A Leishmaniasis Vaccine Breakthrough: Are We Almost There?

1

Jude Uzonna, DVM, Ph.D.Professor & Research Manitoba Chair,Department of ImmunologyUniversity of Manitoba

Simple Cutaneous Leishmaniasis• Caused by Leishmania major, mexicana, tropica, etc

• Normally self-healing

• DTH, T cell proliferation

• Low antibody responses

• Healing results in solid immunity

Diffuse cutaneous leishmaniasis

• No DTH response

• High Antibody response

• Chronic, resistant to drug treatment

Mucocutaneous leishmaniasis

• Very strong DTH

• Intense inflammatory response

• Chronic, resistant to drug treatment

• Very few parasites at inflammatory sites

Visceral leishmaniasis

• Leishmania donovani• No DTH response• Very high Ab response• Impaired T cell proliferation in response to antigen

Cutaneous Leishmaniasis

Visceral Leishmaniasis

Leishmaniasis: A global problem

Epidemiology:• 14 million active cases• 2 million new cases yearly• 340 million people at risk• 88 countries, 5 continents• Neglected Tropical Disease• > 3500 cases of cutaneous Leishmaniasis among gulf veterans

•Sources: WHO, CDC

• Resistance to L. major is mediated

by IFN--producing CD4+ Th1 cells

• IFN- activates macrophages

leading to intracellular parasite

killing via:

Nitric oxide (NO) production

ROI and RNI radicals

Immunity to cutaneous Leishmaniasis (simplified)

Summary:• CD4+ Th1 cells• IFN- production

• Healing of cutaneous leishmaniasis in humans and mice is usuallyself-resolving

• This is associated with long-lasting resistance to reinfection

Suggests existence of anti-Leishmania memory cells

Infection-induced immunity

Lesi

on

siz

e (m

m)

0

0.5

1.5

2

0 5 10 15 20 25Weeks

1

Secondary Challenge

2 3 4 5 6 7 8Parasite titer (log10)

ImmuneNaive

*

Infection-induced Resistance

Adoptive Transfer of Protection

Primary

Secondary

-log (Parasite Titer)0 2 4 6 8

No Cells

Naïve Cells

Immune CD4+ T Cells

-log (Parasite Titer)0 2 4 6 8

CD4+ T cells mediate infection-induced immunity

Is there a need for a Leishmania vaccines?

• Epidemiology: Over 14 million active cases 2 million new cases yearly 340 million people at risk 88 countries, 5 continents

• Chemotherapy until recently is very limited high cost, toxicity and route of administration

• Elimination can likely only be achieved through widespread vaccination

• Vaccination could provide longterm reductions in potential reservoirs

10

Pubmed Search!!

• Leishmania and immunity = 2606

• Leishmania and resistance = 2127

• Leishmania and vaccine = 1649

• Leishmania and vaccination = 689

• Leishmania and effective vaccine = 392

• Human Leishmaniasis and effective vaccine = 227

• Leishmania vaccination trials = 115

Approved Effective Human Leishmania Vaccine = 0

12

Scientific Issue: Why is there no effective human vaccine despite prolific

publications?

• We do not fully understand the correlates of

protective immunity.

Immunologic memory (development, maintenance and loss)

Antigens that mediate memory T cell responses

13

Is Vaccination Feasible?

• Infection-induced immunity: Recovery from virulent Leishmania infection leads to development of strong durable immunity against virulent challenge

• Leishmanization is very effective

• Heat Killed parasites

– First generation vaccines

• Live attenuated/genetically modified

– Second generation vaccines

• Defined protein/subunit vaccines

– Third generation vaccines

• Leishmanization

Leishmaniasis Vaccine Approaches

• Heat-inactivated (Autoclaved) Leishmania parasites

• Several doses or‘boosters’with with different adjuvants (BCG, Alum, CpG)

• Questionable efficacy

• Easy and cheap to make (minimal technology) and very safe

• Does not require understanding of complex immunologic correlates of protective immunity

• Reports of immunotherapeutic effects in New World cutaneous leishmaniasis

Heat-killed (ALM) vaccines

Problems with ALM Vaccine

• Standardization and scale-up issues

• Undesirable immune responses Qualitative differences in the early immune response to live and killed Leishmania major:

Implications for vaccination strategies against Leishmaniasis. Okwor I, Liu D, Uzonna JE.

