Morten Nielsen Center for Biological Sequence Analysis

Department of Systems biology Technical University of Denmark,

and Instituto de Investigaciones Biotecnológicas,

Universidad Nacional de San Martín, Argentina mniel@cbs.dtu.dk

Predicting peptide MHC interactions

Bridging between two worlds

“To be honest, I do not believe in the bioinformatics approach to immunology, ...” XX, 2006

What defines a T cell epitope?

•  Processing (Proteasomal cleavage, TAP)? •  Other proteases •  MHC binding •  MHC:peptide complex stability •  T cell repertoire •  Source protein abundance, cellular

location and function

MHC Class I pathway Finding the needle in the haystack

Figure by Eric A.J. Reits

1/200 peptides make to the surface

Finding the needle in the haystack

Bridging between two worlds

This has changed (I hope) due to the availability of data

•  IEDB (Immune epitope database) has been pivotal for moving the field of in silico T cell epitope prediction forward

•  Prior to the IEDB all analysis were anecdotal •  Identifying features on a small scale, one

ot two proteins •  Only now with the large-scale data sets

can we see the big picture

SLLPAIVEL YLLPAIVHI TLWVDPYEV GLVPFLVSV KLLEPVLLL LLDVPTAAV LLDVPTAAV LLDVPTAAV LLDVPTAAV VLFRGGPRG MVDGTLLLL YMNGTMSQV MLLSVPLLL SLLGLLVEV ALLPPINIL TLIKIQHTL HLIDYLVTS ILAPPVVKL ALFPQLVIL GILGFVFTL STNRQSGRQ GLDVLTAKV RILGAVAKV QVCERIPTI ILFGHENRV ILMEHIHKL ILDQKINEV SLAGGIIGV LLIENVASL FLLWATAEA SLPDFGISY KKREEAPSL LERPGGNEI ALSNLEVKL ALNELLQHV DLERKVESL FLGENISNF ALSDHHIYL GLSEFTEYL STAPPAHGV PLDGEYFTL GVLVGVALI RTLDKVLEV HLSTAFARV RLDSYVRSL YMNGTMSQV GILGFVFTL ILKEPVHGV ILGFVFTLT LLFGYPVYV GLSPTVWLS WLSLLVPFV FLPSDFFPS CLGGLLTMV FIAGNSAYE KLGEFYNQM KLVALGINA DLMGYIPLV RLVTLKDIV MLLAVLYCL AAGIGILTV YLEPGPVTA LLDGTATLR ITDQVPFSV KTWGQYWQV TITDQVPFS AFHHVAREL YLNKIQNSL MMRKLAILS AIMDKNIIL IMDKNIILK SMVGNWAKV SLLAPGAKQ KIFGSLAFL ELVSEFSRM KLTPLCVTL VLYRYGSFS YIGEVLVSV CINGVCWTV VMNILLQYV ILTVILGVL KVLEYVIKV FLWGPRALV GLSRYVARL FLLTRILTI HLGNVKYLV GIAGGLALL GLQDCTMLV TGAPVTYST VIYQYMDDL VLPDVFIRC VLPDVFIRC AVGIGIAVV LVVLGLLAV ALGLGLLPV GIGIGVLAA GAGIGVAVL IAGIGILAI LIVIGILIL LAGIGLIAA VDGIGILTI GAGIGVLTA AAGIGIIQI QAGIGILLA KARDPHSGH KACDPHSGH ACDPHSGHF SLYNTVATL RGPGRAFVT NLVPMVATV GLHCYEQLV PLKQHFQIV AVFDRKSDA LLDFVRFMG VLVKSPNHV GLAPPQHLI LLGRNSFEV PLTFGWCYK VLEWRFDSR TLNAWVKVV GLCTLVAML FIDSYICQV IISAVVGIL VMAGVGSPY LLWTLVVLL SVRDRLARL LLMDCSGSI CLTSTVQLV VLHDDLLEA LMWITQCFL SLLMWITQC QLSLLMWIT LLGATCMFV RLTRFLSRV YMDGTMSQV FLTPKKLQC ISNDVCAQV VKTDGNPPE SVYDFFVWL FLYGALLLA VLFSSDFRI LMWAKIGPV SLLLELEEV SLSRFSWGA YTAFTIPSI RLMKQDFSV RLPRIFCSC FLWGPRAYA RLLQETELV SLFEGIDFY SLDQSVVEL RLNMFTPYI NMFTPYIGV LMIIPLINV TLFIGSHVV SLVIVTTFV VLQWASLAV ILAKFLHWL STAPPHVNV LLLLTVLTV VVLGVVFGI ILHNGAYSL MIMVKCWMI MLGTHTMEV MLGTHTMEV SLADTNSLA LLWAARPRL GVALQTMKQ GLYDGMEHL KMVELVHFL YLQLVFGIE MLMAQEALA LMAQEALAF VYDGREHTV YLSGANLNL RMFPNAPYL EAAGIGILT TLDSQVMSL STPPPGTRV KVAELVHFL IMIGVLVGV ALCRWGLLL LLFAGVQCQ VLLCESTAV YLSTAFARV YLLEMLWRL SLDDYNHLV RTLDKVLEV GLPVEYLQV KLIANNTRV FIYAGSLSA KLVANNTRL FLDEFMEGV ALQPGTALL VLDGLDVLL SLYSFPEPE ALYVDSLFF SLLQHLIGL ELTLGEFLK MINAYLDKL AAGIGILTV FLPSDFFPS SVRDRLARL SLREWLLRI LLSAWILTA AAGIGILTV AVPDEIPPL FAYDGKDYI AAGIGILTV FLPSDFFPS AAGIGILTV FLPSDFFPS AAGIGILTV FLWGPRALV ETVSEQSNV ITLWQRPLV

