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).

Fourth Controlled Human Infection Model (CHIM) meeting, CHIM regulatory issues, May 24, 2023.

Many aspects of Controlled Human Infection Models (CHIMs, also known as human challenge studies and human infection studies) have been discussed extensively, including Good Manufacturing Practice (GMP) production of the challenge agent, CHIM ethics, environmental safety in CHIM, recruitment, community engagement, advertising and incentives, pre-existing immunity, and clinical, immunological, and microbiological endpoints. The fourth CHIM meeting focused on regulation of CHIM studies, bringing together scientists and regulators from high-, middle-, and low-income countries, to discuss barriers and hurdles in CHIM regulation. Valuable initiatives for regulation of CHIMs have already been undertaken but further capacity building remains essential. The Wellcome Considerations document is a good starting point for further discussions.

Immunologic changes are detectable in the peripheral blood transcriptome of clinically asymptomatic Chagas cardiomyopathy patients.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected parasitic disease that affects approximately 6 million individuals worldwide. Of those infected, 20-30% will go on to develop chronic Chagas cardiomyopathy (CCC), and ultimately many of these individuals will progress to advanced heart failure. The mechanism by which this progression occurs is poorly understood, as few studies have focused on early CCC. In this study, we sought to understand the physiologic changes associated with T. cruzi infection and the development of CCC. We analyzed gene expression in the peripheral blood of asymptomatic Chagas patients with early structural heart disease, Chagas patients without any signs or symptoms of disease, and Chagas-negative patients with and without early structural heart disease. Our analysis shows that early CCC was associated with a downregulation of various peripheral immune response genes, with gene expression changes suggestive of reduced antigen presentation and T cell activation. Notably, these genes and processes were distinct from those of early cardiomyopathy in Chagas-negative patients, suggesting that the processes mediating CCC may be unique from those mediating progression to other cardiomyopathies. This work highlights the importance of the immune response in early CCC, providing insight into the early pathogenesis of this disease. The changes we have identified may serve as biomarkers of progression and could inform strategies for the treatment of CCC in its early stages, before significant cardiac damage has occurred.

Evaluation of BNT162b2 Covid-19 Vaccine in Children Younger than 5 Years of Age.

BACKGROUND: Safe and effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in young children. METHODS: We conducted a phase 1 dose-finding study and are conducting an ongoing phase 2-3 safety, immunogenicity, and efficacy trial of the BNT162b2 vaccine in healthy children 6 months to 11 years of age. We present results for children 6 months to less than 2 years of age and those 2 to 4 years of age through the data-cutoff dates (April 29, 2022, for safety and immunogenicity and June 17, 2022, for efficacy). In the phase 2-3 trial, participants were randomly assigned (in a 2:1 ratio) to receive two 3-µg doses of BNT162b2 or placebo. On the basis of preliminary immunogenicity results, a third 3-µg dose (≥8 weeks after dose 2) was administered starting in January 2022, which coincided with the emergence of the B.1.1.529 (omicron) variant. Immune responses at 1 month after doses 2 and 3 in children 6 months to less than 2 years of age and those 2 to 4 years of age were immunologically bridged to responses after dose 2 in persons 16 to 25 years of age who received 30 µg of BNT162b2 in the pivotal trial. RESULTS: During the phase 1 dose-finding study, two doses of BNT162b2 were administered 21 days apart to 16 children 6 months to less than 2 years of age (3-µg dose) and 48 children 2 to 4 years of age (3-µg or 10-µg dose). The 3-µg dose level was selected for the phase 2-3 trial; 1178 children 6 months to less than 2 years of age and 1835 children 2 to 4 years of age received BNT162b2, and 598 and 915, respectively, received placebo. Immunobridging success criteria for the geometric mean ratio and seroresponse at 1 month after dose 3 were met in both age groups. BNT162b2 reactogenicity events were mostly mild to moderate, with no grade 4 events. Low, similar incidences of fever were reported after receipt of BNT162b2 (7% among children 6 months to <2 years of age and 5% among those 2 to 4 years of age) and placebo (6 to 7% among children 6 months to <2 years of age and 4 to 5% among those 2 to 4 years of age). The observed overall vaccine efficacy against symptomatic Covid-19 in children 6 months to 4 years of age was 73.2% (95% confidence interval, 43.8 to 87.6) from 7 days after dose 3 (on the basis of 34 cases). CONCLUSIONS: A three-dose primary series of 3-µg BNT162b2 was safe, immunogenic, and efficacious in children 6 months to 4 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).