Vaccine. 2009 Apr 28;27(19):2554-62.

Inoculation of killed Leishmania major into immune mice rapidly disrupts immunity to a

secondary challenge via IL-10-mediated process. Okwor I, Liu D, Beverley SM, Uzonna JE. Proc

Natl Acad Sci U S A. 2009 Aug 18;106(33):13951-6.

17

Leishmanization

• Deliberate inoculation of live (virulent) parasites to the hidden parts of the body with the aim of inducing protection from natural infection following recovery

• Very Effective

• Undesirable side effects!!!

18

Courtesy: Dr Ali Khamispour

Undesirable effects of Leishmanization

Rationale for Live Vaccine

• Infection-induced Immunity Recovery from infection leads to development of durable immunity

• Persistent parasites are important for maintaining infection-induced immunity Attenuated parasites persist for a long time in infected/vaccinated host

Clearance of parasites leads to loss of protective immunity

• Leishmanization works

Two major strategies for live-attenuated vaccine:

• Virulent parasites (Leishmanization)

• Attenuated parasites

Attenuated Parasites

• Gamma Irradiation– Rivier et al., 1993

• Serial in vitro passage with/without drugs– Daneshvar, et al 2003, Onyalo et al 2005

• Infection with non-pathogenic strain (L. tarentolae)– Breton et al 2005

• Genetically modified organisms– LPG1, LPG2, LPG5A/B, DHFR-Ts, GDP-MP, GP63, etc

0

5

10

15

20

Wk10

Wk4

WT

lpg2

-/-

Ad

d b

ack

LPG2 KO parasites do not cause pathology despite persistence

0

2

4

6

0 5 10 15 20 25

Add Back

lpg2-WT

Weeks post-infection

Lesi

on

siz

e (

mm

)

Uzonna et al 2004

3

6

9

12

0

2

4

6

0 2 4 6 8 10 12 14

lpg2-Naive

Naïve VaccWks post-challenge

Par

asit

e T

ite

r (l

og 1

0)

Lesi

on

siz

e (

mm

)

Vaccination with LPG2 KO parasites induce protection against virulent challenge

Uzonna et al 2004

Problems of live-attenuated vaccine

• Reversion to virulence (due to long-term persistence)– Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes

within macrophages without LPG2-dependent glycoconjugates and its significance to virulence and

immunization strategies. Späth GF, Lye LF, Segawa H, Turco SJ, Beverley SM. Infect Immun. 2004

Jun;72(6):3622-7.

• Contraindicated in immuno-compromised individuals

This vaccination protocol will most likely never be approved

3rd Generation Vaccines

• Subunit vaccines with refined products (recombinant proteins)

• Several recombinant proteins or polyproteins (e.g. Leish-111F, LmSTI1, KSAC, etc) with adjuvants such as MPL

• Some have progressed into clinical trials

• One approved for dogs in South America (Leishmune)

26

Why 3rd Generation Vaccines Fail

• Use of in silico models to predict “protective” vaccine candidates

• Selection of candidates based primarily on those that elicit strong primary immune response

• Skewed and biased immunization protocols: designs favor publication; not necessarily long-term protection– Animal model (BALB/c mice)

– Antigen dose

– Type of adjuvants

– Frequency of immunization

– Time of challenge etc.

28

Objectives:

• To use in vivo approach to identify Leishmania antigens that induce infection-induced immunity (memory CD4+ T cell responses?)

• Assess whether vaccination with these antigens will induce long-lasting protection against virulent challenge

A B

CFSE#

31.5C

ou

nts

IFN-

CD

4

0 1 2 3 4 5 6 7 8 9 10 110

1

2

3

4

weeks postinfection

lesio

n s

ize (

mm

)

C

Healing from primary L. major infection is associated with strong antigen-specific CD4+ T cell proliferation and IFN- production.

