Antibody-Dependent Enhancement of Coxsackievirus B4 Infectivity
of Human Peripheral Blood Mononuclear Cells Results in Increased
Didier Hober,1Wassim Chehadeh,1,2Ahmed Bouzidi,2
and Pierre Wattre ´1
1Laboratoire de Virologie, Centre Hospitalier Re ´gional
et Universitaire, and
de Lille–Centre National de la Recherche Scientifique, Lille, France
2SEDAC Therapeutics, Institut de Biologie
IgG devoid of neutralizing activity and isolated from donor plasma by chromatography
formed immune complexes with coxsackievirus B4 (CVB4) and significantly increased the
infection of peripheral blood mononuclear cells with CVB4. The major host cells for CVB4
infection enhanced with IgG are monocytic CD14+cells. The roles of CVB and adenovirus
receptor and Fcg receptor II and III have been shown. Increased viral replication and the
release of infectious particles were demonstrated when interferon (IFN)–a produced by in-
fected cells was first neutralized by use of antibodies. The CVB4 IgG-induced synthesis of
IFN-a by monocytes reflected entry and uncoating of CVB4 but not of viral replication and
required the presence of CVB4 RNA inside the cells. Thus, CVB4 can infect monocytes by
an antibody-dependent mechanism through interactions between the virus, antiviral antibod-
ies, and specific receptors that result in IFN-a production.
Coxsackieviruses B1–B6 (CVBs) are nonenveloped virusesof
the Picornaviridae family enterovirus group. These plus-sense
RNA viruses are single stranded. They are in the capsid com-
posed of 60 protomers, each consisting of 1 copy of the viral
proteins VP1, VP2, VP3, and VP4. The initial step in virus
infection is binding to specific receptors on the cell surface. A
common receptor for CVB and adenoviruses, called “CAR,”
has been identified [1, 2]. The mechanism of uncoating is not
well known. Inside the cell, in the course of CVB replication,
parental plus-sense RNA strand is transcribed into a minus-
sense RNA strand that serves as a template for transcription
into progeny plus-sense RNA strand. The viral genome serves
as mRNA for the synthesis of a polyprotein precursor cleaved
to yield the viral proteins. Structural proteins such as VP1,
formed by cleavage of the precursor polyprotein, associatewith
positive-strand RNA to form virions .
CVB is associated with a variety of acute diseases in humans
(e.g., myocarditis, meningoencephalitis, Bornholm disease,and
herpangina) , and a role for CVB3 and CVB4 in chronic
diseases such as chronicmyocarditisandinsulin-dependenttype
Received 20 February 2001; revised 5 July 2001; electronically published
12 October 2001.
Financial support: Centre Hospitalier Re ´gional et Universitaire Lille
(AOCHU 98/1911); Ministe `re de l’Education Nationale de la Recherche et
de la Technologie (UPRES EA 1048); Universite ´ Lille II: Nouvelles The ´ra-
peutiques du Diabe `te de Type 1 et Pathoge ´ne `se Virale de la Maladie, which
encompasses the Laboratoire de Virologie.
Reprints or correspondence: Dr. Didier Hober, Laboratoire de Virologie,
CHRU, 59037 Lille Cedex, France (firstname.lastname@example.org).
The Journal of Infectious Diseases
? 2001 by the Infectious Diseases Society of America. All rights reserved.
in the tissues of the respiratory and gastrointestinal tracts, they
can be present in blood and spread to secondary target organs
. A viremia is often detected in hospitalized patients with
enterovirus diseases . We recently detected enteroviral RNA
with a strong homology with CVB3 and CVB4 in the blood of
patients with insulin-dependent type 1 diabetes. This was as-
sociated with increased levels of interferon (IFN)–a in plasma
and the presence of IFN-a mRNA in blood cells .
CVB can be recovered from mononuclear cells . However,
it is not clear whether the virus replicates within these cells.
