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TB vaccines and
diagnostics
Introduction
Global burden of tuberculosis
(The economist’s view)
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
Tuberculosis: Transmission
Primary
Infection
10%
Death ~2 mill
infection
(2 bill,
TB
TB
~ 9 mill/yr)
TB
Exposure/Infection
30%
90%
Latent TB
Reactivation
5-15%
First 2yrs highest chance of developing TB disease
Clearance
70%
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 HIVinfected 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 Africa
linked to HIV/AIDS
per 100 000 population
< 10
10 to 24
25 to 49
50 to 99
100 to 299
300 or more
No Estimate
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 2002
Stop TB Department
TB cases have been rising in
Africa and E Europe
Incidence rate (/100K/yr)
500
rise in incidence slowing
400
Africa - high HIV
300
Africa - low HIV
200
Eastern Europe
100
incidence falling
0
1990
1992
1994
1996
1998
2000
2002
2004
Stop TB Department
Development of Tuberculosis (the clinician’s
view)
Exposure
TB
(5%)
TB
(3%)
TB
(1%)
TB (less than
0.1%/year)
Health
y
(95%)
Health
y
(92%)
Health
y
(91%)
Healthy
(approx. 90%)
Year 1
Year 2
Year 3
thereafter
Response to infection (the immunologist’s
view)
67%
Initial
exposure
33%
These individuals do not
apparently skin-test
convert
Early bacterial growth
arrested at early time
point. May (or may
not) result in latent
infection
Early bacterial growth
not contained. Leads
to clinical illness
9%
Bacterial growth not
contained. Progressive
disease unless treated
These individuals generally
skin-test convert. They
often have characteristic
patterns on X-ray.
24%
Subsequent bacterial
growth contained.
Symptoms abate but
latent infection
established.
2%
Reactivation of latent
infection at a later
point in life
22%
Remain healthy
but latently
infected
TB vaccines (BCG)
• A 60-year follow-up study of American Indians reported the longterm 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
W Pacific
Died
Failed
SE Asia
Defaulted
Transfered
Europe
Not Evaluated
E Med
Americas
Africa
0
10
20
30
Percent of cohort
40
Stop TB Department
TB-specific antigens
M. tuberculosis
M.tuberculosis specific
Antigens (100+):
ESAT-6
Atypical
mycobacteria
Shared TB
complex
Antigens
(4000+)
BCG
Common
mycobacterial
Antigens
(1000+)
Ag85A/B
Rv2031c
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
People who fail to
control bacterial
replication become
ESAT+ and get TB
CFU
“Clinical disease”
threshold
“positivity”
threshold
People who fail to control
initial bacterial replication
become ESAT+, but if they
control later infection,
become latently infected
Time after infection
People who control initial
bacterial replication remain
ESAT-, and may or may not be
latently infected
Response to infection (the immunologist’s
view)
67%
Initial
exposure
33%
These individuals do not
apparently skin-test
convert or become
ESAT-6 positive
Early bacterial growth
arrested at early time
point. May (or may
not) result in latent
infection
Early bacterial growth
not contained. Leads
to clinical illness
9%
Subsequent bacterial
growth contained.
Symptoms abate but
latent infection
established.
22%
Remain healthy
but latently
infected
2%
Bacterial growth not
contained. Progressive
disease unless treated
These individuals generally
skin-test convert and
become ESAT-6 positive.
They often have
characteristic patterns on
X-ray.
24%
Immunologically, little is
known about these
individuals as they cannot be
distinguished from
uninfected individuals
Reactivation of latent
infection at a later
point in life
Immunologically these
individuals tend to express
elevated levels of IL-4 and in
advanced disease, decreased
IFN-g and IL-12
Immunologically, these individuals tend to
express elevated levels of IFN-g and IL-12,
and while IL-4 often remains slightly
increased, its antagonist IL-4d2 is greatly
increased
Bacterial response to infection
Acute infection
infection
Latent
Expression of early phase
genes
genes such as Ag85
and ESAT-6
Expression of late phase
such as a-crystallin and
the DosR regulon
Acute Disease
CFU
Reactivation of
infection
Immune
conversion
Latency?
