Download IFN-γ + CD4 T Cells

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A novel vaccine construct comprising of lipidated
peptide protects against Mycobacterium
tuberculosis by bolstering enduring memory
T cell response
Javed N Agrewala, PhD, FNA
CSIR-Institute of Microbial Technology
Chandigarh, INDIA
[email protected]
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Vaccines-Hyd-2.11..15
Theme of the study
To develop a unique and novel
construct that can function as a
vaccine in TB-endemic regions,
where BCG has failed
Failure of BCG
Despite nine decades of BCG vaccination, TB continues to be a
major global health challenge.
Clinical trials worldwide have proved the inadequacy of the
BCG vaccine in preventing the manifestation of pulmonary TB
in adults.
Ironically, the efficacy of BCG is poorest in TB-endemic
areas.
Factors
such
as
non-tuberculous
or
environmental
mycobacteria and helminth infestation have been suggested to
limit the efficacy of BCG.
Hence, in high TB-burden regions, radically novel strategies
of vaccination are urgently required.
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Gowthaman & Agrewala. Trends Mol Medicine 2012
IMTECH
Antigen presentation
Recent series of studies have suggested that M. tuberculosis and
environmental mycobacteria (EnM) actively inhibit bacterial antigen
processing and presentation by MHC class I and class II pathways,
thus slowing the emergence of protective adaptive immunity (Wolf et
al. 2007; Soualhine et al. 2007; Gehring et al. 2003, Tobian et al,
2003).
BCG and EnM specifically blocks the surface export of mature class
II molecules on APCs (Soualhine et al. 2007).
M. tuberculosis and EnM impairs in vivo antigen processing by DCs
(Wolf et al. 2007).
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Gowthaman & Agrewala. Crit Rev Microbiol. 2014
IMTECH
What ever may be the reason for BCG failure!
 But the fact remains that more people have been
vaccinated with BCG than any other vaccine.
 Still tuberculosis continues to kill some 3 million
people annually.
 2 billion people worldwide are infected with M.
tuberculosis.
Thus, the protective efficacy of the BCG vaccine remains doubtful!
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Any vaccine that require
minimum antigen processing will
work in TB-endemic population
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Peptides can overcome the problem of
antigen processing.
Since they can directly bind to MHC
molecules and require minimum antigen
processing.
Can peptide based vaccine be successful
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choice
in TB-endemic regions?
7
IMTECH
PEPTIDE VACCINES

Comprise of synthetic peptides that represent
immunodominant epitopes

T cells and B cells epitope can be precisely selected

Epitopes are accurately defined; can avoid autoreactive
portions in the antigen

Requires no or minimum processing
Х
Fails to elicit immune response in genetically diverse human
population
Х
Requires adjuvant
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Th -KI
S
I
S
I
Pam2Cys
Th epitope 1: LFAAFPSFAGLRPT FDTRLM
Th epitope 2: SEFAYGSFVRTVSLPVGADE
-K- CTL
I
S
I
S
I
Pam2Cys
Th -K- CTL
I
S
I
S
I
Pam2Cys
Schematic representation of the epitope-based vaccine
candidate. The vaccine will contain Th and CTL epitopes.
In all cases the T-helper (Th) epitope occupies N-terminal
position and is separated from the cytotoxic T cell
epitope (CTL) epitope by a single lysine (K) residue.
Where lipid is attached, this is done through ε-amino
group of the lysine residue such that the self-adjuvant
lipid, linked through two serine residues (S), forms a
branch between the Th and CTL epitope
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Gowthaman & Agrewala. Trends Mol Medicine 2012
IMTECH
Why peptide vaccine may be better than BCG?
• Can overcome problem of antigen processing
due to EM/Mtb/BCG, since no processing is
required.
• No fear of preformed immune response
generated
due
to
exposure
to
BCG/EM/Mtb.
• Selective
epitopes,
autoreactive/suppressive
antigen.
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can
portions
avoid
in the
10
IMTECH
Whether the construct
influences the
maturation of DC?