Sleep duration prior to an enterotoxigenic Escherichia coli challenge predicts diarrhea severity during infection.

BACKGROUND: Acute diarrhea is the most frequent diagnosis among ill travelers. Sleep loss may weaken the body's defense against pathogens and increase susceptibility to infection. The relationship between sleep and infectious diarrhea has not been studied and was assessed utilizing data from a controlled human infection model (CHIM) for enterotoxigenic Escherichia coli (ETEC). METHODS: During a CHIM assessing the efficacy of an immunoprophylactic targeting ETEC against moderate-to-severe diarrhea (MSD) following challenge, we measured sleep via actigraphy over an 8-day inpatient period. We hypothesized better sleep pre-challenge would predict illness symptomatology following challenge. RESULTS: Among 57 participants (aged 34.4 ± 8.1 years, 64% male), there was no relationship between sleep metrics and incidence of MSD. However, longer total sleep time the night preceding ETEC challenge was associated with lower maximum 24 h diarrhea volume (B = -1.80, p = 0.01) and total diarrhea volume (B = -2.45, p = 0.01). CONCLUSIONS: This novel study showed that shorter sleep duration predicted diarrhea severity over the course of an ETEC infection. Future work should experimentally manipulate sleep to further clarify its impact on diarrhea-related outcomes for ETEC and other important enteric pathogens.

Shigella-Controlled Human Infection Models: Current and Future Perspectives.

Shigella-controlled human infection models (CHIMs) are an invaluable tool utilized by the vaccine community to combat one of the leading global causes of infectious diarrhea, which affects infants, children and adults regardless of socioeconomic status. The impact of shigellosis disproportionately affects children in low- and middle-income countries (LMICs) resulting in cognitive and physical stunting, perpetuating a cycle that must be halted. Shigella-CHIMs not only facilitate the early evaluation of enteric countermeasures and up-selection of the most promising products but also provide insight into mechanisms of infection and immunity that are not possible utilizing animal models or in vitro systems. The greater understanding of shigellosis obtained in CHIMs builds and empowers the development of new generation solutions to global health issues which are unattainable in the conventional laboratory and clinical settings. Therefore, refining, mining and expansion of safe and reproducible infection models hold the potential to create effective means to end diarrheal disease and associated co-morbidities associated with Shigella infection.

Case Series of Thrombosis With Thrombocytopenia Syndrome After COVID-19 Vaccination-United States, December 2020 to August 2021.

