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PubMed1984

Thermal stability of different forms of diphtheria toxin.

Diphtheria toxin and its enzymatically active A fragment have been examined by differential scanning calorimetry. The thermal stability was measured for different forms of these molecules, including tryptically nicked and intact, nucleotide-bound and free, cysteine alkylated, and cysteine oxidized and reduced. Three ranges of denaturation temperature have been observed among the different forms of diphtheria toxin studied, and there is a correlation of the thermal stability of these different forms with their biological activity. At the concentrations used for measurement, all forms of the 62,000-Da diphtheria toxin are irreversibly denatured by heating to 100 degrees C, while free A chain is reversibly denatured. In vivo and in vitro activities of the samples were measured before and after heating, and all were found to retain significant degrees of activity after heating.

Diphtheria Toxin↗

PubMed1989

A superactive hormonotoxin prepared with truncated diphtheria toxin.

A chemically truncated form of diphtheria toxin, DT51, which lacks the cell-binding site but retains the membrane-translocating function, was covalently linked to luteinizing hormone (LH) and compared to similar conjugates containing diphtheria toxin (DT) or diphtheria toxin A-chain (DTA). The DT51 hormonotoxin killed cells possessing an LH receptor at concentrations similar to that of DT hormonotoxin and orders of magnitude lower than DTA hormonotoxin. The DTA hormonotoxin exhibited an LD-50 similar to that of previously reported hormonotoxins which employed DTA, ricin A-chain, or gelonin as toxic moieties.

Animals↗

PubMed1992

Lens structures exist transiently in development of transgenic mice carrying an alpha-crystallin-diphtheria toxin hybrid gene.

The development of lens structures in transgenic mice (lnl mice) which carry the diphtheria toxin A chain-coding sequence under the control of the alpha-crystallin promoter is examined here in detail. The initial stages of lens development during embryonic days 10.5 to 12.5 (E10.5-E12.5), including the invagination of the surface ectoderm to form a lens vesicle, closure of the vesicle to form a lens cup, and initial appearance of the lens itself, appeared identical in histologic analyses of lnl mice and genotypically wild-type littermate controls. However, by E12.5, cells in the central posterior lens of developing lnl mice appeared to be vacuolated and undergoing necrosis. This necrosis was quite prominent at E14.5 and the overall lens size was significantly reduced. The lenses of lnl mice continued to be present but were significantly smaller throughout embryonic development. The cells of these lenses were capable of undergoing biochemical differentiation, reacting with antibodies to both alpha- and beta-/gamma-crystallin. alpha-Crystallin expression was initiated at the appropriate time (E10.5) and maintained in most cells of lnl lenses. The expression of beta-/gamma-crystallins was surprising as these crystallins are expressed later in lens development after normal expression of alpha-crystallin and after the anticipated time of expression of the diphtheria toxin transgene. Despite extensive necrosis and cell death, lens structures persisted in lnl mice and disappeared only in the early postnatal period between days 3 and 6. Throughout the perinatal period, the remaining lens cells expressed both alpha- and beta-/gamma-crystallins. Prenatal development of the retina and ciliary body was relatively normal although the eye was significantly reduced in overall size. Some additional developmental defects were noted including persistent hyaloid artery and thickened cornea. In the perinatal period the rapidly expanding retina filled the entire eye leaving essentially no anterior or posterior chamber. These results clearly indicate that lens cells which are the target of diphtheria toxin-mediated cell ablation techniques persist for a significant time during development and thus place limitations on the interpretations of results obtained using this technique.

Animals↗

PubMed1987

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Diphtheria Toxin↗

PubMed1990

Histidine-21 is involved in diphtheria toxin NAD+ binding.

Histidine-21 is the sole histidine present in the A chain of diphtheria toxin and recent evidence suggests that it is involved in NAD+ binding. Fluorimetric assays of NAD+ binding and diethylpyrocarbonate modification performed at different pH values provide further insights on the role of this residue and indicate that its pKa value is 6.3. Conformational changes of subunit A of diphtheria toxin have been detected by analysis of tryptophan fluorescence in the pH 2.5-4 and pH 9-10.5 ranges. This indicates that histidine-21 is unlikely to be involved in the low pH-driven conformational change of diphtheria toxin.