MHC binding motifs

HLA binding motifs

HLA-A0201

http://www.cbs.dtu.dk/biotools/Seq2Logo

HLA specificities

A0201

A0101

A0206

B0702

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

Some 70 MHC molecules covered by binding data

The IMGT/HLA Sequence Database currently encompass more than 1500 HLA class I proteins

Source: http://www.anthonynolan.com/HIG/index.html

HLA polymorphism

0

200

400

600

800

1000

1200

1400

1600

1800

1999 2000 2005 2006 2007

Year

Number HLA-A

HLA-B

HLA-C

Total

5,100 HLA-I proteins release 3.2

HLA polymorphism

•  < 100 HLA alleles are characterized by binding data

•  Reliable MHC class I binding predictions (NetMHC-3.3) for 70 HLA A and B molecules

•  Long way to cover 5000 •  And very little data for non-human MHC’s

HLA polymorphism! B0807 B4804 B0710 B1513 A6817 B5130 A0204 B3503 A2415 B0740 B3929 A0250 B5204 A2420 B1804 B3523 B3502 A3202 B0802 A3601 B4047 A6601 A0268 B0817 B5002 B5602 B3811 B4810 A0103 B1530 B4415 A3111 B7803 A6804 B3520 B3528 A2610 A6802 A2404 A7406 B0744 B3701 B4058 B1803 B1527 B3801 A6826 B5606 B0725 B5603 A0110 B1586 A3205 A0212 B3511 A2603 B5120 A0251 A3106 A6801 B5135 B1567 B4012 A3401 B5106 B3912 B1525 B5703 B4402 B0733 A2901 B0711 A6603 B3907 B4023 B2717 B4507 B4502 B4807 A2438 B1312 B1590 A0258 B5310 B5124 B4103 B0811 B3927 B4104 A1110 B1553 A2621 B5115 B1599 A0102 B5102 A0207 B4444 A3002 A6813 B5709 B5515 B4439 B1561 A2618 B2728 A3404 A6820 A3107 A2430 A0235 A2914 B1301 B4004 A2620 B1573 A0259 B0804 B1548 A2616 B5401 B0707 A2453 A2609 B3554 A0245 B4411 A0220 B1510 A2433 B5512 B5306 B1540 B5114 B3934 B5510 B1521 B0810 B5137 B3932 B4802 B4044 B3709 B3915 B2729 B3810 A0238 B0729 B3537 A2314 B0734 B3702 A0214 B4805 A0269 A3102 B5206 A6819 B3707 A3011 A1123 B1822 A6823 A4301 B3917 B4702 B5118 B3708 A0265 B5203 A3013 B3530 B4701 B4061 A0316 B4814 B2710 A7411 B3930 B0702 B5702 A1107 B7801 A0246 B3534 A0228 B1596 A3305 B2711 B3526 B4445 A0216 B1539 A3308 A2455 A0206 B4605 B2725 A0310 B4037 A1104 A2622 B5607 B4504 B4602 B1598 A3112 B0813 B5113 A0237 A3602 B0805 A6808 B4505 B1544 A0285 A3108 B5402 B6701 A6901 B0730 B4056 B5205 B1310 B5805 B1404 A2435 A2614 A7405 B1520 B3920 A0254 B2702 A6815 A3201 B1570 A0255 B5708 B4033 B4435 A2405 B4007 B4034 B4806 B5615 A0218 B3527 B3512 B0814 B5301 A6829 B4904 B4038 A0304 A7408 B7805 B3549 B1503 B4420 A1120 B1815 B5129 B0801 B0827 B5001 A3402 A0314 B4405 A2305 B4438 B4052 B0823 A8001 B1302 B4021 A2909 B3933 B4408 B4105 B0727 B5508 B4108 A3405 B1315 B3517 A1116 B0731 B4053 B1516 B4704 B1403 A6830 B5610 A3009 B0714 B1303 B1566 B2714 B3923 B5801 A2439 