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secreted antigens of Toxoplasma gondii — their origin and role in the host-parasite interaction
THE IMMUNOBIOLOG
Y OF TOXOPLASMOSIS
Excreted/secreted antigens of Toxoplasma gondii their origin and role in the host-parasite interaction M.F.
Cesbron-Delauw
and A. Capron
Centre d’lmmunologie et de Biologie Parasitaire, Unit& Mixte INSERM U167-CNRS 624, Institut Pasteur, 1, rue du Prof. Calmette, 59019 Lille Cedex (France)
Introduction Recently, efforts have been concentrated on developing a vaccine against toxoplasmosis, the feasibility of which is supported by the long-lasting immunity induced by the primary infection. A vaccine against toxoplasmosis is expected to limit the dissemination of the tachyzoite proliferative stage in order to prevent transplacental contamination of the foetus. Moreover, since, during acute toxoplasmosis, tachyzoites are found either as extracellular invading forms or as intracellular replicative forms, the rationale for such a vaccine had to focus on the killing of both. Although these forms exhibit the same antigenic components, their different “ecological niche” implies that different antigens have to be targeted to induce the appropriate effector mechanisms. Because of their direct accessibility to the immune system, surface antigens probably represent the candidate targets for the killing of the extracellular parasites. Indeed, mAb to the major surface antigen of tachyzoites (P30) lyse the parasite in the presence of complement and are protective by passive transfer. Also, immunization with the purified P30 antigen has recently been reported to protect mice against fatal toxoplasmosis (Biilow and Boothroyd, 1991; and see Kasper’s paper in this Forum). Another range of antigenic structures that have to be evaluated as target antigens are the so-called excreted-secreted antigens (ESA). These products, released as soluble forms by parasites, were first evaluated for diagnostic purposes as powerful indicators of an active infection (circulating antigens). More recently, they have gained growing interest for their role in pathogenesis, immune escape and in immunity. Our interest in ESA as target antigens against toxoplasmosis is based on the working hypothesis that the definitive protection against reinfection ob-
served in natural infection is due to a mechanism of concomitant immunity resulting from the presence of the encysted bradyzoite form in host tissues for a lifetime. The antigens released by the encysted parasites, through repeated stimulation of the immune system, would maintain an immune response against the invading tachyzoite forms. The molecular basis of such immunity implies that common epitopes are expressed by the invading and the encysted parasite and are presented to the immune system as soluble circulating antigens/ESA. Since there is cumulating evidence for the role of soluble antigens of T. gondii in generating cytotoxic T cells (Hakim et al., 1991), the ESA appear to be the candidate targets for limiting the intracellular growth of the tachyzoite.
1) Origin and molecular
characterization
of ESA
There is some controversy regarding the composition of circulating antigens, and a number of hypotheses about their formation in vivo have been proposed. The antigenaemia might result from: a) immune lysis of parasite; b) active secretion by tachyzoites and release into the bloodstream ; c) shedding of membrane components ; or d) a combination of the above. However, the observations that circulating antigens are detected one or two days following infection and that their concentration within the serum is in excess of that expected by parasite lysis strongly suggests that they originate from an active secretory process (Hughes, 1981). The first organelles which were described in active secretory processes of T. gondii were the rhoptries, located at the anterior pole of the parasite (Nichols et al., 1983). They have been shown to contain factor(s) which enhance the infectivity of the parasites both in vivo and in vitro (Lycke and Norby, 1966; Norby, 1971; Schwarzman et al., 1985 ; Werk, 1985). The rhoptries are supposed to secrete
