J. gen. Virol. (1989), 70, 2865-2876.
Printed in Great Britain
2865
Key words: pestivirus/HCV/monoclonal antibodies/epitopes
Topographical and Functional Mapping of Epitopes on Hog Cholera Virus
with Monoclonal Antibodies
By G. W E N S V O O R T
Central Veterinary Institute, Department of Virology, P.O. Box 365, 8200 AJ Lelystad,
The Netherlands
(Accepted 7 July 1989)
SUMMARY
Competitive binding studies and antigen capture assays were done with monoclonal
antibodies (MAbs) raised against hog cholera virus (HCV) to map the corresponding
epitopes. A model was constructed in which the 13 epitopes were situated in four
distinct antigenic domains: A, B, C and D. Domain A was subdivided into A1, A2 and
A3. The functional relevance of this model was assessed by the characterization of
pestivirus strains, by neutralization studies with the MAbs, and by isolation of variants
that escaped neutralization. The topographical arrangement of the epitopes, as
constructed in the model, was corroborated by the functional assays. The MAbs that
defined domains A1 and A2 recognized all 94 HCV strains tested. Domains A3, B, C
and D varied among the HCV strains. Neutralization was observed with MAbs
defining domains A1, B and C. Synergistic neutralization occurred using MAbs
against domains A1 and B, and A1 and C, but not within the domains. With MAbs
defining A1, B or C, variants could be isolated that escaped neutralization and
immunostaining by these MAbs.
INTRODUCTION
Hog cholera virus (HCV) (synonym: swine fever virus), bovine viral diarrhoea virus (BVDV)
and border disease virus (BDV) compose the genus pestivirus in the family Togaviridae
(Westaway et al., 1985). The viruses are closely related structurally and antigenically; infection
with one virus induces, to varying extents, antibodies against the other viruses in this genus
(Darbyshire, 1960; Dinter, 1963; Mengeling, 1963; Osburn, 1973; Wensvoortetal., 1989a). The
three viruses cannot be differentiated when infected monoiayers are immunostained with
polyclonal antisera raised against any of the three, and must be differentiated using crossneutralization tests. The viruses have similar hydrodynamic properties, showing a buoyant
density in sucrose of 1.12 g/cm 3 and a sedimentation coefficient of 138 + 11.5 (Laurie, 1979).
Among several proteins reported, three structural proteins (El, 55K to 57K; E2, 44K to 46K; C,
34K to 36K) have consistently been found (Enzmann & Weiland, 1978; Matthaeus, 1979).
Nothing is known about functional sites on these proteins, or about the location of supposedly
shared antigenic structures.
Hog cholera is a disease of pigs, bovine viral diarrhoea of cattle, border disease of sheep;
however, cross-infections by these viruses do occur (for review, see Van Oirschot, 1983). A
similar feature in the pathogenesis of the three viruses is their ability to induce congenital
infections (Van Oirschot, 1983). Hog cholera is an economically important and notifiable
disease in most pig-producing countries; outbreaks are generally controlled by eradication
programmes. HCV is routinely diagnosed in the laboratory using immunofluorescence tests
(Ressang & Den Boer, 1968). However, BVDV infections in pigs are also detected with
conjugates of polyclonal immunoglobulins against HCV and can be mistaken for HCV
infections (Terpstra & Wensvoort, 1988). To facilitate differential diagnosis of pestivirus
infections in pigs, we produced monoclonal antibodies (MAbs) against HCV strain Brescia
0000-8887 © 1989 SGM
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2866
G. WENSVOORT
( W e n s v o o r t e t al., 1986) a n d c h a r a c t e r i z e d a n u m b e r o f o t h e r p e s t i v i r u s s t r a i n s b y
i m m u n o s t a i n i n g ( W e n s v o o r t et al., 1989 b). T h i s r e p o r t p r e s e n t s t h e results o f t o p o g r a p h i c a l a n d
f u n c t i o n a l studies t h a t w e r e p e r f o r m e d w i t h t h e s e M A b s .
METHODS
Virus, cells and anti-HCV serum. HCV strain Brescia was biologically cloned by three times repeated endpoint
dilution and grown in PK-15 cells in roller bottle cultures. As growth medium, Earle's MEM supplemented with
antibiotics and 5~o foetal bovine serum (FBS) was used. The cells and FBS were free of pestivirus infection. The
FBS was also free of antibodies against HCV and BVDV. Swine anti-HCV hyperimmune serum was produced in
specific pathogen-free pigs (Terpstra et al., 1984). Immunoglobulins were purified by ammonium sulphate
precipitation and conjugated to horseradish peroxidase (HRPO), according to the method of Wilson & Nakane
(1978). The anti-HCV conjugate recognized HCV, BVDV and BDV when used to immunostain infected cell
cultures and tissue sections.
