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Diphtheria antitoxin titres six years after basic immunization of adults.

Diphtheria antitoxin titres were analysed in 160 adults (median age 59 years, range 34-70), who completed basic vaccination with three doses of 7.5 Lf or 15 Lf of diphtheria toxoid (D) in a previous vaccination trial in 1987, in serum samples drawn 6 years later. The median titre had decreased from 3.2 IU ml-1 in the post vaccination samples to 0.2 IU ml-1 after 6 years in the 15 Lf group and 0.1 IU ml-1 in the 7.5 Lf group. An antitoxin titre of < 0.01 IU ml-1, a level usually considered to give no safe protection, was found in 21/73 (29%) individuals, who had received 7.5 Lf and in 12 of 87 (14%), who received 15 Lf diphtheria toxoid (P < 0.05). In the original study, the vaccinees were enrolled as unimmunized based on their own vaccination histories, but many participants had serological evidence of previous immunization. In the subgroup of 48 truly non-immune participants, i.e. without prevaccination titres and without booster response after the first injection, 46% (32% in the 15 Lf group and 58% in the 7.5 Lf group) had antitoxin levels of < 0.01 IU ml-1 6 years after basic vaccination. Therefore, individuals who have received basic vaccination with three doses of diphtheria toxoid need at least one booster injection 5-10 years later.

Adult↗

Immunity to poliomyelitis, diphtheria and tetanus in pediatric patients before and after renal or liver transplantation.

Few studies have considered the safety, efficacy and appropriateness of vaccinations in pediatric patients before and after solid organ transplantation. The aim of this study was to evaluate the immune status after primary vaccination to diphtheria, tetanus and poliomyelitis in pediatric patients before and after hepatic transplantation and to poliomyelitis in pediatric patients before and after renal transplantation. All the patients had received a complete primary immunization schedule for diphtheria and tetanus and poliomyelitis. Immunity to the three polioviruses was evaluated in 56 patients with renal transplant, 27 on chronic dialysis and 33 controls and in 39 patients with hepatic transplant, 25 with chronic hepatic failure and their 36 controls. Immunity to diphtheria and tetanus was evaluated in 52 liver transplant patients, 29 children with chronic hepatic failure and 54 healthy children. Renal transplant patients were less protected and had lower antibody geometric mean titers than healthy controls for polioviruses 1 and 2. Whereas, protection in the children liver transplant patients was similar to that in their controls. Patients with chronic hepatic failure had higher antibody geometric mean titers to diphtheria and polioviruses 1 and 3 than their control group. Immunosuppression after transplantation has a negative influence on the immune status after primary vaccination in children with renal transplant. Whereas children with chronic hepatic failure have higher antibodies than a normal population. When possible, it could be advisable to individualize immunization schedules in patients at high risk.

Adolescent↗

An adult formulation of a five-component acellular pertussis vaccine combined with diphtheria and tetanus toxoids is safe and immunogenic in adolescents and adults.

Pertussis is increasingly being recognized as an important cause of cough illness in adolescents and adults. To evaluate the safety and immunogenicity of an adult formulation of a five-component (pertussis toxoid, filamentous hemagglutinin, pertactin, fimbriae 2 and 3) acellular pertussis vaccine combined with diphtheria and tetanus toxoids, we randomly allocated 749 healthy adolescents and adults from 12-54 years of age recruited from five Canadian communities to receive either tetanus-diphtheria vaccine (Td), acellular pertussis vaccine (aP) or combined diphtheria-tetanus-acellular pertussis vaccine (TdaP). Subjects and personnel were unaware of the vaccine allocation. Antibody levels were measured before and one month postimmunization; adverse events were collected at 24 and 72 h and 8 to 10 days. Adverse events were reported in similar frequency amongst the three vaccine groups. Moderate pain at the injection site was reported less frequently in the aP group than the TdaP group (10.7% compared to 19.4%; relative risk 0.6, 95% confidence interval 0.3-0.9). Chills were reported less frequently after Td (5.3%) than after TdaP (12.5%; relative risk 0.4, 95% confidence interval 0.2-0.9). There were no statistically significant differences between recipients of Td and TdaP in tetanus and diphtheria antitoxin levels achieved. Antibody response against Bordetella pertussis antigens was vigorous in all groups although recipients of aP alone had higher levels of antibody levels against pertussis toxoid, fimbriae, and agglutinins and lower antibody levels against pertactin than did TdaP recipients. We conclude that this adult formulation 5-component acellular pertussis vaccine is safe and immunogenic in adolescents and adults and is a candidate vaccine for adolescent and adult immunization programs.

Adolescent↗

A comparative clinical study of Adsorbed Tetanus Vaccine and Adult-type Tetanus-Diphtheria Vaccine.

