Localized tetanus in immunized mice.

The capacity of tetanus toxin to enhance motor neuron excitability has suggested its potential use as a therapeutic. Widespread active vaccination against tetanus in all developed countries is considered the major obstacle to clinical use of the toxin. We wished to determine the response to localized intramuscular injection of tetanus toxin in both passively and actively immunized animals as an initial exploration into the possible use of tetanus toxin as a clinical therapeutic. Unvaccinated mice (n=18) underwent intramuscular injection of tetanus toxin into the gastrocnemius muscle (0.2 ng, 1 ng, 5 ng). All animals in the lowest dose group developed only local tetanus of the injected limb of at least 2 weeks duration, while all animals in the higher dose groups also rapidly developed generalized tetanus and were euthanized. Another group of mice (n=20) received anti-tetanus immunoglobulin (20-40 IU) at the time of toxin injection. These animals although dramatically resistant to the toxin developed predominantly local tetanus for over one month at doses of 2.5 microg and 5.0 microg. A third group of mice (n=30) underwent active vaccination with tetanus toxoid to induce protective anti-tetanus immunity and then was challenged with high dose toxin injection (5 ng, 50 ng, 0.5 microg, 1.25 microg, 2.5 microg, or 5 microg). All animals developed local tetanus in the injected limb at a dose of at least 0.5 microg. The severity and duration of local tetanus was generally related to dose, but was more variable in the actively vaccinated group than in the naive or passively immunized animals. Response to the toxin over the first few days was predictive of both the duration and maximal severity of the motor response. Although vaccination dramatically increases resistance to tetanus toxin, by virtue of its extremely high potency, the toxin can produce prolonged localized tetanus even in vaccinated animals with relatively small amounts of protein. These results suggest the possible use of tetanus toxin to enhance local motor activity in a variety of neurologic conditions even in immunized humans. This study in uniformly vaccinated animals also illustrates the potential difficulties in determining an appropriate dose of toxin in a human population with variable degrees of immunity.

Immunization does not interfere with uptake and transport by motor neurons of the binding fragment of tetanus toxin.

The nontoxic binding domain of tetanus toxin (fragment C or TTC) readily undergoes retrograde axonal transport from an intramuscular injection site. This property has led to investigation of TTC as a possible vector for delivering therapeutic proteins to motor neurons. However, the vast majority of individuals in the developed world have been vaccinated with tetanus toxoid and have circulating antitetanus antibodies that cross-react with TTC and may block the delivery of a TTC-linked therapeutic protein. However, it is uncertain whether the immune response is capable of completely neutralizing an intramuscular depot of protein prior to its internalization by presynaptic nerve terminals, where it is inaccessible to antibody. We have evaluated uptake of rhodamine-labeled TTC following intramuscular injection in normal animals and animals vaccinated with tetanus toxoid prior to injection of fluorescently labeled TTC. All animals demonstrated uptake of TTC, with fluorescence appropriately localized to the hypoglossal nerve and nucleus. The distribution and intensity of fluorescence within neurons and processes were indistinguishable between the two groups and were characteristic of TTC. Vaccinated animals showed levels of uptake of TTC into the brain comparable to those of immunologically naïve animals as measured by quantitative fluorimetry. All vaccinated animals had protective levels of antitetanus antibodies as measured by ELISA. Uptake of TTC by nerve terminals from an intramuscular depot is an avid and rapid process and is not blocked by vaccination associated with protection from tetanus toxin.

A multi-domain protein system based on the HC fragment of tetanus toxin for targeting DNA to neuronal cells.

One goal of gene therapy is the targeted delivery of therapeutic genes to defined tissues. One attractive target is the central nervous system as there are several neuronal degenerative diseases which may be amenable to gene therapy. At present there is a lack of delivery systems that are able to target genes specifically to neuronal cells. Multi-domain proteins were designed and constructed to facilitate the delivery of exogenous genes to neuronal cells. Neuronal targeting activity of the proteins was achieved by inclusion of the HC fragment of tetanus toxin (TeNT), a protein with well-characterised tropism for the central nervous system. The yeast Gal4 DNA-binding domain enabled specific binding of DNA while the translocation domain from diphtheria toxin (DT) was included to facilitate crossing of the endosomal vesicle. One multi-domain protein, containing all three of these domains, was found to transfect up to 8% of neuroblastoma N18-RE105 cells with marker genes. Monitoring the transfection by confocal microscopy indicated that this protein-DNA transfection complex is to some extent localised at the cell surface, suggesting that further improvements to translocating this membrane barrier may yield higher transfection levels. The demonstration that this multi-domain protein can target genes specifically to neuronal cells is a first step in the development of novel vectors for the delivery of genes with therapeutic potential to diseased neuronal tissues.