Manufacture of Clostridioides difficile spores for experimental infection of human volunteers
1Leiden University Center for Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
2Center for Cell and Gene Therapy, Leiden University Medical Center, Leiden, The Netherlands
3Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands
*To whom correspondence should be addressed: Leiden University Center for Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands. E-mail: w.k.smits@lumc.nl. Phone: +31715261229Abstract
Background
Infections with C. difficile remain a significant global healthcare problem for which novel (preventive) treatment strategies are urgently needed. Controlled human infection models (CHIMs) can contribute to a better understanding of the disease and accelerate (novel) medicinal product development.
Methods
A robust production process for C. difficile spores for experimental human administration, was implemented. Direct-release capsules containing viable C. difficile spores were formulated. The manufacturing process aligns with good manufacturing practices (GMP) guidelines, including quality controls and release by a qualified person (QP).
Results
Selected non-toxigenic and toxigenic C. difficile strains were used to create a master cell bank and working cell bank from which multiple batches of purified active substance were produced. Substance was then used to manufacture the final active product. The active product and its intermediate products passed all quality controls for identity, potency, purity and safety, and were individually released by the QP. Stability of active substance and product is confirmed up to 12 months.
Conclusion
Although there are no clear standardized international guidelines for challenge material production, we demonstrate that it is feasible to produce C. difficile human challenge material for small scale application in CHIM trials. This application of GMP principles to an unconventional production process of a bacterial spore-forming anaerobic challenge agent is an example for the production of challenge material as described in the auxiliary medicinal product guidelines of EMA and FDA.
Article notes
Competing Interest Statement
WKS performs research which is funded in part by a public-private partnership with AcurX Pharmaceuticals unrelated to the work presented here. All other authors declare no competing interests.
Summary of Updates:
Background
Clostridioides difficile is the leading cause of healthcare-associated diarrhoea, causing substantial morbidity and mortality. Globally it is responsible for an estimated 15,600 deaths and 284,000 disability adjusted life years (DALYs), representing the primary cause of diarrhoea-related mortality in high-income countries (1). Concerningly, the incidence of community-acquired C. difficile infections (CDI) (defined as disease in patients not hospitalized during the 12 weeks prior to diagnosis) is on the rise, almost doubling the past years (2). As such CDI causes a substantial clinical, social and economic burden (3).
An acute episode of CDI is treated with antimicrobial therapy, but alternative therapies are urgently needed because of high relapse rates (approximately 20% after treatment of an initial episode and up to 60% after multiple recurrences) and a rise in antimicrobial resistance (4). Microbial restoration therapies, such as faecal microbiota transplantation (FMT) seem to deliver on the promise of high level sustained prevention of recurrent CDI (5-7). However, the complex interactions between C. difficile and the human microbiome remains largely poorly understood, hampering the translation of FMT into a medicinal product. Additionally, animal models cannot fully replicate the human microbiome and its interactions, making preclinical down selection and optimization for novel medicinal products complex. Innovations to accelerate such processes are thus needed.
Controlled human infection models (CHIMs), in which human volunteers are deliberately inoculated with an infectious organism (so-called “challenge agent”) under controlled conditions, are a valuable tool to rapidly evaluate medicinal products (e.g. vaccines) for efficacy in early phase clinical development (8, 9). CHIMs have been developed for a wide variety of pathogens (including parasites, viruses and bacteria) over the past 200 years and CHIM experiments have even led to the registration of a novel cholera vaccine (10). The development of a C. difficile CHIM, in which healthy adult volunteers are experimentally exposed to C. difficile spores in a controlled clinical environment, would provide the opportunity to identify microbiota and immunological targets for novel (preventive) medicinal products, and ultimately test the preliminary efficacy of such products (e.g. vaccines or microbial restoration therapies).
The development of a C. difficile CHIM starts with the production of C. difficile challenge material. To ensure safety of CHIM participants a challenge agent which complies with regulatory requirements for human use must be available. Within the EU, challenge agents are classified as auxiliary medicinal products (AxMPs) by the European Medicines Agency (EMA), defined as a medicinal product used for the needs of a clinical trial as described in the protocol, and should be manufactured according to Good Manufacturing Practice (GMP) equivalent standards. GMP guidelines describe the minimum requirements which medicinal products must meet in their manufacturing process to ensure that the medicinal product is 1) of consistent high quality 2) appropriate for their intended use and meets the requirements of the marketing or clinical trial authorization (11).
