FEMS MicrobiologyImmunology64 (1990)223-234
Publishedby Elsevier
223
FEMSIM00123
Use of baculovirus expression vectors: development of diagnostic
reagents, vaccines and morphological counterparts
of bluetongue virus
Polly Roy
NERC Institute of Virologyand Environmental Microbiology, Mansfield Roa~ Oxfor¢~ U.K.
and University of Alabama at Birmingham School of Public Health, University Station Birmingham AL, U.S.A.
Received28 June 1990
Accepted4 July 1990
Key words: Recombinant proteins; Morphological structures; BTV
1. SUMMARY
The productivity and flexibility of insect baculovirus expression vectors and the ability of the
baculovirus genome to incorporate (and express)
large amounts of foreign DNA allows this system
to be used for both single and multiple gene
expression. Using the system, bluetongne virus
(BTV) gene~ have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic s*ructures of the virions. BTV
which causes disease in ruminants in many parts
of the world, consists of i0 double-stranded RNA
segments enclosed by double capsids that are
composed by 7 structural proteins. Since each
protein is encoded by a single RNA species, DNA
clones of all 10 RNA species were synthesized and
individually expressed in baculovirus vectors at
high levels. This has yielded proteins that have
been shown to be excellent diagnostic and vaccine
Correspondence to: P. Roy, NERC Instituteof Virologyand
EnvironmentalMicrobiology,MansfieldRoad, Oxford OX1
3SR, U.K.
reagents. In addition, multiple expression vectors
have been used to synthesize morphological structures (viral and subviral) representing BTV.
2. INTRODUCTION
Recent advances in biotechnology have macle it
possible to express foreign genes in heterologous
riving organisms. These organisms or expression
vectors include not only bacterial and yeast syst
,Jr a number of mammalian viruses. One of
t..~ ,ost widely used vectors is the vaccinia vector. In recent years the baculoviruses, which are
pathogenic to insects, have received ,-onsiderable
attention because of their potential for use as a
viral insecticide and for their potential use as
vectors for the introduction and expression of
foreign genes in insects and insect cell lines. One
of the major advantages of these invertebrate virus
expression vectors over bacterial, yeast and mammarian expression systems is the very abundant
expression of recombinant proteins which are antigenically, immunologlcallyand functionally similar to their authentic counterparts. In addition,
0920-8534/90/$03.50 O 1990Federationof EuropeanMicrobiologicalSocieties
224
baculoviruses are not pathogenic to vertebrates or
plants and do not employ transformed cells or
transforming elements as do the mammalian expression systems. The baculovirus vector also
utilizes many of the protein modification, processing and transport systems which occur in the
higher eukaryotic cells which may be essential for
the complete biological function of a recombinant
protein. Therefore, this expression system is ideal
for large-scale production of foreign antigens and
has been used successfully to express foreign genes
derived from a variety of sources, from bacteria to
human beings.
We are using this system for development of
not only diagnostic reagents and subunit vaccines
for bluetongue virus infection but also for answering some fundamental questions regarding viral
morphogenesis, for example, how is an architectually complex virus such as bluetongne virus (BTV)
synthesized?
Bluetongue virus is the prototype of the
Orbivirus genus (Reoviridae family) and is vectored to vertebrates by arthropods (Culicoides sp.),
causing disease of economic importance in ruminants in many parts of the world. The genome of
BTV (13 x 103 kDa) consists of 10 doublestranded R N A segments that range in size from
0.5 to 2.7 x 103 kDa [1,2]. The double-stranded
genome is enclosed by a capsid of complex nature
containing 32 well-defined protein capsomers
arranged in icosahedral symmetry. This nucleocapsid structure consists of 5 types of protein, 2
that are major (VP3 and VP7) and 3 that are
minor components (VP1, VP4 and VP6) 13-5].
This icosabedral particle is surrounded by an easily
removed outer capsid structure composed of 2
proteins (VP2 and VPS). This outer capsid lacks
clearly visible morphologlc subunits. Each BTV
segment appears to code for at least a single viral
polypeptide. In addition to the structural proteins,
3 non-structural polypeptides, NS1, NS2 and NS3
[6,7] have been identified in virus-infected cells.