48th FORUM their content during penetration and to release proteolytic enzymes that will modify the host cell membrane. However, it seems unlikely that such materials may have a role as circulating antigens : the localized effect of the PEF (penetrating enhancing factor), the absence of PEF activity in acute sera, as well as the possible regulated secretion of the rhoptries imply that their content might not been discharged in sufficient quantities. More recently, Darcy et al. (1988) have developed a procedure for the obtaining of ESA from tachyzoites in cell-free incubation media containing 10 % of serum. This method has been proven to be a powerful tool in inducing the release of a range of products that have been shown to be highly immunogenic (Darcy et al., 1988; Decoster et al., 1988). These molecules have a subcellular location in agreement with a secretory process (Cesbron-Delauw et al., 1989 ; Charif et al., 1990) but which is distinct from the rhoptry organelles. Various approaches including mAb production, colloidal gold labelling and molecular cloning have led to the characterization of these ESA. These studies revealed that, in the absence of parasite lysis, one minor source of ESA is provided by the surface antigens. Indeed, these have been shown to be anchored in the membrane by a glycosylphosphatidylinositol (Tomavo et al., 1989). Their release as soluble forms after cleavage by a phospholipase is probably the way in which surface antigens are present in the ESA preparation. The other source of ESA was clearly demonstrated by immunocytological studies which revealed the storage of 3 ESA (GP28.5, P23, P21) in the dense granules of Toxoplasma cells (Cesbron-Delauw et al., 1989; Charif et al., 1990). They were further referred to as GRAl (P23), GRA2 (GP28.5) and GRAS (P21) following the nomenclature proposed by Sibley et al. (1991). Other recent studies have also described several proteins in the dense granules, the GRAI and GRA2 (Leriche et Dubremetz, 1990, 1991) and two additional ones, GR43 and GRA4 (Achbarou et al., 1991). The common feature of these antigens is that they are discharged from the dense granules within the parasitophorous vacuole after host-cell invasion (Sibley et al., 1988; Cesbron-Delauw et al., 1989). However, in this particular compartment, GRAl, GRA2 and GRA4 are found to be preferentially associated with the tubular network, whereas GRA3 and GRAS are associated with the vacuole membrane. The molecular structure of GRAl (CesbronDelauw et al., 1989), GRA2 (Mercier et al., in preparation) and GRAS (Lecordier et al., in preparation) has been deduced from cDNA sequences, and these are in agreement with their subcellular location by the presence of typical secretory signal peptide and/or
IN IiWWUNOLOGY membrane spanning domains. Nevertheless, their function has not yet been elucidated. Their contribution to the composition of the vacuolar space at the interface between the parasite and the host’s cell suggests a role in escape mechanisms leading to the intracellular growth of the parasite. Secondary structure analysis of the GRAl amino acid sequence reveals two domains whose structure resembles a calcium-binding site. This structure, called EF hand, is characterized by an alpha helix-loop-alpha helix structure. By Western blotting followed by incubation with calcium 45, the GRAl antigen was shown to bind the calcium (Cesbron-Delauw et al., 1989). Although the calcium-dependent regulation of the T. gondii membrane trafficking during exocytosis remains to be proven, a role for this calcium-binding protein in the packaging of the dense granules products might be considered.
2) Role of ESA in immunity
against toxoplasmosis
In toxoplasmosis, although ESA received little attention compared with the amount of research carried out on surface antigens, several early observations support their role in inducing protective immunity. Hughes and Van Knapen (1982) have demonstrated that actively secreted antigens represent 90 070of the circulating antigens detectable during early infection. These secreted antigens have been reported to induce a higher lymphocyte stimulation than do somatic antigens (Hughes, 1981). Since cellmediated mechanisms are recognized to be the major immune effector, such antigenic stimuli might be the basis for the development of a rapid immune response which would limit the intracellular growth of the tachyzoite forms. On the other hand, the role of ESA in inducing protective immunity is indirectly supported by the observed importance of using viable tachyzoites as antigens either for “in vitro generation” of human T-cell clones (Canessa et al., 1988) or to vaccinate, since vaccines using killed organisms have on the