Download Protection of Mice from Lethal Infection with Aujeszky`s Disease

J. gen. Viral. (1988), 69, 1411-1414.
Printed in Great Britain
1411
Key words: ADV[immunity/gVl
Protection of Mice from Lethal Infection with Aujeszky's Disease Virus by
Immunization with Purified gVI
By H I R O S H I I S H I I , * Y U I C H I K O B A Y A S H I , M A S A H I K O K U R O K I
YOSHIKATU KODAMA
Ghen Corporation, Gifu Laboratory, Sano, Gifu-City 501-11, Japan
AND
(Accepted 3 March 1988)
SUMMARY
Virus envelope glycoprotein gVI (gp50) of Aujeszky's disease virus (ADV) was
purified from a Nonidet P40-solubilized lysate of ADV-infected Vero cells by
immunoaffinity chromatography using a monoclonal antibody against gVI. Mice
immunized by the purified gVI produced virus-neutralizing antibody and were
successfully protected against subsequent challenge with ADV.
Aujeszky's disease virus (ADV), an alphaherpesvirus, causes great economic loss in the swine
industry. It is known that herpesvirus glycoproteins are the principal antigens which induce host
immune responses (Choppin & Scheid, 1980; Glorioso et al. 1984; Norrild, 1980; Weis et al.,
1983), and can be used as protective antigens for subunit vaccines (Ashley et al., 1985; Roberts et
al., 1985). These observations encouraged us to try to prepare a vaccine for ADV.
In ADV, at least six structural glycoproteins [gI, gII, gIII, gIV, gV and gVI (gp50)] have been
identified (Hampl et al., 1984; Wathen & Wathen, 1984). Several monoclonal antibodies
(MAbs) against gI, gIII and gVI have been shown to neutralize ADV (Hampl et al., 1984;
Mettenleiter et al., 1987; Wathen & Wathen, 1984). Therefore, these three glycoproteins must
have epitopes which induce the production of virus-neutralizing antibodies, and these antigens
are candidates for useful subunit vaccines. Among these glycoproteins, however, the ability of
the antigens to induce a protective immune response has not been fully examined. Moreover,
antigenic drift of gI and gIII has been observed among different virus strains (Ben-Porat et al.,
1986). Thus, we focused on the glycoprotein gVI, which has several similarities to glycoprotein
gD (Petrovskis et al., 1986; W athen & Wathen, 1984), a type-common antigen of herpes simplex
virus (Cohen et al., 1978), and has at least one epitope that induces synthesis of a virusneutralizing antibody (Wathen & Wathen, 1984).
The YS-81 virulent strain of ADV (Fukusho et al., 1981) was plaque-purified three times and
propagated in monolayer culture of a swine kidney cell line. Virions were purified according to
the method of Spear & Roizman (1972). MAbs against ADV structural proteins were prepared
as follows. Spleen cells of BALB/c mice, immunized with heat-inactivated (60 °C, 2 h) purified
virus (200 rag), were fused with P3-NS/1-Ag4-1 (NS-1) myeloma cells in the presence of
polyethylene glycol (Galfr6 & Milstein, 1981). Screening of antibody production in the
supernatants of hybridomas was performed by immunofluorescence assay using cold acetonefixed ADV-infected Veto cells as antigens. Positive cells were cloned four times by limiting
dilution and expanded in the peritoneal cavity of mice. It has been confirmed that the MAbs
38-1, J-68 and J-50 recognize gVI, gII and gI glycoprotein, respectively, by immunoprecipitation
methods using a [3H]glucosamine-labelled cell lysate (Fig. 1) (Lamb et al., 1978). MAb 38-1,
which reacts with gVI, neutralized ADV in vitro and the neutralizing effect was enhanced when
fresh rabbit serum (2%) was added as a source of complement. Moreover, if mice were injected
intraperitoneally, 3 h before virus infection with the ascitic fluid of this clone (0.5 ml/mouse)
they were protected from lethal challenge with ADV. The other MAbs did not show virusneutralizing activity in vitro.
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2
3
31K-
45K-
66K-
92K -
116K-
200K-
1
2
3
,0
e
1
2
50K-i dl~
(b)
3
(c)
1
2
3
4
Fig. 2. SDS-PAGE (a) and immunoblotting (b, c) analyses of purified gVI and anti-gVI polyclonal antibody. (a) SDS-PAGE of purified glycoprotein gVI.
Coomassie Brilliant Blue staining (lanes 1 and 2) and periodic acid-Schiff's staining (lane 3). Lane 1, NP40-solubilized lysate of virus-infected Vero cells. Lanes 2
•
" ,
cent swine serum. Lane 1, NP40-solubilized lysate of virus-infected Vero cells; lane 2, purified
d 3 urified VI. (b) Immunoblottmgof purified gV~ ,using conva|e~_..
.
) a d irnmunoprecipitation(lane 4) using sera of mice ]mmumzed
g~I" laPr~e3, Nl~'0-solubilized lysate of mock-infecteo vero cells. (c)h ...... noblottmg (lanes 1 to 3, n
with puri~ed gVI. Lane l, purified virions; lane 2, NP40-solubilized lysate of virus-infected Vero cells; lane 3, purified gVI; lane 4, [3H]gl~cosamine'labelledcell
lysate was immunoprecipitated by the pooled mouse serum immtmized with gVI.
