virology 206, 242--253 (1995)fine mapping of bovine herpesvirus-1 (bhv-1) glycoprotein d (gd)...

VIROLOGY 206, 242--253 (1995)

Fine M a p p i n g of Bov ine H e r p e s v i r u s - 1 (BHV-1) G l y c o p r o t e i n D (gD) N e u t r a l i z i n g Ep i t opes

by T y p e - S p e c i f i c M o n o c l o n a l A n t i b o d i e s and S e q u e n c e C o m p a r i s o n w i t h BHV-5 gD

O. Y. ABDELMAGID,* H. C. MINOCHA,* J. K. COLLINS,t AND S. I. CHOWDHURY *'~

*Department of Pathology and Microbiology, Coflege of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506; and tDepartment of Microbiology, Colorado State University, Fort Coffins, Colorado 80523

Received July 19, 1994; accepted September 30, 1994

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusion proteins in Escherichia coil to map linear neutralizing epitopes defined by BHV-l-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52-126 and 165-216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R64 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92-106 (3402 epitope) and amino acids 202-213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1. © 1995 Academic Press, Inc.

INTRODUCTION

Bovine herpesvirus-1 (BHV-1) causes conjunctivitis, respiratory and genital infections, and occasionally abor- tion and enteritis in cattle (Gibbs and Rweyemamu, 1977; Ludwig, 1983). BHV-1 strains fall into two different groups based on their DNA restriction profiles and pathogenic properties, the respiratory/abortogenic group (IBR, infectious bovine rhinotracheitis virus) and the nonaborto- genic genital group (IPV, infectious pustular vulvovagini- tis). The neurovirulent bovine herpesvirus (previously classified as BHV-1.3) has been reclassified recently as BHV-5 (Roizman et aL, 1992) and is known to cause epi- demics of a uniformly fatal encephalitis in calves (Carillo etaL, 1983a,b; French, 1962; Eugster etaL, 1974; Bartha et aL, 1969). The BHV-1 and BHV-5 virus strains share 85% DNA homology, based on cross-hybridization of entire genomic DNA. However, the DNA restriction maps of BHV-1 and BHV-5 are distinct (Engles et aL, 1987; Bulach and Studdert, 1990), suggesting that differences exist in the nucleotide sequences of these virus strains throughout the genome. Previous reports (Metzler et al., 1985, 1986; Friedli and Metzler, 1987; Collins eta/., 1993) also have demonstrated that genomic differences between these two viruses are reflected in the antigenic properties including the major viral glycoproteins. The

~To whom correspondence and reprint requests should be ad- dressed. Fax; (913) 532-4039.

envelopes of alpha herpesviruses, including herpes simplex viruses (HSV-1 and HSV-2), pseudorabies virus (PRV), and BHV-1, contain a number of functionally and structurally homologous glycoproteins. Herpesvirus glycoproteins are important for the interaction of the virus with its host. The glycoproteins mediate infection of target cells and, thus, influence cell and tissue tropism and are also major antigens recognized by the infected host's immune system (Little etaL, 1981; Highlander etaL, 1987; Johnson and Ligas, 1988; Glorioso eta/., 1984; Lupton and Reed, 1980). Three major glycoproteins, gB (gl), gO (gill), and gD (glV), have been identified on the virus envelope and the plasma membranes of BHV-l-infected cells (Marshall eta/. , 1986; van Drunen Little-van den Hurk et al., 1984; van Drunen Little-van den Hurk and Babiuk, 1986) and are recognized by sera from BHV-1- infected cattle (Collins et al., t984; van Drunen Little-van den Hurk et aL, 1986). Immunization with affinity-purified gB, gO, and gD individually or in combination induces protective immune response against BHV-1 infection (Babiuk eta/., 1987; van Drunen Little-van den Hurk et al., 1990a). However, gB and gD are the only glycoproteins that can induce immunity resulting in significant reductions in viral replication and shedding (Israel et al., 1992; van Drunen Little-van den Hurk et al., 1993). Glycoprotein D also induces a stronger and more consis- tent cellular immune response to BHV-1 than gB and gO (Hutchings et al., 1990). Monoclonal antibodies (MAbs) to gD show the highest complement-independent virus-

0042-6822/95 $6.00 Copyright © 1995 by Academic Press. inc. All rights of reproduction in any form reserved.

242

EPITOPE MAPPING OF BHV-1 gD AND BHV-5 gD SEQUENCE 243

neutralizing activity and inhibit virus adsorption and penetration (Hughes et al., 1988; van Drunen Little-van den Murk eta/., 1990b; Dubuisson etaL, 1992). Herpes simplex virus glycoprotein gD is also involved in the patho- genesis of neurological disease (Izumi and Stevens, 1990). These functional attributes of gD make it one of the most important proteins involved in immunity to BHV-1.

The BHV-1 gD ORF codes for 417 amino acid (aa) residues and a protein with a molecular weight of 75K (Tikoo et al., 1990). Competitive binding assays using gD-specific MAbs have been used to map BHV-1 gD epitopes. Five epitopes, three interrelated and two independent, were reported as targets of neutralizing antibodies (Marshall et aL, 1988). Four antigenic domains have also been described by other investigators (Hughes eta/,, 1988; van Drunen Little-van den Hurk etaL, 1990b). A recent study using truncated and deleted gD-recombinant vaccinia virus proteins located these antigenic determinants on regions of the gD ORF (Tikoo et aL, 1993).