whole, been unsuccessful. The protective role of tachyzoite ESA has also been reproducibly demonstrated in two experimental models of lethal toxoplasmosis, mice and nude rats. Immunization of mice with ESA in the presence of Freund’s incomplete adjuvant led to a 70 070survival rate in the face of a lethal oral challenge with 1,200 cysts of the 76K strain (Darcy et al., 1992). Furthermore, the transfer of either sera or of immunocompetent cells from ESA immunized euthymic ( + / + ) rats led to a significant increase in survival of recipient nude rats receiving a lethal challenge with the virulent RH strain (Darcy et al., 1988; Duquesne et al., 1990). Interestingly, such a level of protection was not obtained when irradiated tachyzoites were used as antigens to immunize (+ /+) Fisher rats
THE IMMUNOBIOLOGY (Darcy et al., 1988). In nude rats passively transferred with ESA immune sera, one day before infection with 5 x lo4 tachyzoites of the RH strain, 40 % of survival was observed at day 90 post-infection, whereas control rats died between days 17 and 19 after infection (Darcy et al., 1988). The role of specific helper cells in inducing protection in nude rats has been shown by adoptive transfer of different doses of ESA-specific T lymphocytes propagated in vitro for one month (Duquesne et al., 1990); 70 070of the nude rats which were transferred by 1 x lo4 and 1 x 10’ ESA-specific T cells were protected against fatal infection and, moreover, against reinfection. In this model, such a level of protection can be obtained by passive transfer of a minimum of 5 x lo6 non-specific T lymphocytes. 3) Characterization of ESA involved in protection against toxoplasmosis The observed importance of ESA in inducing protection in animal models led us to identify the major component of a subunit vaccine, with particular emphasis on antigens shared between the infective tachyzoite and the encysted stage (Capron and Dessaint, 1988). On the basis of these criteria, two antigens GRAl (P23) and GRA2 (GP28.5) have been characterized at the molecular level and used to immunize mice. P23 ESA: GRAl P23 antigen, the calcium-binding protein described above, was initially selected because (i) it presents a peptidic epitope that is cross-reactive with a bradyzoite extract, and (ii) its detection by IgG in the sera of individuals is correlated with chronic infection (Cesbron-Delauw et al., 1989). The P23 antigen (GRAl) has been expressed in E. coli and in eukaryotic cells. The recombinant proteins were immunogenic in mice (producing anti-native P23 antibodies) and antigenic, since recognized by infected sera. In E. coli, the level of expression was very low due to a gradual degradation of rP23. Preliminary protection experiments in mice by immunization with the recombinant vaccinia virus led to a reduction of 50 Vo in the number of brain cysts. However, the obServed exacerbation of Toxoplasma pathogenicity in mice immunized with wild-type vaccinia virus did not enable optimization of protective experiments (M.F. Cesbron, unpublished results). Among the synthetic peptides derived from the sequence, two C-terminal peptides (170-193 and 194-208) were identified as T-cell epitopes, whilst the former is also a B-cell epitope recognized by a mouse TG17-43 mAb (Duquesne et al., 1991). By Western blotting using the
OF TOXOPLASMOSIS
43
TG17-43 mAb, it is possible to detect the P23 antigen in the sera of mice infected with RH strain, confirming its circulating antigen property (Charif, unpublished results).
GP28.5 ESA: GIL42 Success in using an HPLC method to purify native GP28.5 ESA (GRA2) enabled the demonstration of its potentiality as a defined subunit vaccine. Using FIA as adjuvant, GRA2 has been shown to protect 75 Vo of mice from a lethal challenge with 1,200 cysts of the 76K T. gondii strain (Mercier et al., in preparation). Serological studies using human sera and truncated recombinant GRA2 have recently shown that this antigen contains multiple B epitopes (Murray et al., in preparation) ; one of them, the C-terminal epitope, is recognized by the mouse TG17-179 mAb and is probably linear (CesbronDelauw et al., 1992). On the basis of partial cDNA sequences (Cesbron-Delauw et al., 1992) and subcellular distribution, the GP28.5 ESA described in our laboratory appears to be identical to the protective antigen of 28 kDa described using the F3G3 mAb (Sharma et al., 1984; Sibley and Sharma, 1985) and cloned by Prince et al. (1989).