MAbs. Thirteen lines of MAb-producing hybridomas (CVI-HCV-21.1, -21.2, -39.5, -39.6, -39.8, -41.1, -41.4,
-44.3, -44.6, -44.22, -44.38, -44.45 and -44.51) were produced against gradient-purified HCV strain Brescia
(Wensvoort et al., 1986). The cells were multiplied in BALB/e mice. MAbs (numbered 1 to 13, respectively) were
purified from ascitic fluid by ammonium sulphate precipitation. A complete set of anti-mouse immunoglobulin
sera (Nordic) was used to determine the isotype of the MAbs in double immunodiffusion tests. M A b - H R P O
conjugates were produced as described above. MAbs and the conjugates were stored in 5 0 ~ (v/v) glycerol at
20 °C at a protein concentration of 4 mg/ml.
Competitive binding assay (CBA). ELISA plates (Dynatech, M129A) were coated overnight at 4 °C with HCV
antigen, partially purified from HCV Brescia-infected PK-15 cells by centrifuging frozen and thawed cell lysates
through a 25 ~ (w/v) sucrose cushion, as described before (Wensvoort et al., 1986). Plates were washed with 0.05
Tween 80 in demineralized water. M A b - H R P O conjugates were diluted in phosphate-buffered saline (PBS)
containing 0.05~ Tween 80 (PBS-Tw) to a working dilution that resulted in 5 0 ~ to 7 0 ~ of the maximum
absorbance. Each of the unconjugated MAbs was diluted 50-fold in the working dilution of the M A b - H R P O
under study, after which the mixtures were transferred to the wells of ELISA plates. Each mixture and the
conjugates alone were tested in quadruplicate. After incubation for 1 h at 37 °C, the plates were washed as
described above, and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) (Serva) and hydrogen
peroxide were added. The Aa05 was measured after 4 h with a Titertek Multiskan spectrophotometer.
Combinations of MAbs and M A b - H R P O conjugates that showed more than 5 0 ~ reduction of the A4os of the
conjugate alone were retested with 10-2, 10-3 and 10-4 dilutions of the unconjugated MAb.
Antigen capture assay (ACA). Roller bottle (1750 cm 2) cultures of PK-15 cells were infected with HCV Brescia
and incubated for 48 h. The supernatant was discarded and infected cells were harvested by shaking with glass
beads. Cells were suspended in PBS, then frozen and thawed. Cell lysates were sonicated twice for 10 s, after which
Nonidet P-40 (NP40) was added to a final concentration of 1 ~ (v/v). Cell lysates were left for 1 h at room
temperature, then clarified for 15 min at 1000 g. The supernatant was divided up and stored at - 70 °C for use as
HCV antigen in the ACA.
ELISA plates (Costar EIA 3590) were coated for 1 h at 37 °C with 100 gl of a 10-3 dilution of the respective MAb
in 50 mM-NaHCO3 (pH 9.6) per well. Plates were then washed as described above. M A b - H R P O conjugates were
diluted in PBS-Tw and 10~ horse serum to the same dilution as used in the competitive binding studies. Fifty ~1 of
conjugate was added to the wells of the coated ELISA plates. HCV antigen was diluted 1:10 in PBS-Tw and 50 Ixl
was added to each well. Each conjugate was tested in quadruplicate with each MAb as coating antibody. A
negative control was added to each plate, eight wells received 50 ~tl of the corresponding M A b - H R P O conjugate
and 50 ktl PBS-Tw. The plates were incubated for 1 h at 37 °C, washed as described above, and then ABTS and
H20~ were added. Absorbance was read after 17 h at 4 °C. An A from 0 to 0.199 was recorded as 0, from 0.2 to
0.399 as 1, etc.
Characterization ofpestiviruses. Ninety-four HCV strains, i.e. 30 laboratory strains that originated from several
European and American laboratories, 57 Dutch field isolates, seven vaccine strains, and 27 BVDV strains and
four BDV strains were characterized with the anti-HCV-HRPO and the MAb--HRPO conjugates. The viruses
were grown in PK-15 cells or foetal bovine epithelial cells (a cell line established at this Institute). Infected
monolayers were immunostained with various conjugates in an immunoperoxidase monolayer assay (IPMA;
Wensvoort et al., 1986). This paper reports only the number of strains or isolates that were recognized by the
various conjugates; detailed results will be published elsewhere (Wensvoort et al., 1989b).
