Genetic fusion of a non‐toxic heat‐stable enterotoxin‐related decapeptide antigen to cholera toxin B‐subunit

Sánchez, Jesús R.; Svennerholm, A. M.; Holmgren, Jan

doi:10.1016/0014-5793(88)81041-x

Cited by 74 publications

(37 citation statements)

References 23 publications

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“…In agreement with this work, the conformation assumed by the flagellin containing wild-type ST may mask the ST epitopes and the disruption of the two ST disulfide bonds may expose important ST determinants. This result was not expected, since the main reason for using an altered version of ST in this work was to reduce its toxic effect, as was previously described for the genetic fusion of the ST-decapeptide (Cys'-Cys"&) to the cholera toxin B-subunit, in which Cys( was replaced by alanine, resulting in a non-toxic hybrid protein (Sanchez et al, 1988).…”

Section: Discussionmentioning

confidence: 81%

“…ST is a non-immunogenic lowmolecular-mass peptide (2 kDa) ; however, its conjugation to different carriers can lead to the induction of neutralizing antibodies. Recently, a number of efforts have been made to render ST immunogenic, including chemical coupling and genetic fusions to appropriate carrier proteins (Clements, 1990 ;Houghten et al, 1984Houghten et al, , 1985Klipstein et al, 1982Klipstein et al, , 1983Sanchez et al, 1986Sanchez et al, , 1988. In general, chemical coupling reduced ST-associated toxicity as a function of the cross-linking and maintained the immunological determinants of ST. Genetic fusions have, as an advantage, the possibility of being delivered by live attenuated bacterial strains to elicit antibody response on the mucosal surfaces.…”

Section: Introductionmentioning

confidence: 99%

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Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

et al. 2001

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The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA-ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (ST mt ), were delivered to the immune system by three distinct strategies : (i) orally, using an attenuated Salmonella strain expressing FLA-ST ; (ii) intraperitoneally, by injection of purified FLA-ST ; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA-ST. The results showed that the flagellin system can be used as a carrier to generate ST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA-ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-ST mt . Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA-ST mt ) seems to be a promising method to elicit an appropriate response against fusions to flagellin.

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Section: Discussionmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

et al. 2001

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“…Several strategies have been investigated to make STa immunogenic either through chemical conjugation [6,[31][32][33] or recombinant fusion protein [7,[34][35][36][37][38]. These approaches have been assessed based on antigenicity of the final product or complexity of the procedure.…”

Section: Discussionmentioning

confidence: 99%

Novel Heat-Stable Enterotoxin (STa) Immunogen Based on Cationic Nanoliposomes: Preparation, Characterization and Immunization

Aref¹,

Nasr²,

Osman³

2017

J Vaccines Vaccin

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The novelties encountered in the field of formulation have provided promising solutions for the problematic vaccine development of hapten molecules. In the current study, the novel preparation of a cationic nanoliposomal immunogen of the heat stable enterotoxin (STa) was reported. STa was produced from clinically ETEC isolate of diarrheic neonatal calves and purified using RP-HPLC. STa was loaded into the cationic vesicles which were characterized for their particle size, surface charge, morphology, STa loading, and stability. The STa loaded cationic nanoliposome was used for mice immunization and the generation of STa antibody was monitored using ELISA. Results displayed the spherical nature of the STa loaded vesicles, their suitable size and homogeneity represented by a particle size of 228.1 nm and a PDI of 0.202. The surface charge of the STa nanoliposomes was +29.9, demonstrating sufficient stability during refrigeration storage. The STa loaded cationic nanoliposome was able to elicit specific STa antibody response, and to confer effective protection against STa challenge in mice. The STa antibody binding titer and neutralization capacity were 10 5 and 10 4 mouse units/ml serum, respectively. The developed system is a one-step procedure, which overcomes the disadvantage of the complexity of generation of the hapten-carrier conjugate. In conclusion, the developed STa-cationic nanoliposomal immunogen is feasible and has the potential to improve effectiveness against ETEC, suggesting its applicability in preventing the harmful effects of ETEC infection in neonatal calves.

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“…Cell supernatants and total cell somcates were assayed for the presence of STa on a competltlve GM1 ELISA using a recombmant decapeptide-CTB protein as the coating agent [9] and STa specific monoclonal antlbodies [13]. A posltlve sample wab identified by bmdmg inhibItion of native STa to the well as followed by a decrease m absorbance at 492 nm after reactinn with orthophcnylenediamme substrate Release of STa to E colr supernatant as a result of cell lysia was ruled out by testing the same culture supernatants and cell somcatea 266 forp-lactamase activity [14].…”

Section: Drtcwtron Of'stu By Elis4mentioning

confidence: 99%

“…as here shown, definitely rule out that Pre, Pro and other natural genetic sequences are essential for secretion of the toxin. To test if secretion of STa in this fashion was shared by structurally similar peptides conotoxin GI [8] and an STa-related decapeptide [9] were cloned with the same genetic procedures. Neither of the two peptides was secreted by E. coli suggesting that STa exits the bacterial cell via a selective secretion pathway which appears to discriminate even against structurally close relatives.…”

Section: Introduction Enterotoxigenicmentioning

confidence: 99%

Extracellular secretion of STa heat‐stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

1993

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The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domams called Pre (aa I-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxm release by this host. The Pro sequence. however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out If Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretlon m those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E coii heat-labile enterotoxin B-subunit (LTB). ExpressIon of this gene fusion resulted m extracellular secretion of biologically active STa by E. co/i independently of natural STa nelghboring genetlc sequences. Moreover. these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry mto the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretIon in this fashion might be extended to other dlsulfide bond-rich small peptides. the 13 ammo acid conotoxin GI and a non-enterotoxlc STa-related decapeptide were cloned. None of the two peptldes was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteme-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. colr.

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Genetic fusion of a non‐toxic heat‐stable enterotoxin‐related decapeptide antigen to cholera toxin B‐subunit

Cited by 74 publications

References 23 publications

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

Novel Heat-Stable Enterotoxin (STa) Immunogen Based on Cationic Nanoliposomes: Preparation, Characterization and Immunization

Extracellular secretion of STa heat‐stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

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