Immune deviation as a strategy for schistosomiasis vaccines designed to prevent infection and egg-induced immunopathology

Microbes and Infection, 1, 1999, 525−534

Immune deviation as a strategy for schistosomiasis vaccines designed to prevent infection and egg-induced immunopathology Thomas A. Wynn The Schistosomiasis Immunology and Pathology Unit, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

1. Introduction While highly effective drugs are available for treatment, schistosome parasites continue to cause significant morbidity and mortality in the human population. This is due in part to the inability of chemotherapy to prevent new infections and the failure of previously infected individuals to develop an effective immune response against the parasite. Thus, a current focus of schistosomiasis research is to develop a vaccine that will significantly reduce the incidence of severe disease. In Schistosomiasis mansoni, eggs collect in the liver and intestines and induce a granulomatous response which, in a subset of individuals, leads to severe hepatic fibrosis, portal hypertension, bleeding, and ultimately death [14]. Therefore, disease in schistosomiasis is caused, not by the parasites themselves, but rather by the eggs that are produced in mass quantities by the paired adult parasites. There are three main strategies being pursued in the quest for a vaccine for schistosomiasis. The first strategy, which has received the greatest amount of attention, is focused on developing a vaccine to prevent infection [5]. The aim is to identify larval stage antigens that will elicit highly protective immune responses in vaccinated hosts. Such a vaccine should reduce worm numbers and thus egg burdens to a level where little morbidity or mortality is observed. Thus, it is generally believed that a vaccine does not need to be 100% effective to significantly reduce the incidence of disease. Experimental antifecundity vaccines have also been identified [72] and have a similar goal of reducing egg production, and therefore tissue egg numbers, to a less damaging level. The third vaccination strategy is focused on manipulating the outcome of infection rather than relying upon changes in parasite or egg numbers. In this approach, the goal is to alleviate eggassociated pathology by either suppressing the host’s immune response to the eggs [55] or by converting the normally pathogenic response to a less damaging reaction [64]. While numerous parasite antigens have been identified as potential vaccine candidates [5], relatively few have consistently provided sufficient levels of protection to Microbes and Infection 1999, 525-534

warrant further development as a vaccine for humans. The problem with many of the existing experimental vaccines, in fact, is that relatively little is known about their mechanism of action. Indeed, one of the fundamental obstacles in schistosomiasis research is a lack of consensus as to what type of an immune response a vaccine should invoke. There is now clear evidence that a modest level of resistance develops naturally in endemic populations [7, 8]. Moreover, studies with putatively resistant individuals suggest that naturally acquired resistance in humans correlates with IgE Ab [9, 60] and Th2 or Th0/Th2-type cytokine expression (interleukin-4/interleukin-5; IL-4/IL-5) [19]. In addition, rodent studies have demonstrated that the natural progression of schistosome infections is towards a Th2-dominant cytokine pattern [26, 48]. Thus, a vaccine that is based on eliciting this type of an immune response would seem to be an appropriate goal. Nevertheless, studies of vaccine-induced immunity in mice have identified a major role for Th1-associated cytokines (interferongamma/tumor necrosis factor-alpha; IFN-γ/TNF-α) in resistance [29, 54]. Indeed, exceptionally high levels of protection have been achieved in mice exhibiting highly polarized parasite-specific Th1 responses [71]. Thus, both cellular- (Th1 cytokine-associated) and humoral-based (Th2 cytokine-associated) resistance mechanisms have been described for S. mansoni. While a vaccine that elicits both types of protective responses would be highly desirable, it is generally believed that such a vaccine may be difficult to develop. This is primarily due to the mutually antagonistic nature of humoral vs. cell-mediated immunity as well as Th2 vs. Th1-type CD4+ T-cell responses [3, 44]. These issues emphasize only a few of the difficulties encountered in schistosomiasis vaccine development. To better understand the mechanisms of vaccineinduced immunity and the factors leading to the development of egg-induced immunopathology, we have been investigating the immunoregulatory role of cytokines. We have been particularly interested in cytokines associated with the Th1/Th2 paradigm. To study vaccine-induced immunity, we focused on the irradiated cercariae model since this is the gold standard by which all other experimental vaccines are compared [43]. To elucidate the role 525

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of cytokines in granuloma formation, we relied upon an experimental pulmonary granuloma model [59]. Key findings from the pulmonary studies are then extended to the murine infection model where the identification of factors leading to the development of hepatic fibrosis is the primary focus. In nearly all of these studies, we used reverse transcription-polymerase chain reaction (RT-PCR) to elucidate in vivo cytokine patterns. The ultimate goal of these studies is to better understand basic mechanisms so that modern immunologically based strategies might be exploited in the development of highly effective vaccines for schistosomiasis. This review attempts to summarize some of the more recent findings in this area.

