A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model.

Poly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli.

Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant.

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.

[Mechanisms of formation of post-vaccinal immune response in children immunized with APDT and ADT-M preparations].

AIM: Study the mechanisms of formation of cell and humoral immunity against pertussis, diphtheria and tetanus in children immunized with immunobiological preparations (APDT vaccine and ADT anatoxin). MATERIALS AND METHODS: 30 practically healthy children (6 - 9 years of age) immunized with APDT and ADT-M preparations had TLR2, TLR4 expression determined in mononuclear cells (MNC). Vaccine preparations (APDT, ADT-M, AD-M, AT) and Corynebacterium diphtheriae gravis tox+, C. diphtheriae mitis tox- and Bordetella pertussis 345 were used as ligands. Cytokine production was determined in EIA. Content of anti-diphtheria, anti-tetanus and anti-pertussis antibodies--by PHA reaction and EIA. RESULTS: During stimulation with vaccines and B. pertussis 345 strain MNC were characterized by an increase (p < 0.05) of expression level of TLR2 and TLR4 and did not respond to stimulation with C. diphtheriae gravis tox+ and C. diphtheriae mitis tox- strains. Similar results were obtained during study of cytokine production (TNFalpha, IL-1, IL-6). A direct correlation between levels of antitoxic antibodies against diphtheria and tetanus (R = 0.486), antibacterial antibodies against pertussis and diphtheria was detected (R = 0.529). CONCLUSION: Analysis of cytokine production profile and determination of surface TLR expression can be used during evaluation of functional status of innate immunity cells and intensity of post-vaccinal immunity.

Protection against avian necrotic enteritis after immunisation with NetB genetic or formaldehyde toxoids.

NetB (necrotic enteritis toxin B) is a recently identified ß-pore-forming toxin produced by Clostridium perfringens. This toxin has been shown to play a major role in avian necrotic enteritis. In recent years, a dramatic increase in necrotic enteritis has been observed, especially in countries where the use of antimicrobial growth promoters in animal feedstuffs has been banned. The aim of this work was to determine whether immunisation with a NetB toxoid would provide protection against necrotic enteritis. The immunisation of poultry with a formaldehyde NetB toxoid or with a NetB genetic toxoid (W262A) resulted in the induction of antibody responses against NetB and provided partial protection against disease.

Novel vaccine formulations against pertussis offer earlier onset of immunity and provide protection in the presence of maternal antibodies.

Whooping cough is a respiratory illness most severe in infants and young children. While the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, pertussis remains a problem in neonates and adolescents. New vaccines are needed that can provide early life and long-lasting protection of infants. Vaccination at an early age, however, is problematic due to the interference with maternally derived antibodies (MatAbs) and the bias towards Th2-type responses following vaccination. Here we report the development of a novel vaccine formulation against pertussis that is highly protective in the presence of MatAbs. We co-formulated pertussis toxoid (PTd) and filamentous hemagglutinin (FHA) with cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), cationic innate defense regulator (IDR) peptide and polyphosphazene (PP) into microparticle and soluble vaccine formulations and tested them in murine and porcine models in the presence and absence of passive immunity. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, higher anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel(®), one of the commercially available vaccines for pertussis. Most importantly, the vaccines offered protection against challenge infection in the presence of passively transferred MatAbs.

A conformational change of C fragment of tetanus neurotoxin reduces its ganglioside-binding activity but does not destroy its immunogenicity.

The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside G(T1b) and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects.

Development and evaluation of gamma irradiated toxoid vaccine of Salmonella enterica var Typhimurium.

