High efficiency biosynthesis of O-polysaccharide-based vaccines against extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) has presented a major clinical infection emerged in the past decades. O-polysaccharide (OPS)-based glycoconjugate vaccines produced using the bacterial glycosylation machinery can be utilized to confer protection against such infection. However, constructing a low-cost microbial cell factory for high-efficient production of OPS-based glycoconjugate vaccines remains challenging. Here, we engineered a glyco-optimized chassis strain by reprogramming metabolic network. The yield was enhanced to 38.6 mg L-1, the highest level reported so far. MS analysis showed that designed glycosylation sequon was modified by target polysaccharide with high glycosylation efficiency of 90.7 % and 76.7 % for CTB-O5 and CTB-O7, respectively. The glycoconjugate vaccines purified from this biosystem elicited a marked increase in protection against ExPEC infection in mouse model, compared to a non-optimized system. The glyco-optimized platform established here is broadly suitable for polysaccharide-based conjugate production against ExPEC and other surface-polysaccharide-producing pathogens.

Immunogenicity of 987P fimbriae of enterotoxigenic Escherichia coli surface-displayed on Lactobacillus casei.

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.

Vaccines Against Shigella and Enterotoxigenic Escherichia coli: A summary of the 2018 VASE Conference.

PATH hosted the second Vaccines Against Shigella and Enterotoxigenic Escherichia coli (VASE) Conference in Mexico City in June 2018, again providing experts from around the world an opportunity to participate in a highly collaborative forum to discuss progress in the development of new enteric vaccines. Through a combination of plenary sessions and posters, keynote presentations, and workshops, the 2018 VASE Conference aimed to accelerate communication and progress among those working to achieve the goal of licensed vaccines against these two bacterial pathogens. Many presentations recognized the importance of diarrheal disease and long-term sequelae caused by infections with Shigella and enterotoxigenic E. coli (ETEC). Other presentations explored new strategies for vaccine development, including the search for novel, possibly conserved, antigens for more effective vaccines. Much progress is being made as some vaccine candidates are now moving through clinical trials. Research presented in oral and poster presentations at the VASE Conference covered a range of topics, including: the global burden of disease, epidemiology, and health economics; host parameters and genomics that predict responses to infection and disease; preclinical evaluations of vaccine antigens and models of enteric diseases; and vaccine candidates in clinical trials and human challenge studies. This article reviews key points and highlighted research presented in each of the plenary conference sessions and poster presentations at the 2018 VASE Conference.

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.

Technical product attributes in development of an oral enteric vaccine for infants.

Development of an oral enteric vaccine for infants is important for Shigella and enterotoxigenic Escherichia coli (ETEC) vaccine development. At a recent workshop titled "Technical Product Attributes in Development of an Oral Enteric Vaccine for Infants," at the 2nd International Vaccines Against Shigella and ETEC Conference (VASE Conference), the preferred product attributes for development were discussed for these vaccines. The aims of this workshop were to identify gaps and gather opinions from key experts from preclinical, process development, manufacturing, regulatory, and clinical areas to fine-tune and refine key target product attributes for infant oral vaccine development. The workshop used some examples of marketed oral infant vaccines to discuss potential improvements that can be made, such as inclusion of preservatives, multidose vials, and antacid buffer presentation (liquid or lyophilized) in novel oral enteric vaccine development.

Role of antigen specific T and B cells in systemic and mucosal immune responses in ETEC and Shigella infections, and their potential to serve as correlates of protection in vaccine development.

The generation of robust systemic and mucosal antibody and cell-mediated immune (CMI) responses that are protective, long-lasting, and can quickly be recalled upon subsequent re-exposure to the cognate antigen is the key to the development of effective vaccine candidates. These responses, whether they represent mechanistic or non-mechanistic immunological correlates of protection, usually entail the activation of T cell memory and effector subsets (T-CMI) and induction of long-lasting memory B cells. However, for ETEC and Shigella, the precise role of these key immune cells in primary and secondary (anamnestic) immune responses remains ill-defined. A workshop to address immune correlates for ETEC and Shigella, in general, and to elucidate the mechanistic role of T-cell subsets and B-cells, both systemically and in the mucosal microenvironment, in the development of durable protective immunity against ETEC and Shigella was held at the recent 2nd Vaccines against Shigella and ETEC (VASE) conference in June 2018. This report is a summary of the presentations and the discussion that ensued at the workshop.

Clinical endpoints for efficacy studies.