2. The pulmonary granuloma model: evidence of a primary role for Th2associated cytokines in egg-induced granuloma formation In the pulmonary granuloma model, live eggs are purified from the livers of infected mice and then injected intravenously into lungs of either naïve, infected, or eggsensitized mice. In this model, in contrast to the natural infection, eggs lodge in the lungs and initiate a synchronous inflammatory response where changes in cytokine expression can be more closely linked with corresponding changes in granuloma formation [65]. Studies with this and related models have shown that the predominant CD4+ T helper cell response induced by eggs is phenotypically Th2-like [17, 26, 48, 65]. Detailed kinetic experiments on egg-induced granuloma formation have shown that this Th2 response develops through an early transient Th0-like stage [56]. Cell depletion studies demonstrated that the early transient IFN-γ response, which peaks as early as one day following egg challenge, is predominantly derived from NK cells [66] and plays a significant downregulatory role in pulmonary granuloma development. IFN-γ and natural killer (NK) cell depletion experiments suggested that the function of the early IFN-γ response is to suppress the developing Th2-type response, resulting in a reduction in granuloma size. These findings were confirmed in studies of granuloma formation in IFN-γ-deficient mice [67]. A similar conclusion was also derived from studies examining the role of endogenous IL-12. In these experiments, IL-12 neutralization upregulated pulmonary granuloma size as well as the production of several Th2-associated cytokines, while treatment with recombinant IL-12 downregulated the Th2-type response, upregulated IFN-γ production, and markedly decreased granuloma size [66]. More direct evidence of a major role for Th2 cytokines in pulmonary granuloma formation came from IL-4 depletion studies. In these experiments, neutralization of IL-4, but not IFN-γ, resulted in a marked reduction in granuloma size [17, 65, 66] and correspondingly suppressed the Th2-type cytokine mRNA response [65]. While differences in pulmonary versus liver granuloma formation following anti-IL-4 treatment have been described [24], studies in naturally infected mice confirmed 526

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that IL-4 plays a major role in the pathogenesis of hepatic fibrosis and in Th2 response development [13]. Schistosome-infected IL-4-deficient mice (C57BL/6) also developed smaller liver granulomas [42]. Here, the reduction in granuloma size and tissue eosinophilia was again attributed to a decreased Th2 response. Finally, strong evidence for a primary role for Th2-associated cytokines in granuloma formation came from recent studies examining schistosome infection in Stat-6-deficient mice [33]. These mice lack a Th2-type response, and consequently developed pulmonary and hepatic granulomas that were greatly decreased in size when compared with those in control littermates. A marked decrease in liver hydroxyproline content was also observed. In contrast, Stat-4-deficient mice, that are defective in IFN-γ expression, produced Th2-type cytokines in amounts comparable with control mice and displayed a relatively unimpaired granulomatous response. Together, these studies highlight the critical role of Th2-type cytokines in both pulmonary and hepatic granuloma formation and more importantly, in the pathogenesis of hepatic fibrosis in schistosomiasis.

