Traditionally, diphtheria-tetanus-pertussis (DTwP or DTaP) as pediatric vaccines are produced from the corresponding inactivated toxins or whole cell pathogenic bacteria of Corynebacterium diphtheria toxin (Tox), Clostridium tetani toxin (TetX) and Bordetella pertussis. There are major concerns in the classic or acellular DTP (DTaP) vaccine production processes from native live sources of bacteria as it may raise concerns on adverse effects and safety issues, complexity of the purifications for each agent as well as costs. Here, we designated recombinant multi-epitope candidate vaccine by vaccino-informatics study to address mentioned issues and to develop a single trivalent fusion protein as potent recombinant DTP vaccine. To follow these goals, stages of immune-bioinformatics were retrieved by means of proteinaceous toxins sequences, predicting secondary/tertiary structure and transmembrane topology, energy minimization and models validation. Then, conformational and linear B cell epitope prediction by several different servers, mapping consensus linear/discontinuous immunogenic regions and constructing synthetic fusion vaccine candidate in respect to optimal immunogenic, physico-chemical properties and highly expression in prokaryote host were achieved. Finally, reverse translation, codon optimization, addition of cloning tags for pet 28a vector and optimizations of physico-chemical characteristics of synthetic trivalent fusion protein were performed. By different hybrid immune-informatics and structural bioinformatics analysis predicted and experimental epitopes finally, 12 new consensus highly immunogenic linear and discontinuous epitopes in Tox, TetX and PTXa proteins were selected. Peptide sequence of these immunogenic regions were as follow; PTXA (AA34-64, AA184-256 and AA98-116), Tox (AA47-76, AA117-159, AA515-557 and AA245-265) and TetX (AA226-249, AA819-844, AA923-967, AA1009-1067 and AA1225-1315). Moreover, the characteristics of recombinant trivalent fusion construct were; 546 residue length, solvable (Grand average of hydropathicity (GRAVY) was -0.475), estimated half-life was >10 hours in Escherichia coli, pI 5.94 (a little acidic), stable protein (The instability index (II) 35.58) as well as thermal stable (Aliphatic index (AI) 71.67). The putative antigenic epitopes from different organisms in a single protein, as in the current study, possibly will improve the protective effectiveness as novel potent, safe, cheap and broad-spectrum vaccines for better prevention of diphtheria, tetanus and pertussis infections in future.

Le fragment Cest le domaine C-terminal de la chaîne lourde de la toxine tétanique. Ce dernier favorisé la réponse immunitaire contre la dose létale de cette toxine et peut donc être considéré comme un candidat potentiel pour le vaccin contre linfection tétanique causée par Clostridium tetani. Dans cette étude, lexpression du fragment C de la toxine tétanique dans des cellules BL21 (DE3) pLysS dE. coli ayant une tolérance élevée aux toxines a été comparée en utilisant deux vecteurs dexpression différents, pET22b et pET28a, par SDS-PAGE et western blot. Après l’extraction de lADN de la souche Harvard CN49205 de C. tetani, le gène dintérêt a été amplifié par PCR, séquencé, cloné dans les vecteurs dexpression, pET22b et pET28a, transformé dans des cellules compétentes BL21 (DE3) pLysS et enfin exprimé selon un protocole optimisé. Les cellules ont été induites avec IPTG à quatre températures dincubation différentes (37, 33, 30 et 25° C) et trois temps dincubation différents (1 à 3 h). Bien que lexpression du fragment recombinant C (fragment C) ligaturé dans les deux vecteurs dexpression ait été confirmée par les analyses SDS-PAGE et western blot, pET28a a montré une expression plus élevée du fragment r C comparé à pET22b (38,66 mg / l contre 32,33 mg / l, p

Tetanus is a life-threatening neurologic disease caused by an infectious agent, Clostridium Tetani, and is one of the infectious diseases that is preventable by vaccination. Tetanus is characterized by painful muscle spasm and increased muscle tone, that is caused by tetanospasmin, a powerful toxin elaborated by Clostridium tetani. Due to worldwide regular vaccination, this disease rarely seen recently. The case we present here is a patient admitted in Shohadaye khalije Fars hospital with severe generalized tetanus due to incomplete vaccination.

