Immunogenicity and safety in rabbits of a Clostridioides difficile vaccine combining novel toxoids and a novel adjuvant.

Clostridioides difficile infection (CDI) is a serious healthcare-associated disease, causing symptoms such as diarrhea and pseudomembranous colitis. The major virulence factors responsible for the disease symptoms are two secreted cytotoxic proteins, TcdA and TcdB. A parenteral vaccine based on formaldehyde-inactivated TcdA and TcdB supplemented with alum adjuvant, has previously been investigated in humans but resulted in an insufficient immune response. In search for an improved response, we investigated a novel toxin inactivation method and a novel, potent adjuvant. Inactivation of toxins by metal-catalyzed oxidation (MCO) was previously shown to preserve neutralizing epitopes and to annihilate reversion to toxicity. The immunogenicity and safety of TcdA and TcdB inactivated by MCO and combined with a novel carbohydrate fatty acid monosulphate ester-based (CMS) adjuvant were investigated in rabbits. Two or three intramuscular immunizations generated high serum IgG and neutralizing antibody titers against both toxins. The CMS adjuvant increased antibody responses to both toxins while an alum adjuvant control was effective only against TcdA. Systemic safety was evaluated by monitoring body weight, body temperature, and analysis of red and white blood cell counts shortly after immunization. Local safety was assessed by histopathologic examination of the injection site at the end of the study. Body weight gain was constant in all groups. Body temperature increased up to 1 ˚C one day after the first immunization but less after the second or third immunization. White blood cell counts, and percentage of neutrophils increased one day after immunization with CMS-adjuvanted vaccines, but not with alum. Histopathology of the injection sites 42 days after the last injection did not reveal any abnormal tissue reactions. From this study, we conclude that TcdA and TcdB inactivated by MCO and combined with CMS adjuvant demonstrated promising immunogenicity and safety in rabbits and could be a candidate for a vaccine against CDI.

High-resolution structure of native toxin A from Clostridioides difficile.

Clostridioides difficile infections have emerged as the leading cause of healthcare-associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 Å resolution cryo-EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor-binding domain, which likely serves to shield the C-terminal part of the hydrophobic pore-forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the N-terminal part of the pore-forming region suggest that the globular subdomain shields the upper part of the pore-forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore-forming region at neutral pH, as well as the pH-induced inter-domain dynamics.

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile.

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile.

Clostridioides difficile infections (CDI) has emerged worldwide as a serious antimicrobial-resistant healthcare-associated disease resulting in diarrhea and pseudomembranous colitis. The two cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB) are the major virulence factor responsible for the disease symptoms. We examined time-dependent oxidative detoxification of TcdA and TcdB using different molar ratios of protein:Cu2+:H2O2. The metal-catalyzed oxidation (MCO) reaction in molar ratios of 1:60:1000 for protein:Cu2+:H2O2 at pH 4.5 resulted in a significant 6 log10 fold reduction in cytotoxicity after 120-min incubation at 37 °C. Circular dichroism revealed that MCO-detoxified TcdA and TcdB had secondary and tertiary structural folds similar to the native proteins. The conservation of immunogenic epitopes of both proteins was tested using monoclonal antibodies in an ELISA, comparing our MCO-detoxification approach to a conventional formaldehyde-detoxification method. The oxidative detoxification of TcdA and TcdB led to an average 2-fold reduction in antibody binding relative to native proteins, whereas formaldehyde cross-linking resulted in 3-fold and 5-fold reductions, respectively. Finally, we show that mice immunized with a vaccine consisting of MCO-detoxified TcdA and TcdB were fully protected against disease symptoms and death following a C. difficile infection and elicited substantial serum IgG responses against both TcdA and TcdB. The results of this study present copper ion-catalyzed oxidative detoxification of toxic proteins as a method highly suitable for the rapid production of safe, immunogenic and irreversible toxoid antigens for future vaccine development and may have the potential for replacing cross-linking reagents like formaldehyde.