Download Comparison of the immune response elicited by infectious and

1691
Journal of General Virology (1991), 72, 1691-1694. Printed in Great Britain
Comparison of the immune response elicited by infectious and inactivated
foot-and-mouth disease virus in mice
P. G. Piatti,* t A. Berinstein, O. J. Lopez, M. V. Borca, F. Fernandez, A. A. Schudel and
A. M. Sadir
Institute o f Virology, CICV, INTA-Castelar, Argentina
The immune response to foot-and-mouth disease virus
(FMDV) elicited by infection or immunization with
inactivated virus in adult mice was examined. A model
of adoptive transfer of immunocompetent cells was
used for this purpose. The results presented here
indicate that both short- and long-term secondary
immune responses elicited by high doses of inactivated
virus are indistinguishable, at the humoral or cellular
level, from that observed after infection. The responses
to inactivated or infectious virus were both efficiently
mediated by B cells. However, immunization with low
doses of inactivated virus induced a response which,
although effective in aborting infection, was fully
dependent on FMDV-specific T cell cooperation.
These findings suggest that the different immune
responses observed after infection and immunization
are mainly the result of the different viral mass
presented to the immune system in each case.
Foot-and-mouth disease virus (FMDV), the only member of the aphthovirus genus within the Picornaviridae
family (Bachrach, 1977), causes an economically important disease affecting cloven-hoofed animals (Cottral et
al., 1975). In contrast to natural infection, current
FMDV vaccines prepared with inactivated virus and
adjuvants elicit short-lived protection (Morgan et aL,
1980). Some of the possible explanations, as yet
unsupported by experimental data, could be a constant
antigenic boosting due to virus persistence in infected
animals (Gebauer et ai., 1988; our unpublished data), the
induction of more efficient immune mechanisms after
infection (Lopez et al., 1990) or quantitative differences
due to variation in the mass of virus presented to the
immune system after infection or vaccination. The
objective of this research was to evaluate the last
possibility by characterizing the humoral and cellular
immune responses of adult mice to infection and
different doses of inactivated FMDV.
The neutralizing antibody response up to 120 days
post-infection (p.i.) after intraperitoneal (i.p.) immunization with different amounts of inactivated FMDV (0-1,
0.5, 1 or 10 ~tg) without adjuvant is shown in Fig. 1.
Antibody titres were measured by using the constant
serum/variable virus method on suckling mice (Cunha et
al., 1957). Infection of mice was carried out by i.p.
inoculation of 104 suckling mouse 50% lethal doses
(SMLDso) of FMDV subtype O1 Campos (O1C) diluted
in 0-5 ml of PBS per mouse. FMDV was inactivated with
binary ethylenimine, according to Bahnemann (1975)
and virus purification was performed as described by
Bachrach et al. (1964). Mice were immunized by i.p.
inoculation of inactivated virus in PBS. The duration
and magnitude of the immune response correlates
directly with the mass of inactivated virus used for
vaccination. Thus, animals which received 0.1 ~tg of
inactivated virus developed transitory and low titres of
neutralizing antibodies. Immunization with less than 0-1
~tg (0.075, 0.025 and 0-01 ~tg; data not shown) demonstrated that 0.1 ~tg was the minimum dose capable of
eliciting a reproducible response in this particular
system. A clear difference was obtained when 0.5 or 1 ~tg
of inactivated virus was used for immunization. Interestingly, the titres of neutralizing antibody and the
maintenance of antibody titres in animals which
received 10 ~tg of inactivated virus were not significantly
different from those observed after inoculation with
infectious virus. The dose-response relationship of these
results is in agreement with the results of Rweyemamu et
al. (1984) obtained in cattle.
To gain insight into the cellular mechanisms mediating these antibody responses, an experimental model of
adoptive transfer of immunocompetent cells was utilized
(Borca et al., 1986). Recipient mice were irradiated with
580 rad 24 h prior to FMDV infection and 107 spleen
cells from mice infected 8 days previously or from virusimmunized donors were transferred by i.p. inoculation
t Present address: Plum Island Animal Disease Center, USDA,
Greenport, New York 11944, U.S.A.
