Dengue Virus-Specific Antibodies Enhance Brazilian Zika Virus Infection.

TLDR: It is demonstrated that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line and that serum samples from d Dengue-immune pregnant women enhanced ZikV infection.

Abstract: Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections.

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BRIEF REPORT • JID 2017:215 (1 March) • 781
The Journal of Infectious Diseases
The Journal of Infectious Diseases
®
2017;215:781–5
Dengue Virus–Specic Antibodies
Enhance Brazilian Zika Virus Infection
Priscila M.S.Castanha,
1,2
Eduardo J.M.Nascimento,
5,6
CynthiaBraga,
1,3
Marli T.Cordeiro,
1
Otávio V.de Carvalho,
1,4
Leila R.de Mendonça,
1
Elisa A.N.Azevedo,
1
Rafael F.O.França,
1
RafaelDhalia,
1
and
Ernesto T.A.Marques
1,5,6
1
Aggeu Magalhães Research Center, Oswaldo Cruz Foundation (FIOCRUZ),
Recife,
2
Faculty of Medical Science/ Institute of Biological Science, University of
Pernambuco, Recife,
3
Instituto de Medicina Integral Prof. Fernando Figueira, Recife,
and
4
Universidade Federal de Viçosa, Minas Gerais, Brazil; and
5
Center for Vaccine
Research and
6
Department of Infectious Disease and Microbiology, University of
Pittsburgh, Pennsylvania.
Anti-Flavivirus antibodies are highly cross-reactive and
may facilitate Zika virus (ZIKV) infection through the
antibody-dependent enhancement (ADE) mechanism. We
demonstrate that dengue-specic antibodies enhance the infec-
tion of a primary Brazilian ZIKV isolate in a FcγRII-expressing
K562 cell line. In addition, we demonstrate that serum sam-
ples from dengue-immune pregnant women enhanced ZIKV
infection. ese ndings highlight the need for epidemiological
studies and animal models to further conrm the role of ADE
in the development of congenital and neurological complica-
tions associated with ZIKV infections.
Keywords. Zika virus; antibody dependent enhancement;
dengue antibodies.
Zika virus (ZIKV) is currently in the spotlight as a major pub-
lic health concern worldwide [1]. Until recently, ZIKV infec-
tions in humans were associated with either asymptomatic or
self-limiting exanthematic illness. However, the emergence
of ZIKV in Polynesia and Brazil revealed the virus is associ-
ated with more severe clinical manifestations than previously
reported, including neurological complications in adults (eg,
Guillain-Barré Syndrome) and microcephaly in newborns [2–4].
e factors responsible for the large variability of severe
phenotypes [3] of ZIKV infection remain unclear. Dengue
virus (DENV) antibodies have been shown to enhance ZIKV
infection [5, 6]. ese antibodies cross-react with ZIKV struc-
tural proteins, facilitating virus uptake by cells expressing FcγR
receptors through the mechanism of antibody-dependent
enhancement (ADE) [5, 6]. Because of the high rate of dengue
transmission in Brazil, >90% of the adult population has been
previously exposed to DENV, especially in the Northeast region
[7]. Here, we used a panel of serum samples from individuals
with dierent dengue immunity proles to conrm that dengue
antibodies enhance the infection of a Brazilian ZIKV isolate.
MATERIALS AND METHODS
SeraPanel
We included well-characterized serum samples of (1) 6 laborato-
ry-conrmed symptomatic dengue infections (primary and sec-
ondary dengue cases), (2) 20 pregnant women with dierent dengue
immunity proles, and (3) 3 dengue-naive pregnantwomen.
Dengue symptomatic patient serum samples were collected
from individuals enrolled in a cohort of dengue suspected cases
conducted in the city of Recife [8], a large urban center and
hyperendemic area of dengue in Northeast Brazil. Sequential
blood samples were collected from each patient during the
acute (<7days) and convalescent phases (10–15days) as well as
around 30days aer enrollment in the study. Additional sam-
ples were obtained 6 and 12months later. Dengue cases were
laboratory conrmed by the combination of virus isolation in
C6/36 cells and viral RNA detection by reverse-transcriptase
polymerase chain reaction (RT-PCR) with dengue serology
for immunoglobulin M (IgM)/immunoglobulin G (IgG) by
enzyme-linked immunosorbent assay (ELISA). e kinetics of
IgM and IgG response were used to classify the cases as primary
or secondary dengue infections. For this study, we included
serum samples from primary (n = 3) and secondary dengue
cases (n = 3) collected at the following time points: <7 days,
10–15days, 30–40days, and >260days aer infection [8].
