1. Use of Lactic acid Bacteria as Antigen Delivery Vehicle Kim, Tae-Woon Lab. of Food Biochemistry

2. Development of mucosal vaccines based on lactic acid bacteria Mucosal surface – the first line of defense Macdonald et al. (2005) Mucosal vaccine is administrated orally and induces mucosal immune responses to prevent infectious diseases

3. Advantages of a Mucosal Vaccine Conventional vaccineMucosal vaccine Administrated by needle Require professional skill Risk of cross-contamination Defense after the invasion Induce systemic immunity Administrated orally Easy and No stress Defense both at the front of invasion and after the invasion Induce both systemic and mucosal immunity

4. Types of Mucosal Vaccines Pathogen-based Attenuated pathogen Antigen-capsulated ISCOM (immune stimulating complex) Liposome Chitosan microparticles ADA (antigen delivery agent) - mediated Plant ---------------------------Potato, tomato etc. Commensal bacteria--------Lactic acid bacteria

5. Lactic acid Bacteria Generally regarded as safeAdjuvant effects Lactic acid bacteria are promising antigen delivery agents!!! Mucosal vaccine Lactococcus Lactobacillus Bifidobacterium Protective antigen

6. Membrane Cell Wall Cytoplasm Extracellular P T Target gene SS P T Target gene SS CWA P T Target gene (A) Cytoplasm (B) Supernatant (C) Cell wall anchor - Expression of heterologous protein on lactic acid bacteria -

7. Listeria monocytogenes Intracellular pathogen Production of listeriolysin O (LLO) encoding hlyA (1.5 kb) LLO Hemolysis - Example of construction of mucosal vaccine -

8. 1500bp 1000bp500bp 5’3’ Hemolysis + - - 1. LLO 2. LLOmut 3. LLOshort Mutation LLO- L. caseiL. casei L. monocytogenes hlyA - Construction of LLO-expressing L. casei - Safe delivery vehicle + Non-toxic antigen Safe delivery vehicle + Non-toxic antigen

9. Salmonella enterica serovar Enteritidis (SE) Salmonella enterica serovar Enteritidis (SE) Construction of FliC-expressing Lc. lactis Egg-associated salmonellosis SE contamination in/on egg SE-carrier hens SE-carrier hens Vaccination of hens control of SE contamination control of SE contamination

10. antigen delivery vehicle Generally regarded as safe (GRAS) (GRAS) Lactococcus lactis protective antigen SE Flagellin (FliC) Major immunogen of Salmonella TLR-5 mediated immune responses The mucosal vaccine which confers both systemic and local immunity Construction of FliC-expressing Lc. lactis

11. Bacterial strain IFN-γ, IL-2, TNF-α IL-4, IL-6, IL-10 Cell-mediated immunity Humoral immunity Lactococcus lactis Th 1 cells Th 2 cells Th 1 Th 2 Lactococcus lactis + FliC Th 0

12. PSSfliCAnchor EcoRVKpn1 - Construction of Lc.lactis expressing fliC on its surface - fliC : M84974 pALC1 (5.957 Kb) rep ermA P SS Anchor MCS Transformation Selection PCR & Sequencing

13. - The result of Western blot - kDa 150 100 75 1 2 1 2 1 2 1 2 G53 H45H61527 M : Marker 1. Whole cell lysates of strains expressing fliC on cell surface 2. Whole cell lysates of strains containing vector only M

14. - The results of flow cytometry (Lc.lactis expressing fliC on the cell surface) - L.lactis H45 : Th 1, TNF-α(-) L.lactis H61 : Th 2 L.lactis 527 : Neutral L.lactis G53 : Th 1, TNF-α(+)

15. Lc.lactis exprssing fliC on cell surfaceLc.lactis containing vector only - Bright field images -

16. - Immunofluorescence images - Lc.lactis exprssing fliC on cell surfaceLc.lactis containing vector only

17. - Caco-2 cells -

18. FliC Control FliC Control Invasion into Caco-2 cellsIL-8 secretion in Caco-2 cells

19. Immunization of mice with Lactococcus lactis expressing fliC Immunization of mice with Lactococcus lactis expressing fliC anti-FliC protection IFN-γ

20. Conclusion 1. Possibility 2. Problem - Thank you for your attention !!! -