Veterinary Microbiology, 24 (1990) 1-10 Elsevier Science Publishers B.V., Amsterdam

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Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins Gerardo Iglesias I*, Thomas Molitor 1, David Reed 2 and James L'Italien 2 1Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN (U.S.A.) 2Molecular Genetics Incorporated, Minnetonka, MN (U.S.A.) (Accepted 10 January 1990)

ABSTRACT Iglesias, G., Molitor, T., Reed, D. and L'Italien, J., 1990. Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins. Vet. Microbiol., 24: 1-10. Antibodies to Aujeszky's disease virus (ADV) glycoproteins glI, gllI, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gill. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gill. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gill or vaccinated with MLV, whereas sera from pigs immunized with gll or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.

INTRODUCTION

Aujeszky's disease virus (ADV) also known as pseudorabies virus is a m e m b e r of the herpesvirus group and a widely spread pathogen of swine. The genes for several ADV glycoproteins have sequence homologies with herpes *Present address: Dr. Gerardo Iglesias, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606 (U.S.A.).

0378-1135/90/$03.50

© 1990 Elsevier Science Publishers B.V.

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simplex virus (HSV) glycoproteins genes (Ben-Porat et al., 1983; Robbins et al., 1986a, 1987 ). In ADV-infected cell cultures, there are eight glycoproteins and more than 30 nonglycosylated proteins induced by the virus (Hahn and Hahn, 1987 ). It is assumed that the glycoproteins have the major role in stimulating an immune response by the host, as is the case with HSV (Glorioso et al., 1984). Hampl et al. (1984) reported that gI, gII, gIII, gIV, and gV glycoproteins are present in the virus envelope and are all present in the form of complexes. These complexes are joined by disulfide bonds (Table 1 ). Other ADV glycoproteins that are known to be antigenic are gp50, gp63, and gpX. Glycoproteins I, gp63, and gpX are nonessential for virus replication, since it is known that attenuated strains of ADV that have deletions in regions of the genome where some of these glycoproteins are encoded are still able to replicate in vitro and in vivo (Lomniczi et al., 1987 ). The European vaccine strain, Bartha, is deleted in regions coding for gI and gp63, while the BUK strain is deleted in regions coding for gI, but not gp63 (Petrovskis et al., 1986b). Additionally, the gIII gene could be deleted from the ADV genome and this deletion did not affect its ability to replicate in vitro (Robbins et al., 1986b). The antigenicity of selected ADV glycoproteins has been studied, but primarily in laboratory animals. Hampl et al. ( 1984 ) compared the efficiency of mouse monoclonal antibodies directed against either gI complex, gII complex, or gIII for neutralizing virus infectivity. The results showed that virus neutralization in vitro was achieved by antibodies directed against gIII, and to a lesser extent and only in the presence of complement by antibodies against gII complex, while the antibodies against gI complex did not neutralize the virus even in the presence of complement. Wathen and Wathen (1984) reported that ADV was neutralized by monoclonal antibodies directed against gp50, either in the presence or absence of complement. More recently it was TABLEI Antigenic glycoproteins present in ADV infected cells Name

Molecular weight (kDa)

HSV equivalent

References a

gl complex (gI, gIV, gV other) gIl complex (IIa, IIb, IIc) gill complex (IIIa, IIIb, IIIc) Gp50 Gp63 GpX b

130, 98, 62, I 15 135, 74, 58 98, 67, 58 50 44 99

gE gB gC gD US7 gG

l, 2 1, 2, 3 1, 4 5 6 7, 8

"References: (1) Hampl et al. (1984); (2) Lukacs et al. (1985); (3) Robbins et al. (1987); (4) Robbins et al. ( 1986a); (5) Petrovskis et al. ( 1986a); (6) Petrovskis et al. ( 1986b); (7) Thomsen et al. ( 1987); (8) McGeoch et al. (1987). bHighly abundant in supernatants from infected cultures.