In the present study, the coding sequences of two BHV-1 gD-specific epitopes recognized by neutralizing MAbs were mapped precisely by expressing overlapping gene fragments as trpE-gD fusion protein and by synthetic peptide mapping. In addition, the BHV-5 gD sequence was determined and analyzed by comparison with the BHV-1 gD sequence. BHV-1 gD-specific MAbs were reacted first with trpE-gD fusion proteins to localize the sites to regions in the glycoprotein. Precise sites then were predicted by analyzing differences, in the predicted amino acid sequences and antigenicity and hydropathicity plots, of BHV-1 and BHV-5 gD regions that likely corresponded to epitopes (BHV-1) and lack of epitopes (BHV-5). Finally, synthetic peptides were prepared and tested for epitope specificity.

MATERIALS AND METHODS

Virus strains and cell lines

The Oooper (Colorado-I) strain of BHV-1 was obtained from the American Type Culture Collection (ATCC; Rock- ville, MD). The TX-89 strain of BHV-5 (isolated from a case 0fviral encephalitis (d'Offay eta/., 1993) was provided by Dr. d'Offay from Oklahoma State University. The two viruses were propagated and titrated in Madin-Darby bovine kidney (MDBK) cells grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

M0noclonal antibodies

MAbs 3402 and R54 are both gD-specific (Marshall et aL, 1986; Chowdhury, unpublished data); however, MAb R54 was previously characterized to be gC-specific (Tre- panieretaL, 1986; Abdelmagid etaL, 1992) and has now been reevaluated in our laboratory to have gD specificity.

MAb 3402 was provided by Dr. G. Letchworth from the University of Wisconsin at Madison.

SDS-PAGE and Western blot analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of viral or bacterially expressed fusion proteins was performed under reducing condi- tions as described earlier (Laemmili, 1970; Yei et aL, 1990). Following electrophoresis, proteins were trans- ferred electrophoretically to nitrocellulose (NO) membranes. NC membranes were blocked in 3% nonfat milk in TBS (20 mM Tris-HCI, pH 7.5, 0.5 M NaCI) or PBS and then reacted overnight with MAbs, rabbit, or bovine antisera diluted in TBS or PBS containing 1% gelatin. After being washed with TBS or PBS containing 0.05% Tween 20, blots were reacted with biotinylated second- ary antibodies for 1 to 2 hr at room temperature (RT), followed by incubation for 30 min at RT with avidin- peroxidase complex (VectaStain; Vector Laboratories). Bound antibody was visualized by development in horse- radish peroxide substrate solution according to the manufacturer's protocol (Pierce Biochemical).

Construction of recombinant pATH expression plasmids and preparation of fusion proteins

The 1.3-kb Mael/Mael fragment containing the whole BHV-1 gD ORF (Tikoo et al., 1990) was blunt ended (Klenow) and cloned into the Smal site of a pATH 23 plasmid (Koerner et aL, 1991) to yield the inframe trpE- gD fusion gene (plasmid clone pgD1-417). The recombinant plasmid was used to transform Escherichia coli Re- print requests cells, and insert-containing transformants were selected by colony hybridization using 32p-nick- translated-specific probes, A clone containing the gD coding sequences in the correct orientation (verified by sequencing) was selected and tested for gD expression (described below). All subsequent clonings spanning portions of the gD OBF were generated either directly or indirectly from this clone.

Construction ofpgDl-216. Plasmid pgD1-417 was digested with Xbal and Sinai. The 746-bp fragment was gel purified and inserted into Xbal/Smal-digested pATH23 plasmid.

Construction of pgDl-164. Plasmid pgD1-216 was digested with Narl and treated with the Klenow enzyme. The DNA was redigested with Smal and the larger fragment was purified and religated.

Construction of pgD1-49. Plasmid pgd1-164 was digested with Apal and Sacl and blunt end repaired with T4 DNA polymerase and the large fragment was purified and religated.

Construction ofpgD52-164. Plasmid pgD1-164 was digested with Apal and blunt end repaired with T4 DNA polymerase. The DNA was redigested with Sacl and the

244 ABDELMAGID ET AL.

smaller fragment (350 bp) was purified and inserted into Smal/Sacl-digested pATH23 plasmid.

Construction of pgD129-216. Plasmid pgD1-216 was digested with Banll and blunt end repaired with T4 DNA polymerase. The 280-bp fragment was purified and inserted into Smal-digested pATH 23 plasmid (in right orientation).

Construction of pgD117-164. Plasmid pgD1-164 was digested with Sphl, blunt end repaired with T4 polymerase, and then digested with Xmalll and treated with the Klenow enzyme, The large fragment was purified and religated.

Construction of pgD165-216. Plasmid pgD1-216 was digested with Sphl and blunt end repaired with T4 polymerase. The DNA was redigested with Narl and treated with the Klenow enzyme, and the large fragment was purified and religated.

Construction of pgD52-126. Plasmid pgD52-164 was digested with Banll and blunt end repaired with T4 DNA polymerase. The large fragment was purified and religated.