Serum samples were obtained from healthy pregnant women
included in a prospective dengue birth cohort study carried
out in the same setting [9, 10]. ese samples were collected
between 2011 and 2012 and represent the dengue epidemiolog-
ical prole of women at reproductive age shortly before the cir-
culation of ZIKV in this area. Dengue serological proles of the
mothers included in the cohort were determined by ELISA and
plaque reduction neutralization test (PRNT) [9, 10]. To investi-
gate ADE of ZIKV infection by DENV antibodies, 2 groups plus
a control group of mothers were selected based on their PRNT
status: Group Iwas comprised of serum samples from mothers
with a monotypic DENV-immune prole (PRNT
50
>20 to only
1 serotype; n=10); and Group II included sera from mothers
with multitypic immunity (PRNT
50
>20 to >1 DENV serotype;
n=10) (Supplementary Table2). Serum samples from DENV-
naive mothers enrolled in the cohort were also included in this
study (n= 3). Samples were conrmed as DENV-negative by
IgM/IgG serology and PRNT. Details of the study design and
data collection of both cohort studies have been previously
BRIEF REPORT
© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
DOI: 10.1093/infdis/jiw638
Received 15 October 2016; editorial decision 19 December 2016; accepted 21 December 2016;
published online December 30, 2016.
Correspondence: E. T.A. Marques Jr, MD, PhD, Center for Vaccine Research, 3501 5th Ave,
9052 Biomedical Science Tower 3, Pittsburgh, PA 15261 (marques@pitt.edu).
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782 • JID 2017:215 (1 March) • BRIEF REPORT
described [8, 9]. Adetailed description of the serological char-
acterization of each sera included in this study has been pro-
vided in Supplementary Tables 1 and2.
Viruses
Zika virus PE/243 and DENV-2 16681 (prototype strain)
were used in the ADE assay. Zika virus PE/243 was isolated
from a Zika case diagnosed in Pernambuco state, Northeast
Brazil [11]. e virus strains were propagated in Vero cells, as
described elsewhere [10]. e virus-containing supernatants
were harvested from infected Vero cells and claried by centrif-
ugation (930g, 10 minutes, 4°C). Virus particles were precip-
itated with 50% polyethylene glycol (PEG 3100)in Dulbecco’s
modied Eagle medium (DMEM). Briey, the supernatant/
PEG mixture (proportion of 1:4) was incubated overnight
at 4°C. Aer centrifugation (1500 g, 30 minutes, 4°C), the
supernatant was discarded, and the pellet containing the virus
particles was resuspended in one hundredth of the original
volume of supernatant/PEG in DMEM with 25mM of HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Zika
virus PE/243 was titrated by plaque assay, whereas DENV-2
16681 virus titer was determined by focus-forming assay [10].
Antibody-Dependent EnhancementAssay
FcγRII-expressing K562 cell line were exposed to ZIKV
PE/243 in the presence of either a Flavivirus-naive (AB human
serum) or a dengue-immune serum sample (DENV-3 immune
serum). Antibody-dependent enhancement was determined
by ow cytometry, as previously described [10]. Antibody-
dependent enhancement was measured as the n-fold increase
in the percentage of virus-infected cells relative to that in a
Flavivirus-naive serum. DENV-2 16681 was used as a control
for the assay.
Quantitative Real-Time Polymerase Chain Reaction
Zika virus RNA was extracted from culture supernatants using
QIAamp Viral RNA extraction kit following the manufacture’s
specications. Quantitative RT-PCR was conducted by using
the QuantiTect Probe RT-PCR Kit with amplication in the
Applied Biosystems 7500 real-time PCR system following the
manufacturer’s protocol. Zika virus primers (ENV1086F:5’-
CCGCTGCCCAACACAAG-3’ and ENV1162R:5’-CCACT
AACGTTCTTTTGCAGACAT-3’) and probe (5’-VIC AGCCTA
CCTTGACAAGCAGTCAGACACTCAA-BHQ1-3) sequences
for the quantitative virus detection assay were designed accord-
ing to Lanciotti etal (2008) [12]. e relative quantication of
ZIKV RNA was assessed using the 7500 Soware v2.0.6.
Ethical Statement
e protocol was approved by the Ethical Committee of Aggeu
Magalhaes Research Center (CAAE-0061.0.095.000-10) and of
the Brazilian Ministry of Health (CONEP 25000.119007/2002–
03; CEP68/02).
Statistical Analysis
Student’s t test was used to compare percentage of infected
cells and enhancing activity between 2 groups. e correlation
between previous dengue immunity and ADE was determined
using the Spearman test. e level of signicance was set at .05.
Statistical analysis was performed using Graph Pad Prism, ver-
sion 6.0e.
RESULTS
We postulated that nonneutralizing, cross-reactive, den-
gue-specic antibodies enhance ZIKV infection of FcγR
receptor-bearing phagocytes through the mechanism of ADE.
