3. Laboratory of Enteric and Sexually Transmitted Diseases Historical Perspective Origin: Began in 1994 - Established to review the rapidly increasing number of enteric disease products and expected STD products. Origin: Began in 1994 - Established to review the rapidly increasing number of enteric disease products and expected STD products. Mission: Mission: a. Conduct basic/applied research molecular bases of pathogenesis molecular bases of pathogenesis host’s immune responses to infection host’s immune responses to infection develop models to measure vaccine safety, immunogenicity and efficacy develop models to measure vaccine safety, immunogenicity and efficacy b. Utilize knowledge base enhance product manufacturing analyses enhance product manufacturing analyses better assess product safety and efficacy better assess product safety and efficacy Program Review: Program Review: Last scientific program site-visit review - Aug. 2002

4. Laboratory of Enteric and Sexually Transmitted Diseases Regulatory Responsibilities Responsible for review of IND’s and BLA’s for bacterial enteric, UTI and STD products and certain other assigned products. Responsible for review of IND’s and BLA’s for bacterial enteric, UTI and STD products and certain other assigned products. LESTD is responsible for covering products related to intestinal disease agents (e.g. Shigella, Salmonella spp., all pathogenic Escherichia coli’s, Campylobacter, Vibrio cholerae, Helicobacter pylori, hookworm), the use of live attenuated enteric bacteria as vaccine vectors or as anti-cancer therapy, UTI pathogens, probiotic/LBP therapy (e.g. Tx cancers, IBD, CF), L- aspariginase, bacteriophages or bovine/chicken immunoglobulin concentrates for therapeutic use, and genetic hybrid plant vaccines. [ Involves oral, parenteral, intrarectal, intravaginal, intranasal, transcutaneous routes + new adjuvants]. LESTD is responsible for covering products related to intestinal disease agents (e.g. Shigella, Salmonella spp., all pathogenic Escherichia coli’s, Campylobacter, Vibrio cholerae, Helicobacter pylori, hookworm), the use of live attenuated enteric bacteria as vaccine vectors or as anti-cancer therapy, UTI pathogens, probiotic/LBP therapy (e.g. Tx cancers, IBD, CF), L- aspariginase, bacteriophages or bovine/chicken immunoglobulin concentrates for therapeutic use, and genetic hybrid plant vaccines. [ Involves oral, parenteral, intrarectal, intravaginal, intranasal, transcutaneous routes + new adjuvants]. Annual workload - Annual workload - New candidate products = >1/month Review time commitment: Stibitz and Kopecko - ~50% review effort Carr and Osorio - 25-30% review effort Bhattacharyya - ~ 80% review effort

5. Personnel and Budget - (2003-2006) Laboratory of Enteric and Sexually Transmitted Diseases Personnel and Budget - (2003-2006) Personnel Personnel - Molec. Pathogenesis Gp. - increased from 2 to 7 Gene Regulation Gp. - declined from 5 to 3 STD Gp. - inactivated due to lack of STD vaccine activity and departure of C. Deal. Immune Mechanisms Gp. - created in 2002, new research initiatives being developed Budget Budget - FDA intramural research budget continued its decrease. Fortunately, outside grant support replaced the intramural budget decline. Problem: lack of internal funding consistency, outside grantors limited in number (e.g. special NIH funding).

6. Public Health Issue: Public Health Issue: Enteric bacterial diseases cause ~350 million US episodes of diarrhea/yr Enteric bacterial diseases cause ~350 million US episodes of diarrhea/yr kill more than 2 million children worldwide annually kill more than 2 million children worldwide annually Limited data on pathogenesis and immune responses has slowed the development of enteric vaccine products Limited data on pathogenesis and immune responses has slowed the development of enteric vaccine products Research Relevance to DHHS and FDA Priorities: LESTD research programs are entitled: Research Relevance to DHHS and FDA Priorities: LESTD research programs are entitled: “ Enteric Bacterial Pathogens – Improving Safety and Efficacy of Combination Vaccines for Diarrheal Diseases and Select Agents” “ Enteric Bacterial Pathogens – Improving Safety and Efficacy of Combination Vaccines for Diarrheal Diseases and Select Agents” “Bacterial Vaccine Safety: Biomarkers of Virulence and Attenuation in Bordetella pertussis (Whooping Cough) and Anthrax Bacteria.” “Bacterial Vaccine Safety: Biomarkers of Virulence and Attenuation in Bordetella pertussis (Whooping Cough) and Anthrax Bacteria.” Laboratory of Enteric and Sexually Transmitted Diseases Relevance of Research Program To CBER’s Public Health Mission