Premise: Identification of the antigens that induce and maintain infection-induced

resistance and the corresponding T cells is critically important for development of effective vaccine and vaccination strategy against leishmaniasis

Reverse Immunology

2

Secondary Challenge

1

3

4

Lesion

size (mm)

Weeks post-infection5 10 15 20

BMDC

L. major

Infected

Flow Cytometry

Purify CD4+ T cells

CFSE

Healed mice

Strategy to confirm stimulatory ability of L. major-infected BMDCs for CD4+ T cells

Uninfected

Co-culture

Assess:1. Proliferation

2. IFN- production

Spleen

Naïve Mice

Infected BMDC

Uninfected BMDC

IFN

-γ

CFSE

Healed Mice

Source of T cells

0.8±0.1

3.0±1.8

0.7±0.3

1.9±0.4

0.7±0.3

6.3±0.6

6.5±1.9**

24.7±3.9*

Infected

DC

Uninfected

DC

L. major-infected DCs induce proliferation and IFN- production in CD4+ T cells from healed mice

Source of APC

Membrane cutoff:

10K

Protein A beads

Anti-MHC II Ab

lyophilization

Lysis buffer

MHC-II bound Peptides

Programs:GPMProteinPilot

Databases:1. L. major2. Mouse

Infected BMDCs

QSTAR® Elite Hybrid

LC/MS/MS

Database

Searching

Proteomic identification of L. major antigenic peptides that bind to MHC II molecules

Dis

so

cia

te

MH

C/P

ep

tid

e

Source Protein accession MrLog(e) in GPM

Sequence in GPM Sequence in ProteinPilotScore in PP

RNA binding protein, putative LmjF32.0750 25.2 -53.1 KAAPKAAAPAARPASGSSN; KAAPKAAAPAARPASGSS;

KAAPKAAAPAARPASG;AAPKAAAPAARPASGS; AAPKAAAPAARPASG

KAAPKAAAPAARPASGSSN;KAAPKAAAPAARPASGSS; KAAPKAAAPAARPASG;

AAPKAAAPAARPASGSS;AAPKAAAPAARPASGS;AAPKAAAPAARPASG

13.1

Putative uncharacterized protein

LmjF08.1100 42 -25.4 KAAEASAHSPQASQSAGDG;AAEASAHSPQASQSAGDG;

AEASAHSPQASQSAGD

KAAEASAHSPQASQSAGDGDR;AAEASAHSPQASQSAGDG;

AEASAHSPQASQSAGDG; AEASAHSPQASQSAGD; AEASAHSPQASQSAG;

ARIHQRDDAPVEEKRAAH;

13.2

Glycosomalphosphoenolpyruvatecarboxykinase, putative

LmjF27.1805 58.2 -21.4 NDAFGVMPPVARLTPEQ;DAFGVMPPVARLTPEQ; DAFGVMPPVARLTPEQ; DAFGVMPPVARLTPE

NDAFGVMPPVARLTPEQ;DAFGVMPPVARLTPEQ; DAFGVMPPVARLTPE

6.24

ATP-dependent RNA helicase, putative

LmjF35.3100 100.2 -13.5 APAPAPAAAPTPASAPVS; APAPAPAAAPTPASAPV

APAPAPAAAPTPASAPVS;APAPAPAAAPTPASAPV

4.33

Dihydrolipoyl dehydrogenase LmjF32.3310 50.5 -10 HATHLYHDAHANFAQYG; ATHLYHDAHANFAQYG

HATHLYHDAHANFAQYG; IPGVIYTNPEVAQVGET 5.25

Cytochrome c LmjF16.1310 12.2 -3.5 RPSGKVEGFTYSKANAESG RPSGKVEGFTYSKANAESG 2

Aconitase, putative LmjF18.0510 97.4 -3.2 DSITTDHISPAGNIAKDSPA DSITTDHISPAGNIAKDSPA 2.07

Summary of Leishmania peptides identified by both GPM and ProteinPilot.

34

eP1 P2 P3 P4FT-Ag

CFSE

No peptide P1 P2 P3 P4

IFN

-γ

b

c7.9±0.1 6.6±2.1 9.5±4.0 22.6±6.4* 9.1±2.1

5.2±2.2 4.8±0.6 6.6±0.4 37.7±5.6* 6.5±3.4

d

0

2000

4000

6000

8000

FT-Ag P1 P2 P3 P4

IFN

-γ(p

g/m

l)

Sequence

P1 KAAPKAAAPAARPASGSSN

P2 AAEASAHSPQASQSAGDG

P3 NDAFGVMPPVARLTPEQ

P4 APAPAPAAAPTPASAPVS

a

CD4

FT-Ag P1 P2 P3 P4

Sp

ots

/we

ll

f

0

20

40

100

200

300

Synthetic (PEPCK335-351, P3) peptide stimulates proliferation and IFN-production by CD4+ T cells from healed mice

