Characterization of virus-specific and cross-reactive monoclonal antibodies to Herpesvirus simiae (B virus)

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1 J~urnal~ f`general`~vir l gy~!~9.96)~7~7.~ ~2~7. 8~-~-2793:~P.[!nted~.in Gre~a!Br!!ai~n... Characterization of virus-specific and cross-reactive monoclonal antibodies to Herpesvirus simiae (B virus) Earl Linwood Blewett, Darla Black and R. Eberle Department of Veterinary Parasitology, Microbiology and Public Health, College of Veterinary Medicine, Oklahoma State University, Stillwater, 0K , USA A panel of 13 monoclonal antibodies (HAbs) was produced that detect B virus (BV) proteins. Several of these MAbs were highly specific for BV, while the remainder cross-reacted in varying degrees with the other primate alphaherpesviruses. Utilizing western blot and radioimmunoprecipitation analysis, the HAbs were found to detect at least four distinct BY-infected cell antigens, several of which were composed of multiple polypeptides. One target antigen has been identified as the BV glycoprotein B (gb) homologue and was recognized by both virusspecific and cross-reactive HAbs. Although gb is an essential protein, none of the anti-gb HAbs neutralized infectious virus. ntroduction Herpesviruses parasitize a wide variety of vertebrate species including primates (McCarthy & Tosolini, 1975; Roizman & Baines, 1991). The pathogenic effects of these viruses range from no detectable disease to extreme lethality. Herpesvirus simiae (B virus; BV) spans this range, causing little mortality in its natural host, macaques, but high mortality in other species including humans (Sabin & Wright, 1934; Weigler, 1992). BV mortality in humans is generally limited to persons having direct contact with macaque monkeys, and the increasing utilization of non-human primates in drug testing and biomedical research has raised the incidence of zoonotic transmission of BV. The extreme severity of human BV infections necessitates rapid diagnosis and immediate initiation of chemotherapy to reduce mortality. Attempts are being made to establish specific pathogen free (SPF) macaque colonies as sources of BV-free animals (Ward & Hilliard, 1994; Zwartouw eta]., 1984). This goal has been difficult to achieve due to a lack of sensitive assays for detection of infected monkeys. BV, like most alphaherpesviruses, can establish latent infections in sensory nerve ganglia (Kalter & Heberling, 989). PCR procedures have been described that can specifically identify BV (Scinicariello et al., 1993 a, b; Slomka eta]., 1993). However, only animals actively shedding virus can be identified by PCR; latently infected monkeys not shedding virus are not detected. dentification of Author for correspondence: Earl Linwood Btewe~. Fax blewett@osuunx.ucc.okstate.edu infected monkeys by serological testing is also unreliable, as anti-bv titres may drop to undetectable levels despite the presence of latent virus in neurons. Thus, introduction of monkeys identified as virus-negative and antibody-negative into BV-free colonies has resulted in subsequent outbreaks of BV in the colony (Zwartouw et al., 1984; Zwartouw & Boulter, 1984). Four other alphaherpesviruses which infect primates [herpes simplex virus (HSV) types 1 and 2 in humans, SA8 in African green monkeys and HVP2 in baboons] are closely related to BV. All five viruses share a number of conserved genes and antigenic determinants (Cropper et al., 1992; Eberle & Hilliard, 1995; Ueda et al., 968). Antibodies induced against any of these viruses will cross-react with BV proteins, making specific detection and/or identification of BV antibodies in sera very difficult (Katz et a., 1986; Palmer, 1987). This poses a particular problem in specific diagnosis of BV infection in humans where pre-existing anti-hsv antibody is common. The ability to develop extremely sensitive and specific assays using monoc[onal antibodies (MAbs) represents one potential method of overcoming some of these difficulties. n this report we describe the production and characterization of MAbs to BV. Hethods Virus strains. BV strain E2490, SA8 strain B264, HSV- strain KOS and HSV-2 strain 86 were used in this study (Hilliard et al., 989). HVP2 strain X313 was originally isolated from a baboon at the Southwest Foundation for Biomedical Research, San Antonio, Texas (Eberle et at., 995; Levin e~ al., 988). H. saimiri (HSV-) strain MV-5-4, H. ateles SGM!78~

