Mathematical model of the infection process in diphtheria for determining the therapeutic dose of antitoxic anti-diphtheria serum

It is known that administration of horse serum against diphtheria toxin can cause autoimmune and allergic complications. Therefore it is important for improvement of serotherapy to develop methods of prediction of disease course and quantity of diphtheria toxin and antitoxic antibodies in a serum. We have developed the mathematical model of diphtheria infection, which consists of six differential equations describing dynamics of diphtheria toxin and antitoxic antibodies in a serum, quantity of infection agent and macrophages in a site of inflammation. This mathematical model allows to predict the course of infectious process, the level of diphtheria toxin and antitoxic antibodies in the sera of people with diphtheria and to calculate the individual therapeutic dose of antitoxic serum for each patient.     

Indices of diphtheria (antitoxic) immunity in the dynamics of the disease and its treatment in adults

A total of 1,034 serum samples from 618 persons, including patients with different forms of diphtheria, carriers of the toxigenic forms of Corynebacterium diphtheriae, and angina patients, were studied. Analysis of the incidence of antibodies to C. diphtheriae toxin and their titers revealed that in more than half of all diphtheria patients no antibodies to C. diphtheriae toxin were detected upon admission to hospital. At the same time in 26% of the patients no antibodies were detected during the whole period of the disease; in such patients the toxic and subtoxic forms of diphtheria were registered twice as often as in seropositive patients. In 31% of the patients seronegative by the moment of hospitalization a rapid increase in the titers of antibodies C. diphtheriae toxin was observed in the course of the disease, which was indicative of the secondary character of immune response in patients who had been immunized earlier.     

Entry of diphtheria toxin-protein A chimeras into cells

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.     

Development of monoclonal latex diagnostic kit for diphtheria toxin and toxoid detection

Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).     

Production and characteristics of monoclonal antibodies to the diphtheria toxin

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.     

The estimation of the amount of diphtheria toxin and its activity by various tests in the dynamic cultivation of Corynebacterium diphtheriae PW8]

Direct correlation between the results of tests for the biological activity of diphtheria toxin, carried out in vivo guinea pigs and in vitro in the microcytotoxicity test in CHO cell culture, has been established, which makes it possible to use the latter as one of the methods for the rapid, reproducible and economic evaluation of diphtheria toxin. The qualitative and quantitative evaluation of diphtheria toxin in the enzyme immunoassay with the use of monoclonal antibodies and in the microcytotoxicity test demonstrates that these two tests, when used for controlling cultivation processes, have essential advantages over the flocculation test as regards their specificity and information content.     

Recombinant fluorescent models for studying the diphtheria toxin

Diphtheria toxin is a major factor of the pathogenicity of the causative agent of diphtheria Corynebacterium. Due to a small size, it is of considerable interest as the basis for the development of synthetic protein molecules with a transport function, e.g., immunotoxins. In this work we describe the expression and characterized nontoxic recombinant fluorescent derivatives of the diphtheria toxin and its nontoxic CRM197 mutant. The proteins obtained can be used for studying receptor-binding and transport functions of the toxin in cells, evaluation of the expression level of the toxin proHB-EGF receptor membranes, immunization and generation of specific antibodies against the toxin, as well as for the development of diagnostic test-systems for the diphtheria toxin and antitoxic antibodies.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.     

Antitoxic properties of monoclonal antibodies against diphtheria toxin

Antitoxin properties of monoclonal antibodies to diphtheria toxin were studied in chick fibroblast culture and during the dermonecrotic tests in guinea pigs. Antitoxic properties were shown for antibodies specific to B subunit of the toxin but not for the ones specific to a subunit. The mixture of monoclonal B subunit specific antibodies reveals a higher neutralizing activity as compared with the activity produced by any single monoclonal antibody tested. The protective activity demonstrated by antibodies is connected with the preventing of toxin molecules fixation on the cellular receptors.     