Neutralization by MAbs. Neutralizing titres against HCV Brescia were determined in duplicate in a neutralizing
peroxidase-linked assay (NPLA; Terpstra et al., 1984). Titres were expressed as the reciprocal of the dilution that
prevented multiplication of 100 TCIDs0 of HCV Brescia. Neutralization indices were determined by incubating
10-fold dilutions of HCV Brescia in quadruplicate for 1 h at 37 °C in the absence or presence of MAb diluted 1 : 50.
PK-15 cells were then added and the cultures were grown for 4 days at 37 °C in 5 ~ CO2. Indices were expressed as
-
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Mapping of epitopes on H C V
2867
the log10 reduction of the virus titre by the MAb. Complement-mediated neutralization was studied by
determining neutralization indices for the MAbs in the presence of rabbit complement. After the primary
incubation of the virus-MAb mixtures, lyophilized rabbit serum (Sera-lab) was reconstituted and added to a final
concentration of 0H, 5~ or 10~. The virus-MAb-complement mixtures were incubated for 30 min at 37 °C
before PK-15 cells were added. HCV-positive and -negative pig serum and growth medium were tested as controls.
Neutralization experiments were all performed in PK-15 cells grown in microtitre plates, and infected monolayers
were immunostained with the anti-HCV-HRPO conjugate.
Synergism of MAbs. Synergism of neutralization was detected by titrating MAbs and mixtures of two MAbs, in
the NPLA with approximately 44, 440 and 4400 TCIDso of HCV Brescia. Each MAb was tested in quadruplicate,
alone and in combination with each of the other MAbs. Titres were expressed as described above, plotted on
logarithmic paper against the virus dose, and then interpolated for 100 and 1000 TCIDs0. Synergism of a
combination was expressed as the factor by which the actual titre was increased as compared with the expected
arithmetic mean titre for that combination. Only when a combination had a titre at least four times higher than
expected against both 100 and 1000 TCID5o was synergism considered to have occurred.
Detection of escape variants. To select virus variants that escaped neutralization by MAbs, we mixed 10-fold
dilutions of HCV Brescia in growth medium with neutralizing MAbs 1 to 8 (final dilution 1: 50) and incubated the
mixture for 1 h at 37 °C. Virus-MAb mixtures were added to PK-15 cell suspensions that were then grown in
microtitre plates for 4 days. The supernatants of the cell cultures were collected and stored at - 7 0 °C. The
monolayers were immunostained with the MAb-HRPO conjugate homologous to the selecting MAb, after which
the number and appearance of infected monolayers were recorded. The monolayers were immunostained for a
second time, this time with the polyclonal anti-HCV-HRPO conjugate. Virus variants that were not recognized by
the MAb-HRPO conjugate were found in cultures that stained only on the second occasion, or in cultures in which
many more cells were stained after the second time. From the corresponding supernatants, virus was isolated in
PK- 15 cells. After two cell culture passages without MAb, the escape variants were characterized in the IPMA. To
assess homogeneity, we titrated the isolate in 10-fold dilutions and grew it in PK-15 cells. After 4 days, the virus
titration was immunostained with all 13 MAbs and the polyclonal anti-HCV conjugate. A virus titre of each isolate
was thus determined for each MAb. Only isolates that had a titre at least 10000-fold lower when stained with the
selecting MAb than when stained with other MAbs or with the anti-HCV conjugate, were considered to contain a
variant that lacked the epitope specific for the selecting MAb. These variants were used to determine
neutralization indices for MAbs 1 to 8, as described above.
RESULTS
Topographical analysis
Competitive binding assay
Each M A b , diluted 1:50, was allowed to compete for its epitope on H C V Brescia with all 13
M A b - H R P O conjugates, thus 169 combinations were tested and percentages of inhibition of
the conjugate by the M A b were calculated. This resulted in two groups of M A b / M A b - H R P O
combinations. In one group of 97 combinations, no competition was observed. The m e a n
inhibition of this group was 0 ~ and the standard deviation was 13.2 ~ . In the second group of 72
combinations, competition was observed. T h e mean inhibition of this group was 8 7 ~ and the
standard deviation was 8.2~o. T h e highest inhibition in the first group was 3 8 ~ . In the second
group, the lowest inhibition was 6 2 ~ .