2 _ 2ff Table 1. Characteristics of anti-bv MAbs Western Group MAb ELSA blot RiP VN BV antigens 7C2 * NT 3,5--40 kda Non-glycosylated 8C2 -}- -~- --~ NT NT 20C8 -[- q- -}- -~- q- NT NT 9G kda Non-glycosylated 3E i20 kda Glycosylated 1F glycoprotein B 9B6 -- _ V 2B10 _} }_ NT 130 kda Non-glycosylated 3F2 q- Jr- -{- Jr- NT 4D3 -{- q- q- Jr- NT NT 4D6 -~- q- -}- Jr- NT NT 5Cli q- -{- Jr- -{'-"" "~ NT 12C2 -}'- NT Key:, weak positive;, moderate positive;, strong positive;, very strong positive; -, negative; NT, not tested. (HVA-1) strain Lennette, and pseudorabies virus (PRV) strain Aujeszky were all obtained from the ATCC (Rockville, Maryland). Cell lines. HEp-2, SP-2/0-Ag4 and Vero cells were obtained from the ATCC. HEp-2 and Vero cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 0'1 mg/ml streptomycin, 1"5 % NaHCO 3, 10% fetal bovine serum (FBS) and 2 mm-l-glutamine. SP-2 cells were cultured similarly, except with 20% FBS and further supplementation of pm-2-mercaptoethanol and 10 mm-hepes. Supernatants from healthy SP-2 cultures were centrifuged to remove cells and passed through a 0"22,m filter to prepare conditioned media. Hybridoma cells were maintained in a base DMEM containing 10 mm-2-mercaptoethanol, 10 mm-hepes, 200 U/m penicillin, 0"2 mg/ml streptomycin, 2 mm-l-glutamine, 20% FBS and 20% SP-2 conditioned medium. For hybridoma selection, 0 lam-hypoxanthine, 2 ~M-aminopterin and 100 ~M-thymidine (Sigma) were added to base medium to produce HAT and HT (no aminopterin) medium. Production of MAbs. BALB/c mice were immunized by interperitoneal injection of 0'4 ml of inactivated, denatured (0"5 % SDS) BVinfected Vero ceil antigen (4 106 cells) mixed 1: with complete Freund's adjuvant. Mice were re-immunized 8 weeks later with 0"3 m denatured (0"05 % SDS) BV-infected Vero cel antigen (3 x 106 cells) in incomplete Freund's adjuvant. Three days prior to sacrifice, mice were inoculated intravenously with 10 ~ p.f.u, of infectious BV. The spleen cell/sp-2 fusion and selection of hybridomas were performed using standard methods (Harlow & Lane, 1988). Essentially, spleen cells were mixed 4:1 with SP-2 cells, fusion was induced with 50% PEG-1450 (Sigma) and the cells were plated in 96-well trays at 106 cells/well. After 24 h HAT medium was added. After 10 days of selection, hybridomas were transferred to HT medium and culture supernatants were screened by ELSA to determine positive clones. Putative hybridomas were then cloned by limiting dilution, rescreened by ELSA and positive clones were selected for further characterization. Hybridoma supernatants were screened by ELSA using BV-infected and uninfected HEp-2 cell antigens. Bound antibody was detected using an anti-mouse gg Vectastain kit (Vector). For production of ascites fluid, 6-week-old female BALB/c mice were primed with 0'5 ml of pristane (Sigma) and inoculated intraperitoneally 5-7 days later with 0"5 x x 106 hybridoma cells. After 7-10 days, mice were sacrificed and the fluid was drained, clarified, and stored at -70 C. MAb immunoglobulin class and subclass were identified using a Sigma mmunotype SO kit. mmunoassays. Polyclonal antisera against purified proteins of various herpesviruses were developed in this laboratory as previously described (Eberle et al., 1989, 1995). Hyperimmune antisera to BV were prepared in New Zealand rabbits by repeated monthly iniections of 10 v BV-infected Vero cells mixed : 1 with Freund's adjuvant. Rabbits were bled once a month, beginning 2 weeks after the third immunization. Preparation of virus antigens and procedures for ELSA and western blot (WB) were as described in detail elsewhere (Hilliard et al., 1989; Katz et al., 1986). For radioimmunoprecipitation assays (RP) assays, HEp-2 cells were labelled with 20,Ci/ml 35S-TransLabe], 50 ~tci/ml [14C]glucosamine or 50 ~Ci/ml [ah]glucosamine (CN) from 4-24 h postinfection (p.i.). At 24 h p.i. cells were harvested by scraping into the medium, pelleted by centrifugation, resuspended in H~O at 3 x 06 cells/ml and frozen at - 70 C. Ascites fluid (25 gl) was mixed with antigen overnight at 4 C, then 50 ~tl reconstituted gsorb (The Enzyme Center) charged with rabbit anti-mouse gg (Sigma) was added for 2 h at 4 C. Samples were washed and electrophoresed on an SDS-PAGE gel as previously described (Blewett & Misra, 1991). For virus neutralization (VN) assays 250 p.f.u, of BV in 50 ~1 DMEM was mixed with 25 ~tl of guinea-pig serum (as a complement source) and 25 gl of MAb ascites fluid diluted 1:100, 1:1000 or 1:10000 and incubated at 37 C for 1 h. The virus/mab mixtures were then diluted 1:10 in DMEM, added to Vero cell monolayers for h at 37 C, and overlaid with DMEM containing 1% methyl cellulose until plaques developed. Results The initial screening of hybridoma supernatants produced more than 50 candidate cell lines. Cloning by limiting dilution and subsequent re-screening identified the most reactive '.78~

3 MAb group 1 2 kda V ~ VP5 (capsid) 1~ gb V gd ~: ~ ~ ~:i:- 24 Fig. 1. B virus-infected cell proteins separated by SDS~PAGE. Polypeptides corresponding to target antigens of MAbs are identified on the left of the figure while BV protein counterparts of HSV-1 proteins are identified on the right. Lane 1 contains proteins labelled with [14C]amino acids; lane 2 shows proteins labelled with [14C]glucosamine, cultures which were preliminarily characterized and subsequently used to produce ascites fluid. Table summarizes the characteristics of the MAbs produced by the final 13 stable anti-bv hybridomas produced. All of the MAbs belong to the gg1 subclass, not surprisingly since anti-mouse gg secondary antibody was used to screen for positive hybridomas. A surprisingly large number of the MAbs (10 of 13) reacted with denatured proteins in WB. This, and the fact that the three WB-negative MAbs immunoprecipitated radiolabelled proteins, simplified the identification of the target antigens of all anti-bv MAbs. Fig. 1 shows an SDS-PAGE profile of polypeptides and glycoproteins present in BV-infected HEp-2 cells. The MAb target antigens and other known reference polypeptides are identified. As shown in Table i, the MAbs were divided into four groups based on their reactivity with BV antigens in WB and RP assays. Fig. 2 shows the reactivity of one MAb from each group with BV-infected cell antigen in WB. The polypeptide specificities of the three MAbs that did not react in WB were Fig, 2. Reactivity of MAbs with BV proteins in western blot, Lane 1, MAb 8C2; lane 2, MAb 9G10; lane 3, MAb 19B6; lane 4 NAb 5C11 ;lane 5, normal mouse serum, The arrows indicate the specific reactive band(s) for each MAb. MAbs were used at a dilution of 1:4000. tested by immunoprecipitation (data not shown). Group MAbs reacted in WB with three or more polypeptides ranging from kda in size. RP assays detected only the largest of these polypeptides. The one Group MAb reacted with a single, distinct polypeptide of 46 kda in WB and RP assays. MAbs of Group reacted with two distinct polypeptide bands of kda in both WB and RP assays. Group V MAbs reacted strongly with three or more large polypeptides of approximately 30 kda. To determine whether any of the four BV antigens were glycoproteins, a WB was performed using [14C]glucosaminelabelled BV-infected cell antigens. Polypeptides detected by MAbs from Group, V and possibly, co-migrated with glucosamine-labelled proteins. To further determine which BV antigens were glycoproteins, [3H]glucosamine-labelled and 35S-labelled BV-infected cell extracts were immunoprecipitated and electrophoresed on SDS-PAGE gels (Fig, 3). All MAbs '.78!

