The Porcine Humoral Immune Response against Pseudorabies Virus Specifically Targets Attachment Sites

J OURNAL OF V IROLOGY, 0022-538X/00/$04.00ϩ0Feb.2000,p.1752–1760Vol.74,No.4Copyright©2000,American Society for Microbiology.All Rights Reserved.The Porcine Humoral Immune Response against Pseudorabies Virus Specifically Targets Attachment Sites on Glycoprotein gCBERTRAM T.OBER,†BERTHOLD TEUFEL,‡KARL-HEINZ WIESMU¨LLER,§GU¨NTHER JUNG,§EBERHARD PFAFF,ARMIN SAALMU¨LLER,AND HANNS-JOACHIM RZIHA*Federal Research Centre for Virus Diseases of Animals,Institute of Immunology,D-72076Tu¨bingen,Federal Republic of GermanyReceived11August1999/Accepted22November1999High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus(PRV)(Suid herpesvirus1)are generally observed in the serum of immunized pigs.A known function of the glyco-protein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains(HBDs).Therefore,it was suggested that the virus-neutralizing activity of anti-PRV sera is directed againstHBDs on gC.To address this issue,sera with high virus-neutralizing activity against gC were used tocharacterize the anti-gC response.Epitope mapping demonstrated that amino acids of HBDs are part of anantigenic antibody binding domain which is located in the N-terminal part of gC.Binding of antibodies to thisantigenic domain of gC was further shown to interfere with the viral attachment.Therefore,these results showthat the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance inductionagainst self-proteins,which utilize similar HBDs to promote host protein-protein interactions.Thefindingsindicate that the host’s immune system can specifically block the attachment function of PRV gC.Since HBDspromote the attachment of a number of herpesviruses,the design of future antiherpesvirus vaccines should aimto induce a humoral immune response that prevents HBD-mediated viral attachment.Herpesviruses persist in the natural host for the life of the host,and therefore,they must be able to evade the host’s immune mechanisms directed against the virus and virus-in-fected cells.Besides establishing a latent infection and thereby escaping direct immune attack,herpesviruses employ a variety of strategies to directly interfere with the immune system,e.g., expression of functional homologues or receptors of cytokines, chemokines,and the major histocompatibility complex(MHC) class I molecules(5).Such sophisticated evasion strategies can explain why vaccination against herpesviruses has only limited success.Although vaccines can prevent symptoms of the dis-ease,they do not eliminate the virus from the infected host and do not prevent the establishment of a latent infection. Pseudorabies virus(PRV),a member of the Alphaherpesviri-nae,is the causative agent of Aujeszky’s disease.In pigs,the natural host of PRV,mortality decreases with increasing age, and upon primary infection a latent infection that can be re-activated is generally established(45).Since outbreaks of Au-jeszky’s disease cause important economic losses,attenuated live viruses or inactivated vaccines are widely used for vacci-nation of pigs.Glycoprotein gC of PRV plays an important role in eliciting a protective immune response in pigs.Vacci-nations with protein fractions enriched for PRV gC,recombi-nant gC subunit vaccine,and gC vaccinia virus recombinants as well as vaccination with DNA encoding gC were found to provide protection against PRV challenge(11,16,32). Several immune mechanisms directed against gC have been reported to combat PRV infection.PRV infection can induce MHC class I restricted,gC-specific,cytotoxic T cells(52).It was also shown that gC is involved in priming of porcine T-helper cells(17)and induces MHC class II restricted,PRV-specific memory T-helper cells(30).In addition to its role in the specific cell-mediated immune response,gC also is one of the major viral proteins that trigger the porcine humoral immune response.In pigs,the majority of virus-neutralizing antibodies induced by PRV infection or vaccination with at-tenuated live virus is directed against gC(1).Passive immuni-zation of pigs with gC-specific mouse monoclonal antibodies was sufficient to confer protection(24),indicating that gC-specific antibodies,even in the absence of a cell-mediated immune response,play an essential role in the control of PRV infection.PRV gC,which is a constituent of the viral envelope(34,35), is nonessential for virus growth in tissue culture(34,44)but is