Integration of predictions - cbs.dtu.dk · Integration of predictions ... Technical University of...

Integration of predictions

selection of T cell epitopes by use of an

integrative approach

Wednesday, 9 June 2010

Outline

_Summary of biological processes preceding a CTL response

_Summary of the methods available for predicting

the processes

_Case study

• Obtaining data and generate method

• Application of the method


MHC-I molecules present peptides on the surface of most cells


Healthy cell

Virus-infectedcell

MHC-I

CTL response


Healthy cell

Virus-infectedcell

MHC-I

CTL response


Epitope based vaccine

_Immunization with whole protein induce immune response against immunodominant epitopes.

• Pathogen may evolve to mutate its immunodominat epitopes (immune escape)

_Epitope based vaccine:

• Immunity against epitopes that are subdominant, highly conserved, and critical to the life cycle pf the pathogen

• Epitopes are specifically selected



_Focusing on conserved epitopes• Occuring in 70-80% of different strains

_Example:

• Epitope A conserved in 70% of pathogen strain

• Epitope B conserved in 70% of pathogen strain



>Haemagglutinin - Influenza A virus (A/chicken/Jilin/9/2004(H5N1))MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI



>Haemagglutinin - Influenza A virus (A/chicken/Jilin/9/2004(H5N1))MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSNHEASSGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI




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NetCTLpan

_Goal:

• Predict epitopes as accurate as possible

_Why not just use NetMHC?

• It doesn´t take into account proteosomal cleavage and TAP transport. They are predicted on different peptide length -> this information is lost in netMHC

• Integrated results are proven to be better!


NetCTLpan

_Proteasomal cleavage: NetChop, Artificial Neural Network

Nielsen et al (2005). Immunogenetics 57: 33-41

_TAP transport efficiency:

• Consensus TAP matrix

Peters et al (2003). J. Immunol. 171. 1741-9

_MHC class I affinity:

• NetMHCpan, Artificial Neural Network

Hoof et al. (2009) Immunogenetics 61: 1-13

Combining preexisting prediction methods


Dataset

_NetCTL MV Larsen ~1000 epitopes from SYFPEITHI


Dataset


_NetCTLpan 1300 9mer SYFPEITHI epitopes ~1600 epitopes 8,10,11,12mer


Dataset


_NetCTLpan 1300 9mer SYFPEITHI epitopes ~1600 epitopes 8,10,11,12mer

_The source proteins are found in SwissProt and split into all possible 9mers.

_9mers not known to be epitopes are considered non-epitopes

_optimize the way that epitopes are ”ranked higher”