The synthesis of trpE-gD fusion proteins was induced in modified M9 medium by tryptophan starvation and indoleacrylic acid (IAA) as described (Koerner et aL, 1991). Briefly, modified M9 medium containing 0.5% ca- samino acids, 50 #g/ml ampicillin, and 20 #g/ml tryptophan was inoculated with transformed E. coil RRI cells and grown to midlogarithmic phase. The fusion protein was induced in a 1=10 dilution of this culture in modified M9 medium without tryptophan and by addition of IAA (10 #g/ml). After an additional 4 hr of growth, cell lysates of E. coil producing either trpE or the trpE-gD recombinant proteins were prepared by centrifugation and ho- mogenization of the cell in lysis buffer (0.05 M Tr is- HCI, pH 7.5, 3 mg/ml lysozyme, 0.7% Nonidet P-40) and analyzed in a 10% SDS-PAGE gel.

Preparation of trpE-gD fusion proteins for immunization

A trpE-gD fusion protein antigen was prepared by elution from an NO membrane (Szewczyk and Summers, 1988). Briefly, after preparative electrophoresis and transfer of the protein to the NO membrane, the membrane was washed in PBS containing 0.1% Tween 20 to remove SDS and then air-dried. The portion containing the trpE-gD fusion protein was sliced, dissolved in di- methyl sulfoxide (DMSO), and precipitated by adding an equal volume of 0.05 M sodium carbonate buffer, pH 9.6, and centrifuging. The pellet was resuspended in PBS, emulsified in Freund's complete adjuvant, and used for immunization.

Mapping, cloning, and sequencing of the BHV-5 gD gene

The location of the BHV-5 gD gene on the virus ge- • nome and pertinent restriction sites for subcloning and

the sequencing strategy are illustrated in Fig. 1. A pUC- based plasmid library containing the BamHI genomic fragments of the BHV-5 genomic DNA was developed. To verify the general location of the BHV-5 gD gene, the 1.3-kb MaeI-Mael subfragment subcloned from the Hindlll K fragment (Mayfield et aL, 1983) carrying the gD of BHV-1 was labeled with 32p by nick-translation (Sambrook eta/., 1989) and used as a probe for hybridization (Chowdhury et al., 1990) with the 14.6-kb BamHI C fragment of BHV-5 (Engles et al., 1987; Bulach and Stud- dert, 1990). To precisely map the location of the BHV-5 gD gene, the BamHl-C fragment was digested further with restriction endonucleases, separated on agarose gel, and analyzed by hybridization with the same probe. Based on the hybridization results, a 1.62-kb Mael fragment (MU 0.886-0.899) was identified and cloned. A restriction map of the cloned 1.62-kb fragment was con- structed, and .subclones spanning the entire fragment were generated. DNA was sequenced by using both the chemical method (Maxam and Gilbert, 1977) and the di- deoxy chain termination method (Sanger et al., 1977). Both strands of each fragment were sequenced by Sang- er's method and verified by Maxam and Gilbert's method.

Sequence analysis

DNA sequence data were assembled and analyzed using the Seqaid II sequence analysis software (D. D. Rhodes and D. J. Roufa, Center for Basic Cancer Re- search, Kansas State University (KSU), Manhattan). The predicted amino acid sequences of BHV-5 (this study) and BHV-1 gD (Tikoo et al., 1990) were aligned by using Multalin software (Corpet, 1988). Hydropathicity analyses were performed (Kyte and Doolittle, 1982) using a g- amino-acid Window, and the antigenicity profiles of predicted amino acid sequences were analyzed using the Seqaid II software.

Peptide synthesis

Peptides were synthesized at the Biotechnology Cen- ter of KSU by FMOC chemistry (Barany and Merrifield, 1980) on an ABI Model 431A automated peptide synthe- sizer (Applied Biosystem, Inc., Foster City, CA) according to the manufacturer's protocols. The peptides correspond to the BHV-1 gD protein's predicted aa residues 92-106, 202-225,202-213, and 2t3-225. The peptides were purified by high performance liquid chromatography. To fa- cilitate conjugation to keyhole limpet hemocyanin (KLH), an additional irrelevant cysteine was added at the C- terminus of peptide 92-106. Peptides 92-106 and 202- 213 were conjugated to KLH using succinimidyl 4-(N- maleimidomethyl-cyclohexane)-l-carboxylate as a cross linker.

Immunization of rabbits

New Zealand white rabbits were injected intradermalty at multiple sites along the back and subcutaneously at


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FIG. 1. The BamHI restriction site map of BHV-5 DNA and the cloning and sequencing strategies of BHV-5 gD gene. The genomic organization of BHV-5 (TX89) strain, depicted on top, consists of unique long (UL) and short (Us) regions and two repeat regions (IR and TR). The BamHI restriction endonuclease map was described originally for the (N569) strain of BHV-5 by Engles et al. (1987). The gD gene of BHV-5 was mapped to the BamHl-C (14.6 kb) genomic fragment between MU 0.827 and 0.944 and then to the Mael fragment (1620 bp) between MU 0.886 and 0.899 by subcloning and hybridization with radiolabeled BHV-1 gD gene DNA probe. A restriction site map of the Mael (1620 bp) fragment was generated, and various restriction endonuclease subfragments, indicated as bold lines, were cloned and sequenced. The arrow represents the BHV-5 gD QRF, which is 1251 nucleotides long, starting with the ATG codon and terminating with TGA. The arrowhead shows the direction of transcription.

the inguinal region with a total of 300 fig conjugated synthetic peptides or t rpE-gD fusion protein emulsified in Freund's complete adjuvant. Second and third injec- tions were given intramuscularly at biweekly intervals in incomplete Freund's adjuvant. Rabbits were bled 1 week after each booster dose and sera were analyzed for specific antibodies by Western blot and slot blot ELISA against the corresponding antigen and by virus neutralization.