Fig. 2
Fig. 1
Fig. 1. SDS-PAGE of glycoproteins gVI, gI1 and gI of ADV after immunoprecipitation with MAbs. Vero cells were grown in 3.5 cm plastic dishes and infected
with ADV (YS-81) at an m.o.i, of 50. After adsorption for 1 h at 37 °C, the cells were washed twice with glucose-free medium, and incubated for 8 h. The medium
was replaced with glucose-free medium containing [3H]glucosamine at 0.5 rnCi/ml, and incubated for 3 h. Radiolabelled cells were washed with PBS and lysed with
RIPA buffer, The lysates were immunoprecipitated with MAbs 38-1 (lane 1), J-68 (lane 2) and J-50 (lane 3) which recognize gVI, glI and gI, respectively.
45K-
66K-
92K-
116K-
200K-
1
(a)
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Table 1. Efficacy of active immun&ation of mice with the purified g VI and protection against lethal
infection with A D V
Immunogen
Neutralizingantibody*
gVI (1 i-tg)
gVI (5 ~tg)
PBS + CFA
<2, 2, 2, 4, 4
8, 8, 16, 16, 16
Challenge d o s e
(p.f.u./mouse)
Number of survivors/
total number
104
10"
105
104
103
102
4/10
10/10
0/4
0/4
1/4
3/4
*Each of the serial twofold dilutions of serum was mixed with an equal volume of virus solution (100
TCIDs0/0"I ml). After incubation at 37 °C for 1 h, each mixture (0.2 ml) was inoculated onto a Vero cell culture in
a microplate well. Four wells were used for each dilution. The titres were expressedas the reciprocalsof the highest
serum dilution that neutralized virus in more than 50% of the wells.
In order to examine the efficacy of gVI as a subunit vaccine, the glycoprotein was purified
from virus-infected Veto cells using an immunoabsorbent column, and then injected into mice.
In practice, Vero cells were infected with the virus at an m.o.i, of0-1 and maintained at 37 °C for
18 h. The cells were washed twice in phosphate-buffered saline (PBS pH 7.4) and were scraped
into PBS containing 0.5~ Nonidet P40 (NP40) using a rubber policeman. The solubilized
material was stirred at 4 °C for 10 rain and centrifuged at 20000 r.p.m, for 2 h. The supernatant
was applied to a column of Sepharose 4B (Pharmacia) to which MAb 38-1 had been coupled.
Absorbed material was washed with four column volumes of PBS containing 0-5~ NP40 and
then with the same volume of PBS. Elution was performed with 3 M-potassium thiocyanate
(pH 7.2; Sigma), and eluted material was dialysed against 50 mM-PBS containing 0.05~ NP40.
Following SDS-PAGE (Laemmli, 1970), the material showed one band stained with Coomassie
Brilliant Blue and with periodic acid-Schiff's staining (Zacharius et al., 1969) (Fig. 2a). After
immunoblotting, as described by Towbin et al. (1974), this glycoprotein also reacted with
convalescent serum from a pig inoculated intranasally with ADV (Fig. 2 b). These results suggest
that the purified protein is viral glycoprotein gVI.
Eight-week-old BALB/c mice were immunized with 1 lag or 5 lag of purified gVI emulsified in
complete Freund's adjuvant (CFA), and boosted 2 weeks later by the same injection. Ten days
after the second immunization, five mice of each group were examined for serum antibody titre
and 10 mice were challenged with a lethal dose of ADV. Control mice were immunized with PBS
emulsified in CFA. As shown in Table 1, all the mice which had been immunized with 5 lag of
gVI were protected from lethal infection, whereas all control mice died within 5 days postinfection. The ability to protect against ADV infection, however, was lower in mice immunized
with 1 lag of gVI. The titres of virus-neutralizing antibody were 2 to 4 and 8 to 16 in animals
immunized with 1 lag and 5 lag of gVI, respectively.
Irnmunoprecipitation and immunoblotting experiments were conducted to examine the
specificity of pooled sera of mice immunized with gVI. As shown in Fig. 2 (c), only gVI was
immunoprecipitated from [3H]glucosamine-labelled ADV. Immunoblotting analysis, using
purified virions, NP40-solubilized cell lysate and purified gVI as antigens, also revealed that
gVI specifically reacted with the antiserum. However, a few low M r or diffuse protein bands
were also detected with purified virions and NP40-solubilized lysate. These bands may
represent different glycosylated forms of gVI or degradation products.
The results suggest that glycoprotein gVI might be used as an effective antigen in swine since,
in mice, it induces virus-neutralizing antibody. However, cell-mediated immunity also plays an
important role in preventing herpesvirus infection (Glorioso et al., 1980; Lawman et al., 1980;
Vestergaard, 1980). It will therefore be necessary to elucidate the mechanisms of protective
immune responses induced in swine by gVI from the standpoints of cell-mediated immunity as
well as humoral immunity, if an effective subunit vaccine is to be developed.
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(Received 25 November 1987)
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