Toxoid from Exotoxin A of Pseudomonas aeruginosa: Preparation and Characterization

Pollack, Matthew; Prescott, Richard K.

doi:10.1093/infdis/145.2.688

Cited by 23 publications

(13 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Exotoxin A was partially purified according to the method described by Pollack et al [10] and Homma et al [11]. P. aeruginosa was inoculated into tryptic soy agar and incubated at 37°C for 24 h in ambient conditions.…”

Section: Methodsmentioning

confidence: 99%

Active immunization using exotoxin A confers protection against Pseudomonas aeruginosainfection in a mouse burn model

et al. 2009

View full text Add to dashboard Cite

BackgroundPseudomonas aeruginosa is an important cause of nosocomial infection and may lead to septicemia and death. We evaluated the immunogenicity of semi-purified exotoxin A from the bacterium in a mouse burn model.MethodsThe toxoid was prepared from exotoxin A taken from toxigenic strains of P. aeruginosa (PA 103). 50 mice were immunized with the toxoid, burned with hot metal and infected with 1 × 108 CFU of toxigenic strains of P. aeruginosa (experimental group); 25 non-immunized mice were also burned and infected (control group). The mortality rate and presence of any exotoxin and P. aeruginosa in the sera, liver and spleen were determined.ResultsIn the experimental group, 2 mice died before the burns were administered and were excluded from the study. The remainder (48 mice) were challenged with a lethal dose of P. aeruginosa and followed for 70 days. 3 of these mice died. Neither P. aeruginosa nor exotoxin A was not detected in the liver, spleen or sera of the surviving mice. The protective efficacy of toxoid vaccination was therefore 93.8%. In the control group, all mice died from bacteremia and septicemia, most (80%) within 6 days, and P. aeruginosa and exotoxin A were isolated from sera, spleen and liver.ConclusionActive immunization of mice using a semi-purified exotoxin A derived from P. aeruginosa was 93.8% effective at protecting mice from subsequent P. aeruginosa infections in a mouse burn model.

show abstract

Section: Methodsmentioning

confidence: 99%

Active immunization using exotoxin A confers protection against Pseudomonas aeruginosainfection in a mouse burn model

et al. 2009

View full text Add to dashboard Cite

show abstract

“…ADPRTase assay. The ADPRTase activity of Ex-A was examined by the method of Pollack and Prescott (24). EF-2 was prepared from rat liver by the ammonium sulfate precipitation method (26).…”

Section: Methodsmentioning

confidence: 99%

Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo

et al. 1992

View full text Add to dashboard Cite

Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. In vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin Gl [IgGl]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgGl), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but onl weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgGl), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.

show abstract

“…Thus proteins SF-4 and SF-7 are protective antigens, which are localized in the OM of P. aeruginosa [176]. Tris-EDTA extracts of P. aeruginosa containing proteins with MWs of 9,14,20,25,30,38,44 and 55 kDa (according to electrophoresis in polyacrylamide gel) protect mice against di¡erent immunotypes (serogroups) [169]. Sompolinsky et al [175] was able to resolve 64 antigens in crude water-soluble extracts of P. aeruginosa by crossed immunoelectrophoresis in agarose with polyvalent anti-Pseudomonas immunoglobulin.…”

Section: Outer Membrane Protein Antigensmentioning

confidence: 99%

Pseudomonas aeruginosa antigens as potential vaccines

Stanislavsky

1997

FEMS Microbiology Reviews

View full text Add to dashboard Cite

Pseudomonas aeruginosa is one of the most important opportunistic bacterial pathogens in humans and animals. This organism is ubiquitous and has high intrinsic resistance to antibiotics due to the low permeability of the outer membrane and the presence of numerous multiple drug efflux pumps. Various cell-associated and secreted antigens of P. aeruginosa have been the subject of vaccine development. Among pseudomonas antigens, the mucoid substance, which is an extracellular slime consisting predominantly of alginate, was found to be heterogeneous in terms of size and immunogenicity. High molecular mass alginate components (30^300 kDa) appear to contain conserved epitopes while lower molecular mass alginate components (10^30 kDa) possess conserved epitopes in addition to unique epitopes. Surface-exposed antigens including Oantigens (O-specific polysaccharide of LPS) or H-antigens (flagellar antigens) have been used for serotyping due to their highly immunogenic nature. Chemical structures of repeating units of O-specific polysaccharides have been elucidated and these data allowed the identification of 31 chemotypes of P. aeruginosa. Conserved epitopes among all serotypes of P. aeruginosa are located in the core oligosaccharide and the lipid A region of LPS and immunogens containing these epitopes induce crossprotective immunity in mice against different P. aeruginosa immunotypes. To examine the protective properties of OM proteins, a vaccine containing P. aeruginosa OM proteins of molecular masses ranging from 20 to 100 kDa has been used in pre-clinical and clinical trials. This vaccine was efficacious in animal models against P. aeruginosa challenge and induced high levels of specific antibodies in human volunteers. Plasma from human volunteers containing anti-P. aeruginosa antibodies provided passive protection and helped the recovery of 87% of patients with severe forms of P. aeruginosa infection. Vaccines prepared from P. aeruginosa ribosomes induced protective immunity in mice, but the efficacy of ribosomal vaccines in humans is not yet known. A number of recent studies indicated the potential of some P. aeruginosa antigens that deserve attention as new vaccine candidates. The outer core of LPS was implicated to be a ligand for binding of P. aeruginosa to airway and ocular epithelial cells of animals. However, heterogeneity exists in this outer core region among different serotypes. Epitopes in the inner core are highly conserved and it has been demonstrated to be surface-accessible, and not masked by O-specific polysaccharide. The use of an in vivo selection/expression technology (IVET) by a group of researchers identified a number of P. aeruginosa proteins that are expressed in vivo and essential for virulence. Two of these in vivo-expressed proteins are FptA (ferripyochelin receptor protein) and a homologue of an LPS biosynthetic enzyme. Our laboratory has identified a highly conserved protein, WbpM, and P. aeruginosa with a deficiency in this protein produces only rough LPS and became serum sensit...

show abstract

Toxoid from Exotoxin A of Pseudomonas aeruginosa: Preparation and Characterization

Cited by 23 publications

References 23 publications

Active immunization using exotoxin A confers protection against Pseudomonas aeruginosainfection in a mouse burn model

Active immunization using exotoxin A confers protection against Pseudomonas aeruginosainfection in a mouse burn model

Binding of monoclonal antibody specific for domain Ia/II of Pseudomonas aeruginosa exotoxin A at pH 4 strongly neutralizes exotoxin A-induced cytotoxicity in cell culture and in vivo

Pseudomonas aeruginosa antigens as potential vaccines

Contact Info

Product

Resources

About