Well-established, validated and clinically meaningful primary and secondary endpoints are critical in advancing vaccines through proof of principal studies, licensure and pre-qualification. To that end, the field of vaccine development for Shigella, enterotoxigenic Escherichia coli (ETEC) as well as other enteric pathogens would benefit greatly from a focused review of clinical endpoints and the use of common endpoints across the field to enable study-to-study comparisons as well as comparative assessments between vaccine candidates. A workshop was conducted to review clinical endpoints from controlled human challenge studies, field studies in naïve adult travelers and pediatric studies in low-middle income countries and to develop a consensus on clinical endpoints for future vaccine trials. Following sequential presentations on different study designs (CHIM, travelers' efficacy and pediatric efficacy), workshop participants broke into three simultaneous workgroups focused on those study designs to discuss a number of topics key to clinical endpoints specific to each study design. Previously utilized endpoints were reviewed with an eye towards potentially novel endpoints for future studies and consideration of the disease parameters and spectrum of disease targeted for prevention. The strength of support among workshop participants for the use of various endpoints is summarized as are recommendations for additional endpoints to be considered in future studies. It is anticipated that this report will facilitate endpoint determination in future efficacy trials of vaccine candidates.

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.

How genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions.

Thanks to the modern sequencing era, the extent to which infectious disease imposes selective pressures on the worldwide human population is being revealed. This is aiding our understanding of the underlying immunological and host mechanistic defenses against these pathogens, as well as potentially assisting in the development of vaccines and therapeutics to control them. As a consequence, the workshop "How genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions" at the VASE 2018 meeting, aimed to discuss how genomics and related tools could be used to assist Shigella and ETEC vaccine development. The workshop featured four short presentations which highlighted how genomic applications can be used to assist in the identification of genetic patterns related to the virulence of disease, or host genetic factors that could contribute to immunity or successful vaccine responses. Following the presentations, there was an open debate with workshop attendees to discuss the best ways to utilise such genomic studies, to improve or accelerate the process of both Shigella and ETEC vaccine development. The workshop concluded by making specific recommendations on how genomic research methods could be strengthened and harmonised within the ETEC and Shigella research communities.

The way forward for ETEC controlled human infection models (CHIMs).

In the absence of good animal models, Controlled Human Infection Models (CHIMs) are useful to assess efficacy of new vaccine candidates against Enterotoxic Escherichia coli (ETEC), as well as other preventive or therapeutic interventions. At the 2018 Vaccines Against Shigella and ETEC (VASE) conference, a workshop was held to further review and discuss new challenge model developments and key issues related to further model standardization. During the workshop, invited speakers briefly summarized for attendees recent developments and main agenda issues before workshop participants were divided into four groups for more focused discussions. The main issues discussed were: (1) whether there is a need for more ETEC strains to test a diversity of vaccine candidates, and if so, what criteria/qualities are desirable in strain selection; (2) how ETEC CHIMs could be more standardized to better support ETEC vaccine development; (3) how volunteer selection criteria and screening should be performed, and; (4) how an expanded sample collection schema and collaborative analysis plan may facilitate a more in-depth assessment of the role of antigen-specific humoral and cellular immune responses in ETEC infection, and provide better insights into ETEC pathogenesis and correlates of protection. The workshop concluded that additional challenge strains may need to be developed to better support new vaccines and therapeutics that are advancing in the development pipeline. In this regard, the need for a well characterized ST-only expressing ETEC strain was highlighted as a priority given that promising new heat stable toxoid based vaccine candidates are on the horizon. In addition, further standardization of the ETEC CHIMs was strongly encouraged, noting that it may not be realistic to standardize across all strains. Also, intensified volunteer screening may result in higher attack rates, although more stringent eligibility criteria may contribute to a more limited application of the model and diminish its representativeness. Finally, a sampling schedule and priority list for minimum set of samples was also proposed. Future workshops could be held to further refine standards for ETEC CHIMS and to facilitate more collaborative work on stored sample sets from previous and future ETEC CHIMs to maximize the contribution of these trials to our understanding of ETEC pathogenesis and our development of better prevention and control measures for this important pathogen.

Capturing the true burden of Shigella and ETEC: The way forward.

The mortality and morbidity burden estimation of diarrheal diseases (DD), and Shigella and Enterotoxigenic E. Coli (ETEC) varies among different studies and by the models used for producing these estimates. Understanding the real burden of these important pathogens will guide public health and policy makers to prioritize resources for accelerating interventions against these enteric infections. In addition, long term effects, in the form of growth faltering, cognitive impairment and decreased school performance are important aspects of burden that has not been well captured. Efforts to incorporate these effects and refine their estimation, in the form of Disability Adjusted Life years (DALYs) are very important to inform the burden of diarrheal diseases and Shigella and ETEC specifically. The Institute for Health Metrics and Evaluation (IHME) at the University of Washington conducted a workshop at the VASE 2018 meeting to discuss IHME Global Burden of Diseases (GBD) modelling methods for diarrheal diseases, with a focus on ETEC and Shigella estimates in relation to other pathogens, including limitations, areas of improvements, and IHME plans for future GBD iterations.

A Plant-Produced Candidate Subunit Vaccine Reduces Shedding of Enterohemorrhagic Escherichia coli in Ruminants.