Concluding
remarks
The major conclusion of these studies is that the dense granules are organelles in which a range of highly immunogenic products are stored. These products, which are secreted by both intracellular and extracellular parasites, might provide one of the major sources of the circulating antigens. Although significant progress has been done in the characterization of these dense granule components at the molecular and the subcellular level, little is known about their function in the intracellular growth of the parasite. However, their observed immunogeneity in several experimental models suggests that they might be candidates for a subunit vaccine. One of these, the GRA2 antigen, has been shown to protect mice against a lethal challenge. In the search for such a protective component, it is remarkable that two different approaches, one based on the selection of mAb which were capable of inducing protection by passive transfer (Sharma et al., 1984) and the second based on the identification of ESA expressed both by the tachyzoite and the bradyzoite, have led to the identification of the same protective GRA2 antigen. The immunological mechanisms by which these secreted antigens induce protection will require further investigations. Since CD8+ T lymphocytes appear to be the major effecters of immunity (see Denkers et al., this Forum), presentation of ESA in
48th FOR Uh4 IN IMkfUNOLOG association with MHC class I would be investigated as well. The particular traffic of antigens released by the dense granules suggests that they may be a candidate for presentation to CTL, via processing within infected cells and/or exogenous uptake. References Achbarou, A., Mercereau-Puijalon, 0.. Sadak, A., Fortier, B., Leriche, M-A., Camus, D. & Dubremetz, J.F. (1991), Differential targetting of dense granule proteins in the parasitophorous vacuole of Toxoplasma gondii. Parasitology, 103, 321-329. Btilow, R. & Boothroyd, J.C. (1991), Protection of mice from fatal Toxoplasma gondii infection by immunization with P30 antigen in liposomes. J. Immunol., 147, 3496-3500. Canessa, A., Pistoia, V., Roncella, S., Merli, A., Melioli, G., Terragna, A. & Ferrarini (1988). An in vitro model for Toxoplasma infection in interaction between CD4+ monoclonal T cells and macrophages results in killing of trophozoites. J. Immunol., 140, 3580-3588. Capron, A. & Dessaint, J.P. (1988), Vaccination against parasitic diseases: some alternative concept for the definition of protective antigens. Ann. Inst. Pasteur/ImmunoI., 137, 109-117. Cesbron-Delauw, M.F., Guy, B., Tot-pier, G., Pierce, R. J., Lenzen, G., Cesbron, J.Y ., Charif, H., Lepage, P., Darcy, F., Lecocq, J.P. & Capron, A. (1989), Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii. Proc. nat. Acad. Sci. (Wash.), 86, 7537-7541. Cesbron-Delauw, M.F., Boutillon, C., Mercier, C., Fourmaux, M.P., Murray, A., Miquey, F., Tartar, A. & Capron, A. (1992), Amino acid sequencerequirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28.5 of Toxoplasma gondii. Mol. Immunol. (in press). Charif, H., Darcy, F., Torpier, G., Cesbron-Delauw, M.F. & Capron, A. (1990), Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites. Exp. Parasit., 71, 114-121. Darcy, F., Deslee, D., Santoro, F., Charif, H., Auriault, C., Decoster, A., Duquesne, V. & Capron, A. (1988), Induction of a protective antibody-dependent response against toxoplasmosis by in vitro excreted/secreted antigens from tachyzoites of Toxoplasma gondii. Parasite Immunol., 10, 