Indeed, simple virus-mononuclear association on the cell sur-
face or phagocytosis cannot be excluded. The susceptibility of
peripheral blood cells to CVB is somewhat controversial. It has
been reported that T and B cell lines can be infected withCVB3
but that monocytic cell lines are nonpermissive for infection
. Henke et al.  showed that CVB3 wascapableofinfecting
freshly harvested monocytes; however, Vuorinen et al.  noted
that peripheral blood mononuclearcells(PBMC)fromdifferent
donors did not support CVB3 replication.
Natural infections by a CVB serotype can induce production
of both serotype- and CVB group–specific antibodies . Hu-
mans are constantly exposed to sequential CVB challenges. A
protective role for virus-specific anti-CVB antibodies in pre-
venting or reducing subsequent serotype-specific induced dis-
eases can be inferred from numerous epidemiologic studies,but
this protection is not absolute since contacts of CVB-infected
persons can excrete these viruses in the presence of existing
serotype-specific antibodies .
Viruses can infect cells through the Fc receptor (FcR) in the
presence of virus-specific IgG. Antibodies can facilitate the up-
take of viruses (e.g., dengue, human immunodeficiency, and
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JID 2001;184 (1 November)CVB4 Infectivity of PBMC and IFN-a Synthesis1099
FcR in tissue culture [13–15]. Foot-and-mouth disease virus, a
member of the aphtovirus genus of Picornaviridae [16, 17], can
bind and productively infect cells after binding through theFcg
receptor (FcgR), and Arita et al.  reported enhanced po-
liovirus infection of mouse L cells mediated by the high-affinity
FcR . Human polyvalentIgG enhancestheIFN-a–inducing
capacity of poliovirus in vitro , suggesting that the pro-
duction of IFN-a can result from the internalization of IgG
antibody-complexed poliovirus particles; however,theinfection
of IFN-a–producing cells has not been investigated. Whether
human antibodies can enhance infection with members of the
Picornaviridae family is unknown. Above all, the role of a pre-
vious CVB serotype infection in the outcome of a subsequent
homologous and/or heterologous CVB serotype infection has
not been studied.
In CVB infection, infectious particles and/or viral compo-
nents may be found in circulating blood. Because of the fre-
quency of CVB infections, specific antibodies are present in the
blood of persons with subsequent CVB infection. In this study,
we investigated the role of antibodies in the interactionbetween
CVB4 and cells and the cellular and molecular mechanisms of
infection of PBMC with CVB4.
Materials and Methods
versity ofReading, Reading,UnitedKingdom])wasgrowninHEp-
2 cells (Biowhittaker) in MEM (Gibco BRL) supplemented with
10% heat-inactivated fetal calf serum (FCS; Gibco) and 1% L-
glutamine (Eurobio). Three days after infection, supernatantswere
collected and clarified at 190 g for 10 min. Virus titers were de-
termined by plaque-formation assay on HEp-2 cells. Aliquots of
virus preparations were stored frozen at ?80?C.
Sendai virus was provided byD.Garcin(DepartmentofGenetics
and Microbiology, University of Geneva). The virus was cultivated
as described elsewhere . In brief, it was propagated in eggs,
purified first by clarifying chorioallantoic fluid containing the virus
by centrifugation for 10 min at 1500 g at 4?C, and then pelleted
at 15,000 g for 16 h. Virus titers were assayed by plaque formation
on Hela cells. The virus preparation was stored frozen at ?80?C
Herpes simplex virus (HSV) type 1 (laboratory strain) was cul-
tivated in a Vero cell line (American Type Culture Collection) in
MEM supplemented with 5% FCS and 1% L-glutamine. After 48
h of incubation at 37?C in 5% humidified CO2atmosphere, super-
natants were collected and clarified at 190 g for 10 min. HSV-1
titers were assayed by plaque formation on Vero cells, and virus
preparations were stored at ?80?C.
Plaque infectivity assay.