required for maximum infectivity,as mutant virions lacking gC have been shown to be defective in attachment to target cells (25).The attachment defect of gC-negative virions is due to a failure to bind to heparan sulfate glycosaminoglycan chains on the target cell(28).It was further shown that the heparan-binding protein gC is promoting the initial attachment of the virus(13)and that three functional heparin-binding domains (HBDs),located in the amino-terminal part of gC(Fig.1), mediate this interaction(7,8,21).The single domains are each composed of a cluster of positively charged viral amino acids with homology to the HBD consensus sequence(2).Each separate HBD is able to promote attachment to target cells(8). In the present study,we identified an antigenic,antibody binding domain(ABD)on gC,and we showed that the HBDs, which are located in this ABD,are recognized by the humoral immune response of the natural host,swine.Antibodies bind-ing to HBDs interfered with viral attachment and prevented*Corresponding author.Mailing address:Federal Research Centrefor Virus Diseases of Animals,Institute For Immunology,Paul-Ehr-lich-Strasse28,D-72076Tu¨bingen,Federal Republic of Germany. Phone:49-7071-967253.Fax:49-7071-967303.E-mail:achim.rziha@tue.bfav.de.†Present address:Gwen Knapp Center for Lupus and Immunology Research,Department of Pathology,The University of Chicago,Chi-cago,IL60637.‡Present address:Pathogenix Laboratories GmbH,D-82152Mar-tinsried,Federal Republic of Germany.§Present address:Institute for Organic Chemistry,University of Tu¨bingen,D-72076Tu¨bingen,Federal Republic of Germany.1752infection of target cells.The results demonstrate that the gC-specific neutralization of PRV by the humoral immune re-sponse is linked to the known attachment function of this glycoprotein.Blocking of HBDs is therefore one of the strat-egies the host immune system utilizes to combat PRV infection.MATERIALS AND METHODSViruses and cell culture.The porcine kidney cell line PSEK and the Madin-Darby bovine kidney cell line MDBK were used for virus propagation and titration as described recently (30).The ␤-galactosidase-expressing recombinant PRV PHY-B111is derived from the mutant PHY-EBG,which carries the lacZ gene in a nonessential,intergenic region of the wild-type (WT)PRV strain Phylaxia (9).To improve the lacZ gene expression in PHY-B111,the simian virus 40polyadenylation signal was inserted at the 3Јend of the lacZ gene.In vitro and in vivo,both lacZ mutants of PRV demonstrate the same growth characteristics and virulence as the parental strain Phylaxia (H.-J.Rziha and A.Six,unpublished results).The gE-negative and thymidine kinase-negative live vaccine of PRV (Begonia,Nobi-Porvac live;Intervet,Boxmeer,The Netherlands),an virulent derivative of PRV strain NIA3(29),was kindly provided by N.Visser (Intervet),and it has been reported many times that the induced serum antibodies efficiently neutralize various WT PRV strains.WT PRV strain Kapan (Ka)and the isogenic gC deletion mutant (38)were provided by T.C.Mettenleiter (Federal Research Centre For Virus Diseases of Animals,Insel Riems,Federal Republic of Ger-many).Construction of gC expression plasmids.Fragments of the open reading frame of gC were cloned by standard procedures into the expression vector pEV40,a derivative of plasmid pEx34,as described previously (10,40).Nucleotide se-quence comparison demonstrated the high preservation of the gC gene of var-ious PRV strains (including strains NIA3and Becker),though some minor base differences exist (12).Similarly,in contrast to the published sequence of the PRV strain Becker (34),PRV strain Ka carries a silent nucleotide exchange at nucle-otide 330(G to A),which generates an additional Sac I restriction site.This allowed cloning of the following three gene segments,covering 95%of the gC coding region,as shown in Fig.1:the 335-bp N-terminal Sac I fragment (pOB-EB)comprising amino acids 44to 156and designated gC-N-term;the 402-bp Sac I fragment (pG32-40)comprising amino acids 156to 290and designated gC-middle;and the 448-bp C-terminal Sma I-Bam HI fragment (pOB-SB)com-prising amino acids 309to 459and designated gC-C-term.DNA sequencing proved the correct insertion of each gC fragment (data not shown).Expression,purification,and Western blot analysis of gC fusion proteins.After heat induction,the MS-2polymerase-gC fusion proteins were isolated,and the insoluble protein fraction was dissolved in 7M urea and