Download Protective immunity against bovine leukaemia virus (BLV) induced

Journal o f General Virology (1991), 72, 1887-1892.
Printed in Great Britain
1887
Protective immunity against bovine leukaemia virus (BLV) induced in
carrier sheep by inoculation with a vaccinia virus-BLV e n v recombinant:
association with cell-mediated immunity
Kazue Ohishi, ~ Hidemi Suzuki, 1 Toshiko Yamamoto, 1 Tadashi Maruyama, 1 Keizaburo Mild, 1
Yoji Ikawa, 2 Shigeru Numakunai, 3 Kosuke Okada, 3 Kan-ichi Ohshima 3 and
Masanobu Sugimoto 1.
1Corporate Research and Development Laboratory, Tonen Corporation, 1-3-1 Nishi-Tsurugaoka, Ohi-machL Iruma-gun,
Saitama 354, 2Tsukuba Life Science Center, The Institute of Physical and Chemical Research and 3Department of
Veterinary Pathology, lwate University, Japan
The effects of vaccination of sheep with a recombinant
vaccinia virus (rVV) expressing the bovine leukaemia
virus (BLV) envelope glycoprotein (gp60) were studied
by determining BLV titres in peripheral blood leukocytes after vaccination and challenge. The proliferation
of BLV was suppressed markedly, not only when rVV
was inoculated prior to challenge with BLV, but also
when it was inoculated after challenge. These results
indicate that vaccination with rVV induces protective
immunity that can suppress the growth of BLV in
carrier animals. Since rVV induced a strong anti-BLV
delayed-type hypersensitivity response without producing detectable levels of binding or neutralizing
antibodies, and there was no apparent correlation
between the humoral immune response and BLV
proliferation, a cell-mediated immune response was
assumed to play a major role in protective immunity.
Introduction
responses were carried out. The experiments indicate
that vaccination with rVV suppresses the proliferation of
BLV in carrier animals, presumably by augmenting host
cell-mediated immune responses.
Bovine leukaemia virus (BLV) is the aetiological agent of
enzootic bovine leukaemia; the nucleotide sequence and
gene organization of its genome are similar to those of
human T cell leukaemia virus types I and II (HTLV-I
and -II) (Seiki et al., 1983; Sagata et al., 1984, 1985;
Shimotohno et al., 1985). Since BLV induces leukaemia
in sheep at a high incidence within a relatively short
period of time (Olson & Baumgartener, 1976; Kenyon et
al., 1981), this animal system provides a good disease
model for both BLV and HTLV infection.
A relatively long incubation period exists before the
onset of disease after infection with HTLV-I and -II, and
human immunodeficiency virus (HIV). Therefore, it is
likely that the host immune response against these
viruses is crucial in determining the rate of disease
development.
We have undertaken a series of experiments in sheep
using a recombinant vaccinia virus (rVV) expressing the
BLV envelope glycoprotein (gp60) as an experimental
probe. Vaccination with rVV was done either prior to or
after challenge with BLV, and the BLV titre in
peripheral blood leukocytes (PBLs) was determined to
evaluate the effect of the vaccination. Simultaneous
assessments of humoral and cell-mediated immune
0001-0056 © 1991 SGM
Methods
Animals. Male sheep (3 to 4 years old) were used for the vaccination
and challenge experiments in Expt. 1, 8-month-old male and female
sheep were used for the study of the delayed-type hypersensitivity
(DTH) response induced by rVV inoculation, as well as for the
vaccination and challenge experiments in Expt. II, and 8-month-old
male animals were used in Expt. III. All the animals vaccinated with
rVV were kept in a pen furnished with isolated drainage, and their
excretions were collected and sterilized with an autoclave or
disinfectant. All experiments were conducted according to the
guidelines of The Ministry of Education, Science and Culture, Japan.
All animals were serologically BLV-negative before the experiments.
A cow known to have been BLV-positive since 1984 was used as a
donor of BLV-infected PBLs for challenge; 2500 to 5000 syncytia/106
PBLs were formed. In situ hybridization indicated that about 10 ~ of all
PBLs from this cow were BLV-positive.