Slot blot ELISA

Slot blot ELISA was performed using a microsample blotting apparatus (ABN, Hayward, CA). Each slot was coated with 40 or 8 #g of peptides dissolved in 0.05 M sodium carbonate buffer (pH 9.6) or borate buffer (0.01

sodium borate, 0.15 M NaOl, pH 8) or virus protein and dried at room temperature. The large amount of peptide per slot was necessary because the binding of these peptides to NO membranes was relatively weak. Amounts of bound protein were determined by Ponceau S protein staining (Sigma, St. Louis, MO). The immu- n0blotting assay was carried out as described earlier.

Neutral izat ion a s s a y

The neutralization assay was performed essentially as described by Chowdhury et al., (1988). Briefly, rabbit sera

(inactivated by 56° for 30 min) or MAb ascites fluids were diluted in DMEM containing 5% FBS and incubated with 50-100 PFU of virus for 1 hr at 37 °. Ant ibody-virus mix- tures were added to confluent MDBK monolayers in 24- well plates, allowed to adsorb for 1 hr, and overlaid with 1.2% methylcellulose in DMEM containing 2% FBS. After 2 -3 days of incubation, cells were fixed with 10% formal- dehyde and stained with 0.35% crystal violet; then plaques were counted. Titers were expressed as recipro- cals of the highest dilutions that reduced plaques by 50%.

In vitro neutralization inhibition a s s a y

Serial fivefold dilutions of peptides (in DMEM), starting at a concentration of 25 #g/ml, were incubated with an equal volume of MAb or bovine convalescent serum dilution that gave 50% plaque neutralization and then allowed to react for 1.5 hr at 37 ° . BHV-1 was added to yield an effective dose of 100 PFU/200 ffl of the mixture and incubated for an additional 1 hr at 37 °. Two hundred microli- ters of the mixture was added to each of two wells of confluent MDBK monolayer in a 24-well plate, and the neutralization assay was continued as described above. As controls for the assay, virus was incubated with peptide alone or antibody (Ab) alone. The ability of each peptide to inhibit the neutralizing activity of the corresponding antibody was calculated by the formula


1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0 KD

FIG. 2. SDS-PAGE and immunoblotting analysis of trpE-gD fusion proteins. Coomassie blue-stained gel showing fusion proteins expressed by subfragments of gD ORF using the pATH vector system (A) and immunoreactivity of the same proteins with MAb R54 (B) and 3402 (C). Lane 1, trpE; Lane 2, trpE-gD (1-417); lane 3, trpE-gD (1-216); lane 4, trpE-gD (1-164); lane 5, trpE-gD (52-164); lane 6, trpE-gD (52-126); lane 7, trpE-gD (1-49); lane 8, trpE gD (129-216); lane 9, trpE-gD(t17-164);and lane 10, trpE-gD(165-216).

Average No. of plaques (peptide and Ab) - Average No. of plaques (Ab only)

X 100. Average No. of plaques (peptide only)

- Average No. of plaques (Ab only)

Results were recorded and represented as % virus plaques restored.

RESULTS

Use of recombinant fusion proteins and monoclonal antibodies to define epitope structure on the BHV-1 gD glycoprotein

The 37-kDa trpE protein (vector control) and the larger sized trpE-gD fusion proteins, which were composed of the trpE gene plus the gD coding regions of variable length, are shown in Fig. 2A. The results of immunoblotting with BHV-l-specific gD MAbs R54 and 3402 are shown in Figs. 2B and 20, respectively. The induced fusion proteins were synthesized as single stable poiypeptides, except for the fusion proteins containing the whole gD and the N-terminal half of gD, which showed an additional band(s) detected as a breakdown product of the trpE-gD fusion protein (lanes 2 and 3, respectively, Fig. 2C). The fragment containing the epitope-bearing sequences in the correct reading frame was recognized by the MAbs, thus localizing the epitope region in the glycoprotein. A summary of the reactivity of the fusion proteins with these MAbs and the restriction endonuclease subfragments of the BHV-1 gD ORF specifying these proteins is shown in Fig. 3. trpE-gD fusion plasmids expressing the aa residues 1-417 (entire gD ORF) and the N-terminal half (aa 1-216) reacted on Western blot with both MAbs 3402 and R54. TrpE-gD recombinant plasmids expressing aa residues 129-216 and aa residues 165-216 were recognized by MAb R54 but not by MAb 3402, narrowing the location of R54 epitope to a stretch of 52 amino acids expressed by NarI-Smal subfrag-

ment (Fig. 3). MAb 3402 recognized overlapping trpE-gD- fusion proteins containing gD aa residues 52-164 and aa residues 52-126 but did not react with gD aa residues 117-164, indicating that the location of this epitope is between aa residue 52-116, contained in the recombinant plasmid expressing the ApaI-Banll subfragment (Fig. 3),

Immunogenicity of gD fusion proteins

To determine the immunogenicity of the trpE-gD fusion protein that expressed the N-terminal 216 amino

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FIG. 3. Schematic representation of the BHV-1 gD glycoprotein and its segments that were expressed in pATH vectors. The Mael fragment containing the gD ORF is depicted on top, showing restriction endonuclease sites used for cloning into pATH plasmids. Glycoprotein D pr0- tein segments expressed are shown as solid bars identified by aa numbers on top. The aa indicate restriction sites on the gD QRF. A summary of the immunoreactivity of gD protein segments with MAb R54 and 3402 is shown on the table to the right, with (+) to denote reactivity and (-) for no reactivity.