used for Western blot analysis as described previously (10).Synthesis and analysis of synthetic peptides and lipopeptides.Overlapping peptide amides (15amino acids in length,overlapping each other by 10amino acids)and the lipopeptide {N -palmitoyl-S -[2,3-bis(palmitoyloxy)-(RS )-propyl]-(R )-cysteinyl}of peptide 65-79(covering amino acids 65to 79of gC)were synthesized according to the gC sequence of PRV strain Ka (34)by using Fmoc chemistry (multiple peptide synthesizer SMPS 350A;Zinsser,Frankfurt,Ger-many)as described recently (30).The synthesized peptides and lipopeptides were analyzed by amino acid analysis (ABI 420A;Applied Biosystems,Weiter-stadt,Germany),analytical high-performance liquid chromatography (System Gold;Beckman,San Ramon,Calif.)on a Nucleosil C 18column (Grom,Herren-berg,Germany),and ion-spray mass spectrometry (API III Triple-Quatrupol Ionspray-MS;Grom).The peptides displayed a purity of at least 80%.Animals and antisera.Porcine anti-PRV sera were obtained by immunization of three d/d haplotype National Institutes of Health minipigs (inbred 1,2,and 3)(37)and two outbred pigs (German landrace)(outbred 1and 2)with the PRV live vaccine Nobi-Porvac live (Intervet).The vaccine was administered twice at an interval of 10days,as recommended by the manufacturer.Mouse antisera directed against the region comprising amino acids 65to 79of PRV gC (peptide 65-79)were generated using two different protocols.First,BALB/c mice were immunized with 100␮g of lipopeptide 65-79together with lipopeptide adjuvant three times at 2-week intervals as described previously (6).Second,peptide 65-79was coupled through an additional cysteine at the N terminus to the carrier protein keyhole limpet hemocyanin (KLH)using m -maleimidobenzyl-N -hydroxysuccinimide ester (23).The mice were initially primed with 50␮g of peptide-coupled protein in complete Freund’s adjuvant and boosted with 50␮g of peptide-coupled protein in incomplete Freund’s adjuvant at 2-week intervals.Production and characterization of the polyclonal goat antiserum against PRV,which neutralized PRV in the presence of complement up to a serum dilution of 1:20,000and in the absence of complement in a serum dilution of 1:5,000,has been described previously.Serum samples of the immunized pigs were collected before and 1week after each immunization.Prior to use,serum complement was inactivated by heating of the sera for 1h at 56°C.ELISA.Microtiter plates (96-well)were coated with synthetic peptides and purified PRV at concentrations of 200and 5␮g per ml of phosphate-buffered saline,respectively,and standard enzyme-linked immunosorbent assay (ELISA)was performed as described previously (22).Bound antibodies were detected with rabbit anti-porcine,anti-goat,or anti-mouse immunoglobulin-coupled horseradish peroxidase (Dianova,Hamburg,Federal Republic of Germany).The ELISA titer of the sera was determined as the highest dilution of the serum displaying an optical density at a wavelength of 490nm (OD 490)that was ap-proximately twice that of the corresponding preimmune serum.Virus neutralization test.The virus-neutralizing activity of the anti-PRV sera was tested after preincubation with PRV for 1h at 37°C in the absence of complement on MDBK cells (26).Virus adsorption was allowed for 1h on ice to delay virus penetration (50).After removal of excess virus by washing with media,the cells were incubated at 37°C.The neutralization titer was determined from triplicates and expressed as the dilution of serum resulting in 95to 100%plaque reduction.Dilutions of preimmune sera from the inbred and outbred pigs as well as medium were used as controls.Competitive attachment inhibition assay.The heat-inactivated goat anti-PRV serum was diluted 1:50in medium,the peptide was added at the indicated concentrations,and the mixtures were incubated overnight at 4°C.Thereafter,the lacZ gene-expressing PRV strain PHY-B111(1.5ϫ107PFU)was added,and the mixture was incubated at 37°C for 30min and then used for infection of 106MDBK cells at 2°C.After 1h of adsorption,the cells were washed with phos-phate-buffered saline and incubated for an additional 4h at 37°C.After trypsinization,the cells were hypotonically treated with 2mM fluorescein-di-(␤-D -galactopyranoside)(Sigma-Aldrich,Munich,Federal Republic of Germany),as described recently (36),which treatment allows quantitation of ␤-galactosi-dase-expressing cells by flowcytometry.FIG.1.Schematic representation of the gC