Project Summary Cholera is an acute and severe disease caused by the bacterium Vibrio cholerae that is spread primarily through contaminated water sources due to a lack of adequate sanitation infrastructure. The World Health Organization estimates that there are at least 3 million cases globally per year, 40 percent of which are spread through household transmission. Current prevention methods, such as the oral cholera vaccine (OCV) and water, sanitation, and hygiene (WASH) campaigns, require significant investment of resources and time for efficacy, but household contacts of cholera patients often present with cholera symptoms two to three days after the initial patient becomes sick. In addition, the preventive use of antibiotics is not recommended due to widespread resistance and the known negative consequences of dysbiosis. There is a pressing need to develop a targeted clinical intervention to prevent the community spread of cholera using a rapid prophylactic treatment. PhagePro aims to fill this gap with its first product ProphaLytic-VcTM (PVC). PVC is an orally administered bacteriophage (phage) cocktail comprised of Vibriophages ICP1, ICP2, and ICP3. In this Phase II SBIR proposal, we aim to further develop PVC for deployment in real-world settings as part of a cholera toolkit that includes WASH and OCV campaigns. First, we aim to test co-administration of PVC with the OCV, particularly as the WHO Global Task Force on Cholera Control (GTFCC) recommends that National Cholera Control Plans (NCCP) move towards integration of services to reduce logistical burden and costs. In addition, the majority of interviewed stakeholders stated that a holistic approach is critical to cholera control. Second, we have identified that cold chain dependence is a major contributor to OCV campaign costs. Therefore, we aim to build upon our Phase I STTR success to develop PVC in a solid dosage formulation. This will increase the stability of PVC in hot and humid environments, enabling Ministries of Health to stockpile PVC in-country and distribute to identified cholera hotspots without the use of the cold chain. This mechanism will increase timeliness of the cholera outbreak response, which has been identified as the key factor in controlling an outbreak in both endemic and non-endemic settings. Third, we aim to optimize phage ratios in PVC to most effectively kill circulating strains of V. cholerae. We will collect 96 clinical isolates from recent epidemics in South Asia and Africa in the past 3 years to determine host range coverage. Additionally, we will optimize the ratio of phages on 2 representative clinical isolates to best reduce V. cholerae colonization in mice. Lastly, we will establish a post- exposure, pre-symptomatic rabbit model to better simulate clinical settings for the HCs during the high-risk period and demonstrate that PVC can effectively prevent the onset of symptoms. At the successful conclusion of Phase II, we will have a solid dosage f...