0000-9908 © 1991 SGM
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1692
Short communication
I
I
I
Table 1. Transference of the short duration secondary
i
immune response (8 days p.i.) induced by high and low doses
of inactivated FMD V
4
Viraemia
days post-reconstitution
- V / O ~
3
Group*
5
I
2
3
4
5
6
7
8
9
lo
+
+
+
+
+
+
-+
6 (NI)
13
14
+
+
+
+
+
+
+
-
2
\
0
!
0
50
100
150
'
•
200
250
Days p.i.
Fig. 1. Neutralizing antibody response to different doses of purified
inactivated FMDV. Adult BALB/c mice were infected (11) or
immunized i.p. with 10 Ixg (A), I lig (V), 0.5 lig (0) or 0.1 ltg (0) of
purified inactivated FMDV. Results are expressed as neutralizing
indices (NI) and titres represent the mean of two determinations at
each time.
24 h after infection. Spleen cells were o b t a i n e d by gently
h o m o g e n i z i n g the o r g a n i n 199 m e d i u m ( G i b c o ) c o n t a i n i n g 5 % foetal calf serum. E r y t h r o c y t e s were lysed with
T r i s - N H , C1 p H 7.2 a n d a d h e r e n t cells were r e m o v e d b y
i n c u b a t i o n for 1 h at 37 °C (5% CO2 atmosphere). T cells
a n d B cells were purified from the n o n - a d h e r e n t cells by
i n c u b a t i o n for 1 h at 37 °C with r a b b i t a n t i - m o u s e Ig
s e r u m or a n t i - T h y 1.2 m o n o c l o n a l a n t i b o d y ( M A b ) ,
respectively, i n the presence of g u i n e a - p i g c o m p l e m e n t .
T h e purity of T- a n d B-enriched p o p u l a t i o n s was
assessed by direct i m m u n o f l u o r e s c e n c e using fluorescein
isothiocyanate-labelled a n t i - T h y 1.2 M A b a n d antimouse Ig. O n l y cell p r e p a r a t i o n s s h o w i n g m o r e t h a n 97 %
p u r i t y were used. V i r a e m i a was d e t e r m i n e d by intram u s c u l a r i n o c u l a t i o n o f a litter o f six 6-day-old suckling
mice with 0.05 ml o f blood o b t a i n e d from two reconstituted a n i m a l s each day, diluted 1/10 i n PBS. Results are
expressed as positive (at least one out of six mice died) or
n e g a t i v e (no mice died). As c a n be seen in T a b l e 1, n o
significant difference i n either the p r o d u c t i o n of neutralizing a n t i b o d y or the e l i m i n a t i o n of i n f e c t i o n was
observed for a n i m a l s w h i c h received cells 8 days after
sensitization f r o m d o n o r s w h i c h were either infected or
were i m m u n i z e d with 10 ~tg o f i n a c t i v a t e d virus. I n b o t h
cases, r e p o p u l a t i o n o f recipients w i t h u n f r a c t i o n a t e d
spleen cells or purified B cells was equally effective i n
eliciting the p r o d u c t i o n of n e u t r a l i z i n g a n t i b o d y a n d
e l i m i n a t i o n o f viraemia. S i m i l a r results were observed
w h e n the d o n o r cells were o b t a i n e d from a n i m a l s
infected or i m m u n i z e d with 10 Ixg of i n a c t i v a t e d virus
120 days earlier (Table 2).
Both e x p e r i m e n t s show t h a t there is a direct correlation b e t w e e n the ability of t r a n s f e r r e d B cells a n d
+
+
+
+
+
(O.40)
(2.50)
(2-00)
(0.30)
(2.I4)
(0.24)
(0-24)
(2.20)
(2.20)
(o.io)
* Group 1 refers to the transference of unfractionated cells from
uninfected non-immunized donors; group 2, unfractionated ceils from
infected donors; group 3, B cells from infected donors; group 4, T cells
from infected donors; group 5, unfractionated cells from donors
immunized with 0-1 lig of inactivated FMDV; group 6, B cell from
donors immunized as group 5; group 7, T cells from donors immunized
as group 5; group 8, unfractionated cells from donors immunized with
10 lig of inactivated FMDV; group 9, B cells from donors immunized as
group 8; group 10, not reconstituted.
