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Efforts towards a vaccine against Toxoplasma gondii: A review
Zbl. Bakt. Hyg. A 269, 423-436 (1988)
Efforts Towards a Vaccine Against Toxoplasma gondii: A Review KURT HERMENTIN and HORST ASPOCK Abteilung fur Medizinische Parasitologie (Leiter: Univ. Prof. Dr. H. Aspock) des HygieneInstituts der Universitat Wien (Vorstand: Univ. Prof. Dr. H. Flamm)
Received April 22, 1988 . Accepted July 5, 1988
Abstract In a review, past as well as present investigations carried out towards a vaccine against toxoplasmosis are outlined. A historical retrospect of the various immunization experiments is given, recent research projects intending the characterization of antigens that are relevant to host protective immunity are described, and a prospect to future problems and developments expected in the field is drafted.
Zusammenfassung Die Arbeit gibt einen Uberblick iiber die bisher durchgefiihrten und derzeit laufenden Bemiihungen urn die Herstellung einer Vakzine gegen Toxoplasma gondii. Die verschiedenen Ansatze zur Immunisierung gegen den Parasiten werden in einem historischen Riickblick besprochen, gegenwiirtigeForschungsbemiihungen zur Auffindung einzelner Antigene, die eine protektive Immunantwort bewirken sollen, beschrieben, sowie zukiinftige absehbare Probleme und Entwicklungen dargestellt.
1. Introduction Undoubtedly, prophylactic vaccines have to be considered as one of the principal goals of research in immunoparasitology. While vaccines are successfully applied in the control of many viral and bacterial diseases, this success, however, could not be emulated in the battle against parasitic infections. This is due partly to the more complex structures and antigens of these organisms and partly to the fact that many parasites have evolved ways of either living with the immune response of the host or even escaping it. Principal parasitic defense mechanisms are based (a) on the variation of the parasitic antigens, (b) on the concealment of antigens from the host immune-system, e.g. by formation of cysts or by uptake of host antigens (molecular masking), (c) on the immune modulation or immunosuppression of the host response and (d) on mechanisms of antibody clearance (e.g. capping of amoebae). 28 Zbl. Bakt. Hyg. A 269/4
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Among the parasites, the protozoon Toxoplasma gondii deserves special attention as an infection contracted during pregnancy can cause abortion, severe fetal abnormalities, or neonatal death in humans as well as in domestic animals (6), hence making a prophylactic vaccine highly desirable. Efforts towards a vaccine against toxoplasmosis have recently gained further support with the appearance of AIDS and related cerebral complications due to Toxoplasma gondii. The present paper endeavors to give a historical retrospect of the problem, to review recent research projects towards a vaccine against toxoplasmosis, and to outline future problems and developments to be expected in this field. Before sketching the major results of immunization and vaccination experiments, some general statements on the goals and requirements of a vaccine against toxoplasmosis shall be put at the beginning, to elucidate the aspects under which the present review has been made: - First of all, a vaccine to control toxoplasmosis should prevent infection of the unborn during pregnancy, thus excluding fetal disease. - A vaccine against toxoplasmosis would also be beneficial for HIV-positive individuals who are Toxoplasma-seronegative. AIDSpatients once infected with Toxoplasma gondii frequently develop a severe cerebral affection. Therefore, it would be advantageous to prevent HIV-positive individuals from getting infected with this parasite. A vaccine designed merely to protect against disease, however, would not be sufficient. An effective vaccine has to protect against infection, that means it has to prevent Toxoplasma parasites from invading the host and settling in his tissue. Whether such a vaccine will ever become available remains doubtful; presumably it would have to induce immunity at the first barrier, i.e. the mucosal immune system of the gut, and would necessitate a complete protective effect already at this site. - A third goal of a vaccine against toxoplasmosis is the prevention of oocyst excretion in the domestic cat. Such a vaccine would considerably reduce the transmission rate of the parasite and eliminate one of the two major ways of transmission to man. Since it is much more practicable to avoid uptake of raw meat than the ingestion of oocysts dispersed in the environment, it would also facilitate prophylactic measures against toxoplasmosis during pregnancy. - A prospective vaccine against toxoplasmosis, furthermore, has to fulfill the criteria of safeness, guaranteed longterm protection, cost-effectiveness and easy delivery.
2. Historical Review Since 1928, when Levadity and coworkers set up their first immunization experiments against toxoplasmosis with heat-inactivated Toxoplasma parasites (60), a wide variety of immunization and vaccination experiments have been carried out. Many different vaccines including non-virulent strains, attenuated strains, killed parasites, homogenates, soluble extracts, heterologous antigens, and non-specific antigens have been used. In many cases, the experiments were primarily designed to evolve an immune-response that usually was judged by the detection of specific antibodies only; the question of protection was placed into the background. This is contrary to today's research, where the major goal is the identification of antigens that are relevant to protective immunity of the host.
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2.1. Live vaccines Numerous immunization experiments were carried out with live, low-virulent or "non-virulent" strains of Toxoplasma gondii (47, 57, 67, 76, 82, 106). Such vaccines convey the most effective protection against a virulent challenge strain but, strictly speaking, they cause a genuine infection. It cannot be excluded that a vaccine-induced infection may recrudesce and cause multiple lesions, e.g. when the host becomes immunosuppressed or suffers from immunodeficiency. Moreover, the possibility of a parasite-immanent change of virulence has to be taken into account. Thus, such vaccines cannot be applied in human medicine, but may gain some interest in the veterinary field.
2.2. Immunization with killed parasites, homogenates or soluble extracts Vaccines consisting of killed Toxoplasma parasites, homogenates or soluble extracts are far less immunogenic than live ones. They combine two major disadvantages, namely the need for large amounts of antigens (sinceonly parts of the crude antigen are relevant to the protective immune response) and the need for immunopotentiating adjuvants. The results of the various immunization experiments carried out in the past are extremely difficult to compare: different ways of parasite inactivation, different ways of vaccine administration and different laboratory animals were used, different challenge strains and challenge rates were applied (3, 15,27,28,40,58, 60, 66, 75, 101, 105, 106). What can be nonetheless concluded, is that in general these experiments were not able to induce a satisfying protection. Protection against highly virulent strains could never be achieved; only a slight degree of protection could be obtained in some cases against moderately virulent Toxoplasma strains (27, 58, 66, 106), mostly when Freund's adjuvant had been used. Because of its inflammatory action this adjuvant, however, cannot be applied in humans.
