Reassessing potential economic value and health impact of effective Shigella vaccines.

The gram-negative bacterium Shigella is a leading cause of diarrheal morbidity and mortality in children in low- and middle-income countries. Several promising vaccine candidates are in late stages of clinical development against this increasingly antibiotic-resistant pathogen. However, considering the increasingly crowded and costly paediatric immunization schedule, and likely advent of other important new vaccines, it is unclear whether introduction of a Shigella vaccine would represent a high priority for international agencies or health ministries in low- and middle-income countries. To determine whether there is a compelling public health value proposition for a Shigella vaccine, we used the World Health Organization's Full Value of Vaccine Assessment analytic framework and formulated five broad scientific, policy, economic and commercial-related propositions regarding the development of a Shigella vaccine. We also explored the current regulatory, clinical, policy and commercial challenges to a Shigella-containing combination vaccine development and adoption. Through a series of literature reviews, expert consultations, social science field studies and model-based analyses, we addressed each of these propositions. As described in a series of separate publications that are synthesized here, we concluded that the economic and public health value of a Shigella vaccine may be greater than previously recognized, particularly if it is found to also be effective against less severe forms of diarrheal disease and childhood stunting. The decision by pharmaceutical companies to develop a standalone vaccine or a multipathogen combination will be a key factor in determining its relative prioritization by various stakeholders in low- and middle-income countries.

La bactérie à Gram négatif Shigella est l'une des principales causes de morbidité et de mortalité diarrhéiques chez les enfants des pays à revenu faible et intermédiaire. Plusieurs candidats vaccins prometteurs sont en phase avancée de conception clinique contre cet agent pathogène qui connaît une antibiorésistance croissante. Toutefois, compte tenu du calendrier de vaccination pédiatrique de plus en plus chargé et coûteux et de l'arrivée probable d'autres nouveaux vaccins importants, il n'est pas certain que la mise sur le marché d'un vaccin contre Shigella constitue une priorité élevée pour les agences internationales ou les ministères de la Santé des pays à revenu faible ou intermédiaire. Pour déterminer l'existence d'un intérêt convaincant en matière de santé publique pour un vaccin contre Shigella, nous avons utilisé le cadre analytique du cadre d'évaluation de la valeur totale des vaccins de l'Organisation mondiale de la santé et formulé cinq propositions scientifiques, politiques, économiques et commerciales générales concernant la conception d'un vaccin contre Shigella. Nous avons également étudié les défis en matière réglementaire, clinique, politique et commerciale qui se posent actuellement à la mise au point et à l'adoption d'un vaccin combiné contenant des Shigella. Nous avons abordé chacune de ces propositions au moyen d'une série d'analyses documentaires, de consultations d'experts, d'études de terrain en sciences sociales et d'analyses basées sur des modèles. Comme décrit dans une série de publications distinctes résumées ici, nous avons conclu que la valeur économique et sur le plan de la santé publique d'un vaccin contre Shigella pourrait être plus importante que ce qui était considéré précédemment, en particulier s'il s'avère que ce vaccin s'avère également efficace contre les formes moins sévères de maladies diarrhéiques et de retard de croissance chez l'enfant. La décision d'entreprises pharmaceutiques de mettre au point un vaccin autonome ou une combinaison de plusieurs agents pathogènes sera un facteur clé dans la détermination de sa priorité relative par les différentes parties prenantes dans les pays à revenu faible et intermédiaire.

La bacteria gramnegativa Shigella es una de las principales causas de morbilidad y mortalidad por diarrea en niños de países de ingresos bajos y medios. Varias vacunas candidatas y prometedoras se encuentran en las últimas fases de desarrollo clínico contra este patógeno cada vez más resistente a los antibióticos. Sin embargo, teniendo en cuenta el esquema de inmunización pediátrica, cada vez más saturado y costoso, y la probable llegada de otras vacunas nuevas importantes, no está claro si la introducción de una vacuna contra la Shigella representaría una alta prioridad para los organismos internacionales o los ministerios de salud de los países de ingresos bajos y medios. Para determinar si existe una propuesta de valor de salud pública convincente para una vacuna contra la Shigella, utilizamos el marco de análisis Full Value of Vaccine Assessment de la Organización Mundial de la Salud y formulamos cinco amplias propuestas científicas, políticas, económicas y comerciales relacionadas con el desarrollo de una vacuna contra la Shigella. También exploramos los actuales desafíos reglamentarios, clínicos, políticos y comerciales para el desarrollo y la adopción de una vacuna combinada que contenga Shigella. Mediante una serie de revisiones bibliográficas, consultas a expertos, estudios de campo de ciencias sociales y análisis basados en modelos, abordamos cada una de estas proposiciones. Como se describe en una serie de publicaciones separadas que se sintetizan aquí, llegamos a la conclusión de que el valor económico y de salud pública de una vacuna contra la Shigella puede ser mayor de lo que se reconocía anteriormente, en particular si se descubre que también es eficaz contra formas menos graves de enfermedad diarreica y retraso del crecimiento infantil. La decisión de las empresas farmacéuticas de desarrollar una vacuna independiente o una combinación multipatógena será un factor clave a la hora de determinar su prioridad relativa por parte de las diversas partes interesadas en los países de ingresos bajos y medios.