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FIG. 4. Immunogenicity of TrpE-gD fusion protein expressing the N- terminal 216 amino acids. (A) The immuneblot reactivity of anti-trpE- gD (1-216) rabbit sera with trpE protein alone (lane 1), trpE-gD (1 - 216) protein (lane 2), BHV-l-infected cells (lane 3), and mock-infected cells (lane 4). The locations of gD (lane 3) and trpE-gD (lane 2) fusion proteins are indicated by arrows. (B) The immunoblot reactivity of bovine eera against trpE-gD fusion protein. BHV-l-nonneutralizing (lane 1)and neutralizing (lanes 2, 3, and 4) bovine sera. A control MAb 3402 is shown in lane 5.

acids of BHV-1 gD (subfragment Mael-Smal), rabbits were immunized, and BHV-l-neutralizing bovine sera were analyzed. The rabbit serum recognized the authen- tic BHV-1 gD from BHV-l-infected cells (Fig. 4A), but did not neutralize the virus. Bovine sera that had BHV-1- specific neutralizing antibodies reacted with the t rpE- gD (1-216) fusion protein (Fig. 4B).

Analysis of BHV-5 gD ORF and comparison of predicted aa sequences of BHV-5 and BHV-1 gD

The nucleotide sequence of the BHV-5 gD gene has been submitted to the GenBank Databank with the Ac- 0ession No. U14656. The nucleotide sequence analysis of the 1.62-kb Mael fragment (MU 0.886-0.899) coding the gD gene identified only one 1251-bp ORF (from nt 247to nt 1497) coding for 417 aa residues (Fig. 5), large enough to encode the BHV-5 gD gene. Hydropathic analysis of the predicted protein revealed the presence of two prominent hydrophobic peaks similar to the BHV-1 gD, representing the signal sequence and the transmembrane anchor sequence (Fig. 6). Based on empirical rules f0rpredicting signal sequences (von Heinje, 1986), amino acid residues 4 to 18 have the position, length, relative hydrophobicity, and consensus cleavage site character-

istic of a signal sequence. Similarly, amino acid residues 356 to 382 have the position, length, and relative hydrophobicity characteristic of a transmembrane anchor sequence (Figs. 5 and 6).

Alignment of the predicted aa sequences of the BHV- 5 gD gene with the corresponding sequences of BHV-1 (Tikoo et al., 1990) showed that the amino terminal two- thirds of the protein (1-282 of BHV-5 gD) is relatively well conserved; however, the carboxy terminal region (amino acids 283-354) of BHV-5 gD differs. This region is located immediately upstream of the putative transmembrane domain (355-382 of BHV-5 gD) and charac- terizes a major hydrophilic peak in the case of BHV-1. In the BHV-5, several differences corresponding to the BHV- 1 sequences resulted in a series of hydrophilic peaks compared to one broad peak for BHV-1 gD (Fig. 6). A series of negatively charged residues from amino acids 281 to 295 (residue 280-292 of BHV-1) (Tikoo eta/., 1990) are present in this segment of the BHV-5 gD, which exhib- its several mismatches with respect to the BHV-1 sequence. As in other alphaherpesvirus gD homologues, the predicted protein has seven cysteine residues, six of which are located in the putative external domain. All cysteine residues are conserved between the two sequences and two potential N-linked glycosylation sites (Kornfeld and Kornfeld, 1985)located at amino acid positions 102 and 411 of BHV-1 gD are conserved, but a third site at amino acid position 41 of BHV-1 gD is missing in the BHV-5 gD (Fig. 5).

Alignment of BHV-5 gD sequences corresponding to epitope bearing sites of BHV-1 gD show differences

We compared the BHV-5 gD sequences corresponding to the BHV-1 epitope coding regions, defined above using recombinant fusion proteins, to map the epitope-coding sequences specified by MAbs 3402 and R54 precisely. Specific changes in the predicted amino acid sequence and their effect on the hydrophilic (data not shown) and potential antigenic properties (Fig. 7) were analyzed. Because the two MAbs recognize only BHV-1 gD and not BHV-5 (type specific), these epitopes should be in the regions of BHV-1 gD that correspond to the altered amino acids on BHV-5 gD. Antigenicity plot analysis (Fig. 7A) of amino acid sequence 52-115 indicated some potential antigenic regions. A comparison of the amino acid sequences of these regions with the corresponding regions of BHV-5 gD showed that five amino acids were different. Four of the five mismatches are clustered in one region at positions 92, 97, 98, and 100, and one amino acid change occurred at position 57 of BHV-1 gD. An antigenic peak between residues 94 and 101 of BHV-1 gD was lost on the corresponding region of BHV-5 gD (Fig. 7A), probably because of the four amino acid mismatches. Based on these observations, a 12- mer peptide covering residues 92-106 was synthesized and analyzed further.