553-567. Decoster, A., Darcy, F. & Capron, A. (1988), Recognition of Toxoplasma gondii excreted and secreted antigens by human sera from acquired and congenital toxoplasmosis : identification of markers of acute and chronic infection. Clin. exp. Immunol,, 73,375-382. Duquesne, V., Auriault, C., Darcy, F., Decavel, J.P. & Capron, A. (1990), Protection of nude rats against Toxoplasma infection by excreted-secreted antigenspecific helper T cells. Infect. Immun., 58,2120-2126. Duquesne, V., Auriault, C., Gras-Masse, H., Boutillon, C., Darcy, F., Cesbron-Delauw, M.F.. Tartar, A. &
Y
Capron, A. (1991), Identification of T-cell epitopes within a 23 kD antigen (P24) of Toxoplasma gondii. Clin. exp. Immunol., 84, 527-534. Hakim, F.T., Gazzinelli, R.T., Denkers, E., Hieny, S., Shearer, G.M. & Sher, A. (1991), CD8+ T cell from mice vaccinated against Toxoplasma gondii on cytotoxic for parasite-infected or antigen pulsed host cells. J. Immunol., 147, 2310-2316. Hughes, H.P.A. (1981), Characterization of the circulating antigen of T. gondii. Immunol. Letters, 3, 99-101. Hughes, H.P.A. &Van Knapen, F. (1982), Characterization of a secretory antigen from Toxoplasma gondii and its role in circulating antigen production. ht. J. Parasit., 12, 433-437. Leriche, M.A. & Dubremetz, J.F.’ (1990), Exocytosis of dense granules after host-cell invasion by Toxoplasma gondii. Parasit. Res., 76, 559-562. Leriche, M.A. & Dubremetz, J.F. (1991), Characterization of the proteins of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies. Mol. Biochem. Parasit., 45, 249-260. Lycke, E. 8~Norrby, R. (1966), Demonstration of a factor of Toxoplasma gondii enhancing the penetration of Toxoplasma parasites into cultured host ceils. Brit. J. exp. Path., 41, 248-256. Nichols, B.A., Chiappino, M.L. & O’Connor, G.R. (1983), Secretionfrom the rhoptries of Toxoplasmagondii during host cell invasion. J. Ultrastruct. Res., 83, 85-98. Norrby, R. (1971), Immunological study on the host cell penetration factor of Toxoplasma gondii. Infect. Immun., 3, 278-286. Prince, J.B., Araujo, F.G., Remington, J.S., Burg, J.L., Boothroyd, J.C. & Sharma, S.D. (1989), Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii. Mol. Biochem. Parasit., 34, 3-14. Schwartzman, J.D. (1986), Inhibition of penetrationenhancing factor by Toxoplasma gondii by monoclonal antibodies specific for rhoptries. Infect. Immun., 51, 760-764. Sharma, S.D., Araujo, F.G. Kc Remington, J.S. (1984), Toxoplasma antigen isolated by affinity chromatography with monoclonal antibody protects mice against lethal infection with Toxoplasma gondii. J. Immunol., 133, 2818-2820. Sibley, L.D. & Krahenbuhl, J.L. (1988), Modification of host cell phagosomes by Toxoplasma gondii involves redistribution of surface proteins and secretion of a 32 kDa protein. Europ. J. Cell Biol., 41, 81-87. Sibley, L.D. & Sharma, SD. (1987), Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice. Infect. Immun., 55, 2137-2141. Sibley, L.D., Pfefferkorn, E.R. & Boothroyd, J.C. (1991), Proposal for a uniform genetic nomenclature in Toxoplasma gondii. Parasit. Today, 7, 327-328. Tomavo, S., Schwarz, R.T. & Dubremetz, J.F. (1989), Evidence for glycosyl-phosphatidyl-inositol anchoring of Toxoplasma gondii major surface antigens. Mol. Cell. Biol., 9, 4576-4580. Werk, R. (1985), How does Toxoplasma gondii enter host ceils? Rev. infect. Dis., I, 449-457.
Y OF TOXOPLASMOSIS
Excreted/secreted antigens of Toxoplasma gondii their origin and role in the host-parasite interaction M.F.