On the basis of these findings, the M A b / M A b - H R P O combinations were divided into those
with < 5 0 ~ and those with > 50~o competition (Fig. 1). All M A b / M A b - H R P O combinations
from the second group were retested with higher dilutions of the unconjugated MAb. All
combinations of M A b and conjugate that effected more than 50 ~ reduction of the signal of the
conjugate alone in the 1:50 dilution of the M A b also did so in the 1:100 dilution of the MAb. All
unconjugated M A b s reduced the signal of their conjugated homologue by more than 50 ~ at the
1 : 1000 dilution. In most cases, reciprocal competition, in which one M A b competed with the
conjugate of another M A b and vice versa, was detected. Non-reciprocal competition was
effected by M A b s l, 8, 3 and 7 on the conjugated M A b 12, by M A b 5 on the conjugated M A b s 6,
2, 3, 7, 10, 11, 9 and 12, by M A b 2 on the conjugated M A b s 7, 10, 11, 9 and 12, and by M A b 4 on
the conjugated M A b s 10 and 11. Enhanced binding of conjugates by MAbs, as found by Heinz et
al. (1984) in C B A performed with M A b s directed against tick-borne encephalitis virus, was not
seen.
The relations between the competing MAbs, diluted 1 : 50 and 1 : 100, and the conjugates were
used to group the epitopes in four antigenic domains: A, B, C and D (Fig. 2a). The relations
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2868
G. WENSVOORT
6
1
8
5
2
Competing MAb
4 3 7 10 11 9
12 13
6
1
8
5
~ 4
©
~ 3
e~
~I 7
,.Q
< 10
ll
9
12
mmmmmm
immmmmmm
13
D
50% competition
by MAb 1:50
~
>50% competition
by MAb 1:1000
>50 % competition
byMAb 1:100
~
~
>50 % competition
byMAb 1:10000
Fig. 1. Epitope mapping CBA. ELISA plates were coated with partly purified HCV Brescia. Each
MAb was diluted 1 : 50 and allowed to compete for its epitope with each MAb--HRPO conjugate. All
combinations of MAb and conjugate that effected more than 50~ reduction of the signal of the
conjugate alone were retested in the 1:100, 1:1000 and 1:10000 dilution of the competing MAb.
between the competing MAbs, diluted 1 : 1000, and the conjugates permitted the subdivision of
domain A (Fig. 2b).
Within the domains, the MAbs defining the epitopes differed slightly. In domain A, only
MAbs, 9, 10 and 11 had identical relations with each other and with all the other MAbs, and thus
reacted identically with the H C V antigen under these conditions. At the same time, MAbs 2, 3,
4, 7 and 12 reacted differently in the competition assays with all other MAbs, thus
demonstrating that each has a unique relationship with HCV. In domain C, MAbs 1 and 8
competed with each other and competed reciprocally with M A b 5 and non-reciprocally with
M A b 12, while MAb 5 also competed non-reciprocally with seven other MAbs. M A b 5 was thus
unique, whereas MAbs 1 and 8 reacted identically with the H C V antigen under these conditions.
Antigen capture assay
Each M A b was tested as coating antibody with each M A b - H R P O conjugate, and each
combination was judged for its capacity to recognize H C V antigen in infected cell lysate treated
with NP40. A positive signal in A C A meant that both MAbs could bind simultaneously
indicating that the epitopes were located separately from each other. A negative signal meant
either that the two MAbs could not bind simultaneously and thus recognized an overlapping or
adjacent epitope or that the respective epitopes were not located on the same protein (Fig. 3).
When a M A b was used as coating antibody and its homologous M A b - H R P O was used as
conjugate, no H C V antigen was recognized. This demonstrated that aggregate formation by
proteins, in which the same epitope would appear twice on the aggregate, was not a problem in
ACA. Combinations of MAbs were only able to recognize the antigen when the two MAbs were
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Mapping of epitopes on HCV
(a)
(b)
@
2869
@
Domain B
Domain B
Domain C
Domain C
Domain A
D
o
m
®
U
®
Domain D
Domain D
Fig. 2. Grouping of epitopes in domains by CBA. Epitopes were mapped by CBA (see Fig. 1). A circle
around the number of the MAb indicates that the MAb competed with its conjugated homologue.
MAbs that competed reciprocally with each other's conjugates are connected by solid lines. Nonreciprocal competition is not shown. In (a) more than 50~ competition by the competing MAbs diluted
1 : 50 and 1 : 100 was considered significant, which resulted in the grouping of the epitopes in four
domains. In (b), the level was raised to the 1 : 1000 dilution of the competing MAb, in which way domain
A was divided in two.
