ns1 blockade-of-binding elisa distinguishes between dengue and zika … · 2019-09-13 · ns1...
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NS1 blockade-of-binding ELISA distinguishes between dengue and Zikavirus antibodies
Davide Corti, PhD
CSO
Humabs Biomed SA
Presentation to
WORKSHOP: New and Innovative Approaches to Laboratory Diagnosis of Zika, Dengue and other
Arboviruses. May 3rd, 2017 – Annecy
Page 2
Humabs at a Glance
Swiss privately held biotech company, located in Bellinzona, Switzerland, spin-off of the
Institute for Research in Biomedicine (IRB)
Leader in selecting effective human monoclonal antibodies for prophylaxis and/or
therapy of infectious diseases
Two successful clinical-stage partnered programs: Novartis, in phase 2, and
MedImmune (FluA), in phase 1b/2a
Diversified portfolio
Internal development of three programs
Four programs in partnership with MedImmune
“Public Health” programs
Page 3
Making use of human mAbs
Modified from Corti and Lanzavecchia, Ann. Rev. Immunol. 2013
Identification of HCMV receptor
of trimer and pentamer
PDGFR-alpha (Kabanova et
al. Nat. Micr. 2016)
HCMV pentamer receptor
recently identified
Development of an assay
for Zika virus serological
diagnosis (Balmaseda et
al., PNAS in revision)
Page 4
The serological detection of ZIKV infections is challenging
The high level of cross-reactivity among flaviviruses and their co-circulation has
complicated the use of serological assays to differentially detect clinical and
subclinical ZIKV and dengue virus (DENV) infections
ZIKV could be considered as a fifth member of the DENV serocomplex
Barba-Spaeth, Nature 2016
Page 5
Reactivity of plasma from ZIKV-infected donors to ZIKV and DENV NS1
101 102 103 104 1050
1
2
3
4
Plasma dilution
Bin
din
g to Z
IKV
NS
1 (
OD
)
101 102 103 104 1050
1
2
3
4
Plasma dilution
Bin
din
g to D
EN
V1 N
S1 (
OD
)
ZA
ZB
ZC
ZD
101 102 103 104 1050
1
2
3
4
Plasma dilution
Bin
din
g to D
EN
V2 N
S1 (
OD
)
101 102 103 104 1050
1
2
3
4
Plasma dilution
Bin
din
g to D
EN
V3 N
S1 (
OD
)
101 102 103 104 1050
1
2
3
4
Plasma dilution
Bin
din
g to D
EN
V4 N
S1 (
OD
)
ZIKV-infected individuals pre-exposed
to DENV have high levels of plasma
NS1 cross-reactive antibodies
41 MAbs vs NS1 isolated
from 4 ZIKV-infected
patients
Stettler et al., Science 2016
Page 6
Antigenic site mapping of specific and cross-reactive NS1-mAbs
Octet (Bio-Layer Interferometry) cross-competition
studies were used to generate an antigenic map of NS1
mAbs
None of the DENV cross-reactive mAbs competed with
ZKA35 mAb (S2 antigenic site)
Stettler et al., Science 2016
Page 7
ZKA35 mAb binds to a conserved, but ZIKV-specific, epitope on NS1
ZKA35 does not bind to heat-denatured NS1, likely targeting a discontinuous or
conformational epitope
ZKA35 mAb was isolated from an individual infected with ZIKV on a trip to
Guatemala and El Salvador in November 2015
ZKA35 binds equally well to recombinant NS1 from a 2016 Suriname ZIKV strain as
well as from the prototype Ugandan strain MR766 produced in either insect or
mammalian cells (data not shown). In addition, we found that ZKA35 reacts by flow
cytometry with cells infected with the Asian ZIKV strain H/PF/2013 (data not shown).
Additional studies will be required to map in more detail the epitope of the ZKA35
mAb.