Aligning the manufacturing process of C. difficile challenge material with GMP guidelines, presents several key challenges, particularly in meeting the quality attributes of identity, purity, potency and safety, as outlined in the technical white paper on the quality considerations for challenge agent production for use in CHIM studies (9). Here, we addressed these challenges and describe a robust manufacturing process for challenge material derived from cultures of C. difficile, performed in an academic laboratory by trained personnel with an appropriate quality control strategy. The described process for producing encapsulated viable C. difficile spores serves as an example of the production of auxiliary medicinal products under the European Medicines Agency (EMA) or U.S. Food and Drug Administration (FDA) regulations.
Methods
Manufacturing a C. difficile challenge product
Production was performed at a dedicated laboratory for C. difficile production of the Experimental Bacteriology research group of the Leiden University Center for Infectious Diseases in the Leiden University Medical Center (LUMC) under the quality management system (QMS) of LUMC’s department of Clinical Pharmacy and Toxicology (KFT) and Center for Cell and Gene Therapy (CCG). The production process of C. difficile spores has been established using known and established methods and protocols at the LUMC (12).
The manufacturing process was developed to be adaptable to diverse C. difficile strains, including production of non-toxigenic as well as toxigenic C. difficile strains. Here, the process to produce challenge material for the non-toxigenic C. difficile strain L-NTCD03 (PCR ribotype 416, ST39, clade 4, complete genome GenBank NZ_OX637968.1) and the toxigenic C. difficile strain L-TCD-01 (PCR ribotype 020, ST2, clade 1, complete genome GenBank NZ_OY731254.1) is described. Spores were produced in a dedicated anaerobic isolator cabinet (Whitley A135GMP, Don Whitley Scientific, Bingley, UK) in a restricted-access biosafety level 2 laboratory, using food-grade culture media (Soy Peptone A3X (SA3X), Organotechnie, La Courneuve, France) in batch fermentations in sterile single-use polyethylene terephthalate glycol-modified (PETG)-flasks (Sterile Disposable PETG Flask 2000 ml, Thermo Scientific, Rochester, US). Food-grade SA3X was selected as the production medium based on its absence of animal-derived components and its demonstrated capacity for robust growth and high spore yields (data not shown). Spores were purified using aerobic washing with phosphate-buffered saline (PBS) (Fresenius Kabi, Graz, Austria) supplemented with 0.1% Tween 80 (Polysorbatum 80, Duchefa, Haarlem, The Netherlands). Dose-adjusted aliquots (104 and 107 colony forming units (CFU) C. difficile spores in buffered 70/30% glycerol (Duchefa, Haarlem, The Netherlands) and PBS solution) were filled into liquid-filled direct-release capsules (Lonza Vcaps® Plus Capsules size 0, Lonza, Colmar, France) using an autoclavable capsule filling system (ProFiller1100, Torpac, Heerlen, The Netherlands). A detailed risk assessment of the C. difficile manufacture process and impurities has been conducted. All raw materials used in the manufacturing process are released for their use by a Quality Control (QC) officer. The qualification of all compendial and non-compendial reagents are listed in Supplementary Table 1. Non-compendial reagents are qualified based on vendor Certificate of Analysis (CoA) and meet pre-defined acceptance criteria (Supplementary Table 2 and 3). The production process was formalized in Standard Operating Procedures (SOPs), authorized by QC, QP and Head of Production.
Quality and in-process controls (QC and IPCs) are described further below and are based on well-established protocols of the Dutch National Expertise Center for C. difficile infections (13-15) as well as guidance documents of the European Centers for Disease Control and Prevention (12) and the European Society for Clinical Microbiology and Infectious Diseases (16, 17). All IPCs and QCs were validated or qualified for their intended use. Where possible, IPCs and QCs were performed according to GMP requirements or, if not possible, under ISO15189 standards at the clinical microbiology laboratory of the LUMC. If during culture steps a QC analysis was required, this was performed in a Whitley A35 anaerobic cabinet (Don Whitley Scientific). All QC test results were independently reviewed by a QC officer, and thereafter the master cell bank, working cell bank, active substance and active product were released by a Qualified Person (QP). To verify the robustness of the manufacturing process ≥3 preclinical test runs were performed per challenge strain.