3. IL-4 and IL-10 are both essential to the development of a polarized eggspecific Th2-type cytokine response Because Th2-associated cytokines correlated with maximal granuloma formation and hepatic fibrosis, an important issue stemming from these observations was to identify the factors that regulate Th2 response development in schistosomiasis. Indeed, many factors have been shown to influence Th2 cell development in vivo including Ag dose, route of Ag delivery/exposure, expression of specific costimulatory molecules, activation of different Ag-presenting cell populations, genetic factors, and probably most importantly, the cytokine milieu [50]. While IL-4 is recognized as an almost indispensable factor in Th2 cell development, the identification of its cellular sources during the early phases of an immune response has been an important area of study. Several sources of early IL-4 have been described including CD4+ /NK1.1+ T cells, γδ T cells, non-B, non-T /FceR+ cells, mast cells, basophils, and eosinophils [4, 15, 51, 57, 62]. Recently, Sabin and Pearce examined this question in schistosomiasis using an intraperitoneal egg injection model [51]. In these studies, they observed a transient elevation in IL-4 production as early as 2 to 12 h after egg exposure. The pattern was also observed in egg-injected CD4+ T-cell-depleted and nude mice, which strongly suggested that a non-T cell was the source of the early IL-4. Subsequent studies showed that the peak in early IL-4 production was accompanied by a local eosinophilia and a disappearance of mast cells. Further studies in IL-5 and mast-cell-deficient mice indicated that the early eosinophilia was dependent on both mast cells and IL-5. More importantly, the egg-induced IL-4 response was absent in animals lacking the early peritoneal eosinophilia. They concluded from these studies that a rapid egg-induced mast-cell-derived IL-5 response recruits eosinophils to the site of antigen deposiMicrobes and Infection 1999, 525-534

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tion and that eosinophils provide the early burst of IL-4 which is required for subsequent Th2 cell development. While the formation of these tissue lesions is clearly dependent upon an IL-4-driven Th2 response, we have also noted a marked repression in IL-12 production soon after egg injection [66]. Since IL-12 is a key cytokine driving Th1 cell differentiation, this observation suggested that the development of a polarized Th2-type cytokine response may, in addition to an early IL-4 response, be equally dependent on the active suppression of cytokines or signals involved in Th1 cell differentiation. We have hypothesized that downmodulation of IL-12 synthesis is a critical step in establishing the polarized Th2-type response and that IL-4 and IL-10 are the direct mediators of this inhibition. To formally define the role of IL-4 and IL-10 in Th2 response development in vivo, we studied the generation of egg-induced Th2 responses in mice deficient in IL-10, IL-4, or doubly deficient in both mediators. In IL-10-deficient mice, a marked increase in IFN-γ expression was observed in the granulomatous tissue and in the lung draining lymph nodes after i.v. egg challenge; nevertheless, the expression of several Th2-associated cytokines was similar to or, in most experiments, increased in the cytokine-deficient mice [69]. Thus, the animals appeared to develop a less polarized or mixed Th1/Th2-type cytokine response. IL-4-deficient mice, while showing a markedly reduced Th2 response, displayed only a slight increase in IFN-γ expression, findings that were also recently reported in the spleens of S. mansoni-infected IL-4deficient mice [42]. Only animals deficient in both IL-4 and IL-10 showed dramatic increases in IFN-γ and simultaneous reductions in Th2-type cytokines both in vitro and in vivo [69]. Moreover, the repression in IL-12 production was totally reversed in the double cytokine-deficient mice. Apparently, IL-4-deficient mice fail to default to a robust Th1 response because they maintain a significant non-Tcell-derived IL-10 response. Together, these results demonstrate that the early production of IL-4 and IL-10 are equally important to Th2 response polarization in vivo, with IL-4 driving Th2 development and IL-10 polarizing the response by downregulating IL-12 and Th1-type cytokine expression. Interestingly, egg-injected IL-10-deficient mice treated with anti-IL-12 mAb showed more than a 10-fold increase in lymph node and spleen cell production of IL-4 and IL-5 upon in vitro activation [69]. This was also associated with a significant increase in the number of IL-4-producing spleen cells. The highly polarized and exacerbated Th2 response was not seen in wild type mice treated with anti-IL-12 mAb, which suggests that endogenous IL-10 in these animals serves as a potent inhibitory signal not only for Th1 cytokine expression as was previously demonstrated, but for Th2 responses as well. Thus, two main pathways for suppressing Th2 responses were demonstrated in these experiments; the counterregulatory Th1type IFN-γ response and IL-10, with the latter mechanism being better appreciated in animals only after elimination of both inhibitory pathways. Together, these results demonstrate that IL-10 plays a critical function in polarizing Th2 responses as well as quantitatively controlling their evolution (see figure 1). Microbes and Infection 1999, 525-534

