Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin

Abstract The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.

Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

Equine hyperimmune serum protects mice against Clostridium difficile spore challenge

Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.

Comparison between the two-step and the three-step algorithms for the detection of toxigenic Clostridium difficile.

Purpose: To evaluate usefulness of applying either the two-step algorithm (Ag-EIAs and CCNA) or the three-step algorithm (all three assays) for better confirmation of toxigenic Clostridium difficile. The antigen enzyme immunoassays (Ag-EIAs) can accurately identify the glutamate dehydrogenase antigen of toxigenic and nontoxigenic Clostridium difficile. Therefore, it is used in combination with a toxin-detecting assay [cell line culture neutralization assay (CCNA), or the enzyme immunoassays for toxins A and B (TOX-A/BII EIA)] to provide specific evidence of Clostridium difficile-associated diarrhoea. Materials and Methods: A total of 151 nonformed stool specimens were tested by Ag-EIAs, TOX-A/BII EIA, and CCNA. All tests were performed according to the manufacturer's instructions and the results of Ag-EIAs and TOX-A/BII EIA were read using a spectrophotometer at a wavelength of 450 nm. Results: A total of 61 (40.7%), 38 (25.3%), and 52 (34.7%) specimens tested positive with Ag-EIA, TOX-A/BII EIA, and CCNA, respectively. Overall, the sensitivity, specificity, negative predictive value, and positive predictive value for Ag-EIA were 94%, 87%, 96.6%, and 80.3%, respectively. Whereas for TOX-A/BII EIA, the sensitivity, specificity, negative predictive value, and positive predictive value were 73.1%, 100%, 87.5%, and 100%, respectively. With the two-step algorithm, all 61 Ag-EIAs-positive cases required 2 days for confirmation. With the three-step algorithm, 37 (60.7%) cases were reported immediately, and the remaining 24 (39.3%) required further testing by CCNA. By applying the two-step algorithm, the workload and cost could be reduced by 28.2% compared with the three-step algorithm. Conclusions: The two-step algorithm is the most practical for accurately detecting toxigenic Clostridium difficile, but it is time-consuming.

Prevalence of Helicobacter pylori and cytotoxin-associated gene A in Iranian patients with non-erosive and erosive reflux disease.

Background: The prevalence of Helicobacter pylori (H. pylori) infection and its virulent strain (cytotoxin-associated gene A: CagA) has not been evaluated in patients with gastroesophageal reflux disease (GERD) subgroups (erosive and non-erosive) in Iran. AIMS: This study was conducted to evaluate H. pylori infection and anti-CagA status in GERD. Setting and Design: One hundred fifty-four patients (71 male and 83 female; mean age, 42.6± 15.5 years) were evaluated for symptoms - heartburn and acid regurgitation - compatible with GERD. Materials and Methods: The esophagus was diagnosed endoscopically whether it was with erosion (erosive reflux disease, ERD) or without erosion (non-erosive reflux disease, NERD). Rates of anti-H. pylori and anti-CagA-IgG (immunoglobulin G) antibodies were determined for each subject by ELISA. H. pylori was considered by ELISA if the rapid urease test proved positive. Statistical Analysis: anti-H. pylori and anti-CagA statuses were compared in ERD and NERD patients with and without related IgG antibodies. Mean values and P values were calculated using SPSS (version 12). Results: The overall prevalence of H. pylori infection was 75.3% (116/154). Anti-CagA antibody was found in 60.3% (70/116) of infected patients. The prevalence of H. pylori infection in patients with NERD and ERD was 81.2% (56/69) and 70.6% (60/85), respectively (OR= 0.56; 95% CI [confidence interval]= 0.26-1.20; P= .14). The prevalence of anti-CagA antibody in infected NERD and ERD patients was 58.9% (33/56) and 61.7% (37/60), respectively (OR= 1.16; 95% CI= 0.49-1.70; P= 0.83). Conclusion: The results showed that more than 70% of the patients, either NERD or ERD, acquire H. pylori infection. Anti-CagA antibody is also common in patients with reflux disorders in our community.

