Enterotoxigenic E. coli virulence gene regulation in human infections.

Enterotoxigenic Escherichia coli (ETEC) is a global diarrheal pathogen that utilizes adhesins and secreted enterotoxins to cause disease in mammalian hosts. Decades of research on virulence factor regulation in ETEC has revealed a variety of environmental factors that influence gene expression, including bile, pH, bicarbonate, osmolarity, and glucose. However, other hallmarks of the intestinal tract, such as low oxygen availability, have not been examined. Further, determining how ETEC integrates these signals in the complex host environment is challenging. To address this, we characterized ETEC's response to the human host using samples from a controlled human infection model. We found ETEC senses environmental oxygen to globally influence virulence factor expression via the oxygen-sensitive transcriptional regulator fumarate and nitrate reduction (FNR) regulator. In vitro anaerobic growth replicates the in vivo virulence factor expression profile, and deletion of fnr in ETEC strain H10407 results in a significant increase in expression of all classical virulence factors, including the colonization factor antigen I (CFA/I) adhesin operon and both heat-stable and heat-labile enterotoxins. These data depict a model of ETEC infection where FNR activity can globally influence virulence gene expression, and therefore proximity to the oxygenated zone bordering intestinal epithelial cells likely influences ETEC virulence gene expression in vivo. Outside of the host, ETEC biofilms are associated with seasonal ETEC epidemics, and we find FNR is a regulator of biofilm production. Together these data suggest FNR-dependent oxygen sensing in ETEC has implications for human infection inside and outside of the host.

Consensus Report on Shigella Controlled Human Infection Model: Conduct of Studies.

Shigella causes morbidity and mortality worldwide, primarily affecting young children living in low-resource settings. It is also of great concern due to increasing antibiotic resistance, and is a priority organism for the World Health Organization. A Shigella vaccine would decrease the morbidity and mortality associated with shigellosis, improve child health, and decrease the need for antibiotics. Controlled human infection models (CHIMs) are useful tools in vaccine evaluation for early up- or down-selection of vaccine candidates and potentially useful in support of licensure. Over time, the methods employed in these models have become more uniform across sites performing CHIM trials, although some differences in conduct persist. In November 2017, a Shigella CHIM workshop was convened in Washington, District of Columbia. Investigators met to discuss multiple aspects of these studies, including study procedures, clinical and immunological endpoints, and shared experiences. This article serves as a uniform procedure by which to conduct Shigella CHIM studies.

Human Experimental Challenge With Enterotoxigenic Escherichia coli Elicits Immune Responses to Canonical and Novel Antigens Relevant to Vaccine Development.

Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development. Methods: Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens. Results: Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies. Conclusions: Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.

Rifaximin Fails to Prevent Campylobacteriosis in the Human Challenge Model: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Campylobacter species are a leading cause of diarrheal disease globally with significant morbidity. Primary prevention efforts have yielded limited results. Rifaximin chemoprophylaxis decreases rates of travelers' diarrhea and may be suitable for high-risk persons. We assessed the efficacy of rifaximin in the controlled human infection model for Campylobacter jejuni. Methods: Twenty-eight subjects were admitted to an inpatient facility and randomized to a twice-daily dose of 550 mg rifaximin or placebo. The following day, subjects ingested 1.7 × 105 colony-forming units of C. jejuni strain CG8421. Subjects continued prophylaxis for 3 additional days, were followed for campylobacteriosis for 144 hours, and were subsequently treated with azithromycin and ciprofloxacin. Samples were collected to assess immunologic responses to CG8421. Results: There was no difference (P = 1.0) in the frequency of campylobacteriosis in those receiving rifaximin (86.7%) or placebo (84.6%). Additionally, there were no differences in the clinical signs and symptoms of C. jejuni infection to include abdominal pain/cramps (P = 1.0), nausea (P = 1.0), vomiting (P = .2), or fever (P = 1.0) across study groups. Immune responses to the CG8421 strain were comparable across treatment groups. Conclusions: Rifaximin did not prevent campylobacteriosis in this controlled human infection model. Given the morbidity associated with Campylobacter infection, primary prevention efforts remain a significant need. Clinical Trials Registration: NCT02280044.

Genetic relatedness of Vibrio cholerae isolates within and between households during outbreaks in Dhaka, Bangladesh.