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4. Pulmonary egg-induced granuloma formation is mediated by the dual effects of IL-4 and IL-13 IL-4 neutralization studies, both in infected mice [13] and in animals intravenously challenged with schistosome eggs [17, 65], clearly demonstrated an important role for this cytokine in driving the egg-induced Th2 response, granuloma formation, as well as the accompanying rise in serum IgE titers. Nevertheless, in these reports as well as in studies performed recently in IL-4-deficient mice, it has become clear that a reduced but significant Th2-type cytokine response can develop and that marked, albeit diminished, granulomatous inflammation is observed even in the absence of IL-4 [42, 49, 69]. These findings suggested that the host response to schistosome eggs may not be totally dependent upon IL-4 and that a second Th2-associated cytokine, IL-13, might serve as an important mediator of the egg-induced granulomatous response. The latter hypothesis stemmed from two recent observations. Firstly, IL-13 was shown to share numerous functional activities with IL-4 [73] and secondly, a significant level of IL-13 mRNA expression was induced in the lungs of both WT- and IL-4-deficient mice after intravenous challenge with schistosome eggs [66, 69]. The second observation in particular suggested that IL-13 might possess important granuloma-inducing activities that are independent of IL-4. To examine the role of IL-13, the functional activity of the cytokine was blocked in vivo by treating mice with sIL-13Ra2-Fc. This protein, which effectively blocks IL-13 but not IL-4 from binding to its receptor in vivo [23], was used to dissect the contribution of IL-13 to pulmonary granuloma formation in both WT and IL-4-deficient mice. In these studies, IL-13 blockade significantly reduced the size of pulmonary granulomas in both unsensitized as well as egg-sensitized WT mice [18]. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly however, IL-13 blockade did not effect the evolving egginduced Th2-type cytokine response. Indeed, Th2associated cytokine levels were indistinguishable in control Fc- and sIL-13Ra2 Fc-treated animals, thus confirming that IL-13 plays little or no role in Th2 response development [73]. The smaller granulomas in sIL-13Ra2-Fctreated animals were also phenotypically like control Fctreated mice, displaying a similar eosinophil content. Subsequent studies in IL-4-deficient mice demonstrated that IL-13 was produced in response to eggs, but at levels much lower than in WT mice, while IL-4 secretion was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development as well as the pulmonary eosinophilia. These studies showed an important effector role for IL-13 in pulmonary egg-induced granuloma formation and demonstrated that IL-13 plays an equally, if not more, substantial role as IL-4. Perhaps more importantly, however, these findings suggested that therapeutic strategies based on blocking both IL-4 and IL-13 might prove highly effective at ameliorating a host of 527

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Figure 1. Polarizing the cytokine response to S. mansoni eggs. As proposed schematically in figure 1, WT mice require both an IL-4 and an IL-10 response to polarize an egg-specific Th2 response. Depletion of IL-12 alone has only a minimal effect on Th2 development, since essentially little or no IL-12 production is triggered by schistosome eggs. Because IL-4-deficient mice appear to maintain a significant non-T-cell-derived IL-10 response, they develop only a very weak Th1- and reduced Th2-type response to eggs, while IL-10-deficient mice develop a codominant Th1/Th2 or Th0-like cytokine pattern. Only mice doubly deficient in IL-12/IL-10 or IL-4/IL-10 develop highly polarized and exacerbated Th2 or Th1-type cytokine responses, respectively. Therefore, these double cytokine-deficient mice should provide a valuable tool for studying the mechanisms of resistance and egg-induced immunopathology that develops on both poles of the immune response.

related maladies, such as allergy and asthma, which are also triggered by Th2-associated cytokines.

5. Characterization of an IL-12-based vaccination strategy that reduces egginduced pathology and Th2-type cytokine expression The previous sections highlighted the important role of the Th2-associated cytokines IL-4, IL-10, and IL-13 in mediating many aspects of schistosome egg-induced pathology and disease. As also described above, treatment of egg-injected mice with recombinant IL-12 has proven to be a highly effective strategy for suppressing antigenspecific Th2 responses as well as granuloma formation in both naïve and egg-sensitized animals [66]. The ability of IL-12 to suppress both primary and secondary egginduced Th2 responses suggested the possibility of prophylactically immunizing mice against granulomatous pathology by immune deviation. Indeed, sensitizing with egg antigens in the presence of exogenous IL-12 was shown to significantly inhibit pulmonary granuloma formation in mice subsequently challenged with eggs [66]. As predicted, the pulmonary cytokine response in egg/IL-12sensitized animals deviated from the normal Th2 dominant response to one characterized by expression of high levels of IFN-γ. 528