Cesbron-Delauw
and A. Capron
Centre d’lmmunologie et de Biologie Parasitaire, Unit& Mixte INSERM U167-CNRS 624, Institut Pasteur, 1, rue du Prof. Calmette, 59019 Lille Cedex (France)
Introduction Recently, efforts have been concentrated on developing a vaccine against toxoplasmosis, the feasibility of which is supported by the long-lasting immunity induced by the primary infection. A vaccine against toxoplasmosis is expected to limit the dissemination of the tachyzoite proliferative stage in order to prevent transplacental contamination of the foetus. Moreover, since, during acute toxoplasmosis, tachyzoites are found either as extracellular invading forms or as intracellular replicative forms, the rationale for such a vaccine had to focus on the killing of both. Although these forms exhibit the same antigenic components, their different “ecological niche” implies that different antigens have to be targeted to induce the appropriate effector mechanisms. Because of their direct accessibility to the immune system, surface antigens probably represent the candidate targets for the killing of the extracellular parasites. Indeed, mAb to the major surface antigen of tachyzoites (P30) lyse the parasite in the presence of complement and are protective by passive transfer. Also, immunization with the purified P30 antigen has recently been reported to protect mice against fatal toxoplasmosis (Biilow and Boothroyd, 1991; and see Kasper’s paper in this Forum). Another range of antigenic structures that have to be evaluated as target antigens are the so-called excreted-secreted antigens (ESA). These products, released as soluble forms by parasites, were first evaluated for diagnostic purposes as powerful indicators of an active infection (circulating antigens). More recently, they have gained growing interest for their role in pathogenesis, immune escape and in immunity. Our interest in ESA as target antigens against toxoplasmosis is based on the working hypothesis that the definitive protection against reinfection ob-
served in natural infection is due to a mechanism of concomitant immunity resulting from the presence of the encysted bradyzoite form in host tissues for a lifetime. The antigens released by the encysted parasites, through repeated stimulation of the immune system, would maintain an immune response against the invading tachyzoite forms. The molecular basis of such immunity implies that common epitopes are expressed by the invading and the encysted parasite and are presented to the immune system as soluble circulating antigens/ESA. Since there is cumulating evidence for the role of soluble antigens of T. gondii in generating cytotoxic T cells (Hakim et al., 1991), the ESA appear to be the candidate targets for limiting the intracellular growth of the tachyzoite.
1) Origin and molecular
characterization
of ESA
There is some controversy regarding the composition of circulating antigens, and a number of hypotheses about their formation in vivo have been proposed. The antigenaemia might result from: a) immune lysis of parasite; b) active secretion by tachyzoites and release into the bloodstream ; c) shedding of membrane components ; or d) a combination of the above. However, the observations that circulating antigens are detected one or two days following infection and that their concentration within the serum is in excess of that expected by parasite lysis strongly suggests that they originate from an active secretory process (Hughes, 1981). The first organelles which were described in active secretory processes of T. gondii were the rhoptries, located at the anterior pole of the parasite (Nichols et al., 1983). They have been shown to contain factor(s) which enhance the infectivity of the parasites both in vivo and in vitro (Lycke and Norby, 1966; Norby, 1971; Schwarzman et al., 1985 ; Werk, 1985). The rhoptries are supposed to secrete
48th FORUM their content during penetration and to release proteolytic enzymes that will modify the host cell membrane. However, it seems unlikely that such materials may have a role as circulating antigens : the localized effect of the PEF (penetrating enhancing factor), the absence of PEF activity in acute sera, as well as the possible regulated secretion of the rhoptries imply that their content might not been discharged in sufficient quantities. More recently, Darcy et al. (1988) have developed a procedure for the obtaining of ESA from tachyzoites in cell-free incubation media containing 10 % of serum. This method has been proven to be a powerful tool in inducing the release of a range of products that have been shown to be highly immunogenic (Darcy et al., 1988; Decoster et al., 1988). These molecules have a subcellular location in agreement with a secretory process (Cesbron-Delauw et al., 1989 ; Charif et al., 1990) but which is