BACKGROUND: Household contacts of cholera patients have a 100 times higher risk of developing a cholera infection than the general population. To compare the genetic relatedness of clinical and water source Vibrio cholerae isolates from cholera patients' households across three outbreaks, we analyzed these isolates using whole-genome-sequencing (WGS) and multilocus variable-number tandem-repeat analysis (MLVA). RESULTS: The WGS analyses revealed that 80% of households had source water isolates that were more closely related to clinical isolates from the same household than to any other isolates. While in another 20% of households an isolate from a person was more closely related to clinical isolates from another household than to source water isolates from their own household. The mean pairwise differences in single nucleotide-variant (SNV) counts for isolates from the same household were significantly lower than those for different households (2.4 vs. 7.7 p < 0.0001), and isolates from the same outbreak had significantly fewer mean pairwise differences compared to isolates from different outbreaks (mean: 6.2 vs. 8.0, p < 0.0001). Based on MLVA in outbreak 1, we observed that the majority of households had clinical isolates with MLVA genotypes related to other clinical isolates and unrelated to water source isolates from the same household. While in outbreak 3, there were different MLVA genotypes between households, however within the majority of households, the clinical and water source isolates had the same MLVA genotypes. The beginning of outbreak 2 resembled outbreak 1 and the latter part resembled outbreak 3. We validated our use of MLVA by comparing it to WGS. Isolates with the identical MLVA genotype had significantly fewer mean pairwise SNV differences than those isolates with different MLVA genotypes (mean: 4.8 vs. 7.7, p < 0.0001). Furthermore, consistent with WGS results, the number of pairwise differences in the five MLVA loci for isolates within the same household was significantly lower than isolates from different households (mean: 1.6 vs. 3.0, p < 0.0001). CONCLUSION: These results suggest that transmission patterns for cholera are a combination of person-to-person and water-to-person cholera transmission with the proportions of the two modes varying within and between outbreaks.

Efficacy Evaluation of an Intradermally Delivered Enterotoxigenic Escherichia coli CF Antigen I Fimbrial Tip Adhesin Vaccine Coadministered with Heat-Labile Enterotoxin with LT(R192G) against Experimental Challenge with Enterotoxigenic E. coli H10407 in Healthy Adult Volunteers.

BACKGROUND: Enterotoxigenic E. coli (ETEC) is a principal cause of diarrhea in travelers, deployed military personnel, and children living in low to middle-income countries. ETEC expresses a variety of virulence factors including colonization factors (CF) that facilitate adherence to the intestinal mucosa. We assessed the protective efficacy of a tip-localized subunit of CF antigen I (CFA/I), CfaE, delivered intradermally with the mutant E. coli heat-labile enterotoxin, LTR192G, in a controlled human infection model (CHIM). METHODS: Three cohorts of healthy adult subjects were enrolled and given three doses of 25 µg CfaE + 100 ng LTR192G vaccine intradermally at 3-week intervals. Approximately 28 days after the last vaccination, vaccinated and unvaccinated subjects were admitted as inpatients and challenged with approximately 2 × 107 cfu of CFA/I+ ETEC strain H10407 following an overnight fast. Subjects were assessed for moderate-to-severe diarrhea for 5 days post-challenge. RESULTS: A total of 52 volunteers received all three vaccinations; 41 vaccinated and 43 unvaccinated subjects were challenged and assessed for moderate-to-severe diarrhea. Naïve attack rates varied from 45.5% to 64.7% across the cohorts yielding an overall efficacy estimate of 27.8% (95% confidence intervals: -7.5-51.6%). In addition to reducing moderate-severe diarrhea rates, the vaccine significantly reduced loose stool output and overall ETEC disease severity. CONCLUSIONS: This is the first study to demonstrate protection against ETEC challenge after intradermal vaccination with an ETEC adhesin. Further examination of the challenge methodology is necessary to address the variability in naïve attack rate observed among the three cohorts in the present study.

Validation of a Human Challenge Model Using an LT-Expressing Enterotoxigenic E. coli Strain (LSN03-016011) and Characterization of Potential Amelioration of Disease by an Investigational Oral Vaccine Candidate (VLA1701).