35

• PEPCK: phosphoenolpyruvate carboxykinase• Plays an essential role in glyconeogensis

Sci Transl Med. 2015 Oct 21;7(310)

Expansion, contraction and maintenance of PEPCK-specific CD4+ T cells in L. major-infected mice

a

I-A

b-P

EP

CK

335-3

51

CD44

Footpad

Naïve 2wk 5wksI-

Ab-P

EP

CK

335-3

51

3wks 7wks

dLN

Spleen

14wks 60wks

0 0

0.05 0

0.06 0.18

0.03 0.17

0.08 0.86

0.13 5.76

0.01 0.31

0.03 0.69

0.04 0.21

0.01 0.15

0.01 0.25

0.02 0.09

0.27

0.77

0.11

0.07

CD44

Blood

0 0 0.25 1.22 0.06 2.18 0.16 12.26 0.04 2.95 0.03 0.23

0 5 10 150.00.1

5

10

15

0 5 10 15 40 600.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 40 600.0

0.1

0.2

2

4

6 Spleen

bdLN

Weeks post-infectionWeeks post-infection

cBlood

Weeks post-infection

d

I-A

b-P

EP

CK

335-3

51(%

)

I-A

b-P

EP

CK

335-3

51

(%)

I-A

b-P

EP

CK

335-3

51(%

)

e

12.3

36Sci Transl Med. 2015 Oct 21;7(310)

PEPCK-specific CD4+ T cells are polyfunctional effector cytokine producers

37

I-A

b-P

EP

CK

335-3

51

CD4 IL-2

TN

F

IL-1

7

88.2±6.1

5.0±3.4

25.5±2.7

2.6±2.0

0.1±0.1

0.0

IFN-γ IFN-γ IFN-γ

IL-1

0

IFN-γ

0.1±0.1

0.3±0.1

IL-2

TN

F

IL-1

7

IL-1

0

Sci Transl Med. 2015 Oct 21;7(310)

6

7

8

9

10 CD4+Tet+(2×104)

CD4+Tet- (2×105)

CD4+Tet- (4×106)

Naive CD4+ (4×106)

Para

sit

e b

urd

en

(lo

g1

0)

**n.s.

**

a

Pre-sort

I-A

b-P

EP

CK

335-3

51

CD4

Post-sort

b

100

0

PEPCK-specific CD4+ T cells Protect naïve mice against L. major challenge

38Sci Transl Med. 2015 Oct 21;7(310)

Pro

ma

sti

go

te

Am

as

tig

ote

10

ng

rP

EP

CK

50

ng

rP

EP

CKa

DAPI PEPCK DAPI/PEPCK White light

c

0 mm 7.5

0 mm 7.5

Promastigote

Amastigote

10

0 k

D

65

kD

50

kD

40

kD

30

kD

25

kD

20

kD

b

Phosphoenolpyruvate carboxykinase (PEPCK) is expressed by promastigotes and amastigotes

39

PEPCK elicits strong T cell response in L. major-infected healed human patients

Control

Infected

40

1 20

1

2

3

4

56

1014

a b c

1 20

2

4

6

8

10

12

SLA rPEPCK

Ind

ex o

f sti

mu

lati

on

SLA rPEPCK

IFN

-γ(n

g/m

l)

*

1 20.1

1

10

100

1000

SLA rPEPCK

Grz

B (

ng

/ml)

*

1 20

2

4

6

8

10

12*

Challenge:L. major

L. donovani

Immune

response

Peptide Vaccine:

1) PBS

2) CpG control

3) Peptide + CpG

Does Vaccination with PEPCK Protect Against Virulent Challenge?