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reported that in a comparative assessment of 108 clones of monoclonal antibodies that reacted against ADV, 12 clones displayed complement-independent neutralizing activity. All of the 12 clones were specific for gp50. Passive protection experiments carried out in the same study showed that mice could be protected against ADV lethal challenge by injection of clones reacting with gp50 or gIII (Eloit et al., 1988). It is clear that gII, gIII, and gp50 are major ADV glycoproteins and, thus, likely to be exposed to the immune system. It was, therefore, decided to compare the activities of specific anti-ADV antibodies of pigs immunized with single glycoproteins, or vaccinated with a modified-live vaccine using several serological tests based on different principles, i.e., serum neutralization of virus in vitro is a parameter for the prevention of infection, while lysis of infected cells through antibodies and complement or polymorphonuclear cells provides information on the role of antibodies in the course of the disease. MATERIALS AND METHODS

Virus glycoproteins purification The ADV glycoproteins (glI, glII, and gp50) were purified from ADV-infected cell lysates using immunoaffinity chromatography as described by L'Italien (1987). Briefly, ADV-infected porcine cells (pkl 5 ) were isotonically washed, frozen, and then thawed in cell lysis buffer ( 100 m M Tris, 150 m M NaCI, 1% NP-40, 1% DOC, pH 7.5 ). The cells were briefly sonicated to ensure complete disruption prior to removal of cell debris by centrifugation. The resulting cell lysate supernatant was then sequentially applied to individual immunoaffinity columns for each of the three ADV glycoproteins. The approach enabled the recovery of all three ADV glycoproteins from the same lysate. Each of the immunoaffinity columns used in these studies were prepared using a single monoclonal antibody that was purified from ascities by hydroxylapatite chromatography. The immunoaffinity supports were prepared by immobilization of the purified monoclonal antibody to a preactivated agarose support (Affi-10, Bio-Rad). This was accomplished by quickly rinsing the Affi-10 support with 100 m M H E P E S (pH 7.5) to remove the stabilizing solvent in which the support is packaged, and then adding the purified monoclonal antibody at 3-5 m g / m l in 100 m M HEPES (pH 7.5 ) per ml of support. The coupling reaction was allowed to proceed for 4-8 h at 4°C with agitation. The support was then washed with 100 m M HEPES (pH 7.5) to remove unbound antibody prior to blocking excess reactive sites with 1 M ethanolamine in 100 m M HEPES for 4-16 h at 4 ° C. The resulting immunoaffinity support was packed into a small column equipped with flow adaptors that were adjusted to eliminate all dead space over the packed bed. The packed immunoaffinity column was equilibrated with cell lysis buffer prior to application of the infected cell lysate supernatant

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(described above). The cell lysate supernatant was passed over each column one or more times, depending upon the volume of cell lysate and the column capacity, until all antigen corresponding to the immunoaffinity column in use was depleted from the supernatant. The immunoaffinity column to which the antigen was attached was washed with 100 m M Tris, 500 m M NaC1, 1% NP40, 1% DOC, (pH 7.5) and then with water prior to elution with 50 m M triethylamine (pH 11 ). Following the elution step, the column was reequilibrated with cell lysis buffer for additional chromatography, or with cell lysis buffer plus 0.02% thimerosal for storage. The proteins that were eluted in triethylamine were either lyophilized prior to dialysis, or dialyzed directly versus PBS and then frozen until use. The proteins that were purified in this way were determined to be > 90% pure by densitometry following SDSPAGE. Western blots probed with the serum of a PRV-convalescent pig showed that each one of the eluted proteins had a single antigenic component. Animals immunization Landrace × large white 6-week-old pigs, free of anti-ADV antibodies, were divided into four groups and immunized with either 10/~g/pig of purified ADV glycoprotein II, III, or gp50 with adjuvant formulated as described by Babiuk et al. (1987), or with a commercial modified-live vaccine (PRVMarker, Syntrovet). The virus making up the Syntrovet vaccine consists of deletions in gpX and Tk regions of the ADV genome. All the inoculations were made by the intramuscular route. The pigs immunized with the single glycoproteins were boosted at 14 days postimmunization using the same dose of protein. Fourteen days following booster injection, animals were bled and their sera evaluated for ADV specific antibodies. Animals vaccinated with the modified-live product were bled 28 days after the vaccination. Serological tests A microserum neutralization test (SNT) procedure was performed. Serial twofold dilutions of inactivated sera were mixed with an equal volume of virus (100-150 TCIDso). The incubation for the serum-virus mixture was 1 h at 37°C. Each serum was tested in quadruplicate. Titers are expressed as the last dilution where virus infectivity was neutralized. Complement facilitated serum neutralization test (C'SNT) was performed as above, but the sera samples were diluted in media containing 10% guinea pig serum (Jakubik and Wittman, 1987 ). Antibody-dependent cellular cytotoxicity (ADCC) test was performed with fiat-bottom microtiter plates using the methods described by Rouse et al. ( 1976 ). Target cells were ADV-infected Vero cells labelled with Na2-51CrO4. Effector cells were polymorphonuclear cells collected from age-matched, noninfected pigs. Each well contained 0.1 ml of target cells ( 5 × 104 ) and 0.05 ml of diluted sera, after 1 h of incubation at 37°C the effector cells were added

