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GLOBVAC-Global helse- og vaksin.forskn

Development of a vaccine for enterotoxigenic E. coli based on the heat-stable toxin

Alternative title: Utvikling av en vaksine for enterotoksigene E. coli basert på varmestabilt toksin

Awarded: NOK 24.6 mill.

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Project Period:

2014 - 2020


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Diarrhea is one of the main causes of ill health, malnutrition, and death among young children living in low- and middle-income countries. One of the most important causes of diarrhea among these children is infection with enterotoxigenic E. coli (ETEC) strains that secrete the heat-stable toxin, ST. Despite over 40 years of substantial efforts to develop vaccines against ETEC, no effective vaccines have so far been developed. The main goal of our ETECvac project has been to develop a new vaccine that could induce protection against the toxic activities of ST itself, and we have now developed a vaccine candidate that is ready for testing on humans. During an ETEC infection, secreted ST molecules bind to and activate the guanylate cyclase C receptors on the cell surfaces of the small intestinal wall. The activity of these receptors is normally regulated by the body's own peptide hormones, but ST overstimulates the receptors, triggering diarrhea. Developing ST-based vaccines has in the past been hindered by the facts that ST is a small peptide that does not induce an immune response by itself, it has a complex structure, and it is very toxic. Replacing amino acids in the ST molecule to make it less toxic often changes its shape, which makes it unsuitable for use in vaccines. In addition, ST is structurally similar to the above-mentioned hormones, complicating the process of developing vaccines that only target ST, and not also the hormones. To overcome these challenges, in ETECvac, we identified variants of ST that are 1) non-toxic, 2) still structurally similar to native ST, and 3) less similar to the body's own hormones. By coupling these variants to larger protein carriers and immunizing mice, we were able to induce antibody responses that hindered ST-binding to the receptor but that didn't interfere with the activities of the peptide hormones. The choice of carrier proteins had a large impact on the quality and strength of the immune response. We have obtained patent protection for these ST variants and their use in vaccines, and the next step will be to examine if the vaccine candidate is safe for use in humans and to test that it induces protection against ETEC diarrhea. For this purpose, we also undertook experimental infection studies where adult human volunteers were infected with different wild-type ETEC strains. The purpose of these experiments was to identify a suitable ETEC strain and a dose that will give volunteers diarrhea. When testing our vaccine candidate, we can use this setup to check whether volunteers who are vaccinated with our ST-based vaccine candidate are protected against diarrhea during infection with the selected ETEC strain. ETECvac has been a collaboration between the University of Bergen, NORCE, and Haukeland University Hospital in Bergen, Norway, Institut Pasteur in Paris, France, and the Indian Institute of Science in Bangalore, India.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea both among young children living in low- and middle-income countries as well as among travelers to these countries. In ETECvac, we have completed the pre-clinical development of a vaccine candidate aimed at protecting humans against the most severe forms of ETEC diarrhea. We have, furthermore, established the systems needed to evaluate this candidate in clinical trials. If shown to be efficacious in clinical trials, either alone or in combination with antigens that target other pathogens, such as rotavirus, the vaccine could contribute substantially to reducing the diarrheal burden of humans living in and traveling to areas where ETEC infects endemically.

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable enterotoxin (ST) is among the most important causes of moderate to severe diarrhea, causing ill health, malnutrition and death in children less than 5 years of age in low- and middle-incom e countries. Current available prototype ETEC vaccines target colonization factors and the heat-labile toxin, but not ST. They have so far failed to confer adequate protection, and development of an ST toxoid-based vaccine has a high priority. An ST-base d vaccine has remained elusive despite more than three decades of research. The challenge is to make ST non-toxic and immunogenic in a way that engenders a response with antibodies that neutralize native ST. Adding to that challenge is the fact that ST sh ares epitopes with uroguanylin, an endogenous ST-like peptide that activates the same receptor as ST in the gut. This project proposes an extensive structural vaccinology approach for designing an ST toxoid-based vaccine. With funding from GLOBVAC we have , in the EntVac-project, identified and ranked the best detoxifying ST mutations by screening a library of all 361 possible single-amino acid ST mutants. The top candidates were recently included in a patent application. We will chemically synthesize a se lection of the most promising ST mutants, conjugate them to the B subunit of the heat-labile toxin, and conduct immunization experiments in rabbits and rats. To avoid engendering an immune response with antibodies that cross-react with uroguanylin, we wil l generate a panel of neutralizing monoclonal antibodies and map the epitopes that are unique to ST. The information on desired epitopes will be used to select optimal conjugation chemistry and mutations to generate a vaccine prototype, ready for toxicity testing with already secured funding. In addition to developing an ST toxoid-based vaccine, we will develop the challenge model for initial evaluation of the efficacy of these vaccine candidates in clinical trials.

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GLOBVAC-Global helse- og vaksin.forskn