Tetanus is an important disease which created by tetanospasmin toxin of Clostridium tetani. In this study we surveyed six important factors including LF, KF, MLD, pH, OD and total protein assay of Harvard 52 (H52), G5 (HG5) and 49205 (H49205) strains of the bacterium to determine which of them were more suitable for use in vaccine production. The mentioned strains were seperatedly reactivated in thioglycolate medium, 2ml of this suspension was used to inoculate the Muller-Miller medium of each of sixteen 500 ml Erlenmeyer flasks (7,6 and 3 flasks for H52, HG5 and H49205 respectively), where the fermentation runs were performed. Over a period of seven days of experiment, several tests for evaluation of the six mentioned factors on samples of medium cultures were carried out. Results revealed that H52 strain had significantly lower values in LF and OD compared to strains HG5 & H49205 (P<0.001 and P<0.01 respectively) while its MLD and pH were better than other strains (P< 0.05 and P< 0.02 respectively). In conclusion, it seems that HG5 & H49205 strains have been greater toxin producer than H52 strain and as a result, we hope that with some complementary works, these two strains such as H52 strain, be used for routine tetanus vaccine production.

Clostridium botulinum is a spore-forming bacterium that produces lethal neurotoxin, the causative agent of a paralytic disease known as botulism. Diagnosis of botulism is obtained by detecting the neurotoxin or bacterium in a suspected food sample. The standard method for detecting the toxin is the mouse bioassay. It is highly sensitive and specific, but costly, time-consuming, laborious, and requires handling of laboratory animals thus, only a limited number of samples can be analyzed at one time. Other methods such as ELISA technique were used for detection of the toxin, but the antibody raised against one type had cross reactivity with other types. In this study, a Multiplex Polymerase Chain Reaction (PCR) assay was developed for detection of C. botulinum types A, B, and E. C. botulinum types A, B, and E have formerly been confirmed by the biochemical methods as well as mouse bioassay. For detection by PCR, three primer pairs with equal melting temperatures were designed, each being specific to botulinum neurotoxin gene type A, B, or E and enables a simultaneous detection of the three serotypes. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, and 389 bp for type E alone were obtained and confirmed by digestion enzymes. Clostridium perfringens was used as negative control and did not yield a PCR product. This method is rapid, sensitive, specific and can be used for detection of three types of Clostridium botulinum human pathogen.

Tetanus is caused by the toxin secreted by Clostridium tetani. Due to the rapid infection with this bacterium, it is so important to investigate the tetanus immunity of people. Therefore, electrochemical biosensors, as one of the most effective tools in this regard, have demanded characteristics such as being fast, simple, cost-effective and portable. However, their detection sensitivity is not sufficient. Hereon, silver enhancement of gold-nanoparticles was proposed for the improvement of detection. Hence, the current study applied gold-nanoparticles as label, following with silver enhancement, to investigate the yes/no electrochemical detection of anti-tetanus toxoid antibodies in the indirect immunoassay utilizing glassy-carbon electrodes modified by carbon nanotubes. The analytical procedure consists of the reactions of the tetanus toxoid with the antibody at electrode, so that followed by the interaction of gold-labeled secondary antibody and then silver enhancement process. In this study, the cyclic-voltammeter variation and difference of gold to silver signal based on silver ions fluctuations were also investigated. The results indicated that ∆Ep increased from 0.24 V before silver enhancement reaction to 0.57 V after the silver enhancement. The results also demonstrated that after silver enhancement, current significantly increased and current plot at Ecp transferred to positive potentials and at Eap moved to negative potentials. In conclusion, this method increases the detection sensitivity and can simply use to other bio-molecules detection.

Among the bacterial agents, the most common cause of diarrheal disease is Entrotoxigenic Escherichia coli. Lebal toxin B (LTB) subunit of LT toxin could induce six-month immunity. Tetanus toxin of Clostridium tetani causes the fatal disease, tetanus. Tetanus can be prevented by vaccination with tetanus toxoid. This toxoid induces ten years immunity in humans. Binding domain of tetanus toxin (THc), of the toxoid is considered as immunogenic part of tetanus toxin. Clostridium botulinum causes botulism disease. The protectional effect of this toxoid is only two years against this disease. It seems that the immunogenicity potency of these three subunits may influence on the memory longevity. The aim of this study is the assessment of LTB, BoNT/A and THc immunogenicity in mouse. The transgenic E. coli BlDE3 with pET28a vector, containing recombinant ltb, thc and bont/A-Hc genes separately were used for expression of recombinant proteins. All mentioned proteins were derived, purified and evaluated on SDS-PAGE gel. Finally, mice immunization were carried out and antibody titration of all recombinant proteins were evaluated and compared using t-Test in SPSS software. The result of SDS-PAGE gel evaluation showed a proper expression. The immunoassay results of serum showed that the antibody titer against the recombinant protein of THc was higher than those for the recombinant protein of BONT/A-Hc and LTB. A difference in antibody titer also was observed between two proteins i.e. BoNT/A-Hc and LTB (P value <.0001). The differences in the antibody titer may be related to the longevity of memory cells. However, the result needs more studies