Pradeep K Rai CSIR-IMTECH, Chandigarh

Future peptide vaccine for TB endemic regions: Challenges and solutions

Epidemiology of Tuberculosis

Total no. of new TB cases 9 million

Previously treated 85%

New cases 15%

480,000

MDR: Multi drug resistant

WHO Global tuberculosis report , 2014

Total no. of deaths 1.5 million

Best Prophylactic measure

Vaccine

BCG

BCG Failure

Drugs

DOTs Treatment

Side effects

Emergence of drug resistant

Reasons for BCG failure

Lack of enduring immunologic memory (Andersen and Doherty 2005, Narayanan 2006, Orme 2010, Singh et al 2010, 2011)

Interference by environmental mycobacteria (Masking and Blocking hypotheses) (Andersen and Doherty 2005, Verma and Grover 2009, Flaherty et al 2006, Young et al 2007, Brant et al 2002)

Antigen processing and presentation- Blocking phagosome maturation and export of major histocompatibility complex (MHC) molecules (Soualhine et al 2007, Pancholi et al 1993, Sendide et al 2005, Sun et al 2007, Fulton et al 2004, Sendide et al 2004)

Helminth infections (Malhotra et al 1999, Elias et al 2001, 2005, Black et al 2002, Rook et al 2004)

Regulatory T cells (Hanekom 2005, Ahrens et al 2009, Rook and Kim 2006, Akkoc et al 2010)

Limitations of DOTs

Time consuming (3-12 months), so chances of intermittent therapy increases, resulting in increased risk of drug resistance. Side Effects. No cure for latent infection.

Urgent need to develop: A potent construct with two edge sword (Prophylactic and Therapeutic potential)

Future TB vaccine: Epitope based vaccine

Minimal antigen processing Minimal presence of mycobacterial components (except antigenic portions) Robust Th1 response (even in a Th2 bias environment) Enduring T cell memory No preformed antibodies in the population

Vaccine candidate with both prophylactic and therapeutic properties

Х Poor immunogenicity

Х Binds to very few HLA-alleles

HLA DRB1 Binding Affinity

F91-110

0101 ++++

0103 ++++

0301 ++

0401 ++++

0701 ++++

1101 ++++

1301 +++

1501 ++++

1601 ++++

++++ indicates high-affinity binding (IC50<10µM); +++ intermediate affinity (10 µM<IC50<100µM); and ++ indicates low affinity (100–1000µM).

Agrewala and Wilkinson 1997, 1998, 1999

Which peptide to choose? Is promiscuous peptide right choice?

Pam2Cys S-[2,3-is(palmitoyloxy)propyl]cysteine induces effective

Th1 immune responses, by evoking DCs to secrete IL-12 (Thoma et al 2000, Jackson et al 2004, Zho et al 2004, Ghosh et al 2006)

Dendritic cells copiously express TLR2 so Pam2Cys (TLR2 agonist)

can be used as vaccine delivery module (Jackson et al 2004, Zho et al 2004)

Pam2Cys has ability to mature and activate DCs (Hertz et al 2001, Jackson et al 2004, Zho et al 2004)

Safe for Human use (Zeng et al 2002)

-K I S I S I

Pam2Cys

SEFAYGSFVRTVSLPVGADE (91-110)

L91

Can coupling promiscuous peptides to Pam2Cys render them

immunogenic?

CD4

MΦ

Induction of MHC expression

Induction of microbicidal activity

NOS2 generation

Mechanism of L91

IFNγ , IL-17, TNFα

L91 confers better protection than BCG in Guinea pig model of tuberculosis

Placebo L91 BCG F91 LH

Whether L91 has therapeutic potential?