Controlled Human Infection Models To Accelerate Vaccine Development.

The timelines for developing vaccines against infectious diseases are lengthy, and often vaccines that reach the stage of large phase 3 field trials fail to provide the desired level of protective efficacy. The application of controlled human challenge models of infection and disease at the appropriate stages of development could accelerate development of candidate vaccines and, in fact, has done so successfully in some limited cases. Human challenge models could potentially be used to gather critical information on pathogenesis, inform strain selection for vaccines, explore cross-protective immunity, identify immune correlates of protection and mechanisms of protection induced by infection or evoked by candidate vaccines, guide decisions on appropriate trial endpoints, and evaluate vaccine efficacy. We prepared this report to motivate fellow scientists to exploit the potential capacity of controlled human challenge experiments to advance vaccine development. In this review, we considered available challenge models for 17 infectious diseases in the context of the public health importance of each disease, the diversity and pathogenesis of the causative organisms, the vaccine candidates under development, and each model's capacity to evaluate them and identify correlates of protective immunity. Our broad assessment indicated that human challenge models have not yet reached their full potential to support the development of vaccines against infectious diseases. On the basis of our review, however, we believe that describing an ideal challenge model is possible, as is further developing existing and future challenge models.

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.

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.

Hyperimmune Bovine Colostral Anti-CS17 Antibodies Protect Against Enterotoxigenic Escherichia coli Diarrhea in a Randomized, Doubled-Blind, Placebo-Controlled Human Infection Model.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) commonly cause diarrhea in children living in developing countries and in travelers to those regions. ETEC are characterized by colonization factors (CFs) that mediate intestinal adherence. We assessed if bovine colostral IgG (bIgG) antibodies against a CF, CS17, or antibodies against CsbD, the minor tip subunit of CS17, would protect subjects against diarrhea following challenge with a CS17-expressing ETEC strain. METHODS: Adult subjects were randomized (1:1:1) to receive oral bIgG against CS17, CsbD, or placebo. Two days prior to challenge, subjects began dosing 3 times daily with the bIgG products (or placebo). On day 3, subjects ingested 5 × 109 cfu ETEC strain LSN03-016011/A in buffer. Subjects were assessed for diarrhea for 120 hours postchallenge. RESULTS: A total of 36 subjects began oral prophylaxis and 35 were challenged with ETEC. While 50.0% of the placebo recipients had watery diarrhea, none of the subjects receiving anti-CS17 had diarrhea (P = .01). In contrast, diarrhea rates between placebo and anti-CsbD recipients (41.7%) were comparable (P = 1.0). CONCLUSIONS: This is the first study to demonstrate anti-CS17 antibodies provide significant protection against ETEC expressing CS17. More research is needed to better understand why anti-CsbD was not comparably efficacious. Clinical Trials Registration. NCT00524004.

Consensus Report on Shigella Controlled Human Infection Model: Clinical Endpoints.

The Shigella controlled human infection model (CHIM) is valuable for assessing candidate Shigella vaccine efficacy and potentially accelerating regulatory approval. The Shigella CHIM is currently being conducted at 3 sites in the United States using Shigella flexneri 2a strain 2457T and Shigella sonnei strain 53G. Shigellosis can present variably as watery diarrhea alone or with dysentery, and can be accompanied by manifestations including fever, abdominal cramps, tenesmus, and malaise. For comparability, it is important to harmonize the primary clinical endpoint. An expert working group was convened on 2 February 2018 to review clinical data from Shigella CHIM studies performed to date and to develop a consensus primary endpoint. The consensus endpoint enabled "shigellosis" to present as severe diarrhea or moderate diarrhea or dysentery. The latter 2 criteria are met when concurrent with fever of 38.0°C and/or vomiting, and/or a constitutional/enteric symptom graded at least as "moderate" severity. The use of a blinded independent committee to adjudicate the primary endpoint by subject was also regarded as important. As safety of volunteers in challenge studies is of paramount importance and treatment timing can affect primary outcomes, a standard for early antibiotic administration was established as follows: (1) when the primary endpoint is met; (2) if a fever of ≥39.0°C develops; or (3) if the study physician deems it appropriate. Otherwise, antibiotics are given at 120 hours postinfectious challenge. The working group agreed on objective and subjective symptoms to be solicited, and standardized methods for assessing subject-reported severity of symptoms.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

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.