protein.The location of the three fusion proteins gC-N-term,gC-middle,and gC-C-term is indicated by the respective amino acid positions.The positions delineating the HBDs HBD 1(H1),HBD 2(H2),and HBD 3(H3)are shown as described previously (8),as is that of the identified antigenic ABD.Segments found to react with the porcine immune sera are shaded.V OL .74,2000SERUM ACTIVITY AGAINST PRV TARGETS HBDs ON gC 1753RESULTSPRV gC represents a target for virus-neutralizing antibod-ies.Several reports indicate the importance of gC and gC-specific antibodies in PRV neutralization.To gain a better understanding of the natural humoral immune response,which is directed against gC during PRV infection,we generated anti-PRV sera of three d/d haplotype inbred pigs (Table 1,inbred pigs 1to 3)and two German landrace pigs (Table 1,outbred pigs 1and 2).All immune sera displayed PRV-specific antibody titers of more than 1:10,000as determined by ELISA (data not shown).The presence of gC-specific virus-neutraliz-ing antibodies was tested by comparing the capacities of the different antisera to neutralize WT PRV and an isogenic gC deletion mutant in a plaque-reduction assay.Since gC is in-volved in viral attachment,virus adsorption was performed in the cold in order to prevent premature penetration of PRV during the infection.Excess virus was subsequently removed and incubated at 37°C to allow virus multiplication.The swine anti-PRV sera neutralized WT PRV in the absence of com-plement up to a serum dilution of 1:480or 1:960(Fig.2;Table 1).In contrast,a reduction in serum neutralization titer was reproducibly found when the gC-negative PRV was used (Fig.2;Table 1).The reduction of virus-neutralizing serum antibody titers ranged from 4-fold (serum from outbred pig 1)to 48-fold (serum from inbred pig 2).Using the preimmune sera from inbred and outbred pigs,no plaque reduction was found.These results therefore demonstrate that the immunization led to the induction of a humoral virus-neutralizing antibody response directed against gC.Major B-cell epitopes are located in the N-terminal part of gC.To elucidate the gC-specific reactivity of the different anti-PRV sera,three fragments of gC encoding the amino acids 44to 156(gC-N-term),156to 290(gC-middle),and 309to 459(gC-C-term)(Fig.1)were expressed as insoluble bacterial MS-2polymerase-gC fusion proteins (see Materials and Meth-ods).The Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)gel showed that the fusion proteins gC-C-term and gC-middle were abun-dantly expressed,whereas gC-N-term (23kDa in size)could be induced only to moderate amounts (Fig.3A).These specific protein bands were not detected in bacteria containing the vector plasmid without insertion or in noninduced bacterial lysates (data not shown).To map the gC-specific humoralantibody response,the three fusion proteins were tested by Western blot analysis with the different antisera.The results are shown representatively in Fig.3B to D,which were derived from different Western blotting experiments.Whereas the swine preimmune sera did not react with the gC-specific fusion proteins (Fig.3B),all tested immune sera displayed two dis-tinct patterns of reactivity with gC-N-term and gC-middle,respectively.First,inbred 1and outbred 2reacted strongly with gC-N-term and only weakly or not at all with gC-middle (Fig.3C;Table 1).Second,inbred 2,inbred 3,and outbred 1pig sera showed a strong reactivity with gC-middle but only weaklyTABLE 1.Reactivity of anti-PRV seraAnti-PRV serumVirus neutralization a Result of:Western blot analysis bPeptide ELISA cWTgC-negativegC-N-termgC-middlegC-C-term65-7980-9485-99Inbred pig 1960120ϩϩϩϪϪϩϩϩϪϪ296020ϩϩϩϩϪϪϩϩϩϩϩϩ3ND d ND ϩϩϩϪϪϪϪOutbred pig 1480120ϩϩϩϪϩϩϩϩϩ248060ϩϩϩϪϪϩϩϩϩϩϩGoat5,000640ϩϩϩϩϪϩϩϩϩϩa Values are the reciprocal serum dilutions which resulted in complete virus neutralization (100%plaque reduction)of WT or gC-negative PRV.bWestern blot analysis of gC fusion proteins,which were not (Ϫ),weakly (ϩ),moderately (ϩϩ),or strongly (ϩϩϩ)recognized by the different immune sera.The goat anti-PRV serum was used in a dilution of 1:5,000.cResults from ELISAs with peptides 65-79,80-94,and 85-99,showing the difference in OD 490between preimmune and immune serum (1:100dilution),as follows:Ͻ0.1(Ϫ),0.1to 0.25(ϩ),0.25to 0.5(ϩϩ),and Ͻ0.5(ϩϩϩ).The goat anti-PRV serum was diluted 1:5,000.dND,notdetermined.FIG.2.Porcine immune sera exhibit reduced capacity to neutralize gC-neg-ative PRV compared