The 2022 Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference: Summary of abstract-based presentations.

The global nonprofit organization PATH hosted the third Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Washington, DC, on November 29 to December 1, 2022. With a combination of plenary sessions and posters, keynote presentations, and breakout workshops, the 2022 VASE Conference featured key updates on research related to the development of vaccines against neglected diarrheal pathogens including Shigella, enterotoxigenic Escherichia coli (ETEC), Campylobacter, and Salmonella. The presentations and discussions highlighted the significant impact of these diarrheal pathogens, particularly on the health of infants and young children in low- and middle-income countries, reflecting the urgent need for the development and licensure of new enteric vaccines. Oral and poster presentations at the VASE Conference explored a range of topics, including: the global burden and clinical presentation of disease, epidemiology, and the impact of interventions; the assessment of the value of vaccines against enteric pathogens; preclinical evaluations of vaccine candidates and models of enteric diseases; vaccine candidates in clinical trials and human challenge models; host parameters and genomics that predict responses to infection and disease; the application of new omics technologies for characterization of emerging pathogens and host responses; novel adjuvants, vaccine delivery platforms, and immunization strategies; and strategies for combination/co-administered vaccines. The conference agenda also featured ten breakout workshop sessions on topics of importance to the enteric vaccine field, which are summarized separately. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2022 VASE Conference.

The role of the minor colonization factor CS14 in adherence to intestinal cell models by geographically diverse ETEC isolates.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low- to middle-income countries. ETEC adheres to small intestinal epithelia via colonization factors (CFs) and secretes heat-stable toxin and/or heat-labile toxin, causing dysregulated ion transport and water secretion. There are over 30 CFs identified, including major CFs associated with moderate-to-severe diarrhea (MSD) and minor CFs for which a role in pathogenesis is less clear. The Global Enteric Multicenter Study identified CS14, a class 5a fimbriae, as the only minor CF significantly associated with MSD and was recommended for inclusion in ETEC vaccines. Despite detection of CS14 in ETEC isolates, the sequence conservation of the CS14 operon, its role in adherence, and functional cross-reactivity to other class 5a fimbriae like CFA/I and CS4 are not understood. Sequence analysis determined that the CS14 operon is >99.9% identical among seven geographically diverse isolates with expanded sequence analysis demonstrating SNPs exclusively in the gene encoding the tip adhesin CsuD. Western blots and electron microscopy demonstrated that CS14 expression required the growth of isolates on CFA agar with the iron chelator deferoxamine mesylate. CS14 expression resulted in significantly increased adherence to cultured intestinal cells and human enteroids. Anti-CS14 antibodies and anti-CS4 antibodies, but not anti-CFA/I antibodies, inhibited the adherence of a subset of ETEC isolates, demonstrating CS14-specific inhibition with partial cross-reactivity within the class 5a fimbrial family. These data provide support for CS14 as an important fimbrial CF and its consideration as a vaccine antigen in future strategies. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) infection causes profuse watery diarrhea in adults and children in low- to middle-income countries and is a leading cause of traveler's diarrhea. Despite increased use of rehydration therapies, young children especially can suffer long-term effects including gastrointestinal dysfunction as well as stunting and malnutrition. As there is no licensed vaccine for ETEC, there remains a need to identify and understand specific antigens for inclusion in vaccine strategies. This study investigated one adhesin named CS14. This adhesin is expressed on the bacterial surface of ETEC isolates and was recently recognized for its significant association with diarrheal disease. We demonstrated that CS14 plays a role in bacterial adhesion to human target cells, a critical first step in the disease process, and that adherence could be blocked by CS14-specific antibodies. This work will significantly impact the ETEC field by supporting inclusion of CS14 as an antigen for ETEC vaccines.

Genomic, transcriptomic, and phenotypic differences among archetype Shigella flexneri strains of serotypes 2a, 3a, and 6.

IMPORTANCE: Given the genomic diversity between S. flexneri serotypes and the paucity of data to support serotype-specific phenotypic differences, we applied in silico and in vitro functional analyses of archetype strains of 2457T (Sf2a), J17B (Sf3a), and CH060 (Sf6). These archetype strains represent the three leading S. flexneri serotypes recommended for inclusion in multivalent vaccines. Characterizing the genomic and phenotypic variation among these clinically prevalent serotypes is an important step toward understanding serotype-specific host-pathogen interactions to optimize the efficacy of multivalent vaccines and therapeutics. This study underpins the importance for further large-scale serotype-targeted analyses.

