Awards

Sumiti Vinayak of the University of Georgia in the U.S. will develop a genetic tool to rapidly turn genes off using light in order to study the function of essential genes in the intestinal parasite Cryptosporidium and accelerate drug discovery. Cryptosporidium causes chronic diarrhea and can lead to death in young children. There is currently only one drug available and it is not effective in many patients. New drugs can be developed based on a detailed understanding of the function of essential proteins, however this has been challenging in Cryptosporidium because it is not possible to control when a protein is degraded. They will develop a construct that fuses a protein of interest to a light-inducible domain carrying a hidden degradation signal. When exposed to blue light, this signal is activated, leading to protein degradation at a selected time. They will first optimize their system in vitro using the nanoluciferase gene and C. parvum sporozoites. They will then test it on a candidate essential protein by establishing a stable transgenic parasite cell line to infect mice. Oocysts containing the recombined constructs will then be isolated from the mice and used to infect a human intestinal cell line. After exposing these cells to blue light, they will use microscopy to analyze the effect of degrading the essential protein on the parasite's life cycle.

Gregory Goldgof and Elizabeth Winzler of the University of California, San Diego in the U.S. will use a genetically modified drug-sensitive yeast strain to quickly and inexpensively identify the cellular target of compounds that can kill the parasite Cryptosporidium, which is a major cause of diarrhea-associated deaths of young children in developing countries. Currently, there is only one treatment available and it is of limited use in some of the more severe cases. The yeast strain has been modified to lack transporter proteins that remove toxic compounds from the yeast cells. By treating the modified yeast with a selection of compounds that can kill Cryptosporidium, they hope to drive some of the yeast cells to develop resistance by mutating genes that are responsible for the drugs activity. By sequencing the resistant yeast using whole genome sequencing, they can then discover the likely target of the drug and how it kills the parasite. This would help to develop new drugs that may be more effective.

Pradip Maiti of Immunimed Inc. in Canada will provide passive immunotherapy using chicken-egg-derived polyclonal antibodies against key proteins of the intestinal parasite Cryptosporidium. This orally-administered immunotherapy will prevent the chronic diarrhea and potentially lethal infection caused by this parasite. Treating patients directly with antibodies against a pathogen is quicker than using traditional vaccination methods that induce individuals to make their own antibodies, which takes days to weeks and can also be difficult in malnourished children. This immunotherapy is an alternative to antibiotics, which are becoming less effective. The immunotherapy will be in the form of an egg powder that can be ingested, which is also easier and safer than using injections. The resulting antibodies will be tested for specificity in vitro, and for their ability to treat the disease using a mouse model of Cryptosporidiosis.

Nigel Yarlett of Pace University in the U.S. will determine whether a virus that infects the intestinal parasite Cryptosporidium is a valuable target for developing drugs against the associated chronic diarrheal disease, which causes substantial morbidity and mortality in young children in low-resource settings. The double-stranded RNA Cryspoviruses are not harmful to the parasite, and instead likely enhance the parasite's ability to infect and harm humans. They will create a continuous in vitro culture of the Cryptosporidium parasite carrying viruses that will be genetically modified to fluoresce for easy monitoring, and also use RNA interference to generate a parasite strain that does not contain any viruses. These tools will be used to study the role of the virus in the growth and development of the parasite. Ultimately, a drug targeting the virus, rather than the parasite itself, may be less toxic to the good bacteria in the human gut, and less likely to drive the development of drug resistance.

Honorine Ward of Tufts Medical Center in the U.S. will develop a three-dimensional model of the human intestine for rapid screening of drugs targeting the parasite Cryptosporidium, which causes potentially lethal diarrhea in young children in developing countries. Developing drugs against Cryptosporidium has been particularly difficult, partly because of the limited understanding of the parasites behavior in the human intestine, and particularly of the effect of malnutrition, which commonly co-occurs with infection and likely contributes to disease severity. They will build a three-dimensional model of the human intestine using a scaffold of silk proteins and a hollow lumen structure lined with cells derived from human intestinal stem cells supported by underlying human myelofibroblasts. They will infect their cell model with fluorescently labelled Cryptosporidium to evaluate how the parasite affects the intestine, and to determine its capacity for high-throughput drug screens.

