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Journal of Virology, October 2001, p. 9986-9990, Vol. 75, No. 20
Istituti di
Microbiologia,1 Clinica
Medica3 and Patologia
Medica,6 Facoltà di Medicina e Chirurgia,
Università Cattolica del S. Cuore, 00168 Rome, Istituti
di Microbiologia4 and Medicina
Interna,5 Facoltà di Medicina e Chirurgia,
Università di Ancona, 60020 Ancona, and Department of
Biomedical Sciences, Università di Trieste, Via Giorgieri 22,
34100 Trieste,7 Italy, and Laboratory of
Immunology, College of Pharmacy, Seoul National University, Seoul
151-742, Korea2
Received 16 April 2001/Accepted 12 July 2001
Clinical and experimental evidence indicates that the hepatitis C
virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate
for the development of an effective anti-HCV vaccine. Identification of
the human epitopes that are conserved among isolates and are able to
elicit protective antibodies would constitute a significant step
forward. This work describes the mapping of the B-cell epitopes present
on the surface of HCV/E2, as recognized by the immune system during
infection, by the analysis of the reciprocal interactions of a panel of
human recombinant Fabs derived from an HCV-infected patient. Three
unrelated epitopes recognized by antibodies with no
neutralization-of-binding (NOB) activity were identified; a fourth,
major epitope was defined as a clustering of minor epitopes recognized
by Fabs endowed with strong NOB activity.
Hepatitis C Virus (HCV) is the major
causative agent of blood-borne non-A, non-B hepatitis (12,
19). The tendency of HCV infection toward chronicity
(9), with persistent and continuous viral replication
(4), suggests that in the majority of cases the host
immune response is unable to tackle and eradicate the infection. The
commonest way of controlling viral diseases is by developing vaccines
able to prevent viral spreading typically by eliciting neutralizing
antibodies. In the case of HCV, although specific humoral immunity can
be readily detected and the demonstration of anti-HCV antibodies
establishes a serologic diagnosis of infection (3), it is
controversial whether the humoral response affords any protection
(5, 13, 14, 20, 26, 29). However, recent reports
describing the dynamics of intrahost evolution in an HCV-positive
population during primary infection have shown that a crucial phase for
disease outcome lies at a time point corresponding to the production of
antibodies by the infected host (15, 23). These data
suggest an important role for antibodies in the evolution of HCV infection.
An important viral structure studied as an antibody response target is
the HCV E2 envelope glycoprotein (HCV/E2). Successful protection of
chimpanzees by immunization with glycoproteins E1 and E2 has been
ascribed to the induction of specific anti-E2 antibodies
(11) that seem to be able to neutralize the binding of E2
to susceptible cells. These molecules are commonly referred to as
antibodies with neutralization-of-binding (NOB) activity (28). Although the assessment of the efficacy of this
class of antibodies in inhibiting HCV infection and replication has been hampered by the poor growth efficiency of HCV in cell culture, high titers of NOB antibodies have been seen to correlate with the
natural resolution of chronic HCV infection (18).
These considerations show that the study of the antibody response
against HCV/E2 can greatly contribute to the development of an
effective vaccine. This goal is usually pursued by using panels of
mouse monoclonal antibodies. Since in the case of this viral pathogen
the murine model is not consistent with the human antibody response
(1), the generation from an infected patient of human
monoclonal antibodies representing discrete parts of the immune
response is more suitable to the study of this aspect of virus-host
interplay (10).
Cloning of the immune repertoire of an HCV-infected patient on phage
display combinatorial vectors and generation of recombinant monoclonal
Fab fragments (7, 27) have demonstrated that inhibition of
binding of HCV/E2 to cells varies widely from one antibody clone to another.
The failure of traditional approaches such as peptide scanning
(16) to identify the epitopes recognized by these
molecules is probably connected with the fact that, when assayed by the phage display technology, the most important part of the in vivo antiviral response is usually directed against conformational and
heavily glycosylated regions (17), a finding confirmed by the recent work of Allander et al. (2). An alternative
approach consists of analyzing the reciprocal interactions of
recombinant Fab pairs assuming that Fabs inhibiting each other's
binding are directed against overlapping parts of the E2 molecules and
that Fab pairs that do not interact define two discrete B-cell
epitopes. Two Fabs with identical inhibition patterns would thus be
likely to define the same B epitope.