1 ............... +++ 59

BHV-I.gD -MQGPTLAVLGALLAVAVS~PTPAPRVTVYVDPPAYPMPRYNYTERWHTTGPIPSPFADG : ::::::: : :::::::::::::::::: ::::: ::::::::::::: ::

BHV-5.gD MRRLALLSVLGALLAAAAGLPTPAPRVTVYVDPPAYPPPRYNYIERWHTTGPIPSPFQDG 1 ............. 60

+++ 119

BHV-I.gD REQPVEVRYATS~MLALIADPQVGRTLWEAVRRHARAYNATVIWYKIES..~:~RPLY ................ ::: ..... ::::::: :::: : :::::::::::::::::::

B.v-sgD Ii6 i i ; . Lii; QVG TLWGAV ERTY"ATVlWYKIES Y * +++ 120

179

BHV-I.gD YMEYT:E~EPRKHFG:~:YRTPPFWDSFLAGFAYPTDDELGLIMAAPARLVEGQYRRALYI ........ ::: ::::::::: ::::::::::::::: ::::::: ::::::::::

BHV-5.gD YMEYS~PKKfiFGY~RYRTppFWDGFLAGFAYPTDDELGLVMAAPARLAEGQYRRALYI , . * 180

239

BHV-I.gD DGTVAYTDFMVSLPAG:~:FSKLGAARGYTFG:~:PARDYEQKKVLRLTYLTQYYPQEAH ......... ..., ................. • ......... • ............

, 240

297

BHV-I.gD KAIVDYWFMRHGGVVPPYFEESKGYEPPPAADGGSPAPPGDD--EAREDEGETEDGAAGR ::::::::::: :::::::::::::::::: : ::::::$: :: : :::: :

BHV-5.gD KAIVDYWFMRHEGVVPPYFEESKGYEPPPAVEGASPAPPGDDDGEAHGEGGGEEDGAGGQ * * * 300

357

BHV-I.gD EGNGGPPGPEGDGESQTPEANGGAEGEPKPGPSPDADRPEGWPSLEAITHPPPAPATPAA : : :: : : : : :: : ::::::::::::: ::: :::::

BHV-5.gD ETGGEGEGPAAAGPDGAPP--GEPRPGP-GGPGADVDRPEGWPSLEAITRPPPTPATPAP * 357

+++ 417

BHV-I.gD PDAVPVSVGIGI~I:~:~GAYFVYTRRRGAGPLPRNPKKLPAFGNVNYSALPG BHV-5.gD PTALPVGIGVGIAAAVIA~VAAAGAAAYFVYTRRLGAGPLPKKAKKLLAFGNVNYSALPG

, . * +++ 417

FIG. 5. Comparison of the predicted amino acid sequence of the BHV-5 gD with the predicted amino acid sequences of BHV-1 gD (Tikoo et a/., 1990). The predicted amino acid sequences of the BHV-1 and BHV-5 gD glycoproteins were aligned by using Multalin so~ware (Corpet, 1988). Amino acid numbering sta~s at the first methionine, and numbers are shown to the right, Amino acids aligned identically between the two sequences are indicated by two dots, consewative substitutions are marked by an asterisk (*), and conse~ed cysteine residues are boxed. Potential N-linked glycosylation sites are indicated (+ + +). The presumptive signal sequences and the transmembrane anchor sequences are shown by broken and solid lines, respectively. The position of BHV-1 gD signal peptide cleavage site between amino acids 18 and 19 (Tikoo et al., 1990)is marked ($).

Antigenicity plot analysis of aa residues 165-216 and comparison and analysis with the corresponding regions of BHV-5 gD showed a total of five amino acid mismatches. Two of these (at positions 205 and 216) affected the antigenicity relative to the significant antigenic peaks on BHV-1 gD (Fig. 7B). Two additional amino acid mismatches (at positions 217 and 222) could affect the antigenicity of the protein. Based on these observations, three synthetic peptides covering amino acids 202-226, 202-213, and 213-225 were made and analyzed for epitope activity by neutralization inhibition of MAbs.

Specificity of synthetic peptides in defining the epitopes (MAbs 3402 and R54)

Peptides 202-225 (data not shown) and 202-213 (Fig. 8) both showed specificity for MAb R54 but peptides 213-225 did not show specificity for MAb R54 (Fig. 8), and peptides 92-106 showed specificity for MAb 3402 (Fig. 8). At the 25 #g/ml peptide concentration (optimum concentration) and MAb concentration sufficient to re-

duce 50% of the plaques of the challenge dose (100 PFU), the highest percentage (65-68%) of the virus plaques was restored (Fig. 8). When peptide 202-213 (specific for MAbR54) was tested for the ability to block the BHV- 1-neutralizing capacity of MAb 3402, it failed to show any blocking effect (data not shown). Thus, the blocking of MAb neutralization was a specific interaction of peptide and MAb and not due to inactivation of irrelevant antibody by these peptides. To determine if any of the virus- neutralizing activity in bovine convalescent serum is against the epitopes recognized by the MAbs, similar experiments, using bovine serum and peptides (202-213 and 92-106), were performed. The results revealed only 20-25% (highest) virus plaques restoration at the 25 #g/ ml peptide concentration (data not shown).