B2719 A0219 A2602 A2413 B1821 A0260 B4410 A6605 B1309 B8202 B4426 A2623 B4042 B1805 B3902 A2503 B1536 A0302 A3209 A0205 B2715 B5131 A0262 A6805 B5201 A1119 B1402 A0270 A2450 A1111 A3008 B3806 A6822 A0202 B5503 B0826 B3926 A2428 A1114 A2414 A3301 A0239 B4054 B0825 A0308 B3563 A0305 B4036 B1589 B1314 B1563 B4005 A3104 B4440 B5122 A3206 B7804 B0718 B4446 B4905 B9509 A0112 A0256 A6604 B4029 B1807 B5901 A2906 B1304 B3501 A2502 B5509 B4107 B2707 A0117 B4032 B3914 B3509 A3306 A6602 B1504 B5611 A2904 B3535 A2447 B6702 B1572 A2417 B1811 A2452 B3542 A2612 B1542 B1507 B5406 B3911 A2421 A2443 B4404 A3015 B5704 B4437 B4427 B8101 B4002 B3901 A1103 B3928 A2408 A6827 B1517 B0824 B1576 B4601 A2303 B4811 B4003 A2605 B1505 B4808 A7407 B1809 A0222 B4031 B1511 B4429 B1564 A2406 B1515 B5601 A2301 B4101 B3506 A0113 B5710 A7404 B3531 A0201 B4902 B1581 A2907 B4431 A0252 B4102 A2601 A6825 B5116 B5608 B4201 B5110 B4422 B2720 B2727 A3304 B1306 A2425 B5501 A0233 B0736 A2423 B1549 A1109 B3558 B5134 B5139 A0289 B5121 B4208 A0271 B2705 A2407 B4501 B3550 A2410 B2706 B1552 A1101 A0273 B1546 B3905 B4409 B5808 A2313 B0706 B1534 B5138 B0803 A2429 B5507 A6810 B1405 B2713 B3547 B4013 A3003 B5119 A3010 B0726 A3204 B3552 B3802 A3105 B4062 B4018 B4403 B1550 A0317 B4432 B4433 B3551 B9505 B8201 A3303 B5804 B4008 A0208 A0230 B1819 B2726 B3533 B4428 B5404 A0267 B1529 B4046 A0106 B9507 B3505 B4016 B3922 A7410 B1509 B0822 A3012 A0319 B4503 B5207 B1531 B3904 A2910 B5613 B0717 A2403 A2912 B3510 B0818 B5806 B0724 B7802 B3561 B0728 B1585 B2730 B4030 B4604 B3513 B3809 B5403 B3529 A2617 A3110 B5128 B3504 B3924 B3539 B5511 B5103 B5109 B5604 B1575 A3007 A2627 B3536 A2437 B3805 B4812 A1113 B5518 B3803 A0313 B3514 B9502 A6816 B3808 A2911 A0108 B1524 A2606 B1578 B1538 A2504 B1813 B4407 A0244 B1556 B5307 A0272 A2608 B2723 A2913 A2619 A0231 B2721 B4051 B1551 B5112 B4035 B2701 A0209 B0806 B4418 A2454 A2902 B8301 B4057 B5520 A2903 A6824 B1545 A0275 B4417 A0114 B3548 A0322 B0732 B4059 B3918 A0241 B5132 A2444 B4430 B0739 A3006 B2724 B1818 A2418 A3103 B5514 B0723 A2456 B4060 B5308 B3559 B1547 B5616 B4205 A7402 B4421 B4001 B1597 B5101 B1308 B4406 B4015 A2309 B8102 B0720 B4813 B3557 A6812 A2419 A0277 B4703 B5605 B9506 B3545 A0261 A2615 B5504 B4436 A7403 B1502 B3935 A2312 B4441 A3307 B1592 B0703 B4803 B0708 B5133 B1587 A0225 B5311 B0745 B5519 A0263 B1562 A2458 A2501 B4020 B4009 A6803 A0278 A3004 B4606 B1574 B1535 B1583 B1820 B3909 A2427 B5208 A0234 B0715 B0743 B0709 B5305 A0236 A0274 A2310 B4901 B5706 A2441 B5126 A2426 A1102 A2446 A0307 B1554 A0318 A3001 B1588 B3524 B3936 B3519 B4603 A2442 B1812 A0227 A2424 B0741 A1117 B3546