The human B epitopes present on HCV/E2 and recognized by our panel of
Fabs were thus analyzed by a competitive enzyme-linked immunosorbent
assay (ELISA) using FLAG-labeled Fabs against unlabeled Fabs. For
production of the above-mentioned FLAG-labeled Fabs (FLAG-Fabs), Fab
genes were inserted in the pComb3/FLAG vector (R. Burioni, unpublished
data), adding an epitope (FLAG) to the carboxy-terminal end of the
heavy-chain fragment recognized specifically by a mouse anti-FLAG
monoclonal antibody (Sigma, Saint Louis, Mo.).
For competition assays, ELISA plates (Costar, Corning, N.Y.) were then
coated with recombinant HCV/E2 (genotype 1a, strain H) (7, 22,
24) and blocked with phosphate-buffered saline (PBS)-1% bovine
serum albumin for 1 h at 37°C; subsequently, 50 µl of a
purified preparation of a competing Fab at known concentrations (Fig.
1) was added to the wells and the mixture
was incubated for 2 h at 37°C. After this step, an appropriate
amount of FLAG-Fabs was added directly to the wells to obtain a final
concentration giving approximately 60% of the maximum optical density
at 450 nm (OD450) in the ELISA (Table
1) and the mixture was incubated for an
additional 30 min. Plates were then washed 10 times with PBS-0.05%
Tween, and binding of the FLAG-Fab probe to the antigen was revealed
with anti-FLAG M2 mouse monoclonal antibody (Sigma; 10 µg/ml in PBS)
and demonstrated, after another wash, by addition of
peroxidase-conjugated anti-mouse immunoglobulin serum (Sigma; 1:700 in
PBS). After a final wash, 100 µl of substrate (Sigma) was added and
the OD450s of the plates were read after 30 min at room
temperature in the dark. A negative control sample containing an excess
of a purified control human Fab directed against herpes simplex virus
glycoprotein D (8) and corresponding to 0% inhibition was
included. Wells with no labeled Fab were always included to rule out
nonspecific reactivity of the FLAG-Fab detection system.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9986-9990.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mapping B-Cell Epitopes of Hepatitis C Virus E2
Glycoprotein Using Human Monoclonal Antibodies from Phage
Display Libraries
ABSTRACT
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FIG. 1.
Inhibition of binding of probe FLAG-Fab antigen by
previous binding of different concentrations of purified unlabeled Fabs
used as competitors.
TABLE 1.
Human recombinant Fabs used in this
studya
Final results were determined as percent inhibition with the following
formula: percent inhibition = 100 × [(OD450 of
probe FLAG-Fab alone 
OD450 of probe FLAG-Fab with
competitor Fab)/OD450 of probe FLAG-Fab alone]
(Fig. 1).
At least six different epitopes were characterized. As they did not
inhibit each other, Fabs e137 and e8 were defined as recognizing two
distinct epitopes on HCV/E2 and since they had no NOB activity, these
epitopes were defined as nonneutralizing. A third, different epitope
was recognized by e10-B, as expected since this Fab had been selected
by using antibody-coated HCV/E2 (6). In brief, after
binding of recombinant HCV/E2 to ELISA plates and blocking of the wells
with PBS-3% bovine serum albumin 70 µl of a mixture containing
purified human monoclonal recombinant anti-HCV/E2 Fabs e8, e20, e137,
e301, and e509 (10 µg of each clone per ml) was added to the wells
and incubated for 1 h at 37°C. Antibody-coated HCV/E2 was then
used to select Fab e10-B. Competition binding experiments demonstrated
that, as expected, the binding of Fab e10B to HCV/E2 is not inhibited
by any other Fab. By contrast, previous binding of Fab e10B inhibited
the binding of Fab e8 to HCV/E2. This effect was asymmetric, as e10B
inhibited e8 but was not itself inhibited (6). This may be
due to a modification of the structure of the glycoprotein subsequent
to the binding of e10-B. The epitopes defined by these families of Fabs
(e8, e137, and e10-B) were thus completely distinct. The other three Fabs (e20, e301, and e509) recognized three overlapping but different epitopes, which are probably clustered to form a major epitope on the
surface of HCV/E2. These three Fabs belong to families endowed with
strong NOB activity. The analysis of inhibition data allowed to observe
the reciprocal reactivity of all Fabs in the matrix shown in Table
2. It is important to stress that Fab
e509, which had the highest NOB titer (Table 1), seems to define the minimal region that needs to be recognized for neutralization of
binding. By using these data, a two-dimensional surface map of the
human epitopes on HCV/E2 was also drawn (Fig.
2).
