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Correlates of Protection against Enterotoxigenic Escherichia coli Firdausi Qadri Infectious Disease Division Correlates of enteric vaccine induced protection Fondation Merieux, Annecy 22 March, 2016

Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

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Page 1: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Correlates of Protection against

Enterotoxigenic Escherichia coli

Firdausi Qadri

Infectious Disease Division

Correlates of enteric vaccine induced protection

Fondation Merieux, Annecy

22 March, 2016

Page 2: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Importance of enterotoxigenic E. coli (ETEC) in

children in developing countries

ETEC is the most common cause of bacterial diarrhea in children ≤ 3 years (10- 20% of all cases)

Repeated ETEC diarrheas

can cause malnutrition, growth

retardation and stunting

In Bangladesh, acute dehydrating diarrhea is

common and epidemics seen at least two times every year

and additionally peaks during natural disasters

Page 3: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Num

ber

of E

TE

CDistribution of ETEC cases in 2% surveillance systemof the icddr,b Dhaka Hospital

March, 2016, 18% of ETEC in the 2% Surveillance system of of the icddr,b hospital

Page 4: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

VC: 46 (7%)

ETEC: 29

(4.41%)

VC: 33 (8.13%)

ETEC: 10

(2.46%)

DMC -

VC: 4 (2.48%)

ETEC: 9

(5.59%)

VC: 40 (11.83%)

ETEC: 15

(4.44%)

VC: Positive : 2

(0.43%)

ETEC: Positive : 6

(1.28%)

VC: 24 (4.87%)

ETEC: 24

(4.87%)

BITID -

VC: 145

(26.08%)

ETEC: 23

(4.14%)

VC: 5 (1.47%)

ETEC: 2

(0.59%)

VC: 26 (5.34%)

ETEC: 11

(2.26%)

Uttara -

VC: 8 (5.1%)

ETEC: 7

(4.46%)

Nationwide

surveillance

on ETEC in

Bangladesh

carried out in the

Upazilla hospitals

and other health

facilities

Study in 7

divisions of

Bangladesh

10 sites (2013-

2015; ongoing)

Page 5: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

ETEC diarrheal infections trigger mucosal immune responses which are not

easy to study using invasive procedures such as small intestinal biopsies

Surrogate markers of local responses can be studied in secretions and in blood

to decipher the nature of the B and T cell response

The potential destination of circulating antigen specific antibody secreting cells

can be investigated by studying the expression of gut homing markers

A more targeted immune response can be associated with longer lasting

immunity

Towards bettter understanding ETEC infections and protective

mechanisms for vaccine evaluation

Page 6: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

D2 D7 D30 D2 D7 D300

20

40

60

80

LTB IgA

Study day

Fold increase

Response(%)3.0 1.5 1.4 1.0

60 30 35 10

LT or LT/ST ETEC

ST ETEC

*

*

Pla

sm

a a

nti

-LT

B I

gA

(EL

ISA

Un

it)

D2 D7 D30 D2 D7 D300

100

200

300

400

Study day

Fold increase 4.3 1.7 1.4 1.1Response(%) 60 43 26 19

CFAI groupETEC

Other ETEC

CFAI IgA

*

Pla

sm

a a

nti

-CF

A/I

Ig

A

(EL

ISA

Un

it)

D2 D7 D30 D2 D7 D300

500

1000

1500

Study day

Fold increaseResponse(%)

20 3.4 1.3 1

100 55 28 18

CS6 IgA

CS6 ETEC Other ETEC

*

*Pla

sm

a a

nti

-CS

6 I

gA

(EL

ISA

Un

it)

D2 D7 D30 D2 D7 D300

20

40

60

LTB IgG

Study day

Fold increase

Response(%)2.6 2 1.2 1.2

55 50 18 11

LT or LT/ST ETEC

ST ETEC

* *

Pla

sm

a a

nti

-LT

B I

gG

(EL

ISA

Un

it)

D2 D7 D30 D2 D7 D300

100

200

300

Study day

Fold increase

Response(%)

3.0 1.6 1.2 1.3

55 43 21 22

CFAI IgG

CFA/I group ETEC

Other ETEC

*

*

Pla

sm

a a

nti

-CF

A/I

Ig

G

(EL

ISA

Un

it)

D2 D7 D30 D2 D7 D300

1000

2000

3000

Study day

Fold increase

Response(%)

4.2 3.3 1.1 1.1

75 72 11 29

CS6 ETEC Other ETEC

CS6 IgG

*

*

Pla

sm

a a

nti

-CS

6 I

gG

(EL

ISA

Un

it)

