Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

Implications and measurement of herd protection (indirect effects) for enteric vaccine development.

Diarrhea remains one of the top five causes of disease and death among young children in developing nations. Fortunately, scientists are making progress developing vaccines against enterotoxigenic E. coli (ETEC) and Shigella, two of the leading diarrhea pathogens. As vaccine developers start to consider field efficacy trials of these vaccines, they should be aware of the importance of evaluating not only vaccine direct effects on the immunized, but also the herd effects that vaccination can afford to the unimmunized in a community. In a workshop held at the conference titled "Vaccines against Shigella and ETEC (VASE)", we described to participants what herd effects are and we presented on methods used in cholera and rotavirus studies that could be useful for future ETEC and Shigella vaccine trials conducted in low and middle-income nations. We also presented evidence on the effects of vaccine herd effects for estimates of vaccine cost-effectiveness.

Assessment of disease specific immune responses in enteric diseases using dried blood spot (DBS).

BACKGROUND: Blood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV). METHODOLOGY/PRINCIPLE FINDINGS: We processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively. CONCLUSION: Our results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings.

Using advocacy to increase investment in enteric vaccine development.

Catalyzing and sustaining momentum for long-term research investments can be a challenge, especially for enteric pathogens like ETEC andShigella that are most threatening to the health of children in low-resource areas, and whose vaccines would not be for global use. The 2018 Vaccines Against Shigella and ETEC (VASE) Conference included a workshop focused on building the capacity of scientists to communicate about their own research and advocate for additional attention and funding for enteric disease and vaccines research. Workshop presenters shared best practices and examples of advocacy, communications, and messaging tactics that have been used successfully during early stages of vaccine development research for other pathogens. The presentations were followed by an interactive, hands-on training for real-life communication opportunities for scientists that could result in increased research funding, including developing resonant messaging for relevant audiences and practicing interviews.

Global burden of diarrheal diseases among children in developing countries: Incidence, etiology, and insights from new molecular diagnostic techniques.

The Global Enteric Multicenter Study (GEMS) demonstrated that Shigella and enterotoxigenic Escherichia coli (ETEC) producing heat stable toxin (ST) (either alone or in combination with heat labile toxin) are among the most important pathogens associated with moderate-to-severe diarrhea (MSD) in children younger than 5years of age living in developing countries. To inform the design of vaccines and other interventions, we reviewed published data and new results from GEMS characterizing the burden of Shigella and ST-ETEC infections. Clinical parameters were assessed to examine the value of various case definitions as indicators of MSD associated with Shigella and ST-ETEC for use in clinical trials. We discussed advantages and disadvantages of culture-based and culture-independent molecular diagnostics for detecting clinically and epidemiologically relevant disease. Shigella serotyping data from GEMS were examined to identify desirable components of Shigella and ETEC vaccines likely to confer broad protection. These findings can inform the development and implementation of vaccines to prevent these important infections among infants and children in developing countries.

Development and Efficacy Assessment of an Enteric Coated Porous Tablet Loaded With F4 Fimbriae for Oral Vaccination of Piglets against F4+ Escherichia coli Infections.

Enterotoxigenic Escherichia coli (ETEC) infection is one of the major causes contributing to the development of diarrhoea and mortality in new born, suckling and newly weaned piglets. To date, no preventive/treatment strategy showed promising results, which could be due to the lack of potent vaccines, and/or due to the development of resistance of ETEC to antibiotics. Therefore, in the present investigation, a novel porous sodium alginate (SA) tablet formulation loaded with F4 fimbriae antigen was developed and tested for efficacy against ETEC infections in piglet models. Precompression parameters of the powder mixes and post compression parameters of tablets have been evaluated and results were found to be satisfactory. Loading of F4 fimbrial antigens into the tablets was achieved by inducing pores in the tablets via the sublimation of camphor followed by incubation with purified F4 fimbriae. The loaded tablets have been coated with Eudragit L100 to protect the F4 fimbriae from (a) highly acidic gastric environment; (b) proteolytic cleavage by pepsin; and (c) to promote subsequent release in the intestine. Evaluation of developed F4 fimbrial tablets in a Pig model demonstrated induction of mucosal immunity, and a significant reduction of F4+ E. coli in faeces. Therefore, F4 fimbriae loaded porous tablets could be a novel oral vaccination candidate to induce mucosal and systemic immunity against ETEC infections.

Antibody-mediated disruption of the mechanics of CS20 fimbriae of enterotoxigenic Escherichia coli.

Preventive vaccines against enterotoxigenic Escherichia coli (ETEC) are being developed, many of which target common fimbrial colonization factors as the major constituent, based on empirical evidence that these function as protective antigens. Particularly, passive oral administration of ETEC anti-fimbrial antibodies prevent ETEC diarrhea. Little is, however, known regarding the specific mechanisms by which intestinal antibodies against ETEC fimbriae function to prevent disease. Using coli surface antigen 20 (CS20) fimbriae as a model ETEC colonization factor, we show using force spectroscopy that anti-fimbrial antibodies diminish fimbrial elasticity by inhibiting their natural capacity to unwind and rewind. In the presence of anti-CS20 antibodies the force required to unwind a single fimbria was increased several-fold and the extension length was shortened several-fold. Similar measurements in the presence of anti-CS20 Fab fragments did not show any effect, indicating that bivalent antibody binding is required to reduce fimbrial elasticity. Based on these findings, we propose a model for an in-vivo mechanism whereby antibody-mediated disruption of the biomechanical properties of CS20 fimbriae impedes sustained adhesion of ETEC to the intestinal mucosal surface. Further elucidation of the role played by intestinal antibodies in mechanical disruption of fimbrial function may provide insights relevant to ETEC vaccine development.

An assessment of enterotoxigenic Escherichia coli and Shigella vaccine candidates for infants and children.

Despite improvements to water quality, sanitation, and the implementation of current prevention and treatment interventions, diarrhea remains a major cause of illness and death, especially among children less than five years of age in the developing world. Rotavirus vaccines have already begun making a real impact on diarrhea, but several more enteric vaccines will be necessary to achieve broader reductions of illness and death. Among the many causes of diarrheal disease, enterotoxigenic Escherichia coli (ETEC) and Shigella are the two most important bacterial pathogens for which there are no currently licensed vaccines. Vaccines against these two pathogens could greatly reduce the impact of disease caused by these infections. This review describes the approaches to ETEC and Shigella vaccines that are currently under development, including a range of both cellular and subunit approaches for each pathogen. In addition, the review discusses strategies for maximizing the potential benefit of these vaccines, which includes the feasibility of co-administration, consolidation, and combination of vaccine candidates, as well as issues related to effective administration of enteric vaccines to infants. Recent impact studies indicate that ETEC and Shigella vaccines could significantly benefit global public health. Either vaccine, particularly if they could be combined together or with another enteric vaccine, would be an extremely valuable tool for saving lives and promoting the health of infants and children in the developing world, as well as potentially providing protection to travelers and military personnel visiting endemic areas.