2.3. Immunization with attenuated parasites Vaccines prepared from attenuated Toxoplasma parasites (i.e. parasites which cannot proliferate and cause chronic infection) show a better induction of protectivity compared to killed parasites because the organisms are alive for a short, restricted period. Two types of parasite attenuation have been described for Toxoplasma gondii: 2.3.1. Temperature-sensitive mutants In 1976, Pfefferkorn and Pfefferkorn established several temperature-sensitive mutants of strain RH by the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (83). The defect of such a temperature-sensitive mutant is supposed to be due to an altered amino acid sequence of an essential protein. The altered protein is functional at the permissive temperature but not at the restrictive temperature. One of these mutants, called ts-4, did not persist in the host beyond two months and no cysts could be found (107). The mutant has been patented to be used as a vaccine for the immunization of animals and man. The vaccine consists of live tachyzoites in saline, at least 20 of them have to be administered subcutaneously. The mutant has been deposited in the American Type Culture Collection in Maryland with the accession number 40.050. Vaccination with the ts-4 mutant proved to be one of the most encouraging animal experiments of the past: Hamsters vaccinated with the ts-4 strain developed complete protection against challenge exposure to the most pathogenic RH-strain used (24).
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Lesions due to the infection with ts-4 were limited to mononuclear inflammation and a scar at the injection site. There was no evidence of chronic infection when tissues were controlled for persistent Toxoplasma parasites (23). Yet, some points still need more detailed investigation: a) The mechanism of parasite elimination is not exactly known. The restrictive temperature of ts-4 is around 38°C and it has been speculated that a febrile response after the infection might restrict the multiplication of the tachyzoites (83). But - as Waldeland et al. have stated (107) - this cannot account for the elimination of infection because parasites sometimes were found only up to three or four days after infection, sometimes up to one or two months after infection. b) More detailed and long-term studies on the protective effect of the vaccine are desirable. Challenge experiments have been carried out till week 24 after infection with the RH strain. The survival rate of the vaccinated animals was 100% in weeks 2, 4 and 16, but 70% in week 8 and 60% in week 24 (24). The essential drawback of the ts-4 mutant results from its consisting of live tachyzoites which makes a cooling-chain absolutely necessary, so that delivery of the vaccine is rather difficult. 2.3.2. Irradiation of parasites Several investigators have exposed parasites either to ultraviolet irradiation (25,29), X-rays (62, 65, 93) or y-rays (12, 104) in order to impair the parasites' chromosomes. This should guarantee a limited duration of "infection" while excluding the threat of multiplication and exacerbation. In some cases, however, it could not be excluded that some Toxoplasma parasites escaped destruction and continued to multiply, thus forming a live vaccine which caused a genuine infection. The protective effect of vaccines consisting of irradiated parasites reportedly varies between 10 and 100% (12, 104). The duration of protection achieved seems to be short and so far has not been well documented; moreover, reports on optimal irradition doses differ considerably. When comparing the results, we have to differentiate clearly between immunization experiments with irradiation doses designed to attenuate the parasites (25,29,62) and such with doses meant to completely kill the parasites (21,56,65).
2.4. Heterologous immunization Heterologous immunization experiments take advantage of non-pathogenic organisms, which are closely related to Toxoplasma gondii and are able to provide crossprotection due to cross-reactive antigens. Cross-protection against Toxoplasma gondii has been demonstrated for Hammondia hammondi by Christie and Dubey (13, 18). Hammondia hammondi appears to be non-pathogenic to non-immune animals and was used by Dubey to immunize goats against Toxoplasma gondii and prevent congenital toxoplasmosis (19). It could be demonstrated that goats vaccinated with Hammondia hammondi developed a protective immunity against lethal doses of Toxoplasma gondii (20); a complete protection, however, could not be achieved. Toxoplasma was isolated from all kids of vaccinated goats by the mouse inoculation test, the kids of one out of five vaccinated does died (19). Recently Araujo et al. (1) showed antigenic similarity to exist between Toxoplasma gondii and Hammondia hammondi, and]ohnson and coworkers (43) reported that the rRNA of Hammondia hammondi is identical with that of Toxoplasma gondii. This reveals the extraordinarily close relationship between the two protozoa; a vaccine made of Hammondia hammondi would resemble a so-called "non-virulent" live vac-
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cine with Toxoplasma organisms involving the problems of an alteration of virulence and of exacerbation after immunosuppression or in cases of immunodeficiency. We do not know anything about the possibility of vaccinating humans with Hammondia hammondi but because of the above mentioned threats the use of Hammondia should be confined to veterinary medicine.
2.5. Non-specific immunization Non-specific immunization against Toxoplasma gondii in the absence of the parasite could be effected by various microorganisms or biologic substances : Infections with Corynebacterium paruum (102), Listeria monocytogenes (89), Besnoitia iellisoni (18, 36, 87) or with Mycobacterium sp. (10, 103) as well as immunization with complete Freund's adjuvant (87) or synthetic adjuvant (59, 70) evoked an immune response and resulted in a partial protection against infection with Toxoplasma gondii. In contrast to heterologous immunization, non-specific immunization is induced by a general nonparasite-specific mechanism, in which sensitized lymphocytes produce macrophageactivating factors which in turn mobilize and activate macrophages. Although (35, 91) in this case the induction of immunity is very specific, its expression is non-specific. This mechanism has been termed "acquired cellular immunity" (35). It means that the specific inducing antigen must persist in order to maintain a non-specific immune response.