Table 2. Transference of long lasting secondary immune
response (120 days p.i.) induced by high and low doses of
inactivated FMD V
Viraemia
days post-reconstitution
Group*
5
1
2
3
4
5
6
7
+
+
+
+
+
8
+
6 (NI)
+
+
(2.40)
(2.50)
(2.14)
(2.20)
(2.20)
(0.30)
(0-20)
+ (0.10)
7
.
.
.
.
.
+
+
+
8
.
.
.
.
.
9
.
.
.
.
.
.
+
+
+
10
+
14
.
.
.
.
NVt
ND
ND
ND
ND
ND
.
.
.
.
.
+
+
11
.
.
.
.
+
+
+
* Group 1 refers to transference of unfractionated ceils from infected
donors; group 2, B cells from infected donors; group 3, unfractionated
cells from immunized donors (10 lig); group 4, B cells from immunized
donors (10 lig); group 5, unfractionated cells from immunized donors
(0.1 ~tg); group 6, B cells from immunized donors (0.1 lag); group 7,
unfractionated cells from untreated mice; group 8, non-repopulated
mice.
ND, Not determined.
u n f r a c t i o n a t e d cells to a b o r t i n f e c t i o n a n d the presence
o f n e u t r a l i z i n g a n t i b o d y . However, w h e n 0.1 p.g of
i n a c t i v a t e d virus was used to p r i m e donors 8 days before
the cell transfer, r e c o n s t i t u t i o n with T cells or B cells
alone did n o t result i n a n efficient i m m u n e response
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Short communication
14( I.~
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Table 3. Collaborative effect of T cells in mice immunized
with small doses of inactivated FMD V (0.1 I~g) at 8 days p.i.
12
Viraemia
days post-reconstitution
•~ lO
-~
O
8
6q
•
O.
~0
4
2
0
0
I
I
10
20
I
30
T cells (%)
I
I
40
50
60
Fig. 2. Relationship between immunizing dose and neutralizing
antibody response in T cell-B cell collaboration. Donor mice were
infected (O) or i m m u n i z e d with 0.1 ~tg of inactivated purified virus (O)
8 days before reconstitution. Receptors were reconstituted with 107
cells using different proportions of T cells. The presence or absence of
viraemia was determined as described in the text.
(Table 1). To determine whether this was due to the
absence of T cells, mice were reconstituted with purified
B cells and different proportions of T cells. The addition
of T lymphocytes to the cell suspension used for
repopulation reduced the period of viraemia to a degree
which correlated directly with the proportion of T ceils
present. When equal amounts of T cells and B cells were
used (5 x 106 T cells plus 5 x 106 B cells), viraemia
became undetectable at the same time post-reconstitution as observed for animals repopulated with untreated cells obtained from infected animals (Fig. 2). This T
cell-dependence in a dose-response fashion raised the
question of how effective the T cells could be in
amplifying the B cell response.
Purified B ceils and T cells were obtained from mice
8 days post-immunization with 0-1 p.g of purified inactivated antigen. Different quantities of B cells were-added
to 5 x 106 T cells (this number o f T cells was shown to be
sufficient to reduce viraemia to 6 days in the experiment
described in Fig. 2) and the mixtures were used for
repopulation. As shown in Table 3, even the smallest
number of B cells tested (5 x 105) was able to eliminate
the infection. Thus, doses of B ceils 20 times lower than
those shown to be insufficient to abort viraemia by
themselves, are effective when T cells are present.
These data indicate that for 0.1 Ixg of inactivated virus
the mechanism of virus elimination is mediated by B
cells, since purified T cells were previously shown to be
ineffective, but that T cells play an important role in
enhancing the B cell response. Thus the quantity of B
cells sensitized by immunization with 0.1 p.g of inactivated virus is insufficient to produce an effective
immune response and requires amplification which is
Group*
5
6
1
+
+
2
3
+
+
.