BACKGROUND: Thrombosis with thrombocytopenia syndrome (TTS) is a potentially life-threatening condition associated with adenoviral-vectored COVID-19 vaccination. It presents similarly to spontaneous heparin-induced thrombocytopenia. Twelve cases of cerebral venous sinus thrombosis after vaccination with the Ad26.COV2.S COVID-19 vaccine (Janssen/Johnson & Johnson) have previously been described. OBJECTIVE: To describe surveillance data and reporting rates of all reported TTS cases after COVID-19 vaccination in the United States. DESIGN: Case series. SETTING: United States. PATIENTS: Case patients receiving a COVID-19 vaccine from 14 December 2020 through 31 August 2021 with thrombocytopenia and thrombosis (excluding isolated ischemic stroke or myocardial infarction) reported to the Vaccine Adverse Event Reporting System. If thrombosis was only in an extremity vein or pulmonary embolism, a positive enzyme-linked immunosorbent assay for antiplatelet factor 4 antibodies or functional heparin-induced thrombocytopenia platelet test result was required. MEASUREMENTS: Reporting rates (cases per million vaccine doses) and descriptive epidemiology. RESULTS: A total of 57 TTS cases were confirmed after vaccination with Ad26.COV2.S (n = 54) or a messenger RNA (mRNA)-based COVID-19 vaccine (n = 3). Reporting rates for TTS were 3.83 per million vaccine doses (Ad26.COV2.S) and 0.00855 per million vaccine doses (mRNA-based COVID-19 vaccines). The median age of patients with TTS after Ad26.COV2.S vaccination was 44.5 years (range, 18 to 70 years), and 69% of patients were women. Of the TTS cases after mRNA-based COVID-19 vaccination, 2 occurred in men older than 50 years and 1 in a woman aged 50 to 59 years. All cases after Ad26.COV2.S vaccination involved hospitalization, including 36 (67%) with intensive care unit admission. Outcomes of hospitalizations after Ad26.COV2.S vaccination included death (15%), discharge to postacute care (17%), and discharge home (68%). LIMITATIONS: Underreporting and incomplete case follow-up. CONCLUSION: Thrombosis with thrombocytopenia syndrome is a rare but serious adverse event associated with Ad26.COV2.S vaccination. The different demographic characteristics of the 3 cases reported after mRNA-based COVID-19 vaccines and the much lower reporting rate suggest that these cases represent a background rate. PRIMARY FUNDING SOURCE: Centers for Disease Control and Prevention.

Evaluation of the BNT162b2 Covid-19 Vaccine in Children 5 to 11 Years of Age.

BACKGROUND: Safe, effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in children younger than 12 years of age. METHODS: A phase 1, dose-finding study and an ongoing phase 2-3 randomized trial are being conducted to investigate the safety, immunogenicity, and efficacy of two doses of the BNT162b2 vaccine administered 21 days apart in children 6 months to 11 years of age. We present results for 5-to-11-year-old children. In the phase 2-3 trial, participants were randomly assigned in a 2:1 ratio to receive two doses of either the BNT162b2 vaccine at the dose level identified during the open-label phase 1 study or placebo. Immune responses 1 month after the second dose of BNT162b2 were immunologically bridged to those in 16-to-25-year-olds from the pivotal trial of two 30-µg doses of BNT162b2. Vaccine efficacy against Covid-19 at 7 days or more after the second dose was assessed. RESULTS: During the phase 1 study, a total of 48 children 5 to 11 years of age received 10 µg, 20 µg, or 30 µg of the BNT162b2 vaccine (16 children at each dose level). On the basis of reactogenicity and immunogenicity, a dose level of 10 µg was selected for further study. In the phase 2-3 trial, a total of 2268 children were randomly assigned to receive the BNT162b2 vaccine (1517 children) or placebo (751 children). At data cutoff, the median follow-up was 2.3 months. In the 5-to-11-year-olds, as in other age groups, the BNT162b2 vaccine had a favorable safety profile. No vaccine-related serious adverse events were noted. One month after the second dose, the geometric mean ratio of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing titers in 5-to-11-year-olds to those in 16-to-25-year-olds was 1.04 (95% confidence interval [CI], 0.93 to 1.18), a ratio meeting the prespecified immunogenicity success criterion (lower bound of two-sided 95% CI, >0.67; geometric mean ratio point estimate, ≥0.8). Covid-19 with onset 7 days or more after the second dose was reported in three recipients of the BNT162b2 vaccine and in 16 placebo recipients (vaccine efficacy, 90.7%; 95% CI, 67.7 to 98.3). CONCLUSIONS: A Covid-19 vaccination regimen consisting of two 10-µg doses of BNT162b2 administered 21 days apart was found to be safe, immunogenic, and efficacious in children 5 to 11 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).

A site assessment tool for inpatient controlled human infection models for enteric disease pathogens.

The use of the controlled human infection model to facilitate product development and to advance understanding of host-pathogen interactions is of increasing interest. While administering a virulent (or infective) organism to a susceptible host necessitates an ongoing evaluation of safety and ethical considerations, a central theme in conducting these studies in a safe and ethical manner that yields actionable data is their conduct in facilities well-suited to address their unique attributes. To that end, we have developed a framework for evaluating potential sites in which to conduct inpatient enteric controlled human infection model to ensure consistency and increase the likelihood of success.