Production and Characterization of Anti-Staphylococcal Toxic Shock Syndrome Toxin-1 Monoclonal Antibody / 대한진단검사의학회지

BACKGROUND: Recently the association between the virulence factors of Staphylococcus aureus and the outcome of the patients infected with the organism appears to be the subject of active investigation. Toxic shock syndrome toxin-1 (TSST-1) is thought to be a clinically more significant virulence factor than other staphylococcal toxins. We attempted to produce and characterize monoclonal antibodies to staphylococcal TSST-1. METHODS: An important epitope of TSST-1, amino acids 1-15 region, was synthesized into a peptide antigen, and Balb/c mice were immunized by intraperitoneal injection of the synthetic antigen. Hybridomas were produced by fusing immunized murine splenocytes with immortal myeloma cells. Hybridomas were cloned through a limiting dilution method. Stable cultured hybridoma was injected into the peritoneal cavity of Balb/c mice, and peritoneal fluid containing the monoclonal antibody was produced. RESULTS: One IgG2b type monoclonal antibody and two IgM type monoclonal antibodies were obtained. The IgG2b type monoclonal antibody was able to detect 5 microgram of TSST-1 with Western blot analysis and showed a strong reactivity to TSST-1 with ELISA. CONCLUSIONS: Highly immunoreactive anti-TSST-1 monoclonal antibody was produced by the use of synthesized peptide antigen. Diagnostic and protective capacity of this monoclonal antibody should be evaluated in the future.

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli.

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.

Development of two novel nontoxic mutants of Escherichia coli heat-labile enterotoxin

Escherichia coli heat-labile enterotoxin (LT) is composed of catalytic A and non-catalytic homo-pentameric B subunits and causes diarrheal disease in human and animals. In order to produce a nontoxic LT for vaccine and adjuvant development, two novel derivatives of LT were constructed by a site-directed mutagenesis of A subunit; Ser63 to Tyr63 in LTS63Y and Glu110, Glu112 were deleted in LT delta 110/112. The purified mutant LTs (mLTs) showed a similar molecular structural complex as AB5 to that of wild LT. In contrast to wild-type LT, mLTs failed to induce either elongation activity, ADP-ribosyltransferase activity, cAMP synthesis in CHO cells or fluid accumulation in mouse small intestine in vivo. Mice immunized with mLTs either intragastrically or intranasally elicited high titers of LT-specific serum and mucosal antibodies comparable to those induced by wild-type LT. These results indicate that substitution of Ser63 to Tyr63 or deletion of Glu110 and Glu112 eliminate the toxicity of LT without a change of AB5 conformation, and both mutants are immunogenic to LT itself. Therefore, both mLTs may be used to develop novel anti-diarrheal vaccines against enterotoxigenic E. coli.

Superantígenos y su comportamiento en la respuesta inmunitaria / Superantigens and role in immune responses

Los superantígenos son moléculas derivadas de virus, bacterias o parásitos, que una vez que se unen al complejo pricipal de histocompatibilidad y al receptor de células T tienen la capacidad de estimular en forma potente a los linfocitos T, los que liberan masivamente linfocinas y pueden causar daño tisular. Los superantígenos también pueden provocar anergia clonal, por efecto selectivo sobre las subpoblaciones de las células CD4 (Th0/Th1/Th2)

Estudo em camundongos (CBA x C57B1/10) F1 da influência da enterotoxina termoestável (STa) de Escherichia coli na fagocitose / Study in mice (CBA x C57B1/10) F1 of the influence of termostable (STa) enterotoxin from Escherichia coli in phagocytosis

Camundongos (CBA x C57B1/10) F1 foram utilizados para se estudar a atividade fagocitária de seus macrófagos após a inoculaçäo, nos animais, da enterotoxina termoestável (STa) de Escherichia coli, obtida a partir de um amostra origem humana (sorogrupo 0128), semipurificada de acordo com Ricci e col (1982). Foi utilizada a técnica de Depuraçäo de Carbono Coloidal em camundongos inoculados com 250 unidades tóxicas (UT) de STa, em diferentes intervalos de tempo. Os resultados mostraram que 25OUT de STa aumentaram a velocidade de depuraçäo do carbono coloidal quando inoculados com a toxina 24, 48, 72 e 96 horas antes da administraçäo da substância coloidal. Em incoulaçöes feitas antes e após estes tempos näo houve interferência signficativa da toxina, sendo que a injeçäo de STa 48 horas antes do carbono coloidal, correspondia ao pico de depuraçäo do mesmo