Two peptides, 92-106 and 202-213, were injected into rabbits for further evaluation. Antisera from these rabbits were tested for peptide and viral antigen specificity by slot blot ELISA and virus neutralization. Each antipeptide serum reacted specifically to the immunizing peptide; however, the reactivity of both of the sera with


BHV-5 gD 35 A' -1-

¢ BHV-1 gD ,_o r ~

L 1 ~ , , fl i}il~ ~ ,J ,-r

Vv' ' ' r t " " ' ,o

" I -

50 i00 150 200 250 300 350 400

Amino acid number (1-417)

FiG. 6. Comparison of the hydropathicity plots of the amino acid sequences of BHV-1 and BHV-5 glycoproteins D. The BHV-5 and BHV- 1 gD amino acid sequences were analyzed for hydropathicity character- istios (Kyte and Doolittle, 1982) using a 9-amino-acid window. The BHV- 5gD is shown on top, and the BHV-1 gD (Tikoo etaL, 1990) is shown on the bottom of the figure. The hydropathic scores and the amino acid numbers are shown on the vertical and horizontal axes, respectively.

the virus antigen was stronger (Fig. 9A). On Western blots, both the antipeptide (92-106 and 202-213) sera recognized strongly a 30K protein band from BHV-I-infected MDBK cell lysate (Fig. 9B). A control MAb R54 (Fig, 9B, lane 5) reacted with the whole gD and the 30K bands. No reaction was detected by antipeptide sera against mock-infected MDBK cells. In the plaque reduc- tion assay, both of the antipeptide sera showed low neutralizing activity (titer 1,4) against BHV-1.

DISCUSSION

Several approaches have been used to identify antigenic sites on BHV-1 glycoprotein D, including competition binding assays (Marshall eta/., 1988; Hughes eta/., 1988; van Drunen Little-van den Hurk et aL, 1984) and expression of mutant forms of the protein in a mammalian expression system (Tikoo et al., 1993). Although the latter approach has mapped epitopes within amino acid regions 164-216 and 320-355, the precise amino acid sequences of these epitopes were not identified. The main purpose of this study was to identify the precise location and amino acid sequences of linear antigenic determinants of BHV-1 gD that reacted with neutralizing BHV-1 gD-specific MAbs (MAbs 3402 and R54). To achieve this goal, the location of linear antigenic determinants was identified in segments of the BHV-1 gD ORF by expressing genomic subfragments in E. coil and then determining their reactivity with the MAbs. Second, anti-

genic and hydrophilic properties of the predicted amino acids encoded by the epitope-bearing fragments were analyzed and compared with the corresponding regions of the BHV-5 gD sequence (BHV-5 is resistant to neutralization by MAbs 3402 and R54). Third, specific differences in the predicted amino acid sequences that likely corresponded to recognition or lack of recognition by MAbs 3402 and R54 were identified in the BHV-5 gD ORF, and their effects on the antigenicity and hydrophilic- ity were determined. Based on these analyses, peptides corresponding to BHV-1 aa sequences were synthesized and tested for their specificity against MAbs in competition neutralization assays with virus. Previous studies have located three linear (conformational-independent) epitopes (la, Ilia, and IV) on three separate antigenic domains of BHV-1 gD protein (Tikoo et aL, 1993; van Drunen Little-van den Hurk eta/., 1984, 1990b). The present resul{s showed that neutralizing MAb R54 recognized a trpE-gD fusion protein (165-216) specified by restriction fragment Nar I -Smal . A linear gD epitope specified by MAb 9D6, was identified in this region (Tikoo et al., 1993). Whether this is the same linear epitope should be examined directly. Competition with peptides 202-213 further identified the location by reducing the neutralizing activity of MAb R54 in a competition neutralization inhibition assay.

MAb 3402 neutralizes BHV-1 by blocking viral penetration into susceptible cells (Dubuisson eta/., 1992). Based on competition binding assays, it has been assigned to an antigenic site (V) of BHV-1 gD (Marshall et al., 1988). We have mapped the 3402 epitope to a linear antigenic site between amino acid residues 92 and 106. MAb 3402 recognized a trpE-gD (aa 52-126) fusion protein specified by the Apa I -Ban l l subfragment of the gD ORF. Anti- genic analysis and comparison of region aa 52-115 with BHV-5 gD revealed five amino acid mismatches. Four were nonconservative amino acid changes (between residues 94 and 101) and corresponded to an antigenic peak on the BHV-1 gD that was absent on BHV-5 gD. A synthetic peptide covering region 92-106 reduced the neutralizing activity of MAb 3402.

Both peptides 92-106 (MAb 3402) and 202-213 (MAb R54) induced rabbit antisera that showed neutralizing activity against BHV-1 and recognized a 30-kDa band (probably a breakdown product of gD) in immunoblots containing viral lysates. Similar antipeptide sera reactive with lower molecular size bands have been observed for gD of the herpes simplex virus (Weijer eta/., 1988). Both peptides partially reduced virus neutralization by their respective MAbs. Neutralizing activity of MAbs could not be inhibited completely, even by increasing the peptide concentration (data not shown). The inability of the synthetic peptides to induce highly neutralizing antibody and to completely inhibit virus neutralization by MAbs could be explained by variations in "conformationar' properties of the peptides with respect to the native protein struc-


A .o BHV-5

I BHV-1 O i

52 92 106 115

B .£1BHV-5

~ o

.o BHV-1

165 202 213 226

FIG. 7. Comparison of antigenicity plots of epitope-bearing amino acid regions of BHV-1 gD with the corresponding regions of BHV-5 gD. Antigenic analyses of amino acid regions of BHV-1 gD recognized by MAb 3402 (A) and MAb R54 (B) are shown on the bottom, whereas the corresponding regions of BHV-5 gD antigenicity analyses are shown on the top of each figure. The antigenicity score and amino acid sequences are shown on the vertical and horizontal axes, respectively. Amino acid changes occurred on BHV-5 gD are indicated by arrows. Synthetic peptide sequences bearing epitopes for MAb 3402 (92-106) and MAb R54 (202-213) are underlined.

ture and, thus, reduce antibody binding. Both peptides were much less effective in inhibiting virus-neutralizing activity of bovine convalescent serum compared to neutralization by gD-specific MAbs 3402 and R54. This could be attributed to a broad range of specificities of neutralizing antibodies against other viral glycoprotein epitopes (gD as well).

