Christina Sylvester-Hvid, IMMI, Panum “Experimental and Theoretical description of Peptide-MHC...

Christina Sylvester-Hvid, IMMI, Panum

“Experimental and Theoretical description of

Peptide-MHC binding”

“Theoretical and Experimental description of

Peptide-MHC binding”


Generation of Recombinant MHC Generation of Recombinant MHC Class I and Characterization of the Class I and Characterization of the

Interaction withInteraction with

- Contributions to the Human MHC project- Contributions to the Human MHC project

PeoplePeoplePeptidePeptide


MHC class I with peptideMHC class I with peptide

PeptideBinding groove


Peptides are bound to MHC class I molecules by their ends

N C


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

N C


Peptide binding grove

Lauemøller, S.L. and Buus, S. 2001

Hydrogen bondsHydrogen bondsHydrogen bondsHydrogen bonds

Salt bridgeSalt bridge

PocketsPockets

1 2

7

4 68

5

39

A

B

C

DE

F

TcR


Peptides bind to MHC molecules through structurally related anchor residues

Two MHC class I alleles (P2 (Y), (Pc): V,I, L)(P2 (Y), (Pc): V,I, L)


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


Comparison of the cleft architectures of murine class I alleles, Kb and Kk

Peptide: RGYVYQGL Peptide: FESTGNLI

Stryhn A, et al., 1996, PNAS

The peptide is deeply embedded


Most of the peptide is hidden in the groove - only a minor part is available for the TcR.

Top view Mouse class I Kk

in complex with peptide FESTGNLI

Available for the TCR

FES T GN L I


Peptide binding grove

Lauemøller, S.L. and Buus, S. 2001

Hydrogen bondsHydrogen bondsHydrogen bondsHydrogen bonds

Salt bridgeSalt bridge

PocketsPockets

1 2

7

4 68

5

39

A

B

C

DE

F

Peptides can be anchor optimized, affinity can increased X 10, Does not changes the T cell specificity!

G K


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 immunogensFrom proteins to immunogens

1/200 peptides ends up in the MHC binding groove

1/200


Translating genomes to immunogensTranslating genomes to immunogens


HLA polymorphism - alleles

A total of245 HLA-A480 HLA-B117 HLA-C

class I alleles have been assigned.

A total of3 HLA-DRA, 380 HLA-DRB22 HLA-DQA1, 52 HLA-DQB120 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 specificityHLA 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

Number of peptides

< 5000 nMKD = < 50 nM < 250 nM < 500 nM < 50000 nM

TB: ~ 4000 proteins ~ 1.33 mill. aa ~ 4 mill. 8-10´mersHIV ~ 9 proteins ~ 3000 aa ~ 9000 8-10´mers

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)

TAP difficient cell line

At 37 °C At 26 °C

Add peptide

Measure T cell activation


Experimental description of peptide-MHC binding

Hans-GeorgRammensee et al.,

www.syfpeithi.de

Søren Buus et al.,

www.cbs.dtu.dk/services/NetMHC/

How to examine HLA specificity?

”What the HLA has bound in vivo” 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


Prediction of binding, web based servicesPrediction of binding, web based services

(non quantitative)(non quantitative)

”What the HLA has bound in vivo”


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

Scanning the genome of Chlamydia pneumonia for CTL epitopesKk binding Efficacy

Acc # position sequence C-flush Survivalgi|8163394 7 EEHGSTTI 2 0.73 0.94 3gi|7189942 850 EELDASAI 2 0.89 0.26 9gi|7189608 120 EEVYMGTI 2 0.28 0.29 26gi|8163474 96 TELLAEYI 2 0.19 0.35 33gi|7189405 40 AEQLASEI 2 0.74 0.08 44gi|8163540 496 EEVFSGFI 2 0.03 0.88 72gi|7189969 101 EELWAAEI 2 0.16 0.16 75gi|7189995 360 EEAKSAFI 2 0.04 0.60 107gi|8163481 149 EELRAVSI 2 0.13 0.10 161gi|7189705 31 EEIRYRII 2 0.01 0.30 537gi|7189594 238 DERYASWI 2 0.01 0.45 591gi|7189985 407 EELGSEAI 2 0.03 0.10 604gi|7189979 159 LERLAGFI 2 0.01 0.32 720gi|7189388 269 FELVAHIG 2 0.03 0.08 740gi|7190008 364 EERLAIFI 2 0.00 0.89 789gi|7189561 203 WEVVSHFI 2 0.00 0.43 962gi|7189544 318 DEVIARIH 2 0.00 0.15 1662gi|7189647 52 FEVLCRDI 2 0.00 0.02 2023gi|8163547 296 EEFRQGYI 2 0.00 0.38 2100gi|7189892 56 EELFADFI 2 0.00 0.53 2300