Binding Sites↗

PubMed1985

Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells.

Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin.

Animals↗

PubMed1989

Expression of a poliovirus type 1 neutralization epitope on a diphtheria toxin fusion protein.

The diphtheria toxin (DT) secreted by Corynebacterium diphtheriae is used after formolization as an efficient vaccine against diphtheria. In an attempt to evaluate its capacity to present heterologous peptide sequence in a recognized form, we created in-phase insertion in the gene encoding the non-toxic mutant protein CRM228 of DT. The sequence chosen for insertion was the synthetic DNA fragment encoding a poliovirus neutralization epitope. Tripartite fusion proteins comprising the mutant DT, the poliovirus peptide and beta-galactosidase were obtained in E. coli and purified by affinity chromatography. These fusion proteins reacted both with antibodies directed against the DT and a poliovirus specific monoclonal antibody. Moreover, these hybrid toxins induced protective antibodies against the lethal effect of DT and neutralizing antibodies against poliovirus. We conclude that the modification of highly immunogenic DT may provide a means for the presentation of foreign peptide sequences to the immune system.

Animals↗

PubMed1995

Immunity to diphtheria in the 4-70 year age group in Italy.

Diphtheria antibody level in serum samples obtained from 270 subjects aged 4-70 was measured by in vitro seroneutralization test on Vero cells. Of the studied population, 27.8% had an antibody titre below the protective level (< 0.01 IU/ml). The prevalence of susceptible subjects showed a significant age-related increase (p < 0.01), with the highest value (53.8%) in the 31-40 age group. Mean antibody titre was maximum in individuals aged 13-14, then decreased reaching the minimum level in the 41-50 age group. These data suggest that in individuals aged 30-50, diphtheria immunity is not satisfactory, both for prevalence of immune subjects and for antibody levels. Therefore, a revaccination of adults with reduced doses of diphtheria toxoid may be advisable.

Adolescent↗

PubMed1995

Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

Antigens, Bacterial↗

PubMed1995

Cloning and sequence analysis of the Corynebacterium diphtheriae dtxR homologue from Streptomyces lividans and S. pilosus encoding a putative iron repressor protein.

The iron-regulated promoter involved in desferrioxamine B synthesis of Streptomyces pilosus contains a region homologous to the iron repressor (DtxR)-binding site of the diphtheria toxin gene promoter in Corynebacterium diphtheriae [Günter et al., J. Bacteriol. 175 (1993) 3295-3302]. Here, we report the cloning and sequencing of the putative Streptomyces iron repressor gene, homologous to dtxR of C. diphtheriae. The N-terminal 139 amino acids of the deduced protein are 73% identical to DtxR.

Amino Acid Sequence↗

PubMed2004

Novel potentiometry immunoassay with amplified sensitivity for diphtheria antigen based on Nafion, colloidal Ag and polyvinyl butyral as matrixes.

A novel potentiometry immunoassay with amplified sensitivity has been developed for the detection of diphtheria antigen (Diph) via immobilizing diphtheria antibody (anti-Diph) on a platinum electrode based on Nafion, colloidal Ag (Ag), and polyvinyl butyral (PVB) as matrixes in this study. The modified procedure was further characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The influence and factors influencing the performance of resulting immunosensor were studied in detail. The resulting immunosensor exhibited sigmoid curve with log Diph concentrations, high sensitivity (51.4 mV/decade), wide linear range from 8 to 800 ng ml(-1) with a detection limit of 1.5 ng ml(-1), rapid potentiometric response (<3 min) and long-term stability (>6 months). Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting diphtheria antigen in the clinical diagnosis.

Antigens↗

PubMed2006

Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli.

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.

Animals↗

PubMed1988

Specific cleavage of diphtheria toxin by human urokinase.