The Controlled Human Infection Model for Enterotoxigenic Escherichia coli.

The controlled human infection model (CHIM) for enterotoxigenic Escherichia coli (ETEC) has been instrumental in defining ETEC as a causative agent of acute watery diarrhea, providing insights into disease pathogenesis and resistance to illness, and enabling preliminary efficacy evaluations for numerous products including vaccines, immunoprophylactics, and drugs. Over a dozen strains have been evaluated to date, with a spectrum of clinical signs and symptoms that appear to replicate the clinical illness seen with naturally occurring ETEC. Recent advancements in the ETEC CHIM have enhanced the characterization of clinical, immunological, and microbiological outcomes. It is anticipated that omics-based technologies applied to ETEC CHIMs will continue to broaden our understanding of host-pathogen interactions and facilitate the development of primary and secondary prevention strategies.

Exploring Changes in the Host Gut Microbiota During a Controlled Human Infection Model for Campylobacter jejuni.

Campylobacter jejuni infection is a leading cause of foodborne disease, common to children, adult travelers, and military populations in low- to middle-income countries. In the absence of a licensed vaccine, efforts to evaluate prophylactic agents are underway. The prophylactic efficacy of a twice-daily, 550 mg dose of the antibiotic rifaximin demonstrated no efficacy against campylobacteriosis in a controlled human infection model (CHIM); however, samples from the CHIM study were utilized to assess how the human gut microbiome responds to C. jejuni infection, and if a 'protective' microbiota exists in study participants not developing campylobacteriosis. Statistically significant, but minor, differences in study participant beta diversity were identified during the challenge period (p = 0.002, R2 = 0.042), but no significant differences were otherwise observed. Pre-challenge alpha diversity was elevated in study participants who did not develop campylobacteriosis compared to those who did (p < 0.001), but alpha diversity declined in all study participants from the pre-challenge period to post-discharge. Our work provides insight into gut microbiome shifts observed during a C. jejuni CHIM and following antibiotic treatment. This study utilized a high dose of 1.7 x 105 colony-forming units of C. jejuni; future work could include CHIM studies performed with inocula more closely mimicking natural exposure as well as field studies involving naturally-occurring enteric infections.

Shigella-Specific Immune Profiles Induced after Parenteral Immunization or Oral Challenge with Either Shigella flexneri 2a or Shigella sonnei.

Shigella spp. are a leading cause of diarrhea-associated global morbidity and mortality. Development and widespread implementation of an efficacious vaccine remain the best option to reduce Shigella-specific morbidity. Unfortunately, the lack of a well-defined correlate of protection for shigellosis continues to hinder vaccine development efforts. Shigella controlled human infection models (CHIM) are often used in the early stages of vaccine development to provide preliminary estimates of vaccine efficacy; however, CHIMs also provide the opportunity to conduct in-depth immune response characterizations pre- and postvaccination or pre- and postinfection. In the current study, principal-component analyses were used to examine immune response data from two recent Shigella CHIMs in order to characterize immune response profiles associated with parenteral immunization, oral challenge with Shigella flexneri 2a, or oral challenge with Shigella sonnei. Although parenteral immunization induced an immune profile characterized by robust systemic antibody responses, it also included mucosal responses. Interestingly, oral challenge with S. flexneri 2a induced a distinctively different profile compared to S. sonnei, characterized by a relatively balanced systemic and mucosal response. In contrast, S. sonnei induced robust increases in mucosal antibodies with no differences in systemic responses across shigellosis outcomes postchallenge. Furthermore, S. flexneri 2a challenge induced significantly higher levels of intestinal inflammation compared to S. sonnei, suggesting that both serotypes may also differ in how they trigger induction and activation of innate immunity. These findings could have important implications for Shigella vaccine development as protective immune mechanisms may differ across Shigella serotypes. IMPORTANCE Although immune correlates of protection have yet to be defined for shigellosis, prior studies have demonstrated that Shigella infection provides protection against reinfection in a serotype-specific manner. Therefore, it is likely that subjects with moderate to severe disease post-oral challenge would be protected from a homologous rechallenge, and investigating immune responses in these subjects may help identify immune markers associated with the development of protective immunity. This is the first study to describe distinct innate and adaptive immune profiles post-oral challenge with two different Shigella serotypes. Analyses conducted here provide essential insights into the potential of different immune mechanisms required to elicit protective immunity, depending on the Shigella serotype. Such differences could have significant impacts on vaccine design and development within the Shigella field and should be further investigated across multiple Shigella serotypes.