Latent
infection
Elimination?
1-3
4-50
Years after exposure
Alteration of antigen recognition as
disease progresses (ET)
10000
1000
100
IFN-g (pg/ml)
10
TB
HHC
6000
LTBI
RV2031c response
ESAT-6 response in clinical groups
p<0.001
p<0.001
4000
R2 = -0.097
R2 = 0.3688
2000
10000
R2 = -1.0634
0
0
1000
2000
3000
ESAT-6 response
1000
100
10
TB
HHC
LTBI
Rv2031c response in clinical groups
4000
5000
CC
HHC
TB
Linear (CC)
Linear (HHC)
Linear (TB)
Alteration of antigen recognition as
disease progresses (Ga and NL)
Slope of linear regression
no. of spots from ESAT-6
stimulation vs Rv2031c
0.35
3.0
2.5
2.0
1.5
1.0
0.5
0.0
TB
HHC
CC
Clinical status of participants from Ethiopia
0.30
0.25
0.20
0.15
0.10
0.05
0.00
TB
HHC
0.0005
0.0000
TB
CC
Clinical status of participants from The Gambia
0.0010
Slope of linear regression
IFN-g from ESAT-6
stimulation vs Rv2031c
Slope of linear regression
no. of spots from ESAT-6
stimulation vs Rv2031c
3.5
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
People who fail to
control bacterial
replication become
ESAT+ and get TB
Time after infection
Rv2031c
ESAT-6
Rv2031c
ESAT-6
Rv2031c
ESAT-6
CFU
“Clinical disease”
threshold
“positivity”
threshold
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
Response to infection (the immunologist’s
view)
67%
Initial
exposure
33%
These individuals do not
apparently skin-test
convert or become
ESAT-6 positive
Early bacterial growth
arrested at early time
point. May (or may
not) result in latent
infection
Early bacterial growth
not contained. Leads
to clinical illness
9%
Subsequent bacterial
growth contained.
Symptoms abate but
latent infection
established.
22%
Remain healthy
but latently
infected
2%
Bacterial growth not
contained. Progressive
disease unless treated
These individuals generally
skin-test convert and
become ESAT-6 positive.
They often have
characteristic patterns on
X-ray.
24%
Immunologically, little is
known about these
individuals as they cannot be
distinguished from
uninfected individuals
Reactivation of latent
infection at a later
point in life
Immunologically these
individuals tend to express
elevated levels of IL-4 and in
advanced disease, decreased
IFN-g and IL-12. They weakly
recognise Rv2031c
Immunologically, these individuals tend to
express elevated levels of IFN-g and IL-12,
and while IL-4 often remains slightly
increased, its antagonist IL-4d2 is greatly
increased. They strongly recognise Rv2031c
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/povertydiseases/projects/110_en.htm
Sheila Tang
Cellular immune response
Tubercle bacilli
enter aveoli
IFN-g
Tubercle bacilli
TB peptide
Infect*
macrophages
TCR
Within few weeks
Lysosome
Th1 immune response
+TB
ER
CD4+/CD8+ T
cells
Recruit to lung
Cytokines:
IL-2, TNFa and
IFN-y
CD8 T cells
MACROPHAGE
Cellular immune response
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
http://www.granuloma.homestead.com/tb_microscopic.html
• 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
TB genome. Where to look?