Development of a single effective vaccine against non-typhoidal salmonellosis is very challenging due to the presence of hundreds of serovars of Salmonella which are antigenically different from each other. The Salmonella enterotoxin (Stn), a common virulence factor occurring amongst a wide range of serovars, used as a formalized toxoid vaccine has been found to be effective against homologous and heterologous serovars. However, the process of formalization has its own drawbacks. Gamma radiation (γ) on the other hand is widely used as a safe and convenient method of sterilization worldwide. In this experiment we used gamma rays to inactivate the partially purified Stn of Salmonella enterica serovar Typhimurium (DT 193). The toxoid obtained was tested for its immunogenicity and loss of toxicity and then used to formulate a gamma irradiated toxoid vaccine (ITST). The efficacy of the developed ITST was tested in Kuroiler, a Broiler breed, against homologous and heterologous challenges (S. Typhimurium and S. Gallinarum) administered intra-peritoneally and orally. Birds in groups challenged with S. Typhimurium by both routes recorded protective indices (PI) of 100% while birds in groups challenged intra-peritoneally with S. Gallinarum recorded PI of 83.33% and those challenged orally scored 100%. The overall protective index (PI) being 95.83%. The antibody titres calculated as geometric mean with standard error at 1:10(-4) dilutions showed a steep rise after the first dose and peaked at week 6 post primary vaccinations. Thus the ITST was found very effective in protecting poultry against both the challenge organisms tested.

Antibody responses in adult and neonatal BALB/c mice to immunization with novel Bordetella pertussis vaccine formulations.

A balanced or Th-1 type immune response is required for effective clearance of many pathogens such as Bordetella pertussis, the causative agent of whooping cough. Since current acellular pertussis vaccines induce limited Th-1 type immune responses, novel vaccine formulations are needed to induce protective immunity in the infant in the earliest stages of life. Here, we developed a novel vaccine platform consisting of genetically detoxified pertussis toxoid (PTd) with multiple adjuvant components including CpG oligodeoxynucleotides, polyphosphazenes, and cationic innate defence regulator peptides. Co-formulation with these immunomodulators increased the serum IgG2a and IgG1 antibody titres in adult mice when compared to immunization with each of the selected adjuvants or immunization with PTd antigen alone. When used in combination, these adjuvants were able to induce a superior IgG2a response in both adult and neonatal mice, when compared to antigen alone or commercial vaccines. The increased response observed when using this adjuvant formulation was also initiated earlier and, moreover, was maintained over a period of greater than 22 months. The adjuvant platform also showed an ability to induce an immune response in a greater number of mice as compared to antigen alone. This suggests that this uniquely adjuvanted vaccine induces a stronger and more balanced immune response with an earlier onset of this response than vaccination with PTd antigen alone.

A novel vaccine adjuvant comprised of a synthetic innate defence regulator peptide and CpG oligonucleotide links innate and adaptive immunity.

There has been an increased demand for the development of novel vaccine adjuvants that lead to enhanced induction of protection from infectious challenges and development of immunological memory. A novel vaccine adjuvant was developed comprising a complex containing CpG oligonucleotide and the synthetic cationic innate defence regulator peptide HH2 that has enhanced immune modulating activities. The complex of HH2 and the CpG oligonucleotide 10101 was a potent inducer of cytokine/chemokine expression ex vivo, retained activity following extended storage, had low associated cytotoxicity, and upregulated surface marker expression in dendritic cells, a critical activity for a vaccine adjuvant. Immunization of mice with a coformulation of the HH2-CpG complex and pertussis toxoid significantly enhanced the induction of toxoid-specific antibody titres when compared to toxoid alone, inducing high titres of IgG1 and IgG2a, typical of a balanced Th1/Th2 response, and also led to high IgA titres. This study demonstrates the potential application of the HH2-CpG complex as a vaccine adjuvant.

Enhancement of intranasal vaccination with recombinant chain A ricin vaccine (rRV) in mice by the mucosal adjuvants LTK63 and LTR72.

