1. Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop

2. Overview Intro Lactococcus lactis Interleukins (cytokines) Quick summary of general process Relative Importance Materials and Methods Outline of what was done Specific techniques used Results and Discussion How and what results were generated What was learned

3. Lactococcus lactis Gram positive, cocci, 0.5-1.5μm Used in the production of fermented milk products (buttermilk, cheese, etc…) Noninvasive (can’t multiply in vivo), nonpathogenic Can serve as an antigen delivery vehicle

4. Interleukins (cytokines) Cytokines (secreted signaling molecules) Immune system depends on them Can be useful immune response modulators for vaccines Administration of IL-2 and IL-6 has been shown to increase antibody responses to antigens

5. What was done Constitutive expression strains were engineered to accumulate a test antigen (TTFC) and murine interleukins (cytokines) IL-2 and IL-6 Mice were intranasally immunized with variations of these expression strains (as well as controls) Some recombinants were treated (killed) with mitomycin C prior to immunization Both systemic and mucosa anti-TTFC antibody responses were measured

6. Relative Importance Treatment of disease At this point, virtually all recombinant delivery systems had been derived by infectious agents (Salmonella spp., Mycobacterium bovis, etc…) Cytokine delivery could enable tailored vaccines against particular pathogens.

7. Materials and Methods

8. Recombination of DNA PCR amplification using Vent polymerase DNA-modifying enzymes and restriction endonucleases used under standard conditions L. lactis transformed by electroporation of cells grown in presence of glycine Recombinant strains expressing TTFC, IL- 2 and IL-6

10. Immunoblotting Cell walls were digested using a buffer solution and separated from the cells by centrifugation Proteins extracted using electrophoresis and electroblotted Transfer of TTFC and murine cytokines detected by immunoblotting (western blot)

11. Preparation for Immunization The bacterial strains which carried the desired insert components were cultured, washed and resuspended prior to immunization

12. Mitomycin Pretreatment Prior to immunization, cultured cells were treated with mitomycin C. After treatment, fewer than 1 in 10 4 cells remained viable.

13. Immunization Groups of lady mice, 6-8 weeks old, were intranasally immunized with the modified L. lactis Pretend this is a mouse.

14. Detection of Antibodies The presence of antibodies was detected using enzyme-linked immunosorbent assay (ELISA). This method was developed using antibodies which react with serum antigens or antibodies and signal their presence

15. This was done for both TTFC-specific antibodies and antilactococcal antibodies. L. lactis TTFC

16. Assay of IgA Fecal pellets from the subjects were assyed for IgA concentration. IgA is the main antibody (immunoglobulin) in mucosal tissues

17. Results Feces of mice examined to determine if cytokines would influence IgA in mucosal tissues IgA levels in mice inoculated with different strains of recombinant L. lactis showed the same results as the control groups Therefore, IgA levels in gastrointestinal tract were unaffected. But, blood serum antibody levels were affected….

18. Mean anti-TTFC IgG levels Increased levels in: - TTFC + IL-2 - TTFC + IL-6 Recombinant bacterial strains from left to right: TTFC, TTFC + IL-2, TTFC + IL-6, control non-expressor strain, control nonvaccinated group.

19. Mean Anti-TTFC IgA Levels Increased levels in: - TTFC + IL-6 Recombinant bacterial strains from left to right: TTFC, TTFC + IL-2, TTFC + IL-6, control non- expressor strain, control nonvaccinated group.

20. Anti-TTFC antibody levels a: IL-2 b: IL-6 -Inoculated mice with viable strains and mitomycin C killed strains of L. lactis -TTFC antigen delivery does not require bacterial viability - IL-2 and IL-6 cytokine delivery does require bacterial viability

21. Results So... By demonstrating an increase in levels of IgA and IgG antibodies in mice inoculated with TTFC + IL-2 and TTFC + IL-6, it has been shown that… The viable recombinant lactococci were successful in delivering cytokines to the immune system.

22. Conclusions L. lactis can be used an effective method for delivering cytokines to the immune system via intranasal inoculation. This strategy may potentially be applied as a method of vaccination for a particular pathogen of interest.