2.6. Immunization with Toxoplasma RNA: Araujo and Remington (3) investigated the immunizing potential of Toxoplasma RNA in mice, since a number of studies suggested that bacterial RNA confers speciesspecific resistance to infection. A significant protective effect was seen when 200 ug of Toxoplasma RNA or 50 !1g of RNA incorporated into Freund's incomplete adjuvant were administered. Yet, no specific antibodies against Toxoplasma were detected. It could be shown that the administration of polynucleotides to mice induces a nonspecific protective capacity by non-specific activation of macrophages. 3. Recent Research
Present research is primarily focused on the identification and characterization of the antigens of Toxoplasma gondii. This involves ultrastructural and biochemical studies of the immunohistochemistry and the topographical localization of distinct antigens by the use of monoclonal antibodies as well as analysis of Toxoplasma antigens by means of polyspecific sera from clinically and parasitologically well defined individuals. The purpose of these investigations and the present major task in the efforts to produce a vaccine against toxoplasmosis are to select antigens that induce a beneficial immune response (i.e. host-protective or disease-inhibitory effects) from those that are irrelevant to protection (e.g. antigens which are essential for immunodiagnosis only) or that are even counterproductive (i.e, immunopathologic or proparasitic) (73). Only some of the recent approaches in Toxoplasma immunology shall be named here: Analysis of Toxoplasma antigens were performed by Erlich et al. (26), Hughes and Balfour (38), Partanen et al. (80, 81), Potassmann et al. (84), and Sharma et al. (98), at different stages of infection and by various methods. Efforts to characterize circulating antigen of Toxoplasma gondii were undertaken by several investigators (5,
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14,32, 37, 39, 41, 55, 86). Further studies on the immunochemistry and cellular localization of Toxoplasma antigens with the aid of monoclonal antibodies were done by Araujo et al. (2), Handman et al. (30, 31), Johnson et al. (42), Ogata et al. (78), and Sethi et al. (95): Kaspar and coworkers purified a major membrane protein of Toxoplasma gondii (called P30) by immunoabsorption with a monoclonal antibody (52). The P30 antigen was further characterized by Rodriguez et al. (90). This protein, however, did not confer protection when it was used as a vaccine in mice. The P30 antigen seems to be a tachyzoite antigen which is not expressed by bradyzoites (53). Hauser and Remington (33) and Sethi et al. (94) showed that treatment of intact Toxoplasma tachyzoites with several monoclonal antibodies directed against membrane-associated antigenic determinants facilitated the phagozytosis of Toxoplasma and also prepared the parasite for intracellular destruction by macrophages. Antigens that are able to induce a beneficial immune response have been identified by Araujo and Remington (4), Johnson et al. (46), Sharma et al. (97), and by Sibley and Sharma (99). Johnson et al. injected monoclonal antibodies to antigen of Toxoplasma gondii into mice (46). Two monoclonals directed against a 35kD and a 14kD antigen conferred a total protection against a moderatel y virulent challenge. Immunization against a highly virulent strain resulted in a significant protection as indicated by a prolonged interval until death. Araujo and Remington (4) eluted antigens from SDS polyacrylamide gels and immunized mice. Two of the antigens with molecular rates of 14 and 35 kD, surprisingly being identical with the above mentioned ones, protected mice against a lethal infection by the C56 strain . Sharma et al. (97) demonstrated that passive transfer of a monoclonal antibody (F3G3 ) which recognizes cytoplasmic antigens of Toxoplasma gondii protects mice against a challenge with the C56-strain. Immunization of mice with a single antigenic component, isolated by affinity chromatography with the monoclonal antibody F3G3, resulted in a significant resistance against a lethal challenge (29% of mice died) and in a complete protection when the antigen was administered in combination with Freund's incomplete adjuvant. The epitope recognized by F3G3 as well as by an analogous monoclonal antibody was located beneath the Toxoplasma cell surface membran e. The antigens recognized by both monoclonal antibodies were antigenically related proteins of 58 and 28 kD (99). Scbtoartzmann (92) reported the inhibition of a penetration-enhancing factor of Toxoplasma gondii by various monoclonal antibodies specific for rhoptries . Another important contribution of today 's research towards a vaccine is the recombinant DNA technology. Two groups of investigators have isolated and translated mRNA from tachyzoites of the RH strain: The Remington group in the United States and the Johnson group in Australia. The group of Remington has translated mRNA in a wheat germ system (85). Tachyzoite ant igens synthesized in vitro were recognized by Toxoplasma gondii-specific antibodies obtained from rabbit s, mice and humans. The Johnson group has translated mRNA in a cell-free system derived from rabbit reticulocyte lysate (44).11 out of 16 translated polypeptides were immunoprecipitated by antiToxoplasma mouse and human sera. The next step, just now under investigation, is the screening of the complementary DNA library for expression of Toxoplasma gondii antigens by the use of monospecific antibody probes and the cloning and expression of genes coding for antigens protective against infection with Toxoplasma gondii. In order to identify antigenic determinants, mutants of Toxoplasma gondii selected by coincubation with monoclonal antibodies will become important. Two different mutants lacking membrane proteins of 22 kD and 30 kD that are present on the wild type have already been established (49, 51).
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A completely different approach towards a vaccine might be found by induction of immunity at the mucosal sites of the gut. Since the beginning of this decade, knowledge about the gut-associated lymphoid tissue (GALT) and the gut immune system has enormously increased. Several efforts towards an orally administered vaccine have been made against viral and bacterial infections using killed organisms (Poliovirus,
Shigella flexneri, Vibrio cholerae, Streptococcus mutans, Escherichia coli, Salmonella spp.). Regarding parasitic infections, antigen-exclusion directed against whole organisms by specific secretory IgA in combination with other factors has been described (48,61). Davis et al. (16, 17) have shown a secretory IgA-mediated in vitro inhibition of the cell penetration and intracellular development of sporozoites of Eimeria tenella, a parasite closely related to Toxoplasma gondii, by a synergism of specific secretory IgA antibodies and non-specific factors. Secretory IgA specific to Toxoplasma gondii after oral infection of mice with bradyzoites has been described by McLeod and Mack (71). We have reported the induction of a humoral immune response in rabbits after enteral administration of killed Toxoplasma tachyzoites (34). Further investigations will be necessary to determine the specific secretory IgA response at the gut mucosal site after oral or enteral immunization with antigen of Toxoplasma gondii. As Toxoplasma gondii is acquired in nature by the oral route, the gut mucosal immune system acts as the first barrier against infection. Immune defense at this site would hit the parasite in a very vulnerable life cycle stage (after excysting from cysts or oocysts) and possibly might lead to (partial) antigen exclusion. Induced protectivity would be of great value against an acquisition of Toxoplasma gondii for HIV-positive, Toxoplasma-negative individuals to protect them completely against infection and, secondly, to inhibit Toxoplasma multiplication in the eat's intestine and thus prevent oocyst shedding. Although it remains doubtful today whether specific secretory IgA antibodies in combination with unspecific lytic factors ever will be able to provide sufficient immunity to act as defense against an enteral invasion by Toxoplasma gondii, an orally administered vaccine, no matter whether of molecular structure or not, will presumably support immune defense in cooperation with a parenterally applied vaccine.