F91: free peptide
L91: lipidated peptide
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F91
L91
Number of conjugates
Untreated
12
F91
10
L91
8
6
4
2
0
1DC:2T Cells
1DC:3T Cells
1DC:4T Cells
1DC:>5T Cells
L91 augments IL-12 production by DCs
Unstimulated
L91
3
55
T cells exposed to L91 treated DCs secreted more IFN-γ
30000
IFN-γ (pg/ml)
T Cell Proliferation
15000
Gowthaman & Agrewala. J Infect Dis. 2011
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0
Medium
Pam2Cys
F91
L91
L91 Enhances Activation and Maturation of DCs
CD40
100
CD80
CD86
90
Medium
Percentage
80
70
60
50
40
30
20
LPS
10
0
L91
P91
p91
LPS
Untreated
L91
L91
CD74
CD74
IAd
IAd
P91
CD80
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CD40
CD86
13
Whether the peptides works
across MHC barriers?
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IMTECH
Immunization
L21, P21, L91, P91
[BALB/c, C57BL/6, C3He, FVB]
Booster 7-14d
Sacrificed 240 days
In vitro challenge with peptides
Free peptides
were emulsified
in IFA.
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T cell response
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IMTECH
Peptide works across MHC barriers
T cell proliferation
30000
30000
BALB/c
C57BL/6
20000
20000
10000
10000
0
0
0.003
0.01
0.03
0.1
0.3
1
3
0.003
0.01
0.03
0.1
0.3
1
3
T cell proliferation
60000
C3He
4000
FVB
40000
3000
2000
20000
1000
0
0
0.003
0.01
0.03
L21
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0.1
0.3
1
3
0.003
Peptides (nmoles)
L91
0.01
P91
0.03
0.1
0.3
1
3
P21
16
L91 induces proliferation and activation of CD4 T cells
Control
4.1%
Pam2Cys
4.2%
F91
16%
L91
52%
T Cell Proliferation
Control
Pam2Cys
4
4
F91
L91
6
13
CD44
A
B
CD69+ CD44+ CD4 T
cells (Fold Change)
CD69
3.5
3
2.5
2
1.5
1
17
0.5
0
Control
Pam2Cys
F91
L91
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Immunization with L91 elicits mainly secretion of IFN-γ
A
A
IL-4 (O.D)
F91
L91
0.25
0.2
n.s
0.15
0.1
0.05
Control
IL-5 (pg/ml)
C
Pam2Cys
F91
200
L91
n.s
150
B5
4
3
2
1
0
Control
Pam2Cys
F91
L91
100
50
Control
Pam2Cys
F91
0.3
0.2
0.15
Control
Pam2Cys
F91
Pam2Cys
11
n.s
0.25
0.1
Control
L91
TNF-α
IL-10 (O.D)
25
10
CD4
0
D
L91
IFN-γ
Pam2Cys
IFN-γ+ CD4 T cells (Fold Change)
IFN-γ (pg/ml)
0
F91
8
8
15000
Control
B
Pam2Cys
Medium
30000
L91
11
F91
L91
15
36
IFN-γ
18
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L91 immunization evokes IL-17 secretion
Medium
Immunization
L91
L91
1.4
9.9
2.0
1.5
1.4
3.1
Pam2Cys
2.3
2.4
F91
2.6
2.8
Saline
CD4
BCG
IL-17
1.5
3.4
L91 immunization engenders T cell memory
Control
11
F91
12
4
34
6
CD44
3
L91
CD62L
Fold induction
B
3.5
3
2.5
2
Central Memory
Effector Memory
1.5
1
0.5
0
Untreated
Control
F91
L91
F91
L91
A
B
CD127 (Fold induction)
93
130
315
CD127
6
5
4
3
2
1
0
Control
F91
L91
20
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L91 Vaccination Protects Mice Against Mtb
Experimental design for protection studies in mice. Different groups of BALB/c mice were immunized with L91 or controls (F91, LH, BCG and placebo).
Peptides were given at a dose of 20 nmol (primary immunization) and 10 nmol (booster). BCG (106 CFU/animal) was given as control. Mice were rested
for 75 days. Animals were challenged with a low dose aerosol of M. tb H37Rv (~100 CFU/mouse) 75 days post immunization. Thirty days after infection,
animals were sacrificed and studied for immune response and pathology.