Hypothetical protein: MPADNSELVISISAL

Peptide Proteasomal Cleavage TAP Transport MHC-I Affinity Combined NetCTL score

MPADNSELV 0.43 -0.70 0.04 0.06

PADNSELVI 0.22 0.93 0.54 0.48

ADNSELVIS 0.76 -0.85 0.33 0.32

DNSELVISI 0.88 1.32 0.22 0.36

NSELVISIS 0.99 2.91 0.50 0.67

SELVISISA 0.01 -0.63 0.22 0.14

ELVISISAL 0.95 -0.24 0.18 0.27




MPADNSELV 0.43 -0.70 0.04 0.06

PADNSELVI 0.22 0.93 0.54 0.48

ADNSELVIS 0.76 -0.85 0.33 0.32

DNSELVISI 0.88 1.32 0.22 0.36

NSELVISIS 0.99 2.91 0.50 0.67

SELVISISA 0.01 -0.63 0.22 0.14

ELVISISAL 0.95 -0.24 0.18 0.27




MPADNSELV 0.43 -0.70 0.04 0.06

PADNSELVI 0.22 0.93 0.54 0.48

ADNSELVIS 0.76 -0.85 0.33 0.32

DNSELVISI 0.88 1.32 0.22 0.36

NSELVISIS 0.99 2.91 0.50 0.67

SELVISISA 0.01 -0.63 0.22 0.14

ELVISISAL 0.95 -0.24 0.18 0.27

Calculation of the combined NetCTL score:0.15 * Prot + 0.05 * TAP + 1* MHC-I




MPADNSELV 0.43 -0.70 0.04 0.06

PADNSELVI 0.22 0.93 0.54 0.48

ADNSELVIS 0.76 -0.85 0.33 0.32

DNSELVISI 0.88 1.32 0.22 0.36

NSELVISIS 0.99 2.91 0.50 0.67

SELVISISA 0.01 -0.63 0.22 0.14

ELVISISAL 0.95 -0.24 0.18 0.27

Calculation of the combined NetCTL score:0.15 * Prot + 0.05 * TAP + 1* MHC-I


Comparison


Comparison


NetChop C-term

TAP MHC-I Integrated method

0.789 0.786 0.933 0.948

AUC-values

What does the numbers mean? For an experimentalist aiming at identifying new epitopes he/she has to test 30% fewer peptides to find the same amount of epitopes

Comparison – small numbers, but large difference


DTADVVHPF 0.87872ETIRNIPHL 0.865696

DIIMPPLPF 0.838448

MVNQEMLNM 0.822888

EVSSCIPKI 0.657364DVVHPFFLA 0.656453YLYKHDIIM 0.550085ELAENILKW 0.494939ILAEESYLY 0.389173EIYQKNLEI 0.316719EAVYHRYMV 0.286636

Integratedmethod

NetMHC


Webtool


Application

_HIV 114/184 epitopes (62%)

_Influenza 21/131 epitopes (16%)

_West Nile Virus

26/175 epitopes (15%)


Technical University of Denmark - DTUDepartment of systems biology

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Sylvester-Hvid et al, Tissue Antigens. 2004

Case I: SARS



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Supertype Method Number tested Binding <500nM A1 ANN 15 13 A2 ANN 15 12 A3 ANN 15 14 A24 - 0 - B7 ANN 15 10 B27 PSSM 13 2 B44 - 0 - B58 PSSM 15 13 B62 PSSM 14 12

75% of predicted peptides were binding with an IC50 <500 nM

Sars virus HLA ligands


CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS


Wang et al., Vaccine 2007

Case II:Discovery of conserved Class I epitopes in Human Influenza

Virus H1N1



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Pox Strategy



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Bioinformatics search strategy

1. Read a GenBank file.• Extract the proteins from the file.

2. Calculate conservation• Find homologues to all proteins in user specified database with Blast

(using E<0.05 as threshold ), and build a multiple alignment based on the Blast alignments using the program mview.

• Cluster sequences using 98% sequence identity as a threshold for similarity, and calculate the probability for the different amino acids at each position

• Calculate probability p9 of conservation of 9mers for all 9mers in the reference strain

3. Calculate combined score using the NetCTL algorithm



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>polymerase“

MERIKELRDLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITAD

KRIMEMIPERNEQGQTLWSKTNDAGSDRVMVSPLAVTWWNRNGPTTSTVHYPKVYKTYFE

KVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSE

SQLTITKEKKEELQDCKIAPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCW

EQMYTPGGEVRNDDVDQSLIIAARNIVRRATVSADPLASLLEMCHSTQIGGIRMVDILRQ

NPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTNGSSVKKEEEVLTGNLQTLKIKVHEGY

EEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNF

...

and 9 other proteins

MERIKELRDERIKELRDLRIKELRDLMIKELRDLMSKELRDLMSQELRDLMSQSLRDLMSQSRRDLMSQSRTDLMSQSRTRLMSQSRTRE

and 4376 other 9mers

Proteins

9mer peptides

>Segment 1agcaaaagcaggtcaattatattcaatatggaaagaataaaagaactaagagatctaatgtcgcagtcccgcactcgcgagatactaacaaaaaccactgtggatcatatggccataatcaagaaatacacatcaggaagacaagagaagaaccctgctctcagaatgaaatggatgatggcaatgaaatatccaatcacagcagacaagagaataatggagatgattcctgaaaggaat

and 13350 other nucleotides on 8 segments

Genome

Influenza A virus



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Influenza

• We selected the Influenza peptides with the top 15 combined scores with conservation p9 > 70% for each pf the 12 supertypes.

