Genome – and HLA-wide scanning and validation

for cytotoxic CD8 T cell responses against

Mycobacterium tuberculosis

EU6-FRAME PROJECT

Sheila Tuyet Tang, Ph.D stud. Biologist

2. Vaccine4TBI) peptide prediction

II) Human cytotoxic CD8 T cell responses Human cytotoxic CD8 T cell responses against novel TB epitopesagainst novel TB epitopes

Outline

1. Introduction to Tuberculosis (TB)

Introduction

l

Introduction - Pandemic

Burden ranking 1.India 2.China 3.Indonesia 4.Nigeria 5.South Africa 6.Bangladesh 7.Pakistan 8.Ethiopia 9.Philippines 10.Kenya 11.Democratic Republic of

Congo 12.Russian Federation 13.Viet Nam 14.Tanzania 15.Uganda •Brazil 1.Afghanistan 2.Thailand 3.Mozambique 4.Zimbabwe 5.Myanmar 6.Cambodia

http://www.stoptb.org/countries/

What is tuberculosis?

Tuberculosis disease (TB) is caused by a bacterium

Mycobacterium tuberculosis (Mtb, also known as tubercle bacillus)

Eradication of tuberculosis is difficult since Mtb is able to remain in the host for a long time as latent or chronic state

Bacille Calmette Guérin (BCG), the current vaccine prevent the spread of TB but do not give full protection against pulmonary TB in adult( the initial infection)

BCG is given to infant in endemic areas

Thorough immunological knowledge of BCG and Mtb are lacking and necessary for the development of a more efficacious TB vaccine

www.sudantribune.com

Tuberculosis: Transmission

Exposure/Infection

10%

Clearance70%

TB

TB

TB

Infection

(2 bill,

~ 9 mill/yr)

Primary

infectionDeath ~2 mill

30% Latent TB

90%Reactivation

5-15%

First 2yrs highest chance of developing TB disease

Treatment with several drugs for 6 months or more can cure more than 95% of patients

If not treated 60 % dies

Cellular immune response

Tubercle bacilli enter aveoli

Infect* macrophag

es

Within few weeks

Th1 immune response

CD4+/CD8+ T

cells

Recruit to lung

Cytokines:

IL-2, TNFa and IFN-y

Tubercle bacilli

MACROPHAGE

Lysosome

+TB

ER

TB peptide

TCR

CD8 T cells

IFN-g

Granulomas prevent spread of infection by confining bacteria within a compact collection of several types of immune cells and activated macrophages

Role of these cells:

specific ways to isolate

inhibit the replication of, and destroy the bacteria

Cellular immune response

• Bacilli engulfed by macrophages

• Replicate within the macrophages 2-3 weeks before spreading throughout the body

• 95% contain the bacteria in macrophages

But due to Mtb. complex waxy cel wall the bacteria are protected inside the macrophages

http://www.granuloma.homestead.com/tb_microscopic.html

2. Vaccine4TBI) peptide prediction

II) Human cytotoxic CD8 T cell responses Human cytotoxic CD8 T cell responses against novel TB epitopesagainst novel TB epitopes

Outline

1. Introduction to Tuberculosis (TB)

Vaccine4TB:

Peptide prediction

Bioinformatics strategy

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

2. Predict binding to HLA supertypesA2 (A0201), A3 (A0301), B7 (B0702)

(coverage approx. 80% of the worlds population)

3. Predict proteasome cleavage and TAP binding

4. Calculate combined score using the method of Larsen et al., 2005

- have been used to predict and select potential epitopes based on a number of different criteria

TBVAC epitopes(14

)

Best predicted epitopes in the

entire proteom(67)

Proteins with CD8

epitopes(25)

Proteins from vaccine trials

(Michel Klein WP3)

Selected in proteins -previously

described by other groups to have CTL

epitopes

(Michel Klein WP3)

Selected as the best predicted

epitopes

In

the entire Mtb proteome

Selection criteria for potential vaccine candidates….(1)

3 epitopes/protein used in vaccine

trials (21)

Additional epitopes from proteins with CD8 epitopes (43)

Conserved

epitopes (69)

Selected as the most conserved epitopes with ANN prediction

>0.42(<500nM) binding affinity

Conservation defined from

multiple alignment that have the 9mer

epitope:

LAGS/Dos regulon sequences (71)

Selected as best predicted epitopes

in

the LAGS/Dos regulon

(Michel Klein

Fatima Kazi - WP3)

T cell epitopes

in

proteins with B cell epitopes (60)

These proteins found by Ugur

Sahims

group

WP4

Selection criteria for potential vaccine candidates….(2)

New set of peptides selected (after meeting in Leiden)

Exp verified secreted

epitopes (67)

Predicted secreted

epitopes (68)

Selected as best

predicted from

TubercuList secreted

epitopes (67)

proteins containing

a predicted signal peptide

or

predicted to be secreted by other

pathways

TBVAC epitopes (14)

File: TBVAC.epitopes.xls

protein residue peptide supertype affinity proteasome TAP Combined score

Ag85A 6 RVRGAVTGM B7 0.6485 0.9927 0.5970 20.266 Ag85A 78 ALYLLDGLR A3 0.4357 0.9692 16.900 13.165 Ag85A 113 MPVGGQSSF B7 0.4462 0.9977 24.020 16.705

