Safety and efficacy of the rSh28GST urinary schistosomiasis vaccine: A phase 3 randomized, controlled trial in Senegalese children.

BACKGROUND: Urinary schistosomiasis, the result of infection by Schistosoma haematobium (Sh), remains a major global health concern. A schistosome vaccine could represent a breakthrough in schistosomiasis control strategies, which are presently based on treatment with praziquantel (PZQ). We report the safety and efficacy of the vaccine candidate recombinant 28-kDa glutathione S-transferase of Sh (rSh28GST) designated as Bilhvax, in a phase 3 trial conducted in Senegal. METHODS AND FINDINGS: After clearance of their ongoing schistosomiasis infection with two doses of PZQ, 250 children aged 6-9 years were randomized to receive three subcutaneous injections of either rSh28GST/Alhydrogel (Bilhvax group) or Alhydrogel alone (control group) at week 0 (W0), W4, and W8 and then a booster at W52 (one year after the first injection). PZQ treatment was given at W44, according to previous phase 2 results. The primary endpoint of the analysis was efficacy, evaluated as a delay of recurrence of urinary schistosomiasis, defined by a microhematuria associated with at least one living Sh egg in urine from baseline to W152. During the 152-week follow-up period, there was no difference between study arms in the incidence of serious adverse events. The median follow-up time for subjects without recurrence was 22.9 months for the Bilhvax group and 18.8 months for the control group (log-rank p = 0.27). At W152, 108 children had experienced at least one recurrence in the Bilhvax group versus 112 in the control group. Specific immunoglobulin (Ig)G1, IgG2, and IgG4, but not IgG3 or IgA titers, were increased in the vaccine group. CONCLUSIONS: While Bilhvax was immunogenic and well tolerated by infected children, a sufficient efficacy was not reached. The lack of effect may be the result of several factors, including interference by individual PZQ treatments administered each time a child was found infected, or the chosen vaccine-injection regimen favoring blocking IgG4 rather than protective IgG3 antibodies. These observations contrasting with results obtained in experimental models will help in the design of future trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00870649.

Safety and immunogenicity of rSh28GST antigen in humans: phase 1 randomized clinical study of a vaccine candidate against urinary schistosomiasis.

BACKGROUND: Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment. Therefore, therapeutic vaccines represent an attractive alternative control strategy. The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium (rSh28GST) in healthy volunteers, and to determine its immunogenicity. METHODOLOGY: Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide (Alum) as adjuvant (n = 8), or Alum alone as a comparator (n = 8), twice with a 28-day interval between doses. A third dose of rSh28GST or Alum alone was administered to this group at day 150. In view of the results obtained, another group of healthy volunteers (n = 8) received two doses of 300 µg of rSh28GST, again with a 28-day interval. A six-month follow-up was performed with both clinical and biological evaluations. Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production, the capacity of sera to inhibit enzymatic activity of the antigen, and in vitro cytokine production. PRINCIPAL FINDINGS: Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration. Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects. No hematological nor biochemical evidence of toxicity was detected. Immunological analysis showed that rSh28GST was immunogenic. The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST. CONCLUSIONS: rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response. Together with previous preclinical results, the data of safety, tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.

Schistosomes: the road from host-parasite interactions to vaccines in clinical trials.

Insights over recent years into the interactions between helminths, including schistosomes, and the immune system have generated new concepts in immunology and significant advances in vaccine strategies. Here, we report recent advances that substantially increase our understanding of the nature of the host innate and adaptive responses to schistosomes and on strategies elaborated by the parasite to manipulate such responses. We also describe the long road that has allowed us to move from the identification of an anti-schistosome vaccine candidate, a 28kDa glutathione-S-transferase, to its recent evaluation in human clinical trials.

Specific isotype immune response in the diagnosis of human schistosomiasis pathology?

Since the few indirect markers available for assessing the development and the stage of intestinal schistosomiasis morbidity are weakly specific, endoscopy is still the only method able to detect severe forms of pathology. Therefore, we evaluated the isotype antibody response to the current schistosome antigen preparation (soluble egg antigens [SEA]) in 142 Senegalese patients infected with Schistosoma mansoni. They were stratified into three different stages of pathology according to ultrasonographic, endoscopic, and clinical parameters (stage 1 = no detectable pathology; stage 2 = moderate morbidity; stage 3 = severe forms of pathology). Only median specific IgG4, IgE, and IgA responses changed according to the stage of pathology. The IgA level was significantly higher in stages 2 and 3 compared with stage 1, and the IgE level was higher in stage 3 compared with stage 1. A high specific IgG4 level was observed only in stage 3 and was significantly different compared with stage 2. We show an association between the variability of the specific response to SEA and the degree of morbidity, and demonstrate that IgA and IgG4 responses could be combined markers to easily discriminate the different stages of pathology due to infection with S. mansoni.

