Genetically detoxified tetanus toxin for use in vaccines

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Summary Tetanus and diphtheria toxoids are highly effective vaccines for preventing diseases. As “carrier proteins”, tetanus and diphtheria toxoids enhance the immunogenicity of small molecules and polysaccharides. However, tetanus toxoid (TTxd) represents only 20-70% of the protein in the TTxd vaccine and the TTxd vaccine contains hundreds of ‘un-intended/contaminant’ clostridial proteins. Purification is often needed prior to TTxd use a conjugate vaccine carrier. TT is detoxified with formaldehyde, using an over 30-day incubation that blocks a subset lysines that cannot then be used for conjugation with antigens. Collaborators at the Medical College of Wisconsin have engineered a full-length, atoxic tetanus toxin (M8TT) with 8 independent mutations reducing catalysis, translocation, and binding functions. Here, Fina Biosolutions (FinaBio) proposes to develop and manufacture M8TT in a proprietary engineered E. coli strain that has a unique oxidative environment. This strain has been used successfully to produce multi-grams/L amounts of CRM197, a mutated form of diphtheria toxin that has been successfully used as a recombinantly expressed vaccine protein carrier. This proposal uses recombinant DNA technology, biotechnology, biochemistry and immunological approaches to produce and test the immunological potency of this next generation conjugate tetanus vaccine platform. The Specific Aims for Phase I are to: subclone and scale up production and purify > 1 g/L of M8TT at >95% purity; and to test the immunological properties of M8TT versus conventional TTxd to produce a conjugate of Hemophilus influenzae subtype b polyribitol phosphate sugar PRP conjugated to M8TT and TTxd to determine if PRP-TTxd is a more potent conjugate vaccine to PRP and TT than PRP-TTxd. If successful, Phase II studies will optimize the M8TT manufacturing process to 50 L production scale and characterize the product for safety and efficacy in pre-clinical trials. In addition, the utility of M8TT as a vaccine carrier protein will be further explored with additional antigens, including small molecules and other polysaccharides. Ultimately, a superior tetanus vaccine protein will be commercialized by advancing a 50-year old industrial technology with a new, modernized, economic, effective, and safe conjugate TT vaccine platform.