Antibody-Dependent Enhancement (ADE) of Ebola Virus Infection
Ayato Takada, Heinz Feldmann, [...], and Yoshihiro Kawaoka
Abstract
Most
strains of Ebola virus cause a rapidly fatal hemorrhagic disease in
humans, yet there are still no biologic explanations that adequately
account for the extreme virulence of these emerging pathogens. Here we
show that Ebola Zaire virus infection in humans induces antibodies that
enhance viral infectivity. Plasma or serum from convalescing patients
enhanced the infection of primate kidney cells by the Zaire virus, and
this enhancement was mediated by antibodies to the viral glycoprotein
and by complement component C1q. Our results suggest a novel mechanism
of antibody-dependent enhancement (ADE) of Ebola virus infection, one that
would account for the dire outcome of Ebola outbreaks in human
populations.
Ebola
virus infection of primates is generally characterized by severe
hemorrhagic manifestations and produces higher mortality rates than any
of the other viral hemorrhagic fevers (6, 24). There are four distinct Ebola virus species—Zaire, Sudan, Ivory Coast, and Reston (6, 24).
Among these, the Zaire strain seems to be the most virulent, with a
mortality rate for infected persons of up to 90%, while the Reston
strain has been less pathogenic than other species in experimentally
infected nonhuman primates (7, 24)
and has not been associated with symptomatic infection in humans.
Despite extensive research, the molecular basis for the extreme
virulence of the Zaire virus remains elusive.
Ebola
virus is a filamentous, enveloped, negative-strand RNA virus. Its genome
encodes eight proteins, with the fourth gene from the 3′ end of the
genome encoding two glycoproteins (GPs) (5)-the envelope GP, which is responsible for receptor binding and fusion of the virus with host cell membranes (28, 32), and the nonstructural secretory GP, which is released from infected cells (25, 31). Both GPs are thought to play important but still undefined roles in Ebola virus infection (27).
Takada
et al. have demonstrated previously that the infectivity of vesicular
stomatitis virus (VSV) pseudotyped with the Zaire GP was enhanced by
mouse anti-Zaire GP sera produced by DNA immunization, while
substantially weaker enhancement was observed with anti-Reston GP sera (29).
Here we present evidence indicating that antibodies able to enhance
infectivity of authentic Ebola Zaire virus are produced in humans and
propose a novel mechanism for this antibody-dependent enhancement (ADE)
in which the complement protein C1q mediates the enhancement.
MATERIALS AND METHODS
Viruses.
Ebola
virus (Zaire strain Mayinga) was propagated in Vero E6 cells and stored
at −80°C until use. All infectious materials were handled in the
biosafety level 4 facility at the Canadian Science Centre for Human and
Animal Health. VSV pseudotyped with Ebola GP, expressing green
fluorescent protein (GFP), was generated as described previously (28). 293 and Vero E6 cells were grown in Dulbecco's minimal Eagle's medium complemented with 10% fetal bovine serum, l-glutamine, and antibiotics.
Monoclonal antibodies, human plasma, and sera.
Mouse monoclonal antibodies (MAbs) were produced as described previously (29).
The hybridomas producing MAbs 12/1.1 (immunoglobulin G2a [IgG2a]),
662/1.1 (IgG2a), and 746/16.2 (IgG2a), which enhance the infectivity of
VSV pseudotyped with the Zaire GP, were grown in PFHM II (GIBCO BRL,
Grand Island, N.Y.), and using protein A agarose columns (Bio-Rad
Laboratories, Hercules, Calif.), the antibodies were purified from the
supernatants. Convalescent human plasma (patients 2 to 7) and serum
(patients 1 and 8) samples were obtained 51 to 135 days after onset
during the 1995 outbreak in Kikwit, Democratic Republic of the Congo. In
some experiments, samples of human plasma or serum were treated with
0.05 M egtazic acid (EGTA) for 30 min at room temperature.
Complement and anticomplement antibody.
Human
complement components C1 and C1q (Sigma, St. Louis, Mo.) and chicken
IgG purified from antiserum to human C1q (Immunsystem, Uppsala, Sweden)
were used for enhancement and enhancement inhibition assays and for flow
cytometric analysis.
Immunofluorescent assay (IFA).
