Program Director/Principal Investigator (Last, First, Middle): Barry, Eileen M. Project Summary Shigella and enterotoxigenic Escherichia coli (ETEC) are two of the most important diarrheal pathogens worldwide, causing significant disease in young children in developing countries and in U.S. travelers, including military personnel, who visit developing countries. Both pathogens have been designated by WHO, CDC and NIAID as priorities for the development of new interventions due to the significant disease burden of these increasingly antimicrobial resistant organisms. A combined vaccine provides additive value by targeting two important enteropathogens with a single formulation. We have developed 2 lead prototype Shigella-ETEC vaccine candidates that include the most important components of a broadly protective vaccine. CVD 1208S- 321 consists of an attenuated Shigella flexneri 2a strain engineered to express ETEC colonization factor CFA/I and the LTA2 and B subunits of heat-labile enterotoxin from genes integrated into the Shigella chromosome. CVD 1233-SP::CS2-CS-V2 consists of attenuated S. sonnei engineered with a novel stabilized virulence plasmid modification that provides a transformative advantage for stable and consistent vaccine production and immunogenicity. This strain is engineered for optimal expression of ETEC antigens CS2 and CS3 from the chromosome. Animal models confirmed the induction of protective immune responses by both vaccine strains against these two important human diarrheal pathogens. The overall goal of this proposal is to translate these prototype Shigella-ETEC vaccine constructs from academic research laboratory vaccine candidates to potential human vaccines ready to enter Phase 1 clinical trials (under other funding). Several industrial partners have been engaged to supply the required expertise to assure success of this process. The Walter Reed Army institute of Research Pilot Bioproduction Facility (WRAIR PBF), a world expert in Shigella vaccine production, will produce cGMP cell banks and pilot vaccine lots with documentation required for IND submission. CVD will perform pre- clinical IND-enabling studies with supporting documentation sufficient for vaccine advancement. Experts from Biologics Consulting will oversee all steps in the development and documentation process to assure compliance with requirements for FDA INDs. Vaccine Company Inc., a newly formed company that is committed to the rapid development and deployment of vaccines for global use, will provide consultation and overall strategic guidance for the ultimate expedient deployment of these vaccines. The collective expertise of the assembled team will ensure expeditious development of Shigella-ETEC vaccine candidates with requisite FDA conformity for rapid advancement to clinical trials and deployment to target populations. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page

Program Director/Principal Investigator (Last, First, Middle): Barry, Eileen M. Project Narrative: This project proposes to perform cGMP production, and IND-enabling pre-clinical studies of two lead live, attenuated Shigella-ETEC vaccine candidates that constitute essential components of a broadly protective vaccine against two priority enteropathogens. This work is required for advancement to clinical trials and eventual deployment to multiple at-risk populations in urgent need of novel interventions. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page