Download Monoclonal antibodies to equine arteritis virus proteins identify the

Journal of General Virology (1994), 75, 2439-2444. Printed in Great Britain
2439
Monoclonal antibodies to equine arteritis virus proteins identify the
protein as a target for virus neutralization
GL
Dirk Deregt,'* Antoine A. F. de Vries, 2 Martin J. B. R a a m s m a n , 2 Lindsay D. Elmgren 1
and Peter J. M . Rottier z
1Animal Diseases Research Institute, Agriculture Canada, P.O. Box 640, Lethbridge, Alberta, Canada T1J 3Z4 and
2 Institute o f Virology, Department o f Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University,
Yalelaan 1, 3584 CL Utrecht, The Netherlands
Monoclonal antibodies (MAbs) to equine arteritis virus
(EAV) proteins were produced and characterized. The
protein specificifies of eight MAbs were determined
definitively by immunoprecipitation of EAV proteins
expressed from vaccinia virus recombinants (WRs).
Included were two new W R s produced for this study,
expressing the M and the G~ proteins, respectively.
Three MAbs were determined to be N-specific and five
MAbs recognized the G Lprotein. One GL-Specific MAb,
17F5, of the IgA class, efficiently neutralized EAV
infectivity. In competitive binding assays (CBAs), the N-
specific MAbs defined a single antigenic domain on this
protein. Four GL-specific MAbs, including MAb 17F5,
demonstrated strong reciprocal competition in binding
to the G L protein but differed in their virus-neutralizing
ability. Thus the antigenic domain defined by these
MAbs is probably composed of overlapping or closely
adjacent epitopes. The fifth GL-specific MAb, a nonneutralizing antibody, may define an epitope adjacent to
this antigenic domain as reciprocal CBAs demonstrated
lower competition.
Equine arteritis virus (EAV) causes both subclinical
infections and clinical disease with symptoms that
include fever, anorexia, oedema of the ventral body,
serous nasal discharge, lacrimation, conjunctivitis, and
abortion in pregnant mares (Huntington et al., 1990).
The virus was first isolated in 1957 from an aborted fetus
after an abortion epidemic among Standard bred horses
in Bucyrus, Ohio, U.S.A. (Doll et al., 1957a, b).
Transmission is probably airborne, occurring primarily
via the respiratory route, but the virus is also shed in the
semen of persistently infected stallions and can be
transmitted venereally (Timoney et al., 1986).
EAV belongs to a group of small, enveloped, positivestranded RNA viruses that also includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and
respiratory syndrome virus and simian haemorrhagic
fever virus (den Boon et al., 1991; Godeny et al., 1993;
Meulenberg et al., 1993). Based on physicochemical
properties, polarity and size (12.7 kb) of the genome,
nucleocapsid morphology (isometric), and size (45 to
70 nm) of the virions, EAV was originally classified as a
togavirus (Porterfield et al., 1978). However recent
studies of EAV demonstrate a replication strategy
resembling that of corona- and toroviruses. Notably,
EAV transcribes a 3'-coterminal nested set of six
subgenomic RNAs with common leader sequences and
expresses the 3' half of the polymerase gene by ribosomal
frameshifting (de Vries et al., 1990; den Boon et al.,
1991).
The structural proteins of EAV consist of a phosphorylated nucleocapsid (N) protein with an M r of 14K,
encoded by ORF 7, and three envelope proteins
designated M, G L and G s, encoded by ORFs 6, 5 and 2,
respectively (Hyllseth, 1973; Zeegers et al., 1976; van
Berlo et al., 1986; den Boon et al., 1991 ; de Vries et al.,
1992). The M protein is an unglycosylated membrane
protein with an M~ of 16K, whereas GL and G s are Nglycosylated envelope proteins of 30K to 42K and 25K,
respectively. Although both glycoproteins contain a
single N-glycan and travel through the same intracellular
compartments as part of a virion, G L becomes heterogeneously glycosylated with N-acetyl-lactosamine (de
Vries et al., 1992) whereas G s acquires a normal complextype oligosaccharide side-chain (A. A. F. de Vries et al.,
unpublished).
To investigate further the antigenicity and function of
the structural proteins of EAV, we produced a panel of
EAV-specific monoclonal antibodies (MAbs). In this
study we describe their characterization and the finding
that the G L protein can induce the production of virusneutralizing antibodies.