293
cells infected with Ebola virus were fixed with 2% paraformaldehyde 1
day after infection and treated with 0.1% Triton X-100 in PBS. To detect
virus-infected cells, we used rabbit antiserum to VP40 of Ebola Zaire
species (12) as a primary antibody to abolish any cross-reactivity with the mouse MAb.
Virus titration.
Ebola
virus infectivity was quantified by counting IFA-positive cells in 5 to
10 microscopic fields. The infectivity of VSV pseudotyped with Ebola GP
was determined by counting the GFP-positive cells as described
previously (28). Infectivity-enhancing tests were done as described previously (29).
Untreated and EGTA-treated samples of convalescent human plasma or
serum (1:10 dilution) were incubated with Ebola Zaire virus for 1 h at
room temperature and then inoculated into 293 cells. The relative
percentage of infected cells was determined as the number of infected
cells in the presence of normal mouse or human serum alone
(approximately 20 to 50 IFA-positive cells per microscopic field) set to
100%.
RESULTS
Infectivity of Ebola Zaire virus is enhanced by MAbs to GP.
Takada
et al. previously demonstrated that immunization of mice with the Zaire
GP induces antibodies that enhance the infectivity of VSV pseudotyped
with this protein and that heat-labile serum factors are required for
ADE (29).
To test the relevance of ADE for authentic Ebola virus, we infected
human embryonated kidney (293) cells with the Zaire virus in the
presence or absence of an anti-GP mouse MAb (MAb 12/1.1) (29). As shown in Fig. Fig.1A,1A,
the infectivity of the virus was markedly enhanced in the presence of
MAb 12/1.1 and normal mouse serum in contrast to results with a control
sample lacking the antibody. Although ADE was also observed with monkey
kidney cells and human umbilical vein endothelial cells, the extent of
the enhancement varied with the type of cells (data not shown),
suggesting that cellular molecules contribute to the enhancing effect.
ADE
of Ebola virus infection. (A) 293 cells were infected with the Zaire
virus in the presence or absence of purified MAb 12/1.1 (final
concentration, 2 μg/ml) and fresh mouse serum (final concentration, 2%).
Using rabbit anti-VP40 antiserum, ...
Infection of humans with Ebola Zaire virus induces infectivity-enhancing antibodies.
We
next tested whether authentic Ebola Zaire virus infection in humans
stimulates the production of antibodies capable of enhancing viral
infectivity (Fig. (Fig.1B).1B).
Two of eight samples of convalescent plasma or serum (no. 2 and 6)
collected from patients infected with the Ebola Zaire virus during the
1995 outbreak in Kikwit, Democratic Republic of the Congo, strikingly
enhanced the infectivity of Zaire virus. This effect required prior
treatment of the clinical samples with EGTA, which promotes the activity
of a critical serum factor for ADE (29)
(see below). Less prominent but still substantial increases in
infectivity were noted for samples from three other patients (no. 1, 7,
and 8). A similar pattern of ADE was seen in studies testing VSV
pseudotyped with the Zaire GP (Fig. (Fig.1B).1B).
None of the samples showed appreciable neutralizing activity. These
results indicate that human infection with authentic Ebola Zaire virus
can induce antibodies that enhance viral infectivity.
Complement component 1 mediates ADE of Ebola Zaire virus infection.
We
have shown that ADE of infection by VSV pseudotyped with the Zaire GP
requires a heat-labile serum factor that can interact with the Fc
portion of the antibodies, since protein A or heat treatment reduced the
infectivity-enhancing activity of antiserum to the GP (29). Although activation of the complement pathway is not involved in the ADE of Zaire virus infection (29),
complement component 1 (C1), an initial component of the classical
complement pathway, seemed to be a reasonable candidate for the
requisite serum factor since it is heat labile, binds to
antibody-antigen complexes via the Fc portion of the antibodies, and
interacts with cell surface molecules (3, 16, 23). The C1 complex consists of C1q and two serine protease proenzymes, C1r and C1s (see Fig. Fig.5A).5A). Under physiologic conditions, these three molecules associate with each other in a Ca2+-dependent manner and thus can be separated by EGTA, leading to increased C1q binding to its cell surface ligands (3, 16, 23). Separation of C1r and C1s from C1q is also mediated by a C1 inhibitor in plasma (3, 16, 23) when C1q binds to an activator (3, 16). Prohaszka et al. (20)
suggested that C1q-mediated ADE might underlie human immunodeficiency
virus (HIV) infection, but the mechanism of enhancement remains elusive,
since C1q directly binds to HIV gp41 (3, 15). Thus, we tested purified C1q for its ability to mediate ADE of Ebola Zaire virus infection.
