The anaerobic, Gram-positive Clostridium difficile is a big problem. It causes rampant diarrhea and tissue necrosis, with more than 150,000 annual cases in the United States alone. Many of the disease manifestations of C. difficile are mediated by two exotoxins that C. difficile produces: TcdA and TcdB. Researchers have long been working at toxin inhibition as an approach to disarm C. difficile and improve treatment, and new research in Clinical and Vaccine Immunology shows promise in blocking toxin activity in vivo.
Both TcdA and TcdB toxins are glucosyltransferases that transfer sugars to several targeted host molecules, including Ras family members involved in cell signaling and actin regulation. Cells exposed to TcdA/TcdB experience a rapid change in cell shape as F-actin concentrations decrease (see figure, left). Structural damage increases tight junction permeability, and fluid secretion from epithelial cells accumulates, leading to edema. Ultimately, many of the cells die by apoptosis.
To exert these effects, the TcdA and TcdB toxins are released by C. difficile into the extracellular space, and must interact with host cell receptors for uptake by endocytosis. A team of scientists led by Saul Tzipori generated a molecule, based on recombinant antibody technology, that blocks TcdA and TcdB uptake. Rather than treat C. difficile infection with an entire antibody, which has been successful but is quite expensive, the team generated a VHH, which is the variable region of the immunoglobulin heavy chain (see figure, right). This small fragment can be recombinantly expressed from E. coli, decreasing cost and increasing yield. Because the molecule is based on a toxin-recognizing antibody, it can potentially neutralize these molecules.
What’s better than a single VHH? Multiple VHHs! The team had previously characterized VHHs that neutralize against TcdA and TcdB, and used this data to create a construct of two VHHs that neutralize each toxin (four total), connected by linker sequences. This tetra-specific, heteromultimeric VHH-based neutralizing agent, called VNA2-Tcd, was the molecule tested against C. difficile pathogenesis in two forms: one, as a purified protein expressed from E. coli, and one, as a gene therapeutic in an adenovirus vector.
The first proof-of-principle was to look at the ability of purified VNA2-Tcd to protect against toxin. Host cells were exposed to TcdA/TcdB, which resulted in a classic round phenotype, as the actin scaffold within the cell disintegrated. As expected, increasing doses of VNA2-Tcd protected more cells from rounding. A dose of VNA2-Tcd was also able to protect mice from subsequent toxin challenge, demonstrating in vivo efficacy.
But the toxins are a problem in the context of disease, so the researchers moved into three animal models of C. difficile infection: mouse, hamster, and pig. The rodent models were first used to test the efficacy of the purified VNA2-Tcd. Mice were challenged with C. difficile spores, and mice receiving purified VNA-Tcd2 treatment were injected at 4, 24, and 48 hours after infection. Untreated mice uniformly became sick with diarrhea, but no treated mice became moribund (although one mouse did develop diarrhea, which resolved after a day), and the treatment protected all animals from death (see figure, left). Hamsters were less well protected: treated animals were delayed in symptom onset, but all animals (except one treated hamster) developed and eventually succumbed to disease symptoms.
Pigs were then tested with both purified protein and adenovirus therapies. Just like the mice, all piglets were infected with C. difficile spores. Both the purified VNA2-Tcd and the adenovirus gene therapy treatment were able to lessen (though not prevent) disease. Histology showed that lesions were smaller in the treated groups, demonstrating the positive effect of toxin blockage on tissue damage. When looking at the adenovirus-treated animals, the research team observed a correlation between animals with a high serum VNA2-Tcd concentration and mild to moderate symptoms – more production equaled more protection.
The VNA2-Tcd treatments were administered to the animals systemically, but ideally sick human patients would take their medication orally. However, even orally-administered purified protein therapy would need to continue as long as the patient remained colonized with high levels of C. difficile bacteria, because the VHHs neutralize toxins but have no antibacterial activity. The gene therapy treatment success in piglets is a promising way to address this problem: patients’ own cells generate the VNA2-Tcd VHH constructs to mediate prolonged protection. Patients would still experience C. difficile overgrowth, which may still need addressing through other clinical treatments. But eliminating the bacterial toxins' effects through neutralization may be the first step to turn this deadly pathogen into a bothersome nuisance.
-- Julie Wolf