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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
IMTECH
Vaccines-Hyd-2.11..15
1 19:48
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.
Gowthaman & Agrewala. Trends Mol Medicine 2012
3 19:48
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).
IMTECH
Gowthaman & Agrewala. Crit Rev Microbiol. 2014 4 19:48
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!
IMTECH
5 19:48
Any vaccine that require
minimum antigen processing will
work in TB-endemic population
6 19:48
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
choice in TB-endemic regions? 7 19:48
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
IMTECH
8 19:48
Th epitope 1: LFAAFPSFAGLRPT FDTRLM Th epitope 2: SEFAYGSFVRTVSLPVGADE
-K- I S I S I
Pam2Cys
-K- I S I S I
Pam2Cys
-K- 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
Th CTL
Th CTL
Gowthaman & Agrewala. Trends Mol Medicine 2012
9 19:48
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, can avoid autoreactive/suppressive portions in the antigen.
IMTECH
10 19:48
Whether the construct
influences the
maturation of DC?
F91: free peptide
L91: lipidated peptide
IMTECH
11 19:48
Untreated F91 L91
0
2
4
6
8
10
12
1DC:2T Cells 1DC:3T Cells 1DC:4T Cells 1DC:>5T Cells
F91
L91
3 55
T Cell Proliferation
Unstimulated L91
Num
ber
of c
onju
gate
s
Gowthaman & Agrewala. J Infect Dis. 2011
12
19:48
L91 augments IL-12 production by DCs
T cells exposed to L91 treated DCs secreted more IFN-γ
IFN
-γ (
pg/m
l)
0
15000
30000
Medium Pam2Cys F91 L91
0
10
20
30
40
50
60
70
80
90
100
L91 p91 LPS Untreated
Perc
en
tag
e
CD40 CD80 CD86
L91 Enhances Activation and Maturation of DCs
IAd IAd
P91 L91
CD74 CD74
CD80 CD40 CD86
Medium
LPS
L91
P91
13 19:48
Whether the peptides works
across MHC barriers?
14 19:48
Immunization L21, P21, L91, P91
Booster 7-14d
In vitro challenge with peptides
T cell response
Sacrificed 240 days
IMTECH
[BALB/c, C57BL/6, C3He, FVB]
Free peptides were emulsified
in IFA.
15 19:48
0
10000
20000
30000
0.003 0.01 0.03 0.1 0.3 1 3
0
1000
2000
3000
4000
0.003 0.01 0.03 0.1 0.3 1 3
0
20000
40000
60000
0.003 0.01 0.03 0.1 0.3 1 3
IMTECH Peptide works across MHC barriers
0
10000
20000
30000
0.003 0.01 0.03 0.1 0.3 1 3
BALB/c
T c
ell p
rolife
ration
T c
ell p
rolife
ration
C3He FVB
C57BL/6
Peptides (nmoles)
L21 L91 P91 P21
16 19:48
Control
4.1%
Pam2Cys
4.2%
F91
16%
L91
52%
13 6 4
CD
44
4
Control Pam2Cys F91 L91 A
0
0.5
1
1.5
2
2.5
3
3.5
CD69
Control Pam2Cys F91 L91
CD
69
+ C
D4
4+
CD
4 T
cell
s (F
old
Ch
an
ge)
B
L91 induces proliferation and activation of CD4 T cells
17
19:48
T Cell Proliferation
0
15000
30000
0.05
0.1
0.15
0.2
0.25
0
50
100
150
200
0.1
0.15
0.2
0.25
0.3
A
B
C
D
IL-5
(p
g/m
l)
IFN
-γ (
pg
/ml)
IL
-4 (
O.D
) IL
-10 (
O.D
)
Control Pam2Cys F91 L91
n.s
n.s
n.s
Control Pam2Cys F91 L91
Control Pam2Cys F91 L91
Control Pam2Cys F91 L91
Immunization with L91 elicits mainly secretion of IFN-γ
L91 F91 Medium Pam2Cys
CD4
IFN
-γ
8 8 10 25
0
1
2
3
4
5
A
B
Control Pam2Cys F91 L91
IFN
-γ+
CD
4 T
cell
s (F
old
Ch
an
ge)
11 11 15 36
Control Pam2Cys F91 L91
IFN-γ
TN
F-α
18
19:48
1.4 2.3 2.6 9.9
1.4 3.1 1.5 3.4
2.0 1.5 2.4 2.8
L91 immunization evokes IL-17 secretion
Immunization
L91
BCG
Saline
Medium L91 Pam2Cys F91
IL-17
CD
4
11 3
Control F91 L91
12 4 34 6
CD62L
CD
44
0
0.5
1
1.5
2
2.5
3
3.5
Central Memory
Effector Memory
B
Untreated F91 L91
Fold
induct
ion
93 130 315
0
1
2
3
4
5
6
A
B CD127
Control F91 L91
Control F91 L91 CD
127 (
Fold
in
du
ctio
n)
L91 immunization engenders T cell memory
20
19:48
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.
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.