Page 8
Blockade-of binding (BOB) assay
Plasma or serum are diluted 1:10 (i.e. 5 µl/sample) and the probe ZKA35 mAb is used at
only 10 ng/ml
Binding of the plasma
polyclonal antibodies to multiple
sites of the coated ZIKV NS1
1 hr
Binding of
ZKA35 is not
blocked
wash
+
STV-AP
ZK
A35
Biotinilated ZKA35 mAb
Plasma polyclonal
antibodies (even undiluted)Coated ZIKV
NS1
STV-AP
Signal
No signal
ZK
A35
Probe Ab is added
without washing
5-15 min wash
+
substrate
(pNPP)
Reading
with a
spectrophotometer
Binding of
ZKA35 is
blocked
Plasma is added
to ZIKV NS1
coated wells
Stettler et al., Science 2016
*NS1 used at 1 µg/ml (Meridian or NAC)
Page 9
Low specificity of ELISA methods based on total IgG binding to NS1
ZIKV
DENV1
DENV2
DENV3
DENV4
WNVYFV
0
1
2
3
4
5
Bin
din
g to N
S1 (
OD
)
DENV-naiveDENV-immuneUnknowns
ZIKV
DENV1
DENV2
DENV3
DENV4
WNVYFV
0
1
2
3
4
5
Bin
din
g to N
S1 (
OD
)
UnknownSec. DENV
ZIKV
DENV1
DENV2
DENV3
DENV4
WNVYFV
0
1
2
3
4
5
Bin
din
g to N
S1 (
OD
) The four plasma samples from ZIKV-infected DENV-immune donors crossreacted with
DENV1-4 and WNV NS1
A large fraction of plasma from ZIKV-naive DENV-immune donors (36-40%) cross-reacted
with ZIKV, WNV and YFV NS1 Ags (all collected before the appearance of ZIKV)
Plasma were tested by
ELISA for binding of IgG Abs
to solid-phase NS1 from
different flaviviruses.
Samples were tested at a
fixed dilution of 1:100
N=18 N=44 N=30
Balmaseda et al., in revision
Page 10
Characteristics of samples from Nicaragua
Year 1 Year 2 Year 3 Year 4
YearlyHealthySamplesConvalescent
Sample
DOS
AcuteSample
*
The Pediat ric Dengue Cohort Study (3,500 children 2-14 y/o)(Dengue 2004-2020; Chikungunya 2014+; Zika 2016+)
DOS
LongitudinalSamples
3 months 6 months 12 months 18 months
The Hospital-based Study (Dengue 1998/2005-2022; Chikungunya 2014+, Zika 2016+)
ConvalescentSample
2 wks
Enrolled at presentation to National Pediatric Reference Hospital
AcuteSamples
Page 11
ROC analysis to establish the optimal inhibition cutoff in the NS1 BOB ELISA
The ROC analysis established 50% as the most effective cut-off value for ZKA35 binding
inhibition
DENV ZIKV
-20
0
20
40
60
80
100
BO
B (
%)
N=146 N=112
0 20 40 60 80 1000
20
40
60
80
100
100% - Specificity%
Sensitiv
ity%
> 0.55 > 10.55 > 22.8 > 40 > 55.5 > 68.85 > 80.80
20
40
60
80
100
BOB (%) cut-off
Perc
enta
ge (
%)
Sensitivity%Specificity%
>50.3
Nicaraguan samples from the latest time-points available from subjects diagnosed by
RT-PCR for ZIKV or DENV infection were used to perform a ROC analysis to identify
the cut-off giving optimum sensitivity and specificity
Balmaseda et al., in revision
The NS1 BOB assay was established in the National Virology Laboratory of the
Ministry of Health in Managua, Nicaragua
Page 12
NS1 BOB analysis of Nicaraguan samples from ZIKV-confirmed patients
The kinetics of the NS1 BOB assay results indicated that inhibiting Abs persisted for over 5
months after ZIKV infection
0 30 60 90 120 150180
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
0 30 60 90 120 150180
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
0 20 40 60 80 100
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
DENV(N
)
DENV(I)
N/A
-20
0
20
40
60
80
100
BO
B (
%)
32/37 33/38 35/37
Testing of the samples collected at least 10 days after symptom onset in the NS1 BOB assay
indicated that 100 of 112 scored positive (89.3%)
In this sample set, we did not observe a significantly different rate of positivity in the NS1 BOB assay
in samples between DENV-naïve and DENV-immune patients
