A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.
Adjuvants, Immunologic ; Aluminum ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19/prevention & control* ; COVID-19 Vaccines/genetics* ; Humans ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/genetics* ; SARS-CoV-2/genetics* ; Spike Glycoprotein, Coronavirus/genetics* ; Tetanus Toxin ; Tuftsin ; Vaccines, Subunit ; Viral Vaccines

Synthetic cannabinoids are one of most abused new psychoactive substances. The recreational use of abused drug has aroused serious concerns about the consequences of these drugs on infection. However, the effects of synthetic cannabinoid on resistance to tetanus toxin are not fully understood yet. In the present study, we aimed to determine if the administration of synthetic cannabinoids increase the susceptibility to tetanus toxin-induced motor behavioral deficit and functional changes in cerebellar neurons in mice. Furthermore, we measured T lymphocytes marker levels, such as CD8 and CD4 which against tetanus toxin. JWH-210 administration decreased expression levels of T cell activators including cluster of differentiation (CD) 3ε, CD3γ, CD74p31, and CD74p41. In addition, we demonstrated that JWH-210 induced motor impairment and decrement of vesicle-associated membrane proteins 2 levels in the cerebellum of mice treated with tetanus toxin. Furthermore, cerebellar glutamatergic neuronal homeostasis was hampered by JWH-210 administration, as evidenced by increased glutamate concentration levels in the cerebellum. These results suggest that JWH-210 may increase the vulnerability to tetanus toxin via the regulation of immune function.
Animals ; Cannabinoids ; Cerebellar Diseases* ; Cerebellum ; Glutamic Acid ; Homeostasis ; Immunosuppression* ; Mice* ; Neurons ; R-SNARE Proteins ; T-Lymphocytes ; Tetanus ; Tetanus Toxin

PURPOSE: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus. MATERIALS AND METHODS: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed. RESULTS: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin. CONCLUSION: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.
Animals ; Antibody Formation ; Flagellin* ; Mice ; Tetanus ; Tetanus Toxin* ; Tetanus Toxoid ; Toll-Like Receptor 5 ; Vaccines ; Vaccines, Subunit ; Vibrio vulnificus

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.
Adult ; Animals ; Bromodeoxyuridine ; Cell Proliferation ; Dentate Gyrus ; Exotoxins ; Fluoresceins ; Gliosis ; Humans ; Immunohistochemistry ; Mice ; Neurogenesis ; Neurons ; Tetanus ; Tetanus Toxin

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to A1a and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to > 95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1 x 10(3) 50% lethal doses (LD50s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.
Escherichia coli ; genetics ; metabolism ; Mutant Proteins ; biosynthesis ; genetics ; immunology ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Protein Conformation ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Tetanus ; prevention & control ; Tetanus Toxin ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

Tetanus is a neurologic disorder caused by a tetanospasmin released from Clostridium tetani and usually occurs following a stab wound or dirty abrasion. Tetanus is uncommon in Korea due to the introduction of vaccination programs. Furthermore, tetanus associated with a gangrenous perforation of the small bowel is extremely rare. We report a case of tetanus developed in a patient who was diagnosed with a gangrenous perforation of the small bowel. This is the first reported case in Korea.
Clostridium tetani ; Digestive System Surgical Procedures ; Humans ; Intestinal Perforation ; Korea ; Metalloendopeptidases ; Nervous System Diseases ; Tetanus ; Tetanus Toxin ; Vaccination ; Wounds, Stab

OBJECTIVEExpression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein (CT-1/TTC) in BL21 (DE3) E. coli, examined whether tetanus toxin C fragment mediate the cardiotrophin-1 target delivery to the central nervous system and the cardiotrophin-1 has the neurotrophic ability.

METHODSInduction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method. And select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl's staining.

RESULTSAfter induction, the fusion protein was about 15% of the total protein and the soluble part was predominant. Purified by GST fusion protein column, the interest protein's concentration is 2.7 g/L. The CT-1/TTC fusion protein was found in lumbar intumescentia by immunohistochemistry method. And after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate.

CONCLUSIONSThe recombinant CT-1/TTC protein can be expressed and purified in BL21 (DE3) E. coli. This fusion protein has two biological activities of targeting delivery to central nervous system and protecting the cornu anterius medullae spinalis motoneurons.

Animals ; Animals, Newborn ; Cytokines ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Disease Models, Animal ; Escherichia coli ; metabolism ; Female ; Motor Neurons ; metabolism ; pathology ; Peptide Fragments ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Protein Transport ; drug effects ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Sciatic Nerve ; injuries ; Spinal Cord ; pathology ; Tetanus Toxin ; biosynthesis ; genetics ; isolation & purification ; pharmacology

Tetanus is a neurologic disease which features the muscle spasm as the hallmark. It is an infectious disease with high mortality rate triggered by tetanospasmin produced by Clostridium tetani. This report concerns incidence of tetanus consequent to oriental medical care such as acupuncture and moxibustion. Although the tetanus occurrence has shown a remarkable decline since nationwide vaccinations in some of the developed countries, including Korea, it still remains to be an important issue, to be dealt within Korea, as the majority of the patients are old aged and Korean population is rapidly becoming an aging society. Furthermore, since more elders are coming to rely on Oriental medicine in Korea, the Korean elders are at a higher risk than elsewhere. The lack of medical experiences, including those in oriental medical field, has been hindering early diagnosis of Tetanus. This study aims to encourage rapid and accurate decisions in diagnosis and treatment through reviewing symptoms particularly specific to tetanus, and also to arouse attention to the riskiness of invasive procedures involving skin puncture.
Acupuncture ; Aged ; Aging ; Clostridium tetani ; Communicable Diseases ; Developed Countries ; Early Diagnosis ; Humans ; Incidence ; Korea ; Medicine, East Asian Traditional ; Metalloendopeptidases ; Moxibustion ; Punctures ; Skin ; Spasm ; Tetanus ; Tetanus Toxin ; Vaccination

OBJECTIVETo obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.

METHODSThe gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity.

RESULTSThe 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1:25 600, and the anti-serum could specifically bind to tetanus toxin.

CONCLUSIONHighly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.

Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Tetanus Toxin ; biosynthesis ; genetics ; immunology ; Tetanus Toxoid ; immunology

OBJECTIVETo develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).

METHODSTo allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.

RESULTSThree 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.

CONCLUSIONPCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.

Base Sequence ; Cloning, Molecular ; Genes, Synthetic ; genetics ; Lactococcus ; genetics ; Peptide Fragments ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; metabolism ; Tetanus Toxin ; genetics ; metabolism