Vaccine 19 (2001) 54±58

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In¯uence of antigenic forms and adjuvants on protection against

a lethal infection of Aujeszky's disease virus
Shigeji Katayama*, Kenji Oda, Toshiaki Ohgitani
Division of Veterinary Microbiology, Kyoto Biken Laboratories, 24-16 Makishima-cho, Uji, Kyoto 611-0041, Japan
Received 9 November 1999; received in revised form 10 April 2000; accepted 11 April 2000

Abstract

The in¯uence of antigenic forms and adjuvant types on protection against a lethal infection of Aujeszky's disease virus (ADV)
in mice was investigated. Antiviral IgG2a antibody response against particulate (inactivated ADV) and soluble antigen (ADV
solubilized with deoxychorate±Na) in approximate order of extent was ISA70 > QS-21 > positively charged liposome >
negatively charged liposome > weak negatively charged liposome > ISA25 > lablabside F saponin > aluminum phosphate gel >
non adjuvant. Particulate antigen induced higher IgG2a antibody production than soluble antigen. Particulate antigen combined
with ISA70, ISA25 or positively charged liposome gave 100, 50 and 40% protection to mice, respectively. In contrast, soluble
antigen plus ISA70 conferred 30% protection on mice. Immunogens using the other adjuvants gave R20% protection to mice.
These results indicate that a combination of particulate antigen and an appropriate adjuvant e€ectively induces the production
of antiviral IgG2a antibody and provides protection against a lethal ADV infection in mice. 7 2000 Elsevier Science Ltd. All
rights reserved.

Keywords: Adjuvant; Particulate antigen; Protection; Soluble antigen

1. Introduction body response. Therefore, it is very important to elicit

Murine antibody of the IgG2a subclass plays an im-
portant role in protection against a variety of patho-
gens [1±4]. The infection of Aujeszky's disease virus
(ADV) in mice was inhibited by antiviral IgG2a anti-
body [5]. IgG2a antibody has been associated with
antibody-dependent cell-mediated cytotoxicity (ADCC)
[6±8], antibody-dependent complement-mediated cyto-
toxity (ADCMC) [9±11], and virus neutralization [12].
Soluble antigens such as peptide [13,14], recombi-
nant protein [15,16] or puri®ed antigen [17,18] are
often used in candidate vaccines for pathogen(s) to
ensure their safety. The soluble antigen frequently
induces immunotolerance or the production of IgG1
antibody in mice but does not stimulate an IgG2a anti-
* Corresponding author. Tel.: +81-774-22-4518; fax: +81-774-24-
1407.
the production of IgG2a antibody by the selection of
an adjuvant in order to protect mice against an infec-
tion of ADV.
Quillaja saponaria QS-21 [19,20], Freund's complete
adjuvant (FCA) [21,22], copolymer [16,23], and
immune stimulating complexes (ISCOMs) [24] promote
IgG2a antibody production. Aluminum gel, which
enhances IgG1 antibody response, stimulates IgG2a
antibody production in combination with Bordetella
pertussis [10] or interleukin-12 [25].
We demonstrated previously that the soluble antigen
of ADV with ISA70 adjuvant stimulates an antiviral
IgG2a antibody response, but particulate antigen with
aluminum phosphate gel does not [26]. The aim of this
study is to examine whether antigenic forms and adju-
vant types in¯uence antiviral IgG2a antibody response
and protection against virulent ADV infection.

