Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools.

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (-169.70 kcal/mol), solubility (-0.755), and better tertiary structure (-0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions.

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC- with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC-. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin-cell interaction using primary hAEC.

Multiantigenic Nanotoxoids for Antivirulence Vaccination against Antibiotic-Resistant Gram-Negative Bacteria.

Infections caused by multidrug-resistant Gram-negative bacteria have emerged as a major threat to public health worldwide. The high mortality and prevalence, along with the slow pace of new antibiotic discovery, highlight the necessity for new disease management paradigms. Here, we report on the development of a multiantigenic nanotoxoid vaccine based on macrophage membrane-coated nanoparticles for eliciting potent immunity against pathogenic Pseudomonas aeruginosa. The design of this biomimetic nanovaccine leverages the specific role of macrophages in clearing pathogens and their natural affinity for various virulence factors secreted by the bacteria. It is demonstrated that the macrophage nanotoxoid is able to display a wide range of P. aeruginosa antigens, and the safety of the formulation is confirmed both in vitro and in vivo. When used to vaccinate mice via different administration routes, the nanotoxoid is capable of eliciting strong humoral immune responses that translate into enhanced protection against live bacterial infection in a pneumonia model. Overall, the work presented here provides new insights into the design of safe, multiantigenic antivirulence vaccines using biomimetic nanotechnology and the application of these nanovaccines toward the prevention of difficult-to-treat Gram-negative infections.

Orally administered recombinant Lactobacillus casei vector vaccine expressing ß-toxoid of Clostridium perfringens that induced protective immunity responses.

Clostridium perfringens types B and C cause enteritis and enterotoxemia in animals. The conventional vaccine production systems need time-consuming detoxification and difficult quality control steps. In this study, a modified ß-toxoid gene was synthesized, cloned into the pT1NX vector, and electroporated into Lactobacillus casei competent cells to yield L. casei-ß recombinant strain. Surface expression of the recombinant ß-toxoid was evaluated by ELISA and confirmed by immunofluorescence microscopy. Vaccinated BALB/c mice with L. casei-ß induced potent humoral and cell-mediated immune responses that were protective against lethal challenges with 100 MLD/mL of the ß-toxin. Safety and efficacy of the recombinant clone was evaluated and the presumptive toxicity of L. casei-ß was studied by toxicity test and histopathological findings, which were the same as negative controls. Our results support the use of L. casei as a live oral vector vaccine, and that the recombinant L. casei-ß is a potential candidate for being used in the control of enterotoxemia diseases caused by C. perfringens types B and C.

Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model.

Treatment of Staphylococcus aureus infections has become complicated owing to growing antibiotic resistance mechanisms and due to the multitude of virulence factors secreted by this organism. Failures with traditional monovalent vaccines or toxoids have brought a shift towards the use of multivalent formulas and neutralizing antibodies to combat and prevent range of staphylococcal infections. In this study, we evaluated the efficacy of a fusion protein (r-ET) comprising truncated regions of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST-1) in generating neutralizing antibodies against superantigen induced toxicity in murine model. Serum antibodies showed specific reactivity to both SEA and TSST-1 native toxins. Hyperimmune serum from immunized animals protected cultured splenocytes from non-specific superantigen induced proliferation completely. Passive antibody administration prevented tissue damage from acute inflammation associated with superantigen challenge from S. aureus cell free culture supernatants. Approximately 80% and 50% of actively and passively immunized mice respectively were protected from lethal dose against S. aureus toxin challenge. This study revealed that r-ET protein is non-toxic and a strong immunogen which generated neutralizing antibodies and memory immune response against superantigen induced toxic effects in mice model.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Divalent toxoids loaded stable chitosan-glucomannan nanoassemblies for efficient systemic, mucosal and cellular immunostimulatory response following oral administration.