The previous findings were derived from studies using the model of pulmonary egg granuloma formation. It was important to determine whether this ‘anti-pathology’ vaccination approach would protect animals from granuloma formation and hepatic fibrosis resulting from natural schistosome infections. For these studies, mice were immunized three times, either with live eggs or soluble egg antigens in the presence or absence of IL-12 and then challenged with cercariae, the infective stage of the parasite. Eight to 16 weeks later, the mice were sacrificed and the effects of vaccination on hepatic pathology were assessed. While the immunization protocol had no effect on worm or egg burdens and only modestly reduced granuloma size in the liver, it had dramatic effects on the development of hepatic fibrosis [64]. Compared with control mice, mice sensitized with eggs/IL-12 showed reductions in hepatic hydroxyproline (a chemical measure of collagen content) of between 58 and 72% by week 12 postinfection. There was also nearly a 10-fold reduction in the levels of induced expression of liver type I and type III collagen mRNAs relative to unsensitized mice. Again, the reduced pathology, as previously observed in the pulmonary model [66], was associated with a marked increase in Th1-associated cytokines including IFN-γ, TNF-α, and IL-12 and a significant reduction in several Th2-associated cytokines. We hypothesized that the reduction in fibrosis observed in the egg/IL-12-sensitized mice resulted from both the Microbes and Infection 1999, 525-534

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reduced expression of collagen-inducing cytokines such as transforming growth factor-beta (TGF-β) and IL-4, and a corresponding increase in cytokines like IFN-γ and/or TNF-α, which are known to inhibit collagen synthesis [21]. Recently, we examined the issue of whether the induced expression of IFN-γ, IL-12, or TNF-α was contributing to the changes in pathology [28]. In these studies, egg/IL-12-sensitized mice were infected percutaneously and five weeks later, at the start of egg production, injected twice weekly with neutralizing mAbs to IFN-γ, IL-12, or TNF-α. The animals were sacrificed three weeks later at the peak of granuloma formation. With this protocol, the effector function of the various Th1-associated cytokines could be examined in mice with an established eggspecific Th1-type memory response. Interestingly, in contrast to egg/IL-12-sensitized animals which showed marked decreases in pathology, mice similarly sensitized but depleted of IFN-γ, IL-12, or TNF-α at the time of egg laying, developed granulomas which were similar to the non-IL-12-treated control group. There was also a significant increase in hepatic fibrosis in each of the anticytokine treated animals. Interestingly, while all three groups exhibited a strong Th1-type cytokine response in antigen-stimulated lymphocyte cultures, the expression of several Th2-associated cytokine mRNAs was markedly restored at the site of granuloma formation. The latter observation likely explained the restoration of egginduced pathology and tissue eosinophilia in the anticytokine treated egg/IL-12-sensitized mice. These findings demonstrated that IFN-γ, IL-12, and TNF-α all contribute to the pathology-reducing effects afforded by the egg/IL-12 sensitization protocol. Moreover, they suggested that highly polarized Th1-type cytokine responses generated against strong Th2-inducing stimuli are potentially unstable, and require multiple Th1-associated cytokines for their maintenance in vivo. Although reducing egg-associated pathology is an attractive approach for ameliorating disease in schistosomiasis, it should be pointed out that while granulomas are potentially pathogenic they are also essential to the survival of the infected host. Indeed, nude and SCID mice, which fail to form effective granulomas, develop severe liver necrosis and die early during infection [2, 11]. The increased mortality in these animals was attributed to the hepatotoxic effects of soluble factors released by the eggs. Interestingly, infected C57BL/6 IL-4-deficient mice, which are also unable to mount efficient Th2-responses, displayed enhanced morbidity and died soon after egg laying began [6]. The authors of the latter study hypothesized that Th2 cytokines were required to downregulate expression of the proinflammatory mediators IFN-γ, TNF-α, and nitric oxide (NO) and that the overproduction of these factors, in infected IL-4 deficient mice, led to inflammation in the intestine, leading to more severe disease. Thus, Th2-associated cytokines appear to serve both a hostprotective and pathogenic role in schistosomiasis, by both suppressing proinflammatory mediators and inducing hepatic fibrosis. Therefore, it is generally believed that disease in schistosomiasis may best be controlled by carefully regulating the balance between Th1 and Th2-associated cytokines. Microbes and Infection 1999, 525-534