B1513 B3811 A3106 B3912 B5102 A3107 B3709 A2314 A7411 A0216 A3108 A2405 B4052 B4408 B4426 A0302 B4036 B5901 A2904 A3001 B1515 B4422 A0273 B4403 B5207 B3514 B1578 A6824 B2724 B5605 A2458 B0709 A2442

HLA polymorphism!

X

Predicting the specificity Align A3001 (365) versus A3002 (365). Aln score 2445.000 Aln len 365 Id 0.9890 A3001 0 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFSTSVSRPGSGEPRFIAVGYVDDTQFVRFDSDAA ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: A3002 0 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFSTSVSRPGSGEPRFIAVGYVDDTQFVRFDSDAA A3001 65 SQRMEPRAPWIEQERPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSD :::::::::::::::::::::::::::: ::::: ::::::::::::::::::::::::::::: A3002 65 SQRMEPRAPWIEQERPEYWDQETRNVKAHSQTDRENLGTLRGYYNQSEAGSHTIQIMYGCDVGSD A3001 130 GRFLRGYEQHAYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARWAEQLRAYLEGTCVEWLRRY ::::::::::::::::::::::::::::::::::::::::::::: ::::::::::::::::::: A3002 130 GRFLRGYEQHAYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARRAEQLRAYLEGTCVEWLRRY A3001 195 LENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPA ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: A3002 195 LENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPA A3001 260 GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGA ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: A3002 260 GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGA A3001 325 VVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV :::::::::::::::::::::::::::::::::::::::: A3002 325 VVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV

HLA-A*3001

HLA-A*3002

 

HLA supertypes

Clustering in: O Lund et al., Immunogenetics. 2004 55:797-810

Supertype Selected allele A1 A*0101 A2 A*0201 A3 A*1101 A24 A*2401 A26 (new*) A*2601 B7 B*0702 B8 (new*) B*0801 B27 B*2705 B39(new*) B*3901 B44 B*4001 B58 B*5801 B62 B*1501

The truth about supertypes!