4. Future Prospects Current research projects allow a clear and concrete view of future developments. Efforts in toxoplasmosis research and in other domains of parasitology are predominantly directed towards a molecular vaccine. The advantages are striking: a molecular vaccine is precisely defined, therefore quality control and monitoring of stability of the vaccine are facilitated. The probability of inducing undesirable immune responses, toxic reactions and harmful side effects following vaccination is reduced. Antigens of life cycle stages that cannot easily be obtained in large quantities (e.g. antigens of sporozoites or of bradyzoites) may be synthesized in a sufficient amount. The immuneresponse in the vaccinee can be more accurately measured and the efficacy of the vaccine more precisely determined. A molecular vaccine, however, may also entail some disadvantages. These derive from the restricted antigenic composition of such a vaccine (in contrast to crude extracts or whole organisms which present multiple antigens to the immune system) and from the biological adaptability of the pathogenic organisms. Theoretically, the
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use of a molecular vaccine might select "antigen-negative" parasites - i.e. parasites that do not express the antigens or epitopes which were used for vaccination (74). Furthermore, we have to take into account that there are stage-specific antigenic differences between Toxoplasma tachyzoites, bradyzoites, and sporozoites (9,50,54, 63, 64) and that therefore the human immune-response to Toxoplasma gondiipresumably will vary depending on the antigens and stages which have been used for immunization. We have to consider also that antigen analysis by SDS-PAGE and the molecular cloning technique are confined to a detection of protein antigens of Toxoplasma gondii, while host-protective antigens of a carbohydrate or glycolipid composition would not be detected by this means. We know, however, that membrane of Toxoplasma gondii does contain antigenic glycoconjugates. Pande et al. (79) first described a heteroglycan of Toxoplasma gondii. In contrast to previous findings by Handman et al. (30) and Sethi et al. (96), Mauras et al. (69) revealed the presence of carbohydrates on the surface membrane of Toxoplasma tachyzoites. Subsequent studies by Johnson et al. (45) have shown that at least three glycosylated polypeptides are present in the insoluble fraction of disrupted parasites. Hughes and Balfour (38) described one out of nine distinct antigens (recognized by human antibodies) to be a lipopolysaccharide and one, a glycoprotein. Mineo et al. (72) used a Toxoplasma polysaccharide fraction in an ELISA and found a preferential binding of specific IgM antibodies. Naot et al. (77) reported that polysaccharides extracted from tachyzoites reacted with both human IgM and IgG antibodies and that lipids were not recognized by either IgM or IgG antibodies. Another point we have to pay attention to is the heterogeneity of the parasite population, that means strain differences of different geographical isolates or in different host reservoirs (7, 8, 68). Antigenic diversity among Toxoplasma strains and differences in the antibody response have been described recently by Ware and Kasper (108), and Weiss et al. (109). Likewise, we have to pay attention to genetically determined differences in the host population and hence to differences in the immune response to antigens (100). No investigations have been carried out so far to determine whether antigen variation occurs with Toxoplasma gondii. At any rate, a critical subset of antigens or epitopes will have to be found that is not labile or changeable and that is capable of inducing protection in the majority of individuals in a genetically diverse host population. Another strategy to maintain an adequate level of protective immunity would be the replacement of antigens in the vaccine at regular intervals (74). The latter, however, seems to be far away from practical use due to the high costs it would involve. Although the humoral branch of the immune system is by far more easily accessible to investigation than the cellular one (which is expressed by the fact that the majority of studies is concerned with the B-cell-derived immune response) we have to keep in mind that cell-mediated immunity is the predominant effector mechanism in antiToxoplasma defence. Besides numerous experiments dealing with macrophage activation and adoptive transfer of immunity by lymphocytes, three recent studies call for a more detailed reflection: Reyes and Frenkel (88) attempted to characterize specific soluble mediators of Toxoplasma immunity and suggested a positive correlation between the rate of appearance of Toxoplasma mediator and the expression of antiToxoplasma tissue immunity, yet did not rule out the possibility that antibodies might have contributed to parasite elimination. Eisenhauer et al. (22) demonstrated a reduction of the numbers of Toxoplasma cysts in the brains of mice by a joint action of both
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humoral and cellular (non-specifically induced) mechanisms, whereas Brinkman et al. (11) concluded from their results on the correlation between antibody response, antigen-specific T-cell activation and brain cyst formation that the major part of protective immunity against Toxoplasma gondii was cell-mediated and that high humoral response did not inhibit, but more likely favoured cyst formation. It is evident that more detailed information about the effector mechanisms of anti-Toxoplasma defence is required and that we will have to decide whether an oligopeptide vaccine should be favoured which consists of antigenic determinants that predominantly stimulate T-cell immunity. Foreseeabledifficulties will emerge in the search for such determinants and their presentation to the immune system. Once an immunizing antigen has been defined it possibly will not convey the complete spectrum of immunity required for host protection. It might be necessary to condense several individual protective antigens in to one single vaccine. Because of its molecular structure, the vaccine will be highly dependent upon an antigen delivery system (e.g. liposomes) and on immunopotentiating adjuvants which elicit humoral and/or cell-mediated immunity. Of course the adjuvants have to be free from sideeffects to be acceptable for human use. The above mentioned points make it evident that even when a protection-inducing molecule will have been characterized and produced, it will be a long way to go until a vaccine becomes available which induces the desired protection and fulfills the requirements for application in human medicine.
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53. Kasper, L. H., K. M. Currie, and M. S. Bradley: An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii. J. Immunol. 134 (1985) 3426-3431
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78. Ogata, K., T. Kasabara, K. Shioiri-Nakano; I. Igarashi, and M. Suzuki: Immunoenzymatic detection of three kinds of 43,000-molecular-weight antigens by monoclonal antibodies in the insoluble fraction of Toxoplasma gondii. Infect. Immun. 43 (1984) 1047-1053 79. Pande, P. G., R. R. Shukla, and P. C. Sekariah: A heteroglycan from Toxoplasma gondii. Nature 190 (1961) 644-645 80. Partanen, P., H. ]. Turunen, R. Paasiuuo, E. Forsblom, J. Suni, and P. O. Leinikki: Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique. FEBS 158 (1983) 252-254 81. Partanen, P., H. ]. Turunen, R. T. A. Paasiuuo, and P. O. Leinikki: Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulins G, M, and A antibodies at different stages of infection. J. Clin. Microbio!' 20 (1984) 133-135 82. Pestre-Alexandre, M. and M. Mounier: Immunization active avec les souches avirulentes. Lyon Med, 248, Supp!. (1982) 95-99 83. Pfefferkorn, E. R. and L. C. Pfefferkorn: Toxoplasma gondii: Isolation and preliminary characterization of temperature-sensitive mutants. Exp. Parasito!' 39 (1976) 365-376 84. Potasman, 1., F. G. Araujo, G. Desmonts, and]. S. Remington: Analyses of Toxoplasma antigens recognized by human sera obtained before and after acute infection. J. Infect. Dis. 154 (1986) 650-657 85. Prince.]. B., M. A. Koven-Quinn,]. S. Remington, and S. D. Sharma: Cell free synthesis of Toxoplasma gondii antigens. Mo!' Biochem. Parasito!' 17 (1985) 163-170 86. Rahman, R. E. and F. A. Neva: Detection of circulating antigen in acute experimental infections with Toxoplasma gondii. J. Infect. Dis. 132 (1975) 44-48 87. Remington, ]. S., ]. L. Krahenbuhl, and]. W. Mendenhall: A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6 (1972) 829-834 88. Reyes, L. and J. K. Frenkel: Specific and nonspecific mediation of protective immunity to Toxoplasma gondii. Infect. Immun. 