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Interestingly, mice sensitized with eggs and IL-12 appear to be a good example in which the balance between type 1 (IFN-γ) and type 2 (IL-10) cytokine-associated expression may have been achieved. We have not observed any increase in morbidity in these animals and they display a striking reduction in the development of hepatic fibrosis, the major cause of disease in schistosomiasis [64]. We have also noted little tissue necrosis in or surrounding the granulomas, suggesting that the animals are effectively walling off and neutralizing the harmful substances released by the eggs. While the sensitized animals do show a marked elevation in the expression of IFN-γ and TNF-α during the acute stage of infection, these responses are downmodulated in the chronic stage of the disease [64]. More importantly, the animals fail to develop the unusual pathology that was reported in one study of IL-4-deficient mice [6]. We hypothesize that the increased expression of IL-10 in the granulomatous tissues of the egg/IL-12-sensitized mice [64] is an important counterregulatory signal that controls the overproduction of IFNγ/TNF-α, and NO in these animals. It is suspected that this IL-10 is derived from cells other than CD4+ Th2 cells, since little or no IL-4, IL-5, or IL-10 is produced by antigenactivated lymphocytes [64]. Thus, the sensitized animals develop an increased IFN-γ response, which likely explains the marked decrease in collagen synthesis and hepatic fibrosis [21]. Perhaps more importantly, however, they maintain the balance in this protective response by upregulating the expression of the counterregulatory cytokine IL-10. We are currently using neutralizing mAbs to IL-10 as well as IL-10-deficient mice to further dissect the role of this important immunoregulatory cytokine in egg/IL-12-sensitized animals. Minimally, these data argue that immune deviation might be exploited as a strategy for reducing pathology in schistosomiasis.

6. The irradiated cercariae model: characterization of the resistance mechanisms in singly and multiply immunized mice An important model used in the study of schistosome immunity is the induction of resistance in mice by immunization with radiation-attenuated cercariae. After a single exposure to this vaccine, animals eliminate 60 to 80% of the worms ordinarily developing from a challenge infection and only a modest increase in protection is observed after multiple exposures to the irradiated parasites [10]. Nevertheless, the mechanisms of resistance in both singly (1X) and multiply immunized (2X) animals appear to be at least partially distinct. Resistance in the 1X model is not easily transferred to naïve mice with serum, is highly dependent on CD4+ T lymphocytes [58], and appears to be associated with the function of Th1 rather than Th2 type cytokines. Thus, treatment of 1X-vaccinated mice with antibodies to IFN-γ causes a marked reduction in immunity, while antibodies against IL-4 and IL-5 are without effect [52, 53]. In contrast to these findings, immunity induced by the 2X model associates with a mixed 529

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Th1/Th2-type response [10], is less dependent on IFN-γ, and involves antibody since significant protection can be passively transferred with serum [40, 71]. Similar to the granuloma studies described above, we have been using both the 1X and 2X models to investigate the contribution of specific cytokines to the development of protective immunity. The ultimate goal of this work is to elucidate the major mechanisms of resistance in this experimental model so that defined subunit vaccines might be developed which elicit similar protective mechanisms. While multiple mechanisms likely participate in the process of parasite elimination [22, 34, 63], several lines of evidence suggest that cell-mediated immunity involving IFN-γ-activated effector cells is a major mechanism of protective immunity in the 1X model. Indeed, previous observations of a correlation between macrophage activity and resistance to S. mansoni infection in the 1X model [32], along with the larvicidal function of NO produced by IFN-γ-activated macrophages and endothelial cells [31], and the reduction of resistance resulting from in vivo treatment with an inhibitor of NO production [70], led us to postulate that a major effector mechanism of vaccine-induced resistance involves Th1associated immune responses leading to NO production. Surprisingly, however, two recent studies examining vaccine-induced immunity in inducible NO synthase(iNOS)-deficient mice have suggested that NO plays only a partial role in immunity to S. mansoni [20, 30]. Although significantly reduced when compared with vaccinated WT animals, the iNOS-deficient mice maintained a marked level of vaccine-induced immunity [30]. Interestingly, however, the decrease in protection was associated with marked increases in the expression of nearly every other mediator of resistance that has been proposed in this model, including IFN-γ, TNF-α, and IgG antibodies. Thus, in addition to its direct anti-parasite effector function, NO appears to serve as a potent immunoregulatory factor as well. Increased spleen cell proliferation and/or release of IFN-γ in the absence of NO in vivo has been reported in mouse models of leishmaniasis [61], toxic shock syndrome [39], bacterial septic arthritis [41], and herpes simplex virus infection [38]. Exaggerated production of TNF-α has also been observed in several of these reports, including our study of the 1X-irradiated cercariae vaccine. Therefore, it remains unclear whether the results obtained with iNOS-deficient mice accurately reflect the role of NO in resistance in WT animals. These results suggest that NO plays a dual and contradictory role in immunity to S. mansoni, by participating in both the effector mechanism of resistance and by downregulating the Th1-type cytokine response, which is ultimately required for NO production. Thus, NO, like IL-4 described above [6], appears to exhibit both positive and negative effects. While NO is clearly a potent anti-parasite effector molecule [31], it may also play an important role in parasite survival by downregulating the induction of related protective Th1type responses. These results reveal the difficulties inherent in interpreting results obtained solely from knockout animals. Interestingly, however, as suggested recently [38], these studies indicate that iNOS-inhibitors may, in fact, be useful adjuvants for vaccination where an 530