A limited assessment of immunity to diphtheria revealed that only 44.8% of adults had protective levels (greater than 0.01 U/ml) of diphtheria antitoxin in their sera. In the light of this information, it was decided to assess the suitability of Adult-type Tetanus-Diphtheria Vaccine as a replacement for Adsorbed Tetanus Vaccine in occupational health schemes. A comparative study of these vaccines showed that a single dose of each produced an equivalent rate and level of tetanus antitoxin responses. Adult-type Td Vaccine elicited more than fourfold increases of diphtheria antitoxin in 76% of vaccinees. No statistically significant differences were observed in the clinical reactivity of the vaccines under test. However, the combined vaccine caused a slightly higher incidence of local reactions (pain, redness and swelling) while recipients of Adsorbed Tetanus Vaccine more frequently experienced pain.

Adolescent↗

Seroepidemiology of diphtheria and pertussis in Luxembourg in 2000.

A large serosurvey was carried out in Luxembourg in 2000-2001, to determine the population immunity against a number of vaccine-preventable infections including diphtheria and pertussis. Immunity to diphtheria and pertussis was assessed using an in-house neutralization assay and a commercial ELISA test respectively. Mean pertussis antibody activity decreased from 4 to 8 years of age, reflecting the effects of waning of vaccine-induced immunity. Mean pertussis antibody activity increased during adolescence due to infection in previously vaccinated individuals and levelled out after approximately 20 years of age. For adults>25 years age, a statistically significant 30% difference in mean antibody activity between men and women was observed. The proportion of seronegatives for diphtheria among children and adolescents aged<20 years was 2.5% reflecting the high vaccination coverage. The proportion seronegative for diphtheria tended to increase with age such that 42% of individuals aged>40 years were seronegative. Our study supports the recently introduced acellular pertussis vaccine booster at 6 years to reduce pertussis transmission in school-aged children and adolescents.

Adolescent↗

Localization of the active site of diphtheria toxin.

Information about the location of the active site of diphtheria toxin was derived from proteolysis studies and an analysis of its sequence. It was found that a specific trypsin cleavage within whole diphtheria toxin occurs at Lys-39. Therefore, Lys-39 appears to be a surface residue. Furthermore, protection from proteolysis could be obtained upon binding of either the substrate beta-nicotinamide adenine dinucleotide (oxidized form) (NAD+) or a competing ligand, adenylyl(3'-5')uridine 3'-phosphate (ApUp). The protection by ApUp, which binds to the toxin very tightly, required only stoichiometric levels. The most likely explanation of these results is that both NAD+ binding and ApUp binding block trypsin either through a steric mechanism or through a local conformational change, suggesting Lys-39 may be near the active site. Further evidence supporting this conclusion comes from comparison of the previously determined sequences of diphtheria toxin and of Pseudomonas exotoxin A, a protein that catalyzes an identical reaction. We find a significant degree of homology between the N-terminal halves of the catalytic domains of these two proteins, which apparently represents active-site residues, and that Lys-39 is in the center of the homologous sequence. Furthermore, the location of the amino acid that is the homologue of Lys-39 within the crystal structure of Pseudomonas exotoxin A is also in agreement with a location in or near the active site. Other unusual features in the sequences of diphtheria toxin and Pseudomonas exotoxin A are also described, and on the basis of the experiments presented, a possible function for ApUp is considered.

ADP Ribose Transferases↗

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin.

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗

A randomized study on donor immunization with tetanus-diphtheria, Haemophilus influenzae type b and inactivated poliovirus vaccines to improve the recipient responses to the same vaccines after allogeneic bone marrow transplantation.

The HLA-identical sibling donors of 111 bone marrow transplantation (BMT) recipients were randomised to receive or not to receive tetanus-diphtheria (T-d), Haemophilus influenzae type b (Hib), and inactivated poliovirus (IPV) vaccines 2-10 weeks before BM harvest. Fifty-three (DV+ group) recipients received the graft from a vaccinated donor and 58 (DV- group) from an unvaccinated donor. All recipients were vaccinated with the T-d, Hib and IPV vaccines at 3, 6 and 12 months after BMT. Diphtheria and Hib antibody concentrations were consistently higher in the DV+ than in the DV- group from 6 months post transplantation onwards. The differences were significant at 6 and 13 months for diphtheria and at 12 months for Hib antibody concentrations. Tetanus, PV1, PV2 and PV3 antibody levels were similar in both groups. Patients transplanted from donors with high tetanus, diphtheria and Hib antibody concentrations had higher respective antibody concentrations after BMT than those transplanted from donors with low antibody concentrations. Especially patients whose donors have low-specific antibody concentrations may benefit from donor vaccination with protein and conjugate vaccines.

Adult↗

Diphtheria toxin endocytosis and membrane translocation are dependent on the intact membrane-anchored receptor (HB-EGF precursor): studies on the cell-associated receptor cleaved by a metalloprotease in phorbol-ester-treated cells.

Preincubation of Vero cells with 1 microM phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-(DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl)-L-3-(2' - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.

Animals↗

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin.

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.

Animals↗

A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol.

A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB(389)IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 10(4)-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit beta to this region of the diphtheria toxin transmembrane domain.

Amino Acid Sequence↗

Investigations into the relationship between structure and function of diphtheria toxin.

