Clostridioides difficile infection (CDI) has emerged as the leading cause of healthcare-associated infectious diarrhea in the western world, causing tens of thousands of related deaths each year. Disease is mediated by two secreted homologous toxins, toxin A (TcdA) and toxin B (TcdB), which can cause a wide spectrum of disease symptoms and in severe cases, death. CDI is currently treated with antibiotics such as metronidazole and vancomycin, but around 25% of patients experience recurrence and this calls for alternative treatment options. Toxoid-based vaccines against CDI have shown promise in preventing disease symptoms and several vaccine candidates have reached clinical trials, although one of them using formaldehyde cross-linking for detoxification of TcdA and TcdB recently failed in a phase III clinical trial. This thesis focuses on developing a new method for detoxification of TcdA and TcdB for producing a toxoid-based vaccine against CDI.
The new detoxification method is based on copper ion-catalyzed oxidation (MCO) of TcdA and TcdB at low pH, and was inspired by a previous oxidation-based detoxification method for preparing pertussis toxoid (whooping cough) at Statens Serum Institut. Detoxification of TcdA and TcdB using this optimized MCO method, reduced the cytotoxicity of both toxins by more than 6 log10 fold relative to native toxins, as determined by a toxin-sensitive Vero cell assay. In addition, circular dichroism (CD) spectroscopy was used to assess the changes in secondary and tertiary structure of the MCO-detoxified toxoids of TcdA and TcdB, indicating relatively similar structural folds compared to the native proteins, with only slight changes in the CD spectra likely due to some degree of precipitation. Furthermore, a panel of toxin-specific monoclonal antibodies (mAbs) were used to evaluate the conservation of several epitopes on MCO-detoxified TcdA and TcdB in an ELISA, and compared to TcdA and TcdB toxoids prepared using conventional formaldehyde cross-linking. The binding affinities of the mAbs to the MCO-detoxified toxoids were on average 2-fold reduced relative to native toxins, whereas formaldehyde cross-linking led to an average of 3-fold and 5-fold reductions in binding to TcdA and TcdB toxoids, respectively. The structure of single particles in a sample of MCO-detoxified TcdA was also analyzed by negative stain electron microscopy, which despite results from CD measurements revealed significant structural changes and possibly partial denaturation compared to the native TcdA structure. Nonetheless, the final and pivotal evaluation of the MCO detoxification was performed using an in vivo mouse model of CDI, by immunizing a group of mice with a bivalent toxoid vaccine comprising TcdA and TcdB detoxified by MCO, prior to a C. difficile challenge. All mice in the immunized group were fully protected against disease symptoms and death, and raised substantial serum antibody responses against both TcdA and TcdB. The efficacy parameters of the MCO-detoxified toxoid vaccine were overall comparable to a similar vaccine prepared using formaldehyde cross-linking. Moreover, lethal toxin from Clostridium sordellii (TcsL) and diphtheria toxin from Corynebacterium diphtheriae (DT) were likewise significantly detoxified using the MCO method developed in this thesis. However, further studies are needed to improve the parameters of the method to ensure optimal preservation of the epitopes on TcsL and DT, as significant reduction in mAb binding affinities were observed to both toxins after MCO detoxification.
To enable the extensive studies mentioned above requiring large amounts of C. difficile toxins, the expression and purification of native TcdA and TcdB were optimized, and a complete protocol for obtaining high yields of highly purified toxins is presented in this thesis. During this optimization study it was found that the presence of zinc and glucose in C. difficile culture medium dramatically increases the toxin production, supporting previous in vivo mouse studies showing exacerbated CDI severity when exposed to excess dietary zinc. Furthermore, differential scanning fluorimetry was used to find improved storage buffer conditions demonstrating higher melting temperatures of TcdA and TcdB, by screening a large number of buffers, pH ranges and stabilizing additives. Finally, the long-term stability of TcdA and TcdB was evaluated by SDS-PAGE and Vero cell assay at various storage temperatures. The study revealed that TcdA is highly resistant to degradation and loss of activity at storage temperatures between 25 to -80 °C for up to 28 days, while TcdB is stable at 5 °C or colder for up to 21 days, and highly prone to degradation at 25 °C.
The findings in this thesis provide a novel method for detoxification of TcdA and TcdB using low pH-dependent MCO, to prepare safe and immunogenic vaccine antigens for use against CDI. Additionally, an improved protocol for the production and purification of native TcdA and TcdB is also presented.