MAbs were generated by intraperitoneal immunization of BALB/c mice with 100 lal (108 TCIDa0) of
sucrose gradient-purified EAV (Bucyrus strain; Doll et
0001-2121 © 1994SGM
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Table 1. Characteristics of EA V-specific M A b s
Protein
specificity
Clone
designation
N
llG10
16G10
17D3
4B2
17B7
17F3
17F5
19D7
5C7
7E5
GI,
UK~
Isotype
ELISA
titre
(log10)*
Neutralization
titret
IgGl
IgG1
IgG1
IgG1
IgG1
IgG1
IgA
IgG1
IgG1
IgGl
6
6
7
6
5
6
6
5
6
7
< 10
< 10
< 10
20
< 10
< 10
320
< 10
160
320
* Ascites fluid.
t Reciprocal of the highest dilution of MAb that completely
neutralized 100 TCIDs0 of EAV.
;~ UK, unknown. In CBAs these MAbs competed with all G w
specific probe MAbs.
al., 1957a) mixed with an equal volume of Freund's
complete adjuvant. The mice were boosted 2 weeks later
with 100 lal of virus in Freund's incomplete adjuvant
(FIA) and a final injection of 500 pl of virus without
adjuvant was given 3 days prior to fusion. Splenocytes
were fused with P3/NSI/1-Ag4-1 (NS-1) cells (K6hler &
Milstein, 1976) essentially as described previously
(Deregt et al., 1987). An indirect immunoperoxidase test
against EAV-infected and mock-infected cells was used
to screen hybridoma-spent media for EAV-specific
MAbs. Positive clones were subcloned by the limiting
dilution method and ascites fluids were produced by
intraperitoneal injection of 106 to 107 hybridoma ceils
into mice that had been primed with FIA. Isotypes were
determined by an enzyme immunoassay (Hyclone
Laboratories).
From a total of 20 hybridomas secreting EAV-specific
MAbs, the protein specificities of eight MAbs could be
determined by immunoprecipitation (see below). Three
were found to be N-specific and five MAbs recognized
the G L protein (Table 1). Two other MAbs, 5C7 and
7E5, were possibly Gwspecific MAbs as they competed
with all GL-specific probe MAbs in the competitive
binding assays (CBAs) described below. With the
exception of MAb 17F5, which was an IgA antibody,
these 10 MAbs were of the IgG1 isotype. ELISA titres of
the MAbs in ascites fluids ranged from 105 to 107 and all
MAbs reached the maximum absorbance over at least
two 10-fold dilutions.
We utilized three vaccinia virus recombinants (VVRs)
expressing the EAV proteins N, M or G L, designated
vAVE07, vAVE16 and vAVE25, respectively, to determine definitively the protein specificities of our MAbs.
To clone ORF 6 into the vaccinia virus insertion vector
pSCll (Chakrabarti et al., 1985; obtained from B.
Moss) the plasmid pAVI16 (de Vries et al., 1992) was
linearized with BamHI, blunt-ended and then digested
with PvuII. The desired 0-5 kb gel-purified fragment was
ligated into SmaI-digested pSCll to yield pAVE16.
ORF 5 from pAVI15 (de Vries et al., 1992) was cloned
into the BglII site of a pSC11 derivative (Krijnse Locker
et al., 1992) to generate pAVE25. Before its insertion, a
cryptic poxvirus early transcription termination signal
(Rohrmann et at., 1986) located in the middle of the gene
at nucleotide positions 11543 to 11549 (den Boon et al.,
1991) was removed by mutagenesis (Kunkel et al., 1991)
using the oligonucleotide 5' GGTAGTACTGATAGGAAGAACAGAACTAAAACG 3'. The VVRs vAVE16
and vAVE25 were then generated similarly as described
previously for vAVE07 (de Vries et al., 1992).