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 S. Katayama et al. / Vaccine 19 (2001) 54±58
2. Materials and methods
2.1. Virus and cell
ADV Iwate strain [27] was propagated and titrated
in Crandell feline kidney (CRFK) cells.
The cells were cultured in Eagle's minimal essential
medium (Eg-MEM) supplemented with 5% fetal calf
serum (FCS).
2.2. Preparation of antigens
Particulate and soluble antigens derived from puri-
®ed ADV were prepared according to the method
described previously [26].
2.3. Adjuvants
Aluminum phosphate gel (alum) was purchased
from Superfos Biosectors Co. Ltd (Denmark). QS-21
was puri®ed from Quillajanin (Maruzen Pharmaceuti-
cals, Japan) extracted from the bark of the South
American tree Quillaja saponaria Molina by the
method of Kensil et al. [28]. Lablaboside F saponin of
Dolichos lablab origin was kindly provided by Dr M.
Yoshikawa (Kyoto Pharmaceutical University, Japan).
Oil-based adjuvants, ISA25 (oil-in-water type) and
ISA70 (water-in-oil type), were purchased from Seppic
Co., Ltd (France). Three di€erently charged (positive,
weak negative and negative) liposomes; COATSOME
EL (lyophilized), were purchased from Nippon Oil and
Fat Co. Ltd (Tokyo, Japan).
2.4. Preparation of immunogens
Immunogens were prepared as previously described
[26]. Each antigen with virus titer of 1.0  107 plaque
forming units (pfu)/dose before inactivation was used.
The respective antigen was adsorbed onto alum (15 w/
v%) at pH 6.5 at room temperature. The saponins
were mixed with each antigen at concentrations of
20 mg per dose. The lyophilized liposomes were resus-
pended in 1 ml (10 doses) of each antigen suspension
according to the attached manual. Each antigen was
emulsi®ed together with ISA25 (o/w) at a ratio of
7.5:2.5 or ISA70 (w/o) at a ratio of 3:7, respectively.
Antigen alone was served as control immunogen.
2.5. Animal experiments
Four-week-old female Balb/c mice were purchased
from Nippon SLC Inc. (Shizuoka, Japan). Eighteen
groups of 15 mice each were injected once intramuscu-
larly with 0.1 ml of each immunogen. Four weeks
after the injection, 10 mice of each group were chal-
lenged with 100 LD50 of ADV Iwate strain. Serum
55
samples were collected from the remaining ®ve mice at
the same time. The survival rate (%) was determined
14 days after the challenge.
2.6. Antibody measurement by enzyme-linked
immunosorbent assay (ELISA)
To measure the titer of anti-ADV IgG subclass anti-
body, an ELISA was carried out as previously
described [26]. Five serum samples per group were
diluted 1/400. ELISA titer was revealed as optical den-
sity at a wavelength of 492 nm.
2.7. Statistical analysis
SD values for the mean antibody titers, obtained
with various combinations of antigenic forms and
adjuvants, were calculated by Student two-tailed t-test.
3. Results
3.1. Anti-ADV antibody responses stimulated by a
combination of antigenic forms and adjuvants
The result of ELISA titer of total IgG antibody is
shown in panel A of Fig. 1. The production of total
IgG against both particulate and soluble antigen was
enhanced by the following adjuvants; QS-21, positively
charged liposome (PL), negatively charged liposome
(NL), ISA25, ISA70 and alum. In all cases except the
combination of particulate antigen plus alum, these re-
sponses were signi®cant ( p < 0.05). Particulate antigen
plus ISA70 induced the largest response of total IgG.
Soluble antigen plus alum induced the next largest re-
sponse.
The result of ELISA titer of IgG1 antibody is
shown in panel B of Fig. 1. The production level of
IgG1 antibody was signi®cantly increased by particu-
late antigen plus ISA70, ISA25, PL and NL ( p < 0.01)
in that order, and by soluble antigen plus alum, PL,
ISA70, ISA25 and NL ( p < 0.05) in that order. Sol-
uble antigen plus alum and particulate antigen plus
ISA70 stimulated the largest IgG1 antibody responses,
respectively.
The result of ELISA titer of IgG2a antibody is
shown in panel C of Fig. 1. Antiviral IgG2a antibody
responses against both antigens were greatly stimulated
by ISA70, QS-21, PL, NL and weak negatively
charged liposome (WNL), in that order. Signi®cant
di€erences ( p < 0.05), compared to the control, in
antibody production were observed. Particulate anti-
gen plus ISA70 stimulated the largest antibody re-
sponse, and then antigen plus QS-21 the next.
56 S. Katayama et al. / Vaccine 19 (2001) 54±58

Fig. 1. ELISA titer of IgG subclass after injection of a combination of antigenic form and adjuvant. Particulate (open bar) or soluble antigen
(solid bar) plus an adjuvant was injected once into a group of ®ve mice. After 4 weeks, the ELISA titer of IgG to antigen was measured in
serum samples. Each panel reveals ELISA titer of total IgG (A), IgG1 (B) or IgG2a (C) antibody. Statistical comparisons between particulate
and soluble antigen and between each adjuvant and control are made by the Student two-tailed t-test.