Detection of diphtheria toxin and its degradation products by using immunoenzyme analysis

An enzyme immunoassay system for the detection of diphtheria toxin and the products of its degradation has been developed on the basis of the enzyme-linked immunosorbent assay. To sensitize the assay plates, bivalent F(ab)2-fragments of purified antidiphtheria antibodies at a concentration of 10.0 micrograms/ml, obtained from the blood serum of hyperimmunized rabbits, have been used. Specific conjugates have been prepared with the use of F(ab)2-fragments of purified antidiphtheria antibodies obtained from the blood serum of hyperimmunized horses. The optimum time and temperature conditions of the assay have been established. The new enzyme immunoassay system permits the detection of diphtheria toxin and the products of its degradation in biological substrates at a concentration of 10.0-5.0 ng/ml.     

The diagnostic effectiveness of an immunoenzyme test system for determining diphtheria toxin antigens in the blood serum in a clinical laboratory trial

The data on the approbation of the diagnostic value of the enzyme immunoassay (EIA) system for the determination of diphtheria toxin in the blood sera of diphtheria patients and persons suspected for diphtheria are presented. The EIA system was prepared on the basis of F(ab)2 fractions of purified antidiphtheria antibodies. 240 serum samples from diphtheria and tonsillitis patients and from healthy persons were studied. Diphtheria toxin was determined in all patients with the toxic form of diphtheria and in 41.3% of patients with its localized forms. Blood was taken mainly of the first week of the disease. In healthy persons the results of EIA were negative. Thus, the trial of the assay system in a clinical laboratory showed its good diagnostic effectiveness. The use of this EIA system in medical practice is believed to be quite promising.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.     

Monoclonal antibodies labeled with colloidal gold for immunochromatographic express analysis of diphtheria toxin

A one-step immunochromatographic method for the detection of the diphtheria toxin in different water samples (phosphate buffer, milk, and human nasopharyngeal swab) was developed using a conjugate of monoclonal antibodies labeled with colloidal gold. The detection limit of diphtheria toxin was 10 ng/ml, the time of the analysis was 15 min. The use of silver to enhance the reaction sensitivity and scanning equipment to evaluate the immunochromatography results decreased the detection limit to 1.25 ng/ml.     

The coagglutination reaction for detecting diphtheria toxin

High-titer antidiphtheria antitoxic rabbit serum has been obtained, and on the basis of this serum a coagglutinating diagnosticum has been developed. The sensitivity of the test has been found to depend on the content of antitoxic antibodies in the serum and on its purity. Diagnostica prepared from native serum containing 500 I. U./ml (a titer of 1:51, 200 in the passive hemagglutination test) permit the detection of 0.02-0.03 Lf/ml of diphtheria toxin. A decrease in antibody titer to 5-25 I. U./ml leads to a drop in sensitivity to 0.2-2 Lf/ml. The use of LgG fraction and pure antibodies increases the sensitivity of the test to 0.002-0.003 Lf/ml. The possibility of detecting toxin in Corynebacterium diphtheriae strains is shown.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

Infectious allergy. XIV. Histochemical changes in redox enzymes in cells of the small intestine smooth muscle during sensitization with diphtheria exotoxin]

Administration to guinea pigs of 1/Dlm of diphtheria exotoxin twice at an interval of 1 and 24 hours, or once in a dose of 25/100 Dlm caused changes in the activity of the redox enzymes. The character of their changes in the animals to which the whole dose of the exotoxin was administered once or repeatedly was directly reverse: in the first case the activity of the enzymes was uniformly increased, and in the second--ununiformly depressed; besides, there was a marked disturbance of coordination between the enzymes. An increased sensitivity of the infectious allergy type following injection of low doses of diphtheria exotoxin was not accompanied by immunity formation. Specific antibodies against diphtheria exotoxin formed only in the organism of animals to which 1/100 Dlm of diphtheria toxin was injected once a day for 15 days, but in this case as well no immunity against diphtheria exotoxin was observed.     

Development of recombinant scFv-antibodies against diphtheria toxin using phage display system

Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

Enzyme treatment of KB cells: the altered effect of diphtheria toxin

Treatment of KB cells with trypsin, phospholipase C and pronase at concentrations which did not affect viability or rate of incorporation of amino acids into protein lowered significantly the inhibition of protein synthesis by diphtheria toxin. Chymotrypsin was effective but only at concentrations toxic to the cells. Lipase and neuraminidase had no effect. It is evident that enzyme-sensitive cell surface components with specificity for diphtheria toxin are present on the KB cell.     

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.