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L91 evokes enduring memory CD4 T cell response in Mtb infected mice
Untreated
274
Pam2Cys
F91
263
Untreated
488
525
CD69
Pam2Cys
30
L91
27
24
20
36
Central: CD44hi/CD6Lhi
Effector: CD44hi/CD62Llo
CD44
19
L91
CD62L
Untreated
Pam2Cys
3
5
L91
19
22
CD127
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L91 immunization induces better lung immunity than BCG
BCG
24
22
F91
L91
26
32
TNF-α
Placebo
CD4
Placebo
FoxP3
5
CD4
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Placebo
5
BCG
8
BCG
F91
L91
9
6
4
Placebo
1424
F91
6
L91
15
IFN-γ
BCG
1175
F91
2334
PD-1
L91
1056
23
Log10 CFU
L91 immunization engenders better protection than BCG in mice
Placebo
BCG
L91
F91
LH
L91 immunization engenders better protection than BCG in mice. Mtb load in lungs was enumerated by CFU plating. Results are depicted as bar graphs
with mean ± SD (log10 value). Data shown are from 3 independent experiments. '*' indicates p<0.05, '**' p<0.01, '***' p<0.001.
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A novel therapeutic strategy to reinforce the potency of drugs to kill
Mycobacterium tuberculosis by concurrently bolstering host immunity by
promiscuous peptide conjugated to TLR-2 agonist
10 immunization
L91 (20nmol)
Aerosol challenge
(100 CFU)
20 immunization
L91 (10nmol)
Second dose of drug
First dose of drug
(INH 25mg/kg orally)
(INH 25mg/kg orally)
4 wk
2 wk
2 wk
12 wk
CFUs: lungs, spleen
Lungs (log10 CFU/g)
***
***
**
ns
***
***
INH+RFP in drinking water
Lungs (log10 CFU/g)
*
* * *
* *
ns
*
INH orally with two dose
*
***
***
***
***
IL-17 (ng/ml)
IFN-γ (ng/ml)
***
***
***
***
Spleen
Spleen
***
***
**
**
**
***
IL-17 (ng/ml)
IFN-γ (ng/ml)
**
**
Lungs
Lungs
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IFN-γ+ IL-17
+
IFN-γ+ TNF-α
CD4 T cells (%)
**
**
*
**
CD4 T cells (%)
+
+
+
CD4 T cells (%)
IFN-γ+ IL-17
IFN-γ+ TNF-α
CD4 T cells (%)
**
*
*
**
Spleen
**
**
**
**
Spleen
*
Lungs
Lungs
*
*
*
28
PBMCs of TB patients on drug therapy on in vitro stimulation with L91
induces secretion of IFN-γ
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ns
***
***
IL-17A+ CD4 T Cells (%)
IFNγ+ CD4 T Cells (%)
***
***
ns
29
Medium
F4.8
L4.8
3.3±0.3
2.8±0.1
10.6±1.6
6.6±0.3
9.0±0.2
14±0.1
IFN-γ
2.8±0.1
Pam2Cys
CD8
IFN-γ
3.9±0.1
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*
*
*
IFN-γ+ CD4 T Cells (%)
IFN-γ+ CD8 T Cells (%)
CD4
***
**
**
30
L91 expands central memory pool of
CD4 T cells of TB patients
Untreated
CD45RO
3.6±1.54
n= 14
16 kDa
3±2.3
n= 3
F91
4.4±2.64
n= 13
IMTECH
L91
13±3.2
n= 14
CD45RA
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L91 Protects Guinea Pigs from Mtb better than BCG
Experimental design for the protection studies in Guinea pigs. Different groups of Duncan-Hartley Guinea pigs were immunized with L91 or controls (F91,
LH, BCG and placebo). Peptides were given at a dose of 100 nmol (primary immunization) and 50 nmol (booster). BCG (106 CFU/ animal) was given as a
control. Animals were rested for 75 days and challenged with a low dose aerosol of M. tb H37Rv (~30 CFU/animal), 75 days post immunization. Thirty
days after infection, animals were sacrificed and studied for bacterial burden and pathology.
0.0178
6
CFU Load of Lung
0.0004
0.0042
5.5
Log CFU
5
0.0316
4.5
4
3.5
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3
Placebo
BCG
L91
p91
FLU
L91 immunized exhibit constrained pathology upon Mtb challenge in Guinea pigs
Placebo
L91
BCG
F91
LH
L91 immunized Guinea pigs exhibit constrained pathology upon M. tb challenge in Guinea pigs. The protection studies were performed as described in
Fig. 21:49
3.4. Representative photomicrographs of formalin fixed H & E stained histopathology lung sections of Guinea pigs (left and center panel).
33Arrows
indicate regions of granulomas or necrosis. Right panel shows photographs of gross pathology in lungs of immunized and infected animals. Data are
representative of 2 comparable experiments with a minimum of 5 animals per group.