55 (1987) 856-863 89. Rezai, H. R., N. C. Behforouz, K. Bahar, and S. Ardehali: Immunity to Toxoplasma and Listeria induced by homologous and heterologous organisms. Acta Trop. 37 (1980) 21-29 90. Rodriguez, c., D. Afchain, A. Capron, C. Dissous, and F. Santoro: Major surface protein of Toxoplasma gondii (p30) contains an immunodominant region with repetitive epitopes. Eur. j. Immuno!' 15 (1985) 747-749 91. Ruskin,J., J. McIntosh, and j. S. Remington: Studies on the mechanisms of resistance to phylogenetically diverse intracellular organisms. J. Immuno!' 103 (1969) 252-259 92. Schu/artzmann.]. D.: Inhibition of penetration-enhancing factor of Toxoplasma gondii by monoclonal antibodies specific for rhoptries. Infect. Immun. 51 (1986) 760-764 93. Seah, S. K. K. and G. Hucal: The use of irradiated vaccine in immunization against experimental murine toxoplasmosis. Can. j. Microbio!' 21 (1975) 1379-1385 94. Sethi, K. K., T. Endo, and H. Brandis: Toxoplasma gondii trophozoites precoated with specific monoclonal antibodies cannot survive within normal murine macrophages. Immuno!' Lett. 2 (1981) 343-346 95. Sethi, K. K., Y. Omata, and H. Brandis: Topographical localization of distinct antigenic domains of Toxoplasma gondii with the aid of monoclonal antibodies. Z. Parasitenk. 70 (1984) 699-707
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96. Sethi, K. K., A. Rahman, B. Pelster, and H. Brandis: Search for the presence of lectinbinding sites on Toxoplasma gondii. ]. Parasitol. 63 (1977) 1076-1080 97. Sharma, S. D., F. G. Araujo, and]. S. Remington: Toxoplasma antigen isolated by affinity chromatography with monoclonal antibody protects mice against lethal infection with Toxoplasma gondii.]. Immunol. 133 (1984) 2818-2820 98. Sharma, S. D.,]. Mullenax, F. G. Araujo, H. A. Erlich, and]. S. Remington: Western blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. ]. Immunol. 131 (1983) 977-983 99. Sibley, 1. D. and S. D. Sharma: Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice. Infect. Immun. 55 (1987) 2137-2141 100. Siqueira, M., 1. S. Drumond, M. Gennari, V. C. Ferreira, M. H. Reis, and G. Biozzi: Effects of genetic modification of antibody responsiveness on resistance to Toxoplasma gondii infection. Infect. Immun. 48 (1985) 298-302 101. Strannegard, o.. The formation of Toxoplasma antibodies in rabbits. Acta path. microbiol. scand. 71 (1967) 439-449 102. Swartzberg, ]. E., ]. 1. Krahenbuhl, and]. S. Remington: Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated with Corynebacterium parium. Infect. Immun. 12 (1975) 1037-1043 103. Tabbara, K. F., G. R. O'Connor, and R. A. Nozik: Effect of immunization with attenuated Mycobacterium bovis on experimental toxoplasmic retinochoroiditis. Am. j. Ophthalmol. 79 (1975) 641-647 104. Tran Manh Sung, R.: Les essais de radio-vaccins dans la toxoplasmose murine. Lyon Med. 248, Suppl. (1982) 101-105 105. Tsuchimoto, M.: Protective antigenicity of cell homogenate and centrifuged cell fractions of Toxoplasma gondii in mice. jap. ]. Parasitol. 27 (1978) 433-443 106. Waldeland, H. and]. K. Frenkel: Live and killed vaccines against toxoplasmosis in mice. j, Parasitol. 69 (1983) 60-65 107. Waldeland, H., E. R. Pfefferkorn, and]. K. Frenkel: Temperature-sensitive mutants of Toxoplasma gondii: Pathogenicity and persistence in mice. ]. Parasitol. 69 (1983) 171-175 108. Ware, P. 1. and 1. H. Kasper: Strain-specific antigens of Toxoplasma gondii. Infect. Immun. 55 (1987) 778-783 109. Weiss, 1. M., S. A. Udem, H. Tanountz, and M. Wittner: Western blot analysis of the antibody response of patients with AIDS and Toxoplasma encephalitis: Antigenic diversity among Toxoplasma strains. J. Infect. Dis. 157 (1988) 7-13 Dr. Mag. K. Hermentin, Univ.Prof. Dr. H. Aspock, Dept. of Med. Parasitology, Institute of Hygiene, Kinderspitalgasse 15, A-1095 Vienna, Austria
Efforts Towards a Vaccine Against Toxoplasma gondii: A Review KURT HERMENTIN and HORST ASPOCK Abteilung fur Medizinische Parasitologie (Leiter: Univ. Prof. Dr. H. Aspock) des HygieneInstituts der Universitat Wien (Vorstand: Univ. Prof. Dr. H. Flamm)
Received April 22, 1988 . Accepted July 5, 1988
Abstract In a review, past as well as present investigations carried out towards a vaccine against toxoplasmosis are outlined. A historical retrospect of the various immunization experiments is given, recent research projects intending the characterization of antigens that are relevant to host protective immunity are described, and a prospect to future problems and developments expected in the field is drafted.
Zusammenfassung Die Arbeit gibt einen Uberblick iiber die bisher durchgefiihrten und derzeit laufenden Bemiihungen urn die Herstellung einer Vakzine gegen Toxoplasma gondii. Die verschiedenen Ansatze zur Immunisierung gegen den Parasiten werden in einem historischen Riickblick besprochen, gegenwiirtigeForschungsbemiihungen zur Auffindung einzelner Antigene, die eine protektive Immunantwort bewirken sollen, beschrieben, sowie zukiinftige absehbare Probleme und Entwicklungen dargestellt.
1. Introduction Undoubtedly, prophylactic vaccines have to be considered as one of the principal goals of research in immunoparasitology. While vaccines are successfully applied in the control of many viral and bacterial diseases, this success, however, could not be emulated in the battle against parasitic infections. This is due partly to the more complex structures and antigens of these organisms and partly to the fact that many parasites have evolved ways of either living with the immune response of the host or even escaping it. Principal parasitic defense mechanisms are based (a) on the variation of the parasitic antigens, (b) on the concealment of antigens from the host immune-system, e.g. by formation of cysts or by uptake of host antigens (molecular masking), (c) on the immune modulation or immunosuppression of the host response and (d) on mechanisms of antibody clearance (e.g. capping of amoebae). 28 Zbl. Bakt. Hyg. A 269/4
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Among the parasites, the protozoon Toxoplasma gondii deserves special attention as an infection contracted during pregnancy can cause abortion, severe fetal abnormalities, or neonatal death in humans as well as in domestic animals (6), hence making a prophylactic vaccine highly desirable. Efforts towards a vaccine against toxoplasmosis have recently gained further support with the appearance of AIDS and related cerebral complications due to Toxoplasma gondii. The present paper endeavors to give a historical retrospect of the problem, to review recent research projects towards a vaccine against toxoplasmosis, and to outline future problems and developments to be expected in this field. Before sketching the major results of immunization and vaccination experiments, some general statements on the goals and requirements of a vaccine against toxoplasmosis shall be put at the beginning, to elucidate the aspects under which the present review has been made: - First of all, a vaccine to control toxoplasmosis should prevent infection of the unborn during pregnancy, thus excluding fetal disease. - A vaccine against toxoplasmosis would also be beneficial for HIV-positive individuals who are Toxoplasma-seronegative. AIDSpatients once infected with Toxoplasma gondii frequently develop a severe cerebral affection. Therefore, it would be advantageous to prevent HIV-positive individuals from getting infected with this parasite. A vaccine designed merely to protect against disease, however, would not be sufficient. An effective vaccine has to protect against infection, that means it has to prevent Toxoplasma parasites from invading the host and settling in his tissue. Whether such a vaccine will ever become available remains doubtful; presumably it would have to induce immunity at the first barrier, i.e. the mucosal immune system of the gut, and would necessitate a complete protective effect already at this site. - A third goal of a vaccine against toxoplasmosis is the prevention of oocyst excretion in the domestic cat. Such a vaccine would considerably reduce the transmission rate of the parasite and eliminate one of the two major ways of transmission to man. Since it is much more practicable to avoid uptake of raw meat than the ingestion of oocysts dispersed in the environment, it would also facilitate prophylactic measures against toxoplasmosis during pregnancy. - A prospective vaccine against toxoplasmosis, furthermore, has to fulfill the criteria of safeness, guaranteed longterm protection, cost-effectiveness and easy delivery.
