ns1 blockade-of-binding elisa distinguishes between dengue and zika … · 2019-09-13 · ns1...

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

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

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)

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

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

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)


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.

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


*NS1 used at 1 µg/ml (Meridian or NAC)

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

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

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


The NS1 BOB assay was established in the National Virology Laboratory of the

Ministry of Health in Managua, Nicaragua

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)

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

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)

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)

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


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

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

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

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

Filippo Riva, CEO & [email protected]

Davide Corti, [email protected]

Humabs BioMed SA

Via Mirasole 1

CH – 6500 Bellinzona

Switzerland

www.humabs.com

ns1 blockade-of-binding elisa distinguishes between dengue and zika … · 2019-09-13 · ns1...

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