Studies on the structure-function relationship of diphtheria toxin are reported. New methods are described for the preparation of pure intact ("unnicked") toxin and for the preparation of the individual A and B chains. A biological assay method for the B chain is also presented, as well as a method for the labelling of "nicked" (one peptide bond broken) diphtheria toxin with 131I such that the label is confined to only one of the two polypeptide chains. Alterations of diphtheria toxin with specific reagents reveal that modifications of the tryptophan, methionine, and arginine residues did not result in a significant loss in toxicity, whereas treatment of the toxin with omicron-phthalaldehyde or by photooxidation with rose bengal results in a complete loss of the toxic activity. Modification of tyrosine by iodination results in active toxin, whereas modification by tetranitromethane causes a loss in activity. Preliminary results also indicate that the isolated A chain is about an order of magnitude more active in incorporating adenosine diphosphoribose into translocase (elongation factor 2) than whole or nicked toxin is under identical conditions. The observed structural properties are discussed in view of the functional activity of diphtheria toxin in cell-free systems as well as in cell cultures. Evidence is presented indicating that the B chain binds to membranes: it inhibits the action of nicked toxin on HeLa cells.

Biological Assay↗

Binding of diphtheria toxin to phospholipids in liposomes.

Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine/cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of diphtheria toxin to cells.

Cardiolipins↗

Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro.

Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to gamma-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.

Animals↗

Anti-idiotypic antibodies that protect cells against the action of diphtheria toxin.

An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of 125I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of 125I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of approximately 15,000. These results are consistent with the hypothesis that the anti-idiotypic serum contains antibodies that carry an internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.

Animals↗

Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol.

Diphtheria toxin belongs to a group of toxic proteins that enter the cytosol of animal cells. We have here investigated the effect of NH2-terminal extensions of diphtheria toxin on its ability to become translocated to the cytosol. DNA fragments encoding peptides of 12-30 amino acids were fused by recombinant DNA technology to the 5'-end of the gene for a mutant toxin. The resulting DNA constructs were transcribed and translated in vitro. The translation products were bound to cells and then exposed to low pH to induce translocation across the cell membrane. Under these conditions all of the oligopeptides tested, including three viral peptides and the leader peptide of diphtheria toxin, were translocated to the cytosol along with the enzymatic part (A-fragment) of the toxin. Neither hydrophobic nor highly charged sequences blocked translocation. The results are compatible with a model in which the COOH-terminus of the A-fragment first crosses the membrane, whereas the NH2-terminal region follows behind. The possibility of using nontoxic variants of diphtheria toxin as vectors to introduce peptides into the cytosol to elicit MHC class I-restricted immune response and clonal expansion of the relevant CD8+ cytotoxic T lymphocytes is discussed.

Amino Acid Sequence↗

Monensin blocks the transport of diphtheria toxin to the cell cytoplasm.

Lysosomotropic amines are believed to inhibit the transport of diphtheria toxin to the cell cytoplasm by raising the pH within intracellular vesicles. If so, then other drugs that dissipate intracellular proton gradients should have a similar effect on toxin transport. We found that monensin, a proton ionophore unrelated to lysosomotropic amines, is a potent inhibitor of the cytotoxic effect of diphtheria toxin. Monensin appears to block the escape of endocytosed toxin from a vesicle to the cytoplasm. Monensin fails to protect cells from the effects of diphtheria toxin that is bound to the cell surface and exposed to acidic medium, suggesting that the step normally blocked by the drug is circumvented under these conditions. The inhibition of toxin transport caused by monensin could not be relieved when monensin was replaced by ammonium chloride, nor when ammonium chloride was again replaced by monensin. This suggests that both drugs block the same step of toxin transport. The effect of monensin on the transport of diphtheria toxin to the cytoplasm is consistent with the proposal (Draper and Simon. 1980. J. Cell Biol. 87:849-854; Sandvig and Olsnes. 1980. J. Cell Biol. 87:828-832) that the toxin is endocytosed and then, in response to an acidic environment, penetrates through the membrane of an intracellular vesicle to reach the cytoplasm.

Animals↗

Studies on the mode of action of diphtheria toxin. II. Protein synthesis in primary heart cell cultures.

Primary heart cell cultures of embryonic guinea pigs and the neonatal rat were established and incubated with purified diphtheria toxin. The rat heart cell cultures were refractory to the effects of the toxin; protein synthesis proceeded normally as measured by the incorporation of tritiated leucine into cell proteins; beating heart cells continued to contract; and the cell monolayers remained intact after exposure to the toxin for periods as long as 72 hr. These findings are compatible with the species resistance of the rat to diphtheria toxin. The guinea pig heart cell cultures were found to be extremely sensitive to the toxin. Protein synthesis was inhibited by approximately 50% after incubation with small quantities of toxin for 3 hr. Increasing the concentration of the length of exposure to the toxin did not increase this inhibition significantly. In addition, diphtheria toxin exerted a true cytopathic effect on the guinea pig heart cells. Monolayers were destroyed when incubated with the toxin for 2 to 3 days. The results show that the heart cells reflect species resistance or sensitivity to diphtheria toxin in the absence of neural or endocrine influences and suggest further that the toxin exerts a direct toxicity to muscle cells of the heart. It is not yet possible to explain in biochemical terms why the toxin seems to act specifically on cardiac tissues.

Animals↗