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

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

MHC class I with peptide Binding groove

Peptides are bound to MHC class I molecules by their ends

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

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

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

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

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

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

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

Peptides - prime targets of immune recognition TcR Peptide HLA

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

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

Translating genomes to immunogens

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 (http://www.anthonynolan.com/HIG/index.html)

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) 50:201-212

Bindings affinity vs. number of epitopes TB: ~ 4000 proteins ~ 1.33 mill. aa ~ 4 mill. 8-10´mers HIV ~ 9 proteins ~ 3000 aa ~ 9000 8-10´mers Number of peptides < 5000 nM KD = < 50 nM < 250 nM < 500 nM < 50000 nM Increasing peptide affinity

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; 24:385-392 2) Buus, S et al., J. Immunol. 1982; 129:1883-1891. 3 ) Buus, S. and Werdelin, O. J. Immunol. 1986; 136: 459-465. 4) Babbitt, B.P et al., Proc. Natl. Acad. Sci. USA. 1986; 83:4509-4513. 5) Buus, S., et al., Cell. 1986; 47:1071-1077. 6) Luescher, I. F et al., Proc. Natl. Acad. Sci USA. 1988; 85:871-874. 7) Townsend, A., et al., Nature. 1989; 340:443-448. 8) Bouillot, M. et al.,Nature. 1989; 339:473-475. 9) Busch, R. and Rothbard, J. B. J. Immunol. Meth. 1990: 134:1-22. 10) Townsend, A., et al., Cell 1990:62:285-295. 11) Parker, K.C. et al., J. Immunol. 1992; 49:1896-1904. 12) Joosten, I. Et al., Trans. Proc. 1993; 25:2842-2843. 13) Khilko, S.N. et al., J. Biol. Chem. 1993; 268:15425-15434. 14) Regner, M. et al.,Exp Clin Immunogenet. 1996; 13:30-35.

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

Experimental description of peptide-MHC binding How to examine HLA specificity? ”What the HLA has bound in vivo” Hans-Georg Rammensee et al., www.syfpeithi.de  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., www.cbs.dtu.dk/services/NetMHC/

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

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

Scanning the genome of Chlamydia pneumonia for CTL epitopes  Søren Buus et al., www.cbs.dtu.dk/services/NetMHC/ Scanning the genome of Chlamydia pneumonia for CTL epitopes (nM)

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 = (10-15-10-6 M) Peptide [M] Cold Peptide Log [M] Log IC50

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

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

How to determine the peptide binding motif

Specificity description of A*0204 (matrix)

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

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

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 10-15 M MHC class I complex !

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 2002. 59: 259

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

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.

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