Helminth infections and allergic diseases: from the Th2 paradigm to regulatory networks.

Both helminth infections and allergic diseases are characterized by a Th2 response. The former mostly affects developing countries while the later is steadily increasing in westernized countries, since most parasitic diseases have been eradicated. However, besides the cytokine pattern, both pathologies display striking similarities regarding the cell types involved in the process, the mucosal component of the inflammatory reaction and the immunoglobulin isotypes produced, IgE and IgA. Recent advances also suggest that allergic pathologies and some other inflammatory diseases arise from a similar dysfunction of the regulatory mechanisms, existing within the immune system. Such a dysregulation leads to a rupture of tolerance towards allergen. Chronic and moderate helminth infections might be beneficial to the induction of various regulatory mechanisms, involving specific regulatory cell populations (dendritic cells and lymphocytes), inhibitory receptors, blocking antibodies and two prominent cytokines: IL-10 and TGF-b, which would decrease the incidence of allergic diseases. This mechanism could provide a new vision of the so-called "hygiene hypothesis".

Pivotal roles of the parasite PGD2 synthase and of the host D prostanoid receptor 1 in schistosome immune evasion.

Prostaglandins (PG) are important modulators of immune and inflammatory responses. We recently demonstrated that the production of PGD(2) by the helminthic parasite Schistosoma mansoni inhibits the migration of epidermal Langerhans cells (LC) to the draining lymph nodes (DLN). Here, we identify the responsible parasite enzyme as being a 28-kDa glutathione-S-transferase (termed Sm28GST). Intradermal injection of Sm28GST in wild-type (WT), but not in D prostanoid receptor (DP) 1-deficient mice abrogates the departure of LC from the epidermis after TNF-alpha or FITC treatment. During infection, DP1 deficiency restores LC migration, but does not enhance the rate of T cell proliferation in the skin DLN. However, relative to WT mice, DLN cells from DP1-deficient infected mice produce dramatically less IFN-gamma and IL-10, but equal amount of IL-4. Interestingly, infected DP1-deficient mice develop a more Th2-biased humoral immune response, a significantly reduced parasitemia and a decreased egg-induced inflammatory response in the liver and intestines. Taken together, we propose that DP1 activation by the Sm28GST-derived PGD(2) could represent a strategy for the schistosome to evade host immune defenses. We also suggest that DP1 is important in the Th1/Th2 balance of the immune response and in inflammatory reactions during infection.

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium.

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.

A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection.

We have constructed and efficiently produced and purified a candidate vaccine against schistosomiasis consisting of a novel hybrid protein in which two dominant T- and B-cell epitopes from Schistosoma mansoni 28 kDa glutathione-S-transferase (Sm28GST) antigen (a.a 24-43 and 191-212) are fused to cholera toxin B subunit (CTB). Intranasal treatment of S. mansoni-infected mice with the hybrid protein, which similar to native CTB was assembled into receptor binding pentamers, significantly reduced total worm burden and liver egg counts due to the induction of Sm28GST-specific antibodies. Immunopathologic granuloma formation in the liver was also significantly suppressed and there was an almost complete suppression of delayed-type hypersensitivity reactions to both Sm28GST and to total soluble egg antigen in infected animals. The results suggest that this type of hybrid protein could be used as a combined anti-immunopathology and anti-infection vaccine against schistosomiasis.

Prospects for a schistosome vaccine.

After some 20 years experience it is generally agreed that chemotherapy against schistosomiasis, a parasitic disease which should be considered a consequence of a chronic infection, does have significant limitations. In particular, chemotherapy does not affect transmission of the infection or the high re-infection rates and so limits the success by demanding frequently re-scheduled mass treatments. For this reason, a complementary approach that can be integrated and could sustain chemotherapy-based control programs, i.e. vaccination, is very much needed. The rationale is that drug treatment would provide short-term reduction of worm burdens and vaccination, long-term protective immune response. Vaccination can either be targeted towards the prevention of infection or to the reduction of parasite fecundity. A reduction in worm numbers is the "gold standard" for anti-schistosome vaccine development but, as schistosome eggs are responsible for both pathology and transmission, a vaccine targeted on parasite fecundity and egg viability also appears to be entirely relevant. This review considers various aspects of anti-schistosome protective immunity that are important in the context of vaccine development. The current status in the development of vaccines against the African (Schistosoma mansoni and S. haematobium) and Asian (S. japonicum) schistosomes is then discussed as the new approaches that may improve on the efficacy of the available vaccines and aid in the identification of new targets for immune attack.