Protein Proteasome

(nM)

Søren Buus et al.,

www.cbs.dtu.dk/services/NetMHC/


How to determine peptide affinityHow to determine peptide affinityLaw of mass action

[R] + [L] [RL]

koff

kon

KD = koff (S-1)/kon (M-1S-1)

Peptide [M]

Bin

ding

KD = (10-15-10-6 M)

100%

50%

Peptide Log [M] Cold Peptide Log [M]

Log IC50B

ind i

n g h

o t p

eptid

e

Bi n

ding

[MHC] + [P] [P*MHC]

koff

kon

Saturation assay

Inhibition assay


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


A spun column binding assayA spun column binding assay

G50

b2m

MHC

peptide

Binding test peptid

Non binding test peptid


How to determine the peptide binding motif


Specificity description of Specificity description of A*0204A*0204 (matrix)(matrix)


PROSPROS

• Truly quantitativeTruly quantitative

• Can address affinities in Can address affinities in

the low nM levelthe low nM level

• ReproducibleReproducible

CONS

• Radioactive

• Not a standard method

• Waste problem

The radioactive biochemical binding assay


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

• Pros:• Reasonably simple, sensitive and quantitative• Does not depend on labeled peptide• It is easily adaptable to other laboratories

• Disseminated protocol and standard reagents

L.O.Pedersen et al., , EJI. 2001, 31: 2986


DevelopmentDevelopment

Sensitivity below 0.1 nM or 5 x 10Sensitivity below 0.1 nM or 5 x 10-15-15 M M MHC class I complex !MHC class I complex !

Strategy for the assay

• Step II: Detection of de novo folded MHC class I molecules by ELISA

IncubationIncubation

• Step I: Folding of MHC class I molecules in solution


nM M

HC

com

ple

x d

etec

ted

nM peptide offerednM peptide offered

Fitted in PrismFitted in Prism® ® 4.0 GraphPad4.0 GraphPad

Concentrations of de novo folded MHC complexes, plotted as function of the concentrations of peptide offered

KKDD: Peptide affinity: Peptide affinityincluding 95% confidence intervalincluding 95% confidence interval

RR22: Precision of the fit: Precision of the fit

Data out put:Data out put:

BBMAXMAX : Amount of detected complex : Amount of detected complex

including 95% confidence intervalincluding 95% confidence interval

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….

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.

MHC class I molecule preferably binds peptides of 8-11 aa

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

The Radioactive binding assayThe Radioactive binding assay or or The quantitative ELISA The quantitative ELISA assayassay can measure the exact affinity in nM for the best known can measure the exact affinity in nM for the best known bindersbinders

Measurements of affinity can be used to generate tools Measurements of affinity can be used to generate tools (ANN) for prediction of peptide binding to any MHC class I (ANN) for prediction of peptide binding to any MHC class I molecule of human interest.molecule of human interest.

Peptide binding can be addressed in a qualitative or Peptide binding can be addressed in a qualitative or quantitative matter.quantitative matter.

Subsequently to validation, peptides can be directly used Subsequently to validation, peptides can be directly used in peptide based vaccines or rationally optimized to increase in peptide based vaccines or rationally optimized to increase their immunogenesitytheir immunogenesity

Christina Sylvester-Hvid, IMMI, Panum “Experimental and Theoretical description of Peptide-MHC...

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