N=37 DENV-naïve N=38 DENV-immuneN=37 unknown
PDCS
Balmaseda et al., in revision (analysis perfomed in Nicaragua, A. Balmaseda, D. Collado, J.V. Zambrano, S. Saborio, and E. Harris)
Page 13
NS1 BOB analysis of Nicaraguan samples from DENV-confirmed patients
BOB assay is highly specific in primary DENV patients and in the great majority (about 4 of
5) of patients who experienced secondary DENV infections
0 10 20 30 40
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
0 10 20 30 40
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
0 30 60 90 120150180
-20
0
20
40
60
80
100
days after symptoms
BO
B /%
)
Prim
. DEN
V1
Prim
. DEN
V2
Prim
. DEN
V3
Sec
. DEN
V1
Sec
. DEN
V2
Sec
. DEN
V3
-20
0
20
40
60
80
100
BO
B (
%)
0/1
6
0/2
1
1/2
5 6/3
1
0/2
2 10/3
1
Samples from subjects diagnosed as primary DENV1, DENV2 or DENV3 infections during 2010 to
2014 (prior to the introduction of ZIKV into Nicaragua) were analyzed none of these samples
scored positive
Samples from subjects with secondary DENV1, DENV2 or DENV3 infections from 2005 to 2016 were
evaluated. In 17/86 secondary DENV infections, the NS1 BOB assay scored positive
N=58 Prim DENV N=87 Sec DENV
Balmaseda et al., in revision (analysis perfomed in Nicaragua, A. Balmaseda and Eva Harris)
N=14 Prim DENV
N=14 Sec-DENV
Page 14
NS1 BOB analysis of Brazilian samples from ZIKV-confirmed patients
0 10 20 30 40
-20
0
20
40
60
80
100
days after symptoms
BO
B (
%)
T1 T2 T30
20
40
60
80
100
BO
B (
%)
0-5 6-10 >10 d
25/2622/288/45
Mea
sles
/
Rub
ella
YFV
vac
cine
es
CM
V/H
AV
HBV/H
CV/H
EV
-20
0
20
40
60
80
100
BO
B (
%)
0/26 0/120/14
The NS1 BOB assay was
transferred to the Flavivirus
Laboratory at the Oswaldo Cruz
Foundation in Rio de Janeiro
29/31 samples (93.5%) collected more than 10 days after symptoms onset scored positive
Samples collected between 2002 and 2013 (prior to ZIKV introduction into Brazil) from 82
patients diagnosed with acute DENV infection by RT-PCR: only 4 samples scored positive
(specificity of 95.1%)
62 samples collected from 2000 to 2014 from patients diagnosed with infection with
different viruses, as well as YFV vaccinees, all scored negative
Balmaseda et al., in revision (analysis perfomed in Brazil, A.M. Bispo de Filippis, P. Sequiera and D. Brown)
Page 15
NS1 BOB analysis of samples from European travelers
0 20 40 60 8000
20
40
60
80
100
days after symptoms
BO
B (
%)
ZIKV
Prim
. DENV
Sec
. DENV
WNV
YFV-v
accine
es
CHIK
V
Sys
tem
ic illnes
s
Pre
gnan
t
Blood
don
ors
-40
-20
0
20
40
60
80
100
BO
B (
%)
2/1
0
0/8
1/3
4
0/1
16
14/1
5
0/3
0
1/1
02
1/3
9
0/1
7N=23
31/32 samples (96.9%) from WNV patients collected more than 10 days after symptom onset scored
negative. The only positive was obtained from a sample collected in 2016.
2/27 samples from DENV patients collected more than 10 days after symptom onset scored positive.
The two positive samples were derived from secondary DENV infections.
None of the samples derived from CHIKV patients or YFV-vaccinees scored positive
Only two samples from a wide set of samples from healthy adults (blood donors, pregnant women
and travelers) (n=257) scored positive. The two positive samples were collected in 2015 and 2016
Balmaseda et al., in revision (analysis perfomed in Italy, UK and CH, Fausto Baldanti, Maria Zambon and Humabs)
Of ZIKV RT-PCR confirmed infections,
21/22 samples (95.5%) collected more
than 10 days after symptom onset scored
positive (UK and Italy)
Page 16
Characteristics of subjects included in the study
Population Loc.* N
subjects/
samples
Gender
(% fem.)