Ag85B 9 RAWGRRLMI B7 0.5542 0.2306 10.120 16.971 Ag85B 30 GLAGGAATA A2 0.4276 0.9973 -0.6310 11.107 Ag85B 75 AVYLLDGLR A3 0.5697 0.6129 18.090 15.771

ESAT-6 28 LLDEGKQSL A2 0.4922 0.9976 0.8560 14.379

HBHA 49 RVEESRARL B7 0.4946 0.9987 12.470 16.729 HBHA 113 QSFEEVSAR A3 0.4322 0.6731 16.010 12.540 HBHA 153 KLVGIELPK A3 0.5658 0.9984 0.8230 15.128 HBHA 170 APAKKAAPA B7 0.5951 0.2629 -0.5250 16.393 HBHA 186 APAKKAAAK B7 0.4250 0.9677 0.4220 13.794

Mtb72f 13 WLLSVLAAV A2 0.5541 0.9942 0.3790 15.322 PPE 126 LLGQNTPAI A2 0.4217 0.8156 0.5360 12.034

3 epitopes /protein used in vaccine trials (21)

File: TBVACII.epitopes.xls Protein description residue peptide supertype affinity scaled_affinity

Proteasome cleavage TAP

Combine score

Ag85A 201 AMGPTLIGL A2 0.5115 11.119 0.9985 10.080 13.776

Ag85A 210 AMGDAGGYK A3 0.4388 11.928 0.9986 0.6150 14.133

Ag85B 15 LMIGTAAAV A2 0.6219 13.519 0.8923 0.6410 15.595

Ag85B 207 AMGDAGGYK A3 0.4388 11.928 0.9987 0.6150 14.134

ESAT-6 28 LLDEGKQSL A2 0.6187 13.451 0.9976 0.8560 15.932

ESAT-6 49 EAYQGVQQK A3 0.1930 0.5246 0.9216 0.5540 0.7265

ESAT-6 75 TISEAGQAM B7 0.3144 0.8722 0.9939 0.5400 10.834

Mtb72f 13 WLLSVLAAV A2 0.6701 14.568 0.9942 0.3790 16.495

Mtb72f 261 TVHIGPTAF A3 0.1868 0.5078 0.6391 25.370 0.8954

Mtb72f 310 APINSATAM B7 0.6627 18.387 0.9963 0.1830 20.092

PPE 180 GLLEQAAAV A2 0.6511 14.154 0.9990 0.1430 15.817

PPE 221 SSKLGGLWK A3 0.5365 14.585 0.8300 0.4110 16.302

PPE 270 APAAAAQAV B7 0.6787 18.830 0.9924 0.0660 20.394

HspX 13 SLFPEFSEL A2/A24 0.6291 13.675 0.9991 12.710 16.635

HspX 33 PTFDTRLMR A3 0.3366 0.9152 0.8937 12.270 11.904

HspX 32 RPTFDTRLM B7 0.5063 14.046 0.9956 0.2560 15.834

HBHA 63 DLPEQLTEL A2 0.3558 0.7734 0.9987 0.4760 0.9779

HBHA 153 KLVGIELPK A3 0.3714 10.096 0.9984 0.8230 12.540

TB10.4 4 IMYNYPAML A2 0.6064 13.183 0.9991 12.370 16.104

TB10.4 61 AMEDLVRAY A3 0.1325 0.3601 0.9751 29.970 0.8511

TB10.4 31 EIAVEQAAL B7 0.3178 0.8816 0.9738 10.500 11.484

Protein residue peptide supertype affinity scaled_affinity nM combined score

Rv0570 641 YVAAWKAKV A2 0.4416 10.674 420.634 11.325

Rv0570 450 GLAELLAAL A2 0.4752 11.487 292.428 13.960

Rv0570 419 FLDDVIDVS A2 0.5090 12.303 202.859 0.9823

Rv0570 663 QVLSYAAPK A3 0.4564 10.231 358.393 11.870

Rv0570 353 LVFLDTINR A3 0.4430 0.9932 414.311 13.262

Rv0570 518 VAPTGTISL B7 0.4529 12.635 372.226 15.407

Rv0570 496 FPAFTDSRF B7 0.5284 14.740 164.45 18.888

Rv0570 488 RLAEERGAF B7 0.5049 14.085 212.061 17.624

Rv0574c 328 RVSRMRLQR A3 0.4560 10.222 359.948 13.181

Rv1736c 611 ALWPFTRLV A2 0.5555 13.427 122.657 15.552

Rv1736c 494 GLSEGAYHV A2 0.5754 13.909 988.969 15.759

Rv1736c 623 SAPIGYLFR A3 0.4357 0.9768 448.362 11.475

Rv1736c 454 QLYESRLLR A3 0.5076 11.380 205.955 14.150

Rv1737c 316 GVFAWVARR A3 0.4309 0.9660 472.263 12.823

Rv1812c 348 AVYTEGWER A3 0.5125 11.489 195.321 15.162

Rv1997 836 GMFANRWII A2 0.4462 10.785 400.212 12.146

Rv1997 784 LLVASAWWL A2 0.5048 12.203 212.29 14.550

Rv1997 728 ILPTQILWI A2 0.4514 10.911 378.316 11.525

Rv1997 716 ILAAIAVGV A2 0.4451 10.759 405.003 12.650

Rv1997 659 AMGRGGTEV A2 0.4752 11.487 292.428 13.516

Rv1997 650 ALRQANIGV A2 0.4441 10.735 409.409 12.671

Rv1997 630 ALQARGHVV A2 0.5621 13.587 114.203 15.541

Rv1997 580 GLLDNTEPA A2 0.4249 10.270 503.939 11.034

Rv1997 745 GLMLAFEPK A3 0.6323 14.176 534.332 16.144

Rv1997 457 GTDHVVLAK A3 0.5166 11.583 186.845 12.973

Rv1997 415 GAMLVVAAK A3 0.4568 10.241 356.846 11.953

