a novel vaccine construct comprising of lipidated peptide ...€¦ · 2.8±0.1 3.3±0.3 2.8±0.1...

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]

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


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

)


n.s

n.s

n.s




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


IFN

-γ+

CD

4 T

cell

s (F

old

Ch

an

ge)

11 11 15 36


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


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

a novel vaccine construct comprising of lipidated peptide ...€¦ · 2.8±0.1 3.3±0.3 2.8±0.1...

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