Vaccine development against schistosomiasis from concepts to clinical trials.

Schistosomiasis is still a major helminth infection at the beginning of the 21st century and an important public health problem in many non-industrialised countries. As the second major parasitic disease in the world after malaria, schistosomiasis affects 200 million people, 800 million being exposed to the risk of infection. It is also estimated that 20 million individuals suffer from severe consequences of this chronic and debilitating disease responsible for at least 500,000 deaths per year.

Antigen presentation by CD1d contributes to the amplification of Th2 responses to Schistosoma mansoni glycoconjugates in mice.

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.

Functional specific binding of testosterone to Schistosoma haematobium 28-kilodalton glutathione S-transferase.

During parasitic disease such as schistosomiasis, sex hormones have an important influence on the age- and gender-dependent level of infection. Since mammal glutathione S-transferase (GST) has the ability to bind hormones and particularly sexual steroids to influence their transport, metabolism, and physiological action, we have evaluated the capacity of testosterone to bind the 28-kDa GST of the Schistosoma haematobium parasite (Sh28GST). For the first time, we have demonstrated a specific binding of testosterone to parasite GST protein with high affinity (K(d) = 2.57 x 10(-7) M). In addition, we have assessed the effect of this binding on Sh28GST enzymatic activity, a mechanism closely associated with the reduction of Schistosoma fecundity. We showed that testosterone has the functional ability to inhibit the Sh28GST enzymatic activity in a dose-dependent manner, suggesting that this hormone could be directly involved in an antifecundity mechanism. This effect seemed to be related to the binding of testosterone to one peptide involved in the enzymatic site (i.e., amino acids 24 to 43). During human infection, binding of sexual hormones to Schistosoma Sh28GST could play a key role in parasite metabolism, especially the decrease of fecundity, and could be involved in the sex-dependent immune response to Sh28GST that we have previously observed in infected adults.

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccines strategies to generate efficient protection against infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazon parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

From allergy to schistosomes: role of Fc receptors and adhesion molecules in eosinophil effector function

The dual function of eosinophils has been evidenced in protective immunity against parasites as well as in pathological manifestations during allergic disorders. We have demonstrated that a new class of IgE receptors, Fc etha RII/CD23, was involved in the functional duality of eosinophils and other proinflammatory cells. More recently, we have shown that Fc etha RI, the high affinity IgE receptor thought to be only expressed by basophils and mast cells, was involved in eosinophil-mediated cytotoxity against schistosomes as well as in mediator release. These reults favour the view that both IgE and its receptors have been primarily associated to a protective immune response, rather than to pathology. Nor only IgE receptors but also members belonging to the family of adhesion molecules can participate as co-receptors in eosinophil effector function. The inhibitory role of monoclonal antibodies to Lewis X (Le X,CD15) or to selectins in eosinophil-mediated cytotoxicity towards schistosomes and the detection of Le X and 'selectin-like' molecules on schistosomula surface indicate a double interaction mediated by selectins and their carbohydrate ligands between eosinophils and schistosomula. These results suggest new functions for these adhesion molecules, previously known to be involved mainly in cell infiltration.

Development of a vaccine strategy against human and bovine schistosomiasis. Background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output.(ABSTRACT TRUNCATED AT 250 WORDS)

Effector functions of eosinophils in schistosomiasis

The dual function of eosinophils is clearly illustred in schistosomiasis. Well equipped in membrane receptors for immunoglobulins and complement, and due to the presence of granule basic proteins, eosinophils can become cytotoxic for parasite larvae and thus participate to protective immunity. However mediators can also exert their cytolytic effect on normal cells or tissues, inducing therefore pathology. Through ADCC mechanisms against schistosome larvae in vitro involving different antibody isotypes (IgG, IgE and IgA) and also in experiments performed in vivo, eosinophils have been clearly involved in protective immunity. Although no direct evidence of the protective role of eosinophils were brought in humans, the striking association of eosinophil-dependent cytotoxic antibody isotypes with resistance to reinfection (for instance IgE and IgA antibodies), whereas in vitro blocking antibody isotypes (IgG4, IgM) were detected in susceptible subjects, strongly, suggested the participation of eosinophils in antibody-dependent protective immune response. However eosinophils could also participate to granuloma formation around S. mansoni eggs and consequently to the pathological reactions induced by schistosomiasis