The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs.

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations.

The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

Sequence variations in the ETEC CS6 operon affect transcript and protein expression.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrheal disease in developing nations where it accounts for a significant disease burden in children between the ages of 0 to 59 months. It is also the number one bacterial causative agent of traveler's diarrhea. ETEC infects hosts through the fecal-oral route and utilizes colonization factors (CF) to adhere within the small intestine. Over 25 CFs have been identified; 7 are considered major CFs and a vaccine targeting these is predicted to provide protection against up to 66% of ETEC associated disease. Coli Surface Antigen 6 (CS6) is a major CF and is associated with disease-causing ETEC isolates. Analysis of the CS6 operon sequence led to the identification of two regions of variability among clinical isolates which we predicted exert effects on CS6 transcript and protein expression. A total of 7 recombinant E. coli strains were engineered to encode the CS6 operon in wild-type, hybrid, and mutant configurations. Western blot analysis and RT-qPCR provided evidence to support the importance of an intergenic hairpin structure on CS6 expression. Our results reveal the significance of CS6 sequence selection regarding ETEC vaccine development and present novel information regarding CS6 sequence variation in WT ETEC strains.

Evaluation of a Live Attenuated S. sonnei Vaccine Strain in the Human Enteroid Model.

Shigella is a leading cause of bacillary dysentery worldwide, responsible for high death rates especially among children under five in low-middle income countries. Shigella sonnei prevails in high-income countries and is becoming prevalent in industrializing countries, where multi-drug resistant strains have emerged, as a significant public health concern. One strategy to combat drug resistance in S. sonnei is the development of effective vaccines. There is no licensed vaccine against Shigella, and development has been hindered by the lack of an effective small-animal model. In this work, we used human enteroids, for the first time, as a model system to evaluate a plasmid-stabilized S. sonnei live attenuated vaccine strain, CVD 1233-SP, and a multivalent derivative, CVD 1233-SP::CS2-CS3, which expresses antigens from enterotoxigenic Escherichia coli. The strains were also tested for immunogenicity and protective capacity in the guinea pig model, demonstrating their ability to elicit serum and mucosal antibody responses as well as protection against challenge with wild-type S. sonnei. These promising results highlight the utility of enteroids as an innovative preclinical model to evaluate Shigella vaccine candidates, constituting a significant advance for the development of preventative strategies against this important human pathogen.

Deletion Mutants of Francisella Phagosomal Transporters FptA and FptF Are Highly Attenuated for Virulence and Are Protective Against Lethal Intranasal Francisella LVS Challenge in a Murine Model of Respiratory Tularemia.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We evaluated the attenuation and protective capacity of LVS derivatives with deletions of the fptA and fptF genes in the C57BL/6J mouse model of respiratory tularemia. LVSΔfptA and LVSΔfptF were highly attenuated with LD50 values of >20 times that of LVS when administered intranasally and conferred 100% protection against lethal challenge. Immune responses to the fpt mutant strains in mouse lungs on day 6 post-infection were substantially modified compared to LVS and were associated with reduced organ burdens and reduced pathology. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1ß in the BALF versus LVS, and increased numbers of B cells, αß and γδ T cells, NK cells, and DCs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of Ft and the modulation of the host immune response.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Identification of an Attenuated Substrain of Francisella tularensis SCHU S4 by Phenotypic and Genotypic Analyses.

Pneumonic tularemia is a highly debilitating and potentially fatal disease caused by inhalation of Francisella tularensis. Most of our current understanding of its pathogenesis is based on the highly virulent F. tularensis subsp. tularensis strain SCHU S4. However, multiple sources of SCHU S4 have been maintained and propagated independently over the years, potentially generating genetic variants with altered virulence. In this study, the virulence of four SCHU S4 stocks (NR-10492, NR-28534, NR-643 from BEI Resources and FTS-635 from Battelle Memorial Institute) along with another virulent subsp. tularensis strain, MA00-2987, were assessed in parallel. In the Fischer 344 rat model of pneumonic tularemia, NR-643 and FTS-635 were found to be highly attenuated compared to NR-10492, NR-28534, and MA00-2987. In the NZW rabbit model of pneumonic tularemia, NR-643 caused morbidity but not mortality even at a dose equivalent to 500x the LD50 for NR-10492. Genetic analyses revealed that NR-10492 and NR-28534 were identical to each other, and nearly identical to the reference SCHU S4 sequence. NR-643 and FTS-635 were identical to each other but were found to have nine regions of difference in the genomic sequence when compared to the published reference SCHU S4 sequence. Given the genetic differences and decreased virulence, NR-643/FTS-635 should be clearly designated as a separate SCHU S4 substrain and no longer utilized in efficacy studies to evaluate potential vaccines and therapeutics against tularemia.

