Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests.

In the hope of developing a vaccine against Clostridium difficile based on its toxin(s), we have developed a fermentation medium for the bacterium that results in the formation of Toxin A and contains no meat or dairy products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Soy Peptone A3, have been found to replace both the meat/dairy product tryptone in the preparation of working cell banks and seed media, and NZ-Soy BL4 does the same in the fermentation medium. These replacements yield even higher toxin titers.

Novel polysaccharide-protein conjugates provide an immunogenic 13-valent pneumococcal conjugate vaccine for S. pneumoniae.

Pneumonia remains the single leading cause of childhood death worldwide. Despite the commercial availability of multiple pneumococcal conjugate vaccines (PCVs), high dosage cost and supply shortages prevent PCV delivery to much of the developing world. The current work presents high-yield pneumococcal conjugates that are immunogenic in animals and suitable for use in human vaccine development. The 13-valent pneumococcal conjugate vaccine (PCV-13) investigated in this research incorporated serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Pneumococcal polysaccharides (PnPSs) and CRM197 carrier protein were produced and purified in-house, and used to prepare PnPS-CRM conjugates using unique, cyanide-free, in vacuo glycation conjugation methods. In vitro characterization confirmed the generation of higher molecular weight PnPS-CRM conjugates low in free protein. In vivo animal studies were performed to compare PnuVax's PCV-13 to the commercially available PCV-13, Prevnar®13 (Pfizer, USA). A boost dose was provided to all groups post-dose 1 at t = 14 days. Post-dose 2 results at t = 28 days showed that all 13 serotypes in PnuVax's PCV-13 were boostable. Per serotype IgG GMCs demonstrated that PnuVax's PCV-13 is immunogenic for all 13 serotypes, with 10 of the 13 serotypes statistically the same or higher than Prevnar®13 post-dose 2. As a result, the novel polysaccharide-protein conjugates developed in this work are highly promising for use in human PCV development. The in vacuo conjugation technique applied in this work could also be readily adapted to develop many other conjugate vaccines.

The role of reduced iron powder in the fermentative production of tetanus toxin.

When tetanus toxin is made by fermentation with Clostridium tetani, the traditional source of iron is an insoluble preparation called reduced iron powder. This material removes oxygen from the system by forming FeO(2) (rust). When inoculated in a newly developed medium lacking animal and dairy products and containing glucose, soy-peptone, and inorganic salts, growth and toxin production were poor without reduced iron powder. The optimum concentration of reduced iron powder for toxin production was found to be 0.5 g/l. Growth was further increased by higher concentrations, but toxin production decreased. Inorganic iron sources failed to replace reduced iron powder for growth or toxin formation. The iron source that came closest was ferrous ammonium sulfate. The organic iron sources ferric citrate and ferrous gluconate were more active than the inorganic compounds but could not replace reduced iron powder. Insoluble iron sources, such as iron wire, iron foil, and activated charcoal, were surprisingly active. Combinations of activated charcoal with soluble iron sources such as ferrous sulfate, ferric citrate, and ferrous gluconate showed increased activity, and the ferrous gluconate combination almost replaced reduced iron powder. It thus appears that the traditional iron source, reduced iron powder, plays a double role in supporting tetanus toxin formation, i.e., releasing soluble sources of iron and providing an insoluble surface.

Effective levels of tetanus toxin can be made in a production medium totally lacking both animal (e.g., brain heart infusion) and dairy proteins or digests (e.g., casein hydrolysates).

We have developed a fermentation medium for Clostridium tetani that results in the formation of tetanus toxin and contains no meat (e.g., beef heart infusion) or dairy (e.g., casein digest) products, thus obviating the problem of possible prion diseases. Particular preparations of hydrolyzed soy proteins, especially Quest Hy-Soy, have been found to replace both the meat extract and casein digest components of traditional tetanus toxin production media and to yield even higher toxin titers. The comparison of the traditional versus the new medium has been carried out repeatedly by us and the superiority of our medium has been consistently observed. To our knowledge, this is the first time that such a medium has been devised.