In this study, PCR technique was used with 2 primer pairs for amplifying DNA fragments at 5’ and 3’ of the gene encoding diphteria toxine of the strains Corynebacterium diphtheriae. After the cloning and sequencing, 2 separated fragments were joined to form a complete gene. The sequence of gene was translated into protein and the results were submitted into Gene Bank Database
Diphtheria Toxin ; Genes ; Corynebacterium diphtheriae

BACKGROUND: Diphtheria epidemics in Russia have spread to all the other independent states of the former Soviet Union and East European countries around 1990s. One of the most important measures in preventing diphtheria is to maintain high levels of immunity in the population. We studied the diphtheria antibody levels of 1,086 participants to investigate herd immunity in Korea. METHODS: The tested 1,086 serum specimens were collected from healthy individuals from September 1995 to March 1996. Diphtheria antitoxin titers were measured by a micro cell culture method using Vero cells. Antibody titer of 0.01 IU/ml to neutralize diphtheria toxin is an internationally accepted protective level. RESULTS: We studied the diphtheria antitoxin titer levels of 1,086 cases consisting of 579 males and 507 females. The proportion of protective antitoxin level to diphtheria is 69.2%. Diphtheria antitoxin levels showed no significant difference between males and females. The highest seropositive rate was observed in the 5 to 9-year old age group(95.8%). The seropositivity rate declined with age. The lowest seropositive rate was observed in the 20~39 years of age, maximally 43.4 %. Over 40 years of age, the seropositive rates increased again. CONCLUSION: The antibody titers in the Korean population declined from 95.8% to below 50% with age in the 1~39 year-old age group. To maintain the rate of population with protective antibodies to diphtheria, we recommend Td booster immunization to adults with low antitoxin titers and continuous survey for antitoxin titers.
Adult ; Antibodies ; Cell Culture Techniques ; Child ; Diphtheria Antitoxin ; Diphtheria Toxin ; Diphtheria* ; Female ; Humans ; Immunity, Herd ; Immunization, Secondary ; Korea ; Male ; Russia ; USSR ; Vero Cells

PURPOSE: We conducted the study to evaluate the immunogenicity and safety of three component DTaP vaccine (Infanrix(R)) in a group of Korean healthy infants on a three-dose primary vaccination. And we compared the immunogenicity of this DTaP vaccine with two component DTaP vaccine which has been widely used in Korea. METHODS: We enrolled one hundred fifty one healthy infants aged 8-9 weeks. These infants were vaccinated at age 2, 4 and 6 months of age with three component DTaP vaccine. Solicited adverse events were actively monitored for 72 hours following each vaccination, and all adverse events after each vaccination were observed for three weeks. Anti-diphtheria toxoid Ab., anti-tetanus toxoid Ab., anti-pertussis toxin Ab., anti-filamentous hemagglutinin Ab., and anti-pertactin Ab. were measured using ELISA for assessing immunogenicity of study vaccine in 60 infants. Immunogenicity analysis of two component DTaP vaccine was performed with same methods in 14 infants as control. RESULTS: The seroconversion rates of anti-diphtheria toxoid Ab, anti-tetanus toxoid Ab. anti- filamentous hemagglutinin Ab. were 100% in both group. Seroconversion rate of anti-pertactin Ab in study group was 100%, but the rate in control group was 50%. However, geometric mean concentration of anti-pertussis toxin Ab. was higher in control group. Mild local and systemic reactions were observed within three days after vaccination, and no serious adverse events related study vaccine were happened during study period. CONCLUSIONS: Our study results suggest that three component DTaP vaccine (Infanrix(R)) is a well- tolerable and high immunogenic vaccine, especially anti-Pertactin Ab. of the study vaccine is very immunogenic. It can be available as routine DTaP vaccination in our infants.
Diphtheria-Tetanus-acellular Pertussis Vaccines* ; Enzyme-Linked Immunosorbent Assay ; Hemagglutinins ; Humans ; Infant* ; Korea ; Pertussis Toxin ; Vaccination

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Bacterial Proteins ; Diphtheria Toxin/genetics* ; Escherichia coli/genetics* ; Humans ; Molecular Chaperones/genetics* ; Recombinant Proteins/genetics*