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(0.1 ml) the concentration of polymorphonuclear cells was adjusted according to the required effector: target ratio. Plates were then incubated for 18 h at 37°C in an atmosphere of 5% CO2 and humidified air. After incubation, 0.1 ml of supernatant was collected from each well and counted in a gamma counter. The results were expressed as % of cytotoxicity after dividing the test counts minus the spontaneous release by the total counts minus the spontaneous release. The total release counts were obtained after the addition of 1% Triton X-100 to target cells. The spontaneous release counts were from wells that contained target and effector cells only. A complement-mediated cytolysis test was performed using ADV-infected 51Cr labelled Vero cells as target cells following the procedures described by Norley and Wardley (1982). The test consisted of adding 51Cr-labelled infected Vero cells (0.1 ml), diluted sera to be tested (0.1 ml) and nonheatinactivated newborn pig serum diluted 1:5 (0.05 ml) as a source of complement. The incubation and evaluation of 51Cr release was carried out as previously described for ADCC. In both ADCC and complement-mediated cytolysis test, all samples were tested in quadruplicate and the means calculated. In all tests each sample was tested at least twice on different days. A known negative serum was included as control in the C'SNT, ADCC, and complement-mediated cytolysis test. RESULTS

Neutralization of virus infectivity as measured by SNT was consistently recorded in sera from pigs immunized with gp50, as well as in some samples from pigs immunized with gill, but it was negative in all samples from pigs immunized with glI. The addition of guinea pig complement to the test, i.e., C'SNT, produced a two to fourfold enhancement of the neutralizing activity of samples from pigs immunized with gp50. It did not affect the neutralization capabilities of sera from pigs immunized with gill. Whereas, the neutralizing activity of samples from animals immunized with glI was drastically changed, those samples consistently had higher C'SNT titers than the samples from pigs inoculated with gp50. Sera samples from the vaccinated animals had titers in both tests that were comparatively higher than the titers of animals inoculated with single glycoproteins. Table 2 shows the results of an assay in which all the samples were tested in both tests on the same day. Antibody-dependent cellular cytotoxicity test, showed a marked difference among the groups immunized with single glycoproteins. The animals immunized with glII were consistently higher in their cytotoxicity values than pigs immunized with glI or gp50 even at high serum dilutions and under stringent conditions, i.e., low effector: target cells ratios (Figs. 1 and 2). In all the experiments the known negative sera produced 5XCr release values (counts per minute) that were either below the spontaneous release counts or just above,

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TABLE 2 ADV antibody titers in pigs immunized with single ADV glycoproteins or a modified-live vaccine gIl

gIIl

ADV glycoproteins Pig no. 888 SNT