4 iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iiii! i!iiiiiii!iiiiiiiiiiiii (a) (b) kda kda N i m 66 m all Group V MAbs reacted with SA8 and HVP2 as well as with BV antigen. n addition, two of seven Group V MAbs also reacted with HSV-1 and HSV-2, albeit at a much lower level. This high level of cross-reactivity was also observed for both Group V MAbs tested by WB (Fig. 4 b). n contrast, the only MAb of Group (9G10) was very specific for BV in both ELSA and WB assays. All Group MAbs reacted with two polypeptides of approximately kda. As described above, these two polypeptides represent the pgb/gb homologues of BV. Two of the anti-gb MAbs (11F6 and 19B6) were very strong and BV-specific in ELSA assays while the third MAb (3E8) crossreacted with HVP2 and SAS. Of these three MAbs, only 19B6 recognized proteins in WB assays, where it cross-reacted with a single HSV-2 protein. Based on the relative size of this protein it may be HSV-2 gb (Hilliard et al., 1989). Multiple polypeptides of approximately kda from BV-infected cells were recognized by Group MAbs. When examined in WB assays, the MAbs of this group cross-reacted with HVP2 and SA8 polypeptides of kda. The relative size and kinetics of the immune response to these BV proteins resembles that of the HSV-1 p40 homologues (Eberle & Mou, 983 ; E. L. Blewett, D. Black and R. Eberle, unpublished observations). Fig. 3. Radioimmunoprecipitation of (a) [3H]glucosamine-labelled and (b) [35S]methionine-labelled BV proteins. Lane, rabbit antiserum R012 raised against BV-infected cells; lane 2, MAb 7C2; lane 3, MAb 9G O; lane 4, V]Ab 5C ; lane 5, MAb 19B6; lane 6, rabbit antiserum R26 directed against purified HSV- gb. precipitated 35S-labelled proteins, but only Group MAbs precipitated polypeptides labelled with [~H]glucosamine. Thus, only MAbs of group were directed against a glycoprotein. To further identify this glycoprotein, polyclonal antisera against purified HSV-, SA8 and HVP2 glycoproteins were used in WB assays with [14C]glucosamine-labelled BV antigen. Antisera against the gb glycoproteins of SAS, HVP2 and HSV- 1 cross-reacted with the BV gb in WB. When anti-hsv-1 gb sera and MAb 19B6 were used in RP, identical polypeptides were immunoprecipitated (Fig. 3b). These results clearly identified the target of the Group MAbs as the BV gb homologue. However, none of the three anti-gb MAbs was able to neutralize infectious BV (Table 1). A major aim of this project was development of BV-specific MAbs to aid in identifying BV-infected primates. Since extensive antigenic cross-reactivity exists among BV, SA8, HVP2, and to a lesser extent HSV-1 and HSV-2, the reactivity of the BV MAbs with these other viruses was examined. As shown in Fig. 4 (a) and summarized in Table 2, Group V MAbs directed against the largest antigen, three polypeptides of approximately 130 kda, were highly cross-reactive. n ELSA, Discussion n this investigation a number of potentially useful MAbs were developed, many of which reacted in WB. The surprisingly large number of WB-positive MAbs that were produced may be reflective of the immunization protocol employed. Due to the biohazardous nature of BV and its lethality in mice, SDS-denatured antigen was used for all but the last immunization. The epitopes exposed in this denatured antigen may well be similarly exposed in SDS-denatured BV antigen bound to nitrocellulose in the WB procedure. The degree of MAb cross-reactivity with other primate herpesvirus proteins reflected the phylogenetic relationships among these viruses (McGeoch et a., 1995; Eberle eta]., 1995). MAbs cross-reacted most strongly with HVP2 of baboons, followed by SA8 of green monkeys, and least with HSV-2 and HSV- of humans. When used at low dilutions in ELSA assays, there may also have been slight reactivity of several MAbs with the platyrrhine monkey viruses, HVA-1 and HVS-. No reactivity was seen with the more distantly related PRV, a varicellovirus of swine. Relationships of viral DNA and protein sequence comparisons support this hierarchy of homology (Eberle & Black, 1993; Eberle & Hilliard, 1995; McGeoch et al., 1995). A large panel of MAbs directed against BV have been described by Cropper et al. (1992). These MAbs were tested against a wide variety of strains of BV, SAS, HSV-1 and HSV- 2. Although reactivity with specific BV polypeptides was not performed, their MAbs were similar to those we produced in 179(