CD4
CD8
Pathogen
IL-6, IL-12, IFN-γ
IFN-Y, TNFα, IL-17, IL-2
Cytokine ReceptorCM: Costimulatory molecule
Gowthaman & Agrewala. Trends Mol Medicine 2012
CONCLUSION
Lipidated peptide vaccine may be a
future prophylactic strategy to
eradicate TB from TB-endemic zones
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Juraj Ivanyi, TB & RI Unit, Hammersmith Hospital, London
David Jackson, University of Melbourne, Australia
Pushpa Gupta and UD Gupta, JALMA, India
AK Janmeja, Government Medical College and Hospital, India
IMTECH, CSIR and DBT for Financial Support
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IMTECH
Fails to elicit immune response in
genetically diverse human population!
Answer: Promiscuous Peptides?
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IMTECH
Promiscuous Peptide Selection
We generated 141 over lapping peptides from:
16kDa, 19kDa, 30kDa, 38kDa, 65kDa, CFP-10, ESAT-6
All the individuals generated robust T cell proliferation and
predominant secretion of IFN-g by PBMCs of the PPD+ healthy
individuals against 16 kDa (Acr1) antigen of Mtb.
1
20
MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRA
ELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADE
DDIKATYDKGILTVSVAVSEGKPTEKHIQI RSTN (141 AAs)
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Identification of T-Helper Cell Activating
Promiscuous Peptides
Buffy coat of 20 PPD+ healthy volunteers with
no history of TB were selected and tested
using promiscuous peptides for:
(i) Peptides binding to HLA-class II molecules
(ii) T cell proliferation
(iii) Secretion of IFN-g
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IMTECH
Peptides of sequence 21-40 and 91-110 showed permissive binding
HLA DRB1
Binding Affinity
p21-40
Binding Affinity
p91-110
0101
+++
++++
0103
++
++++
0301
+
++
0401
+++
++++
0701
++
++++
1101
+++
++++
1301
++
+++
1501
++++
++++
1601
+++
++++
The HLA type of donors was performed by low resolution PCR with sequence-specific primers (PCR-SSP). All
presenting HLA-DR-homozygous L-BCL were chosen to conform at least to the donor’s major serological, and
usually to molecular, type. The affinity of the presenting MHC class II molecule for peptides p21-40 and p91110 was determined by a competitive MHC binding assay. ++++ indicates high-affinity binding (IC50<10µM); +++
intermediate affinity (10 µM<IC50<100µM); and ++ indicates low affinity (100–1000µM).
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Agrewala et al. 1997, 1998, 1999
IMTECH
Peptides of sequence 21-40 (p21-40) and 91-110 (p91-110)
showed T cell proliferation in all the tested individuals.
p91-110 induces mainly secretion of IFN-g.
Th1 clones (IFN-g): 90%
Th0 clones (IFN-g +IL-4): 10%
p21-40 promotes production of IFN-g and IL-4.
Th0 clones (IFN-g + IL-4): 75%
Th2 clones (IL-4): 25%
p21-40: LFAAFPSFAGLRPTFDTRLM
p91-110: SEFAYGSFVRTVSLPVGADE
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Agrewala et al. 1997, 1998, 1999
Peptides are weak immunogens!
Can conjugation with Pam2Cys
increase the immunogenecity of
peptides?
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IMTECH
Why Pam2Cys?
S-[2,3-is(palmitoyloxy)propyl]cysteine
Ligand for TLR2, induces signaling through it.
Has self-adjuvanting properties when conjugated with peptides.
Induces robust Th1 and Th17 type immune responses.
Long-term protective effect.
Potent immunogenicity can be explained by its ability to mature and
activate dendritic cells (suggesting that vaccines containing this lipid
moiety may interact directly with DCs to promote immune responses).
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Jackson et al. 2004, Zhu et al. 2004, Düesberg et al. 2002, Zeng et al. 2002
IMTECH
Why TLR2?
Incorporating the Pam2Cys into peptide structures
effectively triggers the TLR2 and secretion of IL-12 by
DCs.
TLR2 is copiously present on DCs, monocytes, and lung
epithelia.
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IMTECH
Why DCs?
DCs are decisive for efficient immune response.
Only cells, capable of activating naïve T cells.
Skews immune response towards Th1 and Th17 cells.
Constitutive expression of MHCs and costimulatory
molecules.
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IMTECH
Advantage of Vaccine
Totally synthetic
No adjuvant is required
Can activate CD4 and CD8 T cells
Skews immune response to Th1 and Th17 type
Can activate naïve T cells
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