L91 Vaccination Protects Mice Against Mtb
21
19:48
Untreated Pam2Cys L91
19
30
20
27 24
36
CD62L
CD
44
Untreated Pam2Cys F91 L91
274 263 488 525
CD69
Untreated Pam2Cys L91
3 5 19
CD127
L91 evokes enduring memory CD4 T cell response in Mtb infected mice
Central: CD44hi/CD6Lhi
Effector: CD44hi/CD62Llo
22
19:48
Placebo BCG F91 L91
24 22 26 32
CD4
TN
F-α
IFN-γ
5 8 6 15
Placebo BCG F91 L91
CD4
Fox
P3
5 9 6 4
Placebo BCG F91 L91
L91 immunization induces better lung immunity than BCG
1424 1175 2334 1056
Placebo BCG F91 L91
PD-1 23 19:48
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.
Log
10 C
FU
Placebo BCG L91 F91 LH
L91 immunization engenders better protection than BCG in mice
24 19:48
10 immunization L91 (20nmol)
First dose of drug
(INH 25mg/kg orally) Aerosol challenge
(100 CFU)
4 wk 2 wk 2 wk
CFUs: lungs, spleen
20 immunization L91 (10nmol)
Second dose of drug (INH 25mg/kg orally)
12 wk
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
Lun
gs (log 1
0 C
FU/g
)
*** ***
** ***
***
ns
* * * * *
* *
* ns
INH+RFP in drinking water
INH orally with two dose
Lun
gs (log10 C
FU/g
)
Lungs
IFN-γ
(ng/
ml)
**
** **
***
Spleen
IFN-γ
(ng/
ml)
***
*** ***
***
Lungs
IL-17
(ng/
ml)
***
** **
***
Spleen
IL-17
(ng/
ml)
***
*** ***
***
28 19:48
** *
** *
Spleen
IFN-γ+
TNF-α +
CD4 T
cells
(%)
** **
**
*
Lungs
IFN-γ+
TNF-α +
CD4 T
cells
(%)
** **
** **
Spleen
IFN-γ+
IL-17
+ C
D4 T
cells
(%)
* *
* *
Lungs
IFN-γ+
IL-17
+ C
D4 T
cells
(%)
IFNγ
+ C
D4
T C
ells
(%
)
*** ***
ns
29 19:48
PBMCs of TB patients on drug therapy on in vitro stimulation with L91 induces secretion of IFN-γ
IL-1
7A
+ C
D4 T
Cel
ls (
%)
ns
*** ***
CD8
IFN
-γ
CD4
IFN
-γ
IFN-γ+
CD8 T
Cells
(%)
IFN-γ+
CD4 T
Cells
(%) ***
** **
* *
*
Medium F4.8 Pam2Cys L4.8
2.8±0.1 3.3±0.3 2.8±0.1 10.6±1.6
3.9±0.1 6.6±0.3 9.0±0.2 14±0.1
30 19:48
IMTECH L91 expands central memory pool of CD4 T cells of TB patients
3.6±1.54 n= 14
4.4±2.64 n= 13
13±3.2 n= 14
3±2.3 n= 3
CD45RA CD
45
RO
Untreated 16 kDa F91 L91
31 19:48
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.
L91 Protects Guinea Pigs from Mtb better than BCG
3
3.5
4
4.5
5
5.5
6
Placebo BCG L91 p91 FLU
Lo
g C
FU
0.0178
0.0004
0.0316
0.0042
CFU Load of Lung
32 19:48
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. 3.4. Representative photomicrographs of formalin fixed H & E stained histopathology lung sections of Guinea pigs (left and center panel). Arrows
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.
L91 immunized exhibit constrained pathology upon Mtb challenge in Guinea pigs
33 19:48
CD8
CD4
Pathogen IL-6, IL-12, IFN-γ IFN-Y, TNFα, IL-17, IL-2 Cytokine Receptor CM: 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
35 19:48
IMTECH, CSIR and DBT for Financial Support
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
36 19:48
37 19:48
Fails to elicit immune response in
genetically diverse human population!
Answer: Promiscuous Peptides?
IMTECH
38 19:48
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.
IMTECH
1 20
MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRA
ELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADE
DDIKATYDKGILTVSVAVSEGKPTEKHIQI RSTN (141 AAs)
39 19:48
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
Agrewala et al. 1997, 1998, 1999
40 19:48
Peptides of sequence 21-40 and 91-110 showed permissive binding
IMTECH
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 p91-110 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).
Agrewala et al. 1997, 1998, 1999
41 19:48
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
Agrewala et al. 1997, 1998, 1999
IMTECH
42 19:48
Peptides are weak immunogens!
Can conjugation with Pam2Cys
increase the immunogenecity of
peptides?
43 19:48
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).
Jackson et al. 2004, Zhu et al. 2004, Düesberg et al. 2002, Zeng et al. 2002
IMTECH
44 19:48
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.
Jackson et al. 2004, Zhu et al. 2004, Düesberg et al. 2002, Zeng et al. 2002
IMTECH
45 19:48
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.
IMTECH
46 19:48
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
IMTECH
47 19:48