We have been involved in developing rational
strategies for the identification, control and prevention of BTV infections. Our approach has been
to obtain c D N A clones of each R N A species and
express the D N A clones in baculovirus expression
vectors. Using these approaches we have synthesized BTV proteins and structural entities that are
discussed in this report.
3. M E T H O D S A N D RESULTS
3.1. The baculovirus expression system and expression of BTV genome segments in insect cells using
recombinant baculoviruses
The availability of complete c D N A clones representing the 10 discrete d s R N A segments and
the availability of their sequence database have
been further exploited for understanding the
structure-function relationships of the BTV proteins (Table 1).
Each of the c D N A clones has been manipulated and expressed to high levels in insect cells
using recombinant baculoviruses. Studies with
these recombinant viruses and the expressed proteins have only begun to contribute to our under-
Table 1
Coding assignments (proteins) of the BTV-10viral RNA species
Segment
No. of
bp
Size
(Da)
Proteins
No. of
aa's
Size
Location
L1
3954
1302
149, 588
111,112
core
2 926
2772
2011
1639
1769
1156
1123
1046
822
2.7 X 10 e
2.0 x 10e
VPl
L2
L3
M4
M5
M6
$7
$8
$9
SI0
1.9 × 106
1.4 x 106
1.0x 10e
1.2z 106
7,8 X 105
7.6 x 10~
7.1 x 105
5.0 x l0 s
VP2
VP3
VP4
VP5
NS1
VP7
NS2
VP6
NS3
956
901
654
526
552
349
357
328
229
103, 433
76, 433
59, 163
64, 445
38, 548
40, 999
35, 750
25, 572
outer shell
core
core
outer shell
nonstructural
core
nonstructural
core
nonstructural
225
standing of the structure-function relationships of
various BTV gene products. In addition, by
manipulating the vector systems, we have embarked on defming the architecture of the complex
morphology of the viral capsids. The description
of those BTV proteins which are relevant for
development of diagnostic reagents and subunit
vaccines for BT disease, as well as morphological
structures which have proved of particular interest, will be discussed below following a brief
summary of the baculovirus expression technology.
This expression system utilizes the major late
promoter of the polyhedrin gene in Autographa
californica nuclear polyhedrosis virus (AcNPV) [8].
The life cycle of this virus is characterized by the
production of 2 forms of progeny: extracellular
virus particles (ECV) and occluded virus particles
(OV). ECVs are produced relatively early in infection (from 12 h onwards) and are released by
budding from the cell surface. They mediate the
systemic infection of the insect and also account
for the mode of infection in cell culture. Later in
the infection cycle (from 18 h onwards) viral progeny is occluded into a paracrystalline matrix composed primarily of a 29-kDA protein called polyhedrin (Fig. 1). These occlusions, called polyhedra,
protect the progeny virus during horizontal transmission and effect their release in a new host by
dissolving in the alkaline environment of the insect gut. Polyhedra accumulate in infected cells
for 4-5 days until cell lysis, by which time polyhedrin may constitute up to 50% of the total cell
protein. Because cell-to-cell infection is propagated by ECVs, the synthesis of polyhedrin protein is a non-essential function for the replication
of AcNPV in cell culture. The use of AcNPV as an
expression system therefore involves replacement
of the polyhedrin gene with a foreign gene which,
due to the control of the polyhedrin promoter, has
the potential to be expressed to a high level.
A number of transfer vectors are available to
construct such recombinant baculoviruses. One
which has found particular favour is pAcYM1 [9].
This, in common with most other baculovirus
expression vectors, consists of a restriction enzyme
fragment of the AcNPV genome encompassing the
polyhedrin gene, cloned into a high copy-number
q)
Transfer vector
-
LONA exWactlen
l
Fore~n DNA
Co-trw~sfectlon
[[•mmJ•
Ret;;.w,btnation
2-4 days incubation
!
Sup.