Rv1997 326 STTVICADK A3 0.5748 12.887 99.541 14.647

Rv1997 751 EPKEAGIMT B7 0.4653 12.979 325.491 12.861

Rv1997 726 LPILPTQIL B7 0.4503 12.562 382.846 15.067

Rv1997 718 AAIAVGVAL B7 0.4329 12.076 462.153 14.638

Rv1997 648 APALRQANI B7 0.6561 18.304 413.024 19.917

Rv1997 632 QARGHVVAM B7 0.6082 16.967 693.516 18.612

Rv1997 542 PPRAAAASA B7 0.5003 13.957 222.882 13.549

Rv1997 486 RPLDRATVL B7 0.6006 16.756 752.954 19.358

Continue....

LAGS/Dos regulon sequences (71)

from 13 proteins

Summary

• 505 peptides of the 800 to be selected in this project have been chosen – binding affinity have been measured and received in Leiden

What next ?

• new selection of the remaining 295 peptides have recently been made in Mainz

- Diagnostic peptides- New antigens dicovered by Ganymed- More LAGS

Verification of prediction - Wet lab experiments

pre

dic

tion

s

2. Vaccine4TBI) peptide prediction

II) Human cytotoxic CD8 T cell responses Human cytotoxic CD8 T cell responses against novel TB epitopesagainst novel TB epitopes

Outline

1. Introduction to Tuberculosis (TB)

Vaccines4TB

Genome- and HLA-wide scanning and validation of cytotoxc CD8 T cell responses against Mycobacterium tuberculosis

WP3 Human cytotoxic CD8 T cell responses WP3 Human cytotoxic CD8 T cell responses against novel TB epitopesagainst novel TB epitopes

Fatima Kazi, Pascale van Weeren, Corine Prins, Tom Ottenhoff, Michèl Klein

Department of Immunohematology and Blood TransfusionLeiden University Medical Center Leiden, The Netherlands

• Establishment of blood panel for screening peptides

• Design of assay for testing immunogenicity of peptides

• Analysis of CD8 response to peptides

Identification of novel CD8 T cell epitopesIdentification of novel CD8 T cell epitopes

• 41 Buffy coats – more coming up

• Approx 50% PPD+ (purified protein derivative cell culture extract used as the classical Tubeculin/Mantoux test)

• non-BCG vaccinated

• A2+, A3+, B7+ (full HLA-typing details)

• IFN- ELISA testing for PPD response

• 6 day assay• > 100 pg/ml = PPD+

Blood panelBlood panel

PBMC(Peripheral blood mononuclear cells)

CFSE labelled FACs

(cfse /CD56/CD3/CD8)

7 days

Acquire 10,000

events in live CD8

gate

Assay for peptide screeningAssay for peptide screening

1.5x105 cells/well+

10ug/ml peptide

0 200 400 600 800 1000FSC-H

R1

100 101 102 103 104

FL4-CD8 APC

R5

R1=gate on lymphocytes

FSC

SSC

CD8

SSC

+

R2=gate on CD8 T cells

Analysing peptide screeningAnalysing peptide screening

0 200 400 600 800 1000FSC-H

R1

100 101 102 103 104

FL3-CD3 Pcp

R4

R1=gate on lymphocytes (PBMCs)

CD3

CD8

MEDIUM

100 101 102 103 104

FL1-cfse

M1

PPD

100 101 102 103 104

FL1-cfse

M1

PHA

100 101 102 103 104

FL1-cfse

M1

Mtb/p51

100 101 102 103 104

FL1-cfse

M1

CFSE

CD8

SSC

FSC

MEDIUM

100 101 102 103 104

FL1-cfse

PPD

100 101 102 103 104

FL1-cfse

PHA

100 101 102 103 104

FL1-cfse

Mtb/p51

100 101 102 103 104

FL1-cfse

1.33%

9.96%

74.7%

9.73%

medium

peptide

PPD

PHA

Blood panelBlood panel

• 21 donors tested (cfse)• PPD+ = 9 donors (43%)• PPD- = 12 donors

• 7/21 (33%) donors responded to Mycobacteria specific Ags

CD8 T cell proliferation in response to PPD

0

2

4

6

8

10

12

14

16

18

PPD+ PPD-

% C

FS

E

PPD (ELISA) HLA-type PPD (cfse) Ag85B ESAT6 CFP101 donor BCPP1 ++ A2 - - - -2 donor BCPP2 + A3 - - - -3 donor BCPP3 - A24 donor BCPP4 + A35 donor 25 - A26 donor 26 - A27 donor 27 - A28 donor 28 - A39 donor 29 ++ B7

