strategy in our studies, we have revealed the existence of two distinguishable activities in protective serum from S. mansoni-resistant rats following its absorption with the schistosomula stage of the parasite. One activity is responsible for protection while the other appears to interfere with the protective activity (B.S. Srivastava, R.H. Barker and P.M. Knopf, manuscript in preparation). Both activities can be attributed to antibodies in the serum. Another strategy employs antigen-labeling methods. Extrinsic radiolabeling of surface macromolecules and biosynthetic radiolabeling techniques have been used (Brink, 1977; Hayunga et al., 1979). In my laboratory, we are using biosynthetic radiolabeling of protein antigens of different schistosome life cycle stages (cercariae, schistosomula, 4-week-old worms). Soluble, radiolabeled proteins released by freeze--thaw or detergent lysis are subjected to immunoprecipitation with either protective or nonprotective rat antischistosome sera. We have identified a subset of protein antigens uniquely precipitated by antibody in protective rat serum and are currently developing procedures for isolating these antigens in sufficient quantities for immunization studies (R.H. Barker, B.S. Srivastava and P.M. Knopf, manuscript in preparation).
Monoclonal antibody technology Even before target or protective antigens have been identified, a collection of monoclonal antibody producing cells may be generated. One method for achieving this is accomplished by hybridization of myeloma cells (in continuous culture) with antibody-producing cells from immunized donor animal (Kohler and Milstein, 1976). The limitation is the development of a highly effective selection technique for identifying continuously growing cells producing the desired antibody. The stability of antibody production by clones derived from the selection procedure appears to be a matter of chance at the present time. Monoclonal anti-schistosome antibodies have been produced in several laboratories, with the exciting claim that certain of these monoclonal antibodies are capable of protecting mice against schistosome infections. Protective anti-sporozoite monoclonal antibody has been documented in rodent malaria (Yoshida et al., 1980).
258
Recombinant DNA technology If the target or protective antigens are proteins, recombinant DNA technology may be applied for generating plasmid-converted bacteria, hopefully capable of producing this parasite protein. These techniques are being applied to solve mass production problems of human hormones (insulin, growth hormone) and human interferon (Denniston and Enquist, 1981). The production of a viral antigen capable of inducing protective immunity has been reported and establishes the validity of this approach to vaccine production (Kupper et al., 1981). The goal of this procedure is to develop ("engineer") a plasmid vector into which has been inserted the double-stranded DNA coding, partially or completely, for amino acid sequences of the target or protective antigen(s). A variety of approaches for accomplishing this goal are available (Denniston and Enquist, 1981). The antigen-coding DNA sequences are inserted in the vicinity of a promoter site for m R N A transcription and the sequences are embedded between the appropriate start/stop signals for translation of the genetic information into polypeptide chains. This recombinant plasmid vector also contains genetic information for initiation of DNA replication and drug resistance genes. Incorporation of this recombinant plasmid into a suitable bacterial host (drug sensitive) is followed by growth of these bacteria in a drug containing medium. The resultant drug resistant bacteria are plated and colonies tested for production of antigen, utilizing monoclonal Ab. If successful, a source of the target or protective antigen is available. If the antigen proves to be a glycoprotein and the polysaccharide is essential for antigenicity, it may be possible to glycosylate the protein in vitro with appropriate enzyme systems. There are other critical problems, e.g., that the coding information of the parasite template is in phase and correctly translated, and that the protein is stable within the bacteria. The protein may only contain a portion of the antigenic determinants of the target or protective antigen, depending upon the composition of the parasite DNA inserted into the plasmid. Some of these technical problems may be solved using recombinant plasmids for transformation of eukaryote cells (e.g., yeast, cultured mammalian cells).
Immunization protocol The assumption that proteins of the parasite will be sufficient for induction of protection is essential to this approach. Some of the experiments required to satisfy this assumption must certainly proceed before the investment in recombinant DNA technology be undertaken. There are several strategies which can be attempted without the availability of large quantities of purified protein. It may prove necessary to immunize with several proteins to achieve sufficient protection. Inclusion of safe adjuvants or incorporation of protein into liposomes may be required to improve immuno-
259
genicity of the proteins. The cellular products of the recombinant DNA plasmid, if not precisely identical to the parasite antigen, would have to be tested to ascertain their efficacy. ACKNOWLEDGEMENT
Supported by a research grant from the Edna McConnell Clark Foundation.
REFERENCES Brink, L.H., 1977. Schistosoma mansoni: Characterization and comparison of the surface of developing schistosomula and adult worms. Ph.D. Thesis, University of London, London School of Hygiene and Tropical Medicine, pp. 148--205. Butterworth, A.E., 1977. The eosinophil and its role in immunity to helminth infection. In: W. Arber, W. Henle, P.H. Hofschneider, J.H. Humphrey, J. Klein, P. Koldovsky, H. Koprowski, D. Maal6e, F. Melchers, R. Rott, H.G. Schweiger, L. Syru~ek and P.K. Vogt (Editors), Current Topics in Microbiology and Immunology. Vol. 77, SpringerVerlag, Berlin/Heidelberg, pp. 127--168. Denniston, K.J. and Enquist, L.W., (Editors) 1981. Recombinant DNA. In: Benchmark Papers in Microbiology. Vol. 15, Academic Press, NY, 400 pp. Hayunga, E.G., Murrell, K.D., Taylor, D.W. and Vannier, W.E., 1979. Isolation and characterization of surface antigens from S c h i s t o s o m a mansoni. Parasitology, 65: 488--506. Knopf, P.M. and Cioli, D., 1980. S c h i s t o s o m a mansoni: resistance to an infection with cercariae induced by the transfer of live adult worms to the rat. Int. J. Parasitol., 10: 13--19. Kohler, G. and Milstein, C., 1976. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol., 6: 511--519. Kupper, H., Keller, W., Kurz, C., Forss, S., Schaller, H., Franze, R., Strohmaier, K., Marquardt, O., Zaslavsky, V.G. and Hofschneider, P.H., 1981. Cloning of cDNA of major antigen of foot and mouth disease virus and expression in E. coli. Nature, 289: 555-559. Maddison, S.E. and Kagan, I.G., 1979. Adoptive transfer of protective immunity in experimental schistosomiasis in the mouse. J. Parasitol., 65: 515--519. Marigold, B.L. and Knopf, P.M., 1981. Host protective immune responses to S c h i s t o s o m a mansoni infections in the rat. I. Kinetics of hyperimmune serum-dependent sensitivity and elimination of schistosomes in a passive transfer assay. Parasitology, 83: 559--574. Phillips, S.M. and Colley, D.G., 1978. Immunological aspects of host responses to schistosomiasis: resistance, immunopathology and eosinophil involvement. Prog. Allergy, 24: 49--182. Phillips, S.M., Reid, W.A., Bruce, J.I., Hedlund, K., Colvin, R.C., Campbell, R., Diggs, C.L. and Sadun, E.H., 1975. The cellular and humoral immune response to S c h i s t o s o m a mansoni infection in inbred rat. I. Mechanism during initial exposure. Cell. Immunol., 19: 99--116. Phillips, S.M., Reid, W.A. and Sadun, E.H., 1977. The cellular and humoral immune response to S c h i s t o s o m a mansoni infections in inbred rats. II. Mechanisms during reexposure. Cell. Immunol., 28: 75--89. Smithers, S.R. and Terry, R.J., 1967. Resistance to experimental infection with S. mansoni in rhesus monkeys induced by the transfer of adult worms. Trans. R. Soc. Trop. Med. Hyg., 61: 517--533. Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V. and Aikawa, M., 1980. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science, 207: 71--73.