A product dossier was created according to EMA/CHMP/BWP/534898/2008 Rev. 2. for pharmacological products. All raw materials, disposables and excipients were quarantined until release was done by a QC officer.
The product was characterized as an auxiliary medicinal product (regulation EU No 536/2014 (CTR)). The principles of GMP as outlined in Regulation (EU) 536/2014 along with the white paper “Considerations on the Principles of Development and Manufacturing Qualities of Challenge Agents for Use in Human Infection Models” (9) were applied to the production process wherever possible and audited as such.
Results
Master cell bank (MCB) and working cell bank (WCB)
The C. difficile challenge strains were selected based on considerations previously published (18). The NTCD strain was isolated from faecal material of an asymptomatic nursing-home resident and the TCD strain from faecal material of a symptomatic patient with uncomplicated CDI disease. The selected isolated challenge strains were used to form a master cell bank (MCB), the starting material for the production of the active product (Figure 1). MCB has been prepared under good laboratory practice (GLP) conditions according to the SOPs of the Dutch National Expertise Center for C. difficile infections, which is an ISO-15189 certified laboratory. In short, the MCB was created through enrichment culture in CDEB MOD (C. difficile enrichment modified broth, Mediaproducts BV, Groningen, The Netherlands), inoculated onto CLO plates (Clostridioides difficile selective agar plates, bioMérieux, Marcy-l’Étoile, France), followed by re-culturing on a tryptic soy sheep blood (TSS) plate (bioMérieux, Marcy-l’Étoile, France), after which a colony was transferred to glycerol for storage at -80°C.
To produce the working cell bank (WCB) intermediate product (Figure 1), MCB was thawed and plated out on a SA3X plate for expansion of C. difficile. After incubation of the plate, colonies were picked and subsequently the WCB was stocked in a glycerol/SA3X solution and stored in a -80°C freezer.
The MCB and WCB were released for further processing after undergoing successful quality control testing for confirmation of identity, potency, purity and safety of the products and passing all acceptance criteria (Table 1). NGS testing additionally showed stability of the challenge strain genome, with no evidence of laboratory-driven adaptation of the original strain (data not shown).
Active substance (AS)
To produce purified spores in solution (Figure 1), which constitute the active substance (AS), the WCB was recovered on a SA3X agar plate and re-streaked on a fresh SA3X plate to increase cell yield. Subsequently, a starter culture was generated in liquid SA3X medium under anaerobic conditions using the anaerobic isolator cabinet. The optical density (OD) of the cultures was then measured and diluted in fresh SA3X medium to a starting OD of 0.05. The diluted cultures were incubated in shaking Erlenmeyer flasks for 14 days to produce spores. In the production medium, spore yield increases during the first week (from 0 CFU/mL to >106/mL); a 14-day time frame was selected to ensure maximum reproducibility of spore yield. We found no significant difference in spore yield between different culture volumes up to a liter-scale, demonstrating robustness of the method (data not shown). After 14 days, cells were pelleted by centrifugation and washed 5 times with PBS/0.1% Tween 80 to remove residual vegetative cell debris. All batches of the active substance yielded a high spore count (Figure 2), meeting the acceptance criterion for spore count (>1.6×106 spores/mL). Active substance was stored at -80°C.
The active substance was released for further processing after undergoing successful quality control testing and passing all acceptance criteria as indicated in Table 1.
Active Product (AP)
The active product consists of a 500-μl suspension containing 104 or 107 CFU C. difficile spores formulated in buffered 70/30% (v/v) glycerol and PBS encapsulated in liquid-filled direct-release capsules for oral administration. The dosing of 104 and 107 CFU spores were selected based on previously published experimental NTCD studies (19, 20).
To produce the active product, active substance was thawed and adjusted to the right concentration in formulation buffer after which 500-μl volumes were dispensed in liquid-filled direct-release Lonza Vcaps®. Capsules were stored at room temperature (15-25°C) and quarantined until release.
The active product was checked for identity, potency, purity and safety, and released after passing all acceptance criteria (Table 1).