6
1
~I"~'~"'"
' : :"~: ' ~ : [
8
MAb used as coating antibody
5 2 4 3 7 10 11 9
x.:.:.:.x.:.
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iiiii:~iiiiill
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8
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........
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Z
4 [iiii~! 9 L,':J~:~,::d
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::::.~-~:'
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0.6 to 0.8
0-8 to 1.0
1.0 to 1-2
1.2 to 1.4
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1.4 to 1.6
11
~. .~,.~.',.
9 ~ l~l
l l'~~~!,,.i
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1 2 I ~ [ ~ d d 9 I= 4 1 3
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Fig. 3. Epitope mapping by ACA. All MAbs were used as coating antibody in all combinations with
the MAb-HRPO conjugates. Combinations were tested for their capacity to recognize HCV antigen in
1% NP40-pretreated infected cell lysate.
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2870
G. WENSVOORT
directed against two different domains, as defined in the CBAs. Exceptions to this were the
combination o f M A b 2 and MAb 12 and, to a lesser extent, combinations of MAbs 3, 4, 7 and 12.
Again, as in the CBA, the epitopes could be grouped into four antigenic domains. Since each
MAb against a domain reacted positively in the ACA with all MAbs directed against each of the
other domains, all domains are located on the same protein. Domain A could be subdivided into
domains A1, A2 and A3, since the combination of MAbs 2, 3, 4 and 7 (A1) and MAb 12 (A3)
gave positive signals in the ACA.
Functional analyses
Characterization of pestiviruses
Immunostaining (IPMA) of cell cultures that were infected with 125 different pestivirus
strains or isolates revealed that MAbs 2, 3, 4 and 7 (subdomain A1), and 9, 10 and 11 (subdomain
A2) consistently recognized all 94 HCV strains or isolates tested (Table 1). MAbs 6 (domain B),
1, 5 and 8 (domain C), 12 (subdomain A3) and 13 (domain D) did not label several HCV strains
or isolates; thus the epitopes defined by these MAbs were absent from these viruses. None of the
latter MAbs detected the same variation; however, MAbs 1 and 8 were very similar, MAb 8
stained the same viruses as MAb 1, and one strain more. None of the 27 BVDV or the four BDV
strains or isolates was detected by the 13 MAbs, whereas the polyclonal anti-HCV conjugate
recognized all 125 pestiviruses. No reaction was observed with uninfected monolayers, thus all
MAbs were specific for HCV as grown in cell culture. The MAbs detected a high level of
epitopic variability among HCV. This finding confirms the findings of an earlier study
(Wensvoort & Terpstra, 1986), in which five different variants were isolated from the blood of a
pig infected with one virulent HCV strain.
Neutralization by MAbs
MAbs 1 to 8 (domains A1, B and C) neutralized strain Brescia to varying extents (Table 1).
Two MAbs (2 and 6) had low titres and indices, two (3 and 4) had moderately high titres and
indices, and four (l, 5, 7 and 8) had high neutralizing titres and indices. MAbs 9 to 13 did not
neutralize strain Brescia. Neutralization indices were not enhanced by the addition of
complement.
Synergism of MAbs
To study the synergistic effect of MAbs in neutralization tests, titres of individual MAbs and
combinations of MAbs were determined against HCV Brescia. Synergism was observed only
with MAbs 1 to 8, and only specific pairs interacted synergistically. One member of each pair
was always MAb 2, 3, 4 or 7 (domain A1), and the other member was always MAb 1, 5, 8
(domain C) or 6 (domain B) (Table 2).