A2

A24

A26

A3

A1

Supertypes. What are they good for? •  Alleles with in supertypes present the

same set of peptides! •  Is this really so?

–  Less that 50% of A6802 binders will bind to A0201!

–  Less than 33% of A0201 binders will bind to A6802!

NetMHCpan - the method

NetMHC NetMHCpan

Example Peptide Amino acids of HLA pockets HLA Aff VVLQQHSIA YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.131751 SQVSFQQPL YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.487500 SQCQAIHNV YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.364186 LQQSTYQLV YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.582749 LQPFLQPQL YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.206700 VLAGLLGNV YFAVLTWYGEKVHTHVDTLVRYHY A0201 0.727865 VLAGLLGNV YFAVWTWYGEKVHTHVDTLLRYHY A0202 0.706274 VLAGLLGNV YFAEWTWYGEKVHTHVDTLVRYHY A0203 1.000000 VLAGLLGNV YYAVLTWYGEKVHTHVDTLVRYHY A0206 0.682619 VLAGLLGNV YYAVWTWYRNNVQTDVDTLIRYHY A6802 0.407855

NetMHCpan predicted motifs

www.cbs.dtu.dk/services/NetMHCpan

NetMHCpan output

SKADVIAKY. Known BoLA Tp5 CTL epitope

What is the %rank score?

•  Different MHC molecules have very different number of binders (affinity < 500 nM) –  Take 100,000 random natural 9mer peptides

• HLA-A02:01 will binding 5000 of these • HLA-A01:01 will bind 500 • HLA-C06:01 will bind 5

•  Is this biology? And does it mean that different MHC molecules present peptides at different binding thresholds?

•  The rank score levels this difference

What is the % rank score?

1% rank (percentile) score

1% rank (percentile) score

MHC binding, a (CBS) historical overview - From a few to all in 8 years •  1994, Bimas HLA-A2, B27 motif •  1999, SYFPEITHI •  2003, NetMHC-1.0 (neural network)

–  HLA-A0204, H-2Kk •  2004, NetMHC-2.0 (NN+PSSM)

–  12 HLA supertypes (NN) + 120 PSSM •  2008, NetMHC-3.0

–  43 NN’s, prediction for 8-11mer peptides, non-human primates •  2008, NetMHCpan-1.0

–  Pan-specific prediction for any HLA-A and HLA-B molecule with known protein sequence

•  2011, NetMHCpan-2.4 –  Pan-specific prediction to any MHC molecule with known

protein sequence. Predictions of 8-11mer peptides

I do

not

bel

ieve

I

do b

elie

ve

How good are we at predicting T cell epitopes? Evaluation. MHC ligands from SYFPEITHI

Sort on prediction

score

Top Rank: AUC=1.0

Random Rank: AUC=0.5

ROC curves

0 0.2 0.4 0.6 0.8 1False positive rate

0

0.2

0.4

0.6

0.8

1

Sens

itivi

ty

0 0.1 0.2 0.3 0.4False positive rate

0

0.2

0.4

0.6

0.8

1Se

nsiti

vity

AUC AUC 0.1

AUC 0.1 = 0.85 ~ 1% FP AUC 0.1 > 0.90 ~ 0.5% FP AUC = 0.95 ~ 1% FP AUC = 0.90 ~ 6% FP

Supertypes are no good – Knowing the exact restriction is critical

0.77

0.78

0.79

0.8

0.81

0.82

0.83

0.84

0.85

0.86

0.87

NetMHC (ST) NetMHCpan (ST) NetMHCpan

AUC0.1

0.825

0.85

0.875

0.9

0.925

0.95

0.975

1

HLA-A,B HLA-C

AUC NetMHCpan-2.2

NetMHCpan-2.8

•  2.2 training data 102 alleles, 1 HLA-C allele 3 peptides 3 binders •  2.8 training data 150 alleles, 10 HLA-C alleles 2248 peptides 957

binders

How good are we?

