Progress 09/01/20 to 08/31/23

Outputs
Target Audience:During the project, we engaged with the chief veterinarians of major chicken producers, including Tyson Foods, Wayne-Sanderson, Pilgrim's Pride, Perdue, Fieldale, and Amick farms. We discussed our product GP1191 and its value propositions and agreed to test it in field trials. Tyson, Perdue and Amick farms tested our product in their farms. We demonstrated a dual value proposition: lower mortality by up to 70% due to necrotic enteritis in birds treated with GP1191, and a higher performance with a 6-point lower feed conversion ratio in treated vs. untreated birds. In order to test GP1191 in the field we secured a Food Use Authorization granted by the Center for Veterinary Medicine at the FDA. We opened an Investigational New Animal Drug file and presented data that supported our claim that the risk of recombinant live therapeutics to the public health is low. The FDA granted the FUA to test GP1191 in up to 3 million birds. In order to make material for tests in up to 3 million birds, we optimized manufacturing and scaled it up to thousands of liters of fermentation at the University of Minnesota's Biotechnology Resource Center and at Iowa State University's Center for Carbon Utilization Research. In our development efforts we also engaged with scientists at Elanco, Zoetis, Merck Animal Health, DSM and Cargill. There is continued interest in the progress we are making in the development of recombinant live therapeutics for prevention and control of necrotic enteritis in poultry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the project we answered the major questions needed for registration of GP1191, the final engineered product version: a) Is the LBP safe for poultry? GP1191 is safe for poultry. Tests in over 1 million birds in multiple farms across the US demonstrated a strong safety profile when GP1191 is used according to instructions. GP1191 is manufactured in powder form, which is mixed in the water supply of birds from day 7 until day 28. The daily dose per bird is 10^9 colony forming units. b) Is the LBP safe for producers and consumers? GP1191 is safe for producers and consumers. We presented data to the FDA's Center for Veterinary Medicines demonstrating that GP1191 is non-pathogenic, non-virulent strain of E. coli, which is engineered to express and secrete enterocin A, an antimicrobial peptide with strong activity against Clostridia perfringens. Based on the presented evidence, General Probiotics concludes that consumption of food derived from animals treated at the maximum levels with the minimum withdrawal periods will be consistent with the public health. The estimation is the result of integrating the following assessments: 1. Release assessment: the probability that GP1191 or enterocin A are present in poultry as a consequence of use is low. 2. Exposure assessment: the probability for humans to ingest GP1191 or enterocin A from food derived from chicken broilers is low. 3. Consequence assessment: the probability that human exposure to GP1191 or enterocin A results in an adverse health consequence is low. 4. Microbial resistance assessment: assessed the human food safety with respect to the potential microbiological effects of enterocin A on food-borne bacteria of human health concern. This overall risk is estimated to be low. Ultimately, the FDA concurred with our assessment and granted General Probiotics a Food Use Authorization. c) Is the LBP safe for the environment? GP1191 is safe for the environment. The FDA also granted General Probiotics a Categorical Exclusion claim, which means there is no need for an extended environmental assessment study for GP1191. Based on presented evidence, General Probiotics concludes that no extraordinary circumstances exist as described under 21 CFR 25.21 for the investigational use of a genetically modified Escherichia coli-secreting enterocin A. GP1191 is a benign, non-pathogenic, non-virulent natural isolate, without any extraordinary growth potential. The antimicrobial activity is conferred by a plasmid that encodes for the expression and secretion of enterocin A, a small protein that is generally harmless and degrades rapidly in any environment containing proteases. The sponsor therefore considers the environmental risk of accumulation of GP1191 and enterocin A to be low. 1. Less than 1% of the organisms administered to chickens were found cumulatively in the gut contents of birds, on the surface of birds, or in/on the floor litter. 2. There was steady decline in GP1191 over time under all conditions tested. 3. The half life of enterocin A ranges from a few hours in soil and litter samples to a few seconds inside the GI tract of chickens. d) Is the LBP effective in preventing and/or controlling necrotic enteritis in poultry? GP1191 is effective in preventing necrotic enteritis in poultry. In CRO studies, administration of GP1191 resulted in up to 70% reduction of NE in treated birds compared to untreated ones. e) Can the LPB be manufactured and formulated in a consistent, and cost-effective way, with adequate shelf-life? GP1191 can be manufactured and formulated in a consistent, and cost-effective way, with adequate shelf-life. We now have robust cell banks in-house and we have deposited GP1191 with ATCC. We have developed and validated the analytical tests to verify the quality, purity, strength and stability of GP1191 during the manufacturing process. The media are optimized for batch fermentation processes. We finalized fed-batch tests in-house to determine optimal media use for maximizing cell densities. We have optimized the conditions for batch and fed-batch fermentation. We moved from flasks to 1-liter and 5-liter bioreactors in-house. We conducted experiments at the 10-liter scale at the Center for Carbon Utilization at Iowa State. From early January 2023 to July 2023, we completed three 1,000-liter fermentation batches at CCUR. These runs resulted in very high cell densities. We are working with the Bioresource Center at the University of Minnesota to optimize fed-batch processes at large scale. From January 2023 to Jul 2023, BRC conducted four 300-liter fed-batch runs. We purchased a lab-scale spray dryer in-house and tested hundreds of final formulations of dry GP1191. We worked with GEA Inc., Polar Dry Inc. and Artisan Inc. to optimize formulations for scaled up drying. We determined the best formulation for drying GP1191 at CCUR. The staff at CCUR committed to the success of our project, worked through weekends optimizing the parameters of their equipment, and produced beautiful powders with high densities of viable cells.