BACKGROUND: The incidence of diphtheria has been markedly reduced and almostly eradicated by widespread use of DTP vaccines in developed countries. However, outbreaks of this disease may be occurred under some circumstances of ineffective immunization. In recent time, some studies reported persistent outbreaks of diphtheria in developed countries and indicated the existence of a large pool of susceptible individuals with potential for epidemic infection. In Korea, diphtheria vaccination has been well maintained since 1956 with high acceptant vaccination rates. So, there has been no reported diphtheria patient since 1987. But, there has been few study to diphtheria serosuvey, and no assessment of diphtheria immunization. Also, we do not use Td vaccine in adults for maintenance of diphtheria immunity. In this aspect, we conducted age related seroepidemiology of diphtheria and indirectly assessed the immunity of diphtheria vaccines, used in Korea. METHODS: For the evaluation of age related serosurvey of diphtheria immunity in Korean populations, study subjects below 10 years old aged children were classified into 10 groups (A~J) with one year interval, and beyond this age to 60 years old aged adults were classified into 5 group (K~O) with 10 years interval. And the adults over 60 years old age was classified into the last group (P). The numbers of each group were 100, and sex distributions of each group were almostly equal. And for the indirect assessment of diphtheria immunization in Korean children, children under 15 years old were classified into 6 groups (I~ VI) according to the status of DTaP vaccination. The numbers of this each group were 30, and sex ratio was almostly equal. Detection of specific IgG antibody to diphtheria toxin were determined by ELISA (contained fragment A & B toxin; IBL, Germany). RESULTS: In age related groups, the antibody titers to diphtheria toxin were well maintained until 10 years old age group, thereafter the titers abruptly decreased below 0.1 IU/mL and then slightly elevated after 30 years old age group and then maintained with low levels. In the groups related DTaP vaccine status, the antibody titers were very low (below 0.07 IU/mL) in prevaccination status, but the titers after primary vaccinations were markedly increased and maintained (above 0.6 IU/mL) until 15 years. And diphtheria antitoxin levels in the groups (L, M, N) showed no significant differences between males and females. CONCLUSION: The results of our study showed that the antibody titers to diphtheria toxin in the 20~50 years old aged groups dramatically decreased. This result indicated that vaccine induced diphtheria immunity did not last throughout life, and Td vaccination program in adults should be considered for maintenance of diphtheria immunity. And the immunity to diphtheria in Korean children indicated that 3 timesprimary and 2 times booster diphtheria immunizations were optimal.
Adolescent ; Adult ; Child ; Developed Countries ; Diphtheria Antitoxin ; Diphtheria Toxin ; Diphtheria* ; Diphtheria-Tetanus-acellular Pertussis Vaccines ; Disease Outbreaks ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunization ; Immunoglobulin G ; Incidence ; Korea ; Male ; Middle Aged ; Seroepidemiologic Studies* ; Sex Distribution ; Sex Ratio ; Vaccination ; Vaccines

OBJECTIVE: Immunotoxin therapy is a novel approach for the treatment of tumor, and it has been successfully used in the central nervous system. The purpose of this study is to evaluate the cytotoxicity of OKT9 ScFv-Diphtheria toxin fusion immunotoxin on various human brain tumor cell lines. METHODS: Immunotoxin which was composed of OKT9 ScFv and Diphtheria toxin was made. Its cytotoxicity on glioblastoma cell lines(U87MG, U118MG) and medulloblastoma cell line(TE671) was tested and compared with anti-cancer chemotherapeutic agents. And we also examined the relationship between its cytotoxicity and transferrin receptor expression. RESULTS: It showed most cytotoxicity on U87MG cell line and nearly no effect on U118MG cell line, moderate cytotoxicity on TE671 cell line in sixteen hours exposure experiment. In continuous exposure experiment, it showed moderate cytotoxicity on U118MG cell line, but showed strong cytotoxicity on other cell lines comparable or higher than anti-cancer chemotherapeutic agents. The relationship between its cytotoxicity and transferrin receptor expression was tested using flow cytometry, but no direct relationship could be found. CONCLUSION: Collectively, the result shows the cytotoxic effects of OKT9 ScFv-Diphtheria toxin fusion immunotoxin against various human brain tumor cell lines in continuous exposure experiment. Therefore, we suggest that this immunotoxin could be developed as a potential immunotherapeutic agent in the treatment of various human brain tumor clinically.
Brain Neoplasms* ; Brain* ; Cell Line* ; Central Nervous System ; Diphtheria Toxin ; Flow Cytometry ; Glioblastoma ; Humans* ; Immunotoxins* ; Medulloblastoma ; Receptors, Transferrin