Epitope Prediction
TBVAC epitopes(14)
Proteins with CD8
epitopes(25)
Proteins from vaccine trials
Selected in proteins -previously
described by other groups to have
CTL epitopes
(Michel Klein WP3)
(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 strain
1
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Peptides
(A2)TB-VAC #108-50
(A2)TB-VAC #108-51
(A2)TB-VAC #108-52
(A2)CD8 #108-63
(A2)CD8 #108-64
(A2)CD8 #108-65
(A2)CD8 #108-66
(A2)CD8 #108-67
(A2)CD8 #108-68
(A2)CD8 #108-69
(A2)CD8 #108-70
(A3)TB-VAC #108-53
(A3)TB-VAC #108-54
(A3)TB-VAC #108-55
(A3)TB-VAC #108-56
(A3)CD8 #108-71
(A3)CD8 #108-72
(A3)CD8 #108-73
(A3)CD8 #108-74
(A3)CD8 #108-75
(A3)CD8 #108-76
(A3)CD8 #108-77
(A3)CD8 #108-78
(A3)CD8 #108-79
(B7)TB-VAC #108-57
(B7)TB-VAC #108-58
(B7)TB-VAC #108-59
(B7)TB-VAC #108-60
(B7)TB-VAC #108-61
(B7)TB-VAC #108-62
(B7)CD8 #108-80
(B7)CD8 #108-81
(B7)CD8 #108-82
(B7)CD8 #108-83
(B7)CD8 #108-84
(B7)CD8 #108-85
(B7)CD8 #108-86
(B7)CD8 #108-87
ESAT6
Ag85B
PPE
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
Ag85B
HBHA
Ag85A
HBHA
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
Ag85A
HBHA
Ag85B
HBHA
Ag85A
HBHA
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
Sequence
LLDEGKQSL
GLAGGAATA
LLGQNTPAI
VLMGGVPGV
GLLDVTDNV
SMLPPGYPV
YLAEGHACL
HLSGPLAGV
YIMKLHHLV
LLHDIGKPV
SLYEKSGSZ
AVYLLDGLR
KLVGIELPK
ALYLLDGLR
QSFEEVSAR
TVGYMYIMK
ATFEAVLAK
RTEILGLVK
ATIEAVLAK
KIMDYGKYK
QINELHHSK
KYFVRSTEK
GTFKSVAVK
SVFPFDGTR
RVRGAVTGM
APAKKAAPA
RAWGRRLMI
RVEESRARL
MPVGGQSSF
APAKKAAAK
RARKRGITM
RARKRGITL
RPKPDYSAM
KPIPHRTVL
RVRQAWDTL
TPVEHGLVL
KVRGRLLAL
LPAQLTATA
Antigen
SECRETED L-ALANINE DEHYDROGENASE ALD
PROBABLE NAD-DEPENDENT GLUTAMATE DEHYDROGENASE GDH (NAD-GDH)
PROBABLE CONSERVED TRANSMEMBRANE PROTEIN
hypothetical protein Rv1461
ISONIAZID INDUCTIBLE GENE PROTEIN INIB
DNA-DIRECTED RNA POLYMERASE
hypothetical protein Rv2823c
hypothetical protein Rv1461
DNA-DIRECTED RNA POLYMERASE
hypothetical protein Rv0094c
PROBABLE NAD-DEPENDENT GLUTAMATE DEHYDROGENASE GDH (NAD-GDH)
hypothetical protein Rv1148c
PROBABLE INITIATION FACTOR IF-3 INFC
SUPEROXIDE DISMUTASE [FE] SODA
hypothetical protein Rv1461
60 KDA CHAPERONIN 2 GROEL2, HEAT SHOCK PROTEIN 65
hypothetical protein Rv3378c
hypothetical protein Rv1148c
hypothetical protein Rv0094c
hypothetical protein Rv3378c
hypothetical protein Rv1461
hypothetical protein Rv1073
ISONIAZID INDUCTIBLE GENE PROTEIN INIB
PROBABLE CONSERVED TRANSMEMBRANE PROTEIN
hypothetical protein Rv1148c
Analysing peptide screening
CD8+ antigen
Specific cells
R4
CD8
100
CD3
101
102
FL 3-CD3 Pcp
103
CD4+CD8 cells
104
peptides