The present study reports dual tetanus and diphtheria toxoids loaded stable chitosan-glucomannan nanoassemblies (sCh-GM-NAs) formulated using tandem ionic gelation technique for oral mucosal immunization. The stable, lyophilized sCh-GM-NAs exhibited ~152 nm particle size and ~85% EE of both the toxoids. The lyophilized sCh-GM-NAs displayed excellent stability in biomimetic media and preserved chemical, conformation and biological stability of encapsulated toxoids. The higher intracellular APCs uptake of sCh-GM-NAs was concentration and time dependent which may be attributed to the receptor mediated endocytosis via mannose and glucose receptor. The higher Caco-2 uptake of sCh-GM-NAs was further confirmed by ex vivo intestinal uptake studies. The in vivo evaluation revealed that sCh-GM-NAs posed significantly (p<0.001) higher humoral, mucosal and cellular immune response than other counterparts by eliciting complete protective levels of anti-TT and anti-DT (~0.1 IU/mL) antibodies. Importantly, commercial 'Dual antigen' vaccine administered through oral or intramuscular route was unable to elicit all type of immune response. Conclusively, sCh-GM-NAs could be considered as promising vaccine adjuvant for oral mucosal immunization.

Maternal immunization with vaccines containing recombinant NetB toxin partially protects progeny chickens from necrotic enteritis.

Avian necrotic enteritis is a major economic and welfare issue throughout the global poultry industry and is caused by isolates of Clostridium perfringens that produce NetB toxin. Previously we have shown that birds directly vaccinated with inactivated C. perfringens type A culture supernatant (toxoid) combined with recombinant NetB (rNetB) protein were significantly protected from homologous and heterologous challenge. In the present study the protective effect of maternal immunization was examined. Broiler breeder hens were injected subcutaneously with genetically toxoided rNetB(S254L) alone, C. perfringens type A toxoid and toxoid combined with rNetB(S254L). Vaccination resulted in a strong serum immunoglobulin Y response to NetB in hens immunized with rNetB(S254L) formulations. Anti-NetB antibodies were transferred to the eggs and on into the hatched progeny. Subclinical necrotic enteritis was induced experimentally in the progeny and the occurrence of specific necrotic enteritis lesions evaluated. Birds derived from hens immunized with rNetB(S254L) combined with toxoid and challenged with a homologous strain (EHE-NE18) at either 14 or 21 days post-hatch had significantly lower levels of disease compared to birds from adjuvant only vaccinated hens. In addition, birds from hens immunized with rNetB(S254L) alone were significantly protected when challenged at 14 days post-hatch. These results demonstrate that maternal immunization with a NetB-enhanced toxoid vaccine is a promising method for the control of necrotic enteritis in young broiler chickens.

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.

Identification of a novel pneumococcal vaccine antigen preferentially expressed during meningitis in mice.

Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H(2)O(2). A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.

Effects of heating on the immunogenicity and biological toxicity of Deinagkistrodon acutus venom and its fractions.

To improve toxoid preparation, the effects of selective heat denaturation were assessed on Deinagkistrodon acutus venom. The venom and its fractions (peak 1 and peak 2 separated by gel filtration chromatography) were heated to various temperatures (45-70 degrees C) for 30 min, after which protein concentration, immunoreactivity, lethality, myotoxicity and hemorrhagic and membrane lysis activities of the samples were determined. In addition, the synergistic effects of the venom fractions were evaluated by separate or simultaneous intramuscular injection in mice. The results showed that the peak 1 fraction consisted primarily of proteins in the range of 18 to 105 kDa, while the peak 2 fraction consisted primarily of proteins smaller than 21 kDa. The hemorrhagic activity, immunoreactivity, and protein concentration of heated samples were gradually reduced as the temperature increased from 25 degrees C to 70 degrees C. Bioactivities significantly decreased but immunoreactivity was retained when the crude venom, peak 1 fraction, or peak 2 fraction were heated to the critical temperatures of 60 degrees C, 55 degrees C, or 60 degrees C, respectively. Synergistic effects of two kinds of heated fractions were observed in toxicity and antibody production after the peak 1 and peak 2 injected simultaneously or respectively. The results suggest that venom fractions heated and injected separately could significantly reduce their toxicity and enhance the neutralization of antiserum induced by them.