to WT PRV.Immune sera from inbred pigs 1and 2(numbered circles)and from outbred pigs 1and 2(numbered triangles)were diluted and used for plaque reduction assay (performed in triplicate)with WT PRV and an isogenic gC-negative mutant PRV (gC-minus)as described in Materials and Methods.The reciprocal serum dilutions resulting in 95to 100%plaque reduction are depicted,as are the standard errors (error bars).1754OBER ET AL.J.V IROL .recognized gC-N-term (Fig.3D;Table 1).In contrast,none of the tested immune sera reacted specifically with the fusion protein gC-C-term (Fig.3C and D;Table 1),containing the transmembrane region of gC.Additional,nonspecific protein bands reacted in some Western blotting tests;a protein with an apparent molecular mass of approximately 21kDa (Fig.3C and D)variably appeared in different SDS-PAGE gels and reacted in more and less pronounced manners with nonim-mune (data not shown)and immune swine sera (Fig.3C and D),respectively.Taken together,these results indicate that the N-terminal part of gC is predominantly recognized by gC-specific antibodies of the immunized pigs,which is consistent with the location of the cytoplasmic and transmembrane region in the C-terminal part of gC (34).Identification of an ABD in the N-terminal part of gC.To further characterize the humoral immune response against the N-terminal part of gC,overlapping synthetic peptides of gC (amino acids 1to 310)were tested by ELISA using the porcine anti-PRV sera or the respective preimmune sera.The peptides were 15amino acids in length and overlapped each other by 10amino acids.No PRV-specific reaction was found with pep-tides covering amino acids 1to 54and 105to 310.However,between amino acids 54and 104,three reactive peptides could be identified.While all peptides resulted in ELISA OD 490values of approximately 0.2with the preimmune sera (Fig.4),the peptides 65-79,80-94,and 85-99showed a significant in-crease in reactivity with several of the tested serum samples (Fig.4;Table 1).Peptide 65-79(65-STPPVPPPSVSRRKP-79)showed a strong reaction with the serum from inbred pig 1(OD 490,0.81)(Fig.4A),a weak reaction with the serum from outbred pig 1(OD 490,0.3),and no reaction with the sera from inbred pigs 2and 3and from outbred pig 2(Table 1).Peptides 80-94and 85-99(80-PRNNNRTRVHGDKATAHGRK-99)reacted strongly with the serum from inbred pig 2(OD 490values,0.62and 0.58,respectively)(Fig.4B)and outbred pig 2(OD 490values,2.16and 1.98,respectively),a weaker reaction was found with serum from outbred pig 1(OD 490values,0.48and 0.41,respectively),and no specific reaction was found with sera of inbred pigs 1and 3(Table 1).All three reacting pep-tides map between amino acids 65and 99of gC.ThispeptideFIG.3.Western blot analysis of gC fusion proteins.The fusion proteins gC-N-term (N),gC-middle (M),and gC-C-term (C)were obtained by urea extraction and analyzed by SDS–15%PAGE followed by Coomassie blue staining (A)or Western blot analysis (B to D).The shown results of the Western blot analysis are representative of the reactivities of all sera tested (Table 1),and the porcine preimmune serum (B)and immune sera from inbred pig 1(C)and inbred pig 2(D)were diluted 1:500.The specifically induced proteins are marked by arrowheads.Migration of the molecular weight (MW)standards (in thousands)is indicated to the left.V OL .74,2000SERUM ACTIVITY AGAINST PRV TARGETS HBDs ON gC 1755screening approach therefore allowed us to identify an ABD in the N-terminal region of gC comprising at least amino acids 65to 99(Fig.1).ABD is accessible to humoral immune responses on the surface of PRV.The peptide screening approach did not an-swer the question of whether the identified ABD is exposed on the virion surface and accessible to the humoral immune re-sponse.To answer this question,BALB/c mice were immu-nized with the synthetic peptide 65-79,either as a lipopeptideor coupled to the KLH protein as a carrier.The specificity of the obtained antisera was shown by ELISA as depicted repre-sentatively in Fig.5for one of the mouse anti-peptide 65-79serum samples.As expected,the peptide antisera strongly re-acted with peptide 65-79in serum dilutions higher than 1:1,000(up to 1:10,000),whereas the reaction with the control peptide was as negative as the preimmune serum reaction (Fig.5,insert).More importantly,the generated anti-peptide 65-79antisera,but not the preimmune sera,reacted specifically with purified PRV (Fig.5).This priming of anti-PRV