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The first conclusion that can be derived from the data presented in this paper is that all NOB antibodies recognized a specific region of HCV/E2, which may be the one involved in binding of the virus to the cellular target. Although other explanations are possible, such as an antibody-induced modification of the protein conformation preventing further interaction of HCV/E2 with the cellular target, the antibodies directed against this epitope are useful reagents in view of a better definition of this key step in virus-host interaction. A second aspect is that only a cluster of epitopes was recognized by antibodies with the NOB effect. Immunoglobulins directed against the e137 and e8 epitopes not only lack this activity but can also displace from the virus other antibodies (like e301 and e20) that can inhibit important viral functions. In addition, antibodies binding to the e10-B epitope can reduce the NOB activity of other immunoglobulins even without displacing them (6). The eventual NOB effect is the sum of the interactions among all antibody clones, and the absence of NOB activity in HCV-positive sera can be due to a prevalence of the effect of NOB-inactive clones. Studies are in progress to evaluate these synergies.
To identify the physical location of the epitopes on the surface of HCV/E2, competition experiments were performed with a panel of mouse monoclonal Fabs directed against a specific mouse B-cell epitope present in the HCV/E2 region and spanning amino acid residues 527 to 560 (21). In no experiment was binding of human Fabs inhibited by previous binding of an excess of mouse monoclonal antibody to HCV/E2 (data not shown). This indicates that the human B-cell epitopes defined in this study did not correspond to the murine epitope recognized by the CET monoclonal antibodies (Fig. 2).
Our Fabs were also tested by ELISA, as previously described (7), for the ability to bind a synthetic peptide corresponding to HVR-1 derived from a sequence of HCV/E2 with the 1a genotype (H isolate, amino acids 388 to 409) (16) used to produce the recombinant HCV/E2 employed in this study. As expected, no binding was evidenced since library selection was performed with strategies designed to favor antibodies directed against epitopes conserved among different genotypes. Although these data are not conclusive, as they require testing of reactivity against HCV/E2 with HVR-1 deleted, as well as against different HVRs expressed in the context of a whole E2 glycoprotein, they leave open the possibility that NOB antibodies directed against regions in which HVR-1 is not directly involved as an epitope might be present in the repertoire of infected patients.
Finally, to exclude the artifactual nature of our Fabs, we
evaluated by competitive ELISA the abilities of HCV-positive and HCV-negative sera to inhibit Fab binding to the antigen. As shown in
Fig. 3, the results indicate that all of
the positive patients, albeit to different extents, had antibodies able
to bind to the epitopes recognized by our Fabs. Remarkably, in some
cases, inhibition of FLAG-Fab binding was noted in serum dilutions of
up to 1:500 (data not shown). Previous binding to HCV/E2 of a mixture
of the six Fabs (10 µg of each clone per ml) described in this work
was able to inhibit the binding to HCV/E2 of sera obtained from
HCV-positive patients (1:200 dilution) up to 75% in an ELISA format
(R. Burioni, unpublished data). An assay able to measure the amount of
serum antibodies directed against the different epitopes is being
developed. No binding to HCV/E2 and no inhibition were demonstrated in
the HCV-negative sera, even at the highest concentrations (data not shown).
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Although additional studies are needed for the fine characterization of human B-cell epitopes, this paper provides useful data for a better understanding of virus-host interplay in this infection. The absence of an in vitro neutralization model requires further investigations aimed at correlating NOB and true neutralization activity. If this is proved, antibodies recognizing neutralizing and conserved viral epitopes similar to the ones described in this study will have the potential for therapeutic use. The availability of a panel of human recombinant Fabs and of detailed information on the epitopes recognized by them would allow easy in vitro evaluation of antigens (25), which would be a considerable advance for an infection lacking an animal model. Molecules demonstrated in vitro to be able to stimulate selectively the production of neutralizing antibodies in immunized patients will be the best candidates for further in vivo studies.
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ACKNOWLEDGMENTS |
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We thank Silvio Bighi, Mario Perotti, and Cristiano Scalpelli for valuable assistance. We are deeply grateful to Giovanni Gasbarrini for critical help and continuous and enthusiastic support.
This work was supported by grants from the Istituto Superiore di Sanità to R.B. and M.C.
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FOOTNOTES |
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* Corresponding author. Mailing address: Servizio di Virologia, Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Università di Ancona, Via Conca, 60020 ANCONA, Italy. Phone: 39 071 5964849. Fax: 39 071 5964852. E-mail: r.burioni{at}libero.it.
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Journal of Virology, October 2001, p. 9986-9990, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9986-9990.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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