L91 efficiently decreases bacterial burden in the lungs and spleen in conjunction with anti-TB drugs

Lu

ng

s (

log

10

CF

U/g

)

*** ***

** ***

***

ns

*** ***

**

***

*** ns

Sp

leen

(lo

g1

0 C

FU

/g)

0 4 6 8 20 weeks

Mtb

infection

INH+RIF

L91 L91 CFU count

T cell response

L91 immunization reduces Mtb induced lung pathology

Placebo

L91

Drug

Drug-L91

20X 100X

L91 has enough therapeutic potential

to confine bacterial growth in lungs

Further, L91 restricts dissemination

of bacteria in spleen

Mechanism of protection?

iNOS

ACTIN

L91 stimulation elicits innate immunity

NF-kB

FB US F91 Pam L91 LPS

L91 immunization elicits secretion of protective cytokines against Mtb

3

4

5

6

7

8

9

PBS Drug L91 Drug+L91

IL-1

β (

S.I

) **

**

0

5

10

15

20

25

PBS Drug Drug+L91 L91

IL-1

7A

(S

.I)

***

**

0

5

10

15

20

25

30

PBS Drug L91 Drug+L91

IFNγ

(S.I

)

*** ***

ns

Lungs Single cell

suspension

In vitro stimulation

with L91

T cell response

72h

**

*

IFN

-γ+ T

NF

-α +

CD

4 T

cells (

%)

* *

IL-1

7+IF

N-γ

+ C

D4

T c

ell

s (

%)

L91 immunization induces generation of multifunctional CD4 T cells

*

*** C

XC

R3

+ C

CR

6+

IF

N-γ

+ IL

17

A+

CD

4 T

Ce

lls

(%

)

L91 immunization generates specific subtype of Th17 cells

PD

-1 (

iMF

I)

** *

**

*

ns

L91 immunization rescues CD4 T cells from exhaustion

CD

44

hi C

D62

Lh

i CD

4 T

ce

lls

(%

)

* **

**

**

CD

127

+ C

D4 T

cells (

%)

L91 immunization engender long lasting memory CD4 T cells

L91 immunization eradicate bacteria by

eliciting strong Th1 and Th17 response

L91 immunization generates unique subset

of IFN-γ+IL-17+ polyfunctional Th17 cells

L91 engenders memory generation

Whether immunotherapy with L91

elicits immune response in TB

patients?

Lipidated peptide exhibits T cell proliferation in PBMCs isolated from TB patients and their close contacts

T c

ell p

rolife

rati

on

(S

tim

ula

tio

n i

nd

ex) ****

n= 53 (TB patients)

PBMCs (TB patients) + L91 + IL-2 96h Immune response

9.7±1.5 5.2±0.8

L91 stimulation elicits IFN-γ secretion by the CD4 T cells of TB patients

CD

4+IF

Nγ

+ T

cells (

%)

3.6±0.6

Medium Pam2Cys L91

IFNγ

CD4 *** **

ns

L91 stimulation induces IL-17 secretion by the CD4 T cells of TB patients

CD

4+

IL-1

7+ T

cells (

%)

5.7±1.6 4.9±0.4 IL

-17

CD4

12.5±1.2

Medium Pam2Cys L91

*** **

ns

L91 stimulation generates polyfunctional Th17 cells

IFN

-γ +

IL-1

7A

+ C

D4

T c

ell

s (

%)

***

**

ns

2.8±0.4

(n=14)

3.7±0.6

(n=12)

8.3±1.2

(n=14)

Medium Pam2Cys L91

IFN

-γ

IL-17A

L91 expands the pool of memory T cell

Medium Pam2Cys L91

CD45RA

CD

45R

O

3.8±0.4

(n=28)

4±0.4

(n=15)

7.7±0.8

(n=21)

CD

45

RA

+C

D4

5R

O+

CD

4 T

ce

lls

(%

) ***

**

ns

F91 specific antibodies are absent in TB endemic population

Conclusions

Promiscuous lipidated peptide can elicit robust Th1 and

Th17 response in human PBMCs isolated from TB patients and their closed contacts

L91 immunization induces unique subset of co-expressing CCR6 and CXCR3 Th17 cells, which secrets both IFN-γ and IL-17

No anti-peptide antibodies in population so L91 has

great potential to be a future vaccine candidate for TB endemic area

Immunotherapy with L91 may reduces risk of generation

of drug resistant Mtb

Thanks

Acknowledgment Dr. Javed N Agrewala Professor David C Jackson Professor Ashok K Janmeja CSIR DBT