TB vaccines and diagnostics. Introduction An estimated 15 million active cases, leading to….. An...

TB vaccines anddiagnostics

Introduction

An estimated 15 million active cases, leading to…..

An estimated 9 million new infections

Approx 2 million deaths

Approx 2 Billion USD in direct control costs

And an uncounted indirect cost in lost lives and productivity

Global burden of tuberculosis(The economist’s view)

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

TB

• At present tuberculosis kills more people than any other infectious disease about 3 million people a year, including almost 300,000 children under 15, and is producing over 7,000 deaths and over 24,000 new cases every day.

• No new drugs have been added to the first-line treatment regimen for TB for >30 yrs.

• There is a clear synergy between M. tuberculosis and HIV, and active TB increases HIV-related immunodeficiency and mortality.• TB remains the largest attributable cause of death in HIV-infected individuals and is responsible for 32% of the deaths of HIV-infected individuals in Africa.

• The neediest populations, in countries where TB incidence is highest, do not have access to treatment and, furthermore, in many cases, anti-TB drugs are ineffective.

Highest TB rates per capita are in Africalinked to HIV/AIDS

25 to 49

50 to 99

100 to 299

< 10

10 to 24

300 or more

No Estimate

per 100 000 population

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.

© WHO 2002Stop TB DepartmentStop TB Department

TB cases have been rising in Africa and E Europe

0

100

200

300

400

500

1990 1992 1994 1996 1998 2000 2002 2004

Incidence rate (/100K/yr)

Africa - high HIV

Africa - low HIV

Eastern Europe

incidence falling

rise in incidence slowing

Stop TB DepartmentStop TB Department

Exposure

Healthy

(95%)

Year 1 Year 2 Year 3 thereafter

TB (5%)

Healthy

(92%)

TB (3%)

Healthy

(91%)

TB (1%)

Healthy (approx. 90%)

TB (less than

0.1%/year)

Development of Tuberculosis (the clinician’s view)

Early bacterial growth arrested at early time point. May (or may not) result in latent infection

Initial exposure Early bacterial

growth not contained. Leads to clinical illness

Subsequent bacterial growth contained. Symptoms abate but latent infection established.

Bacterial growth not contained. Progressive disease unless treated

Reactivation of latent infection at a later point in life

33%

67%

9%

24%

2%

Remain healthy but latently infected

22%

These individuals do not apparently skin-test convert

These individuals generally skin-test convert. They often have characteristic patterns on X-ray.

Response to infection (the immunologist’s view)

TB vaccines (BCG)

• A 60-year follow-up study of American Indians reported the long-term efficacy of BCG to be 52%. The reasons for the low efficacy of the BCG vaccine may be generic differences in the BCG strains, differences in immunological properties of study populations or exposure to environmental factors such as mycobacteria.

• Today, most of the world's population is vaccinated with BCG. It is generally accepted that BCG protects against childhood TB but this immunity wanes with age, resulting in no or insufficient protection against TB.

• Among new vaccine candidates are live attenuated Mycobacterium tuberculosis vaccines, recombinant BCG, DNA vaccines, subunit vaccines and fusion proteins with novel adjuvants and delivery systems. Some of these vaccines are now in clinical trials.

Reasons for failure:Treatment outcomes are worst in

Africa and Europe

0 10 20 30 40

Africa

Americas

E Med

Europe

SE Asia

W Pacific

Percent of cohort

Died

Failed

Defaulted

Transfered

Not Evaluated

Stop TB DepartmentStop TB Department

TB-specific antigens

M. tuberculosis

Atypical mycobacteria

BCG

M.tuberculosis specific Antigens (100+):

ESAT-6

Common mycobacterial Antigens (1000+)

Ag85A/BRv2031c

Shared TB complex Antigens (4000+)

TB diagnostics

• Left untreated, each person with active TB disease will infect on average between 10 and 15 people every year.

Risk of TB in ESAT+ healthy contacts from Ethiopia

Doherty et al., JCM , Feb. 2002

PPD: skin test (Purified Protein Derivative)

High ESAT-6 immune reactivity reflects high levels of M. tuberculosis replication

CF

U

Time after infection

“positivity” threshold

People who fail to control bacterial replication become ESAT+ and get TB

People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected

People who control initial bacterial replication remain ESAT-, and may or may not be latently infected

“Clinical disease” threshold







33%

67%

9%

24%

2%


22%

These individuals do not apparently skin-test convert or become ESAT-6 positive

These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray.

Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN- and IL-12

Immunologically, these individuals tend to express elevated levels of IFN- and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-42 is greatly increased

Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals


Acute infection Latent infection

Expression of early phase Expression of late phase genesgenes such as Ag85 such as -crystallin and and ESAT-6 the DosR regulon

CF

U

Acute Disease

Reactivation of infection

Years after exposure

1-3 4-50

Elimination?

Latent infection

Immune conversion

Latency?

