1. “Theoretical and Experimental description of Peptide-MHC binding”
“Experimental and Theoretical description of Peptide-MHC binding”

2. Generation of Recombinant MHC Class I and Characterization of the Interaction with - Contributions to the Human MHC project
Peptide
People

3. MHC class I with peptide
Binding
groove

4. Peptides are bound to MHC class I molecules by their ends

5. Peptides are bound to MHC class II molecules by interactions along the length of the binding groove

6. Peptide binding grove TcR
Hydrogen bonds
Salt bridge
Pockets
Lauemøller, S.L. and Buus, S. 2001

7. Peptides bind to MHC molecules through structurally related anchor residues
Two MHC class I alleles
(P2 (Y), (Pc): V,I, L)

8. Peptides that bind to MHC-II molecules are variable in length

9. The peptide is deeply embedded
Comparison of the cleft architectures of murine class I alleles, Kb and Kk
Stryhn A, et al., 1996, PNAS
Peptide: RGYVYQGL
Peptide: FESTGNLI
The peptide is deeply embedded

10. Most of the peptide is hidden in the groove - only a minor part is available for the TcR.
FES T GN L I
Available for the TCR
Top view Mouse class I Kk
in complex with peptide FESTGNLI

11. Peptide binding grove G  K
Peptides can be anchor optimized, affinity can increased X 10,
Does not changes the T cell specificity!
G  K
Hydrogen bonds
Salt bridge
Pockets
Lauemøller, S.L. and Buus, S. 2001

12. Peptides - prime targets of immune recognition
TcR
Peptide
HLA

13. Determining primary protein structures
is like
charting the landscape of the immune system

14. From proteins to immunogens
1/200
1/200 peptides ends up in the MHC binding groove

15. Translating genomes to immunogens

16. HLA polymorphism - alleles
A total of
245 HLA-A
480 HLA-B
117 HLA-C
class I alleles have been assigned.
3 HLA-DRA, 380 HLA-DRB
22 HLA-DQA1, 52 HLA-DQB1
20 HLA-DPA1, 97 HLA-DPB1
class II alleles have been assigned.
As of April 2002 (

17. HLA polymorphism - supertype specificity
Supertype Specificity Av. frequency
P2 Pc
A1 TI (LVMS) FWY 25 %
A2 AILMVT AILMVT 42 %
A3 AILMVST RK 44 %
A24 YF (WIVLMT) FI (YWLM) 40 %
B7 P AILMVFWY 50 %
B27 RHK FYL (WMI) 23 %
B44 E (D) FWYLIMVA 37 %
B58 ATS FWY (LIV) 10 %
B62 QL (IVMP) FWY (MIV) 18 %
Sette et al, Immunogenetics (1999) 50:

18. Bindings affinity vs. number of epitopes
TB: ~ 4000 proteins ~ 1.33 mill. aa ~ 4 mill. 8-10´mers
HIV ~ 9 proteins ~ 3000 aa ~ ´mers
Number of peptides
< 5000 nM
KD = < 50 nM
< 250 nM
< 500 nM
< nM
Increasing peptide affinity

19. Experimental description of peptide-MHC binding
Many different peptide-MHC-binding assays have been suggested over the years ; without being exhaustive:
1) Olsen, A.C., et al., Eur. J. Immunol. 1994; 24:
2) Buus, S et al., J. Immunol. 1982; 129:
3 ) Buus, S. and Werdelin, O. J. Immunol. 1986; 136:
4) Babbitt, B.P et al., Proc. Natl. Acad. Sci. USA. 1986; 83:
5) Buus, S., et al., Cell. 1986; 47:
6) Luescher, I. F et al., Proc. Natl. Acad. Sci USA. 1988; 85:
7) Townsend, A., et al., Nature. 1989; 340:
8) Bouillot, M. et al.,Nature. 1989; 339:
9) Busch, R. and Rothbard, J. B. J. Immunol. Meth. 1990: 134:1-22.
10) Townsend, A., et al., Cell 1990:62:
11) Parker, K.C. et al., J. Immunol. 1992; 49:
12) Joosten, I. Et al., Trans. Proc. 1993; 25:
13) Khilko, S.N. et al., J. Biol. Chem. 1993; 268:
14) Regner, M. et al.,Exp Clin Immunogenet. 1996; 13:30-35.