Effects of adrenocorticosteroid in the rabbits given repeated sublethal doses of diphtheria toxin were studied with respects to survival time, electrocardiographic behavior, change in serum potassium, and histopathology of the heart, kidney and adrenal gland. Experiments were performed twice with different doses of toxin and prednisolone and with different methods of administration. In the first experiment, all fourteen rabbits received daily subcutaneous injections of 0.1 M.L.D. of toxin per kg of body weight for three consecutive days, and eight of them received daily intramuscular injections of 6mg of prednisolone per kg of body weight beginning soon after the first injection of toxin until death. In the second experiment, all sixteen rabbits received subcutaneous injections with daily doses of 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 M.L.D. of toxin per kg of body weight in this order for six consecutive days, and nine of them received daily intramuscular injections of 10mg of prednisolone per kg of body weight beginning two days prior to the first injection of toxin until death. As control groups, six rabbits in the first experiment and seven in the second, received daily intramuscular injections of normal saline for the same period. Electrocardiograms were taken in six rabbits given intravenous KCI solution only to compare the findings with those of the first and second experimental animals. The results are as follows: 1. In the first experiment, mean survival times were 118.024.6 hours in the prednisolone-treated group and 145.842.6 in the control and in the second experiment, 159.226.1 in the prednisolone-treated group and 176.324.1 in the control. The difference was not statistically significant in the first experiment while significant in the second. 2. Major electrocardiographic findings consisted of tenting of T wave, change in S-T segment, inversion of T wave, widening of QRS duration and P-R interval, deviation of QRS axis, disappearance of P wave and decrease in heart rate. No significant differences were found between the prednisolone-treated and control groups. In general, electrocardiographic patterns showed marked similarities to those of progressive hyperpotassemia. 3. After toxin injection, hyperpotassemia developed and progressed to 11.2 mEq/1 (mean) just before death. No significant differences were found between the prednisolone-treated and control groups. 4. Electrocardiographic findings in six rabbits given intravenous KCI solution only were similar to those of the toxin-treated rabbits with or without prednisolone administration, in which left deviation of QRS axis and inversion of T wave in lead I and V6 were also observed in addition to other hyperpotassemia findings. 5. Gross findings at autopsy showed; a) Multiple petechiae were frequently observed on the surface of the kidney. b) Brownish black discoloration of the adrenal gland probably due to hemorrhage and necrosis was found mainly in the rabbits of preliminary study group given relatively large doses of toxin. c) Abnormalities were rare in the heart. 6. Microscopically, the kidney showed more pronounced change than those of the heart and the adrenal gland. In the kidney, severe dilatation of the glomerular capillary with fibrin thrombi, many casts in the tubule, and relatively mild tubular degeneration and dilatation were observed in most cases. Interstitial hemorrhage was also seen in some cases, but necrosis was rare. In the heart, granular degeneration of the muscle cell and interstitial hemorrhage were relatively prominent in most cases. Mild myocytolysis and fibrin thrombi in interstitium were also observed frequently, but interstitial infiltration, loss of striation and myocardial necrosis were rare. Fatty change of the muscle cell, although found in most, was very mild. In the adrenal gland, cortical hemcorrhage and necrosis were found mainly in the rabbits of preliminary study group. Among the findings mentioned above, dilatation of the glomerular capillary with fibrin thrombi and interstitial hemorrhage in the kidney were more pronounced in the prednisolone-treated groups than in the control. 7. Fibrin thrombi observed in the kidney and the heart suggest that disseminated intravascular coagulation was developed during toxemia.
Adrenal Glands ; Animals ; Autopsy ; Axis, Cervical Vertebra ; Body Weight ; Capillaries ; Dilatation ; Diphtheria Toxin* ; Diphtheria* ; Disseminated Intravascular Coagulation ; Electrocardiography ; Fibrin ; Heart ; Heart Rate ; Hemorrhage ; Hyperkalemia ; Injections, Intramuscular ; Injections, Subcutaneous ; Kidney ; Muscle Cells ; Necrosis ; Potassium ; Prednisolone ; Purpura ; Rabbits* ; Survival Rate ; Toxemia

Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.
Angiogenesis Inhibitors ; biosynthesis ; Diphtheria Toxin ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Immunotoxins ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

OBJECTIVETo test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells.

METHODSThe diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed.

RESULTSThe beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells. Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ.

CONCLUSIONSelective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.

Diphtheria Toxin ; genetics ; Enhancer Elements, Genetic ; Genes, Immunoglobulin ; Genetic Therapy ; methods ; Humans ; Immunoglobulin kappa-Chains ; genetics ; Neoplasms ; therapy ; Peptide Fragments ; genetics ; Promoter Regions, Genetic ; Tumor Cells, Cultured

PURPOSE: There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. MATERIALS AND METHODS: The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. RESULTS: When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. CONCLUSION: We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.
Diphtheria ; Enzyme-Linked Immunosorbent Assay ; Hemagglutinins ; Immunoassay ; Immunoglobulin G ; Korea* ; Methods* ; Pertussis Toxin ; Pertussis Vaccine ; Vaccination ; Vaccines* ; Whooping Cough* ; World Health Organization