Prediction of 10- and 11mers using 9mer prediction tools

How can we benefit from the many 9mers when predicting binding of a

10mer?

Prediction of 10- and 11mers using 9mer prediction tools

Figure by Melani Zolfagharian Khodaie and Mikael Holm Thomsen

Performance for different peptide length

Pan-specific predictions

•  Pan-specific MHC peptide binding

prediction is the single most important recent (in silico) development for understanding presentation of T cell epitopes/ligands

T cell epitopes lacking MHC restriction

T cell epitopes lacking MHC restriction

Trimming prior to binding

BoLA Class I epitopes, Work by Ivan Morrison and co-workers

-10

0

10

20

30

40

50

60

70

80

90

100ng 10ng 1ng 0.1ng 0.01ng

SKFPKMRMGSKFPKMRMGKGSKFPKMRMGKKFPKMRMGKSKFPKMRMKFPKMRMG

SKFPKMRMG – Rank 7% SKFPKMRM - Rank 0.1%

Non-binding CTL epitopes

Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs.

Using NetMHCpan predictions

Tetra-mers is the way to go

Tetra-mer validations

Conclusions, part I

•  High resolution MHC typing is essential for understanding cellular immune-responses

•  Pan-specific MHC prediction method is the single most important (in silico) contribution to the understanding of cellular immune-responses

•  Proper definition of T cell epitopes is essential for epitope identification. –  All T cell epitopes bind to one or more of the host

MHC molecules –  Tetra-mer validation is the optimal solution for

understanding T cell immunogenicity (MHC restrictions element and minimal epitope)

What defines a T cell epitope?

•  Processing (Proteasomal cleavage, TAP)? •  Other proteases •  MHC binding •  MHC:peptide complex stability •  T cell repertoire •  Source protein abundance, cellular

location and function

MHC Class I Assay Technology

Affinity

D A

MHC

β2m

Peptide

Stability

Affinity

10-3

10-2

10-1

100

101

102

103

104

105

0.00

0.05

0.10

0.15

0.20

0.25High affinityLow affinity

KD = 10 nM

KD = 2000 nM

[Peptide], nM

[pM

HC

], nM

Stability

0 20 40 60100

1000

10000

StableUnstable

T½ = 1 hours

T½ = 25 hours

Δt, hours @ 37°C

CPM

SA

Harndahl M, Rasmussen M, Roder G, Buus S.J Immunol Methods. 2010 Oct 31.

MHC:peptide Stability

Two peptides, one T-cell epitope (FLTSVINRV, filled circles ) and a non-immunogenic peptide (NQNDNEETV, open circles) were compared with respect to affinity and stability. A) Peptide titration of two peptides binding with equal affinity to HLA-A*02:01, KD = 7nM. B) The stability of the same two peptides measured at 37°C. M.N Harndahl et al. Eur J Immunol. 2012 Jun;42(6)

MHC:peptide Stability

Stability of 12 pairs (paired on affinity) of immunogenic and non-immunogenic peptides.

M.N Harndahl et al. Eur J Immunol. 2012 Jun;42(6)

Predicting stability – affinity match peptides

NetMHCstab

NetMHCstab

0.8

0.85

0.9

0.95

1

AUC0.1 AUC0.1 AUC AUC

T-cell epitopes Ligands T-cell epitopes Ligands

Comb

NetMHCcons

NetMHCstab

Class II MHC binding

•  Binds peptides of length 9-18 (even whole proteins can bind!) •  Binding cleft is open •  Binding core is 9 aa •  Binding motif highly generate •  Amino acids flanking the binding core affect binding •  Peptide structure might determine binding

The problem. Where is the binding core?