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enhanced Th1-type cellular and/or humoral immune response is required for protective immunity. Such an approach might generate a more polarized Th1-type response, as was observed in iNOS-deficient mice [30], but at the same time allow for the downstream anti-parasite activity of NO.

7. Evidence of a mixed Th1/Th2-type response in the lungs and lymph nodes of vaccinated and challenged mice Mice vaccinated with irradiated cercariae display a significant reduction in parasite burden following challenge infection, whereas animals exposed to normal parasites develop little immunity. Because the lungs are the primary site where parasite attrition occurs in immunized animals, we compared the changes in pulmonary cytokine mRNA expression during a primary exposure to normal and attenuated parasites [68]. Interestingly, the most obvious difference observed in these experiments was in the kinetics of IFN-γ induction. Although unattenuated parasites triggered an IFN-γ mRNA response, the increase was significantly delayed when compared with irradiated cercariae and followed the induction of multiple Th2associated cytokines. Irradiated parasites, by contrast, simultaneously induced a codominant or mixed Th1/Th2type response. A mixed response was also reported in vaccinated mice after administration of a challenge infection [70]. While neutralization of IFN-γ in these animals resulted in a decrease in IL-12 p40, TNF-α, and IFN-γ mRNA expression, it simultaneously upregulated the expression of several Th2 cytokines including IL-4, IL-5, and IL-13 [70]. The shift in the dominant cytokine profile from Th0- to Th2-like in the anti-IFN-γ-treated mice correlated with a significant decrease in immunity. Thus, while vaccinated mice develop a cytokine microenvironment in the lung that is conducive to the activation of anti-parasite effector cells [70], the significant expression within the same tissues of IL-4, IL-10, and IL-13 suggested that Th1associated effector mechanisms may not be operating optimally in vivo. Indeed we hypothesize that the coexpression of several Th2-associated cytokines, which are known to inhibit macrophage/endothelial cell activation [47], might partially explain the inability of the 1Xattenuated cercariae vaccine to provide complete protection.

8. IL-12 enhances immunity induced by the irradiated cercariae vaccine by boosting type-1-associated humoral and cell-mediated immune responses against the parasite In an attempt to increase the efficacy of the irradiated cercariae vaccine, we used IL-12, a potent inducer of IFN-γ production [35], as an adjuvant to drive Th1responses and thereby suppress Th2 cell differentiation. In Microbes and Infection 1999, 525-534