Viruses and cells. We used an rVV (mO-HA/ATI) expressing the
BLV envelope glycoprotein under the control of a promoter of the
A-type inclusion body protein gene of cowpox virus (Funahashi et al.,
1988), and an attenuated vaccinia virus strain, LC16mO (kindly
donated by the Chiba Serum Institute), as a vector (Hashizume et al.,
1985). The construction of rVV and its immunogenicity in rabbits have
been reported (Ohishi et al., 1990). Rabbit kidney cells (RK13) were
grown at 37 °C in Eagle's MEM supplemented with 10% calf serum
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K. Ohishi and others
and used for the growth of rVV and LC16mO. Ovine embryonic cells
(KTO-1) (kindly donated by Dr H. Koyama, Kitasato University,
Japan) were cultured in Dulbecco's MEM (DMEM) supplemented
with 10% foetal calf serum and were used as indicator cells in the
syncytium formation assay.
Isolation of PBLs. Briefly, buffy coat cells were obtained by
centrifuging heparinized blood at 1600g for 20 min at 4 °C. The PBL
fraction was obtained by centrifuging buffy coat cells in 60% Percoll
solution at 1600g for 20 min at room temperature.
3
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Determination of neutralizing antibody titre by a syncytium inhibition
assay. The assay was performed as described above except that sample
serum (2% final concentration) was added, allowing its neutralizing
activity to be determined. When bovine anti-BLV serum and BLVinfected cells were added to this system, syncytium formation was
inhibited completely (data not shown).
ELISA. Titration of anti-BLV antibodies was carried out using a
standard ELISA method. Briefly, BLV virions in 0.1% Triton X-100
were adsorbed to each well of a 96-well plate. After coating with 0.5 %
casein in PBS, ovine serum diluted 1/100 was added to each well and
incubated at 37 °C for 1 h; peroxidase-conjugated donkey anti-sheep
IgG or IgM was used as a secondary antibody. The absorbance at
492 nm was read using an automatic ELISA reader.
Immunodiffusion. This was carried out using partially purified BLV
virions as the antigen and sera from cattle with leukaemia as a positive
control, both from a commercial kit obtained from the Kitasato
Institute, Tokyo, Japan.
DTH responsesof sheep ~aecinated with rVV. Sheep were vaccinated
with 108 p.f.u, rVV, or with parental VV LC16mO as a control, and 7
days after vaccination they were challenged intradermally with BLV
(2 units/0.1 ml, as determined using the immunodiffusion kit) or
keyhole limpet haemocyanin (KLH) (0.5 rtg/0"l ml) as a control. Skin
biopsy was carried out 3, 24, 48 and 72 h after challenge, and biopsies
were fixed with 10% formalin and embedded in paraffin; thin sections
were stained with haematoxylin and eosin, and observed under light
microscopy.
Results
Determination o f challenge dose
B L V - i n f e c t e d b o v i n e P B L s (8 × 102, 8 × 103 o r 8 × 104)
were inoculated subcutaneously into groups of two sheep.
Infection of sheep PBLs with BLV was assessed using a
s y n c y t i u m f o r m a t i o n assay, as well as b y an i m m u n o d i f -
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Syncytiumformation assay. The assay was performed according to the
procedure described by Itohara & Mizuno (1984). Ovine embryonic
cells (105) were grown for 24 h in a 35 mm dish with 2 mm grids and
inoculated with 106 BLV-infected ovine PBLs in DMEM containing
1% DMSO and 4 Ixg/ml polybrene. Cells were cultured for 24 h, PBLs
were removed and culturing was continued for another 5 days, after
which the cells were fixed with 2% paraformamide solution, and
stained with May-Gruenwald and Giemsa stain. Cells containing more
than five nuclei were counted as syncytia. There was a roughly linear
relationship between the number of syncytia formed and doses of PBLs
between 105 and 106 cells (data not shown). To confirm the
reproducibility of the assay system, we assessed the number of syncytia
formed by an aliquot of a standard BLV suspension obtained from the
supernatant of a culture of BLV-producing foetal lamb kidney cells in
Expt. II and III; the number of syncytia formed was relatively constant
(between 2430 and 3410 in Expt. II, and 2030 and 2620 in Expt. III).
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Fig. 1. Suppressive effect of vaccination with rVV on the growth of
BLV in PBLs of sheep challenged with 105 BLV-infected PBLs. (a and
e) Sheep [no. 12 (O), 13 (O), 18 (A) and 20 (z~)] inoculated
intradermally with 108 p.f.u, rVV; (b and d) sheep [no. 17 (O), 19 (C)),
21 (Ak), 23 (A) and 35 ( I ) ] inoculated with 108 p.f.u. LC16mO. At 20
weeks p.i., all sheep were challenged subcutaneously with 105 BLVinfected bovine PBLs. (a and b) The BLV titre was determined by a
syncytium formation assay; (e and d) the antibody response of each
sheep was assessed by ELISA. Vac, inoculation with rVV or LC 16toO;
Ch, challenge with BLV-infected bovine PBLs.
f u s i o n test. A l l s h e e p w e r e B L V - p o s i t i v e in o n e o f t h e
tests by d a y 60 ( d a t a n o t s h o w n ) , i n d i c a t i n g t h a t 8 x 10 z
BLV-infected bovine PBLs could infect sheep. Based on
t h e s e results, w e i n o c u l a t e d s h e e p w i t h 10 s P B L s in E x p t .
I a n d I I I , a n d 2 × 103 P B L s in E x p t . II.
Effects o f vaccination with r V V on the growth o f B L V in
naive sheep challenged with a large dose o f BLV-infected
PBLs
In Expt. I sheep were divided into two groups: group A
(four s h e e p ) w a s v a c c i n a t e d i n t r a d e r m a l l y w i t h 108 p.f.u.
r V V ; g r o u p B (five s h e e p ) w a s i n o c u l a t e d w i t h 108 p.f.u.
L C I 6 m O . A t 20 w e e k s p o s t - i n o c u l a t i o n (p.i.), all s h e e p
w e r e s u b c u t a n e o u s l y c h a l l e n g e d w i t h 105 b o v i n e P B L s .
S y n c y t i a w e r e d e t e c t e d in all s h e e p in g r o u p B 3 w e e k s
p o s t - c h a l l e n g e (p.c.) (Fig. l b ) , t h e s e v a l u e s r e a c h e d
p l a t e a u l e v e l s at a b o u t 6 w e e k s . I n c o n t r a s t , t h e n u m b e r
o f s y n c y t i a d e t e c t e d in a n i m a l s in g r o u p A (Fig. 1 a) w a s
v e r y l o w ; t h e n u m b e r o f s y n c y t i a f o r m e d p e r 106 P B L s
d e t e c t e d at 26 w e e k s p.c. w a s b e t w e e n 12 a n d 17, w h e r e a s
t h a t in a n i m a l s in g r o u p B w a s b e t w e e n 60 a n d 340.
The antibody response was monitored simultaneously.
Fig. l(c and d) shows the anti-BLV IgG response
a s s e s s e d by E L I S A . V a c c i n a t i o n w i t h r V V a l o n e d i d n o t
induce a detectable antibody response. Challenge with
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Protective immunity against B L V in sheep
BLV-infected bovine PBLs caused the antibody titre in
animals in group A to increase sharply within 3 weeks
p.c., whereas those of the controls (group B) increased
rather slowly. Thus, animals in group A showed a
maximal antibody response more quickly than those in
the control group, although the responses at 26 weeks p.c.
did not differ very much, suggesting that rVV inoculation stimulated helper T cells without inducing detectable antibodies. One animal (no. 19) which had a
remarkably high titre of BLV had antibody titres similar
to those of the other animals. Anti-BLV IgM responses
were not observed in either group during the experimental period. A significant neutralizing antibody titre,
assessed using the syncytium inhibition assay, was not
detected in any serum 20 weeks after vaccination with
rVV (syncytium inhibition was 20.1 ~ for group A and
18.7 ~o for the control group). The titre was also negligible
9 weeks p.c. (25.8~o for group A, 31.8~ for controls).
Thus, there was no apparent correlation between the
humoral antibody response and the proliferation of BLV
in PBLs.
Effects of rVV vaccination on the growth of B L V in naive
sheep challenged with a smaller dose of BL V-infected
PBLs
In Expt. II, six sheep were inoculated with rVV (group
C) and six sheep were inoculated with LC 16mO (group
D); 9 weeks later they were challenged with 2 x 103
BLV-infected PBLs. As shown in Fig. 2(a and b), no
syncytia were detected in two animals in group C, or one
animal in group D during the experimental period.
Although syncytia were detected in four sheep in group
C, the number was relatively small. As shown in Fig. 2(c
and d), inoculation with rVV did not induce detectable
anti-BLV antibodies, as was the case in Expt. I.
Challenge with BLV-infected PBLs produced antibody
responses of the secondary type in animals infected with
BLV in group C, whereas those of the primary type were
observed for those in group D.
Effects of rVV inoculation on the growth of B L V in BLVcarrier sheep
In Expt. III, the effect of vaccination with rVV on sheep
carrying BLV was investigated. Ten sheep were challenged with 105 BLV-infected PBLs, and the number of
PBL syncytia was determined 5 weeks later. These sheep
were then divided into two groups (E and F), group E
was inoculated with rVV, whereas group F was
inoculated with LC16mO. As shown in Fig. 3(a and b),
syncytium formation in three group F animals continued
to increase, whereas that in group E animals increased
only slightly or decreased. The effect of the vaccine could
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30
40
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Time p.i. (weeks)
Ch
Time p.c. (weeks)
Fig. 2. Suppressive effect of vaccination with rVV on the growth of
BLV in PBLs of sheep challenged with 2 x 103 BLV-infectedPBLs. (a
and c) Sheep [no. 46 (O), 47 (O), 49 CA), 66 (A), 67 (11) and 74 (F-l)]
inoculated intradermally with 108 p.f.u, rVV; (b and d) sheep [no. 50
(O), 51 (O), 52(A), 69 (Z~), 70 (11) and 72 (n)] inoculated with 108
p.f.u. LC16mO. At 9 weeks p.i., all sheep were subcutaneously
challenged with 2 x 103 BLV-infected bovine PBLs. See legend to
Fig. 1.
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Time p.c. (weeks)
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10
20
30 I 40
Ch Vac
Time p.c. (weeks)
Fig. 3. Suppressive effect of vaccination with rVV on the growth of
BLV in PBLs of carrier sheep challenged with 105 BLV-infectedPBLs
9 weeks previously. (a and c) Sheep [no. 54 (O), 55 (O), 57 (A), 58 (A)
and 62 (11)] inoculated intradermally with 108 p.f.u, rVV; (b and d)
sheep [no. 56 (O), 59 (O), 60 (A), 61 (/~) and 63 (11)]inoculated with
108 p.f.u. LC16mO. See legend of Fig. 1.
not be determined for two animals in each group because
the number of syncytia formed was too low. After
challenge with BLV-infected PBLs, anti-BLV antibody
titres increased in all animals of both groups, although
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1890
K. Ohishi and others
Fig. 4. DTH skinreactions.Sheepwerevaccinatedwith 108p.f.u, rVV
and challengedintradermallywith BLV virions(a) or KLH (b) 7 days
later. Skin biopsy was done 48 h after challenge and sections were
stained with haematoxylinand eosin. Bar markers represent 100 ~tm.
the variation from animal to animal was relatively large
(Fig. 3 c and d) and there was no significant difference
between the two groups. Vaccination of animals carrying
BLV with rVV did not induce a secondary antibody
response. These results suggest that antibodies directed
against the BLV envelope glycoprotein did not contribute to the suppression of BLV growth.
D T H skin reactions o f sheep vaccinated with r V V
Although the induced humoral immune response was
poor, vaccination with rVV did induce a marked DTH
response. Two sheep were inoculated with rVV and
challenged intradermally with BLV virions 7 days later;
strong DTH skin reactions were observed. This reaction
was not seen in two animals vaccinated with LC16mO.
These clinical results were confirmed by histological
examination (Fig. 4): skin lesions of animals vaccinated
with rVV showed intensive infiltration by mononuclear
cells, but poor infiltration by polymorphonuclear leukocytes 48 and 72 h p.c., and no infiltration was apparent
3 h p.c. This result is consistent with the fact that antigp60 antibodies were virtually absent from the animals.
The DTH skin reactions were highly antigen-specific; no
reaction was observed when animals vaccinated with
LC16mO were challenged with BLV virions, nor when
animals vaccinated with rVV were challenged with
KLH.
Discussion
This study has demonstrated that vaccination with rVV
expressing BLV gp60 induces protective immunity,
suppressing the growth of BLV in carrier animals. First,
although the inoculation of naive sheep with rVV does
not protect the animals completely against BLV challenge, it does induce protective immunity in the resultant
carrier animals, consistently suppressing the proliferation of BLV over a relatively long time period. Second,
vaccination with rVV is effective in sheep which already
carry BLV.
Humoral immunity seems unlikely to play a major role
in the protective immunity induced by vaccination with
rVV because (i) rVV inoculation itself does not induce a
detectable antibody response, (ii) rVV inoculation of
animals carrying BLV suppresses the growth of BLV
with virtually no effect on the antibody response and (iii)
antibody titres do not correlate with the degree of BLV
proliferation in PBLs.
Unexpectedly, rVV itself was not able to induce
detectable anti-BLV antibodies in sheep, despite the fact
that the same rVV has been shown to be immunogenic in
rabbits (Ohishi et al., 1990). This failure may be ascribed
to poor expression of gp60, probably due to the low
proliferative capability of rVV in sheep skin; the skin
lesion at the site of inoculation was much smaller in
sheep than it was in rabbits (unpublished observation).
Portetelle et al. (1991) have reported recently that sheep
inoculated with rVV expressing the BLV envelope
glycoprotein produce neutralizing antibodies to BLV.
This discrepancy may be because they used the Copenhagen strain of VV. It is generally accepted that a
relatively large amount of antigen is necessary for the
induction of the humoral immune response, although
much less antigen is sufficient to induce T cell-mediated
immunity, including helper T cell and DTH T cell
responses (Ohishi et al., 1988). We have shown that
inoculation of sheep with rVV induces a strong DTH
response and stimulates helper T cells, as deduced from
the memory effect for the antibody response. This fact
strongly suggests that the cell-mediated immune response plays a major role in the suppression of BLV
growth in carrier animals. The mononuclear cells which
infiltrated the skin lesions in the DTH response were
about 65 % CD8 ÷ and 25 % CD4 ÷ 48 h after challenge
(K. Okada, S. Numakunai & K. Ohshima, personal
communication), suggesting that CD8 ÷ cytotoxic T
lymphocytes suppress BLV proliferation by killing BLVinfected cells. Interestingly, Doherty et al. (1990) argue
that CD8 ÷ T cells play a major role in the DTH reaction
to lymphocytic choriomeningitis (LCM) in mice infected
with LCM virus. It is also possible that CD4 ÷ T cells are
involved in the suppression of the growth of BLV by
releasing lymphokines such as interferon ~. Some reports
do support a role for cell-mediated immunity in
retrovirus infection in vivo, e.g. Earl et al. (1986) have
reported circumstantial evidence that a cell-mediated
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Protective i m m u n i t y against B L V in sheep
immune response is involved in the protection of mice
against Friend leukaemia virus (FLV), as determined by
vaccination with an rVV expressing the FLV envelope
glycoprotein.
The question arises of which cells are the putative
target of the cell-mediated immune response because
BLV-infected cells do not usually express gp60 on their
cell surface. We believe that some BLV-infected cells in
carrier animals could express the antigen in an unidentified tissue because antibodies against BLV gp60 remain
at relatively high levels throughout the life-span of BLVinfected animals once induced.
The fact that the level of proliferation of BLV varies
greatly between individual animals suggests that the host
immune response differs. We believe that this individual
variability should not be ascribed to experimental failure
because the titres of BLV and antibodies in an animal
showed consistent patterns during the experimental
period. Animals which had not been vaccinated with
rVV but which had very low BLV titres during the
experimental period may have an immune response
suppressing the proliferation of BLV similar to that in
animals vaccinated with rVV. The variability in the
immune response to BLV gp60 may be explained by
some feature of the immune response (Ir) gene of the
major histocompatibility complex. For instance,
Miyazawa et al. (1988) have reported that proliferative
murine T cell responses to the FLV envelope glycoprotein are highly dependent on the haplotype of the Ir gene.
Takahashi et al. (1988) have also reported that murine
cytotoxic T lymphocyte responses to the HIV envelope
glycoprotein are dependent on the H-2 haplotype.
BLV is integrated into the host genome as proviral
D N A , so antibodies mainly effective against free virus
would be unlikely to eliminate the virus from carrier
animals, although they can destroy virus-infected cells by
antibody-dependent cytotoxicity reactions. Miller and
colleagues (Miller & Van der Maaten, 1978; Miller et al.,
1983; Miller, 1986) first demonstrated that vaccination
with BLV virions together with adjuvant partly protected animals against BLV infection. The importance of the
antibody response in protection against BLV infection
has been demonstrated further by other investigators
(Onuma et al., 1984; Kono et al., 1986). For complete
protection against BLV, a vaccine must induce strong
immune responses, both humoral and cell-mediated. It
still remains to be determined whether other viral
antigens, such as core proteins, can induce protective
immunity. The results showing that vaccination with
rVV is effective even in carrier animals have very
important implications for the prevention of the development of disease in retrovirus carriers in general, e.g.
carriers of HIV.
1891
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