The titers of infectious virus particles
in cultures were determined, by thestandardplaque-formationassay,
2 cells by use of 100 mL of a 10-folddilutionperwell.Beforeinocula-
tion, the cell suspensions were frozen and thawed 3 times to release
the virus and were clarified by low-speed centrifugation. In case of
virus detection and titration in culture supernatants, the latter were
only clarified by low-speed centrifugation. CPE was read on postin-
CVB4 (JBV strain, provided by J. W. Almond [Uni-
fection day 7, and results were expressed as the number of plaque-
forming units per milliliter at end-point titers.
Plasma samples were obtained from 5 healthy
subjects with anti-CVB B4 antibodies in their blood detected by
neutralization assay (titer ?256). Blood samples were obtained
from 5 additional donors for isolation of PBMC. The 10 donors
were adults (5 men and 5 women; median age, 28 years [range,
23–45 years]) with no suspected immunologic, infectious, or meta-
bolic disease at the time the blood was obtained. Blood samples
were also obtained from 6 children (4 boys and 2 girls; median age,
6 months [range, 6–12 months]). Neutralization assay results for
anti-CVB antibodies were negative.
PBMC and monocyte isolation.
, PBMC from heparinized blood were separated over a ficoll-
paque solution (Diatrizoate ficoll; Eurobio). Themononuclearcells
were then collected and washed 3 times with RPMI 1640. They
were then adjusted to a concentration of
medium supplemented with 10% FCS and 1% L-glutamine and
distributed as 0.1-mL aliquots into 96-well tissue-culture plates.
A monocyte isolation technique based on the Optiprep density-
gradient medium has been described . In brief, PBMC were
isolated from whole blood as described above. We mixed 4 mL of
Optiprep (Sigma) with 10 mL of PBMC before an overlay with
7.5 mLof a 1.078 g/mL lymphocyte-specificdensitylayer.Thelatter
was overlaid with 20 mL of 1.068 g/mL solution and 0.5 mL of
HEPES-buffered saline (Sigma). Centrifugation was done at 600 g
for 25 min at room temperature. Fractions of the resulting phases
were collected. The first fraction corresponded to monocytic cells
at the 1.068-g/mL layer; the second fraction included the lympho-
cyte-trapping layer at the interface of the 1.078- and 1.068-g/mL
layers. Each fraction was washed once with PBS and twice with
RPMI 1640, adjusted to a concentration of
RPMI medium supplemented with 10% FCS and 1% L-glutamine,
Of the isolated cells, 90%–95% at the 1.068-g/mL layer wereCD14?
monocytes, as determined by fluorescein-isothiocyanate (FITC)
monoclonal anti-CD14 conjugated antibody (data not shown).
IgG were isolated from plasma from
5 healthy subjects. Then 2 mL of plasma was diluted 2-fold with
PBS at pH 7.0 and passed through a 0.45-mm filter. Each plasma
sample was passed at a flow rate of 0.1 mL/min over a 1.6 ? 20–cm
protein A–Sepharose CL-4B column (Pharmacia Biotech) buffered
with PBS. The column was washed extensively with the samebuffer
until the UV absorbance (at 280 nm) of the effluent was 0 and
then was eluted at pH 3.0 at 1 mL/min with elution buffer con-
taining 50 mM glycine (Sigma) and 150 mM NaCl (E. Merck,
Darmstadt, Germany). For each subject, fractions containing the
eluted immunoglobulins were pooled and concentrated on a
Biomax-30 membrane (Centricon Plus-20 system; Millipore). To
preserve the activity of acid-labile IgG, the eluted fractions were
neutralized by adding 50–100 mL of 1 M Tris-HCl at pH 9 (Sigma).
IgG concentrations were estimated by using an assumed extinction
coefficient of 1.43 for 1 mg/mL solution at 280 nm.
To isolate specific anti-CVB IgG antibodies from each subject,
IgG-eluted fractions obtained through protein A affinity chroma-
tography were passed over a divinylsulfone (DVS; Sigma)–activated
Sepharose CL-4B column coupled to CVB4. Sepharose CL-4B was
activated with DVS as described elsewhere . CVB4 (
By the technique of Bo ¨yum
cells/mL in RPMI
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