specific anti-bodies by peptide 65-79immunization demonstrated that the identified ABD on gC is exposed on the virion surface and accessible to gC-specific antibodies.This is corroborated by computer-aided prediction of the secondary structure of gC,indicating that the region of the ABD (amino acids 65to 99)forms a loop structure with a high surface probability (data not shown).Amino acids of the HBD consensus sequence are directly recognized by anti-PRV sera.The identified ABD overlapped with two of the three known HBDs of gC (Fig.1).Since the HBD consensus sequence was shown to mediate viral attach-ment,we asked whether amino acids of ABD can be directly recognized by the anti-PRV sera.A direct involvement of HBD 2in binding of the antisera appears unlikely,since both peptides 80-94and 85-94reacted with most immune sera (Ta-ble 1),but only peptide 85-94is overlapping HBD 2.To test for direct binding of the serum antibodies to HBD 1,shortened and mutated versions of peptide 65-79,which includes 4of the 6amino acids of HBD 1(76-RRKPPR-81),were synthesized (Fig.1).Due to the strong reactivity with peptide 65-79,the antiserum of inbred pig 1was used for ELISA with the differ-ent mutated peptides.The removal of amino acids 65and 66,which do not overlap with the HBD,led to a markedly de-creased serum reactivity (Fig.6,peptide 67-79).However,theFIG.4.Identification of linear B-cell epitopes in the N-terminal part of gC.Overlapping synthetic peptides spanning amino acids 55to 114of gC were coated onto 96-well plates and tested by ELISA with diluted (1:200)immune sera (open columns)of inbred pig 1(A)and inbred pig 2(B)and with the respective preimmune sera (gray columns).Peptide numbers correspond to the first amino acid position of each peptide in the gC sequence.Each experiment was performed in triplicate,with the standard deviations shown by error bars;no error bar is shown where the standard deviation was less than the extent of thebar.FIG.5.The identified ABD is exposed on the surface of PRV virions.Mouse antisera against peptide 65-79(open symbols)and preimmune sera (closed symbols)were tested by ELISA for reactivity with peptide 65-79(insert,squares)and an irrelevant control peptide (insert,circles),or with purified PRV (squares)and bovine serum albumin (circles).Each experiment was performed in tripli-cate,with the error bars indicating the standard deviations.The results are representative for five different mouse anti-peptide 65-79serum samples.1756OBER ET AL.J.V IROL .loss of amino acids 78and 79of the HBD completely pre-vented binding of the antisera (Fig.6,peptide 65-77).In ad-dition,amino acid exchanges at position 66(T to G,unrelated N-terminal amino acid)or at position 71(P to G,central amino acid of the predicted loop structure in this region)had either no effect on antibody binding (Fig.6,peptide 65-79/66G)or led to a serum reactivity only slightly reduced (Fig.6,peptide 65-79/71G)compared to that of the authentic peptide 65-79.In contrast,a change of the central amino acid of HBD 1,amino acid 78,from K to E resulted in the complete loss of reactivity of the anti-PRV serum (Fig.6,65-79/78E).There-fore,these results demonstrate that amino acids constituting the HBD 1domain are critical for the recognition of peptide 65-79by the immune sera.Consequently,it indicates that the humoral immune response against gC can be directed against HBD 1on the surface of PRV.Antibodies against ABD can block viral attachment to target cells.The findings that the ABD is expressed on the surface of PRV and that amino acids of HBD 1are targets for the PRV-specific humoral immune response indicate that gC-specific antibodies directed against the ABD can interfere with attach-ment of PRV by blocking HBD 1.To test this assumption,MDBK cells were incubated with PRV,which was preincu-bated with different dilutions of mouse anti-peptide 65-79se-rum.To prevent virus penetration,the adsorption was per-formed in the cold and thereafter shifted to 37°C to allow virus multiplication.The ability of the sera to interfere with viral attachment was measured in a plaque assay.However,even at very low dilution (1:5)of the anti-peptide 65-79serum,no significant reduction of virus plaques could be observed,either by using the sera generated by lipopeptide injection or by KLH coupling.The failure to prevent PRV attachment with antibodiesagainst peptide 65-79might be explained by the described functional redundancy of