B CELLS AND BLOOD STAGE MALARIA

Sean Elias

MALARIA LIFE CYCLE

ANTIGEN CONSTRUCTS

MSP-1: Merozoite surface protein. 19 fragment contains major B cell epitopes (anchored to RBCs during invasion) Preclinical association of 19 antibodies with protection 33 fragment contains Th cell epitopes

AMA-1: Apical membrane antigen. Required for RBC invasion Preclinical association of antibodies with protection

1 3 5 12 3D7-33 3D7-19

F VO-33 FVO-19

Glycine-Proline linker

MSP-1

Glycine-Proline linker

tPA 3D7 FVO Ectodomain

FVO Terminus

AMA-1

CLINICAL TRIAL STRUCTURE

VAC36 VAC37

Adeno Prime MVA Boost

8wk

CLINICAL TRIAL STRUCTURE

VAC39

VOLUNTEER BLEEDS

Vac1AdCh63

D0 D2 D14 D28 D56 D90S

Vac1AdCh63

D0 D2 D14 D28 D56 C-1 D63 D58 C+21 / DoDS

Vac 2MVA

Vac1AdCh63

D0 D2 D14 D28 D56 D84 D63 D58 D140S

Vac 2MVA

= Blood test

AdCh63 = AdCh63 AMA-1 (VAC36), MSP-1 (VAC37)

MVA = MVA AMA1 (VAC36), MSP-1 (VAC37)

S = Screening

C+7 C+11 C+35 C+90

2A

0 20 40 60 80 1001

10

100

1000

10000

100000004016

MS

P1

AU

2B

0 50 100 1501

10

100

1000

10000

100000006013019022

023

MS

P1

AU

2C

0 50 100 1501

10

100

1000

10000

100000007014017

MS

P1

AU

2C

MEMORY B CELLS AND MALARIA

What is the effect of challenge/infection on mBC expansion?

The P. falciparum-specific human memory B cell compartment expands gradually with repeated malaria infections. Weiss 2010

Long-lived antibody and B cell memory to P. falciparum in low transmission areas. Wipasa 2010

P. Yoelii Can Ablate Vaccine-Induced Long Term protection in mice. Wykes 2005

Kenyan children exposed to malaria may develop antibody but not memory to blood stage antigens (AMA-1 more so than MSP-1) Dorfman 2005

Decreases in memory and naive B cell subsets in Kenyan children with acute P. falciparum infection. Asito 2008

Atypical memory B cells (‘exhausted’) are greatly expanded in individuals living in a malaria-endemic area. Weiss 2009

A Positive Correlation between Atypical Memory B Cells and Plasmodium falciparum Transmission Intensity in Cross-Sectional Studies in Peru and Mali Weiss 2011

VAC37 : MSP-1 ANTIBODY DATA

0 20 40 60 80 1001

10

100

1000

10000

100000III

Time (d)

An

ti-M

SP

119

EL

ISA

un

its

1B

0 50 100 1501

10

100

1000

10000

100000IIIIIIIV

Time (d)

An

ti-M

SP

119

EL

ISA

un

its

1A

0 20 40 60 80 1001

10

100

1000

10000

100000III

Time (d)

An

ti-M

SP

119

EL

ISA

un

its

A B

C D

0 50 100 1501

10

100

1000

10000

100000IIIIIIIVV

Time (d)

An

ti-M

SP

119

EL

ISA

un

its

Group 1A Group 1B

Group 2A Group 2B+2C

0 50 100 1501

10

100

1000

10000

100000IIIIIIIVVVIVIIVIII

Time (d)

An

ti-M

SP

119

EL

ISA

un

its

ELISA DATA: S. Draper

VAC37 : MSP-1 MEMORY B CELL ELISPOT

2C Total Ag Specific mBC

0 28 56 84 112 140 1680

50

100

150

200

250

300

350

400

450

500

III

III

Day

Ag

sp

ecifi

c m

BC

/Mill

ion

Cu

ltu

red

PB

MC

2B Total Ag Specific mBC

0 28 56 84 112 140 1680

50

100

150

200

250

300

350

400

450

500

IVV

VIVII

VIII

Day

Ag

sp

ecifi

c m

BC

/Mill

ion

Cu

ltu

red

PB

MC

2C % mBC of total

0 28 56 84 112 140 1680.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

III

III

Day

Ag

Sp

ecifi

c m

BC

's a

s %

of

To

tal I

gG

BC

's

2B % mBC of total

0 28 56 84 112 140 1680.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

IVV

VI

VII

VIII

Day

Ag

Sp

ecifi

c m

BC

's a

s %

of

To

tal I

gG

BC

's

Challenge

Boost

Prime

Day of Diagnosis

Prime

Boost

VAC37 : MSP-1 MEMORY B CELL ELISPOT

2C Total IgG mBCs

0 28 56 84 112 140 1680

5000

10000

15000

20000

25000

30000

35000

40000

III

III

Day

Ag

sp

ecifi

c m

BC

/Mill

ion

Cu

ltu

red

PB

MC

2B Total IgG mBCs

0 28 56 84 112 140 1680

5000

10000

15000

20000

25000

30000

35000

40000

IVV

VI

VIIVIII

Day

Ag

sp

ecifi

c m

BC

/Mill

ion

Cu

ltu

red

PB

MC

2C % Dip mBC of total

0 28 56 84 112 140 1680.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

III

III

Day

Ag

Sp

ecifi

c m

BC

's a

s %

of

To

tal I

gG

BC

's

2B % Dip mBC of total

0 28 56 84 112 140 1680.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

IVV

VI

VIIVIII

Day

Ag

Sp

ecifi

c m

BC

's a

s %

of

To

tal I

gG

BC

's

VAC37 : MSP-1 MEMORY B CELL ELISPOT

Day 84

0 2000 4000 6000 8000 100000

50

100

150

200

Ab Titre 3D7

mB

C's

/mill

ion

cu

ltu

red

PB

MC

Day 84 (Outlier Excluded)