To conrm that, FcγRII-expressing K562 cell lines were exposed
to the local Brazilian ZIKV isolate PE/243 in the presence of
either a Flavivirus-naive or a dengue-immune serum sample.
Zika virus and DENV-2 infectivity were low on K562 cells
infected without antibodies (1.23%±0.87% and 0.91%±0.39%,
respectively) or in the presence of Flavivirus-naive serum
(2.05% ± 1.09% and 2.11% ± 1.11%, respectively). However,
preincubation of ZIKV with a serum sample from a DENV-3–
immune individual increased the infectivity of both viruses
compared with the Flavivirus-naive sample (9.09%±5.58% for
ZIKV and 3.45%±3.20% for DENV-2). e ADE observed for
ZIKV was 3 times greater than the one observed for DENV-2
(4.35±1.11 and 1.43±0.53, respectively; P=.003). e exper-
iment was independently performed 4 times on dierent days
to assure reproducibility. Arepresentative analysis is shown in
Figure1A and 1B.
Next, we determined the kinetics of ADE of ZIKV infection
in a panel of well-characterized serum samples from primary
(n = 3) and secondary (n = 3) laboratory-conrmed dengue
cases collected from acute (<7days aer onset of symptoms)
and convalescent (10–15days aer onset of symptoms) phases
until aer complete recover (30–40days and >260 days aer
onset of symptoms). Antibody-dependent enhancement of
ZIKV infection was not observed in samples collected during
the febrile and acute phases (before serum conversion) in
patients experiencing dengue primary infections (Figure 2A).
Antibody-dependent enhancement was only observed in this
group at later time points, aer convalescence and recovery
(Figure2A). In contrast, serum samples from dengue secondary
cases induced ADE of ZIKV infection regardless of the phase of
infection analyzed (Figure2A).
We then selected a panel of serum samples from pregnant
women immune to either DENV-3 alone (monotypic; n=10)
or in combination with DENV-4 (multitypic; n=10), as deter-
mined by PRNT assays. All samples from DENV-immune moth-
ers show ADE of ZIKV infection (n=20/20), whereas ADE was
not observed among the DENV-naive mothers (n=0/3). Serum
samples from monotypic and multitypic dengue-immune
mothers induced greater ADE of ZIKV infection (16.41±3.12
and 17.08± 2.86, for monotypic and multitypic, respectively,
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as compared with DENV-2 (1.54 ± 0.62 and 0.76 ± 0.36, for
monotypic and multitypic, respectively) (Figure 2C and 2B).
However, immunity to multiple DENV serotypes inhibited
the infectivity of DENV-2 (P=.003; Figure2B), whereas ADE
of ZIKV was elevated in monotypic and multitypic groups
(P=.62) (Figure2C).
Figure1. Antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection by dengue-specific antibodies. FcγRII-expressing K562 cells were infected with ZIKV
PE/243 in the absence of antibodies or in the presence of either a Flavivirus-naive serum or a dengue virus (DENV)–3 immune serum. Antibody-dependent enhancement was
measured as the n-fold increase in the percentage of virus-infected cells relative to that in a Flavivirus-naive serum. A, Percentage of DENV- and ZIKV-infected K562 cells
in absence or in presence of serum. B, Antibody-dependent enhancement of ZIKV in K562 cells.
Figure2. Antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection by dengue antibodies. A, Longitudinal serum samples from dengue-infected patients were
used to determine the kinetics of ADE of ZIKV infection after dengue exposure in individuals experiencing a primary (dotted lines) or secondary (solid lines) dengue infection.
Antibody-dependent enhancement of dengue virus (DENV) (B) and ZIKV (C) was tested in the presence of a panel of serum samples from pregnant women with different
dengue immune status, as determined by plaque reduction neutralization test: monotypic (DENV-3) (n=10) and multitypic (DENV-3 and DENV-4) (n=10). C, The n-fold increase
in the ZIKV RNA quantification in the presence of monotypic or multitypic dengue immune serum relative to that in a Flavivirus-naive serum. Abbreviations: ADE, Antibody-
dependent enhancement; DENV, dengue virus; ZIKV, Zika virus, vRNA, virus ribonucleic acid.
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784 • JID 2017:215 (1 March) • BRIEF REPORT
Antibody-dependence enhancement of ZIKV infection by
dengue-specic antibodies was also observed aer quantica-
tion of viral RNA by quantitative RT-PCR in culture superna-
tants of infected K562 cells. Viral RNA levels increased up to
7-fold in the presence of dengue immune serum compared with
the Flavivirus-naive serum (Figure2D). ere was no dierence
in the ZIKV RNA levels collected from supernatants harvested
from infected cells in the presence of dengue monotypic or mul-
titypic maternal sera (P=.15).