Publications

Progress 09/01/21 to 08/31/22

Outputs
Target Audience:During this past period, we have engaged with scientists at Elanco, Zoetis, Merck Animal Health, DSM and Cargill. There is continued interest in the progress we are making in the development of recombinant live therapeutics for prevention and control of necrotic enteritis in poultry. We have also interacted with the Center for Veterinary Medicine at the FDA. We have presented data that support our claim that the risk of recombinant live therapeutics to the environment is low. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Please see final report.

Publications

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:During this past period, we have engaged with scientists at Elanco, Zoetis, Merck Animal Health, DSM and Cargill. There is continued interest in the progress we are making in the development of recombinant live therapeutics for prevention and control of necrotic enteritis in poultry. We have also interacted with the Center for Veterinary Medicine at the FDA. We have presented data that support our claim that the risk of recombinant live therapeutics to the environment is low. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We continue developing recombinant live therapeutics. A next big step will involve animal tests. We will work with our collaborators to design studies for demonstrating the effectiveness and safety profile in poultry challenged with Clostridia perfringens.

Impacts
What was accomplished under these goals? In the past few months, we have made significant progress in the maturation of our existing E. coli project targeting C. perf. Additionally, we have achieved major breakthroughs in our development of a new Bacillus-based asset. We continue to work towards the project goals set forth in the previous interim report. Plasmid stabilization with no antibiotic resistance marker Our genetic constructs required for the production of enterocin A are currently located on plasmids which are transformed into the bacterial chassis of choice. Typically, plasmids contain an antibiotic resistance marker. Bacteria are grown in the presence of antibiotics such that the cells are forced to maintain the plasmid over time. However, the presence of an antibiotic resistance gene is unacceptable in our probiotics because of the risk of resistance transfer to other bacteria that they may encounter in the birds or in the farm environment. As mentioned above, we had previously implemented a method to enable plasmid stabilization without requiring an antibiotic resistance marker. We successfully developed two auxotrophic versions of the probiotic. In one of these strains, we knocked out the dapA gene from the chromosome and complemented it on the plasmid. In the other, we knocked out the thyA gene and complemented it on the plasmid. The dapA and thyA versions are referred to as GP1191 and GP1189 respectively. We tested both GP1191 and GP1189 in a large-scale field trial with Elanco the at industry recognized CRO, Blue River (Carthage, IN). The study included eight treatment groups. For the study, birds in groups 5-8 were administered ~1010 CFUs per bird per day for 42 days. Note: CFU is a colony forming unit and can generally be considered one bacterial cell. The study thus required ~2.5x1014 CFU of both GP1189 and GP1191. This amounted to a requirement of over 700 liters of bacterial culture that was carefully prepared and tested in our lab. The study itself was funded by Elanco, however, probiotic production was made possible by support from this NSF Phase II SBIR grant. With the NSF's support, we optimized culture and harvest conditions. Without these efforts, preparation of sufficient, high quality test material would not have been possible. Two different challenge levels of C. perf were tested. One in which birds were inoculated with 105 CFU C. perf per bird and one in which they were inoculated with 106 CFU C. perf per bird. For both challenge models, GP1191 significantly reduced mortality in birds compared to the untreated control (Cp) (51% reduction in mortality in 106 model). Interestingly, there was no significant difference between birds treated with traditional antibiotics and birds