2. Historical Review Since 1928, when Levadity and coworkers set up their first immunization experiments against toxoplasmosis with heat-inactivated Toxoplasma parasites (60), a wide variety of immunization and vaccination experiments have been carried out. Many different vaccines including non-virulent strains, attenuated strains, killed parasites, homogenates, soluble extracts, heterologous antigens, and non-specific antigens have been used. In many cases, the experiments were primarily designed to evolve an immune-response that usually was judged by the detection of specific antibodies only; the question of protection was placed into the background. This is contrary to today's research, where the major goal is the identification of antigens that are relevant to protective immunity of the host.
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2.1. Live vaccines Numerous immunization experiments were carried out with live, low-virulent or "non-virulent" strains of Toxoplasma gondii (47, 57, 67, 76, 82, 106). Such vaccines convey the most effective protection against a virulent challenge strain but, strictly speaking, they cause a genuine infection. It cannot be excluded that a vaccine-induced infection may recrudesce and cause multiple lesions, e.g. when the host becomes immunosuppressed or suffers from immunodeficiency. Moreover, the possibility of a parasite-immanent change of virulence has to be taken into account. Thus, such vaccines cannot be applied in human medicine, but may gain some interest in the veterinary field.
2.2. Immunization with killed parasites, homogenates or soluble extracts Vaccines consisting of killed Toxoplasma parasites, homogenates or soluble extracts are far less immunogenic than live ones. They combine two major disadvantages, namely the need for large amounts of antigens (sinceonly parts of the crude antigen are relevant to the protective immune response) and the need for immunopotentiating adjuvants. The results of the various immunization experiments carried out in the past are extremely difficult to compare: different ways of parasite inactivation, different ways of vaccine administration and different laboratory animals were used, different challenge strains and challenge rates were applied (3, 15,27,28,40,58, 60, 66, 75, 101, 105, 106). What can be nonetheless concluded, is that in general these experiments were not able to induce a satisfying protection. Protection against highly virulent strains could never be achieved; only a slight degree of protection could be obtained in some cases against moderately virulent Toxoplasma strains (27, 58, 66, 106), mostly when Freund's adjuvant had been used. Because of its inflammatory action this adjuvant, however, cannot be applied in humans.
2.3. Immunization with attenuated parasites Vaccines prepared from attenuated Toxoplasma parasites (i.e. parasites which cannot proliferate and cause chronic infection) show a better induction of protectivity compared to killed parasites because the organisms are alive for a short, restricted period. Two types of parasite attenuation have been described for Toxoplasma gondii: 2.3.1. Temperature-sensitive mutants In 1976, Pfefferkorn and Pfefferkorn established several temperature-sensitive mutants of strain RH by the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (83). The defect of such a temperature-sensitive mutant is supposed to be due to an altered amino acid sequence of an essential protein. The altered protein is functional at the permissive temperature but not at the restrictive temperature. One of these mutants, called ts-4, did not persist in the host beyond two months and no cysts could be found (107). The mutant has been patented to be used as a vaccine for the immunization of animals and man. The vaccine consists of live tachyzoites in saline, at least 20 of them have to be administered subcutaneously. The mutant has been deposited in the American Type Culture Collection in Maryland with the accession number 40.050. Vaccination with the ts-4 mutant proved to be one of the most encouraging animal experiments of the past: Hamsters vaccinated with the ts-4 strain developed complete protection against challenge exposure to the most pathogenic RH-strain used (24).
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Lesions due to the infection with ts-4 were limited to mononuclear inflammation and a scar at the injection site. There was no evidence of chronic infection when tissues were controlled for persistent Toxoplasma parasites (23). Yet, some points still need more detailed investigation: a) The mechanism of parasite elimination is not exactly known. The restrictive temperature of ts-4 is around 38°C and it has been speculated that a febrile response after the infection might restrict the multiplication of the tachyzoites (83). But - as Waldeland et al. have stated (107) - this cannot account for the elimination of infection because parasites sometimes were found only up to three or four days after infection, sometimes up to one or two months after infection. b) More detailed and long-term studies on the protective effect of the vaccine are desirable. Challenge experiments have been carried out till week 24 after infection with the RH strain. The survival rate of the vaccinated animals was 100% in weeks 2, 4 and 16, but 70% in week 8 and 60% in week 24 (24). The essential drawback of the ts-4 mutant results from its consisting of live tachyzoites which makes a cooling-chain absolutely necessary, so that delivery of the vaccine is rather difficult. 2.3.2. Irradiation of parasites Several investigators have exposed parasites either to ultraviolet irradiation (25,29), X-rays (62, 65, 93) or y-rays (12, 104) in order to impair the parasites' chromosomes. This should guarantee a limited duration of "infection" while excluding the threat of multiplication and exacerbation. In some cases, however, it could not be excluded that some Toxoplasma parasites escaped destruction and continued to multiply, thus forming a live vaccine which caused a genuine infection. The protective effect of vaccines consisting of irradiated parasites reportedly varies between 10 and 100% (12, 104). The duration of protection achieved seems to be short and so far has not been well documented; moreover, reports on optimal irradition doses differ considerably. When comparing the results, we have to differentiate clearly between immunization experiments with irradiation doses designed to attenuate the parasites (25,29,62) and such with doses meant to completely kill the parasites (21,56,65).
2.4. Heterologous immunization Heterologous immunization experiments take advantage of non-pathogenic organisms, which are closely related to Toxoplasma gondii and are able to provide crossprotection due to cross-reactive antigens. Cross-protection against Toxoplasma gondii has been demonstrated for Hammondia hammondi by Christie and Dubey (13, 18). Hammondia hammondi appears to be non-pathogenic to non-immune animals and was used by Dubey to immunize goats against Toxoplasma gondii and prevent congenital toxoplasmosis (19). It could be demonstrated that goats vaccinated with Hammondia hammondi developed a protective immunity against lethal doses of Toxoplasma gondii (20); a complete protection, however, could not be achieved. Toxoplasma was isolated from all kids of vaccinated goats by the mouse inoculation test, the kids of one out of five vaccinated does died (19). Recently Araujo et al. (1) showed antigenic similarity to exist between Toxoplasma gondii and Hammondia hammondi, and]ohnson and coworkers (43) reported that the rRNA of Hammondia hammondi is identical with that of Toxoplasma gondii. This reveals the extraordinarily close relationship between the two protozoa; a vaccine made of Hammondia hammondi would resemble a so-called "non-virulent" live vac-
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cine with Toxoplasma organisms involving the problems of an alteration of virulence and of exacerbation after immunosuppression or in cases of immunodeficiency. We do not know anything about the possibility of vaccinating humans with Hammondia hammondi but because of the above mentioned threats the use of Hammondia should be confined to veterinary medicine.
