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*

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

Foxp3 is a master regulator of CD4⁺CD25⁺ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4⁺ or CD8⁺ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma.
Adenoviridae ; Airway Remodeling ; Animals ; Asthma* ; Bronchoalveolar Lavage Fluid ; Cockroaches ; Cytokines ; Diphtheria Toxin ; Eosinophils ; Epithelial Cells* ; Goblet Cells ; Hyperplasia ; Hypertrophy ; Immunoglobulin E ; Inflammation* ; Lung* ; Lymphocytes ; Macrophages ; Mice ; Myocytes, Smooth Muscle ; Neutrophils ; Ovalbumin* ; Ovum ; T-Lymphocytes

Osteocytes establish an extensive intracellular and extracellular communication system via gap junction-coupled cell processes and canaliculi, through which cell processes pass throughout bone, and the communication system is extended to osteoblasts on the bone surface. To examine the osteocyte function, several mouse models were established. To ablate osteocytes, osteocytes death was induced by diphtheria toxin. However, any types of osteocyte death result in necrosis, because dying osteocytes are not phagocytosed by scavengers. After the rupture of cytoplasmic membrane, immunostimulatory molecules are released from lacunae to bone surface through canaliculi, and stimulate macrophages. The stimulated macrophages produce interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha), which are the most important proinflammatory cytokines triggering inflammatory bone loss. Therefore, the osteocyte ablation results in necrosis-induced severe osteoporosis. In conditional knockout mice of gap junction protein alpha-1 (GJA1), which encodes connexin 43 in Gap junction, using dentin matrix protein 1 (DMP1) Cre transgenic mice, osteocyte apoptosis and enhanced bone resorption occur, because extracellular communication is intact. Overexpression of Bcl-2 in osteoblasts using 2.3 kb collagen type I alpha1 (COL1A1) promoter causes osteocyte apoptosis due to the severe reduction in the number of osteocyte processes, resulting in the disruption of both intracellular and extracellular communication systems. This mouse model unraveled osteocyte functions. Osteocytes negatively regulate bone mass by stimulating osteoclastogenesis and inhibiting osteoblast function in physiological condition. Osteocytes are responsible for bone loss in unloaded condition, and osteocytes augment their functions by further stimulating osteoclastogenesis and further inhibiting osteoblast function, at least partly, through the upregulation of receptor activator of nuclear factor-kappa B ligand (RANKL) in osteoblasts and Sost in osteocytes in unloaded condition.
Animals ; Apoptosis ; Bone Resorption ; Cell Membrane ; Collagen Type I ; Connexin 43 ; Connexins ; Cytokines ; Dentin ; Diphtheria Toxin ; Gap Junctions ; Interleukin-6 ; Interleukins ; Macrophages ; Mice* ; Mice, Knockout ; Mice, Transgenic ; Necrosis ; Osteoblasts ; Osteocytes* ; Osteoporosis ; RANK Ligand ; Rupture ; Tumor Necrosis Factor-alpha ; Up-Regulation

Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, breaks out in developing countries. However, existing vaccines only induce relatively low protective effects with humoral responses and do not stimulate secondary immune response, especially to young people. The objective of this study is to evaluate the immunogenicity of the vaccine containing virulence capsular polysaccharide (Vi) conjugated with the optimal ratios of non-toxic variant of diphtheria toxin (CRM(197)) in mice. Six-week-old BALB/c female mice were injected intraperitoneally three times at intervals of 14 days and sera were collected on days 0, 14, 28, 42 and 56 post-injection. The efficacy of the vaccine was evaluated by comparing between negative control group injected with PBS and vaccine groups injected with Vi or Vi-CRM(197) conjugate of different ratio. Vi and CRM(197)-specific antibody responses were evaluated using enzyme-linked immunosorbent assay. The result showed that Vi-CRM(197)-1 group revealed the highest and significant Vi-specific IgG immune responses among the other groups and Vi group (p < 0.01). In conclusion, Vi-CRM(197)-1 conjugate vaccine induced the highest humoral immune response in mice and may be used as an effective vaccine to replace the existing typhoid vaccine for infants under 2 years old.
Animals ; Antibody Formation ; Child, Preschool ; Developing Countries ; Diphtheria Toxin ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunity, Humoral ; Immunoglobulin G ; Infant ; Mice* ; Salmonella enterica* ; Salmonella typhi* ; Salmonella* ; Typhoid Fever ; Typhoid-Paratyphoid Vaccines ; Vaccines ; Virulence

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.
Adjuvants, Immunologic ; Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Ehrlich Tumor ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Diphtheria Toxin ; genetics ; immunology ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Immunoglobulin G ; immunology ; Immunotherapy ; Mice ; Neovascularization, Pathologic ; Peptide Fragments ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tandem Repeat Sequences

OBJECTIVETo explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.

METHODSThe mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.

RESULTSThe expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.

CONCLUSIONSThe recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; therapy ; Diphtheria Toxin ; biosynthesis ; genetics ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Genomic Imprinting ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

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

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS); it serves as a model for the human multiple sclerosis (MS). In mice, EAE is mediated by T cells specific for various myelin basic proteins which migrate from the periphery to the CNS. In search of a way to prevent the induction and progression of EAE, we observed the effects of recombinant immunotoxin IP10-DT390 on blocking or eliminating the active T cells in the EAE model. In this paper is presented an experimental gene therapy-based model in which the mice were made resistant to EAE induction by plasmid DNA encoding recombinant immunotoxin that was injected into the leg muscles of mice. The new immuno-biological construct could selectively impair autoreactive T-cell homing while the duration of clinical signs is shorter, and the new construct would not affect other components of the immune response. These data demonstrated the effectiveness of the constructs in the treatment of EAE and suggested its usefulness in the treatment of other autoimmune diseases.
Animals ; Chemokine CXCL10 ; biosynthesis ; genetics ; therapeutic use ; Diphtheria Toxin ; biosynthesis ; genetics ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; pathology ; therapy ; Female ; Genetic Therapy ; Immunoglobulin Fragments ; biosynthesis ; genetics ; therapeutic use ; Immunotoxins ; genetics ; metabolism ; therapeutic use ; Mice ; Mice, Inbred C57BL ; Receptors, CXCR3 ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; therapeutic use ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; T-Lymphocytes ; immunology ; Transfection

OBJECTIVETo evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).

METHODSEAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.

RESULTSEAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.

CONCLUSIONThe immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.

Animals ; Antigens, CD19 ; analysis ; B-Lymphocytes ; cytology ; metabolism ; CD3 Complex ; analysis ; Chemokine CCL3 ; genetics ; metabolism ; Diphtheria Toxin ; genetics ; metabolism ; Disease Models, Animal ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; Female ; Flow Cytometry ; Immunoglobulin Fragments ; genetics ; metabolism ; Immunohistochemistry ; Immunologic Factors ; therapeutic use ; Immunotoxins ; therapeutic use ; Meninges ; chemistry ; pathology ; Mice ; Mice, Inbred C57BL ; Multiple Sclerosis ; drug therapy ; NIH 3T3 Cells ; Receptors, CCR5 ; analysis ; Recombinant Fusion Proteins ; genetics ; metabolism ; therapeutic use ; T-Lymphocytes ; cytology ; metabolism