These epitopes may be important in inducing protec-

tive immunity against BHV-1 infection for the following reasons (i) MAbs specific to these epitopes neutralize BHV-1 in the absence of complement (MAb 3402, Dubuis- sion eta/., 1992; MAb R54, Chowdhury, unpublished data) and (ii) convalescent sera (with neutralization titers against BHV-1) from cattle naturally infected with BHV-1 reacted to the bacterially expressed N-terminal half (1- 216) of gD that includes both epitopes. Epitopes on the


100 m 3402 + P(92-106)

R54 + P(202-213)

R54 + P(213-225)

13 == o 80

~ 6 0 ID ,,q

- 4 0 Q.

~ 2o

25 5 1 0 .2 0 . 0 4

P e p t i d e C o n c e n t r a t i o n ( p g / m l )

FiG. 8. Inhibition of monoclonal antibody neutralization by peptides. FJvefold dilutions of peptides starting at a concentration of 25 #g/ml of DMEM were incubated with equal volumes of limiting MAb dilution lid2,000) and allowed to react for 90 rain at 37 °. BHV-1 was added to a final number of 100 PFU/200 #1 of the mixture and incubated for an additional 1 hr, and the neutralization assay was continued as described under Materials and Methods. The ability of each peptide to reduce the neutralizing activity of MAb was recorded as % virus plaques restored. Each bar represents an average of four readings. The legends are shown on the upper right of the figure.

BHV-1 gD molecule bound by MAbs 3402 and R54 may be critical to the funct ional interaction of the virus with cell membrane and membrane-bound molecules as wel l as being a target for the protect ive immunity in cattle. Further studies are required to determine the funct ions of these epi topes in v i rus -ce l l interactions.

Rabbit ant isera against trpE-fusion protein expressing

the N-terminal half (1-216) of gD recognized gD molecules from infected cell lysate, but did not neutral ize the virus. An E. co~i-expressed BHV-1 gD protein has been shown to el icit a low neutral iz ing ant ibody response in cattle. This was attr ibuted to the lack of glycosylat ion and to conformat ional changes of d iscont inuous epitopes caused by poor dimerizat ion (van Drunen Little- van den Hurk eta/., 1993). In our s tudy the fai lure of rabbit ant ibody to neutral ize BHV-1 could not be expla ined by conformat ional dependence since the fusion protein (1 - 216) conta ined at least two conformat ion- independent neutral iz ing epitopes. An important N-l inked glycosylation site has been reported within this region (aa residue 102) (Tikoo et al., 1993). However, as ev idenced by the reactivity of MAb 3402 with the fusion protein (aa 5 2 - 126), the unglycosylated protein appeared to have no effect! on the structural conformat ion of the epitope.

Our study also showed that, in addi t ion to the differences in the two epi tope-coding regions, other sequence di f ferences occur between BHV-1 and BHV-6 gD genes. The C-terminal amino acids (residue 283-354), immedi-

ately upstream of the putative t ransmembrane domain, showed several di f ferences between the two gD. These amino acids are in the major hydrophi l ic peak of gD and may be important for p ro te in -p ro te in interact ions (Tikoo et aL, 1993). These dif ferences could contr ibute to biological propert ies of BHV-5, including the neuroinvasiveness of BHV-5. P ro l i ne -a lan ine repeats have been descr ibed in approx imate ly the same region of PRV gD (Petrovskis etaL, 1986). This region of BHV-5 gD needs to be further characterized. Future studies of BHV-1 and 5 gD should

KD

205 -

8 0 -

1 2 3 4 5

BHV-1

Pep 92-106

Pep 202-213

Anti-pep 92-106 Anti-pep 202-213 Protein staining P 1 2 ] 1 2 I 1 2 "J

4 9 -

3 2 -

2 7 -

FIG. 9. Immunoreactivity of antipeptide rabbit sera with peptides and BHV-1 proteins. Antipeptide (92-106) and (202-213) rabbit sera were tested for BHV-1 and peptide specificities by slot blot ELISA (A). Each antipeptide serum reacted specifically with its immunizing peptide and with the virus. The slots were coated with 40 #g (lane 1) and 8 #g (lane 2) of peptide or purified virus protein, and the bound proteins were determined by Ponceau S protein staining (Sigma). The antipeptide sera also were tested by Western blot (B) against BHV-l-infected MDBK cells, lane 1 (antipeptide 202-213) and lane 3 (antipeptide 92-106), and against mock-infected ceils, lane 2 (antipeptide 202-213) and lane 4 (antipeptide 202- 213). Positive control reaction of MAb R54 against BHV-l-infected cells is shown in lane 5. The positions of whole gD and the 30-kDa protein bands are marked (*-).


determine the mechanism of gD-mediated resistance of cells to BHV superinfection and its role in infectivity, immunity, and neuropathogenesis.

ACKNOWLEDGMENTS

G. Letchworth is acknowledged for critically reading the manuscript. We thank W. Lawrence for providing us a plasmid clone containing the BHV-1 Hindlll K fragment. This work was supported by USDA Section 1433 formula funds and BRSG funds of KSU to S. Chowdhury. Published as Contribution 94-482-J, Kansas Agricultural Experiment Station.

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