Western blots with lysates of EAV-infected rabbit
kidney (RK-13) cells revealed that the MAbs 17D3,
11G10 and 16G10 recognized a protein of approximately
14K (data not shown). This suggested that these MAbs
were directed against either the N or M protein. To
determine their specificity unequivocally we infected
BHK-21 cells with EAV, with vAVE07 expressing the
14K N protein, or with vAVE16 expressing the 16K M
protein. The cells were labelled in methionine-free
medium for 30 min at 8.5 h post-infection (p.i.) for EAVinfected cells or for 1 h at 5 h p.i. for VVR-infected cells
with 100 or 250 ~tCi/ml of Pro-mix L-[35S] in vitro cell
labelling mix ( > 1000 Ci/mmol; Amersham), respectively. Immunoprecipitations with 3 lal of anti-peptide
sera (de Vries et al., 1992; E.D. Chirnside et al.,
unpublished) or 1 tll of ascites fluids were followed by
incubation with 30 rtl of a 10% suspension of heatinactivated group G Streptococcus sp. cells (Omnisorb;
Calbiochem) to bind immune complexes. Lysates from
vAVE07- and vAVE16-infected cells were subjected to
immunoprecipitations with anti-peptide sera, the three
reactive MAbs, and a control MAb (Fig. 1a). The Nspecific anti-peptide serum (P0 specifically recognized a
14K protein in lysates of vAVE07-infected cells but not
in lysates of vAVE16-infected cells and this protein
comigrated with the N protein synthesized in EAVinfected cells. For the M-specific anti-peptide serum (P6)
the opposite was observed, i.e. a 16K protein comigrating
with the M protein synthesized in EAV-infected cells was
immunoprecipitated from a lysate of vAVE16-infected
cells but not from a lysate of vAVE07-infected cells.
Each of the MAbs 17D3, 11G10 and 16G10, specifically
precipitated the N protein expressed in vAVE07-infected
ceils, although with different efficiencies. As a control
antibody, the Gwspecific MAb 19D7 (see below) failed
to bind a clearly detectable amount of either the
expressed N or M protein. Thus these results confirmed
our conclusion that the MAbs 17D3, llG10 and 16G10
are directed against the N protein.
Immunoprecipitations performed on lysates of EAV-
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VVRs
(a)
M
V
6
7
6
7
6
7
6
7
6
7
6
7
°LI
--
30K
--
14K
G S --
M
m
N--
Ab
c
P6
P7
17D3
IlG10
(b)
16G10
19D7
VVRs
M
V
V
V
7
5
7
5
7
5
7
5
7
5
7
5
7
5
oLE
G s --
M
D
N--
Ab
c
P0
P5
17F5
4B2
17F3
19D7
17B7
17D3
Fig. 1. (a) Characterization of N-specific MAbs. BHK-21 cells were infected with VVR vAVE16 (lanes 6) or vAVE07 (lanes 7)
expressing the M and N proteins, respectively, and labelled with [3~S]methionine. Cell lysates were incubated with N-(pr) or M-specific
(P6) anti-peptide sera or with MAbs 17D3, i 1GI0, 16GI0 and 19D7 using 0.1% SDS in the immunoprecipitation (RIPA) buffer. (b)
Characterization of GL-specific MAbs. BHK-2I cells were infected with VVR rAVE07 (lanes 7) or rAVE25 (lanes 5) expressing the
N and G L protein, respectively, and radiolabelled as in (a). Cell lysates were incubated with GL-specific anti-peptide serum (Ps) or with
MAbs 17F5, 4B2, 17F3, 19D7, 17B7 and 17D3 using 0.25 % SDS in the RIPA buffer. The P5 antiserum and preserum (P0) were also
tested on lysates of EAV-infected (lanes V) cells. In this case, 1 mM-DTT was included in the RIPA buffer to dissociate disulphide-linked
protein complexes. (a and b) The immune complexes were resuspended in sample buffer containing 20 mM-DTT and heated for 5 rain
at 96 °C (a) or incubated for >~ 15 min at room temperature (b) before electrophoresis in an SDS-I 5 % polyacrylamide gel (Laemmli,
1970). The positions o f the EAV proteins and the size limits of the G L protein (bracket) are indicated (left). The position and size o f
marker proteins are also shown (right). For comparison, a cocktail of four anti-peptide sera directed against each of the structural
proteins (c) was applied to lysates of labelled mock- (lanes M) and EAV-infected (lanes V) cells.