distinct from the rhoptry organelles. Various approaches including mAb production, colloidal gold labelling and molecular cloning have led to the characterization of these ESA. These studies revealed that, in the absence of parasite lysis, one minor source of ESA is provided by the surface antigens. Indeed, these have been shown to be anchored in the membrane by a glycosylphosphatidylinositol (Tomavo et al., 1989). Their release as soluble forms after cleavage by a phospholipase is probably the way in which surface antigens are present in the ESA preparation. The other source of ESA was clearly demonstrated by immunocytological studies which revealed the storage of 3 ESA (GP28.5, P23, P21) in the dense granules of Toxoplasma cells (Cesbron-Delauw et al., 1989; Charif et al., 1990). They were further referred to as GRAl (P23), GRA2 (GP28.5) and GRAS (P21) following the nomenclature proposed by Sibley et al. (1991). Other recent studies have also described several proteins in the dense granules, the GRAI and GRA2 (Leriche et Dubremetz, 1990, 1991) and two additional ones, GR43 and GRA4 (Achbarou et al., 1991). The common feature of these antigens is that they are discharged from the dense granules within the parasitophorous vacuole after host-cell invasion (Sibley et al., 1988; Cesbron-Delauw et al., 1989). However, in this particular compartment, GRAl, GRA2 and GRA4 are found to be preferentially associated with the tubular network, whereas GRA3 and GRAS are associated with the vacuole membrane. The molecular structure of GRAl (CesbronDelauw et al., 1989), GRA2 (Mercier et al., in preparation) and GRAS (Lecordier et al., in preparation) has been deduced from cDNA sequences, and these are in agreement with their subcellular location by the presence of typical secretory signal peptide and/or
IN IiWWUNOLOGY membrane spanning domains. Nevertheless, their function has not yet been elucidated. Their contribution to the composition of the vacuolar space at the interface between the parasite and the host’s cell suggests a role in escape mechanisms leading to the intracellular growth of the parasite. Secondary structure analysis of the GRAl amino acid sequence reveals two domains whose structure resembles a calcium-binding site. This structure, called EF hand, is characterized by an alpha helix-loop-alpha helix structure. By Western blotting followed by incubation with calcium 45, the GRAl antigen was shown to bind the calcium (Cesbron-Delauw et al., 1989). Although the calcium-dependent regulation of the T. gondii membrane trafficking during exocytosis remains to be proven, a role for this calcium-binding protein in the packaging of the dense granules products might be considered.
2) Role of ESA in immunity
against toxoplasmosis
In toxoplasmosis, although ESA received little attention compared with the amount of research carried out on surface antigens, several early observations support their role in inducing protective immunity. Hughes and Van Knapen (1982) have demonstrated that actively secreted antigens represent 90 070of the circulating antigens detectable during early infection. These secreted antigens have been reported to induce a higher lymphocyte stimulation than do somatic antigens (Hughes, 1981). Since cellmediated mechanisms are recognized to be the major immune effector, such antigenic stimuli might be the basis for the development of a rapid immune response which would limit the intracellular growth of the tachyzoite forms. On the other hand, the role of ESA in inducing protective immunity is indirectly supported by the observed importance of using viable tachyzoites as antigens either for “in vitro generation” of human T-cell clones (Canessa et al., 1988) or to vaccinate, since vaccines using killed organisms have on the whole, been unsuccessful. The protective role of tachyzoite ESA has also been reproducibly demonstrated in two experimental models of lethal toxoplasmosis, mice and nude rats. Immunization of mice with ESA in the presence of Freund’s incomplete adjuvant led to a 70 070survival rate in the face of a lethal oral challenge with 1,200 cysts of the 76K strain (Darcy et al., 1992). Furthermore, the transfer of either sera or of immunocompetent cells from ESA immunized euthymic ( + / + ) rats led to a significant increase in survival of recipient nude rats receiving a lethal challenge with the virulent RH strain (Darcy et al., 1988; Duquesne et al., 1990). Interestingly, such a level of protection was not obtained when irradiated tachyzoites were used as antigens to immunize (+ /+) Fisher rats