DNA Vaccine:

1) PBS

2) CpG control

3) DNA+ CpG

4) Vector + CpG

Protein Vaccine:

1) PBS

2) CpG control

3) Protein + CpG

4) Protein only

0 1 2 3 4 5 6 8 10wks

12

Challenge:L. major

1 2 3 4 5 60

1

2

3

4PBS

CpG

Peptide vaccine

****** *** ***

Weeks Post-challenge

Les

ion

siz

e (m

m)

B

A

D

C

PB

S

CpG

Vec

tor

DN

A v

acci

ne

2

4

6

8***

Par

asite

bu

rden

(lo

g10

)

PB

S

CpG

Pep

tide

vac

cin

e

4

5

6

7

8

**

Par

asite

bu

rden

(lo

g10

)

1 2 3 4 5 60

1

2

3

4

PBS

CpG

Vector

DNA vaccine

**** ** ** **

Weeks Post-challenge

Les

ion

siz

e (m

m)

Vaccination with the synthetic peptides and DNA vaccine expressing PEPCK induces protection against virulent L. major challenge.

C57BL/6

PB

S

CpG

rPE

PC

K

rPE

PC

K+C

pG

2

4

6

8

10

***

**

Par

asite

bu

rden

(lo

g10

)

BALB/c

1 2 3 4 5 60

1

2

3

4

5PBS

CpG

rPEPCK

rPEPCK + CpG

* ** ** **

*** *** *** *****

Weeks Post-challenge

Les

ion

siz

e (m

m)

PB

S

CpG

rPE

PC

K

rPE

PC

K+C

pG

5

6

7

8

9

Par

asite

bu

rden

(lo

g10

)

***

1 2 3 4 5 60

2

4

6PBS

CpG

rPEPCK

rPEPCK+CpG

***

*

Weeks Post-challenge

Les

ion

siz

e (m

m)

A B

C D

Vaccination with the recombinant PEPCK induces protection against virulent L. major challenge. (6 weeks)

DNA Vaccine Confers Long-term (12 Weeks) Protection

Balb/c

1 2 3 4 50

1

2

3

4

5PBS

CpG

Vector

DNA vaccine

**** **

Weeks Post-challenge

Lesi

on s

ize

(mm

)

PB

S

CpG

Vec

tor

DN

A v

acci

ne

4

6

8

10

*

Par

asite

bur

den

(log 10

)

C57BL/6

1 2 3 4 50

1

2

3

4PBS

CpG

Vector

DNA vaccine

* * *

Weeks Post-challenge

Lesi

on s

ize

(mm

)

PB

S

CpG

Vec

tor

DN

A v

acci

ne2

4

6

8

10

12

14 ***

Par

asite

bur

den

(log 10

)

C

A

D

B

PEPCK Vaccine Cross-Protects Against Experimental Visceral Leishmaniasis

PB

S

Ve

cto

r

DN

A v

ac

cin

e

0

5

10

15

20

*P

ara

sit

e b

urd

en

(´10

7)

PB

S

Ve

cto

r

DN

A v

ac

cin

e

0

1

2

3 **

Para

sit

e b

urd

en

(´10

6)

LIVER SPLEEN

Naturally processed Leishmania major peptides were eluted and identified from MHCII molecules on L. major -infected BMDCs

PEPCK-specific T cells are immunodominant, polyfunctional cytokine producers andprotect against L. major

Vaccination with PEPCK peptides, PEPCK DNA or rPEPCK induces cross-protectionagainst Leishmania challenges

L. major, L. donovani, L. infantum (preliminary evidence)

Summary:

46

Implications for Vaccine Designs and Vaccination Strategies

• Conventional methods of immunogen prediction may be misleading

In silico predictions, early versus memory immune responses

• Approach aimed at identifying antigens that are really relevant

Reverse immunology

• May be relevant in other parasitic infections where concomitant immunity is critical for resistance

Malaria, toxoplasmosis

Next Step:

• Vaccination studies in NHP, Dogs

• Clinical Trials?

• Collaborators and funding wanted!!!

Acknowledgements

Project Leader:

Dr. Zhirong Mou

Uzonna Lab membersJintao LiForough Khadem

Manitoba centre for Protemics and System BiologyDr. John WilkinsPeyman Ezzati

Third Military Medical University, ChinaChuanmin Hu; Weijing Yi; Shufeng Wang

Flow cytometry Core FacilityChristine Zhang

Cancer Care ManitobaLudger Klewes

Collaborators:

Funding:

Pasteur Institute of Tunis, TunisiaThouraya BoussoffaraHechmi Louzir

University of Toyama , JapanHiroyuki KishiHiroshi Hamana

McGill UniversityMomar Ndao

Sichuan University, ChinaJianping Chen

Pasteur Institute of Iran, IranSima Rafati

University of IowaChristine Petersen

49