Development of a Live, Genetically attenuated Cholera Vaccine Prototype that Could

Cholera is an acute, water-borne diarrheal disease caused by the facultative Gram-negative bacterium, Vibrio cholerae of serogroup O1 of the classical and El Tor biotypes and by V. cholerae serogroup O139. The magnitude, severity and duration of recent outbreaks in Angola, Zimbabwe, Vietnam, and Haiti, have strengthened a recommendation by the World Health Organization for the preventive use of oral cholera vaccines to avert outbreaks and control their spread. Several live genetically attenuated cholera vaccine strains such as Peru-15 and V. cholerae 638 have been demonstrated to be clinically safe, immunogenic and protective in phase I and II clinical trials. However, the efficacy of current vaccines in an outbreak scenario has not been tested and could be challenged by the fact that their infectivity and potential for transmission to households and within the community are inferior to that of epidemic strains. Recent studies have shown that V. cholerae cells present in fresh cholera stools or within biofilm aggregates display a hyperinfective phenotype. The discovery of a V. cholerae hyperinfective physiological stage has led to a new epidemiological model for cholera that incorporates human-to-human transmission. <p/> We propose to develop a live, genetically-attenuated vaccine prototype based on strain 638 that could be administered as a hyperinfective biofilm to mimic cholera transmission in outbreaks, enhance immunogenicity, and promote herd immunity. We will first modify strain 638 by deleting the cholera phage attachment site to increase its safety without affecting its colonization capacity. <p/>In Aim 1, we wil introduce additional mutations in this strain to enhance its infectivity, intestinal colonization, nd biofilm formation. Then, we will conduct competition assays in vivo to determine the capacity of planktonic and biofilm-derived cells of the modified vaccine to outcompete strain 638 for colonization of the suckling mouse small intestine. In Aim 2, we will use an oral (intragastric) adult rabbit immunization model to compare the immunogenicity and protective capacity of planktonic and biofilm-derived hyperinfective vaccine candidates to that of the classical vaccine strain 638.