Prophylactic Efficacy of Hyperimmune Bovine Colostral Antiadhesin Antibodies Against Enterotoxigenic Escherichia coli Diarrhea: A Randomized, Double-Blind, Placebo-Controlled, Phase 1 Trial.

Background: Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers. Methods: Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 109 colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge. Results: After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively. Conclusions: Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.

Special Issue on Enterotoxigenic Escherichia coli (ETEC) Vaccines: ETEC Infection and Vaccine-Mediated Immunity.

Enterotoxigenic Escherichia coli (ETEC) is the most prevalent bacterial pathogen causing young children to suffer acute watery diarrhea in Low- and Middle-Income Countries (LMICs) [...].

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).

Oral inactivated whole cell vaccine for mucosal immunization: ETVAX case study.

Oral immunization is an effective strategy for inducing protective immunity against mucosal enteric pathogens. Although live-attenuated as well as subunit approaches have been explored for vaccination against enteric pathogens, inactivated whole bacterial cells may also be effective in introducing protective immunity. Successfully accomplishing this goal with inactivated whole bacterial cells will require that a complex antigenic repertoire be presented in controlled immunogenic amounts, in a safe and relatively simple and self-contained delivery format. The benefit from immunization with whole cell vaccines can be further enhanced through genetic engineering to over-express selected antigens and also by the use of mucosal adjuvants to direct a more robust immunologic response. These steps are being taken for the development of ETVAX, the most clinically advanced vaccine candidate against the major enteric pathogen, enterotoxigenic Escherichia coli (ETEC) with significant positive impact.

Vaccine value profile for Shigella.

Shigella is the leading bacterial cause of diarrhoea and the second leading cause of diarrhoeal mortality among all ages. It also exhibits increasing levels of antibiotic resistance. The greatest burden is among children under five in low- and middle-income countries (LMICs). As such, a priority strategic goal of the World Health Organization (WHO) is the development of a safe, effective and affordable vaccine to reduce morbidity and mortality from Shigella-attributable dysentery and diarrhea, including long term outcomes associated with chronic inflammation and growth faltering, in children under 5 years of age in LMICs. In addition, a safe and effective Shigella vaccine is of potential interest to travellers and military both to prevent acute disease and rarer, long-term sequelae. An effective Shigella vaccine is also anticipated to reduce antibiotic use and thereby help diminish further emergence of enteric pathogens resistant to antimicrobials. The most advanced vaccine candidates are multivalent, parenteral formulations in Phase 2 and Phase 3 clinical studies. They rely on O-antigen-polysaccharide protein conjugate technologies or, alternatively, outer membrane vesicles expressing penta-acylated lipopolysaccharide that has been detoxified. Other parenteral and oral formulations, many delivering a broader array of Shigella antigens, are at earlier stages of clinical development. These formulations are being assessed in alignment with the WHO Preferred Product Characteristics, which call for a 1 to 2 dose primary immunization series given during the first 12 months of life, ideally starting at 6 months of age. This 'Vaccine Value Profile' (VVP) for Shigella is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic and societal value of pipeline vaccines and vaccine-like products. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, government agencies and multi-lateral organizations. All contributors have extensive expertise on various elements of the Shigella VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

Vaccine value profile for enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) is one of the leading bacterial causes of diarrhoea, especially among children in low-resource settings, and travellers and military personnel from high-income countries. WHO's primary strategic goal for ETEC vaccine development is to develop a safe, effective, and affordable ETEC vaccine that reduces mortality and morbidity due to moderate-to-severe diarrhoeal disease in infants and children under 5 years of age in LMICs, as well as the long-term negative health impact on infant physical and cognitive development resulting from infection with this enteric pathogen. An effective ETEC vaccine will also likely reduce the need for antibiotic treatment and help limit the further emergence of antimicrobial resistance bacterial pathogens. The lead ETEC vaccine candidate, ETVAX, has shown field efficacy in travellers and has moved into field efficacy testing in LMIC infants and children. A Phase 3 efficacy study in LMIC infants is projected to start in 2024 and plans for a Phase 3 trial in travellers are under discussion with the U.S. FDA. Licensing for both travel and LMIC indications is projected to be feasible in the next 5-8 years. Given increasing recognition of its negative impact on child health and development in LMICs and predominance as the leading etiology of travellers' diarrhoea (TD), a standalone vaccine for ETEC is more cost-effective than vaccines targeting other TD pathogens, and a viable commercial market also exists. In contrast, combination of an ETEC vaccine with other vaccines for childhood pathogens in LMICs would maximize protection in a more cost-effective manner than a series of stand-alone vaccines. This 'Vaccine Value Profile' (VVP) for ETEC is intended to provide a high-level, holistic assessment of available data to inform the potential public health, economic and societal value of pipeline vaccines and vaccine-like products. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations. All contributors have extensive expertise on various elements of the ETEC VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information.