A B C

D EF

LTB, CFA/I and CS6 specific antibody responses

in plasma of Bangladeshi adults with ETEC diarrhea

Increased levels of plasma IgA and IgG antibodies at day 7 after onset ETEC diarrhea to different antigens

Page 7: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

LTB IgA

d2 d7 d30 d2 d7 d300.0

0.1

0.2

0.3

0.4

0.5*

LT or LT/ST

ETECST ETEC

LT

B s

pecific

Ig

A

per

tota

l Ig

AS

C (

%)

CFA/I IgA

d2 d7 d30 d2 d7 d300.0

0.5

1.0

1.5 *

CFA/I group ETEC Other ETECC

FA

/I s

pecific

Ig

A

per

tota

l Ig

AS

C (

%)

CS6 IgA

d2 d7 d30 d2 d7 d300.0

0.5

1.0

1.5

2.0 *

CS6 ETEC Other ETEC

CS

6 s

pecific

Ig

A

per

tota

l Ig

AS

C (

%)

LTB IgG

d2 d7 d30 d2 d7 d300.0

0.5

1.0

1.5

2.0

LT or LT/ST

ETECST ETEC

*

LT

B s

pecific

Ig

G

per

tota

l Ig

AS

C (

%)

CFA/I IgG

d2 d7 d30 d2 d7 d300

1

2

3

CFA/I group ETEC Other ETEC

*

CF

A/I s

pecific

Ig

G

per

tota

l Ig

AS

C (

%)

CS6 IgG

d2 d7 d30 d2 d7 d300

1

2

3

*

CS6 ETEC Other ETECC

S6 s

pecific

Ig

G

per

tota

l Ig

AS

C (

%)

Circulating ASC responses to LTB, CFA/I and CS6 in

adult Bangladeshi ETEC patients

Increased circulating ASCs of IgA and IgG isotypes at day 7 after onset of ETEC diarrhea

Page 8: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

d2 d30 d2 d300.00

0.05

0.10

0.15

0.20

Responder (%) 41 72 55 57

LT or LT/ST ETEC ST ETEC

LTB IgA

*

% L

TB

-Ig

A m

em

ory

B c

ell p

er

tota

l Ig

A

d2 d30 d2 d300.0

0.2

0.4

0.6

Total Response (%) 35 60 45 40

CFA/I specific (%) 33 50 33 35

CS6 specific (%) 37 70 62 53

CFA/I group and CS6 ETEC Other ETEC

CF IgA

*

% C

F-I

gA

mem

ory

B c

ell

per

tota

l Ig

A

d2 d30 d2 d300.00

0.01

0.02

0.03

0.04

Responder (%) 25 48 40 50

LTB IgG

LT or LT/ST ETEC ST ETEC

*

% L

TB

-Ig

G m

em

ory

B c

ell

per

tota

l Ig

G

d2 d30 d2 d300.00

0.05

0.10

0.15

0.20

0.25

Total Response(%) 21 55 35 34

CFA/I specific (%) 17 42 18 33

CS6 specific (%) 28 75 57 36

CFA/I group and CS6 ETEC Other ETEC

CF IgG

*

% C

F-I

gG

mem

ory

B c

ell

per

tota

l Ig

G

A

DC

B

LTB and CF specific memory B cell responses in

patients infected with ETEC diarrhea

Alam et al., PNTD, 2014

Page 9: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Avidity indices of LTB (A and D), CFA/I (B and E) and CS6 (C and F) specific IgA and IgG antibodies in plasma in Bangladeshi adults infected with ETEC

Alam et al., PNTD, 2014

Avidity indices at day 7 remained elevated up to day 30 in patients infected

with ETEC expressing LT or LT/ST, CFA/I and CS6

Page 10: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Total IgA

HC

Day

2

Day

7

Day

30

0

20000

40000

60000

80000**

***

To

tal Ig

A s

po

ts/ m

illio

n c

ell

Total IgG

HC

Day

2

Day

7

Day

30

0

25000

50000

75000

100000

*

***

To

tal Ig

G s

po

ts/ m

illio

n c

ell

Total gut-homing (7+) ASC responses in ETEC patients

Increased levels of gut homing total immunoglobulin (IgA and IgG) were observed at day 7 after onset of ETEC diarrhea

Bhuiyan et al., CVI, 2015

Page 11: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

HC d2 d7

d30

0

20

40

60

CFA/I

% o

f C

FA

/I s

pecific

Ig

A/

tota

l Ig

HC d2 d7

d30

0

10

20

30

CFA/I

% o

f C

FA

/I s

pecific

Ig

G/

tota

l Ig

CTB and CFA/I specific IgA and IgG (7+) ASC responses were observed on day 7 compared to healthy controls and on study, day 2 and 30