10 donor 30 - A211 donor 31 - A212 donor 32 ++ A213 donor 33 ++ A2 - - - -14 donor 34 ++ -15 donor 35 - A3/B716 donor 36 - A217 donor 37 + A2 - - - -18 donor 38 - A219 donor 39 - A320 donor 40 ++ A2 + - - -21 donor 41 ++ A2 - - - -22 donor 42 ++ A2 + + + -23 donor 43 ++ A2 - - - +24 donor 44 - A225 donor 45 + A2 + - - +26 donor 46 + B7 - - - -27 donor 47 ++ A3 + - - +28 donor 48 - A3/B729 donor 49 - A3/B7 - - - -30 donor 50 ++ A3 - - - -31 donor 51 A2 + - - -32 donor 53 A3/B7 + - - -33 donor 54 A3/B7 - - - -34 donor 55 A3 - - - -35 donor 56 A3 + - + -36 donor 57 A3/B7 - - - -37 donor 59 A3/B7 + + - +38 donor 60 B7 + + - +39 donor 61 A2/B740 donor 62 B741 donor 63 A2/B7

Epitope PredictionEpitope Prediction

TBVAC epitopes(14

)

Proteins with CD8

epitopes(25)

Proteins from vaccine trials

(Michel Klein WP3)

Selected in proteins -previously

described by other groups to have CTL

epitopes

(Michel Klein WP3)

3 epitopes/protein used in vaccine

trials (21)

Additional epitopes from proteins with CD8 epitopes (43)

PeptidesPeptides

TBVAC peptides: Ag85A/B, ESAT6, PPE, HBHA

TB-CD8 peptides: Mycobacteria tuberculosis H37Rv strainPeptides Sequence Antigen

1 (A2)TB-VAC #108-50 ESAT6 LLDEGKQSL2 (A2)TB-VAC #108-51 Ag85B GLAGGAATA3 (A2)TB-VAC #108-52 PPE LLGQNTPAI4 (A2)CD8 #108-63 H37Rv VLMGGVPGV SECRETED L-ALANINE DEHYDROGENASE ALD 5 (A2)CD8 #108-64 H37Rv GLLDVTDNV PROBABLE NAD-DEPENDENT GLUTAMATE DEHYDROGENASE GDH (NAD-GDH)6 (A2)CD8 #108-65 H37Rv SMLPPGYPV PROBABLE CONSERVED TRANSMEMBRANE PROTEIN 7 (A2)CD8 #108-66 H37Rv YLAEGHACL hypothetical protein Rv1461 8 (A2)CD8 #108-67 H37Rv HLSGPLAGV ISONIAZID INDUCTIBLE GENE PROTEIN INIB9 (A2)CD8 #108-68 H37Rv YIMKLHHLV DNA-DIRECTED RNA POLYMERASE

10 (A2)CD8 #108-69 H37Rv LLHDIGKPV hypothetical protein Rv2823c11 (A2)CD8 #108-70 H37Rv SLYEKSGSZ hypothetical protein Rv1461

1 (A3)TB-VAC #108-53 Ag85B AVYLLDGLR2 (A3)TB-VAC #108-54 HBHA KLVGIELPK3 (A3)TB-VAC #108-55 Ag85A ALYLLDGLR4 (A3)TB-VAC #108-56 HBHA QSFEEVSAR5 (A3)CD8 #108-71 H37Rv TVGYMYIMK DNA-DIRECTED RNA POLYMERASE 6 (A3)CD8 #108-72 H37Rv ATFEAVLAK hypothetical protein Rv0094c7 (A3)CD8 #108-73 H37Rv RTEILGLVK PROBABLE NAD-DEPENDENT GLUTAMATE DEHYDROGENASE GDH (NAD-GDH)8 (A3)CD8 #108-74 H37Rv ATIEAVLAK hypothetical protein Rv1148c9 (A3)CD8 #108-75 H37Rv KIMDYGKYK PROBABLE INITIATION FACTOR IF-3 INFC

10 (A3)CD8 #108-76 H37Rv QINELHHSK SUPEROXIDE DISMUTASE [FE] SODA11 (A3)CD8 #108-77 H37Rv KYFVRSTEK hypothetical protein Rv1461 12 (A3)CD8 #108-78 H37Rv GTFKSVAVK 60 KDA CHAPERONIN 2 GROEL2, HEAT SHOCK PROTEIN 6513 (A3)CD8 #108-79 H37Rv SVFPFDGTR hypothetical protein Rv3378c

1 (B7)TB-VAC #108-57 Ag85A RVRGAVTGM2 (B7)TB-VAC #108-58 HBHA APAKKAAPA3 (B7)TB-VAC #108-59 Ag85B RAWGRRLMI4 (B7)TB-VAC #108-60 HBHA RVEESRARL5 (B7)TB-VAC #108-61 Ag85A MPVGGQSSF6 (B7)TB-VAC #108-62 HBHA APAKKAAAK7 (B7)CD8 #108-80 H37Rv RARKRGITM hypothetical protein Rv1148c8 (B7)CD8 #108-81 H37Rv RARKRGITL hypothetical protein Rv0094c9 (B7)CD8 #108-82 H37Rv RPKPDYSAM hypothetical protein Rv3378c