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these experiments, when combined with irradiated cercariae, IL-12 significantly enhanced protection, whereas IL-12 administration alone had no effect on immunity [68]. The decrease in worm burdens correlated with a marked increase in Th1-associated cytokines at both the mRNA and protein level. By contrast, IL-4 and IL-5 production was markedly suppressed, a finding which likely explains the significant reduction in serum IgE levels and tissue eosinophilia in 1X-vaccinated/IL-12-treated mice. A similar Th0- to Th1-like switch in the cytokine profile was also recently reported in multiply immunized mice, when exogenous IL-12 was included during the period of vaccination [71]. In the latter study, the IL-12/vaccinated mice displayed an even more striking increase in protective immunity, with some animals demonstrating complete protection. We have hypothesized that the enhanced protection in the multiply immunized IL-12-treated mice, in contrast to 1X-immunized animals, results from an enhancement in both humoral and cell-mediated protective mechanisms. We observed that these animals develop a strongly polarized Th1-type response, but perhaps more importantly, they also show significant increases in parasite-specific Ab. Moreover, passive transfer experiments demonstrated an enhanced ability of serum from these animals to protect naïve recipients. The animals vaccinated in the presence of IL-12 also developed macrophages with increased NO-dependent killing activity against the parasites. Together, these studies demonstrated that IL-12, which was initially described as an adjuvant for cell-mediated immunity [1], is also highly useful for simultaneously promoting both humoral and cell-mediated protective responses against infection. Importantly, more recent studies have shown that IL-12 can also enhance immunity induced by soluble antigens prepared from lung-stage parasites [45]. The latter observation in particular highlights the potential for developing an IL-12-based protocol to enhance immune responses to some of the defined schistosomiasis vaccine candidates, which to date have yielded variable and often disappointing results [25].

9. Summary and conclusions The data presented here demonstrate that IL-12 can augment vaccine-induced protective immunity against S. mansoni and, when administered together with egg antigens prior to infection, markedly reduce granuloma formation and the development of hepatic fibrosis. Both ‘immune deviation’ protocols appear to operate primarily by suppressing parasite-induced Th2 responses. These findings suggest that IL-12 might be used in the formulation of a combined anti-parasite/anti-pathology vaccine that lessens disease in schistosomiasis by both reducing worm burdens and inhibiting the development of eggrelated tissue pathology. Thus, the use of cytokines as adjuvants offers a rational approach for immunomodulation when the effector mechanism of a particular vaccine is known. Whether similar vaccination strategies, which are based on deviating the natural immune response, can be developed for use with defined schistosome antigens or specific subunit vaccines has yet to be established. MoreMicrobes and Infection 1999, 525-534

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over, while numerous anti-parasite vaccine candidates have been described and can now be tested [25], relatively little information exists regarding egg-specific antigens that might be developed as targets of an antipathology vaccine [16, 27]. Thus, although human and rodent studies have shown that schistosome infection evokes a predominantly Th2type response and epidemiological studies point to a role for Th2 cytokines, IgE, and eosinophils in acquired resistance to infection, we favor the development of vaccines for schistosomiasis which elicit protection through Th1dependent mechanisms. This bias is based primarily on the following observations: i) an extremely high level of protective immunity is induced in mice by a Th1-type response, ii) vaccinated animals benefit from a strong cellular and humoral immune response, iii) egg-induced hepatic fibrosis, the major cause of disease in schistosomiasis, correlates with a Th2 response in mice, iv) the parasite normally exists in a Th2 milieu in both mice and man, and v) if Th2 cytokines mediate egg-related pathology in infected humans in manner similar to that described for mice, it is possible that vaccines based on evoking Th2-dependent protective mechanisms might inadvertently trigger more severe pathology. The last observation is an especially important consideration since no vaccine is expected to give 100% protection from infection and several studies now have clearly demonstrated the existence of adult parasite antigens that can sensitize for egginduced granuloma formation [12, 36, 37]. Nevertheless, recent studies examining cytokine patterns in hepatosplenic patients [46], as well as experiments performed in infected IL-4-deficient mice [6] suggest that infection-related pathology may also correlate with Th1type responses. These observations emphasize the complexity of developing a vaccine to combat this disease and reaffirm the need for more basic studies on the mechanisms of protective immunity and pathogenesis in schistosomiasis, in both mice and man.

Acknowledgments I would like to thank the many people who participated in the work described in this review including Alan Sher, Allen Cheever, Dragana Jankovic, Stephanie James, Pat Caspar, Sara Hieny, Monica Chiaramonte, Karl Hoffmann, Matthias Hesse, Bill Gause, Debra Donaldson, Tammy Neben, Alicia Reynolds, Mette Strand, Isabelle Oswald, Isam Eltoum, Fred Lewis, Barbara Clark, Werner Müller, Ralf Kühn, Sandy Morse, Tanya Scharton-Kersten, and Renate Morawetz.

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