Intranasal (i.n.) vaccination of mice with three doses of 40 microg of rRV stimulated low anti-ricin ELISA and neutralizing antibody responses, which were only marginally protective against aerosol-delivered 5-10 LD(50) of ricin toxin. To enhance the protection, and to reduce the lung injury of vaccinated mice that survived ricin toxin challenge, the mucosal adjuvant LTK63 or LTR72, two mutants of Escherichia coli LT enterotoxin adjuvant was administered with rRV. The safety of intranasally administered LTR63 was assessed as well. With 4, 2, or l microg of LTR63, the anti-ricin ELISA serum immunoglobulin geometric mean titer (GMT) increased up to 147-, 356-, 493-, and 17-fold for IgG, IgG1, IgG2a, and IgA, respectively. The comparable increases for GMTs of IgG and IgG1 in the presence LTR72 were up to 147-, and 617-fold, respectively. All three dose levels of LTK63 enhanced the ELISA GMTs in the lung lavage up to 192-, 22-, 4-, and 5-fold for IgG, IgG1, IgG2a, and IgA, respectively. Compared to GMT of rRV alone, the serum-neutralizing antibody GMTs for the three dose levels were enhanced up to 11-fold with LTK63. LTK63 augmented the ricin-related lymphoproliferative response of the cultured spleen lymphocytes and of the isolated CD4+ T lymphocytes. In the cultured lymphocytes, LTK63 stimulated predominantly TH1 cytokines. While only 10% of the mice that were vaccinated with rRV survived lethal challenge, in the presence of LTK63 or LTR72, the respective survival rates were augmented to 100%. Compared to the surviving mice vaccinated with rRV alone, the vaccine with LTK63 or LTR72 did not attenuate the extent of the ricin-related lung injury at a single or two time-points, respectively. Safety of LTK63 administration was indicated by the absence of histopathological changes in every organ, including the lungs and in the central nervous systems (CNS) of the mice during the entire 92 days of the study. In the nasal passages of the mice that received LTK63, a transient inflammation occurred without permanent epithelial changes. Administration of three dose levels of the adjuvant in the presence of rRV caused no additional changes. LTK63 and LTR72 both were very effective and safe mucosal adjuvants at all three dose levels employed in these studies. Both significantly enhanced the protection of a marginally effective dose of rRV against aerosol-delivered ricin challenge. LTK63 stimulated cytokines, which could be surrogate markers of efficacy, with human relevance potential. In spite of the better efficacy, rRV with LTK63, or with LTR72, failed to reduce the ricin-related lung injury. Most likely, a larger than suboptimal dose could resolve the lung injury of the vaccinated mice in the presence of a larger dose of the mucosal adjuvant.

Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication.

The development of small-animal models is necessary to understand host responses and immunity to emerging infectious diseases and potential bioterrorism agents. In this report we have characterized a murine model of intestinal ricin intoxication. Ricin administered intragastrically (i.g.) to BALB/c mice at doses ranging from 1 to 10 mg/kg of body weight induced dose-dependent morphological changes in the proximal small intestine (i.e., duodenum), including widespread villus atrophy and epithelial damage. Coincident with epithelial damage was a localized increase in monocyte chemotactic protein 1, a chemokine known to be associated with inflammation of the intestinal mucosa. Immunity to intestinal ricin intoxication was achieved by immunizing mice i.g. with ricin toxoid and correlated with elevated levels of antitoxin mucosal immunoglobulin A (IgA) and serum IgG antibodies. We expect that this model will serve as a valuable tool in identifying the inflammatory pathways and protective immune responses that are elicited in the intestinal mucosa following ricin exposure and will prove useful in the evaluation of antitoxin vaccines and therapeutics.

Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin.

Haemophilus ducreyi cytolethal distending toxin (HdCDT) is a tripartite AB toxin, which causes DNA damage in affected cells. We investigated the effects of formaldehyde on the chemical, biological, and immunological properties of the HdCDT complex, which was purified by immobilizing the glutathione S-transferase (GST)-CdtB fusion protein, followed by binding of the CdtA and CdtC recombinant proteins. The HdCDT was treated with increasing concentrations of formaldehyde in the presence of lysine. The treatment of HdCDT at 1 and 0.1 mg protein/ml with 320 and 80 mM of formaldehyde, respectively, resulted in the complete abrogation of cytotoxic activity, loss of DNase activity, and loss of binding capacity to HeLa cells. The toxoid showed protein bands of 75-150 kDa in SDS-PAGE, composed of the three cross-linked CDT components detected by immunoblotting. Three doses of 10 microg protein/mouse of the formaldehyde-treated HdCDT elicited toxin-neutralizing antibodies at titers about 200 times higher than those elicited by the native toxin. The described methodology may be applied to produce immunogenic toxoids from other CDTs, which might be used as candidate components in vaccines against CDT-producing bacteria, including H. ducreyi.