10 (B7)CD8 #108-83 H37Rv KPIPHRTVL hypothetical protein Rv1461 11 (B7)CD8 #108-84 H37Rv RVRQAWDTL hypothetical protein Rv107312 (B7)CD8 #108-85 H37Rv TPVEHGLVL ISONIAZID INDUCTIBLE GENE PROTEIN INIB 13 (B7)CD8 #108-86 H37Rv KVRGRLLAL PROBABLE CONSERVED TRANSMEMBRANE PROTEIN 14 (B7)CD8 #108-87 H37Rv LPAQLTATA hypothetical protein Rv1148c

CD8 T cell proliferation to A2 motif bearing CD8 T cell proliferation to A2 motif bearing peptidespeptides

Donor 40

0

2

4

6

8

10

PP

D

#108-5

0

#108-5

1

#108-5

2

#108-6

3

#108-6

4

#108-6

5

#108-6

6

#108-6

7

#108-6

8

#108-6

9

#108-7

0

peptides

% C

FS

E

Donor 42

0

2

4

6

8

10

PP

D

#1

08

-50

#1

08

-51

#1

08

-52

#1

08

-63

#1

08

-64

#1

08

-65

#1

08

-66

#1

08

-67

#1

08

-68

#1

08

-69

#1

08

-70

peptides

% C

FS

E

donor 45

0

2

4

6

8

10

PP

D

#1

08

-50

#1

08

-51

#1

08

-52

#1

08

-63

#1

08

-64

#1

08

-65

#1

08

-66

#1

08

-67

#1

08

-68

#1

08

-69

#1

08

-70

peptides

% C

FS

E

Donor 33

0

2

4

6

8

10

PP

D

#1

08

-50

#1

08

-51

#1

08

-52

#1

08

-63

#1

08

-64

#1

08

-65

#1

08

-66

#1

08

-67

#1

08

-68

#1

08

-69

#1

08

-70

peptides

% C

FS

E

Peptide Name MHC Pep.no. Sequence 10/10-05(nM)

TB.o42.epitopes A2 11613 A2 11613 YMLDMTFPV 1

TB.o42.epitopes A2 11630 A2 11630 FLQGAKWYL 2

TB.o42.epitopes A2 11633 A2 11633 YLAENTFVV 3

TB.o42.epitopes A2 11623 A2 11623 WMYEGKHVL 5

TB.o42.cons A2 11699 A2 11699 LLDEPTNHL 17

TB.o42.cons A2 11687 A2 11687 FLFGDDDAL 20

TB.O42.CONS A2 11690 A2 11690 VLDEPSIGL 20

TB-CD8(A2) 10864 A2 10864 GLLDVTDNV 22

TB-CD8(A2) 10865 A2 10865 SMLPPGYPV 25

TB-CD8(A2) 10868 A2 10868 YIMKLHHLV 33

TB.o42.cons A2 11691 A2 11691 LLDEPTNNL 33

TB.o42.cons A2 11697 A2 11697 DMWEHAFYL 34

TB-CD8(A2) 10866 A2 10866 YLAEGHACL 46

TB.o42.cons A2 11693 A2 11693 RMWEFLDRL 56

TB-VAC(A2) 10850 A2 10850 LLDEGKQSL 86

TB.o42.cons A2 11689 A2 11689 LLLGGTSEI 105

TB-VAC(A2) 10852 A2 10852 LLGQNTPAI 116

TB-VAC(A2) 10851 A2 10851 GLAGGAATA 853

TB-CD8(A2) 10863 A2 10863 VLMGGVPGV non

TB-CD8(A2) 10867 A2 10867 HLSGPLAGV non

CD8 T cell proliferation to A3 motif bearing CD8 T cell proliferation to A3 motif bearing peptidespeptides