20. RMAS Assay: classical way to measure peptide binding
- However not quantitative (no determination of the affinity)
At 37 °C
At 26 °C
Measure T cell activation
Add peptide
TAP difficient cell line

21. Experimental description of peptide-MHC binding
How to examine HLA specificity?
”What the HLA has bound in vivo”
Hans-Georg
Rammensee et al.,
 Elution and sequencing of natural ligands
 Simpel motif ~ low sensitivity predictions
”What the HLA will, or will not, bind in vitro”
 Determine the binding strength of any peptide
 Extended motif ~ higher sensitivity predictions
Søren Buus et al.,

22. ”What the HLA has bound in vivo”
Prediction of binding, web based services
(non quantitative)

23. www.syfpeithi.de (Hans-Georg Rammensee et al.,)

24. Scanning the genome of Chlamydia pneumonia for CTL epitopes
Søren Buus et al.,
Scanning the genome of Chlamydia pneumonia for CTL epitopes
(nM)

25. How to determine peptide affinity
Law of mass action
koff
koff
[R] + [L] [RL]
[MHC] + [P] [P*MHC]
kon
kon
KD = koff (S-1)/kon (M-1S-1)
Saturation assay
100%
Inhibition
assay
Peptide Log [M]
Binding
50%
Binding hot peptide
Binding
KD = ( M)
Peptide [M]
Cold Peptide Log [M]
Log IC50

26. Non binding test peptide
How to do radioactive biochemical inhibition binding assays
Obtain purified HLA
Or recombinant heavy chain & b2m
Obtain indicator peptide
Perform dose titration of any inhibitory peptide
Separate free from bound peptide
Calculate binding and IC50
Non binding test peptide
Binding test Peptide

27. Non binding test peptid
A spun column binding assay
MHC
b2m
peptide
G50
Binding test peptid
Non binding test peptid

28. How to determine the peptide binding motif

29. Specificity description of A*0204 (matrix)

30. The radioactive biochemical binding assay
PROS
Truly quantitative
Can address affinities in the low nM level
Reproducible
CONS
Radioactive
Not a standard method
Waste problem

31. The Quantitative ELISA Capable of
Determining Peptide-MHC Class I Interaction
Made possible by our recent development of highly active recombinant MHC class I heavy chains
functional equivalents of ”empty” molecules
L.O.Pedersen et al., , EJI. 2001, 31: 2986
Pros:
Reasonably simple, sensitive and quantitative
Does not depend on labeled peptide
It is easily adaptable to other laboratories
Disseminated protocol and standard reagents

32. Sensitivity below 0.1 nM or 5 x 10-15 M
Strategy for the assay
Step I: Folding of MHC class I molecules in solution
Incubation
Step II: Detection of de novo folded MHC class I molecules by ELISA
Development
Sensitivity below 0.1 nM or 5 x M
MHC class I complex !

33. C.Sylvester-Hvid, et al., Tissue Antigens 2002. 59: 259
Concentrations of de novo folded MHC complexes, plotted as function of the concentrations of peptide offered
Fitted in Prism® 4.0 GraphPad
Data out put:
BMAX : Amount of detected complex
including 95% confidence interval
KD: Peptide affinity
including 95% confidence interval
nM MHC complex detected
R2: Precision of the fit
nM peptide offered
C.Sylvester-Hvid, et al., Tissue Antigens : 259

34. Data base of HLA ligands, founded by the NIH (Nat. Inst. Health) USA

35. Take home messages….
 MHC class I molecule preferably binds peptides of 8-11 aa
 Peptides bind to the MHC binding groove by hydrogen bonds, hydrophobic forces and other non-covalent interactions
 MHC binding specificity is obtained through the recognition of peptide- motifs, a recognition mode which requires the presence and proper spacing of particular amino acids in certain anchor positions.
 The binding strength, the affinity - is very important: The higher affinity of a peptide to the class I molecule, the higher chance of being immunogenic.

36. and more….. http://www.ihwg.org/components/peptider.htm
 Peptide binding can be addressed in a qualitative or quantitative matter.
 The Radioactive binding assay or The quantitative ELISA assay can measure the exact affinity in nM for the best known binders
 Measurements of affinity can be used to generate tools (ANN) for prediction of peptide binding to any MHC class I molecule of human interest.
 Subsequently to validation, peptides can be directly used in peptide based vaccines or rationally optimized to increase their immunogenesity