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infected BHK-21 cells suggested that the MAbs 17F5,
4B2, 57F3, 19D7 and 17B7 were directed against the G L
protein (data not shown). To confirm these results,
BHK-21 cells were radiolabelled after infection with
vAVE07 or with vAVE25 and the cell lysates were
subjected to immunoprecipitation with a GL-specific
anti-peptide serum (Ps), the putative GL-specific MAbs,
and a N-specific MAb. To precipitate immune complexes
of MAb 17F5 the Omnisorb was first saturated with
swine anti-mouse IgA antibodies (Nordic). The G Lspecific anti-peptide serum and the corresponding preimmune serum (P0) were first tested on a lysate of
radiolabelled, EAV-infected BHK-25 cells (Fig. 1b). The
anti-peptide serum specifically recognized a 30K protein
and various higher M r species. The 30K band represents
the core-glycosylated form of the G L protein; the larger
species originate from maturation of its N-glycan to a
polylactosaminoglycan (de Vries et al., 1992). The nonspecific precipitation of the N protein from lysates of
EAV-infected cells by the anti-peptide serum and its
corresponding preserum is a common phenomenon that
has been documented by van Berlo et al. (1983). From
lysates of vAVE25-infected cells, the GL-specific antipeptide serum and the MAbs 17F5, 4B2, 57F3, 19D7 and
17B7 specifically immunoprecipitated two polypeptides
of 30K and 24K, respectively (Fig. 1b). The N-specific
MAb 17D3 failed to recognize these two proteins in the
same lysate but efficiently precipitated the N protein
expressed in vAVE07-infected cells. The major 30K
protein comigrated with the G L protein from EAVinfected BHK-21 cells. The minor 24K protein which
was coprecipitated represents a premature termination
product or a discrete degradation product of the G L
protein (A. A. F. de Vries & P. J. M. Rottier, unpublished). Whatever its precise nature, the coprecipitation
of this protein served as an additional identification
criterion of the MAbs as the same protein was coimmunoprecipitated with the GL-Specific anti-peptide
serum. The absence of higher M r forms of the G Lprotein
in vAVE25-infected cells is a consequence of the
inefficient maturation of GL in these cells (A. A. F. de
Vries & P. J. M. Rottier, unpublished). Since neither the
GL-Specific anti-peptide serum nor the MAbs 57F5, 4B2,
17F3, 19D7 and 17B7 bound a 30K protein when applied
to a lysate of vAVE07-infected cells, the results demonstrated that these MAbs are directed against the G L
protein.
To determine which MAbs could neutralize virus
infectivity, ascites fluids were heat-inactivated (56 °C for
30 min) and serial dilutions were incubated with 500
TCIDs0 of EAV without complement in a microneutralization assay. The endpoint was calculated by
determining the maximum dilution at which all c.p.e, was
neutralized. The MAbs 4B2 and 17F5, both reactive to
GL, were found to be neutralizing (Table 1). MAbs 5C7
and 7E5, for which protein specificities were not
definitively determined, also showed virus-neutralizing
activity. The three N-specific MAbs 17D3, 11G50 and
16G10, and the GL-Specific MAbs 17F3, 19D7 and 17B7,
were found to be non-neutralizing.
CBAs were used to determine the topography of
epitopes on the N and G L proteins. To prepare probe
antibodies, the N- and GL-specific MAbs were purified
on columns of immobilized Protein G (Pierce) or
concentrated by ammonium sulphate precipitation (MAb
17F5) and biotinylated using a kit (Amersham). CBAs
were then performed as described (Deregt & Babiuk,
1987) with several modifications. Briefly, purified EAV
(50 gg/ml in 0"5 M-sodium carbonate buffer pH 9"3) was
coated to microtitre plates (Nunc Maxisorp; Gibco)
overnight at 4 °C. The plates were then incubated with
competitor MAbs (as ascites fluids), followed by probe
MAbs, each for 90 rain. The probe MAbs were used at a
dilution that gave an absorbance of > 0"5 in the absence
of competitor antibody (Lussenhop et al., 1988). The
amount of binding of the probe MAbs was determined
with an enzyme immunoassay essentially as described
previously (Deregt et al., 1991). Competition was
calculated using the formula of Kimura-Kuroda & Yasui
(5983) and was regarded as strong when greater than
70 % and intermediate when between 40 and 70 %. For
each assay, a MAb to another EAV protein was used as
a control antibody.