Donor 47

0

5

10

15

20

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

peptides

% C

FSE

Donor 53

0

5

10

15

20

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

peptides

% C

FSE

Donor 59

0

5

10

15

20

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

peptides

% C

FSE

Donor 49

0

5

10

15

20

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

peptides

% C

FSE

Donor 50

0

5

10

15

20

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

peptides

% C

FSE

A3-peptidesBinding versus peptide immunogenicity

TB.o42.cons A3 11705 A3 11705 RVYLQGHGY 3

TB.o42.cons A3 11706 A3 11706 TLLESFLFY 4

TB- CD8(A3) 10878 A3 10878 GTFKSVAVK 29

TB.o42.epitopes A3 11643 A3 11643 RVFGFRTAK 37

TB- CD8(A3) 10875 A3 10875 KIMDYGKYK 41

TB.o42.epitopes A3 11640 A3 11640 RVMPVFAFK 53

TB.o42.epitopes A3 11653 A3 11653 RVYLNGIGK 66

TB.o42.cons A3 11719 A3 11719 ALFDRPAFK 78

TB- CD8(A3) 10874 A3 10874 ATIEAVLAK 104

TB.o42.cons A3 11700 A3 11700 AVHGYYIGY 109

TB.o42.cons A3 11707 A3 11707 KLMALELFK 109

TB.o42.cons A3 11708 A3 11708 KLYPNVDFY 178

TB- CD8(A3) 10871 A3 10871 TVGYMYIMK 318

TB- CD8(A3) 10876 A3 10876 QINELHHSK 335

TB- CB8(A3) 10877 A3 10877 KYFVRSTEK 750

TB.o42.epitopes A3 11648 A3 11648 ATFEVFLAK 913

TB.o42.epitopes A3 11645 A3 11645 AVFPRYHPR 930

TB.o42.epitopes A3 11644 A3 11644 AVFDSFVER 1003

TB.o42epitopes A3 11639 A3 11639 AVMLVHTYY 1328

TB.o42.cons A3 11711 A3 11711 KIGEVIGPK 1500

TB.o42.cons A3 11716 A3 11716 QVFKGVVIR 3258

TB.o42.epitopes A3 11642 A3 11642 YVYPDNLPR 5618

TB.o42.epitopes A3 11634 A3 11634 AVFLSYIGY >5000

TB- VAC(A3) 10855 A3 10855 ALYLLDGLR non

TB- VAC(A3) 10856 A3 10856 QSFEEVSAR non

TB.o42.cons A3 11720 A3 11720 NIMEFCKAY non

3/53/5

donor donor recognitionrecognition

CD8 T cell proliferation to B7 motif bearing CD8 T cell proliferation to B7 motif bearing peptidespeptides