Stability of Active Product
Stability testing was performed on the active substance and active product at multiple timepoints according to a predefined stability plan (1, 3, 6, 12 and 24 months). Stability testing consists of: 1) identity testing by gluD PCR and ribotyping PCR 2) potency testing by spore enumeration (Figure 3) 3) purity testing by visual inspection of colonies on a TSS plate, colorimetric measurement of protein and DNA content and aerobic plating and capsule controls by visual inspection of capsule integrity and measuring fill weight and volume (Table 1).
We observed stability, with limited or no decrease in plating efficiency over a period of 12 months so far for the NTCD active product, and up to 6 months for the TCD active product (Figure 3). All other stability tests also met the predefined criteria (Table 1) at these timepoints. Stability of the active substance aligns with the stability observed of the active product. The observed stability at multiple timepoints across different production batches confirms robustness of stability across the batches. The expiry date of the (N)TCD active product is adjusted to the stability data. Notably, stability of the TCD product to date strongly resembles the NTCD product, suggesting a similar stability profile. Based on these observations, we expect that the actual shelf life of the challenge product extends well beyond the current stability data. Together this demonstrates that the active product has a sufficiently long shelf life (stability) to enable its use in challenge studies following production.
In-process controls and quality controls
The in-process controls (IPCs) and quality controls (QCs) performed to ensure identity, potency, purity and safety during the production process of C. difficile challenge material are scheduled in Figure 1 and Table 1. A short description of the tests is given below.
Identity
Species PCR
Confirmation of the presence of cells/spores belonging to the species C. difficile was obtained by performing a gluD PCR on isolated total DNA. The gluD PCR contains highly specific primers which allow amplification of a 158-bp fragment of glutamate dehydrogenase gene (gluD gene), after which the fragment can be detected using a specific gluD probe in a real time PCR assay (12).
Ribotyping
To identify and confirm the PCR ribotype of the C. difficile challenge material capillary ribotyping PCR was performed, in which the 16S-23S intergenic spacer regions on the C. difficile chromosome are amplified by PCR and visualized on a capillary gel electrophoresis system (14). The resulting banding pattern is matched to a library of reference patterns, using BioNumerics 7.6 software (bioMérieux, Marcy-l’Étoile, France).
Complete genome sequencing
A reference sequence for the challenge strains was generated using long-read circular consensus sequencing (Pacific Bioscience Sequel II) and automated assembly using established protocols (21, 22). Reference assembly of the 150-bp paired-end Illumina reads of this sequence revealed no single nucleotide polymorphisms. Analysis of the complete genome sequence allowed for assessment of genome characteristics such as the presence or absence of specific toxin and antimicrobial resistance genes.
Short read sequencing
Short-read next generation sequencing (Illumina Miniseq, NextSeq 500 or Novaseq Xplus) was additionally performed on the working bank and active substance; the results of a core genome analysis using Ridom SeqSphere+ (Ridom GmbH, Münster, Germany) confirmed that the clade (clonal complex, CC) and complex type (CT) of the strain matches the reference genome sequence.
Potency
Total cell count
To enumerate viable bacterial cells in the master and working cell bank, serial dilutions were cultured on BHI (brain heart infusion) plates. The total cell count is based on the geometric mean minus two times standard deviation of log transformed values obtained from the colony forming unit (CFU) enumeration.
Total spore count
The quantity of viable spores was assessed by plating out serial dilutions of the product onto BHI plates supplemented with 0.1% of the germinant sodium taurocholate (Thermo Fisher Scientific, Lancashire, UK) and enumerating the CFUs after 48h. The total spore count is based on the geometric mean minus two times standard deviation of log transformed values obtained from the CFU enumeration. Production yield of active substance was targeted at > 1.6 x 106 spores/mL, but generally produced >107 spores/mL. In our studies, the active product was formulated in capsules with a targeted dose of 104 or 107 spores.
Purity
Visual appearance of colonies
Routinely, C. difficile cells or spores were anaerobically cultured on a TSS plate for 72h. Plates were visually inspected and all colonies on the plate should macroscopically be morphologically consistent with C. difficile, which required confirmation by a second person.
Contamination with aerobic bacteria
To confirm absence (<10 CFU/mL) of aerobic bacterial contaminants, routine aerobic culture was performed on non-selective TSS agar plates.
Measuring protein and DNA content
To quantify remaining vegetative C. difficile cell remnants, e.g. free protein or DNA, in the active substance and product, colorimetric measurements (Qubit dsDNA HS Assay and Qubit Protein Assay, LifeTechnologies, Eugene, Oregon, US) were performed. The limits set at ∼ 10-fold the minimum value observed after repeated washing (please refer to Table 1).
Safety
Presence of toxin gene
A multiplex PCR for genes encoding toxin A (tcdA), toxin B (tcdB) and binary toxin (cdtA and cdtB) genes was performed on total DNA extracted from the samples. As a DNA isolation control, the 16S rRNA and gluD gene were also tested, which should show a positive result.
Antimicrobial susceptibility
To ensure susceptibility to antibiotics which may be used for rescue treatment (fidaxomicin, vancomycin and metronidazole), antimicrobial susceptibility tests are performed. Susceptibility for fidaxomicin (Selleckchem, Köln, Germany) was tested using the agar dilution method (23) whereas for vancomycin and metronidazole E-tests (bioMérieux, Marcy-l’Étoile, France) were used. Breakpoints are defined based on EUCAST criteria (24).
Microbial contaminants of the master cell bank
To test for contaminants with gastrointestinal pathogens, total DNA from the master cell bank was tested by qPCR for the presence of gastrointestinal pathogens, i.e. the bacterial pathogens Salmonella, Shigella, Campylobacter, Yersinia, Aeromonas and Plesiomonas shigelloides spp. and the viral pathogens adenovirus, norovirus, rotavirus, sapovirus and astrovirus.
Capsule controls
Capsule integrity
Capsules from the beginning, middle and end of the manufacturing process were inspected for integrity by visually inspecting the capsules: both capsule parts should be aligned, show no deformations and no liquid should be leaking out when tilted.
Fill weight and volume
The fill weight of the active product capsules is measured on an analytical balance; each capsule is filled with 500ul which should align with a weight of 700mg +/- 20 gram.
Conclusion and Discussion
To summarize, we demonstrate feasibility to produce C. difficile challenge material for use in CHIM studies at small scale aligning to GMP principles including appropriate quality controls and release. This application of GMP principles to an unconventional production process of a bacterial spore-forming anaerobic challenge agent is a representation for the production of bacterial challenge agents as described in the auxiliary medicinal product guidelines of EMA and FDA.
Challenge material is not classified as a medicinal product and specific international challenge material production guidelines are lacking. The EMA identifies challenge agents as auxiliary medicinal products (AxMPs) and states that when the AxMP is not authorized it shall be manufactured according to GMP or at least an equivalent standard (26). In the United Kingdom, the manufacturing of challenge agents is not regulated. In the US the challenge agent is considered a biological product and is subject to regulation under federal law by the FDA. The challenge agent must therefore 1) be manufactured under GMP conditions where possible, 2) satisfy the FDA regulations of safety, purity and potency and 3) have detailed information on the provenance and manufacture as part of the required Investigational New Drug Application (IND).
To ensure the quality attributes of identity and safety NGS was applied for full characterization of the challenge strain. Additionally, applying NGS for the detection of adventitious agents enhances the QC testing of challenge material facilitating the identification of potential other bacterial pathogens in the active product beyond Ph. Eur. requirements. To safeguard the level of contamination with adventitious agents, acceptance criteria must be predefined. This can be challenging, as metagenomic analysis is prone to spurious identifications (e.g. due to DNA segments shared between bacterial species). This is of particular concern for C. difficile spore challenge material, as DNA isolation from spores is difficult and typically yields low-biomass DNA samples. We established acceptance criteria for detecting adventitious agents through NGS by analysing simulated reads generated from the reference genome using a well-characterized pipeline, resulting in a cut-off of fewer than 0.3% of mapped reads aligning to pathogenic species. Such criteria may require adaptation for other species. In the case of spurious NGS identifications, a quantitative 16S micelle PCR (27) can provide additional value by enabling both the identification and quantification of the bacterial composition.
Secondly, to ensure the quality attribute of safety, food-grade media and GMP-grade raw materials– formulated to avoid the use of animal-derived products where possible, thereby mitigating the risk of potentially important diseases such as transmissible spongiform encephalopathies (TSE) - are required during challenge material production. However, the culture media routinely used for C. difficile growth and sporulation contain animal-derived constituents, and conventional spore purification techniques involve reagents that are not indicated for use in a spore production process or for oral ingestion (e.g. non-ionic X-ray contrast agents). Therefore, during the manufacturing process development, the normally used brain heart infusion (BHI) medium was replaced by a soy peptone (SA3X) medium and for spore purification washing with PBS-0.1% Tween was established. Moreover, the dedicated manufacturing area was adapted to include appropriate control measures, equipment validation and a cleaning protocol to prevent cross-contamination. Although GMP guidelines may stipulate production in a controlled environment (e.g. a clean room), the inherent resistance and persistence of C. difficile spores present significant challenges for decontaminating such facilities. Notably, the guidance document on the production of challenge agents (9) states that a clean room is not mandatory, but the manufacturing area should be a part of the contamination control strategy and the level of environmental control for particulate and microbial contamination should be tailored to the manufacture of the challenge agent and production step, considering the potential level of contamination of starting materials and the risks for the final batch of the challenge agent. Since C. difficile requires anaerobic conditions during production, an anaerobic isolator cabinet was needed as the manufacturing area, and to maintain a robust and reproducible manufacturing process a qualified anaerobe cabinet (GMP grade A) was selected. The isolator underwent full qualification (installation, operational and performance qualification). Thereby this manufacturing area, provided not only the necessary environment for spore production but also ensured environmental control, including regulation of particulate and microbial contamination through controlled, HEPA-filtered, unidirectional airflow.
Thirdly, to ensure the quality attribute of potency, the route of administration, storage and transport conditions of the challenge agent must be carefully considered. Oral capsules were chosen as the route of administration to mimic the natural infection route, while simultaneously maintaining control over the number of spores reaching the gastrointestinal tract. As C. difficile spores need exposure to primary bile acids in the duodenum for germination (28, 29), liquid-filled direct-release capsules (instead of enteric capsules) were chosen as they release the spores in the stomach or shortly after. 70/30% (v/v) buffered glycerol/PBS was selected as the formulation buffer for its optimal balance between feasibility to formulate the spore suspension and prevention of downstream capsule leakage. The capsules were preserved at room temperature (15-25 °C) to avoid structural changes in the content and integrity of the capsules which occur upon freezing. Extensive testing of different capsule types demonstrated best performance of Lonza Vcaps® liquid-filled direct-release capsules in terms of secure containment of liquids and semi-solids, stability and preventing leakage at room temperature.
Applying the GMP principles to C. difficile spore challenge material for clinical use highlights the need for collaboration and thorough risk analysis among various disciplines, including pharmacists, microbiologists, clinicians and technicians. This collaboration has led to a highly controlled production of C. difficile spore challenge material in an academic setting. Building on these efforts, the C. difficile challenge material has been approved by the Medical Research Ethics Committee to use for colonisation (with non-toxigenic C. difficile spores, ClinicalTrials.gov NCT05693077) and infection (with toxigenic C. difficile spores, ClinicalTrials.gov NCT06702345) of healthy volunteers in a proof-of-concept clinical trial to find a safe and infectious dose. Establishing a CHIM for C. difficile will be a critical advancement in improving our understanding of C. difficile infections as well as accelerating (novel) product development to battle the rising incidence and global burden of this complex disease.
Contribuon
Writing-original draft (lead): AH, Writing original draft (supporting): MR and WKS, Writing-review and editing: EK, PS, PM, ES, IA, CH, CV, LP Conceptualization: WKS, MR, EK, ES, PM and AH, Investigation: CH, CV, AG and ME, Methodology: ES, PM, MR, WKS, EK, CH, CV, AG and ME, Resources: PS, PM, IA, ES, EK and WKS Project administration: LP, Validation: ES, PM, IA and PS, Supervision: WKS and MR.
Acknowledgements
We gratefully acknowledge the contributions of all partners involved in Work Package 10 (Subtopic 2) of the Inno4Vac project, an Innovative Health Initiative-funded consortium.
Declaraon of compeng interest
Wiep Klaas Smits performs research which is funded in part by a public-private partnership with Acurx Pharmaceuticals unrelated to the work presented here. All other authors declare no competing interests.
Funding
This project (“Manufacture of Clostridioides difficile spores for experimental infection of human volunteers”) has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 101007799 (Inno4Vac). This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA. This communication reflects the author’s view and that neither IMI nor the European Union, EFPIA, or any Associated Partners are responsible for any use that may be made of the information contained herein.