donor 45
4
0
peptides
#108-65
#108-64
#108-63
#108-70
0
#108-70
2
#108-69
2
#108-69
4
#108-68
6
#108-68
8
#108-67
8
#108-67
Donor 33
#108-66
peptides
#108-66
10
#108-65
peptides
#108-64
10
#108-52
Donor 40
#108-63
0
#108-52
2
#108-51
8
#108-51
8
#108-50
4
PPD
6
% CFSE
10
#108-50
6
% CFSE
#108-70
#108-69
#108-68
#108-67
#108-66
#108-65
#108-64
#108-63
#108-52
#108-51
#108-50
PPD
% CFSE
10
PPD
#108-70
#108-69
#108-68
#108-67
#108-66
#108-65
#108-64
#108-63
#108-52
#108-51
#108-50
PPD
% CFSE
CD8 T cell proliferation to A2 motif bearing
peptides
Donor 42
6
4
2
0
Peptide Name
TB.o42.epitopes A2 11613
MHC
A2
TB.o42.epitopes A2 11630
TB.o42.epitopes A2 11633
A2
A2
11630
11633
TB.o42.epitopes A2 11623
TB.o42.cons A2 11699
TB.o42.cons A2 11687
TB.O42.CONS A2 11690
A2
A2
A2
A2
11623
11699
11687
11690
TB-CD8(A2) 10864
TB-CD8(A2) 10865
A2
A2
10864
10865
TB-CD8(A2) 10868
TB.o42.cons A2 11691
TB.o42.cons A2 11697
TB-CD8(A2) 10866
TB.o42.cons A2 11693
TB-VAC(A2) 10850
TB.o42.cons A2 11689
TB-VAC(A2) 10852
TB-VAC(A2) 10851
TB-CD8(A2) 10863
TB-CD8(A2) 10867
A2
A2
A2
A2
A2
A2
A2
A2
A2
A2
A2
10868
11691
11697
10866
11693
10850
11689
10852
10851
10863
10867
Pep.no.
11613
Sequence 10/10-05(nM)
YMLDMTFPV
1
FLQGAKWYL
2
YLAENTFVV
3
WMYEGKHVL
5
LLDEPTNHL
17
FLFGDDDAL
20
VLDEPSIGL
20
GLLDVTDNV
22
SMLPPGYPV
25
YIMKLHHLV
33
LLDEPTNNL
33
DMWEHAFYL
34
YLAEGHACL
46
RMWEFLDRL
56
LLDEGKQSL
86
LLLGGTSEI
105
LLGQNTPAI
116
GLAGGAATA
853
VLMGGVPGV
non
HLSGPLAGV
non
peptides
0
peptides
peptides
Donor 50
10
peptides
#108-79
#108-78
#108-77
#108-76
#108-75
#108-74
#108-73
#108-72
Donor 53
#108-71
#108-56
#108-55
15
#108-54
% CFSE
15
#108-53
PPD
#108-79
#108-78
#108-77
#108-76
#108-75
#108-74
15
#108-79
0
#108-73
#108-72
#108-71
20
#108-78
0
#108-56
20
#108-77
0
#108-55
20
#108-76
5
#108-75
5
#108-54
10
#108-74
5
#108-73
15
#108-72
Donor 59
#108-71
15
#108-56
20
#108-55
20
#108-54
peptides
#108-53
10
% CFSE
5
#108-53
% CFSE
Donor 47
PPD
#108-79
#108-78
#108-77
PPD
0
#108-76
5
#108-75
#108-79
#108-78
#108-77
#108-76
#108-75
#108-74
#108-73
#108-72
#108-71
10
#108-74
#108-73
#108-72
#108-71
#108-56
#108-55
#108-54
#108-53
PPD
% CFSE
#108-56
#108-55
#108-54
#108-53
PPD
% CFSE
CD8 T cell proliferation to A3 motif bearing
peptides
Donor 49
10
A3-peptides. Binding versus peptide immunogenicity
T B.o42.c ons A3 11705
A3
11705
T B.o42.c ons A3 11706
A3
11706
T B- CD8(A3) 10878
A3
10878
T B.o42.epitopes A3 11643
A3
11643
T B- CD8(A3) 10875
A3
10875
T B.o42.epitopes A3 11640
A3
11640
T B.o42.epitopes A3 11653
A3
11653
T B.o42.c ons A3 11719
A3
11719
T B- CD8(A3) 10874
A3
10874
T B.o42.c ons A3 11700
