1. Experimental methods in classic epitope discovery
T cell epitopes
Experimental methods in classic epitope discovery

2. Pathogens Pathogen specific Humoral Cellular Parasite Bacteria Virus
Bacteria
Virus

3. Adaptive immune response
Signal induced
Pathogens
Antigens
Epitopes
B Cell
T Cell

4. MHC - TCR
Peptide
MHC

5. Germ line genes
© 2001 by Garland Science

6. T-cell receptor α- and β-chain gene rearrangement
© 2001 by Garland Science

7. The development of T cells
© 2001 by Garland Science

8. Killing!

9. Chromium release Interferon gamma FACS CTL Assays Elisa EliSpot
IF-g or IL staining
Tetramer

10. ELISA 1. Coat interferon specific Ab onto microplate
2. Add cell supernatant into each well
3. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate
4. Read absorbance in spectrophotometer with appropriate filter and quantitate relative antigen levels

11. EliSpot

12. EliSpot

13. Tetramer

14. FACS

15. Do the pathogen trigger a CTL response at all !
Epitope discovery
Do the pathogen trigger a CTL response at all !

16. Which proteins induce a CTL response
Antigen discovery
Which proteins induce a CTL response

17. Peptides are eluted from cells with known MHC haplotype
Elution
Peptides are eluted from cells with known MHC haplotype
The eluted peptides is fractionated in HPLC
Each fraction is identified.
Sequencing MS
Identified peptides is mapped back on proteome
Eluted peptides are designated natural ligands
Not neccesary epitopes
Can be verified in CTL assays

18. Overlapping N-mers

19. Pool Methods (Rare events)
Several peptides in each well
De-convolute positive pools
Generally pools lower the signal

20. High(er) amount of expression High(er) N-terminal cleavage
Epitope dominans
High(er) amount of expression
High(er) N-terminal cleavage
Strong(er) TAP affinity
Strong(er) MHC Affinity

21. Immunogens if dominant epitopes are removed
Subdominant epitopes
Immunogens if dominant epitopes are removed
Needs special effort to discover

22. Subdominant vaccine candidates
If:
More conserved among different strains
Lesser probability of escape mutations
Broader HLA coverage