Controlled human infection models are important tools for the evaluation of vaccines against diseases where an appropriate correlate of protection has not been identified. Enterotoxigenic Escherichia coli (ETEC) strain LSN03-016011/A (LSN03) is an LT enterotoxin and CS17-expressing ETEC strain useful for evaluating vaccine candidates targeting LT-expressing strains. We sought to confirm the ability of the LSN03 strain to induce moderate-to-severe diarrhea in a healthy American adult population, as well as the impact of immunization with an investigational cholera/ETEC vaccine (VLA-1701) on disease outcomes. A randomized, double-blinded pilot study was conducted in which participants received two doses of VLA1701 or placebo orally, one week apart; eight days after the second vaccination, 30 participants (15 vaccinees and 15 placebo recipients) were challenged with approximately 5 × 109 colony-forming units of LSN03. The vaccine was well tolerated, with no significant adverse events. The vaccine also induced serum IgA and IgG responses to LT. After challenge, 11 of the placebo recipients (73.3%; 95%CI: 48.0-89.1) and 7 of the VLA1701 recipients (46.7%; 95%CI: 24.8-68.8) had moderate-to-severe diarrhea (p = 0.26), while 14 placebo recipients (93%) and 8 vaccine recipients (53.3%) experienced diarrhea of any severity, resulting in a protective efficacy of 42.9% (p = 0.035). In addition, the vaccine also appeared to provide protection against more severe diarrhea (p = 0.054). Vaccinees also tended to shed lower levels of the LSN03 challenge strain compared to placebo recipients (p = 0.056). In addition, the disease severity score was lower for the vaccinees than for the placebo recipients (p = 0.046). In summary, the LSN03 ETEC challenge strain induced moderate-to-severe diarrhea in 73.3% of placebo recipients. VLA1701 vaccination ameliorated disease severity, as observed by several parameters, including the percentage of participants experiencing diarrhea, as well as stool frequency and ETEC severity scores. These data highlight the potential value of LSN03 as a suitable ETEC challenge strain to evaluate LT-based vaccine targets (NCT03576183).

Evidence for heterozygote instability in microsatellite loci in house wrens.

Microsatellite loci have high mutation rates and high levels of allelic variation, but the factors influencing their mutation rate are not well understood. The proposal that heterozygosity may increase mutation rates has profound implications for understanding the evolution of microsatellite loci, but currently has limited empirical support. We examined 20 microsatellite mutations identified in an analysis of 12 260 meiotic events across three loci in two populations of a songbird, the house wren (Troglodytes aedon). We found that for an allele of a given length, mutation was significantly more likely when there was a relatively large difference in size between the allele and its homologue (i.e. a large 'allele span'). Our results support the proposal of heterozygote instability at microsatellite loci.

Intestinal and systemic inflammation induced by symptomatic and asymptomatic enterotoxigenic E. coli infection and impact on intestinal colonization and ETEC specific immune responses in an experimental human challenge model.

Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.

Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlate of protection.

BACKGROUND: Shigellosis is a major cause of moderate to severe diarrhoea and dysentery in children under 5 years of age in low and middle-income countries. The Flexyn2a vaccine conjugates the O-polysaccharide of Shigella flexneri 2a to Pseudomonas aeruginosa exotoxin A. We describe a Phase 2b proof-of-concept challenge study that evaluated safety, immunogenicity, and efficacy of the Flexyn2a vaccine to protect against shigellosis. METHODS: In this randomized, double blind, placebo-controlled trial, healthy adults were randomized 1:1 to receive Flexyn2a (10 µg) or placebo intramuscularly, twice, 4 weeks apart, followed by challenge 4 weeks later with 1500 colony forming units (CFUs) of S. flexneri 2a strain 2457T. The primary outcome was vaccine-induced protection. S. flexneri 2a lipopolysaccharide (LPS)-specific immune responses were assessed. FINDINGS: Sixty-seven subjects were enrolled, 34 received vaccine and 33 placebo. The vaccine was well tolerated; the majority of adverse events were mild in nature. Thirty vaccinees and 29 placebo recipients received the S. flexneri 2a challenge. Vaccination resulted in a 30.2% reduction in shigellosis compared with placebo (13/30 vs. 18/29; p = 0.11; 95% CI -15 to 62.6). Vaccine efficacy was more robust against severe disease, reaching 51.7% (p = 0.015, 95% CI 5.3 to 77.9) against moderate/severe diarrhoea or dysentery concurrent with fever or severe enteric symptoms and 72.4% (p = 0.07) against more severe diarrhoea (≥10 lose stools or ≥1000 g loose stools/24 h). Vaccinated subjects were less likely to need early antibiotic intervention following challenge (protective efficacy 51.7%, p = 0.01; 95% CI 9 to 76.8). In those who developed shigellosis, vaccinated subjects had a lower disease severity score (p = 0.002) than placebo-recipients. Additionally, LPS-specific serum IgG responses in Flexyn2a recipients were associated with protection against disease (p = 0.0016) and with a decreased shigellosis disease score (p = 0.002). INTERPRETATION: The Flexyn2a bioconjugate vaccine was immunogenic, well tolerated and protected against severe illness after Shigella challenge and is a promising Shigella vaccine construct. We identified a strong association between anti-S. flexneri 2a serum IgG and a reduction in disease outcomes. (Clinicaltrials.gov, NCT02646371.) FUNDING: Funding for this study was through a grant from the Wellcome Trust.