Efficacy of an Enterotoxigenic Escherichia coli (ETEC) Vaccine on the Incidence and Severity of Traveler's Diarrhea (TD): Evaluation of Alternative Endpoints and a TD Severity Score.

The efficacy of an Oral Whole Cell ETEC Vaccine (OEV) against Travelers' Diarrhea (TD) was reexamined using novel outcome and immunologic measures. More specifically, a recently developed disease severity score and alternative clinical endpoints were evaluated as part of an initial validation effort to access the efficacy of a vaccine intervention for the first time in travelers to an ETEC endemic area. A randomized, double-blind, placebo-controlled trial followed travelers to Guatemala or Mexico up to 28 days after arrival in the country following vaccination (two doses two weeks apart) with an ETEC vaccine. Fecal samples were collected upon arrival, departure, and during TD for pathogen identification. Serum was collected in a subset of subjects to determine IgA cholera toxin B subunit (CTB) antibody titers upon their arrival in the country. The ETEC vaccine's efficacy, utilizing a TD severity score and other alternative endpoints, including the relationship between antibody levels and TD risk, was assessed and compared to the per-protocol primary efficacy endpoint. A total of 1435 subjects completed 7-28 days of follow-up and had available data. Vaccine efficacy was higher against more severe (≥5 unformed stools/24 h) ETEC-attributable TD and when accounting for immunologic take (PE ≥ 50%; p < 0.05). The vaccine protected against less severe (3 and 4 unformed stools/24 h) ETEC-attributable TD when accounting for symptom severity or change in activity (PE = 76.3%, p = 0.01). Immunologic take of the vaccine was associated with a reduced risk of infection with ETEC and other enteric pathogens, and with lower TD severity. Clear efficacy was observed among vaccinees with a TD score of ≥4 or ≥5, regardless of immunologic take (PE = 72.0% and 79.0%, respectively, p ≤ 0.03). The vaccine reduced the incidence and severity of ETEC, and this warrants accelerated evaluation of the improved formulation (designated ETVAX), currently undergoing advanced field testing. Subjects with serum IgA titers to CTB had a lower risk of infection with ETEC and Campylobacter jejuni/coli. Furthermore, the TD severity score provided a more robust descriptor of disease severity and should be included as an endpoint in future studies.

Volunteer challenge with enterotoxigenic Escherichia coli that express intestinal colonization factor fimbriae CS17 and CS19.

Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 10(9) organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 10(9) organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials.

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.

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.

Linking inherent O-Linked Protein Glycosylation of YghJ to Increased Antigen Potential.

Enterotoxigenic Escherichia coli (ETEC) is a WHO priority pathogen and vaccine target which causes infections in low-income and middle-income countries, travelers visiting endemic regions. The global urgent demand for an effective preventive intervention has become more pressing as ETEC strains have become increasingly multiple antibiotic resistant. However, the vaccine development pipeline has been slow to address this urgent need. To date, vaccine development has focused mainly on canonical antigens such as colonization factors and expressed toxins but due to genomic plasticity of this enteric pathogen, it has proven difficult to develop effective vaccines. In this study, we investigated the highly conserved non-canonical vaccine candidate YghJ/SsLE. Using the mass spectrometry-based method BEMAP, we demonstrate that YghJ is hyperglycosylated in ETEC and identify 54 O-linked Set/Thr residues within the 1519 amino acid primary sequence. The glycosylation sites are evenly distributed throughout the sequence and do not appear to affect the folding of the overall protein structure. Although the glycosylation sites only constitute a minor subpopulation of the available epitopes, we observed a notable difference in the immunogenicity of the glycosylated YghJ and the non-glycosylated protein variant. We can demonstrate by ELISA that serum from patients enrolled in an ETEC H10407 controlled infection study are significantly more reactive with glycosylated YghJ compared to the non-glycosylated variant. This study provides an important link between O-linked glycosylation and the relative immunogenicity of bacterial proteins and further highlights the importance of this observation in considering ETEC proteins for inclusion in future broad coverage subunit vaccine candidates.