A plant-based oral vaccine to protect against systemic intoxication by Shiga toxin type 2.

Hemolytic uremic syndrome, the leading cause of kidney failure in children, often follows infection with enterohemorrhagic Escherichia coli and is mediated by the Shiga type toxins, particularly type 2 (Stx2), produced by such strains. The challenge in protecting against this life-threatening syndrome is to stimulate an immune response at the site of infection while also protecting against Shiga intoxication at distal sites such as the kidney. As one approach to meeting this challenge, we sought to develop and characterize a prototypic orally delivered, plant-based vaccine against Stx2, an AB5 toxin. First, we genetically inactivated the Stx2 active A subunit gene and then optimized both subunit genes for expression in plants. The toxoid genes were then transformed into the Nicotiana tabacum (tobacco) cell line NT-1 by Agrobacterium tumefaciens-mediated transformation. Toxoid expression was detected in NT-1 cell extracts, and the assembly of the holotoxoid was confirmed. Finally, mice were immunized by feeding with the toxoid-expressing NT-1 cells or by parenteral immunization followed by oral vaccination (prime-boost strategy). The immunized mice produced Stx2-specific mucosal IgA and Stx2-neutralizing serum IgG. The protective efficacy of these responses was assessed by challenging the immunized mice with E. coli O91:H21 strain B2F1, an isolate that produces an activatable variant of Stx2 (Stx2d) and is lethal to mice. The oral immunization fully protected mice from the challenge. Results of this study demonstrated that a plant-based oral vaccine can confer protection against lethal systemic intoxication.

A natural anatoxin, Amm VIII, induces neutralizing antibodies against the potent scorpion alpha-toxins.

In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus mauretanicus mauretanicus, to elicit specific polyclonal antibodies in rabbit. Using liquid-phase radioimmunoassay, we have studied its selectivity and its neutralizing activity both in vitro and in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin of reference AaH II. We have shown that the anti-Amm VIII serum prevents the association of 125I-AaH II with its receptor and is able to remove 125I-AaH II already bound to its site (the half-life of the complex 125I-AaH II-receptor site was 12 min in the absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by millilitre are neutralized, measured by subcutaneous injection.

Development of an approach for the laboratory toxicological evaluation of Bordetella pertussis adenylate cyclase genetic toxoid constructs as multipurpose vaccines.

Detoxified recombinant CyaA constructs have been developed as potential viral and tumoral epitope carriers for immunoprophylactic and therapeutic vaccination and as antigen candidates for inclusion in acellular pertussis vaccines. In this work, we attempt to explore a test system for the laboratory safety evaluation of CyaA preparations. Endotoxin was determined in vitro by the Limulus amoebocyte lysate assay. Cytotoxicity was measured by a tetrazolium salt test and a lactate dehydrogenase release assay using murine and human phagocytic cell lines. Cell viability was < 50% at concentrations > 1 microg/mL of the wild type toxin and concentrations in the nanogram range inhibited the zymosan-induced oxidative burst as measured by chemiluminescence. However, no effects were observed for detoxified and non-acylated forms of CyaA at comparable and higher toxin concentrations. Effects found in the in vitro assays could not be related to the in vivo mouse weight gain test used as a general toxicity test. In vitro cytotoxicity and oxidative burst studies on phagocytes, and the evaluation of endotoxin levels are useful for safety screening of CyaA constructs. However, in vivo specific toxicity tests and potential toxic implications of enzymatic activity-independent effects of CyaA on cell function should be investigated.