PEPTIDE IC50(nM) VPLTDLRIPS 48000 GWPYIGSRSQIIGRS 45000 ILVQAGEAETMTPSG 34000 HNWVNHAVPLAMKLI 120 SSTVKLRQNEFGPAR 8045 NMLTHSINSLISDNL 47560 LSSKFNKFVSPKSVS 4 GRWDEDGAKRIPVDV 49350 ACVKDLVSKYLADNE 86 NLYIKSIQSLISDTQ 67 IYGLPWMTTQTSALS 11 QYDVIIQHPADMSWC 15245

NN-align

PEPTIDE Pred Meas VPLTDLRIPS 0.00 0.03 GWPYIGSRSQIIGRS 0.19 0.08 ILVQAGEAETMTPSG 0.07 0.24 HNWVNHAVPLAMKLI 0.77 0.59 SSTVKLRQNEFGPAR 0.15 0.19 NMLTHSINSLISDNL 0.17 0.02 LSSKFNKFVSPKSVS 0.81 0.97 GRWDEDGAKRIPVDV 0.39 0.45 ACVKDLVSKYLADNE 0.58 0.57 NLYIKSIQSLISDTQ 0.84 0.66 IYGLPWMTTQTSALS 1.00 0.93 QYDVIIQHPADMSWC 0.12 0.11

Predict binding affinity and core

Calculate prediction error

Update method to Minimize prediction

error

GRWDEDGAKRIPVDV

GRWDEDGAK RIP

RWDEDGAKR IPV G

WDEDGAKRI PVD GR

DEDGAKRIP VDV GRW

0.45

0.15

0.03

0.39

0.05

Nielsen et al. BMC Bioinformatics 2009, 10:296

NN-align NetMHCIIpan-2.0 TEPITOPE Allele # # bind P C C AU C P C C AU C AU C DRB1*0101 7 6 8 5 4 3 8 2 0.675 0.825 0.711 0.846 0.727 DRB1*0301 2 5 0 5 6 4 9 0.690 0.855 0.709 0.864 0.718 DRB1*0302 1 4 8 4 4 0.272 0.659 0.525 0.757 DRB1*0401 3 1 1 6 1 0 3 9 0.643 0.833 0.670 0.848 0.762 DRB1*0404 5 7 7 3 3 6 0.565 0.766 0.630 0.818 0.747 DRB1*0405 1 5 8 2 6 2 7 0.710 0.869 0.698 0.858 0.780 DRB1*0701 1 7 4 5 8 4 9 0.718 0.855 0.740 0.864 0.777 DRB1*0802 1 5 2 0 4 3 1 0.518 0.778 0.526 0.780 0.645 DRB1*0806 1 1 8 9 1 0.744 0.902 0.796 0.924 0.884 DRB1*0813 1 3 7 0 4 5 5 0.729 0.878 0.746 0.885 0.750 DRB1*0819 1 1 6 5 4 0.370 0.706 0.608 0.808 DRB1*0901 1 5 2 0 6 2 2 0.597 0.810 0.634 0.818 DRB1*1101 1 7 9 4 7 7 8 0.756 0.873 0.777 0.883 0.793 DRB1*1201 1 1 7 8 1 0.699 0.860 0.764 0.892 DRB1*1202 1 1 7 7 9 0.695 0.866 0.769 0.900 DRB1*1302 1 5 8 0 4 9 3 0.630 0.819 0.634 0.825 0.596 DRB1*1402 1 1 8 7 8 0.623 0.825 0.694 0.860 DRB1*1404 3 0 1 6 0.466 0.661 0.613 0.737 DRB1*1412 1 1 6 6 3 0.680 0.857 0.757 0.894 DRB1*1501 1 7 6 9 7 0 9 0.641 0.815 0.653 0.819 0.731 DRB3*0101 1 5 0 1 2 8 1 0.673 0.843 0.690 0.850 DRB3*0301 1 6 0 7 0 0.604 0.826 0.736 0.853 DRB4*0101 1 5 2 1 4 8 5 0.701 0.854 0.675 0.837 DRB5*0101 3 1 0 6 1 2 8 0 0.735 0.865 0.765 0.882 0.760