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.

US Case Reports of Cerebral Venous Sinus Thrombosis With Thrombocytopenia After Ad26.COV2.S Vaccination, March 2 to April 21, 2021.

Importance: Cerebral venous sinus thrombosis (CVST) with thrombocytopenia, a rare and serious condition, has been described in Europe following receipt of the ChAdOx1 nCoV-19 vaccine (Oxford/AstraZeneca), which uses a chimpanzee adenoviral vector. A mechanism similar to autoimmune heparin-induced thrombocytopenia (HIT) has been proposed. In the US, the Ad26.COV2.S COVID-19 vaccine (Janssen/Johnson & Johnson), which uses a human adenoviral vector, received Emergency Use Authorization (EUA) on February 27, 2021. By April 12, 2021, approximately 7 million Ad26.COV2.S vaccine doses had been given in the US, and 6 cases of CVST with thrombocytopenia had been identified among the recipients, resulting in a temporary national pause in vaccination with this product on April 13, 2021. Objective: To describe reports of CVST with thrombocytopenia following Ad26.COV2.S vaccine receipt. Design, Setting, and Participants: Case series of 12 US patients with CVST and thrombocytopenia following use of Ad26.COV2.S vaccine under EUA reported to the Vaccine Adverse Event Reporting System (VAERS) from March 2 to April 21, 2021 (with follow-up reported through April 21, 2021). Exposures: Receipt of Ad26.COV2.S vaccine. Main Outcomes and Measures: Clinical course, imaging, laboratory tests, and outcomes after CVST diagnosis obtained from VAERS reports, medical record review, and discussion with clinicians. Results: Patients' ages ranged from 18 to younger than 60 years; all were White women, reported from 11 states. Seven patients had at least 1 CVST risk factor, including obesity (n = 6), hypothyroidism (n = 1), and oral contraceptive use (n = 1); none had documented prior heparin exposure. Time from Ad26.COV2.S vaccination to symptom onset ranged from 6 to 15 days. Eleven patients initially presented with headache; 1 patient initially presented with back pain and later developed headache. Of the 12 patients with CVST, 7 also had intracerebral hemorrhage; 8 had non-CVST thromboses. After diagnosis of CVST, 6 patients initially received heparin treatment. Platelet nadir ranged from 9 ×103/µL to 127 ×103/µL. All 11 patients tested for the heparin-platelet factor 4 HIT antibody by enzyme-linked immunosorbent assay (ELISA) screening had positive results. All patients were hospitalized (10 in an intensive care unit [ICU]). As of April 21, 2021, outcomes were death (n = 3), continued ICU care (n = 3), continued non-ICU hospitalization (n = 2), and discharged home (n = 4). Conclusions and Relevance: The initial 12 US cases of CVST with thrombocytopenia after Ad26.COV2.S vaccination represent serious events. This case series may inform clinical guidance as Ad26.COV2.S vaccination resumes in the US as well as investigations into the potential relationship between Ad26.COV2.S vaccine and CVST with thrombocytopenia.

Evaluation of 2 Lateral Flow Rapid Tests in the Diagnosis of Chagas Disease in the Washington Metropolitan Area.

We compared the accuracy of the Stat-Pak and Chagas Detect Plus with a latent class analysis. Sensitivity values of 89.7% and 91.9% and specificities of 97.1% and 80.3%, respectively, were seen in the serodiagnosis of Chagas disease in Hispanic immigrants, revealing the limitations of these tests in diverse populations.

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.