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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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 02:15:57 2440 Short communication 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- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 02:15:57 Short communication 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 02:15:57 2441 2442 Short communication 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 02:15:57 Short communication 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. 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The structural proteins of equine arteritis virus. Virology 73, 200-205. (Received 27 September 1993 ; Accepted 14 March 1994) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 02:15:57