Bacterial response to infection

10

100

1000

10000

TB HHC LTBI

p<0.001

p<0.001

Rv2031c response in clinical groups

10

100

1000

10000

TB HHC LTBI

ESAT-6 response in clinical groups

IFN- (pg/ml)

Alteration of antigen recognition as disease progresses (ET)

R2 = -0.097

R2 = 0.3688

R2 = -1.0634

0

2000

4000

6000

0 1000 2000 3000 4000 5000ESAT-6 response

RV2031c response

CCHHCTBLinear (CC)Linear (HHC)Linear (TB)

Alteration of antigen recognition as disease progresses (Ga and NL)

Slo

pe

of

line

ar

reg

ress

ion

no

. o

f sp

ots

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

TB HHC CC0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Clinical status of participants from The Gambia

TB HHC CC0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Clinical status of participants from Ethiopia

Slo

pe

of

line

ar

reg

ress

ion

no

. o

f sp

ots

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

Slo

pe

of

line

ar

reg

ress

ion

IF

N-

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

0.0000

0.0005

0.0010

TB TST+Clinical status of participants from the Netherlands

A lowered ratio of ESAT-6 immune reactivity to Rv2031c reactivity reflects a shift from acute to latent TB

CF

U

Time after infection

“positivity” threshold

People who fail to control bacterial replication become ESAT+ and get TB

People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected

People who control initial bacterial replication remain ESAT-, and may or may not be latently infected

“Clinical disease” threshold

ES

AT

-6

Rv

20

31

c ES

AT

-6

Rv

20

31

c

ES

AT

-6

Rv

20

31

c







33%

67%

9%

24%

2%


22%

These individuals do not apparently skin-test convert or become ESAT-6 positive

These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray.

Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN- and IL-12. They weakly recognise Rv2031c

Immunologically, these individuals tend to express elevated levels of IFN- and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-42 is greatly increased. They strongly recognise Rv2031c

Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals


Summary

• Immunity to M. tuberculosis is dependent on the generation of Th1 immunity, particularly IL-12, IFN-g and TNF-a

• As the bacteria persists in the face of this Th1 response, it begins to alter its proteome towards a pattern characteristic of latency, downregulating some antigens, upregulating others

• At the same time, a Th2 response seems to develop

• Susceptibility to infection therefore appears to correlate not so much with inability to generate a Th1 response, as with inability to maintain it long term, or perhaps inability to direct it to relevant antigens

• We are starting to see evidence that M. tuberculosis-derived antigens are driving some of this Th2 response

Identification of CD8+ epitopes

• Vaccines4TB• Vaccines against tuberculosis are urgently needed. CD4 T cell responses play a major role in the generation of acquired immunity against M. tuberculosis. However, it is increasingly recognised that CD8 cytotoxic T cells (CTL) also contribute to optimal host defence against mycobacteria.

• Unfortunately, relatively few CTL responses against TB have been identified.

• http://ec.europa.eu/research/health/poverty-diseases/projects/110_en.htm

Sheila Tang

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 cell wall the bacteria are protected inside the macrophages

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

TB genome. Where to look?

Epitope 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)

Peptides

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 Rv14611(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

Analysing peptide screening

100 101 102 103 104

FL3-CD3 Pcp

R4

CD3

CD8

CD4+CD8 cells

CD8+ antigenSpecific cells

CD8 T cell proliferation to A2 motif bearing peptides

Donor 40

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#108-70peptides

% CFSE

Donor 42

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

peptides

% CFSE

donor 45

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

peptides

% CFSE

Donor 33

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

peptides

% CFSE

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 peptides

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

% CFSE

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#108-79peptides

% CFSE

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

% CFSE

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#108-79peptides

% CFSE

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#108-79peptides

% CFSE

A3-peptides. Binding 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 peptides

donor 53

0

5

10

15

20

25

PPD

#108-57#108-58 #108-59#108-60#108-61#108-62#108-80#108-81#108-82#108-83#108-84#108-85#108-86#108-87

peptides

% CFSE

donor 59

0

5

10

15

20

25

PPD

#108-57#108-58 #108-59#108-60#108-61#108-62#108-80#108-81#108-82#108-83#108-84#108-85#108-86#108-87

peptides

% CFSE

donor 60

0

5

10

15

20

25

PPD

#108-57#108-58 #108-59#108-60#108-61#108-62#108-80#108-81#108-82#108-83#108-84#108-85#108-86#108-87

peptides

% CFSE

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#108-70A2 peptides

% CFSE

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#108-79A3 peptides

% CFSE

BCPP2 Donor 56

donor 46

0

2

4

6

8

10

PPD

#108-57#108-58 #108-59#108-60#108-61#108-62#108-80#108-81#108-82#108-83#108-84#108-85#108-86#108-87

B7 peptides

% CFSE

Y

PPD-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 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%)

SUMMARY

• 19/38 predicted peptides induced a CD8

proliferative response

• The frequency of proliferating CD8 T cell

response to peptides varied between

individuals

• Heterogeneous response to peptides

• For A3-peptide responses, 3/5 donors

recognised the same peptide: QINELHHSK

(CD8-#108-76), suggesting it may be

immunodominant peptide

Acknowledgements. Vaccines4TB

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

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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