Diphtheria toxin must undergo a specific cleavage reaction and subsequent reduction to express the enzymatic ADP-ribosyltransferase activity that is responsible for its toxicity. In an effort to identify potential cellular enzymes that might be involved in this process we have found that a human urinary plasminogen activator, urokinase, is capable of specifically cleaving diphtheria toxin to yield an enzymatically active A fragment (more homogeneous than that produced by trypsin cleavage) and a B fragment (with an identical amino-terminal sequence to that produced by trypsin cleavage). The results raise the possibility that urokinase or urokinase-like enzymes play a role in diphtheria toxin-mediated intoxication.

Amino Acid Sequence↗

PubMed1993

Safety and immunogenicity of bacille Calmette-Guérin, diphtheria-tetanus-pertussis, and oral polio vaccines in newborn children in Zaire infected with human immunodeficiency virus type 1.

OBJECTIVE: To determine the safety and immunogenicity of childhood vaccines in children with perinatally acquired human immunodeficiency virus type 1 (HIV-1) infection. DESIGN: Nonrandomized, prospective cohort study; 12-month follow-up period. SETTING: Obstetric wards and outpatient pediatric clinics at two large hospitals in Kinshasa, Zaire. PATIENTS: A total of 8108 pregnant women were screened for HIV-1 antibodies. The 474 children born to 466 seropositive women identified during screening and the 616 children born to 606 seronegative, age- and parity-matched women were vaccinated. INTERVENTION: The following vaccines were administered at the stated ages: bacille Calmette-Guérin (BCG) vaccine (2 days); trivalent oral Sabin poliomyelitis vaccine (2 days and 6, 10, and 14 weeks); and adsorbed diphtheria-tetanus-pertussis (DTP) vaccine (6, 10, and 14 weeks). MEASUREMENTS AND MAIN RESULTS: Protective antibody titers to tetanus and poliovirus types 1, 2, and 3 were achieved in 95% of all children. Among children with HIV-1 infection, 70.8% had protective antibody titers to diphtheria compared with 98.5% of uninfected children (p < 0.05). Geometric mean antibody titers to diphtheria and poliovirus types 1, 2, and 3 were significantly lower in children with HIV-1 infection than in uninfected children. Vaccine-associated side effects were similarly low in all children. CONCLUSIONS: The low incidence of side effects and the high proportion of children with HIV-1 infection who achieved protective postimmunization antibody titers support the continuing use of BCG, DTP, and oral polio vaccines in childhood immunization programs in HIV-1 endemic areas.

Antibodies, Bacterial↗

PubMed1995

Effect of priming with diphtheria and tetanus toxoids combined with whole-cell pertussis vaccine or with acellular pertussis vaccine on the safety and immunogenicity of a booster dose of an acellular pertussis vaccine containing a genetically inactivated pertussis toxin in fifteen- to twenty-one-month-old children. Italian Multicenter Group for the Study of Recombinant Acellular Pertussis Vaccine.

OBJECTIVE: To evaluate the safety and the immunogenicity of a booster dose of recombinant acellular pertussis vaccine combined with diphtheria and tetanus toxoids (DTaP, Biocine SpA) in 15- to 21-month-old children primed in infancy with either whole-cell diphtheria-tetanus-pertussis (DTwP) vaccine or DTaP vaccine. DESIGN: Open-label second phase of a double-masked, controlled trail, with masked analysis of serum samples. PARTICIPANTS AND SETTING: Three hundred fifty children, 15 to 21 months of age, who had been primed at 2, 4, and 6 months of age with either three doses of DTaP vaccine (n = 173) or DTwP vaccine (n = 177). The children were enrolled in eight vaccination centers in Italy. INTERVENTIONS: All children received a booster dose of the DTaP vaccine and were examined for safety at 48 hours and at 7 days after vaccination. Serum samples for evaluation of immunogenicity were obtained from 196 (55%) of the 350 children. MAIN OUTCOME MEASURES: IgG antibodies to pertussis toxin (Ptox), filamentous hemagglutinin, 69-kilodalton protein, and tetanus toxoid were measured by enzyme-linked immunosorbent assay. Pertussis toxin-neutralizing antibodies were measured by the Chinese hamster ovary cell toxin neutralization assay. MAIN RESULTS: Adverse reactions to DTaP were infrequent, and there was no difference in the incidence of local or systemic reactions in children given DTaP as a fourth dose in comparison with a first dose. One month after the DTaP booster vaccination, both groups had 6- to 40-fold increases in serum antibody concentrations to all antigens tested; the concentrations against the three pertussis antigens were higher in the DTaP-primed children (p < 0.05). The antibody titers to diphtheria and tetanus toxoids were higher in the DTwP-primed group (p < 0.05), but both groups had protective titers. The geometric mean ratio of anti-Ptox neutralizing antibody per unit of IgG anti-Ptox antibody was higher in the DTaP-primed group (p < 0.001). CONCLUSIONS: There are quantitative and qualitative differences in booster responses to DTaP vaccine in young children, depending on whether they were given DTaP or DTwP as primary immunization. This DTaP vaccine is safe and highly immunogenic as a booster.

