correlates of protection against enterotoxigenic ... · correlates of protection against...
TRANSCRIPT
Correlates of Protection against
Enterotoxigenic Escherichia coli
Firdausi Qadri
Infectious Disease Division
Correlates of enteric vaccine induced protection
Fondation Merieux, Annecy
22 March, 2016
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
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
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)
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
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
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
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
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
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
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
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
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
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
+
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)
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
Studies on ETEC in an urban setting in Mirpur in Dhaka
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