HC

Day

2

Day

7

Day

30

0

2

430

32

CTB

*

% o

f C

TB

sp

ecific

Ig

A/

tota

l Ig

HC d2 d7

d30

0

4

815

20 *

CTB

% o

f C

TB

sp

ecific

Ig

G/

tota

l Ig

IgA

IgA

IgG

IgG

Page 12: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

HC d7

d30

0

4

820

24

CS60.0949p=

% o

f C

S6 s

pecific

Ig

A/

tota

l Ig

HC d7

d30

0

5

10

1530

35

40

CS6* *

% o

f C

S6 s

pecific

Ig

G/

tota

l Ig

CS6 specific gut homing (7+) ASC responses in ETEC patients

CS6 specific IgA and IgG responses were observed at day 7

Bhuiyan et al., CVI, 2015

IgA IgG

Page 13: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

0 1 2 3 4 50

50

100

150

200

25 30

r =p =

0.62

0.0003

% of CTB specifiic 7+ IgA ASCs at day 7

CT

B Ig

A E

LIS

A U

nit

s a

t d

ay

7

0 1 2 3 4 50

20

40

60

80

25 30

r =p =

0.390.03

% of CTB specific 7+ IgA ASCs at day 7

CT

B Ig

A E

LIS

A U

nit

s a

t d

ay

30

0 5 10 15 20 25 300

50

100

150

200800

1000

r =p =

0.56

0.002

% of CTB specific 7+ IgG ASCs at day 7

CT

B Ig

G E

LIS

A U

nit

s a

t d

ay

7

0 5 10 15 20 25 300

50

100400

500

r =p =

0.37

0.05

% of CTB specific 7+ IgG ASCs at day 7

CT

B Ig

G E

LIS

A U

nit

s a

t d

ay

30

A B

C D

Association of gut-homing ASCs with plasma

antibody titers in patients with ETEC infection

Bhuiyan et al., CVI, 2015

ETEC specific gut-homing IgA and IgG ASCs at day 7 were positively correlated with

specific IgA and IgG antibodies on days 7 and 30 post onset of diarrhea

CTB IgA/D7

CTB IgG/D7 CTB IgG/D30

CTB IgA/D30

Page 14: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Antigen specific Memory helper T cell responses

HC 2 7 30 HC 2 7 30 HC 2 7 30 HC 2 7 30 HC 2 7 300

5

10

LTB dmLT ST EatA G33D mCT

Day

Antigen

***

***

**

***

**

^^

0.0651

**

**

Sti

mu

lati

on

In

de

x,

CD

3+

CD

4+

CD

45

RO

+

Page 15: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Cytokine responses

IFN- ELISA

day2 day7 day2 day7 day2 day70

100

200

300

**

**

LTB dmLT G33D mCT

*

IFN

-g c

on

ce

ntr

ati

on

(p

g/m

L)

IL-13 ELISA

day2 day7 day2 day7 day2 day70

10

20

30

40

*

*

0.3125

LTB dmLT G33D mCT

IL-1

3 c

on

ce

ntr

ati

on

(p

g/m

L)

Page 16: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Birth cohort study on ETEC and relatedness of ETEC diarrhea with blood group

antigen

Studies on children in a birth cohort in Mirpur have shown :

ETEC infections cause stunting in children.

Children with CFA/I ETEC diarrhea are protected from homologous ETEC infection

Children with histoblood group AB and A have increased susceptibility to ETEC

infection

Children with Lewis blood group Le(a+b-) are more susceptible to CFA/I diarrhea

Lewis blood group ‘a’ antigen may be a candidate receptor for ETEC colonization

factor CFA/I

Page 17: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Studies on ETEC in an urban setting in Mirpur in Dhaka

Page 18: Correlates of Protection against Enterotoxigenic ... · Correlates of Protection against Enterotoxigenic Escherichia coli ... ETEC diarrheal infections trigger mucosal immune responses

Conclusions

• ETEC induces memory B cells and high avidity antibodies to LTB

and CFs that could mediate anamnestic responses on re-exposure

to ETEC and may help in understanding the requirements for

designing and following effectiveness of vaccines

• Need to evaluate whether memory responses, affinity maturation

and level of antibodies to these ETEC antigen correlate with

protection following vaccination

• Vaccine studies can now include studies of mucosal gut homing

ASC in young children and infants using low volumes of blood

using the cell sorting and ELISPOT technique

• Measurement of ALS/ASC and plasma antibody levels within 5

days of second/booster dose of oral vaccines is critical for

gauging the peak of the responses in endemic settings

• Relationship of ABO and Lewis blood group and genetic factors

on response to oral vaccines is an area that needs to be given

attention

• ETVAX vaccine is now in the infant phase with encouraging

results of safety in all age groups