In CBAs, the N-specific MAbs 17D3 and 51G10
showed strong reciprocal competition over three 10-fold
dilutions and defined one antigenic domain (CBAs not
shown). The N-specific MAb 16G10 was adversely
affected by biotinylation and was not used as a probe in
CBAs. However as a competitor, this MAb showed
strong competition with both MAb probes 17D3 and
55G50, suggesting that MAb 56G50 also recognized the
same antigenic domain.
The GL-specific MAbs 4B2, 17F3, 17F5 and 59D7, all
showed a strong level of reciprocal competition with
each other over three 10-fold dilutions and also defined a
single antigenic domain. In contrast, MAb 17B7 demonstrated strong competition against G L probes 4B2, 17F5
and 59D7 at the first 10-fold dilution only. In the
reciprocal experiments, 17F3 and 19D7 showed only
intermediate levels of competition with the 17B7 probe.
Hence the epitope recognized by MAb 17B7 probably
exists adjacent to the antigenic domain of the other four
GL-specific MAbs.
To determine whether the neutralizing MAbs 5C7 and
7E5, for which the protein specificity is unknown, could
compete with GL-specific MAb probes, these MAbs were
used in CBAs as competitor antibodies. MAbs 5C7 and
7E5 demonstrated intermediate or strong competition
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with all five GL-specific MAb probes over three 10-fold
dilutions including an intermediate (MAb 5C7) and
strong (MAb 7E5) competition with the neutralizing
MAb 17F5 probe. Thus these neutralizing MAbs may
also be GL-specific.
In this report we described the production and
characterization of MAbs to the N and GL proteins of
EAV. We determined definitively the protein specificities
of these MAbs with VVRs expressing EAV proteins,
including two new VVRs (for which production was
described in this paper). The results presented show that
the major envelope glycoprotein GL can induce the
production of neutralizing antibodies because two of five
MAbs with demonstrated specificity to the G L protein,
namely MAb 17F5 and MAb 4B2, were found to
neutralize virus infectivity in vitro. This implies that the
G L protein has a role in virus infectivity and may
function in attachment to cell receptors and/or in virus
penetration into the cytoplasm of target cells.
The CBAs for the N- and GL-specific MAbs suggest
that these MAbs bind to the same, overlapping, or
adjacent epitopes on their respective proteins. Although
the membrane topology of the G L protein has not been
formally established, its hydropathy profile (de Vries et
al., 1992) and the position of its N-glycosylation site (den
Boon et al., 1991) imply that amino acid residues 19 to
155 are contained within the ectodomain. Since a deletion
mutant which lacks amino acids 116 to 211 is still
recognized by MAb 4B2 (data not shown), we speculate
that the MAbs bind the hydrophilic part of the G L
protein comprising amino acid residues 19 to 115. Within
this region a number of antigenic sites are indeed
predicted using the algorithm of Jameson & Wolf (1988).
In a previous study, Chirnside et al. (1988) produced
four EAV-specific MAbs: two reactive with the N
protein, one probably directed against the G L protein,
and one MAb with undetermined specificity, all of which
were non-neutralizing. The MAbs that we raised against
EAV display specificities and activities very similar to
those previously produced to LDV virions. In the latter
case the majority of antibodies was also directed against
the heterogeneously glycosylated membrane protein
VP3, the putative homologue of the EAV G L protein. A
few antibodies specific for the N or VP1 protein were
obtained and no antibodies directed against the M or
VP2 protein could be isolated (Coutelier et al., 1986;
Harty et aL, 1987). It was shown that only MAbs
directed against VP3 were able to neutralize virus
infectivity (Coutelier & van Snick, 1988; Harry &
Plagemann, 1988).
While this manuscript was in review, Balasuriya et al.
(1993) reported that MAbs directed against an EAV 29K
glycoprotein could neutralize virus infectivity. Although
this protein was not fully identified, it is very likely that
2443
these MAbs also recognize the GL protein based on its
size and the results reported herein.
The authors wish to thank Ms S. Smithson and Mr S. Gilbert for
their excellent technical assistance and Ms E. Blake for assistance with
the preparation of the manuscript. We also thank G. Wertz for kindly
providing the wild-type vaccinia virus strain WR.
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(Received 27 September 1993 ; Accepted 14 March 1994)
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