A3
11700
T B.o42.c ons A3 11707
A3
11707
T B.o42.c ons A3 11708
A3
11708
T B- CD8(A3) 10871
A3
10871
T B- CD8(A3) 10876
A3
10876
T B- CB8(A3) 10877
A3
10877
T B.o42.epitopes A3 11648
A3
11648
T B.o42.epitopes A3 11645
A3
11645
T B.o42.epitopes A3 11644
A3
11644
T B.o42epitopes A3 11639
A3
11639
T B.o42.c ons A3 11711
A3
11711
T B.o42.c ons A3 11716
A3
11716
T B.o42.epitopes A3 11642
A3
11642
T B.o42.epitopes A3 11634
A3
11634
T B- VAC(A3) 10855
A3
10855
T B- VAC(A3) 10856
A3
10856
T B.o42.c ons A3 11720
A3
11720
RVYLQGHGY
TLLESFLFY
GTFKSVAVK
RVFGFRTAK
KIMDYGKYK
RVMPVFAFK
RVYLNGIGK
ALFDRPAFK
ATIEAVLAK
AVHGYYIGY
KLMALELFK
KLYPNVDFY
TVGYMYIMK
QINELHHSK
KYFVRSTEK
ATFEVFLAK
AVFPRYHPR
AVFDSFVER
AVMLVHTYY
KIGEVIGPK
QVFKGVVIR
YVYPDNLPR
AVFLSYIGY
ALYLLDGLR
QSFEEVSAR
NIMEFCKAY
3
4
29
37
41
53
66
78
104
109
109
178
318
335
750
913
930
1003
1328
1500
3258
5618
>5000
non
non
non
3/5
donor
recognition
0
peptides
peptides
donor 60
25
20
15
10
5
peptides
#108-87
#108-86
#108-85
#108-84
#108-83
#108-82
#108-81
#108-80
#108-62
#108-61
#108-60
#108-59
#108-58
donor 53
#108-87
#108-86
0
#108-85
0
#108-84
5
#108-57
20
#108-83
20
PPD
25
#108-82
10
% CFSE
25
#108-81
#108-87
#108-86
#108-85
#108-84
#108-83
#108-82
#108-81
#108-80
#108-62
#108-61
#108-60
#108-59
#108-58
#108-57
15
#108-80
#108-62
#108-61
#108-60
#108-59
#108-58
#108-57
PPD
% CFSE
5
PPD
% CFSE
CD8 T cell proliferation to B7 motif bearing
peptides
donor 59
15
10
B7-peptides
Binding versus peptide immunogenicity
TB.o42.epitopes B7 11669
TB-CD8(B7) 10886
TB.o42.epitopes B7 11658
TB-CD8(B7) 10881
TB.o42epitopes B7 11666
TB-VAC(B7) 10859
TB.o42.epitopes B7 11660
TB.o42.cons B7 11735
TB.o42.cons B7 11729
TB.o42.epitopes B7 11667
TB-CD8(B7) 10883
TB.o42.cons B7 11724
TB-VAC(B7) 10861
TB.o42.epitopes B7 11661
TB.o42.epitopes B7 11656
TB.o42.epitopes B7 11665
TB-CD8(B7) 10884
TB.o42.epitopes B7 11662
TB.o42.epitopes B7 11668
TB-CD8(B7) 10882
TB-VAC(B7) 10858
TB-CD8(B7) 10885
TB.o42.cons B7 11746
TB-CD8(B7) 10880
TB.o42.epitopes B7 11659
TB.o42.epitopes B7 11657
TB.o42.cons B7 11731
TB-VAC(B7) 10857
TB.o42.cons B7 11741
TB-VAC(B7) 10860
TB.o42.cons B7 11733
TB-VAC(B7) 10862
TB.o42.cons B7 11738
TB-CD8(B7) 10887-2
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
B7
11669
10886
11658
10881
11666
10859
11660
11735
11729
11667
10883
11724
10861
11661
11656
11665
10884
11662
11668
10882
10858
10885
11746
10880
11659
11657
11731
10857
11741
10860
11733
10862
11738
10887-2
SPRSRNRSF
KVRGRLLAL
RPRQRGIPF
RARKRGITL
IPRLGGMAF
RAWGRRLMI
RPVFARLPF
GPAFVRTKL
GPRGRHVVL
RPRVAQLTF
KPIPHRTVL
RIRSERPAF
MPVGGQSSF