THE IMMUNOBIOLOGY (Darcy et al., 1988). In nude rats passively transferred with ESA immune sera, one day before infection with 5 x lo4 tachyzoites of the RH strain, 40 % of survival was observed at day 90 post-infection, whereas control rats died between days 17 and 19 after infection (Darcy et al., 1988). The role of specific helper cells in inducing protection in nude rats has been shown by adoptive transfer of different doses of ESA-specific T lymphocytes propagated in vitro for one month (Duquesne et al., 1990); 70 070of the nude rats which were transferred by 1 x lo4 and 1 x 10’ ESA-specific T cells were protected against fatal infection and, moreover, against reinfection. In this model, such a level of protection can be obtained by passive transfer of a minimum of 5 x lo6 non-specific T lymphocytes. 3) Characterization of ESA involved in protection against toxoplasmosis The observed importance of ESA in inducing protection in animal models led us to identify the major component of a subunit vaccine, with particular emphasis on antigens shared between the infective tachyzoite and the encysted stage (Capron and Dessaint, 1988). On the basis of these criteria, two antigens GRAl (P23) and GRA2 (GP28.5) have been characterized at the molecular level and used to immunize mice. P23 ESA: GRAl P23 antigen, the calcium-binding protein described above, was initially selected because (i) it presents a peptidic epitope that is cross-reactive with a bradyzoite extract, and (ii) its detection by IgG in the sera of individuals is correlated with chronic infection (Cesbron-Delauw et al., 1989). The P23 antigen (GRAl) has been expressed in E. coli and in eukaryotic cells. The recombinant proteins were immunogenic in mice (producing anti-native P23 antibodies) and antigenic, since recognized by infected sera. In E. coli, the level of expression was very low due to a gradual degradation of rP23. Preliminary protection experiments in mice by immunization with the recombinant vaccinia virus led to a reduction of 50 Vo in the number of brain cysts. However, the obServed exacerbation of Toxoplasma pathogenicity in mice immunized with wild-type vaccinia virus did not enable optimization of protective experiments (M.F. Cesbron, unpublished results). Among the synthetic peptides derived from the sequence, two C-terminal peptides (170-193 and 194-208) were identified as T-cell epitopes, whilst the former is also a B-cell epitope recognized by a mouse TG17-43 mAb (Duquesne et al., 1991). By Western blotting using the
OF TOXOPLASMOSIS
43
TG17-43 mAb, it is possible to detect the P23 antigen in the sera of mice infected with RH strain, confirming its circulating antigen property (Charif, unpublished results).
GP28.5 ESA: GIL42 Success in using an HPLC method to purify native GP28.5 ESA (GRA2) enabled the demonstration of its potentiality as a defined subunit vaccine. Using FIA as adjuvant, GRA2 has been shown to protect 75 Vo of mice from a lethal challenge with 1,200 cysts of the 76K T. gondii strain (Mercier et al., in preparation). Serological studies using human sera and truncated recombinant GRA2 have recently shown that this antigen contains multiple B epitopes (Murray et al., in preparation) ; one of them, the C-terminal epitope, is recognized by the mouse TG17-179 mAb and is probably linear (CesbronDelauw et al., 1992). On the basis of partial cDNA sequences (Cesbron-Delauw et al., 1992) and subcellular distribution, the GP28.5 ESA described in our laboratory appears to be identical to the protective antigen of 28 kDa described using the F3G3 mAb (Sharma et al., 1984; Sibley and Sharma, 1985) and cloned by Prince et al. (1989).
Concluding
remarks
The major conclusion of these studies is that the dense granules are organelles in which a range of highly immunogenic products are stored. These products, which are secreted by both intracellular and extracellular parasites, might provide one of the major sources of the circulating antigens. Although significant progress has been done in the characterization of these dense granule components at the molecular and the subcellular level, little is known about their function in the intracellular growth of the parasite. However, their observed immunogeneity in several experimental models suggests that they might be candidates for a subunit vaccine. One of these, the GRA2 antigen, has been shown to protect mice against a lethal challenge. In the search for such a protective component, it is remarkable that two different approaches, one based on the selection of mAb which were capable of inducing protection by passive transfer (Sharma et al., 1984) and the second based on the identification of ESA expressed both by the tachyzoite and the bradyzoite, have led to the identification of the same protective GRA2 antigen. The immunological mechanisms by which these secreted antigens induce protection will require further investigations. Since CD8+ T lymphocytes appear to be the major effecters of immunity (see Denkers et al., this Forum), presentation of ESA in
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