A Novel High-Potency Tetanus Vaccine

Abstract

Chemically inactivated tetanus toxoid (CITT) is clinically effective and widely used. However, CITT is a crude nonmalleable vaccine that contains hundreds of Clostridium tetani proteins, and the active component is present in variable and sometimes minor percentages of vaccine mass. Recombinant production of a genetically inactivated tetanus vaccine offers an opportunity to replace and improve the current tetanus vaccine. Previous studies showed the feasibility of engineering full-length tetanus toxin (TT) in Escherichia coli. In the present study, full-length TT was engineered with eight individual amino acid mutations (8MTT) to inactivate catalysis, translocation, and host receptor-binding functions, retaining 99.4% amino acid identity to native tetanus toxin. 8MTT purified as a 150-kDa single-chain protein, which trypsin nicked to a 100-kDa heavy chain and 50-kDa light chain. The 8MTT was not toxic for outbred mice and was >50 million-fold less toxic than native TT. Relative to CITT, 8MTT vaccination elicited a strong immune response and showed good vaccine potency against TT challenge. The strength of the immune response to both vaccines varied among individual outbred mice. These data support 8MTT as a candidate vaccine against tetanus and a malleable candidate conjugate vaccine platform to enhance the immune response to polysaccharides and other macromolecular molecules to facilitate a rapid response to emerging microbial pathogens. IMPORTANCE Chemical inactivation is a clinically effective mechanism to detoxify protein toxins to produce vaccines against microbial infections and to serve as a platform for production of conjugate polysaccharide vaccines. This method is widely used for the production of protein toxin vaccines, including tetanus toxoid. However, chemical modification alters the protein structure with unknown effects on antigenicity. Here, a recombinant full-length tetanus toxin (TT) is engineered with 8 mutations (8MTT) that inactivate three toxin functions: catalysis, translocation, and receptor binding. 8MTT is nontoxic and elicits a potent immune response in outbred mice. 8MTT also represents a malleable platform for the production of conjugate vaccines, which can facilitate a rapid vaccine response against emerging microbial pathogens.

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification

“…According to the densitometric quantification, the purity was 80%. Given that the Ttx is a mixture of forms with different degrees of cross-linking, , it is consistent that a band as wide as that observed by SDS-PAGE is obtained.…”

“…This mixture of the inactivated tetanus toxoid is a nonmalleable crude containing hundreds of C. tetani proteins, and the active component is present in varying percentages and sometimes less than the mass of the vaccine. , During formaldehyde treatment, intra- and interprotein cross-linking could occur.…”

International Nonproprietary Names (INN) for novel vaccine substances: A matter of safety

“…While detailed information on the structure of protein toxins is available, the nature of chemically inactivated toxin is much less precise and is not appropriate for an INN. However, a recombinant genetically inactivated toxin being developed as a vaccine could be amenable to assignment of an INN [28] .…”

Resolving the Molecular Steps in Clostridial Neurotoxin Light Chain Translocation

“…Antibodies that bind the HCN may sterically inhibit toxin association with receptors or distinct intermediates in translocation such as structural rearrangement, pore-formation, or LC delivery. Additionally, design of immunogens that stabilize secondary structural elements or inhibit toxin action without chemical-inactivation will improve current vaccines [54]. Last of all, the screening of current drugs and small molecule libraries may reveal compounds that prevent HCN secondary structure interaction with the membrane or downstream host chaperones.…”