Immune response characterization in a human challenge study with a Shigella flexneri 2a bioconjugate vaccine.

BACKGROUND: Diarrheal diseases are a leading cause of global morbidity and mortality affecting all ages, but especially children under the age of five in resource-limited settings. Shigella is a leading contributor to diarrheal diseases caused by bacterial pathogens and is considered a significant antimicrobial resistance threat. While improvements in hygiene, and access to clean water help as control measures, vaccination remains one of the most viable options for significantly reducing morbidity and mortality. METHODS: Flexyn2a is a bioconjugate vaccine manufactured using novel conjugation methodologies enzymatically linking the O-polysaccharide of S. flexneri 2a to exotoxin A of Pseudomonas aeruginosa. The protective capacity of Flexyn2a was assessed in a controlled human infection model after two intramuscular immunizations. Immune responses pre- and post-immunization and/or infection were investigated and are described here. FINDINGS: Flexyn2a induced lipopolysaccharide (LPS)-specific serum IgG responses post-immunization which were associated with protection against shigellosis. Additionally, several other immune parameters, including memory B cell responses, bactericidal antibodies and serum IgA, were also elevated in vaccinees protected against shigellosis. Immunization with Flexyn2a also induced gut-homing, LPS-specific IgG and IgA secreting B cells, indicating the vaccine induced immune effectors functioning at the site of intestinal infection. INTERPRETATION: Collectively, the results of these immunological investigations provide insights into protective immune mechanisms post-immunization with Flexyn2a which can be used to further guide vaccine development and may have applicability to the larger Shigella vaccine field. FUNDING: Funding for this study was provided through a Wellcome Trust grant.

Refinement of the CS6-expressing enterotoxigenic Escherichia coli strain B7A human challenge model: A randomized trial.

BACKGROUND: Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea. METHODS: An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute fast at doses of 109 or 1010 cfu. A second cohort included naïve and rechallenged subjects who had moderate-severe diarrhea and were given the target regimen. Immune responses to important ETEC antigens were assessed. RESULTS: Among subjects receiving 108 cfu of B7A, overnight fast, or 109 cfu, 90-minute fast, 42.9% (3/7) had moderate-severe diarrhea. Higher attack rates (71.4%; 5/7) occurred in subjects receiving 109 cfu, overnight fast, or 1010 cfu, 90-minute fast. Upon rechallenge with 109 cfu of B7A, overnight fast, 5/11 (45.5%) had moderate-severe diarrhea; the attack rate among concurrently challenge naïve subjects was 57.9% (11/19). Anti-CS6, O148 LPS and LT responses were modest across all groups. CONCLUSIONS: An overnight fast enabled a reduction in the B7A inoculum dose; however, the attack rate was inconsistent and protection upon rechallenge was minimal.

Development of a novel multiplex electrochemiluminescent-based immunoassay to aid enterotoxigenic Escherichia coli vaccine development and evaluations.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.

Live attenuated enterotoxigenic Escherichia coli (ETEC) vaccine with dmLT adjuvant protects human volunteers against virulent experimental ETEC challenge.