Detection of escape variants
Viral variants were isolated that escaped neutralization and were not immunostained by
specific MAbs. With the eight neutralizing MAbs used for selection, 11 non-neutralizable
variants were isolated, seven of which were different when immunostained with the full set of
MAbs in IPMA (Table 3). The neutralization indices of MAbs 1 to 8 were determined for these
eight (Table 3). Viral variants that were isolated with MAbs directed against domain A, B or C
were no longer neutralized or detected in immunostaining by MAbs directed against A, B or C,
respectively, but had normal reactivity patterns with other MAbs. Results obtained with MAbs
1, 5 and 8 directed against domain C permitted a division of this domain. MAb 5 was used to
isolate a variant that was neutralized by MAbs 1 and 8 but not by MAb 5; MAbs 1 and 8 were
used to isolate variants that were neutralized by MAb 5, but not by MAbs 1 and 8. In general, we
found that MAbs 1 to 8 neutralized the viral variants that they recognized in the IPMA. Three
variants, however, were recognized by a MAb in immunostaining, but were not neutralized by
this MAb; these were variant C-lc5 and MAb 8, variant A-4d7 and MAbs 3 and 7, and variant
A-4d12 and MAb 7. Note that escape variants could be found in vitro that were no longer
recognized by MAbs directed against domains A1 and A2. This finding apparently contradicts
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IgG 1
75t
135000§
3.9§
C
IgG 1
33t
5_44§
1.3§
B
1
3.4§
C
12000§
IgG 1
76t
8
4.4§
C
80000§
IgG 1
17t
5
1.9§
AI
58§
IgG2a
94
2
2-8§
AI
1600§
IgG2a
94
4
3
3.0§
A1
1600§
IgG2a
94
MAb
A
3.6§
A1
25600§
IgG2b
94
7
0.9
A2
<25
IgG 1
94
10
0.8
A2
<25
IgG 1
94
11
- 0-1
A2
<25
IgG 1
94
9
Isotype, detection of stra& variation by, and neutralization with 13 MAbs against H C V Brescia
A3
IgG 1
27t
12
1.7/3'5t
0.5/1.4
1-4/4-6
18,/28:~
11/14~:
4.6/9-8:~
9-8/17:~
B
1.2/0.4
2.1/2.3
1.4/0-5
3.2/7-5
1-7/3.5
13/13:1:
C
1
1.7/2.l
6.5/28:~
4.6[20~.
15/52:~
23/104~
C
8
1.2/4-3
2.6/5.3
5-3/30:~
9.2/34~
C
5
MAb
A
1.6/1.3
1.5/1.9
3.2/3.0
A1
2
1.2/2.3
0.7/2.6
A1
4
Synergism of MAbs in neutralization test*
0-8/0.7
A1
3
A1
7
C
A1
AI
A1
A1
C
C
Domain
D
IgG2a
13
* Neutralizing titres of individual MAbs and combinations of MAbs were determined against HCV Brescia. Results are calculated as the factor by which the
actual titre of the M A b mixture was increased, compared with the expected arithmetic mean titre of the two MAbs.
, Results at 100 TCIDs0/1000 TCID50, thus the mixture of equal volumes of MAb 2 and M A b 6 gave an 18-fold increase with 100 TCIDso and a 28-fold increase
with 1000 TCIDs0, as compared with the mean titre of both MAbs titrated separately.
Combinations of MAbs that are considered synergistic. MAbs 9 to 13 did not neutralize or cause synergism.
MAb
1
8
5
2
4
3
7
Domain
6
T a b l e 2.
* Number of HCV strains recognized out of 94 HCV strains tested by immunostaining infected cell cultures in IPMA.
t Underlined indicates detection of variation; none of the 13 MAbs detected any of 25 BVDV strains or four BDV strains tested.
~: Neutralizing titre against 100 TCIDso HCV Brescia.
§ Neutralization occurred,
jj loglo reduction of the titre of HCV strain Brescia grown in the presence of the respective MAb, which was diluted 1:50.
Isotype
Variation*
Neutralization
titre:~
Neutralization
indexll
Domain
• 6
T a b l e 1.
t~
oo
C~
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(2A5)
(4D5)
(4D7)
(4D12)
(3A6)
(7A5)
2
4
4
4
3
7
B
C
+
+
+, 3
+, 3
+
+ , 3.5
-, 0
-, -0-2
+ , 3.5
+ , >3.3
1
C
+
+
+ , 3-2
+, 3
+
+ , 3.7
+ , 0-2§
-, 0
+ , 4.2
+ , >3.3
8
C
+
+
+ , 3.2
+ , 3.2
+
+ , 3-5
+, 3
+ , 3-7
+
- , 0.5
A1
.
.
.
.
-, -0.3
- , 0.2
.
.
-, -0.3
+ , 2-5
+ , 2.5
+
+, 3
+ , >2-3
A1
.
.
-, 0
-, -0.2
.
-, 0
+ , 3.5
+ , 2.8
+
+ , 3.2
+ , >3.3
A1
.
.
.
.
+ , 0.2§
- , 0.2
.
.
- , 0-5
+ , 3.5
+ , 3.5
+
+ , 3.5
+ , >3.3
7
A1
.
.
+ , 1.2§
+ , 1§
.
- , 0.3
+ , 3-8
+ , 3.8
+
+, 4
+ , >3.3
2
4
3
Characterization in IPMA/neutralization index~
~
+ , >3.3
5
MAb
Detection of escape variants
A2
.
A2
+
+
+
+
+
+
11
+
.
.