3.2. E€ect of antigenic forms and adjuvants on

protection

Particulate antigen plus adjuvant conferred e€ective

protection on mice, but soluble antigen plus adjuvant
did not (Fig. 2). Particulate antigen with ISA70, ISA25
or PL gave 100, 50 and 40% protection to mice, re-
spectively. On the other hand, soluble antigen plus
ISA70 conferred 30% protection on mice which was
the maximum in groups injected with soluble antigen.

4. Discussion

Schijns et al. [5] reported that inactivated ADV

alone can induce antiviral IgG2a antibody production
and confer protection against a lethal ADV infection.
Puri®ed glycoproteins of ADV with an adjuvant such
as FCA, Freund's incomplete adjuvant (FIA) and hy-
droxide aluminum can also provide protection to mice
[18].
In this study, each antigen with ISA70 (w/o type)
elicited the highest level of antiviral IgG2a antibody Fig. 2. E€ect of antigenic form and adjuvant on protection against a
lethal ADV infection. Immunogen consisting of particulate (open
production and protection among all combinations of bar) or soluble antigen (solid bar) and adjuvant was injected once
antigenic forms and adjuvants. In general, mineral-oil into a group of 10 mice. After 4 weeks, each group was challenged
based adjuvants have a high potency for immune re- with 100 LD50 of a virulent ADV. Survival rate (%) indicates the
sponse [22,29]. Water-in-oil type emulsions such as result on day 14 after challenge.
 S. Katayama et al. / Vaccine 19 (2001) 54±58
FCA and FIA also tend to elicit antiviral IgG2a anti-
body production for soluble antigen [21,30]. On the
other hand, the result obtained here showed that
ISA25 (o/w type) could not elicit a level of antibody
production and protection as high as ISA70 (w/o
type), indicating that the emulsion type of the immu-
nogen may in¯uence the immune response. We con-
sider this is because antigen adsorbed onto the surface
of oil drops of o/w type emulsion may be released fas-
ter than antigen in w/o type emulsion and then it will
be rapidly treated by immunocompetent cells. Ad-
ditionally, although antigen plus ISA25 showed a
lower level of antiviral IgG2a antibody production
than antigen plus QS21, the former conferred better
protection than the latter, suggesting that the challenge
with a virulent ADV will eciently boost IgG2a anti-
body production in mice injected with the immunogen
containing ISA25.
Our results showed that positively charged liposome
induced a good immune response to each antigen. In
many liposomal vaccine candidates, an antigen has
been encapsulated in the liposome [31,32] and stimu-
lated a Th1 type immune response which involves the
production of IgG2a antibody and interferon-g [31,33].
However, Therien et al. [34] demonstrated that antigen
on the surface of a liposome stimulated a stronger
immune response than antigen encapsulated in the
liposome. These results indicate that properties such as
formulation and the charge of an antigen and liposome
in¯uence immune response(s).
In this study, immunogen with QS21 induced a
higher level IgG2a antibody response than that with
positively charged liposome, and conferred less protec-
tion than the liposome. Wu et al. reported that QS21
has been shown to elicit Th1 type immune responses in
mice, which involve the production of IgG2a antibody
and interferon-g and the induction of CD8+ cytotoxic
T lymphocyte (CTL) [35]. However, our results con-
cerning adjuvanticity of QS21 suggest that the immu-
nocompetent cells involved in defence against ADV
infection cannot be activated by the saponin. In con-
trast, lablabside F saponin, without hemolytic activity,
could not induce high level IgG2a antibody pro-
duction, and the immunogen conferred no protection.
In a previous study, lablabside F plus inactivated
ADV induced a moderate IgG2a antibody response
when the immunogen was injected twice in mice [26].
Therefore, administration times and the amount of
saponin or antigen in the immunogen should be
adjusted to optimize the immune response.
Neither aluminum gel plus particulate, nor alumi-
num gel plus soluble ADV antigen conferred protec-
tion on mice. In our previous study, aluminum gel
plus the antigens each induced the production of IgG1
but not IgG2a antibody [26]. Nakamura et al. [18]
reported that aluminum gel plus puri®ed gB of ADV
57
a€orded protection of mice, but a larger amount of
antigen was required to obtain the same e€ect as FCA
plus antigen. It was indicated that aluminum gel could
not e€ectively induce protective immunity against a
lethal ADV infection.
The combination of an antigenic form and an adju-
vant can induce the production of IgG2a antibody
which plays an important role in protection against
ADV infection. We conclude that vaccines must be
designed to generate an essential immune response for
protection against a pathogen.
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