VPADHRLAF
RPAGARAAF
VPRENATAF
RVRQAWDTL
VPRDRNGTF
LPAEVRAAF
RPKPDYSAM
APAKKAAPA
TPVEHGLVL
TPRIANRLL
RARKRGITM
APRGFRAAF
APRARTAAF
IPAPGLGAL
RVRGAVTGM
MPRLSRNAA
RVEESRARL
TPALATRGF
APAKKAAAK
YPACEAIGL
LPAQLTATA
0,3
0,4
0,4
0,4
0,4
0,4
0,6
0,9
1,0
1,2
1,4
1,5
1,8
1,9
2,0
2,0
2,1
2,6
2,8
2,8
3,0
3,2
3,9
4,5
4,6
5,6
5,9
6,5
10,3
38,0
318,0
346,5
436,0
>5000
B7 peptides
#108-87
#108-86
#108-85
#108-84
2
2
0
0
10
8
4
2
0
Y
6
• No responses to peptides
#108-79
#108-78
#108-77
#108-76
8
#108-75
#108-74
BCPP2
#108-73
#108-72
#108-71
donor 37
#108-56
4
#108-55
A2 donors
#108-54
6
% CFSE
10
#108-53
PPD
#108-70
#108-69
#108-68
#108-67
#108-66
8
#108-83
#108-82
#108-65
BCPP1
#108-81
#108-80
#108-64
#108-63
#108-52
#108-51
#108-50
PPD
% CFSE
Donor 43
#108-62
#108-61
#108-60
#108-59
#108-58
#108-57
PPD
% CFSE
PPD-ve individuals do not respond to peptides
10
Donor 56
A3 donors
6
4
A2 peptides
A3 peptides
donor 46
B7 donors
• PPD responses < 1% cfse+ve
Peptides Recognised by CD8 T cells
B7 peptides
A3 peptides
A2 peptides
1
2
3
4
5
6
7
8
9
10
11
(A2)TB-VAC #108-50
(A2)TB-VAC #108-51
(A2)TB-VAC #108-52
(A2)CD8 #108-63
(A2)CD8 #108-64
(A2)CD8 #108-65
(A2)CD8 #108-66
(A2)CD8 #108-67
(A2)CD8 #108-68
(A2)CD8 #108-69
(A2)CD8 #108-70
4/11
(36%)
ESAT6
Ag85B
PPE
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
1
2
3
4
5
6
7
8
9
10
11
12
13
(A3)TB-VAC #108-53
(A3)TB-VAC #108-54
(A3)TB-VAC #108-55
(A3)TB-VAC #108-56
(A3)CD8 #108-71
(A3)CD8 #108-72
(A3)CD8 #108-73
(A3)CD8 #108-74
(A3)CD8 #108-75
(A3)CD8 #108-76
(A3)CD8 #108-77
(A3)CD8 #108-78
(A3)CD8 #108-79
Ag85B
HBHA
Ag85A
HBHA
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
9/13
(70%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
(B7)TB-VAC #108-57
(B7)TB-VAC #108-58
(B7)TB-VAC #108-59
(B7)TB-VAC #108-60
(B7)TB-VAC #108-61
(B7)TB-VAC #108-62
(B7)CD8 #108-80
(B7)CD8 #108-81
(B7)CD8 #108-82
(B7)CD8 #108-83
(B7)CD8 #108-84
(B7)CD8 #108-85
(B7)CD8 #108-86
(B7)CD8 #108-87
Ag85A
HBHA
Ag85B
HBHA
Ag85A
HBHA
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
H37Rv
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
Michel Klein
Tuberculosis group
Immunological bioinformatic group
Immunohematology and Blood Transfusion
CBS-BioCentrum, DTU
Leiden University Medical Center
Techinical University of Denmark
Leiden, Netherlands
In silico peptide prediction, NetCTL
Proliferation assays, FACS analysis and IFN-g-ELISA
Leucosep Isolation of PBMC
Tom
Søren Buus, MD, Ph.D Prof.
IMMI, University of Copenhagen
MHC binding
Ugur Sahin
Fatima Kazi
Ganymed
Pascale van Weeren
Genetic library
And the rest of the Ottenhoff’s group