treated with GP1191. Impact of GP1191(GP1) and GP1189 (GP2) on mortality in two C. perfringens infection models. GP1191 reduced mortality by 51% compared to the untreated control group. The positive results from these trials have catalyzed further commercialization steps of GP1191. In response to these studies, we initiated discussions with the FDA Center for Veterinary Medicine to better define the subsequent steps in the regulatory pathway. Accumulation of GP1191 in Chicken GI tracts and Poultry Pen Environments Data for these studies was collected from the field trial discussed above performed at Blue River. Sample processing and analysis was supported by this NSF Phase II SBIR. To evaluate the accumulation of GP1191 in the poultry pen, we consider an isolated system that consists of a pen in a poultry farm, the litter on the floor, and the birds in the pen. We assume N=22 birds, but this number can be chosen arbitrarily. Isolated means that the modified E. coli can only enter the system in the water administered to birds. We assume that modified E. coli cannot escape the pen. We know how much E. coli is administered daily to each bird on average. We can count E. coli carriage inside bird organs and on the surface of birds. We can also count E. coli in the pen floor litter. We can then calculate a number balance of E. coli cells over a certain period of time. For the calculation herein we choose 42 days. We administered ~1010 CFU modified E. coli per bird per day in the water for 42 days. A balance equation for the overall counts of modified E. coli over 42 days is: Accumulation= Input + Generation - Output We assume Output = 0 since the pen is considered isolated. We calculate Input = (1010 CFU modified E. coli per bird per day) x (22 birds) x (42 days) =~ (1013 CFU modified E. coli). To calculate the Accumulation term, we add the following: total counts of E. coli found in the litter of the pen on day 42, total counts of E. coli inside birds on day 42, total counts of E. coli on the surface of the birds on day 42. For a) we take twelve independent samples of pen litter (four samples from three pens), count CFUs per gram of litter, average the CFUs and multiply by the total weight of pen litter measured on day 42. For b) we collect the entire intestinal tract (from the duodenal loop to the ceca) from 9 birds on day 42 (three birds from three pens), measure CFUs per gram of intestine, average over 9 birds and then multiply by the total number of birds in the pen. For c) we bathe five birds separately in distilled water, collect the water and count E. coli cells on the surface of each birds. We then average the counts per bird over five birds and multiply be the number of birds in the pen. The results yield Accumulation = 1010.78 CFUs. According to the balance equation, inserting the known values for Input, Output and Accumulation, we calculate Generation = 1010.78 - 1013 CFUs < 0. This fraction is (1010.78 / 1013) = 10-2.22. In other words, less than 1% of all E. coli survives when administered in birds in a farm environment. This result and the negative generation term clearly demonstrate that there is a natural decay in GP1191 in pen environments. The risk of uncontrolled proliferation thus appears minimal. Development of antimicrobial probiotics based on Bacillus subtilis We have made progress in our development of Bacillus-based probiotics targeting C. perf. Over the past six months, we have accomplished the following: Identify protease activity as a bottleneck in peptide production Screen and establish a library of Bacillus chassis candidates Engineer a library of enterocin A constructs for use in various Bacillus isolates Bacillus subtilis is a generally regarded as safe (GRAS) organism that is widely used both as a probiotic as well as for the production of numerous industrial enzymes. We have sought to develop a Bacillus-based probiotic because of their ability to form heat-stable spores. This would enable us to incorporate Bacillus into the feed of birds which undergoes high-temperature processing. The improved shelf-life and logistical benefits of using a spore-former like Bacillus have been expressed on numerous occasions by multiple poultry producers.

Publications