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
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