• 180 peptides selected

• 167 tested for binding and CTL response

• 89 (53%) of the influenza peptides tested have an affinity better than 500nM



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Measured affinity of the predicted peptide binders

HLA supertype KD ≤ 50 50<KD ≤ 500 500< KD ≤ 5000 KD > 5000 Non-binders TOTAL

A1 5 3 8 A2 5 5 1 3 14 A3 4 5 5 1 15 A24 9 3 3 15 A26 1 2 2 8 13 B7 6 6 1 3 1 17 B8 1 3 1 7 12

B27 4 5 2 2 2 15 B39 1 2 5 1 5 14 B44 1 2 3 1 8 15 B58 1 10 1 1 1 14 B62 1 12 2 15 Total 34 55 23 16 39 167

KD, the equlibrium dissociation constant; a measurement of the affinity of peptides binding to the relevant HLA molecules in nM. The lower the value, the stronger the binding

34+55 = 89 binders (53%)

Results



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•Measure number of white blood cells that in vitro produce interferon-γ in response to a peptide

•A positive result means that the immune system have earlier reacted to the peptide (during a response of a vaccine/natural infection)

FLDVMESM

Two spots

FLDVMESM

FLDVMESMFLDVMESMFLDVMESM

FLDVMESM

ELISPOT assay



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•35 normal healthy blood donors

•35-65 years old

•Expected to have had influenza more than 3 times

•Vaccinated as children against

•Smallpox

•TB

•HLA typed by SBT for HLA A and B

Donors



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Protein Supertype Affinity PA SA Comb Cons_9

Polymerase PB1 A1 nt 0.62 3.25 3.74 0.90Nucleoprotein NP A1 nt 0.55 2.89 3.35 0.80Polymerase PB1 A2 51 0.46 1.09 1.25 0.76Polymerase PB1 A26 5 11.3 1.59 2.05 0.98Polymerase PB1 B27 246 0.43 1.54 2.02 0.97Nucleoprotein NP B27 37 0.37 1.34 1.72 0.87Matrix protein M1 B39 nt 7.08 0.99 1.29 0.84Nucleoprotein NP B58 41 0.44 1.51 1.64 0.99Polymerase PB1 B62 178 0.40 0.96 1.47 0.97Polymerase PB1 B62 87 0.46 1.08 1.45 0.92Polymerase PB1 B7 5 0.67 1.87 2.08 0.99Polymerase PA B8 nb 7.81 1.05 1.28 0.77

Protein: Common name for proteinSupertype: HLA supertype that the peptide is predicted to bind toAffinity: Measured affinity (kD in nM)PA: Predicted affinitySA: Scaled affinityComb: Combined score calculated as in Larsen et al., 2005cons_9: Fraction of clusters (with more than 98% sequence identity) that contain the 9mernb: non bindernt: not tested

Peptides positive in ELISPOT assay



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Protein CTL+ Binders Polymerase PB2 0 24 Polymerase PB1 7 43 Polymerase PA 1 30 Hemagglutinin HA 0 1 Nucleoprotein NP 3 12 Neuraminidase NA 0 3 Matrix protein M2 0 1 Matrix protein M1 1 6 Nuclear export protein NEP 0 0 Nonstructural protein NS1 0 0

Peptides positive in ELISPOT assay



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Conservation of epitopes

• Number of 9mers 100% conserved:

• 10/12 conserved in Influenza A virus (A/Goose/Guangdong/1/96(H5N1))

• 11/12 conserved in Influenza A virus (A/chicken/Jilin/9/2004(H5N1))



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EPISELECT

Genotype 1

Genotype 2

Genotype 3

Genotype 4

Genotype 5

Genotype 6

S elect peptide with maximal coverage

S elect peptide with maximal coverage preferring lowes t covered s trains

Top Scoring Peptides

Repeat until the desired number of peptides is selected



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HIV - RESULTS• 185 peptides were selected for testing• 31 patients with different ethnicity infected with different

subtypes were tested for CTL response. • 30 out of 31 had response towards at least one peptide.• 116 of our 185 peptides (62%) did induce a response in at least

one patient. • 21 of the recognized peptides induced a response in >4

patients (13% of the study subjects).


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