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.
Amino Acid Substitution ; Animal ; Bacterial Toxins/toxicity* ; Bacterial Toxins/metabolism ; Bacterial Toxins/immunology* ; Bacterial Toxins/genetics ; CHO Cells ; Cyclic AMP/metabolism ; Enterotoxins/toxicity* ; Enterotoxins/metabolism ; Enterotoxins/immunology* ; Enterotoxins/genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli*/metabolism ; Escherichia coli*/genetics ; Female ; Hamsters ; IgA, Secretory/blood ; Ileum/metabolism ; Immunity, Mucosal ; Mice ; Mice, Inbred BALB C ; Mutagenesis, Site-Directed ; NAD+ ADP-Ribosyltransferase/metabolism ; Recombinant Proteins/toxicity ; Recombinant Proteins/metabolism ; Recombinant Proteins/immunology ; Recombinant Proteins/chemistry

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.
Adult ; Bacteremia/*microbiology ; Bacterial Proteins/genetics ; Bacterial Toxins/*blood ; Exotoxins/*blood ; Female ; Humans ; Korea ; Leukocidins/*blood ; Male ; Methicillin/pharmacology ; Methicillin Resistance/drug effects ; Middle Aged ; Polymerase Chain Reaction/methods ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification

BACKGROUND: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by some Staphylococcus aureus strains and associated with skin and soft tissue infections; these strains are epidemiologically associated with current outbreaks of community-acquired methicillin-resistant S. aureus (MRSA) and with necrotizing pneumonia in healthy adults in USA and Europe. This study was performed to investigate the presence of PVL-positive S. aureus and the significant infections known to be caused by this organism. METHODS: A total of 573 strains of S. aureus blood isolates at university-affiliated hospital during 2002 to 2005 were selected. The presence of PVL was investigated using PCR. Additional 12 staphylococcal toxin genes were also examined in PVL-positive S. aureus strains, and MRSA isolates were typed for the staphylococcal cassette chromosome mec (SCCmec). RESULTS: PVL genes were detected in 5 (0.9%) of 573 S. aureus strains, including 1 MRSA and 4 MSSA. The PVL-positive MRSA isolate was SCCmec type IV, and no other staphylococcal toxins were detected. The median age of the patients infected with PVL-positive S. aureus was 36 yr. Three cases of bacteremia were preceded by skin and soft-tissue infections. CONCLUSIONS: Bacteremia caused by PVL-positive S. aureus strain were detected in 5 patients in Korea, and some of the patients were associated with severe skin and soft-tissue infections. In addition, the PVL-positive MRSA strain of SCCmec type IV, a characteristic of community-acquired MRSA isolates in USA and Europe, also exists in Korea, and can cause the severe infections known to be associated with this organism.
Adult ; Bacteremia/*microbiology ; Bacterial Proteins/genetics ; Bacterial Toxins/*blood ; Exotoxins/*blood ; Female ; Humans ; Korea ; Leukocidins/*blood ; Male ; Methicillin/pharmacology ; Methicillin Resistance/drug effects ; Middle Aged ; Polymerase Chain Reaction/methods ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification

Induced by 42 degrees C, the recombinant engineering bacterial pBV/cpa408 was highly expressed. After having been pelleted by 80% (NH4)2 SO4 and dialysised, the expressed protein was isolated and purified by the gel filtration choromatography. Then according to an amount of 1.0 mg/kg, the Kunming Mice (body weighted 18g) were immuned with the purified protein by intraperitoneal inoculation. One week after the first enhanced immunization, the Kunming Mice were attacked with an amount of 1.0MLD alpha-toxin, in which the eight mice immuned all survive and the control group all died. During the period of immunization, the titre of the mouse's serum antibody was measured by ELISA. One week after the first immunization, the titre of the mice's serum antibody was 1:800, but that of one week after the first enhanced immunization reached to 1:6400.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; biosynthesis ; immunology ; Bacterial Toxins ; immunology ; Calcium-Binding Proteins ; immunology ; Female ; Immunization ; Male ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Type C Phospholipases ; immunology

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.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; Female ; Gene Fusion ; genetics ; Mice ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.
Animals ; Antibodies, Bacterial ; blood ; immunology ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Blotting, Western ; Cloning, Molecular ; Clostridium Infections ; immunology ; microbiology ; prevention & control ; Clostridium septicum ; genetics ; immunology ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Mice ; Polymerase Chain Reaction ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Toxoids ; immunology ; Vaccination

BACKGROUND: Burn wounds lack normal barriers that protect against pathogenic bacteria, and burn patients are easily colonized and infected by Staphylococcus aureus. Toxic shock syndrome (TSS) is a rare but fatal disease caused by S. aureus. A lack of detectable antibodies to TSS toxin-1 (TSST-1) in serum indicates susceptibility to TSS. METHODS: A total of 207 patients (169 men and 38 women; median age, 42.5 yr) admitted to a burn center in Korea were enrolled in this study. The serum antibody titer to TSST-1 was measured by sandwich ELISA. S. aureus isolates from the patients' nasal swab culture were tested for TSST-1 toxin production by PCR-based detection of the TSST-1 toxin gene. RESULTS: One hundred seventy-four (84.1%) patients showed positive results for antibody against TSST-1. All patients aged > or =61 yr (n=28) and <26 months (n=7) were positive for the anti-TSST-1 antibody. S. aureus was isolated from 70 patients (33.8%), and 58.6% of the isolates were methicillin resistant. Seventeen patients were colonized with TSST-1-producing S. aureus. The antibody positivity in these 17 carriers was 88.2%, and the positivity in the non-carriers was 83.7%. CONCLUSIONS: Most burn patients had antibody to TSST-1, and nasal colonization with TSST-1-producing S. aureus was associated with positive titers of anti-TSST-1 antibody. Additionally, patients with negative titers of anti-TSST-1 antibody might be susceptible to TSS.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Bacterial/*blood ; Bacterial Toxins/genetics/immunology/*metabolism ; Burns/blood/*immunology/*microbiology/pathology ; Child ; Child, Preschool ; Enterotoxins/genetics/immunology/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Male ; Middle Aged ; Nasal Cavity/microbiology ; Polymerase Chain Reaction ; Prevalence ; Staphylococcal Infections/epidemiology ; Staphylococcus aureus/isolation & purification/*metabolism ; Superantigens/genetics/immunology/*metabolism ; Young Adult

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.
Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; genetics ; immunology ; Botulinum Toxins, Type A ; biosynthesis ; genetics ; immunology ; Botulism ; immunology ; prevention & control ; Clostridium botulinum type A ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocytes ; immunology ; Vaccines, DNA ; genetics ; immunology