C1q-mediated
ADE of Ebola virus infection (model). (A) Schematic representation of
complement proteins C1 and C1q. One C1q molecule has six globular heads
that bind to the Fc portions of antigen-bound antibodies and one
collagenous region that serves ...
The
infectivity of the Zaire virus in cultures of 293 cells was
significantly enhanced in the presence of MAb 12/1.1 and purified C1q
added in place of serum (Fig. (Fig.2A).2A).
This enhancement was abolished by heat treatment (56°C, 60 min) of the
purified protein. When treated with anti-C1q antibody, the mouse serum
previously shown to be required for ADE of Ebola virus infection
completely lost its activity (Fig. (Fig.2B),2B),
indicating that C1q is in fact the serum factor required and sufficient
for ADE. Addition of purified C1 (a complex of C1q, C1r, and C1s
molecules) similarly enhanced Ebola virus infectivity, while EGTA
treatment of this complex enhanced infectivity still further (data not
shown). These results demonstrate that ADE of Ebola Zaire virus
infection is mediated by the C1q molecule and that dissociation of C1r
and C1s from C1q promotes the ability of C1q to mediate ADE, resulting
in increased infectivity.
C1q
is required for ADE of Ebola virus infection. (A) Zaire virus or VSV
pseudotyped with the Zaire GP was incubated with purified MAb 12/1.1 and
untreated or heat-treated C1q. (B) Viruses were incubated with MAb
12/1.1 and 4% mouse serum pretreated with ...
GP has multiple epitopes recognized by infectivity-enhancing antibodies.
In
addition to MAb 12/1.1, two other MAbs to the Zaire GP (662/1.1 and
746/16.2) enhanced the infectivity of the Zaire virus but not the Reston
virus (data not shown). To identify the epitopes involved, we studied
VSVs pseudotyped with chimeric proteins between Zaire and Reston GPs
(Fig. (Fig.3).3).
MAbs 12/1.1, 662/1.1, and 746/16.2 recognized amino acid positions 418
to 562, 1 to 232, and 304 to 417 of the Zaire GP, respectively. We then
examined the species specificity of ADE. Among three MAbs, 12/1.1 and
746/16.2 enhanced the infectivity of only VSV pseudotyped with Zaire GP,
while 662/1.1 did so with VSV bearing either the Zaire or Ivory Coast
GP but not the GPs of the other two strains (Fig. (Fig.4).4). These results indicate the presence of multiple epitopes in the induction of ADE.
Multiple
epitopes on the Zaire GP are involved in ADE. VSVs pseudotyped with the
chimeric GPs were incubated with MAb 12/1.1, 662/1.1, or 746/16.2 in
the presence of EGTA-treated mouse serum. Other experimental conditions
were the same as those described ...
Species
specificity of the infectivity-enhancing MAbs. 293 cells were infected
with VSV pseudotyped with Ebola Zaire (Z), Sudan (S), Ivory Coast (I),
or Reston (R) GP incubated with MAb 12/1.1, 662/1.1, or 746/16.2 in the
presence of untreated or EGTA-treated ...
DISCUSSION
Some
viruses elicit antibodies that enhance infectivity through the binding
of virus-antibody complexes to cellular Fc receptors (e.g.,
monocytes/macrophages) via the Fc portion of the antibodies (8, 10, 13, 17, 18, 19, 21, 22).
Alternatively, the complement pathway, activated by virus-antibody
complexes, can facilitate virus entry, as demonstrated with the
antibody-dependent, complement-mediated enhancement of HIV infection (8).
However, neither mechanism adequately explains the ADE of Ebola Zaire
virus infection in primate kidney cells, since Fc receptors are
expressed exclusively on the cells of the immune system, such as
monocytes/macrophages, neutrophils, B cells, and granulocytes (4), and complement inhibitors did not reduce the infectivity-enhancing activity of antiserum to the Zaire GP (29).