+
+
+
+
+
10
A2
-
+
+
+
+
+
+
9
A2
+
+
+
+
+
+
+
+
+
+
+
12
D
+
+
+
+
+
+
+
+
+
+
+
•
13
* HCV Brescia was grown in the presence of the respective MAb.
t Virus variants that escaped neutralization were characterized by immunostaining in I P M A with M A b - H R P O conjugates. + represents positive staining; -, no
staining.
~t Variants that differed in I P M A were tested in neutralization tests with MAbs 1 to 8. The neutralization index is given as the log~o reduction of the titre of the
variant by MAb diluted 1:50.
§ MAb did recognize a variant in the IPMA, but showed reduced neutralizing activity with this variant.
Domain
+ , 2.2
+ , 2.7
+
+, 2
+
+
+ , 2.7
+ , 2.4
+
+, 3
- , 0.3
(1C5)
(IC1)
(8C11)
(5B2)
1
1
8
5
6
6* ( 6 C l l t )
•
Escape
variant
T a b l e 3.
o
o
rn
Z
0
to
oo
-.d
to
Mapping o f epitopes on H C V
2873
the finding that these domains are conserved among 94 laboratory strains and field isolates of
HCV. However, it could also indicate that although the variants are suited to a tissue culture
flask environment, their particular genotype does not permit them to predominate in vivo. One of
the viral variants (A-7a5) that was not recognized by MAbs directed against domains A1 and A2
was cloned by endpoint dilution three times and was subsequently grown for seven passages
in cell culture. During these passages, the variant was not recognized in immunostaining with
the MAbs that defined A1 and A2.
DISCUSSION
Thirteen MAbs, produced against HCV strain Brescia, were used to analyse epitopes
topographically and functionally. CBA and antigen capture assays ACA were used for
topographical analyses.
Competition between a MAb and a MAb conjugate in CBA indicates that the epitopes that
are recognized by the two MAbs overlap or are adjacent on the antigen (Stone & Nowinsky,
1980), especially for reciprocally competing MAbs. Non-reciprocal competition may be
explained in several ways. The affinity of the two MAbs may differ greatly; the binding of one
MAb may induce a change in conformation that alters the binding site of the other MAb; the
binding of one MAb may sterically hinder the binding of the other MAb; conjugating one MAb
to peroxidase may alter the ability of the MAb to bind. Since the information gained from nonreciprocal competition was not straightforward, only the results that indicated homologous and
reciprocal competition were incorporated in Fig. 2. Four distinct antigenic domains, A, B, C
and D, were defined (Fig. 2a). Domain A was divided into two when the more discriminative
1 : 1000 dilution of the competing MAbs was used (Fig. 2b). The domains consist of adjacent or
overlapping epitopes.
The ability of pairs of MAbs and MAb--HRPO conjugates to recognize HCV antigen was
determined in ACA. The HCV antigen was obtained by treating HCV-infected cell lysates with
the non-ionic detergent NP40. Because homologous pairs of MAbs and MAb conjugates scored
negative in the ACA, we reasoned that under the test conditions epitopes were available only
once on each protein. The same four domains were defined in ACA as in CBA, but domain A
was divided into subdomains Al, A2 and A3 (Fig. 2 and 4). Fig. 4 can therefore serve as an
abstract model depicting the spatial relations between the 13 epitopes.
The biological relevance of this model was assessed by immunostaining pestivirus strains, by
neutralization studies, and by isolating variants that escaped neutralization by various MAbs.
The topographical arrangement of the domains was fully corroborated by the functional assays
(Fig. 4). The domains were HCV-specific. MAbs used to define subdomains A1 and A2
consistently reacted with all 94 HCV strains or isolates tested. The domains B, C, D and A3
varied among 30 laboratory strains, 57 field isolates and seven vaccine strains of HCV (Table 1).
Because of their specificity for HCV, MAbs can now be used routinely to differentiate between
HCV and BVDV infections in pigs (Terpstra & Wensvoort, 1988). The widely used Chinese
vaccine virus strain (C strain) is always recognized by the MAbs directed against A1, A2 and D,
but lacks the epitopes for the MAbs directed against A3, B and C. The 57 Dutch field strains are
always recognized by the MAbs directed against AI and A2 and by MAbs 1 and 8. Because of
this feature, these MAbs can be used to differentiate between infections caused by field strains
or by the vaccine virus, strain C (Wensvoort et al., 1989b).
The eight MAbs directed against domains A1, B and C neutralized HCV (Table 1). Thus,
domain A1 is conserved among HCV strains and is involved in neutralization.