0 2000 4000 6000 8000 100000

50

100

150

200

Ab Titre 3D7

mB

C's

/mill

ion

cu

ltu

red

PB

MC

P= 0.076 P= 0.0004

D84/D90

1A 1B 2A 2B0

50

100

150

200

Group

mB

C's

/mill

ion

cu

ltu

red

PB

MC

Boost required for significant B cell response

mBC’s correlate with Ab’s

VAC36 : AMA-1 MEMORY B CELL ELISPOT

D84

I II III IV V VI VII VIII0

50

100

150

200

250

Volunteer

mB

C's

/mill

ion

cu

ltu

red

PB

MC

mBC vs AB

0 1000 2000 3000 40000

50

100

150

200

250

III

III

IV

V

VI

VII

VIII

Ab Titre 3D7

mB

C's

/mill

ion

cu

ltu

red

PB

MC

1B

0 20 40 60 80 100 120 1401

10

100

1000

10000I

IIIII

IV

Time (d)

An

ti-A

MA

1 (

3D

7)

EL

ISA

un

its

0 20 40 60 80 100 120 1401

10

100

1000

10000

VVIVII

VIII

2B

Time (d)

An

ti-A

MA

1 (

3D

7)

EL

ISA

un

its

P= 0.0962

ELISA DATA: S. Draper, S. Biswas

VAC39 CONTROLS

3/5 controls had mBC response to MSP-1 at 35 days after challenge

No response to AMA-1

Appears to be rough correlation with Ab titre for MSP-1. Needs more power to confirm relationship.

Only 2/5 volunteers showed low Ab titre to AMA-1 at C+35

Kenyan children exposed to malaria may develop antibody but not memory to blood stage antigens (AMA-1 more so than MSP-1) Dorfman 2005

Controls C+35

MSP-1 AMA-10

10

20

30

40

50

mB

C's

/mill

ion

cu

ltu

red

PB

MC

MSP-1 mBC vs Ab

0 200 400 600 800 10000

10

20

30

40

50

Ab Titre 3D7

mB

C's

/mill

ion

cu

ltu

red

PB

MC

MEMORY B CELL ELISPOTS Conclusions:

Ad/MVA prime boost regime induces mBC formation to both MSP-1 & AMA-1

Following this regime with parasite exposure further boosts mBCs to levels much higher than seen through natural exposure (up to x10?)

mBC derived B cells appear to correlate with Ab titre Activation of mBC by malaria antigens does not appear to activate other

mBC’s (Diphtheria) by bystander effect Blood stage parasitemia appears to deplete circulating mBC’s but this

doesn’t effect boosting when patients drug cured at cut-off peak parasitemia.

Will be interesting to see boost effect of challenge on vaccinees on AMA-1 given lack of seroconversion and mBC’s in controls.

VAC39 aims: Replicate data for challenged volunteers (Group 1) Look for effect of challenge on mBC’s for AMA-1 (Group 2) Study effect of dual antigen vaccination on mBC formation (Group 3)

VAC39 : ASC ELISPOT

Ahmed 2008: Showed that for flu vaccination there was a rapid and robust influenza specific IgG+ antibody secreting plasma cell (ASC) response that peaked at approximately day 7 after boosting.

Questions:- What time after boosting can we see a peak? Limited to D63 with

current protocol.- What % of cells detected are antigen specific. - When during challenge can such cells be seen?- Is there a correlation with Antibody production?

VAC39 : ASC ELISPOT

5 7 8 9 150

50

100

150

200

250

300

Days since boost

AS

C's

mill

ion

PB

MC

13 14 15 16 17 230

10

20

30

40

50

60

Days since boost

AS

C's

mill

ion

PB

MC

Group Effect D63 (Day 7 only)

1 3 40

50

100

150

200

250

300

Group

AS

C/M

illio

n P

BM

C

Group Effect C-1 (all)

1 3 40

10

20

30

40

50

60

Group

AS

C's

mill

ion

PB

MC

VAC39 : ASC ELISPOT Answers?:

- Peak likely to be somewhere between day 5 and 8. - Harder to calculate total IgG secreting cells compared to mBC assay.- No ASC’s detected during challenge (C+7, C+11)- No ASC’s detected in protected volunteer at C+21- No significant correlation with Antibody production Day 63.

Ab vs ASC (Day 7 only)

0 2000 4000 60000

100

200

300

Ab Units

AS

C's

mill

ion

PB

MC

VAC44 : UPCOMING TRIAL

VAC44 : EXPERIMENTAL PLAN

ASC ELIspots: Additional timepoints for detection (1, 4 & 7 days post boost) More regulation on visit windows so these time points more accurate

Can test whether successive boosts shorten time for peak response Can compare effect of boost regimes. Vectors/Protein/Adjuvant.

mBC ELIspots: Can compare effect of boost regimes. Vectors/Protein. Effect of CPG adjuvant (mBC stimulator)

FUTURE WORK

B cell phenotyping Atypical memory B cells (‘exhausted’): Are they induced in our

vaccination regimes or during controlled challenge?

B cell clonality Clonal dissection of mBC repertoire (Pinna 2009).

Clonal burst size Antibody production on clonal level

B cell receptor (BCR) variable region genes will be cloned and sequenced to assess the antibody / BCR repertoire induced by different priming and boosting vaccinations

Does successive vaccinations/challenge give a more directed, higher affinity response?