Age
Mean (range)
Time†
Mean (range)
ZIKV NIC 112/215 65.1 18 (2-66) 32 (1-170)
BR 58/116 58.6 38 (3-67) 7 (1-53)
IT/UK 23/27 39.1 43 (18-68) 104 (3-753)
1° DENV NIC 59/65 54.2 8 (2-13) 44 (1-132)
IT 25/25 52.0 32 (6-58) 24 (2-200)
2° DENV NIC 87/99 48.2 10 (4-14) 31 (1-136)
IT 19/19 33.3 44 (15-68) 11 (3-30)
Acute DENV BR 82/82 50.0 30 (0-80) 4 (1-9)
WNV IT 49/49 12.2 60 (29-93) 35 (3-105)
YFV-vacc. BR 14/14 30.7 18 (1-53) -#
IT 30/30 56.6 45 (12-76) > 1 year
CHIKV IT 10/10 50.0 42 (24-74) 23 (5-55)
Other dis. BR 37/37 56.7 18 (0-74) - #
Healthy CH 116/116 -# -# N/A§
Pregnant UK 102/102 100.0 -# N/A§
Syst. Illness¥ UK 39/39 -# -# >14
Total 873/1069 60.0 27 (0-93)
*Location: NIC, Nicaragua; BR, Brazil; IT, Italy; UK, United Kingdom; CH, Switzerland†Time, days since symptoms onset§NA, not available; § N/A, not applicable¥, Symptomatic systemic illness
Balmaseda et al., in revision
Page 17
Analysis of the sensitivity and specificity of the BOB NS1 ELISA
ZIKV (>
10d)
DENV (>
10d) A
ll-40
-20
0
20
40
60
80
100
BO
B (
%)
N=
158
N=
540
N=
171
>10≥2
0≥5
0
≥100
0
20
40
60
80
100
BO
B (
%)
Sensitivity (%)
91.8
95.0
94.3
95.0
DENV (>
10d)
Flaviviru
ses
+ ot
her i
llnes
ses A
ll
Specificity (%)
91.9
88.9
93.8
95.9
Of the 19 DENV samples that scored positive, all were derived from secondary DENV patients
(mainly from secondary DENV2 and DENV3 infections); if the specificity was restricted to samples
from secondary DENV cases 80.4%Balmaseda et al., in revision
Page 18
Conclusions and future perspectives (1/2)
Additional studies are ongoing to further simplify the ZIKV NS1 BOB assay protocol and to
set up a similar assay for DENV1-4
The strengths of this study include:
Well-characterized samples that enabled distinction between DENV-naïve and
DENV-exposed ZIKV-infected patients
Samples from DENV infections stratified by serotype and immune status
Acute, convalescent and longitudinal samples from the same patients allowed
careful kinetic analyses to be performed
Time-matched comparisons of ZIKV infections versus DENV infections of different
serotypes
The NS1 BOB ELISA was established in laboratories in five countries, including
Nicaragua and Brazil
One limitation of this study is that we could not test samples from subjects
convalescent from DENV4 infections
Page 19
Conclusions and future perspectives (2/2)
The NS1 BOB ELISA could be used in Zika surveillance, sero-prevalence studies
and intervention trials
Important features of the NS1 BOB assay are:
i) its simplicity
ii) its single-assay format
iii) its requirement for only one dilution of samples and low volume
iv) low cost (estimated cost per well of approximately US $0.25)
We tested a large number of well-characterized samples from RT-PCR-confirmed ZIKV
infections
The NS1 BOB ELISA showed high sensitivity and specificity and could be developed as
a novel, low cost, accessible ZIKV NS1 serological assay
Humabs is interested to out-license the NS1 BOB assay to be developed and
commercialized as a low-cost ELISA kit
Page 20
Acknowledgments
Karin Stettler
Stefano Jaconi
Xia Jin
Fabrizia Vanzetta
Siro Bianchi
Martina Beltramello
Elisabetta Cameroni
Eva Harris
Angel Balmaseda
Damaris Collado
José Victor Zambrana
Saira Saborio
David Brown
A.M. Bispo de Filippis
Raquel Medialdea Carrera
Patricia Siqueira
Fausto Baldanti
Elena Percivalle
Francesca Rovida
Luisa Barzon (Un. of
Padova)
Maria Zambon
Richard Tedder
Samreen Ijaz
Ines Ushiro-Lumb
Page 21
Filippo Riva, CEO & [email protected]
Davide Corti, [email protected]
Humabs BioMed SA
Via Mirasole 1
CH – 6500 Bellinzona
Switzerland
www.humabs.com