DISCUSSION
Although ZIKV has been linked to the increased incidence of
congenital microcephaly cases [3, 4], the mechanisms underly-
ing ZIKV transmission from mother to fetus remain unknown.
Here, we demonstrated that the presence of dengue antibodies
increased the infectivity of a primary Brazilian ZIKV isolate in
a human cell line expressing FcγRII receptors.
Antibody-dependent enhancement of ZIKV infection in
mononuclear phagocytes was rst evidenced in the 1980s [13]
and has been recently conrmed by others [5, 6]. Dejnirattisai
etal [5] showed that pooled convalescent serum and monoclo-
nal antibodies derived from DENV-infected patients were able
to promote ADE of ZIKV infection on the monocyte cell line
U937 [5]. Our results conrmed these ndings in a dierent
cell line and also explored the kinetics of ADE on paired sam-
ples taken from the same subjects at dierent time points, con-
tributing to better understanding of how preexisting Flavivirus
immunity inuences ZIKV infection in vitro. We acknowl-
edge that it is not possible to denitively rule out previous
ZIKV exposure of the patients included in our study, although
there were no reports of ZIKV or microcephaly outbreaks in
Brazil between 2004–2006 and 2011–2012, when the samples
were collected. Of note, monoclonal antibodies directed to the
envelope dimer epitope of DENV have been recently demon-
strated to potently neutralize ZIKV infection and inhibit ADE
in vitro [5, 14], pointing to a potential immunotherapy against
ZIKV and opening the venues for the identication of ZIKV
and DENV shared epitopes able to elicit neutralizing antibodies
against both viruses [14].
Interestingly, we showed that sera from pregnant women—
representative of the pregnant population of Recife, which was
the epicenter of the microcephaly epidemic in Brazil— promote
ADE of ZIKV infection in vitro. Noteworthy, increased numbers
of congenital disease associated with ZIKV infection have not
been reported in Southeast Asia countries [15], where dengue
has been hyperendemic for >5 decades and pregnant women are
usually immune to all DENV serotypes. Unlike Asia, pregnant
women from our setting are mostly immune to DENV-3 alone
and probably have lower levels of DENV-associated antibodies
than women exposed to several DENV infections [10]. us, we
cannot exclude the possibility that background immunity and
DENV-specic antibody levels of the population might contrib-
ute to increased disease severity of ZIKV infections. is probably
explains the higher rates of congenital syndrome and neurolog-
ical complications associated with ZIKV infections observed in
Brazil compared with Asian countries [4, 15], although virus vir-
ulence of the circulating ZIKV might also play arole.
In summary, we demonstrated that dengue-specic antibod-
ies dramatically increase ZIKV infectivity in vitro in phago-
cytes expressing FcγRII receptors. is nding might have
implications for the immunopathogenesis of ZIKV infection
in dengue-endemic areas. We acknowledge that the inuence
of ADE in determining severe disease in vivo has been con-
troversial in the dengue eld [10]. To date, there has been no
epidemiologic evidence of enhanced ZIKV illness during the
ongoing epidemic in South and Central America. Although
our data clearly demonstrate the ability of dengue antibodies
to enhance ZIKV infection in vitro, it is important to point out
that the relevance of this mechanism in vivo must be carefully
explored. Additional epidemiological and animal model stud-
ies are needed to elucidate the contribution of previous dengue
immunity in mediating congenital and neurological complica-
tions associated with ZIKV infections.
Supplementary Data
Supplementary materials are available at e Journal of Infectious Diseases
online. Consisting of data provided by the authors to benet the reader,
the posted materials are not copyedited and are the sole responsibility of
the authors, so questions or comments should be addressed to the corre-
sponding author.
Notes
Acknowledgments. We thank Dr Renato Oliveira and Ms Verônica
Gomes (Aggeu Magalhães Research Center) for reagents, help with cytom-
eter, and samples selection. We also thank Dr Donald Burke for advice and
invaluable discussion.
Financial support. is work was supported by the Brazilian Federal
Agency for Support and Evaluation of Graduate Education (CAPES); Center
for Vaccine Research, University of Pittsburgh; Fogarty Training Program
(D43TW006592 Pitt GIDRTP/ 323 NIH to P.M. S.C.); National Council
for Scientic and Technological Development (CNPq) (482915/2010-2
MCT/CNPq-321 14/2010); Strategic Program to Support Health Research/
PAPES VI (322 407697/2012-8); and the National Institute of Allergy and
Infectious Diseases (U19 AI56541).
Potential conicts of interest. All authors. No reported conicts. All
authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
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