2.5. Non-specific immunization Non-specific immunization against Toxoplasma gondii in the absence of the parasite could be effected by various microorganisms or biologic substances : Infections with Corynebacterium paruum (102), Listeria monocytogenes (89), Besnoitia iellisoni (18, 36, 87) or with Mycobacterium sp. (10, 103) as well as immunization with complete Freund's adjuvant (87) or synthetic adjuvant (59, 70) evoked an immune response and resulted in a partial protection against infection with Toxoplasma gondii. In contrast to heterologous immunization, non-specific immunization is induced by a general nonparasite-specific mechanism, in which sensitized lymphocytes produce macrophageactivating factors which in turn mobilize and activate macrophages. Although (35, 91) in this case the induction of immunity is very specific, its expression is non-specific. This mechanism has been termed "acquired cellular immunity" (35). It means that the specific inducing antigen must persist in order to maintain a non-specific immune response.
2.6. Immunization with Toxoplasma RNA: Araujo and Remington (3) investigated the immunizing potential of Toxoplasma RNA in mice, since a number of studies suggested that bacterial RNA confers speciesspecific resistance to infection. A significant protective effect was seen when 200 ug of Toxoplasma RNA or 50 !1g of RNA incorporated into Freund's incomplete adjuvant were administered. Yet, no specific antibodies against Toxoplasma were detected. It could be shown that the administration of polynucleotides to mice induces a nonspecific protective capacity by non-specific activation of macrophages. 3. Recent Research
Present research is primarily focused on the identification and characterization of the antigens of Toxoplasma gondii. This involves ultrastructural and biochemical studies of the immunohistochemistry and the topographical localization of distinct antigens by the use of monoclonal antibodies as well as analysis of Toxoplasma antigens by means of polyspecific sera from clinically and parasitologically well defined individuals. The purpose of these investigations and the present major task in the efforts to produce a vaccine against toxoplasmosis are to select antigens that induce a beneficial immune response (i.e. host-protective or disease-inhibitory effects) from those that are irrelevant to protection (e.g. antigens which are essential for immunodiagnosis only) or that are even counterproductive (i.e, immunopathologic or proparasitic) (73). Only some of the recent approaches in Toxoplasma immunology shall be named here: Analysis of Toxoplasma antigens were performed by Erlich et al. (26), Hughes and Balfour (38), Partanen et al. (80, 81), Potassmann et al. (84), and Sharma et al. (98), at different stages of infection and by various methods. Efforts to characterize circulating antigen of Toxoplasma gondii were undertaken by several investigators (5,
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14,32, 37, 39, 41, 55, 86). Further studies on the immunochemistry and cellular localization of Toxoplasma antigens with the aid of monoclonal antibodies were done by Araujo et al. (2), Handman et al. (30, 31), Johnson et al. (42), Ogata et al. (78), and Sethi et al. (95): Kaspar and coworkers purified a major membrane protein of Toxoplasma gondii (called P30) by immunoabsorption with a monoclonal antibody (52). The P30 antigen was further characterized by Rodriguez et al. (90). This protein, however, did not confer protection when it was used as a vaccine in mice. The P30 antigen seems to be a tachyzoite antigen which is not expressed by bradyzoites (53). Hauser and Remington (33) and Sethi et al. (94) showed that treatment of intact Toxoplasma tachyzoites with several monoclonal antibodies directed against membrane-associated antigenic determinants facilitated the phagozytosis of Toxoplasma and also prepared the parasite for intracellular destruction by macrophages. Antigens that are able to induce a beneficial immune response have been identified by Araujo and Remington (4), Johnson et al. (46), Sharma et al. (97), and by Sibley and Sharma (99). Johnson et al. injected monoclonal antibodies to antigen of Toxoplasma gondii into mice (46). Two monoclonals directed against a 35kD and a 14kD antigen conferred a total protection against a moderatel y virulent challenge. Immunization against a highly virulent strain resulted in a significant protection as indicated by a prolonged interval until death. Araujo and Remington (4) eluted antigens from SDS polyacrylamide gels and immunized mice. Two of the antigens with molecular rates of 14 and 35 kD, surprisingly being identical with the above mentioned ones, protected mice against a lethal infection by the C56 strain . Sharma et al. (97) demonstrated that passive transfer of a monoclonal antibody (F3G3 ) which recognizes cytoplasmic antigens of Toxoplasma gondii protects mice against a challenge with the C56-strain. Immunization of mice with a single antigenic component, isolated by affinity chromatography with the monoclonal antibody F3G3, resulted in a significant resistance against a lethal challenge (29% of mice died) and in a complete protection when the antigen was administered in combination with Freund's incomplete adjuvant. The epitope recognized by F3G3 as well as by an analogous monoclonal antibody was located beneath the Toxoplasma cell surface membran e. The antigens recognized by both monoclonal antibodies were antigenically related proteins of 58 and 28 kD (99). Scbtoartzmann (92) reported the inhibition of a penetration-enhancing factor of Toxoplasma gondii by various monoclonal antibodies specific for rhoptries . Another important contribution of today 's research towards a vaccine is the recombinant DNA technology. Two groups of investigators have isolated and translated mRNA from tachyzoites of the RH strain: The Remington group in the United States and the Johnson group in Australia. The group of Remington has translated mRNA in a wheat germ system (85). Tachyzoite ant igens synthesized in vitro were recognized by Toxoplasma gondii-specific antibodies obtained from rabbit s, mice and humans. The Johnson group has translated mRNA in a cell-free system derived from rabbit reticulocyte lysate (44).11 out of 16 translated polypeptides were immunoprecipitated by antiToxoplasma mouse and human sera. The next step, just now under investigation, is the screening of the complementary DNA library for expression of Toxoplasma gondii antigens by the use of monospecific antibody probes and the cloning and expression of genes coding for antigens protective against infection with Toxoplasma gondii. In order to identify antigenic determinants, mutants of Toxoplasma gondii selected by coincubation with monoclonal antibodies will become important. Two different mutants lacking membrane proteins of 22 kD and 30 kD that are present on the wild type have already been established (49, 51).