Antibodies, Bacterial↗

PubMed1983

The potency testing of diphtheria and tetanus toxoids as determined by the induction of antibody in mice.

An antibody induction (AI) assay in mice has been developed for the potency testing of diphtheria and tetanus toxoids. A diphtheria-pertussis-tetanus served as a reference preparation so that both toxoids could be tested within the same experiment. Results have been compared with those obtained by the conventional lethal challenge tests in guinea pigs (diphtheria) and mice (tetanus). The potency values obtained in the AI tests reflected the differences in the toxoid content of the preparations. Being an economical method, the AI test can be recommended for the potency control of DPT vaccines subject to confirmation of the authors' observations by others.

Animals↗

PubMed1987

The effects of reductions in the numbers of animals used for the potency assay of the diphtheria and tetanus components of adsorbed vaccines by the methods of the European pharmacopoeia.

Forty eight assays of adsorbed diphtheria vaccine and seven assays of adsorbed tetanus vaccine using either a lethal challenge (38 assays) or a serum neutralization test (17 assays) were evaluated for the effects of reductions in the number of animals used at each dilution on the potency values and 95% confidence intervals. The results were assessed in the light of the requirements of the European Pharmacopoeia and the WHO. In the majority of assays, 50% of the number of animals presently required would have sufficed for the determination of a potency within the limits of confidence stipulated by the European Pharmacopoeia and the WHO. Therefore it is concluded that a simplified assay with a reduced number of animals is suitable for the routine potency testing of the diphtheria and tetanus components of the combined vaccines and the monovalent that were examined. Flexibility in the national and international requirements in respect of the numbers of animals used at each dilution is suggested for the routine potency assay of the diphtheria and tetanus components of adsorbed vaccines.

Animal Welfare↗

PubMed1995

Diphtheria immunity in UK blood donors.

Immunity to diphtheria was determined in serum samples from 1000 UK-born blood donors at the North London Blood Transfusion Centre during a three-month period in 1993; 125 women and 125 men were stratified in 10-year age groups, from 20 to 59. A tissue (vero cell)-culture toxin-neutralisation assay was used to measure serum diphtheria antitoxin concentrations. According to internationally accepted definitions (antitoxin < 0.01 IU/mL = susceptibility, 0.01-0.09 IU/mL = basic protection, and > or = 0.1 IU/mL = full protection), 37.6% of donors were susceptible to diphtheria, 31.5% had basic protection, and 30.9% were fully protected. Log-linear modelling of the influence of age and sex on population immunity showed a significant trend (p < 0.001) of decreasing immunity with increasing age: 25.2% of donors aged 20-29 were susceptible compared with 52.8% of those aged 50-59. There was a small sex effect (p = 0.052); similar proportions of men and women were susceptible, but fewer women had full protection. There was no age-sex interaction on immunity (p = 0.454). Our results suggest that booster immunisation of adults is necessary to increase herd immunity of the adult population.

Adult↗