7. Laboratory of Enteric and Sexually Transmitted Diseases Research Programs - 1 Molecular Pathogenesis Section - Molecular Pathogenesis Section - I.Studies of Salmonella Typhi Ty21a as a safe vector for expression of foreign antigens II.Pathogenesis and immune responses to Campylobacter jejuni infection in humans III.Minor research projects - aided by key collaborations.

8. I. Studies of Salmonella Typhi Ty21a as a safe vector for expression of foreign antigens Key Goals Define key attenuating features of Ty21a Define key attenuating features of Ty21a (Genomic sequence and microarray analyses) (Genomic sequence and microarray analyses) Optimizing a temperature-stabilized formulation and delivery system Optimizing a temperature-stabilized formulation and delivery system (NIAID-supported collaboration with Aridis Pharmaceuticals LLD) Demonstrate stable expression of targeted Shigella or anthrax genes in a live platform vector system Demonstrate stable expression of targeted Shigella or anthrax genes in a live platform vector system

9. 2. Pathogenesis and immune responses to Campylobacter jejuni infection in humans Overall goals of project: A. Examine C. jejuni attachment, invasion, and exocytosis events by TEM and SEM. B. Define host signal transduction pathways involved in C. jejuni internalization into epithelial cells C. Study C. jejuni interaction with human DCs for cytokine/chemokine responses involved in inflammation

10. Laboratory of Enteric and Sexually Transmitted Diseases Research Programs - 2 Immune Mechanisms Section Immune Mechanisms Section New Directions – Evaluate approaches to achieving mucosal immunization

11. Immune Mechanisms Section Mucosal Immunization against Enteric Pathogens 1.Study methods useful for inactivation of enteric bacteria 2.Demonstrate immunogenicity of antigen delivery systems a.Transcutaneous immunization b.Bacterial ghosts c.Mucosally delivered whole cells 3.Develop Animal Models for Evaluating Vaccine Efficacy Against Enteric Pathogens

12. Laboratory of Enteric and Sexually Transmitted Diseases Research Programs - 3 Gene Regulation Section - Gene Regulation Section - I. Virulence Gene Regulation - using B. pertussis as model system. II. Development of Genetic Tools for the Analysis and Manipulation of Bacillus anthracis.

13. I. Virulence Gene Regulation - using B. pertussis as model system A. Molecular mechanisms of virulence gene activation by the BvgAS two-component system Progress: Completed a study of eight different BvgA- regulated virulence gene promoters for high-resolution mapping of BvgA-binding sites using BvgA conjugated to Fe-BABE. Future Directions: More detailed studies of specific promoters and comprehensive genetic studies to elucidate critical interactions of BvgA with RNA polymerase.

14. I. Virulence Gene Regulation - using B. pertussis as model system B. Development of Genetic Tools Progress: Have constructed a powerful allelic exchange system for manipulation of unmarked B. pertussis strains (for use in animal studies)

15. I. Virulence Gene Regulation - using B. pertussis as model system C.Use of bioluminescence to characterize B. pertussis infection in a mouse aerosol challenge model. Progress: Have demonstrated utility of in vivo imaging system for examining pertussis infection - can follow individual mice over time. Characteristic growth in upper respiratory (nose) tract followed by growth in lungs and trachea. Future Directions: Implementation to characterize the effect of mutations that affect key/novel virulence genes and regulatory phenotypes.

16. 2. Development of Genetic Tools for the Analysis and Manipulation of Bacillus anthracis 1.Developed routine allelic-exchange procedures 2.Constructed over 70 directed mutants of B. anthracis. Future Directions: 1.Development of additional tools: expression vectors, promoter assay vectors, transposon delivery vectors. 2.Application of tools in a genomic search for new virulence genes ( funded by MARCE developmental grant ) 3.Characterization of underlying causes for sub-optimal stability of rPA anthrax vaccine (funded by CBER/NIAID partnership)