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A completely different approach towards a vaccine might be found by induction of immunity at the mucosal sites of the gut. Since the beginning of this decade, knowledge about the gut-associated lymphoid tissue (GALT) and the gut immune system has enormously increased. Several efforts towards an orally administered vaccine have been made against viral and bacterial infections using killed organisms (Poliovirus,
Shigella flexneri, Vibrio cholerae, Streptococcus mutans, Escherichia coli, Salmonella spp.). Regarding parasitic infections, antigen-exclusion directed against whole organisms by specific secretory IgA in combination with other factors has been described (48,61). Davis et al. (16, 17) have shown a secretory IgA-mediated in vitro inhibition of the cell penetration and intracellular development of sporozoites of Eimeria tenella, a parasite closely related to Toxoplasma gondii, by a synergism of specific secretory IgA antibodies and non-specific factors. Secretory IgA specific to Toxoplasma gondii after oral infection of mice with bradyzoites has been described by McLeod and Mack (71). We have reported the induction of a humoral immune response in rabbits after enteral administration of killed Toxoplasma tachyzoites (34). Further investigations will be necessary to determine the specific secretory IgA response at the gut mucosal site after oral or enteral immunization with antigen of Toxoplasma gondii. As Toxoplasma gondii is acquired in nature by the oral route, the gut mucosal immune system acts as the first barrier against infection. Immune defense at this site would hit the parasite in a very vulnerable life cycle stage (after excysting from cysts or oocysts) and possibly might lead to (partial) antigen exclusion. Induced protectivity would be of great value against an acquisition of Toxoplasma gondii for HIV-positive, Toxoplasma-negative individuals to protect them completely against infection and, secondly, to inhibit Toxoplasma multiplication in the eat's intestine and thus prevent oocyst shedding. Although it remains doubtful today whether specific secretory IgA antibodies in combination with unspecific lytic factors ever will be able to provide sufficient immunity to act as defense against an enteral invasion by Toxoplasma gondii, an orally administered vaccine, no matter whether of molecular structure or not, will presumably support immune defense in cooperation with a parenterally applied vaccine.
4. Future Prospects Current research projects allow a clear and concrete view of future developments. Efforts in toxoplasmosis research and in other domains of parasitology are predominantly directed towards a molecular vaccine. The advantages are striking: a molecular vaccine is precisely defined, therefore quality control and monitoring of stability of the vaccine are facilitated. The probability of inducing undesirable immune responses, toxic reactions and harmful side effects following vaccination is reduced. Antigens of life cycle stages that cannot easily be obtained in large quantities (e.g. antigens of sporozoites or of bradyzoites) may be synthesized in a sufficient amount. The immuneresponse in the vaccinee can be more accurately measured and the efficacy of the vaccine more precisely determined. A molecular vaccine, however, may also entail some disadvantages. These derive from the restricted antigenic composition of such a vaccine (in contrast to crude extracts or whole organisms which present multiple antigens to the immune system) and from the biological adaptability of the pathogenic organisms. Theoretically, the
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use of a molecular vaccine might select "antigen-negative" parasites - i.e. parasites that do not express the antigens or epitopes which were used for vaccination (74). Furthermore, we have to take into account that there are stage-specific antigenic differences between Toxoplasma tachyzoites, bradyzoites, and sporozoites (9,50,54, 63, 64) and that therefore the human immune-response to Toxoplasma gondiipresumably will vary depending on the antigens and stages which have been used for immunization. We have to consider also that antigen analysis by SDS-PAGE and the molecular cloning technique are confined to a detection of protein antigens of Toxoplasma gondii, while host-protective antigens of a carbohydrate or glycolipid composition would not be detected by this means. We know, however, that membrane of Toxoplasma gondii does contain antigenic glycoconjugates. Pande et al. (79) first described a heteroglycan of Toxoplasma gondii. In contrast to previous findings by Handman et al. (30) and Sethi et al. (96), Mauras et al. (69) revealed the presence of carbohydrates on the surface membrane of Toxoplasma tachyzoites. Subsequent studies by Johnson et al. (45) have shown that at least three glycosylated polypeptides are present in the insoluble fraction of disrupted parasites. Hughes and Balfour (38) described one out of nine distinct antigens (recognized by human antibodies) to be a lipopolysaccharide and one, a glycoprotein. Mineo et al. (72) used a Toxoplasma polysaccharide fraction in an ELISA and found a preferential binding of specific IgM antibodies. Naot et al. (77) reported that polysaccharides extracted from tachyzoites reacted with both human IgM and IgG antibodies and that lipids were not recognized by either IgM or IgG antibodies. Another point we have to pay attention to is the heterogeneity of the parasite population, that means strain differences of different geographical isolates or in different host reservoirs (7, 8, 68). Antigenic diversity among Toxoplasma strains and differences in the antibody response have been described recently by Ware and Kasper (108), and Weiss et al. (109). Likewise, we have to pay attention to genetically determined differences in the host population and hence to differences in the immune response to antigens (100). No investigations have been carried out so far to determine whether antigen variation occurs with Toxoplasma gondii. At any rate, a critical subset of antigens or epitopes will have to be found that is not labile or changeable and that is capable of inducing protection in the majority of individuals in a genetically diverse host population. Another strategy to maintain an adequate level of protective immunity would be the replacement of antigens in the vaccine at regular intervals (74). The latter, however, seems to be far away from practical use due to the high costs it would involve. Although the humoral branch of the immune system is by far more easily accessible to investigation than the cellular one (which is expressed by the fact that the majority of studies is concerned with the B-cell-derived immune response) we have to keep in mind that cell-mediated immunity is the predominant effector mechanism in antiToxoplasma defence. Besides numerous experiments dealing with macrophage activation and adoptive transfer of immunity by lymphocytes, three recent studies call for a more detailed reflection: Reyes and Frenkel (88) attempted to characterize specific soluble mediators of Toxoplasma immunity and suggested a positive correlation between the rate of appearance of Toxoplasma mediator and the expression of antiToxoplasma tissue immunity, yet did not rule out the possibility that antibodies might have contributed to parasite elimination. Eisenhauer et al. (22) demonstrated a reduction of the numbers of Toxoplasma cysts in the brains of mice by a joint action of both
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humoral and cellular (non-specifically induced) mechanisms, whereas Brinkman et al. (11) concluded from their results on the correlation between antibody response, antigen-specific T-cell activation and brain cyst formation that the major part of protective immunity against Toxoplasma gondii was cell-mediated and that high humoral response did not inhibit, but more likely favoured cyst formation. It is evident that more detailed information about the effector mechanisms of anti-Toxoplasma defence is required and that we will have to decide whether an oligopeptide vaccine should be favoured which consists of antigenic determinants that predominantly stimulate T-cell immunity. Foreseeabledifficulties will emerge in the search for such determinants and their presentation to the immune system. Once an immunizing antigen has been defined it possibly will not convey the complete spectrum of immunity required for host protection. It might be necessary to condense several individual protective antigens in to one single vaccine. Because of its molecular structure, the vaccine will be highly dependent upon an antigen delivery system (e.g. liposomes) and on immunopotentiating adjuvants which elicit humoral and/or cell-mediated immunity. Of course the adjuvants have to be free from sideeffects to be acceptable for human use. The above mentioned points make it evident that even when a protection-inducing molecule will have been characterized and produced, it will be a long way to go until a vaccine becomes available which induces the desired protection and fulfills the requirements for application in human medicine.
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