Enterohemorrhagic Escherichia coli (EHEC) are commonly present in the gastrointestinal tract of cattle and cause serious infectious disease in humans. Immunizing cattle against EHEC is a promising strategy to decrease the risk of food contamination; however, veterinary vaccines against EHEC such as Econiche have not been widely adopted by the agricultural industry, and have been discontinued, prompting the need for more cost-effective EHEC vaccines. The objective of this project is to develop a platform to produce plant-made antigens for oral vaccination of ruminants against EHEC. Five recombinant proteins were designed as vaccine candidates and expressed transiently in Nicotiana benthamiana and transplastomically in Nicotiana tabacum. Three of these EHEC proteins, NleA, Stx2b, and a fusion of EspA accumulated when transiently expressed. Transient protein accumulation was the highest when EHEC proteins were fused to an elastin-like polypeptide (ELP) tag. In the transplastomic lines, EspA accumulated up to 479 mg kg-1 in lyophilized leaf material. Sheep that were administered leaf tissue containing recombinant EspA shed less E. coli O157:H7 when challenged, as compared to control animals. These results suggest that plant-made, transgenic EspA has the potential to reduce EHEC shedding in ruminants.

Advances in vaccination against avian pathogenic Escherichia coli respiratory disease: potentials and limitations.

Avian pathogenic Escherichia coli (APEC) is one of the most economically devastating pathogens affecting the poultry industry. This group of extra-intestinal E. coli causes a variety of clinical conditions including airsacculitis and cellulitis. The economic impact of APEC is mainly due to mortality, slower growth rates, and carcass downgrading. In commercial broiler operations, APEC infections are controlled indirectly by vaccination against other respiratory diseases and minimizing stress conditions, and directly by administration of antimicrobial agents to suppress the infection in already infected flocks. The fact that most APEC strains possess some common virulence factors suggests that an effective vaccine against APEC is a viable option. The most important virulence factors that have been investigated over the years include type I and P fimbriae, aerobactin iron-acquisition system, and serum resistance traits. Despite the potential for developing an efficacious vaccine to combat this economically important poultry disease, several obstacles hinder such efforts. Those obstacles include the cost, vaccine delivery method and timing of vaccination as the birds should be immune to APEC by 21 days of age. Herein, we review the various attempts to develop an effective vaccine against the respiratory form of APEC diseases in poultry. We also discuss in-depth the potentials and limitations of such vaccines.

Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond.

In the last few years, plants have become an increasingly attractive platform for recombinant protein production. This builds on two decades of research, starting with transgenic approaches to develop oral vaccines in which antigens or therapeutics can be delivered in processed plant biomass, and progressing to transient expression approaches whereby high yields of purified targets are administered parenterally. The advantages of plant-based expression systems include high scalability, low upstream costs, biocontainment, lack of human or animal pathogens, and ability to produce target proteins with desired structures and biological functions. Using transgenic and transient expression in whole plants or plant cell culture, a variety of recombinant subunit vaccine candidates, therapeutic proteins, including monoclonal antibodies, and dietary proteins have been produced. Some of these products have been tested in early phase clinical trials, and show safety and efficacy. Among those are mucosal vaccines for diarrheal diseases, hepatitis B and rabies; injectable vaccines for non-Hodgkin's lymphoma, H1N1 and H5N1 strains of influenza A virus, and Newcastle disease in poultry; and topical antibodies for the treatment of dental caries and HIV. As lead plant-based products have entered clinical trials, there has been increased emphasis on manufacturing under current Good Manufacturing Practice (cGMP) guidelines, and the preparation and presentation to the relevant government agencies of regulatory packages.

Current status of plant-made vaccines for veterinary purposes.

Interest is growing for the use of plant-made vaccines for veterinary purposes since the regulatory landscape still enables delivery of either crude extracts or minimally processed plant materials to animals for medicinal purposes. In this article, we highlight the current research directions taken with four diseases considered as important constraints to international trade in animals: avian influenza, Newcastle disease, foot-and-mouth disease and diarrheal disease caused by enterotoxigenic Escherichia coli. We also discuss appropriate plant production platforms with regards to plant species and transformation methodologies, possible areas of development, and the remaining challenges for plant-made vaccines for veterinary purposes.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins.

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.

[Construction and immunization of an attenuated vaccine candidate enteropathogenic Escherichia coli O45 ].

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.

Plant-based oral vaccines: results of human trials.

Vaccines consisting of transgenic plant-derived antigens offer a new strategy for development of safe, inexpensive vaccines. The vaccine antigens can be eaten with the edible part of the plant or purified from plant material. In phase 1 clinical studies of prototype potato- and corn-based vaccines, these vaccines have been safe and immunogenic without the need for a buffer or vehicle other than the plant cell. Transgenic plant technology is attractive for vaccine development because these vaccines are needle-less, stable, and easy to administer. This chapter examines some early human studies of oral transgenic plant-derived vaccines against enterotoxigenic Escherichia coli infection, norovirus, and hepatitis B.