Regulation of Clostridium tetani Neurotoxin Expression by Culture Conditions.

BACKGROUND: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. METHODS: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. RESULTS: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. CONCLUSIONS: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production.

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology.

Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization-mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.

Toll-like receptor 5 is not essential for the promotion of secretory immunoglobulin A antibody responses to flagellated bacteria.

Toll-like receptor 5 recognizes bacterial flagellin, plays a critical role in innate immunity, and contributes to flagellin-specific humoral immunity. Further, TLR5-expressing dendritic cells play an important role in IgA synthesis in the intestine; however, the contribution of TLR5 to antigen (Ag)-specific mucosal immunity remains unclear. Thus, whether TLR5 is essential for the induction of intestinal secretory (S)IgA antibody (Ab) responses against flagellin and bacterial Ags attached to the bacterial surface in response to an oral flagellated bacterium, Salmonella, was explored in this study. Our results indicate that when TLR5 knockout (TLR5(-/-)) mice are orally immunized with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella-Tox C), tetanus toxoid (TT)- and flagellin (FliC)-specific systemic IgG and intestinal SIgA Abs are elicited. The numbers of TT-specific IgG Ab-forming cells (AFCs) in the spleen and IgA AFCs in the lamina propria (LP) of TLR5(-/-) mice were comparable to those in wild-type mice. rSalmonella-Tox C was equally disseminated in TLR5(-/-) mice, TLR5(-/-) mice lacking Peyer's patches (PPs), and wild-type mice. In contrast, TLR5(-/-) PP-null mice failed to induce TT- and FliC-specific SIgA Abs in the intestine and showed significantly reduced numbers of TT-specific IgA AFCs in the LP. These results suggest that TLR5 is dispensable for the induction of flagellin and surface Ag-specific systemic and mucosal immunity against oral flagellated bacteria. Rather, pathogen recognition, which occurs in PPs, is a prerequisite for the induction of mucosal immunity against flagellated bacteria.

A phase III, randomized controlled study to assess the safety and immunogenicity of a semi-synthetic diphtheria, tetanus and whole-cell pertussis vaccine in Indian infants.

BACKGROUND: Reactions to DTwP vaccine are well known and are a matter of great concern, much for the development of next generation combination vaccines. To avoid such reactions which occur from foreign compounds, WHO suggested manufacture of DTwP vaccine using semi-synthetic medium. The phase III trial reported here was conducted to assess the immunogenicity, tolerability and safety of a new DTwP vaccine manufactured using semi-synthetic medium for both tetanus and diphtheria toxoids in comparison with the routinely manufactured DTwP vaccine. METHODS: In all, 331 infants aged 6-8 weeks were enrolled, out of which 308 completed the study. The vaccination was done at 6-10-14 weeks following EPI/WHO recommended immunization schedule. Blood samples were collected prior to the administration of first dose and one month after the third dose. RESULTS: Postvaccination, geometric mean titres for each component did not differ significantly amongst the two study groups. Though, the immunogenicity results were comparable between the two vaccines, the incidence of adverse events was comparatively low in semi-synthetic vaccine as against the routine vaccine group for all the three doses. CONCLUSIONS: The semi-synthetic DTwP vaccine was immunogenic and showed a significant lower incidence of local adverse events in comparison to the routine vaccine. This vaccine is now being used in the routine vaccination programme both as a triple antigen (DTwP alone) as well as a combination with Hepatitis B and/or Haemophilus influenzae type b vaccine.

New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC.

Vaccines are a proven method of controlling disease. However there are issues with the delivery and administration of vaccines. A particular problem is that the majority of vaccines currently used are injected, which can be unsafe if needles are reused in areas where blood-borne diseases are prevalent. Vaccines targeting the mucosal immune system avoid many of the problems associated with injections. One potential form of mucosal vaccine is based on the expression of vaccine antigens in plants. Current research in this area has focused on the expression of immunogens from the plant's nuclear genome but low expression levels generally achieved using this system have limited progress. In recent work we have used the model antigen, TetC, which confers resistance to Tetanus infection, to demonstrate the feasibility of expressing vaccine antigens at high levels in the plant chloroplast.

[Study on the effect of a biostimulant on the growth and toxigenic function of Clostridium tetani strain Copenhagen-471]. / Izuchenie vliianiia biostimuliatora na rost i toksinoobrazuiushchuiu funktsiiu Clostridium tetani "Kopenhagen-471."

Bakstim, a new biostimulating preparation obtained from the organs of the immune system of animals, was developed. The impact of Bakstim on the growth and toxigenic function of C. tetani production strain Copenhagen-471 was evaluated. The addition of the preparation to Gluzman commercial medium for obtaining tetanus toxoid led to an increase in the yield of bacterial biomass from 1.9 to 4-fold and an increase in the toxoid production from 2 to 2.8-fold. The optimum concentration of this biostimulant ensuring the maximum yield of tetanus toxin from the production culture was determined (1,000 mg/l). Bakstim will supposedly be used as additive to nutrient media for the production of tetanus toxoid.

Profilaxis Antitetánica Posterior a Trauma

El tétanos es una enfermedad grave con alta mortalidad, causada por una toxina producida por el bacilo Gram positivo anaerobio Clostridium tetani. Se produce cuando las esporas que habitan en el medio ambiente penetran en el organismo por cualquier herida, permitiendo que estas eclosionen, proceso en el que se genera la tetanospasmina, sustancia proteica responsable del cuadro clínico. Este se produce por inhibición de la sinaptobrevina II, metaloproteína enzimática que participa en el proceso de degranulación de los axones inhibitorios motores. Esta inhibición es irreversible y solo desaparece con la generación de un nuevo axón. La prevención es posible con la práctica de inmunización previa y sistemática de la población con toxoide tetánico y la aplicación de antitoxina tetánica en los casos en los que el estado inmune del paciente es incierto, o las heridas sean potencialmente tetanógenas

Nitrogen-gas bubbling during the cultivation of Clostridium tetani produces a higher yield of tetanus toxin for the preparation of its toxoid.

We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N2 was bubbled through nine 10-liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis.

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.

Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.