Boris Striepen of the University of Georgia in the U.S. will develop a new, more natural mouse model for cryptosporidiosis, which is a leading cause of severe diarrhea in children, to help identify effective treatments. Unlike previous mouse models of this disease, these mice do not need to be immune deficient as they can be infected by a natural strain of the Cryptosporidium parasite, which they previous isolated from house mice. They will genetically modify this strain so it will fluoresce and can thus be easily located in the mice and within individual cells. These mice also experience symptoms more similar to the human disease, and they will use it to assess the effects of malnutrition, which often co-occurs with infection and appears to worsen symptoms. They will also study the effect of different bacterial communities in the gut on disease progression and the effects of existing and emerging treatments.

Anastasios Tsaousis of the University of Kent in the United Kingdom will build a screening platform to identify drugs that can be used to treat diarrhea caused by the parasite Cryptosporidium, which is the second major cause of death in children under five years old in developing countries. There are currently no effective drugs for treating Cryptosporidium, largely because it cannot easily be grown in the laboratory making it difficult to study and test for new drugs. They have developed a two-dimensional cell culture system using a specific cell type that can be stably infected with Cryptosporidium and cultivated long term. They will use the CRISPR/Cas9 gene modification technique to alter selected Cryptosporidium genes for monitoring parasite growth, and use it in their cell culture system to screen a library of FDA-approved drugs to identify candidate drugs that block Cryptosporidium growth.

Jan Mead of Emory University in the U.S. will develop a mouse model of cryptosporidiosis using human fecal transplants to mimic changes in the bacterial populations (microbiome) in the gut that occur in the human disease, which causes substantial morbidity and mortality in young children from developing countries. Drugs used to eradicate the intestinal parasite Cryptosporidium are thought to be affected by the levels and types of bacteria that populate the human gut, which is of particular importance in malnourished children who most often become infected. They will colonize germ-free mice with human fecal material and infect them with Cryptosporidium. The effect of the infection and of selected drugs on the microbiome will then be evaluated by DNA sequencing.

Samuel Arnold of the University of Washington in the U.S. will develop methods to evaluate drug candidates for treating Cryptosporidium infections, which cause severe diarrhea particularly in young children from developing countries. There are no effective drugs against the Cryptosporidium parasite. This is partly because when it infects humans it becomes isolated in specific cells lining the gastrointestinal tract, which is where a drug would also need to be located at sufficient concentrations to be effective. However, the standard in vitro models are unable to easily and rapidly predict the efficacy of multiple drug candidates at this specific location. To address this, they will build a cell model of the intestinal wall that can also be infected with Cryptosporidium, and measure the permeability of a panel of protein kinase inhibitors that they have developed to kill the parasites. They will also use the software platform Gastroplus that can simulate the behavior of a drug once it has been administered and predict concentrations at multiple sites in the gastrointestinal tract. They will evaluate their method for predicting drug activity in vivo by testing the drugs in a neonatal mouse model of Cryptosporidium infection.

William Witola of the University of Illinois in the U.S. will help develop new drugs for treating children infected with the protozoa Cryptosporidium by using a gene knockdown approach to evaluate candidate drug targets. Found in contaminated water, Cryptosporidium is the second most common cause of potentially lethal diarrhea in young children in developing countries. There are no safe and effective drugs available due largely to the lack of genetic tools for studying Cryptosporidium in the laboratory. Phosphorodiamidate morpholino oligomers (PPMOs) are small molecules that can be designed to bind and silence specific genes also in protozoa. If these genes encode for proteins critical for vital cellular functions, the PPMOs can cause death. To evaluate his approach, he will design PPMOs and test their ability to silence essential Cryptosporidium genes and thereby block chronic infection in mice.