Cholera, an acute diarrheal illness, causes 3-5 million cases and over 100,000 deaths worldwide each year, particularly in resource poor settings. Areas of humanitarian crises are often associated with cholera outbreaks due a breakdown in access to clean water. The mainstay of cholera treatment is oral rehydration, a potentially life-saving intervention, while the best long-term prevention strategy is to improve access to clean water and improved sanitation, infrastructure modifications that can take years to implement. To slow the spread of cholera, particularly in the setting of emerging outbreaks, in 2013 the WHO began stockpiling oral killed vaccines for use in reactive vaccination campaigns. This strategy is demonstrating efficacy; however, one of its limitations is that post-vaccination, it takes 2 weeks to develop protective immunity. Thus, interventions that provide protection during this window are needed. Here, as a stopgap measure to address this issue, we propose to develop designer variants of the probiotic Escherichia coli Nissle 1917 (EcN) that we have equipped with a protein delivery system (T3EcN) to secrete relevant therapeutics into the gastrointestinal lumen of at risk individuals. Interestingly, prior infection with cholera provides protection for ~5 years and humans infected with cholera have been found to primarily generate antibodies that target and block the activity of the two cholera toxin subunits (CTA and CTB), sialidase and the O-specific polysaccharide (OSP) moiety of its LPS. These observations strongly suggest that probiotic-mediated deposition of agents that block the activity of these virulence factors, i.e., single domain antibodies (aka VHH), should also act to prevent the development of cholera. Here, in Aim 1, we propose to identify and develop heterodimers of VHH that neutralize OSP, CTB and Sia. These heterodimers will be engineered to be recognized as secreted proteins by T3EcN, a variant of EcN that encodes a modified type III secretion system, which secretes proteins into its surrounding rather than into host cells.
In Aim 2, using the well-established mouse neonatal cholera model, we will investigate the ability of T3EcN that secrete these VHH to block the development of cholera as well as the deposition of cholera into intestinal epithelial cells. These exploratory studies have the potential to lead to the development of a new low-cost intervention to stem the spread of emerging cholera epidemics.
Reactive vaccination is emerging as an effective strategy to stem the spread of cholera epidemics. However, post-vaccination it takes two-weeks for immunized individuals to mount an effective protective immune response. Here, as a stopgap measure to prevent the acquisition of an infection during this period, we propose to develop single domain antibody-secreting designer probiotics that block cholera the activity of essential Vibrio cholerae virulence determinants.