BACKGROUND: There is no licensed vaccine against enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea-associated morbidity and mortality among infants and children in low-income countries and travelers. The results of this vaccination/challenge study demonstrate strong protection by an attenuated ETEC vaccine candidate, ACE527, when co-administered with a mucosal adjuvant, the double-mutant heat-labile toxin (dmLT) of ETEC. METHODS: Sixty healthy adults participated in a randomized, placebo-controlled, double-blind study with three doses of lyophilized ACE527 (∼3 × 109 of each strain per dose) administered orally with or without dmLT adjuvant (25 µg/dose). Six months later, 36 of these volunteers and a control group of 21 unvaccinated volunteers were challenged with virulent ETEC strain H10407. The primary outcome was severe diarrhea, defined as passing >800 g of unformed stools during the inpatient period following challenge. FINDINGS: The vaccine was well tolerated and induced robust immune responses to key antigens. The protective efficacy (PE) against the primary outcome of severe diarrhea was 65.9% (95% confidence interval [CI] 5.4-87.7, p = 0.003). Among subjects receiving the adjuvanted vaccine, the attack rate of severe diarrhea was 23.1, while in unimmunized controls it was 67.7%. The PE against diarrhea of any severity was 58.5% (95% CI 3.8- 82.1, p = 0.016). There was a strong inverse correlation between shedding of the vaccine strain after either of the first two doses and absence of severe diarrhea upon challenge (RR = 0.29, 95% CI 0.08-1.05, p = 0.041). Challenge strain shedding was 10-fold lower in those receiving the adjuvant than in those receiving vaccine alone. The unadjuvanted vaccine was not protective (PE = 23.1%). INTERPRETATION: The results of this study support further development of ACE527 + dmLT as a vaccine for children in endemic countries and travelers. This is the first clinical demonstration that dmLT can contribute significantly to vaccine efficacy and may warrant testing with other oral vaccines. (ClinicalTrials.gov registration: NCT01739231).

Interrogation of a live-attenuated enterotoxigenic Escherichia coli vaccine highlights features unique to wild-type infection.

Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of severe diarrheal illness in low- and middle-income countries. The live-attenuated ACE527 ETEC vaccine, adjuvanted with double mutant heat-labile toxin (dmLT), affords clear but partial protection against ETEC challenge in human volunteers. Comparatively, initial wild-type ETEC challenge completely protects against severe diarrhea on homologous re-challenge. To investigate determinants of protection, vaccine antigen content was compared to wild-type ETEC, and proteome microarrays were used to assess immune responses following vaccination and ETEC challenge. Although molecular interrogation of the vaccine confirmed expression of targeted canonical antigens, relative to wild-type ETEC, vaccine strains were deficient in production of flagellar antigens, immotile, and lacked production of the EtpA adhesin. Similarly, vaccination ± dmLT elicited responses to targeted canonical antigens, but relative to wild-type challenge, vaccine responses to some potentially protective non-canonical antigens including EtpA and the YghJ metalloprotease were diminished or absent. These studies highlight important differences in vaccine and wild-type ETEC antigen content and call attention to distinct immunologic signatures that could inform investigation of correlates of protection, and guide vaccine antigen selection for these pathogens of global importance.

Impact of lower challenge doses of enterotoxigenic Escherichia coli on clinical outcome, intestinal colonization and immune responses in adult volunteers.

A reliable and effective human challenge model is needed to help down-select the most promising ETEC vaccines currently under development. Such a model would need to reliably induce diarrhea in a high proportion of volunteers using the lowest possible inoculum to maximize safety and sensitivity. Previously we validated a challenge model that utilized a dose of 2x107 CFU of ETEC strain H10407 (LT+, ST+, CFA/I+ and O78+) to induce attack rates for moderate to severe diarrhea (MSD) of ~60-70%. Here we detail efforts to further refine the model in an attempt to determine if a lower challenge dose of H10407 can be used. Thirty subjects were randomized 1:1 to receive an oral administration of H10407 at doses of 106 or 105 CFU in bicarbonate buffer. After challenge, subjects were monitored for signs and symptoms of enteric illness and stool samples were collected to detect shedding of the challenge strain. Systemic and mucosal immune responses were measured using serum, antibody in lymphocyte supernatant and fecal samples. The attack rate was 13.3% (2/15) and 26.7% (4/15) for MSD in the 105 and 106 groups, respectively. Four MSD cases met criteria for early antibiotic treatment. All subjects but one shed the challenge strain in fecal samples. The frequency and magnitude of anti-LT toxin, CFA/I and LPS O78 immune responses were antigen, dose, severity of diarrhea and shedding levels dependent. Notably, although of lower magnitude, there were considerable immune responses in the subjects with no diarrhea. This may indicate that immune responses to asymptomatic infections of ETEC in children in the endemic countries may contribute to protection. Based on this and our prior studies, we conclude that a dose of 2x107 H10407 remains the lowest practical dose for use in future volunteer studies evaluating candidate vaccines and other preventive or therapeutic ETEC interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT00844493.

Campylobacter jejuni transcriptional and genetic adaptation during human infection.

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.

A noninvasive, direct real-time PCR method for sex determination in multiple avian species.

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.