+
4
.
5
6
+
+
.
.
7
+
+
8
+
.
9
+
+
.
7
8
+
+
.
.
+
.
.
+
.
.
.
+
.
.
.
+
10
11
+
+
+
+
--
--
+
+
.
+
.
.
.
.
+
.
+
+
.
.
.
.
.
9
.
.
--
.
.
+
* G r o u p 1 refers to transference o f 107 unfractionated cells from noni m m u n i z e d donors; group 2, 107 unfractionated ceils from F M D V immunized donors; group 3, 107 B cells from F M D V - i m m u n i z e d
donors; group 4 to group 6, different a m o u n t s of B and T cells taken
from F M D V - i m m u n i z e d donors ( 4 : 5 x 106 B and 5 x 106 T; 5:
2.5 x 106 B and 2.5 x 106 T; 6 : 5 x l0 s B and 5 x 106 T); group 7,
5 x 106 B cells from F M D V - i m m u n i z e d donors plus 5 x 106 T cells
from K L H - i m m u n i z e d donors; group 8, 5 x 106 B cells from K L H i m m u n i z e d donors plus 5 x 106 T cells from F M D V - i m m u n i z e d
donors; group 9, not reconstituted. Groups 7 and 8 were inoculated
with 30 ~tg of K L H in PBS i.p. simultaneously with the virus challenge.
mediated by T cells. Surprisingly, T cells sensitized with
0.1 txg of FMDV were also able to help cells obtained
from donors 21 days post-immunization with 30 ~tg of
keyhole limpet haemocyanin (KLH) in Freund's incomplete adjuvant in eliminating the infection. This result
emphasizes the importance of T cells when low doses of
inactivated FMDV are used for immunization. Thus,
FMDV-primed T cells could alter the response from that
of a primary reaction (group 7, Table 3) to a secondary
one (groups 5, 6 and 8, Table 3). This fact could indicate
that even at a very low frequency FMDV B precursor
cells are effective in eliminating the infection if FMDVsensitized T cells are present. The analysis of T celldependence of the secondary response to 0.1 Ixg of
inactivated virus was extended to determine whether this
event requires T cells and B cells to be sensitized by the
same antigen (FMDV-specific response) or whether it
could be mediated by soluble factors released by T cells
sensitized by an unrelated T cell-dependent antigen
(KLH). As can be seen in Table 3, T cells sensitized to
KLH did not drastically modify the neutralizing antibody response observed in mice reconstituted with
purified B cells from animals immunized with 0.1 Ixg of
inactivated FMDV, suggesting that the dependence is
virus-specific. The need for T cell collaboration observed
when low doses of inactivated virus are used can be
compared to those results obtained in an in vitro system
by Collen et al. (1989).
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1694
Short communication
In summary, we have shown that there does not seem
to be any difference in the immune response elicited by
experimental infection or by immunization with high
doses of inactivated FMDV, both being relatively T cellindependent. On the other hand, the response to
infection mediated by cells sensitized to low doses of
inactivated FMDV is efficient only when T cells are
specifically sensitized. These T cells can in turn help B
cells to eliminate infection, even if they were not
previously exposed to FMDV antigen. Although the role
of T cells after infection or immunization with 10 ~tg of
inactivated virus was not examined, its importance as an
amplification factor can not be discounted. Unfortunately no data are available with regard to either the
mass of viral antigen produced during infection of cattle
or the immune response elicited in this species by the
administration of massive amounts of inactivated virus.
The amount of 140S fraction present in commercial
vaccines is approximately 10 ~tg and the amount of viral
particles produced during the FMDV infection is
probably far greater than this. Under the experimental
conditions presented here it seems likely that the
differences observed between the immunization with
inactivated virus could be due solely to the virus mass
that is in contact with the immune system in either case.
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The authors wish to acknowledge the excellent technical assistance
of Ana M. Hernandez, Carmen Maciel and Antonio Varone. P.G.P.
and A.B. are research fellows of the Consejo Nacional de Investigaciones Cientificas y Tecnicas.
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