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens.

Enteric bacterial pathogens cause significant morbidity and mortality globally. Studies in tissue culture and animal models shaped our initial understanding of these host-pathogen interactions. However, intrinsic shortcomings in these models limit their application, especially in translational applications like drug screening and vaccine development. Human intestinal enteroid and organoid models overcome some limitations of existing models and advance the study of enteric pathogens. In this review, we detail the use of human enteroids and organoids to investigate the pathogenesis of invasive bacteria Shigella, Listeria, and Salmonella, and noninvasive bacteria pathogenic Escherichia coli, Clostridium difficile, and Vibrio cholerae. We highlight how these studies confirm previously identified mechanisms and, importantly, reveal novel ones. We also discuss the challenges for model advancement, including platform engineering to integrate environmental conditions, innate immune cells and the resident microbiome, and the potential for pre-clinical testing of recently developed antimicrobial drugs and vaccines.

Characterization of Schu S4 aro mutants as live attenuated tularemia vaccine candidates.

There is a need for development of an effective vaccine against Francisella tularensis, as this potential bioweapon has a high mortality rate and low infectious dose when delivered via the aerosol route. Moreover, this Tier 1 agent has a history of weaponization. We engineered targeted mutations in the Type A strain F. tularensis subspecies tularensis Schu S4 in aro genes encoding critical enzymes in aromatic amino acid biosynthesis. F. tularensis Schu S4ΔaroC, Schu S4ΔaroD, and Schu S4ΔaroCΔaroD mutant strains were attenuated for intracellular growth in vitro and for virulence in vivo and, conferred protection against pulmonary wild-type (WT) F. tularensis Schu S4 challenge in the C57BL/6 mouse model. F. tularensis Schu S4ΔaroD was identified as the most promising vaccine candidate, demonstrating protection against high-dose intranasal challenge; it protected against 1,000 CFU Schu S4, the highest level of protection tested to date. It also provided complete protection against challenge with 92 CFU of a F. tularensis subspecies holarctica strain (Type B). Mice responded to vaccination with Schu S4ΔaroD with systemic IgM and IgG2c, as well as the production of a functional T cell response as measured in the splenocyte-macrophage co-culture assay. This vaccine was further characterized for dissemination, histopathology, and cytokine/chemokine gene induction at defined time points following intranasal vaccination which confirmed its attenuation compared to WT Schu S4. Cytokine, chemokine, and antibody induction patterns compared to wild-type Schu S4 distinguish protective vs. pathogenic responses to F. tularensis and elucidate correlates of protection associated with vaccination against this agent.

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice.

Vaccine studies for Shigella flexneri and enterotoxigenic Escherichia coli have been impaired by the lack of optimal animal models. We used two murine models to show that a S. flexneri 2a bivalent vaccine (CVD 1208S-122) expressing enterotoxigenic Escherichia coli colonization factor antigen-I (CFA/I) and the binding subunits A2 and B of heat labile-enterotoxin (LTb) is immunogenic and protects against weight loss and diarrhea. These findings document the immunogenicity and pre-clinical efficacy effects of CVD 1208S-122 vaccine and suggest that further work can help elucidate relevant immune responses and ultimately its clinical efficacy in humans.

A tale of two bacterial enteropathogens and one multivalent vaccine.

Shigella and enterotoxigenic Escherichia coli (ETEC) are among the top four enteric pathogens that cause diarrheal illness in young children in developing countries and are major etiologic agents of travellers' diarrhoea. A single vaccine that could target both of these pathogens would have significant public health impact. In this review, we highlight the many pivotal contributions of Phillippe Sansonetti to the identification of molecular mechanisms of pathogenesis of Shigella that paved the way for the development of rationally designed, novel vaccines candidates. The CVD developed a series of live attenuated Shigella vaccine strains based on the most prevalent serotypes associated with disease. Shigella vaccine strains were engineered to express critical ETEC antigens to form a broadly protective Shigella-ETEC multivalent vaccine.

Experimental Infection of Human Volunteers with the Heat-Stable Enterotoxin-Producing Enterotoxigenic Escherichia coli Strain TW11681.

Infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-stable enterotoxin (ST) is one of the most important causes of childhood diarrhoea in low- and middle-income countries. Here, we undertook a controlled human infection model (CHIM) study to investigate whether ST-producing ETEC strain TW11681 would be suitable for testing the protective efficacy of new ST-based vaccine candidates in vaccine challenge models. In groups of three, nine volunteers ingested 1 × 106, 1 × 107, or 1 × 108 colony-forming units (CFU) of TW11681. Flow cytometry-based assays were used to measure CD4+ T cell responses and antibody levels targeting virulence factors expressed by the strain. We found that infection with TW11681 elicited few and mild symptoms, including mild diarrhoea in two volunteers, both of whom ingested 1 × 106 CFU. Averaged across all volunteers, the CD4+ T cell responses specific for E. coli YghJ mucinase peaked 10 days after infection (3.2-fold (p = 0.016)), while the CD4+ T cell responses specific for Colonization Factor Antigen I (CFA/I) major fimbrial subunit (CfaB) peaked after 28 days (3.6-fold (p = 0.063)). The serum CfaB-specific anti-IgA and anti-IgG/IgM levels were significantly increased and peaked 3 months after infection. Both remained elevated for the duration of the 12-month follow-up. The corresponding anti-YghJ serological response was strongest after 10 days, although a significant increase was seen only for IgA levels (3.2-fold (p = 0.008)). In conclusion, due to its low diarrhoea attack risk, TW11681 is probably not suitable for testing the efficacy of new vaccines in human challenge studies at doses 1 × 106 to 1 × 108. However, the strain may still be useful in CHIMs for studying ETEC host-pathogen interactions.

Development, Characterization, and Standardization of a Nose-Only Inhalation Exposure System for Exposure of Rabbits to Small-Particle Aerosols Containing Francisella tularensis.

Inhalation of Francisella tularensis causes pneumonic tularemia in humans, a severe disease with a 30 to 60% mortality rate. The reproducible delivery of aerosolized virulent bacteria in relevant animal models is essential for evaluating medical countermeasures. Here we developed optimized protocols for infecting New Zealand White (NZW) rabbits with aerosols containing F. tularensis We evaluated the relative humidity, aerosol exposure technique, and bacterial culture conditions to optimize the spray factor (SF), a central metric of aerosolization. This optimization reduced both inter- and intraday variability and was applicable to multiple isolates of F. tularensis Further improvements in the accuracy and precision of the inhaled pathogen dose were achieved through enhanced correlation of the bacterial culture optical density and the number of CFU. Plethysmograph data collected during exposures found that respiratory function varied considerably between rabbits, was not a function of weight, and did not improve with acclimation to the system. Live vaccine strain (LVS)-vaccinated rabbits were challenged via aerosol with human-virulent F. tularensis SCHU S4 that had been cultivated in either Mueller-Hinton broth (MHB) or brain heart infusion (BHI) broth. LVS-vaccinated animals challenged with SCHU S4 that had been cultivated in MHB experienced short febrile periods (median, 3.2 days), limited weight loss (<5%), and longer median survival times (∼18 days) that were significantly different from those for unvaccinated controls. In contrast, LVS-vaccinated rabbits challenged with SCHU S4 that had been cultivated in BHI experienced longer febrile periods (median, 5.5 days) and greater weight loss (>10%) than the unvaccinated controls and median survival times that were not significantly different from those for the unvaccinated controls. These studies highlight the importance of careful characterization and optimization of protocols for aerosol challenge with pathogenic agents.

Genome and Functional Characterization of Colonization Factor Antigen I- and CS6-Encoding Heat-Stable Enterotoxin-Only Enterotoxigenic Escherichia coli Reveals Lineage and Geographic Variation.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST+) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.

A roadmap for enterotoxigenic Escherichia coli vaccine development based on volunteer challenge studies.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of travelers' diarrhea and of diarrhea among young children in developing countries. Experimental challenge studies in adult volunteers have played a pivotal role in establishing ETEC as an enteric pathogen, elucidating its pathogenesis by identifying specific virulence attributes, characterizing the human immune response to clinical and sub-clinical ETEC infection and assessing preliminarily the clinical acceptability, immunogenicity and efficacy of prototype ETEC vaccines. This review provides a historical perspective of experimental challenge studies with ETEC. It summarizes pioneering early studies carried out by investigators at the University of Maryland School of Medicine to show how those studies provided key information that influenced the directions taken by many research groups to develop vaccines to prevent ETEC. In addition, key experimental challenge studies undertaken at other institutions will also be cited.