the three HBDs of gC (8).For effi-ciently preventing viral attachment,probably all three HBDs have to be blocked simultaneously with an antiserum contain-ing antibodies directed against all ing the overlap-ping peptides,a linear B-cell epitope was not detected in HBD 3(amino acids 133to 141).However,this approach is unable to identify conformation-dependent epitopes.The presence of additional conformation-dependent ABDs on gC can be sug-gested from the Western blot reactivity of the porcine anti-PRV sera with the fusion protein gC-middle (linear B-cell epitopes were not detected in this part of gC)(Table 1;Fig.1).Thus,we wanted to test the involvement of the identified ABD 65-99in binding of virus-neutralizing antibodies.To this end,we established an assay that allowed quantitation of the capac-ity of this peptide to interfere with complement-independent virus neutralization of anti-PRV sera.PRV strain PHY-B111,expressing the lacZ gene in a nonessential intergenic region,was used for infection of MDBK cells in the cold upon prein-cubation with peptide 65-79.After removing unattached virus,virus-infected cells could be detected by flow cytometry through their ␤-galactosidase activity (34).After 4h of incu-bation at 37°C (in the absence of antibodies and peptide),75%of cells were virus infected (Fig.7A).To enable sensitive de-termination of peptide binding with virus-neutralizing antibod-ies,we used a goat anti-PRV serum which showed the same reactivity with the gC-fusion proteins and with the mutated gC peptide 65-79as the serum from inbred pig 1but exhibited a fivefold higher virus-neutralization titer in the absence of com-plement (Table 1).The addition of this antiserum alone neu-tralized the virus and reduced the concentration of infected cells to 11%(Fig.7B).To test the ability of peptide 65-79to interact with virus-neutralizing antibodies,the anti-PRV serum was then prein-cubated with peptide 65-79or with a control peptide and PHY-B111.Preincubation with peptide 65-79clearly reduced the virus-neutralizing capacity of the anti-PRV serum,depending on the concentration of the peptide added.At the highest peptide concentration (500␮g),54%of the cells were PRV infected,and decreasing peptide concentrations resulted in a reduction of infected cells to 23%(Fig.7C).In contrast,pre-incubation with the control peptide did not decrease the virus-neutralizing activity of the serum.In this case,the percentage of virus-infected cells ranged from 15to 18%,not exceeding 25%(Fig.7D),demonstrating the specificity of peptide 65-79to inhibit virus neutralization.These results could be also con-firmed by the use of a conventional PRV plaque assay as a readout (data not shown).These results therefore demonstrate that antibodies directed against the ABD can interfere with the attachment of PRV.Since the ABD on gC was identified by screening of anti-PRV sera,these results indicate further that virus neutralization by gC-specific antibodies,which are in-duced after natural infection or vaccination,is at least partly due to antibody binding and thereby to blocking of HBDs on the viral surface.DISCUSSIONThe glycoprotein gC of PRV is essential for efficient primary attachment of the virus to heparan sulfate glycosaminoglycan chains on target cells (28,49).This attachment is mediated through three independent HBDs located in the N-terminal part of the protein (7,8).Among the virion glycoproteins,which are targets for the humoral immune response of pigs,PRV gC is suggested to represent a primary target for virus-neutralizing antibodies (1,45).The gC-specificvirus-neutral-FIG.6.Amino acids corresponding to HBD 1are essential for specific anti-body recognition of ABD.Ninety-six-well plates were coated with synthetic peptides 65-79,65-77,and 67-79(designated as such because of the amino acids of gC that they comprise)and peptides with mutated amino acid 66(T to G;65-79/66G),amino acid 71(P to G;65-79/71G),or amino acid 77(R to E;65-79/77E)and tested by ELISA with diluted (1:200)immune serum from inbred pig 1(open columns)and the respective preimmune serum (gray columns).Each experiment was performed in triplicate,with the error bars indicating the stan-dard deviations;no error bar is evident where the standard deviation was less than the extent of the bar.Very similar results were obtained with the goat anti-PRV serum diluted at 1:2,000(data not shown).V OL .74,2000SERUM ACTIVITY AGAINST PRV TARGETS HBDs ON gC 1757。