5 (a) Uninfected HVS-1 HVA-1 HVP-2 i BV SA8 HSV-2 HSV-1 l.jmmmm =mmmmmmmm 13mmmmmmmm ~'~,~' ~,~.~, B : l J, - ~ UninfectedHvA_lHvP_2HVS_l BV SA8 HSV-2 HSV-1 J, i 0.2 0, (b) 3F G Fig. 4. Cross-reaction of Group and V MAbs with other herpesviruses by (a) ELSA and (b) western blot. MAbs were used at a dilution of 1:10000 in ELSA and 1:4000 in WB. Lane 1, uninfected cells; lane 2, HSV-1 ; lane 3, HSV-2; lane 4, SA8; lane 5, HVP2; lane 6, BV; lane 7, HVS-1 ; lane 8, HVA-1 ; lane 9, PRV. that they identified three patterns of virus reactivity: BVspecific, SA8 cross-reactive, and cross-reactive with SA8, HSV- and HSV-2. Several of our MAbs directed against BV gb and the kda proteins reacted with BV and HVP2 of baboons, while several of the MAbs against the large 130 kda proteins reacted with HVP2, SAS, HSV-1 and HSV-2 as well. MAbs directed against the BV gb homologue were similar to those directed against other herpesvirus gbs. n HSV-1, MAbs are directed against both continuous and discontinuous epitopes and can be specific to the HSV-1 gb or cross-reactive with the gb homologue of other herpesviruses (Balachandran eta]., 1987; Pereira et a]., 1989). Anti-gB MAbs are not necessarily neutralizing, even though gb is an essential viral protein (Little et al., 1981; Sarmiento et al., 1979). We found that BV-infected monkeys, as well as rabbits and mice immunized with BV, produced very strong antibody responses against the gb polypeptides (unpublished observations) as is the case for HSV-1 and bovine herpesvirus 1 (Bernstein et al, 1990; Eberle & Mou, 1983; Glorioso eta]., 1984; Marshall et al., 1988; van Drunen Little-van den Hurk & Babiuk, 1985). The three anti-gb MAbs in group show much promise, both for diagnostic use and as reagents for large-scale purification of BV gb. The anti-gb MAb 19B6 was rather exceptional. t was highly specific for BV by ELSA but cross-reacted very strongly with a 120 kda protein of HSV-2 in WB. Both laboratory _~7~

6 Table 2. Summary of MAb cross-reactivity with other herpesviruses Group* V Test 7C2 8C2 20C8 9G10 3E8 11F6 19B6 2B10 3F2 4D3 4D6 5Cll 12C2 ELSA HSV HSV SA BV HVP HVA HVS Western blot HSV HSV SA BV HVP HVA HVS PRV..... * Key:, positive reaction; --, no reaction; blank indicates not tested. strains of HSV-2 as well as three clinical isolates of HSV-2 were tested and gave identical results (data not shown). The crossreactive protein was the same apparent size as HSV-2 gb, but MAb 19B6 did not cross-react with HSV-2 in ELSA assays, even when the HSV-2 antigen was SDS-denatured. t is possible that the 19B6-reactive epitope was not exposed in the native state or under mildly denatured conditions but was only exposed when strongly denatured by the further addition of urea, 2-mercaptoethanol and heating at 100 C. Apparently BV gb shares an epitope with HSV-2 that is not present in any of the other viruses tested. The target antigens we identified were consistent with data on BV proteins synthesized in infected Vero cells as described by Hilliard eta]. (1987). They identified a 122 kda viral glycoprotein (CPll) which corresponds to the BV gb homologue we have described. Although CPll is not a clear doublet as seen here, differences in gb polypeptide synthesis in Vero and HEp-2 cells could account for this (Pereira et a., 1982). The protein identified as CP8 (130 kda, non-glycosylated) appears to correspond to one of the large proteins which the Group V MAbs recognize. Similarly, CP32 may be the antigen which the Group MAb reacts with. Finally, the antigen our Group MAbs reacts with appears to be in the CP36-40 range, and may correspond to the p40 proteins (UL26.5 gene product) of HSV-. This work was supported by PHS grant RR References Balachandran, N., Oba, D. E. & Hurt-Fletcher, L. M. (1987). Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus and cytomegalovirus. Journal of Virology 61, Bernstein, D.., Frenkel, L. H., Bryson, Y.. & Myers, M. G. (1990). Antibody response to herpes simplex virus glycoproteins gb and gd. Journal of Medical Virology 30, Blewett, E. L. & Misra, V. (1991). Cleavage of the bovine herpesvirus glycoprotein B is not essential for its function. Journal of General Virology 72, Cropper, L. M., Lees, D. N., Patt, R., Sharp,. R. & Brown, D. (1992). Monoclonal antibodies for the identification of Herpesvirus simiae (B virus). Archives of Virology 123, Eberle, R. & Black, D. (1993). Sequence analysis of the gb gene homologs of two platyrrhine monkey ~-herpesviruses. Archives of Virology 129, Eberle, R. & Mou, S.-W. (1983). Relative titers of antibodies to individual polypeptide antigens of herpes simplex virus type 1 in human sera. Journal of nfectious Diseases 48, Eberle, R. & Hilliard, J. K. (1995). The simian herpesviruses. nfectious Agents and Disease 4, Eberle, R., Black, D. & Hilliard, J. K. (1989). Relatedness of glycoproteins expressed on the surface of simian herpesvirus virions and infected cells to specific HSV glycoproteins. Archives of Virology 109, Eberle, R., Black, D., Upper, S. L. & Hilliard,. K. (1995). Herpesvirus papio 2, an SA8-like ~-herpesvirus of baboons. Archives of Virology 140, Glorioso, J., Schroder, C. H., Kumel, G., Szczesiul, M. & Levine, M. ~.79;

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