Our findings suggest a model (Fig. (Fig.5)5)
in which two or more molecules of monomeric IgG antibodies bind to
specific GP epitopes in close proximity, allowing C1 to bind to the Fc
portion of the antibodies (2).
The resultant complex, consisting of the virus, antibodies, and C1,
then binds C1q ligands at the cell surface, promoting either binding of
the virus to Ebola virus-specific receptors or endocytosis of the target
cells by intracellular signaling via C1q ligands (3, 16).
Indeed, we confirmed by flow cytometric analysis that human C1q
efficiently attaches to 293 cells (data not shown). In accord with this
model, protein A reduced the enhancing activity (29). EGTA treatment enhances ADE because the ligand-binding affinity of C1q increases when Ca2+-dependent association of C1r and C1s with C1q is disrupted (3, 16, 23) (Fig. (Fig.5B).5B).
Under physiologic conditions, separation of C1r and C1s from C1q is
mediated by a C1 inhibitor, normally to control harmful activation of
the classical complement pathway (3, 16, 23).
The requirement for EGTA treatment in the present study was most likely
due to the delayed collection of the clinical samples (>50 days
after disease onset) and the resultant reduction of antibody titers. C1q
ligands have been identified in many cell types, including
monocytes/macrophages and endothelial cells (3, 16), which are preferentially targeted by Ebola virus and seem to be directly involved in viral pathogenesis (26, 27).
Thus, enhanced infection of these cells would be expected to exacerbate
the hemorrhagic disease typically produced by this virus.
Both
human and nonhuman primates develop virus-specific antibodies that can
be detected by immunofluorescent or enzyme-linked immunosorbent assay or
by Western blotting (1, 7, 11, 14).
The infectivity-enhancing activity of convalescent plasma or serum in
this study is likely mediated by IgG antibodies. Importantly, C1 binds
more efficiently to polymeric IgM than to IgG antibodies (2).
Since not only IgG but also IgM antibodies are produced in Ebola
virus-infected patients, even in nonsurvivors, during the early phase of
Ebola virus infection (1, 14),
we suggest that some anti-GP IgM antibodies can contribute to the
extreme virulence of Ebola virus infection. In a recent study, serum
from a monkey experimentally infected with Ebola virus failed to enhance
viral infectivity (9), analogous to our observation that not all samples of human convalescent plasma or serum can mediate ADE (Fig. (Fig.1B).1B).
Possibly, immune responses to the GP epitopes involved in ADE differ
among infected individuals. Previous studies by Takada et al.
demonstrated that mouse MAbs to the GP can be divided into three groups
on the basis of their properties: neutralizing (without C1q component;
unpublished data), enhancing, or nonneutralizing and nonenhancing (29, 30).
Thus, the activities (whether enhancing, neutralizing, or neither) of
the test sera can be determined by the extent of contribution from the
neutralizing and enhancing antibodies and by the cell types used for the
assays.
The demonstration of ADE of
Ebola Zaire virus infection raises fundamental issues about the
development of GP-based Ebola virus vaccines and the use of passive
prophylaxis or treatment with Ebola GP antibodies. Although studies with
animals indicate that GP-based Ebola virus vaccines are effective (27),
the protective effect appears to depend on cytotoxic T-cell rather than
antibody responses. We suggest that such vaccines should be designed to
avoid induction of known infectivity-enhancing antibodies, while
passive prophylaxis with whole GP antiserum should be performed with
caution.
Acknowledgments
We
thank Daryl Dick, Michael Garbutt, Krisna Wells, and Martha McGregor
for excellent technical assistance, John Gilbert for editing the
manuscript, and Yuko Kawaoka for illustrations.
This
work was supported by a Grant-in-Aid for Scientific Research on Priority
Areas from the Ministries of Education, Culture, Sports, Science, and
Technology, Japan, to A.T., in part by the Japan Health Sciences
Foundation (A.T.), by CREST (Japan Science and Technology Corporation)
(A.T. and Y.K.), by National Institute of Allergy and Infectious
Diseases Public Health Service research grants to Y.K., and by a
research grant from the Canadian Institutes of Health Research to H.F.
Article information
J Virol. Jul 2003; 77(13): 7539–7544.
PMCID: PMC164833
Received December 20, 2002; Accepted April 8, 2003.
Copyright © 2003, American Society for Microbiology
This article has been cited by other articles in PMC.
Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)
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