Combining MAbs directed against domain A1 with MAbs directed against either B or C
produced strong synergistic effects in neutralization tests. These were not seen with
combinations of MAbs directed against the same domain (Table 2). Synergism was more
pronounced with high virus doses, when antigen was in relative excess. In CBA, there were no
MAbs that enhanced the binding of other MAbs; therefore, it is unlikely that the synergism can
be explained by enhanced binding of two MAbs to the virion. Synergism occurred only when
MAbs that individually neutralized were combined. This finding is at variance with that of
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2874
G. WENSVOORT
/
D
A
-©
©
A3
Fig. 4. Grouping of epitopes in domains by ACA and synthesis of topographical and functional
analyses with 13 MAbs against HCV Brescia. Combinations of MAbs that scored positive in the ACA
are situated distantly from each other, on different comers of the quadrangle. A negative score in ACA
indicated that the epitopes were adjacent or spatially overlapping, these epitopes are grouped at each
comer. The epitopes were grouped in four antigenic domains: A, B, C and D. Domain A was divided
into subdomains A 1, A2 and A3. By testing the MAbs in neutralization tests it was shown that domains
AI, B and C are involved in neutralization. MAbs directed against domains A1 and A2 bound to all
viral strains and isolates tested. Symbols: ~ , neutralizing site; k~, conserved site; *--~,synergism.
Peiris et al. (1982), who reported that a combination of two non-neutralizing M A b s was able to
neutralize the West Nile flavivirus.
The observed synergistic effect is not simply the result of multiple binding to more 'critical
sites' on the virion, as proposed by DeUa-Porta & W e s t a w a y (1978). N e i t h e r can it be solely
explained by assuming that more and more complex v i r i o n - a n t i b o d y aggregates are formed by
which infection is prevented. N e i t h e r of these explanations accounts for the fact that synergism
does not occur when M A b s that define domains B and C are combined. It appears that antibody
must attach to d o m a i n A1. Hence antibodies against d o m a i n A1 m a y play a specific role in
neutralizing the virus, as a function o f d o m a i n A1 or o f the antibodies directed against A1. All
M A b s defining A1 belong to the I g G 2 isotype, and all M A b s defining domains B and C belong
to the I g G 1 isotype (Table 1). Klaus et al. (1979) found that mouse I g G 2 antibodies are involved
in complement activation, whereas I g G 1 antibodies are not. However, addition of complement
did not enhance neutralization; complement-mediated neutralization can thus not be a cause of
the observed synergism.
Neutralizing M A b s 1 to 8 were used to select seven different variants that escaped
neutralization and recognition in the I P M A . M A b s that were directed against a certain d o m a i n
selected viral variants that escaped neutralization by M A b s directed against that domain. These
viral variants, however, were always neutralized and immunostained by M A b s defining the
other domains (Table 3). This finding further illustrates the independence of the domains.
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Mapping of epitopes on HCV
2875
By using MAbs against domain C to isolate escape variants, we demonstrated that this
domain could be divided. The epitope recognized by MAb 5 was separated from the closely
related epitopes of MAbs I and 8. In agreement with this finding, MAb 5 detected only 17 out of
94 HCV strains (Table 1), whereas MAb 1 detected 75 and MAb 8 detected 76 of the 94 HCV
strains; moreover in the CBA, MAb 5 showed more non-reciprocal blocking of other MAbs than
did MAbs 1 and 8.
Three escape variants (C-lc5, A-4d7 and A-4d12) showed the phenomenon of unimpaired
immunostaining but reduced neutralization by a MAb. In every case, these MAbs belonged to
the same group as the MAb that was used to select the variant. How these variants bind to the
MAb but are no longer neutralized by it is not understood. This phenomenon has also been
observed in studies with poliovirus, in which an escape mutant was recognized by
immunoprecipitation but was no longer neutralized by a selecting MAb (Thomas et al., 1986).
In conclusion, CBA and A C A were used to define an abstract model of an antigenic region of
HCV, in which 13 epitopes form four antigenic domains: A, B, C and D. Domain A was divided
into subdomains A1, A2 and A3. Functional analyses supported virtually all these results (Fig.
4). In a future report (G. Wensvoort & J. Boonstra, unpublished data) we will demonstrate that
these domains are all located on the major envelope protein of HCV.
I am grateful to N. Oei for excellent technical assistance, to J. G. van Bekkum, C. Terpstra, A. L. J. Gielkens and
A. A. M. Thomas for their invaluable comments, to V. Thatcher for editorial assistance and to R. Heuckeroth and
G. Loo for typing the manuscript.
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