Performance

AUC ~ 0.85

MHC class II binding predictions

•  www.cbs.dtu.dk/services/NetMHCII •  15 DR, 5 DP, 6 DQ, 2 H-2

•  www.cbs.dtu.dk/services/NetMHCIIpan •  DR pan-specific

•  www.cbs.dtu.dk/services/NetMHCIIpan-XX •  DR, DQ, and DP pan-specific

NNAlign Server – Output

Binding motif of 14 HLA-DR molecules

Binding motif of HLA-DQ

DQA1*0101-DQB1*0501 DQA1*0102-DQB1*0602

DQA1*0301-DQB1*0302

DQA1*0401-DQB1*0402

DQA1*0501-DQB1*0201 DQA1*0501-DQB1*0301

Sidney J, et al.JI 2010 Oct 1;185(7)

Defining specificities for novel MHC molecules •  Pipeline

–  Construct recombinant MHC protein sequence –  Use Positional Scanning Combinatorial Peptide

Library (PSCPL) to get first estimate of motif –  Screen peptide database for potential

binders using PSCPL and NetMHCpan – Measure binding –  Retraining prediction methods

Defining specificities for novel MHC molecules

BoLA-HD6

Defining specificities for novel MHC molecules

•  Select ~100 peptide using PSCPL •  Select ^100 peptide using NetMHCpan NOT matching the

PSCPL motif

Defining specificities for novel MHC molecules

Peptide MHC Protein Npep NetMHCpan-2.0 NetMHC2.8+NetMHC-3.4 FP Alternative Rank

VGYPKVKEEML HD6 Tp1 2138 0.289 0.016 34

SHEELKKLGML T2b Tp2 662 0.136 0.021 14 EELKKLGML 0.000 DGFDRDALF T2b Tp2 662 0.498 0.216 143 GFDRDALF 0.041

KSSHGMGKVGK T2a Tp2 662 0.014 0.005 3 FAQSLVCVL T2c Tp2 662 0.000 0.023 15 QSLVCVLMK T2a Tp2 662 0.030 0.008 5

KTSIPNPCKW T2a Tp2 662 0.095 0.137 91 KTSIPNPCK 0.015 TGASIQTTL AW10 Tp4 2282 0.034 0.003 7 SKADVIAKY T5 Tp5 586 0.015 0.005 2 EFISFPISL T7 Tp7 2850 0.078 0.065 184 FISFPISL 0.001 CGAELNHFL AW10 Tp8 1726 0.130 0.007 12

AKFPGMKKSK D18.4 Tp9 1302 0.167 0.052 68 Ave 0.124 0.046 0.015

T-parva.

Defining specificities for novel MHC molecules

T-Annulata. Ivan Morrisson

Prediction of epitopes. Summary

•  Cytotoxic T cell epitope: (AROC ~ 0.95) •  Will a given peptide bind to a given MHC

class I molecule •  Helper T cell Epitope (AROC ~ 0.85)

•  Will a part of a peptide bind to a given MHC II molecule

•  B cell epitope (AROC ~ 0.80) •  Will a given part of a protein bind to one of

the billions of different B Cell receptors

Conclusions

•  Data is everything –  IEDB has been pivotal for moving the field of in silico

T cell epitope prediction forward •  Prior to the IEDB all analysis were

anecdotal –  Identifying features on a small scale, one ot

two proteins •  Only now with the large-scale data sets

can we see the big picture

Conclusions

•  Rational epitope discovery is feasible –  Prediction methods are an important guide for

epitope identification –  Given a protein sequence and an HLA molecule, we can

accurately predict the peptide binders •  Pan-specific MHC prediction method can deal

with the immense MHC polymorphism –  Supertypes are a nice simplification, but are often

wrong •  All CTL epitopes have specific MHC restrictions

matching their host –  There is no such thing as a non-binding CTL epitope

•  Computational methods are a highly cost-effect guide for rational epitope discovery