donor 53

0

5

10

15

20

25

PP

D

#108

-57

#108

-58

#10

8-59

#108

-60

#108

-61

#108

-62

#108

-80

#108

-81

#108

-82

#108

-83

#108

-84

#108

-85

#108

-86

#108

-87

peptides

% C

FS

E

donor 59

0

5

10

15

20

25

PP

D

#108

-57

#108

-58

#10

8-59

#108

-60

#108

-61

#108

-62

#108

-80

#108

-81

#108

-82

#108

-83

#108

-84

#108

-85

#108

-86

#108

-87

peptides

% C

FS

E

donor 60

0

5

10

15

20

25

PP

D

#108

-57

#108

-58

#10

8-59

#108

-60

#108

-61

#108

-62

#108

-80

#108

-81

#108

-82

#108

-83

#108

-84

#108

-85

#108

-86

#108

-87

peptides

% C

FS

E

B7-peptidesBinding versus peptide immunogenicity

TB.o42.epitopes B7 11669 B7 11669 SPRSRNRSF 0,3

TB-CD8(B7) 10886 B7 10886 KVRGRLLAL 0,4

TB.o42.epitopes B7 11658 B7 11658 RPRQRGIPF 0,4

TB-CD8(B7) 10881 B7 10881 RARKRGITL 0,4

TB.o42epitopes B7 11666 B7 11666 IPRLGGMAF 0,4

TB-VAC(B7) 10859 B7 10859 RAWGRRLMI 0,4

TB.o42.epitopes B7 11660 B7 11660 RPVFARLPF 0,6

TB.o42.cons B7 11735 B7 11735 GPAFVRTKL 0,9

TB.o42.cons B7 11729 B7 11729 GPRGRHVVL 1,0

TB.o42.epitopes B7 11667 B7 11667 RPRVAQLTF 1,2

TB-CD8(B7) 10883 B7 10883 KPIPHRTVL 1,4

TB.o42.cons B7 11724 B7 11724 RIRSERPAF 1,5

TB-VAC(B7) 10861 B7 10861 MPVGGQSSF 1,8

TB.o42.epitopes B7 11661 B7 11661 VPADHRLAF 1,9

TB.o42.epitopes B7 11656 B7 11656 RPAGARAAF 2,0

TB.o42.epitopes B7 11665 B7 11665 VPRENATAF 2,0

TB-CD8(B7) 10884 B7 10884 RVRQAWDTL 2,1

TB.o42.epitopes B7 11662 B7 11662 VPRDRNGTF 2,6

TB.o42.epitopes B7 11668 B7 11668 LPAEVRAAF 2,8

TB-CD8(B7) 10882 B7 10882 RPKPDYSAM 2,8

TB-VAC(B7) 10858 B7 10858 APAKKAAPA 3,0

TB-CD8(B7) 10885 B7 10885 TPVEHGLVL 3,2

TB.o42.cons B7 11746 B7 11746 TPRIANRLL 3,9

TB-CD8(B7) 10880 B7 10880 RARKRGITM 4,5

TB.o42.epitopes B7 11659 B7 11659 APRGFRAAF 4,6

TB.o42.epitopes B7 11657 B7 11657 APRARTAAF 5,6

TB.o42.cons B7 11731 B7 11731 IPAPGLGAL 5,9

TB-VAC(B7) 10857 B7 10857 RVRGAVTGM 6,5

TB.o42.cons B7 11741 B7 11741 MPRLSRNAA 10,3

TB-VAC(B7) 10860 B7 10860 RVEESRARL 38,0

TB.o42.cons B7 11733 B7 11733 TPALATRGF 318,0

TB-VAC(B7) 10862 B7 10862 APAKKAAAK 346,5

TB.o42.cons B7 11738 B7 11738 YPACEAIGL 436,0

TB-CD8(B7) 10887-2 B7 10887-2 LPAQLTATA >5000

0

2

4

6

8

10

PPD

#108

-50

#108

-51

#108

-52

#108

-63

#108

-64

#108

-65

#108

-66

#108

-67

#108

-68

#108

-69

#108

-70

A2 peptides

% C

FSE

Donor 43 BCPP1 donor 37

0

2

4

6

8

10

PPD

#108

-53

#108

-54

#108

-55

#108

-56

#108

-71

#108

-72

#108

-73

#108

-74

#108

-75

#108

-76

#108

-77

#108

-78

#108

-79

A3 peptides

% C

FSE

BCPP2 Donor 56

donor 46

0

2

4

6

8

10

PP

D

#108

-57

#108

-58

#10

8-59

#108

-60

#108

-61

#108

-62

#108

-80

#108

-81

#108

-82

#108

-83

#108

-84

#108

-85

#108

-86

#108

-87

B7 peptides

% C

FSE

Y

PPD-ve individuals do not respond to peptidesPPD-ve individuals do not respond to peptides

• PPD responses < 1% cfse+ve

• No responses to peptides

A2 donors A3 donors

B7 donors

Peptides Recognised by CD8 T cellsPeptides Recognised by CD8 T cells

1 (A2)TB-VAC #108-50 ESAT6

2 (A2)TB-VAC #108-51 Ag85B

3 (A2)TB-VAC #108-52 PPE

4 (A2)CD8 #108-63 H37Rv

5 (A2)CD8 #108-64 H37Rv

6 (A2)CD8 #108-65 H37Rv

7 (A2)CD8 #108-66 H37Rv

8 (A2)CD8 #108-67 H37Rv

9 (A2)CD8 #108-68 H37Rv

10 (A2)CD8 #108-69 H37Rv

11 (A2)CD8 #108-70 H37Rv

1 (A3)TB-VAC #108-53 Ag85B2 (A3)TB-VAC #108-54 HBHA3 (A3)TB-VAC #108-55 Ag85A4 (A3)TB-VAC #108-56 HBHA5 (A3)CD8 #108-71 H37Rv6 (A3)CD8 #108-72 H37Rv7 (A3)CD8 #108-73 H37Rv8 (A3)CD8 #108-74 H37Rv9 (A3)CD8 #108-75 H37Rv

10 (A3)CD8 #108-76 H37Rv11 (A3)CD8 #108-77 H37Rv12 (A3)CD8 #108-78 H37Rv13 (A3)CD8 #108-79 H37Rv

1 (B7)TB-VAC #108-57 Ag85A2 (B7)TB-VAC #108-58 HBHA3 (B7)TB-VAC #108-59 Ag85B4 (B7)TB-VAC #108-60 HBHA5 (B7)TB-VAC #108-61 Ag85A6 (B7)TB-VAC #108-62 HBHA7 (B7)CD8 #108-80 H37Rv8 (B7)CD8 #108-81 H37Rv9 (B7)CD8 #108-82 H37Rv

10 (B7)CD8 #108-83 H37Rv11 (B7)CD8 #108-84 H37Rv12 (B7)CD8 #108-85 H37Rv13 (B7)CD8 #108-86 H37Rv14 (B7)CD8 #108-87 H37Rv

A2 peptidesA3 peptides B7 peptides

4/11(36%)

9/13(70%)

6/14(43%)

SUMMARYSUMMARY

• 19/38 predicted peptides induced a CD8 proliferative response

• The frequency of proliferating CD8 T cell response to peptides varied between individuals

• Heterogenous response to peptides

•For A3-peptide responses, 3/5 donors recognised the same peptide: QINELHHSK (CD8-#108-76), suggesting it may be immunodominant peptide

SUMMARYSUMMARY

• 19/38 peptides induced a CD8 proliferative response

• The frequency of proliferating CD8 T cell response to peptides varied between individuals

• Heterogenous response to peptides

•For A3-peptide responses, 3/5 donors recognised the same peptide QINELHHSK (CD8-#10876), suggesting it may be immunodominant peptide

Best predicted epitopes in the

entire proteom(67)

Selected as the best predicted

epitopes

In

the entire Mtb proteome

Conserved

epitopes (69)

Screening of new set of peptides

Conservation defined from

multiple alignment that have the 9mer

epitope:

Selected as the most conserved epitopes with ANN prediction

>0.42(<500nM) binding affinity

Visiting Ph.D student

Donor 46(B7) PPD+

0.69%

0.61%

67.62%

77.21%

4.47%

1.74%

0.57% 3.37%

ppd+ in ELISA

CD

8

CFSE

Donor 29(HLA-B7) PPD+

CFSE

16.50%

6.33%

92.45%o.19%

1.01%

85.23% CD

8

Preliminary results:

LUMC No. TBp#

Peptide no. Peptide

Supertype

Affinity

Protein

Description

No. of pts reacted

37 11655 RPRLHSISF B7 0.7651 CAB02075.1 Rv2331 38 11656 RPAGARAAF B7 0.7630 CAA16655.1 Rv3190c 39 11657 APRARTAAF B7 0.7543 CAA17983.1 Rv3661 40 11658 RPRQRGIPF B7 0.7657 CAB09040.1 Rv1129c 41 11659 APRGFRAAF B7 0.7503 CAA18007.1 Rv3685c 42 11660 RPVFARLPF B7 0.7424 CAB08990.1 Rv0523c 43 11661 VPADHRLAF B7 0.7397 CAB01459.1 ilvG 44 11662 VPRDRNGTF B7 0.7308 CAB08502.1 Rv0920c 45 11663 VPAERRGVF B7 0.7339 CAB10058.1 nanT 46 11664 RPRCAYLPF B7 0.7296 CAB02176.1 Rv1394c 47 11665 VPRENATAF B7 0.7214 CAB07815.1 glgC 48 11666 IPRLGGMAF B7 0.7286 CAB06869.1 Rv1043c 49 11667 RPRVAQLTF B7 0.7367 CAB02537.1 Rv0051 50 11668 LPAEVRAAF B7 0.7147 CAA17441.1 nrdZ 1 51 11669 SPRSRNRSF B7 0.7141 CAB05430.1 gpdA2 1 52 11670 RPAFPAGTF B7 0.6970 CAA15987.1 Rv1457c 53 11671 RPAPARLPL B7 0.7566 CAA16264.1 Rv0083 1 54 11672 RPAIVVPAF B7 0.6932 CAB06686.1 Rv0259c 3 55 11673 RPAPATGAL B7 0.7580 CAB06268.1 pknK 2 56 11674 WPRHRRLSI B7 0.7767 CAA17488.1 Rv2184c 57 11675 RPRRASSPF B7 0.7111 CAA98319.1 Rv1543 58 11676 VPPFPRTAF B7 0.6965 CAB02041.1 Rv1491c 59 11677 AVREATAAF B7 0.6703 CAB07068.1 accA3

5/23 peptides B7 ~22%

proliferative CD8+Tcell response

Concerved hypothetical protein

SubCell: Cytoplamic

ProFun:

Structural protein involved in energy metabolism

Preliminary summery

For Best predicted epitopes in the entire proteom(67) peptide set:

No proliferative CD8+ T cell response were observed for A3 peptides from the set of peptide from : Best predicted epitopes in the entire proteom(67)

For the A2 responses => un-going experiments

22% of the B7 peptides were recognized in five of the donors

Two of the B7 peptides were recognized in >1 donors. RPAIVVPAF (#11672) in 3 and RPAPATGAL (#11673) in two of the donors

Preliminary summery

For conserved peptide set(69):

Just initiated the screening last week – finished proliferation assay for two donors:# 49 and 53 (HLA-A3/B7)

Feasible to finish before the end of my study-exchange at LUMC, Leiden, Netherlands

FUTURE WORK and MILESTONESFUTURE WORK and MILESTONES

• Assays for immunological monitoring of cytotoxic CD8 T cells(6 months)

• CFSE assay • Detected CD8 T cell responses to predicted epitopes• Cytokine production (on-going)

•Increase to 10 donor per supertype group

• CD8 T cell epitopes which can elicit HLA class-I restricted cytotoxic T cell responses with potential anti-microbial activity towards M. tuberculosis (6-8 months)

• CD8 T cell lines/clones• Chromium release assay /blocking Abs• Recognise endogenous peptides• Granzyme, CD69/CD40L

FUTURE WORK and MILESTONESFUTURE WORK and MILESTONES

• Understanding the repertoire of CD8 T cell responses induced during (i) natural infection with M. tuberculosis and (ii) BCG vaccination in a cross sectional and retrospective study.

• HLA-tetramers - A2• Frequency• Phenotype• Function

• A2 Transgenic mouse vaccine model (2/3 months : begin mid 2006)

• Test single peptides or multi- peptides as vaccine• Challenge with BCG/Mtb• Examine efficacy

•Tetramers•CD8 expansion•Bacterial burden

Papers planned:Papers planned:

1. A2/A3/B7 screening peptide responses:• proliferation of CD8 T cells• cytokine• selected peptides-MHC restriction and functional studies

2. A2 peptide/Transgenic mice:• Tetramer studies (frequency, phenotype)• Vaccine efficacy

3. CD8 repertoire of BCG vaccinated individuals:• cross-sectional/retrospective study

Acknowledgements

Prof. Dr. Tom Ottenhoff

Tuberculosis group

Immunohematology and Blood Transfusion

Leiden University Medical Center

Leiden, Netherlands

Proliferation assays, FACS analysis and IFN-g-ELISA

Leucosep Isolation of PBMC

Ole Lund, Ph.D.Associate Prof.

Immunological bioinformatic group

CBS-BioCentrum, DTU

Techinical University of Denmark

In silico peptide prediction, NetCTL

Fatima Kazi

Pascale van Weeren

And the rest of the Ottenhoff’s group

Michel Klein

Tom

Søren Buus, MD, Ph.D Prof.

IMMI, University of Copenhagen

MHC binding

Ugur Sahin

Ganymed

Genetic library