Therapeutic vaccine to control stromal tumor-induced immunosuppression in human uterine cervixc cancer.

Cancer cells may escape immune surveillance by secreting in their microenvironment soluble factors that may locally paralyze the stromal effector immune cells. In the human uterine cervix cancer, HPV-16 E7 protein, released in the stroma, should contribute to cancer cells immune escape since this protein inhibits the cellular immune response to recall antigens or alloantigens and strongly enhances the release of immunosuppressive cytokines by APCs. This prompted us to prepare a therapeutic vaccine triggering anti-E7 neutralizing Abs to antagonize the E7-induced stromal immunosuppressive effects and allow cellular immune reaction towards cancer cells including specific CTLs, induced by conventional vaccine, to be effective. Since HPV-16 is a mucosotropic virus, this therapeutic vaccine has been prepared to generate systemic as well as mucosal immunity.

Investigation of cellular and humoral immune responses to whole cell and acellular pertussis vaccines.

New generation acellular pertussis vaccines were compared with the established whole cell pertussis vaccine for the induction of humoral and cellular immune-responses in mice. At the same time, the in vivo protective effect of these two types of vaccine was also compared in both intracerebral (ic) and aerosol challenge models. In general, whole cell vaccine induced lower antibody titres to pertussis toxin, filamentous haemagglutinin and pertactin than the acellular vaccine. Nitric oxide concentration in macrophage cultures was used as a marker for macrophage activation. The nitric oxide concentrations in the macrophage cultures from mice following immunisation with the whole cell vaccine were higher than those from mice immunised with the acellular vaccine, which indicated that the whole cell vaccine was more effective than the acellular vaccine in activating macrophages. This was associated with better protection in vivo after challenge. After ic challenge of mice following immunisation with whole cell or acellular vaccine, 90% of the whole cell vaccine group survived compared with 40% of the acellular vaccine group at the vaccine dose selected. Following aerosol challenge, mice in the whole cell vaccine group showed faster clearance of bacteria from the lungs than those in the acellular vaccine group. Our findings suggest that the different types of pertussis vaccines may achieve protection in different ways and that CMI may play an important role in eliminating bacteria which escape humoral defence mechanisms.

Intranasal administration of a Schistosoma mansoni glutathione S-transferase-cholera toxoid conjugate vaccine evokes antiparasitic and antipathological immunity in mice.

Mucosal administration of Ags linked to cholera toxin B subunit (CTB) can induce both strong mucosal secretory IgA immune responses and peripheral T cell hyporeactivity. In this study, intranasal (i.n. ) administration of CTB-conjugated Schistosoma mansoni 28-kDa GST (CTB-Sm28GST) was found to protect infected animals from schistosomiasis, especially from immunopathological complications associated with chronic inflammation. Worm burden and liver egg counts were reduced in infected animals treated with the CTB-Sm28GST conjugate as compared with mice infected only, or with mice treated with a control (CTB-OVA) conjugate. However, a more striking and consistent effect was that granuloma formations in liver and lungs of mice treated with CTB-Sm28GST were markedly suppressed. Such treatment was associated with reduced systemic delayed-type hypersensitivity and lymphocyte proliferative responses to Sm28GST. Production of IFN-gamma, IL-3, and IL-5 by liver cells was also markedly reduced after i.n. treatment of CTB-Sm28GST, whereas IL-4 production was not impaired. Intranasal treatment of infected mice with CTB-Sm28GST increased IgG1-, IgG2a-, IgA-, and IgE-Ab-forming cell responses in liver in comparison with treatment with CTB-OVA, or free Sm28GST. Most importantly, mucosal treatment with CTB-Sm28GST significantly reduced animal mortality when administered to chronically infected mice. Our results suggest that it may be possible to design a therapeutic vaccine against schistosomiasis that both limits infection and suppresses parasite-induced pathology.