Specific leukotriene receptors couple to distinct G proteins to effect stimulation of alveolar macrophage host defense functions.

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway.

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I-F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I-F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans-homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.

Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases.

BACKGROUND: The present study was undertaken to evaluate anti-proliferative and -apoptotic activities of mahanine, a plant derived carbazole alkaloid, in prostate cancer cells and to determine its molecular mechanism by which it induces apoptotic cell death. METHODS: The growth inhibitory and apoptotic inductive effect of mahanine on prostate cancer cells were examined by measuring cell proliferation and BrdU labeling, caspase activity, DNA fragmentation, and Western blot analyses. RESULTS: Mahanine inhibited growth of PC3 and LNCaP prostate cancer cells in a dose and time-dependent manner. Mechanistically, mahanine inhibited cell-survival pathway by dephosphorylation of PIP3 dependent kinase 1 (PDK1) thereby deactivation of Akt and downregulation of Bcl-xL. In addition, mahanine activated caspase pathway (caspases 9 and 3) and eventually cleavage of DNA repair enzyme, PARP resulting DNA fragmentation and apoptosis. CONCLUSIONS: Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway.

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits.

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

[Inhibition effect of merthiolate used as vaccines preservative on the oxygen-dependent metabolism of neutrophil granulocytes]. / Effekt podavleniia kislorodzavisimogo matabolizma neitrofil'nykh granulotsitov mertiolatom, ispol'zuemym v kachestve konservanta vaktsin.

The influence of the newly developed complex vaccine Pyopol, containing the antigens of opportunistic microorganisms and polyoxydonium used as immunomodulator, on the oxygen-dependent metabolism of neutrophils was studied. The study revealed that the main components of the vaccine, both individually and in association, did not change cellular activity in the range of concentrations used in this study. The inhibition of the oxygen-dependent metabolism of neutrophil granulocytes in the presence of native or weakly diluted vaccine occurred due to the cytotoxic effect of thimerosal used as preservative.

Mutant Escherichia coli heat-labile toxin B subunit that separates toxoid-mediated signaling and immunomodulatory action from trafficking and delivery functions.

The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His-->Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8(+)-T-cell apoptosis, (ii) activate nuclear translocation of NF-kappaB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits.

Effect of booster vaccination with a multivalent clostridial bacterin-toxoid on sudden death syndrome mortality rate among feedlot cattle.

OBJECTIVE: To determine whether booster vaccination with a multivalent clostridial bacterin-toxoid would affect the sudden death syndrome (SDS) mortality rate among feedlot cattle. DESIGN: Field trial. ANIMALS: 83, 115 cattle at a Nebraska feedlot. PROCEDURE: Cattle arriving at the feedlot underwent routine processing according to established protocol. All cattle received a sequentially numbered ear tag and a 2-ml dose of a multivalent bacterin-toxoid designed to protect cattle against Clostridium chauvoei, C speticum, C novyi, C sordellii, and C perfringens types C and D. Approximately 90 days prior to slaughter, growth promotants were implanted in all cattle, and cattle were allocated to a treatment or control group on the basis of the last digits of their ear tag numbers. Cattle in the treatment group received a second 2-ml dose of clostridial bacterintoxoid; control cattle did not. RESULTS: Significant differences between groups in regard to crude, feeding pen, or SDS mortality rates were not detected. Sudden death syndrome mortality rate across both groups was 0.24%. If the SDS mortality rate in midwestern feedlot cattle was reduced > or = 40% by booster vaccination with a multivalent clostridial bacterin-toxoid, this experiment included enough animals to have a 90% probability of detecting that difference. CLINICAL IMPLICATIONS: Booster vaccination with a multivalent clostridial bacterin-toxoid does not affect SDS mortality rate among feedlot cattle.

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment.

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37 degrees C with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected s.c. with 20 micrograms of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 micrograms) was administered i.p. on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice.