“The impressive level of efficacy observed with SER-109 treatment is striking when compared with the high rate of recurrence expected in this population,” said Dr. Stuart H. Cohen, MD, Chief, Division of Infectious Diseases, University of California, Davis. “These results demonstrate the potential of SER-109 to effectively treat recurrent CDI. With current treatment approaches having significant limitations, SER-109 has the potential to fundamentally change the management of this urgent health issue.”
Background. Patients with recurrent Clostridium difficile infection (CDI) have a ≥60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI.
Methods. Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 109 spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 108 spores). The primary efficacy end point was absence of C. difficile–positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed.
Results. Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile–positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non–spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea.
Conclusions. SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.
Clostridium difficile infection (CDI) and its attendant complications, including diarrhea, pseudomembranous colitis, and toxic megacolon, are associated with an estimated 29 000 annual deaths in the United States and is recognized by the Centers for Disease Control and Prevention as an urgent public health priority . Antibiotic exposure is the leading risk factor for CDI, and the risk of recurrent disease is increased among elderly patients and following antibiotic reexposure. Antibiotic therapy for recurrent CDI contributes to persistent disruption of the gut microbiome, which is the first-line defense against colonization and infection by pathogens, including C. difficile [2–5]. The risk of recurrence increases to >60% following a second episode [3, 6, 7].
Research has focused on the potential role that the human microbiome plays in health and disease. In 2008, the National Institutes of Health supported the creation of the Human Microbiome Project to characterize the species composition and function of the healthy microbiome. In the gut, the 2 dominant phyla are Firmicutes (ie, gram-positive organisms, including Bacilli and Clostridia) and Bacteroidetes (ie, gram-negative anaerobes, including Bacteroides, Parabacteroides, and Prevotella) [8, 9]. In contrast, gram-negative Enterobacteriaceae, such as Escherichia coli, make up only a fraction of the healthy microbiome . There is also significant intersubject variability at both the genus and species level, suggesting that the bacterial communities in any one individual are unique, mirroring the complex interplay of diet, host genetics, immune response, and microbial coadaptation. Despite this variation, there are common core species found in a majority of healthy individuals, and metabolic pathways are preserved due to functional redundancy . Thus, a wide range of microbiomes defines a healthy state.
In states of disease, there are also broad patterns that define gut dysbiosis, such as a loss of microbial diversity and increasing representation of gram-negative facultative anaerobes, such as Enterobacteriaceae [11, 12]. Antibiotic-induced dysbiosis underlies colonization and invasion by C. difficile, while repair of the microbiome, through fecal microbiota transplantation (FMT), is associated with efficacy rates of 81%–90% for those with recurrent CDI [13–16]. FMT involves transferring minimally processed, uncharacterized fecal material from a healthy donor to a recipient .
FMT administration is often invasive and requires donor screening and stool preparation. Despite donor screening, stool preparations for FMT have the potential to transmit infections due to pathogens that are present at times outside the period of detectability or for which diagnostic tests are unavailable; there is also the possibility of unwitting transmission of emerging pathogens that have not been identified to date [18, 19]. While there have been recent reports of stool delivered via oral encapsulated FMT or stool enemas, the data demonstrate first-dose efficacy of approximately 52%–70%, which is significantly lower than that for other modes of administration, such as colonoscopy [14, 20, 21]. In recognition of FMT as an experimental biologic, the Food and Drug Administration issued guidance that this intervention should only be used for prevention of recurrent CDI and after receipt of informed consent. An alternative approach for achieving improved safety and convenience with comparable efficacy is urgently needed .
SER-109 is composed of approximately 50 species of Firmicutes spores derived from stool specimens from healthy donors. After demonstrating the preclinical efficacy of SER-109 in rodent CDI models, we formulated it for oral delivery in humans based on the hypothesis that spore-forming organisms would compete metabolically with C. difficile for essential nutrients and/or bile acids [23–27]. In addition, spore purification with ethanol reduces the risk of transmission of other potential pathogens . This initial study was designed to evaluate the efficacy and safety profile of SER-109 for CDI prevention in patients with recurrent infections and to measure alterations in the gut microbiota.
This open-label, single-arm, descending-dose study evaluated the safety, efficacy, and engraftment of SER-109 formulated for oral delivery. The study was sponsored by Seres Therapeutics and conducted at 4 US medical centers: Massachusetts General Hospital (Boston, Massachusetts), Mayo Clinic (Rochester, Minnesota), Miriam Hospital (Providence, Rhode Island), and Emory University Hospital (Atlanta, Georgia). The protocol was developed by investigators at Seres Therapeutics and authors of the current study (E. L. H., D. S. P., and S. K.) and was approved by the institutional review boards of the participating medical centers.
Eligible patients were 18–90 years old and had ≥3 laboratory-confirmed CDI episodes in the previous 12 months, had a life expectancy of ≥3 months, and gave informed consent to receive this donor-derived product. Patients were excluded for a history of acute leukemia; hematopoietic stem cell transplantation, chemotherapy within 2 months and an absolute neutrophil count of <1000 neutrophils/mm3, a history of inflammatory or irritable bowel disease, colectomy, cirrhosis, pregnancy/lactation, severe acute illness unrelated to CDI, antibiotic exposure for a non-CDI indication within 14 days of screening, or prior FMT.
Eligible patients had a clinical response to antibiotic therapy for their current CDI episode immediately prior to dosing and were neither anticipated to require admission to an intensive care unit nor expected to need antibiotics within 6 weeks following study entry.
Screening of Donors
Seven adult donors of stool specimens gave informed consent, underwent a complete medical history and laboratory assessment, and were screened for blood-borne and fecal pathogens, consistent with published protocols [29, 30]. Donors were excluded for being older than 50 years, having a body mass index (BMI; calculated as the weight in kilograms divided by the height in meters squared) of >25, engaging in high-risk behaviors as per a modified American Association of Blood Banks blood donor questionnaire , having a history of acute/chronic gastrointestinal disorders, or using antibiotics (in the previous 6 months), immunosuppressive/antineoplastic agents, or cigarettes (Supplementary Materials).
Preparation of SER-109
SER-109 comprises Firmicutes spores fractionated from stool specimens obtained from healthy donors. Donor stool specimens were processed separately to make unique batches of SER-109. Upon collection, stool specimens were frozen at −80°C. Approximately 150 g was suspended and homogenized in normal saline and filtered through mesh screens. The slurry was centrifuged, and supernatant containing bacterial cells and spores was combined with 100% ethanol to 50% (wt/wt) and incubated at room temperature for 1 hour to reduce risk of pathogen transmission to the recipient . The supernatant was pelleted by centrifugation, washed with saline to remove ethanol, resuspended with sterile glycerol, and filled into capsules (hypromellose DRcaps, Capsugel), which were stored at −80°C.
The product was characterized for spore concentration and absence of residual gram-negative bacteria. Spore content was determined by measuring the dipicolinic acid (DPA) content and normalizing against the DPA content of known numbers of spores representing 3 commensal species . The absence of residual gram-negative bacteria was confirmed by selective plating on MacConkey lactose agar and Bacteroides bile esculin agar. No vegetative microbes were found in any SER-109 preparation within the limit of assay detection (<30 colony-forming units/mL).
Two days prior to dosing, patients discontinued antibiotics for CDI. One day prior to dosing, patients underwent a bowel preparation (to minimize residual antibiotic in the gastrointestinal tract), followed by overnight fasting. Two sites used a regimen of 300 mL of magnesium-citrate (one with Dulcolax), and 2 sites used polyethylene glycol.
Part 1 enrolled 15 patients who each received 30 capsules of SER-109 (observed dose of 15 capsules on day 0 and day 1). The dose of spores varied between 3 × 107 and 2 × 1010, based on natural variations in spore concentration among healthy donors. Based on initial efficacy, 15 additional patients were enrolled in part 2 and treated with SER-109 capsules containing a lower fixed dose of 1 × 108 spores (approximately 17-fold lower than the geometric mean dose administered in part 1 and 3-fold above the minimum dose shown to be effective). Depending on spore content, patients received an observed dose of 1–12 capsules on day 0.
Any patient whose diarrhea recurred between 1 and 8 weeks was eligible for another dose of SER-109, based on data from the conventional FMT literature showing efficacy of a second dose [13, 14]. If a patient elected to receive a second dose of SER-109, the time course of study events was restarted concurrent with the second dose of SER-109.
Adverse events and recurrence of CDI symptoms were monitored through phone calls (on day 4 and weeks 1, 2, and 4) and in-clinic visits (on weeks 8 and 24). Patients were asked to provide a stool sample on day 4 and on weeks 1, 2, 4, 8, and 24 after treatment for genomic and culture-based analysis.
The primary end point was prevention of recurrent CDI during the 8-week follow-up after SER-109. CDI recurrence was defined as a composite end point of >3 unformed bowel movements in a 24-hour period and laboratory confirmation of C. difficile in the stool. Safety was evaluated by monitoring adverse events and assessing changes in laboratory values, vital signs, and physical examination findings over a 24-week period after dosing.
Alterations in Gut Microbiota Composition
The impact of SER-109 on gut microbiota was determined by examining stool samples before and after treatment for (1) engraftment by spore-forming species and (2) augmentation (outgrowth) of commensal bacteria not found in SER-109. Alterations in composition were measured by 16S ribosomal RNA (rRNA) genomic and culture-based analysis of patient fecal samples (Supplementary Materials). Engraftment was defined by newly detected spore formers in the patient after treatment, which were present in SER-109 but not detectable in the patient before treatment. Augmented bacteria were defined as non–SER-109 organisms whose levels increased at least 10-fold after treatment.
Thirty patients were enrolled after therapeutic response to appropriate CDI antibiotics (ie, vancomycin [n = 23], fidaxomicin [n = 5], metronidazole [n = 1], and rifaximin [n = 1]) was documented (Table 1). Patients had a median age of 66.5 years (range, 22–88 years), and the majority of subjects (67%) were female. The median time from the initial C. difficile diagnosis to the most recent recurrence was 23.1 weeks in cohort 1 and 34.3 weeks in cohort 2. In the overall study population, the median number of CDI recurrences was 3 (range, 2–6 recurrences). Infecting C. difficile strains were identified in 10 patients and included types BI, Y, and DH (Supplementary Table 1).
Patient Demographic Characteristics, by Cohort
Complete blood counts and a chemistry panel (including liver function tests and analysis of albumin and creatinine levels) were performed at week 8 (for 27 of 30 patients) and at week 24 or early termination for 20 of 30 patients. No significant changes in laboratory findings were observed, with the exception of those for 1 patient, who had an elevated white blood cell count at week 8 at the time of diagnosis of a urinary tract infection.
Of the 30 patients who received SER-109, 26 (86.7%) achieved the primary end point of no C. difficile–positive diarrhea up to 8 weeks following dosing, with similar outcomes in both dosing cohorts (Figure 1). Of the patients who met the primary end point, 1 required a second dose of SER-109 for recurrence of C. difficile–positive diarrhea on day 26, as per protocol. Four patients who did not meet the primary end point had early onset of symptoms at days 3, 5, 7, and 9 after administration of SER-109 and laboratory confirmation of C. difficile. One of these patients declined a second SER-109 dose and chose not to continue participating in the study. Notably, the other 3 patients were determined by their primary investigator to be recovering from a self-limiting diarrheal episode at the time of stool submission for C. difficile testing. In each case, the investigators advised the patients to refrain from antibiotic use, and all symptoms resolved without any therapeutic intervention; stool samples from these 3 patients were negative for C. difficile carriage at 8 weeks, using a sensitive nucleic acid amplification test for detection of toxins A and B. Thus, 29 of 30 patients (96.7%) achieved clinical resolution of recurrent CDI following SER-109 administration.
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