1
Plaque assay
1
Polyhedrln negative
"clear'plaque
|
1
Rec0nd~inant Virus 0NA
Purification
l
Amplification
Fig. 1. General protocol for the insertion of foreign DNA into
infectious baculovirus vectors and screening for recombinant
plaques.
bacterial plasmid. The polyhedrin gene sequence
has been deleted in pAcYM1 and replaced by a
BamH1 linker to allow for the insertion of a
foreign gene immediately downstream of the polyhedrin promoter. The unchanged wild-type
AcNPV DNA-sequences that flank the inserted
gene mediate homologous recombination when a
cell is transfected with the plasmid DNA and
wild-type AcNPV DNA. Recombinant baculoviruses are selected on the basis of their polyhedrin-negative phenotype.
By means of this in vivo recombination technique, cDNA copies of all 10 BTV segments
(principally from serotype 10) have been expressed in the baculovirus system, as shown in
Fig. 2.
226
O
c
O
~
"~
T
o
I,-
I,-
m
o
m
!
>
I-,-
o
>
a.
Z
O
<
T
>
T
>
T
>
";"
>
T
>
T
>
T
>
T
>
I,m
Ien
I,131
I.m
I,m
I,lib
I,m
I,.m
rJ
<
O
<
O
<
O
<
O
<
O
<
O
(
¢.1
<
VP1
~VP2
"~VP3
~l-VP4
~t-VP5
NS1
NS2
-el- V P6
~'VP7
Polyhedrin -I~
N83
Fig. 2. SDS-PAGE analyses of recombinant baculovimses (AcBTV) that express the 10 8ene products of BTV-10 and compared to
BTV virion proteins and AeNPV proteins. The resolved proteins are stained with Coomassie brilliant blue.
3.2. Recombinant virus.derived B T V proteins as
potential diagnostic reagents
Our studies involving complete sequence analysis of various BTV RNA species as well as cDNARNA hybridization studies indicated that all 5
BTV core proteins (VP1, VP3, VP4, VP6 and VPT)
and the 3 non-structural proteins (NS1, NS2 and
NS3) are highly conserved among all 24 BTV
serotypes. To develop group-specific diagnostic
reagents, some of these BTV recombinant proteins
were subjected to screening BTV antibodies in an
ELISA test and by Western blot analysis.
For ELISA test, either expressed core protein
VP3 or non-structural NS1 protein was used. Recombinant vires-infected cell extracts were absorbed to microtitre plates (represen*,ing some 50
ng of protein per well) and incubated with either
polyclonal BTV-2, BTV-10, BTV-11, BTV-13, or
BTV-17 sheep antisera, or with normal sheep
serum as a control. The derived antigen-antibody
complexes were detected by incubating with antisheep alkaline phosphatase conjugates followed by
the addition of an enzyme substrate. As shown in
Fig. 3, all 5 BTV antisera reacted with both the
recombinant antigens in proportion to the end
point titre of the antisera. No reaction was detected with normal sheep serum. No reactivity was
obtained when each of the BTV antisera was
tested with AcNPV-infected cell extracts.
In another experiment, as shown in Fig. 4, a
mixture of recombinant VP6 and recombinant NS1
was bound to nitrocellulose strips. Each strip was
tested with anti-BTV-10 (lanes 1, 2 and 6), or
anti-BTV-11 (lane 7), or anti-BTV-13 (lane 8), or
227
A
0.6
•
Anli'BTV
2
A
AnU-BTV
13
o ,;::/?,;v ;o
1.2
S
D Antl-aTV
O 0.4
•
Normal
17
shee9
,If
8
¢0
O~
o 0.2
J~
o
OA
~-I-%~
c.~
~??. \
\\
o- - 41- - i - -~. - 4100
,
.
-
,
10-1
10-a 10"~
Antigen d41ulk3n
o
200
400
IO0 I100
- ~r~--.lUl~ql~
~l;lO0 ( [ 4 0 0 1;HIO0
Dilution o f serum
10~
Fig. 3. (A) ELISA (410 rum) using AcNPV antigen (- - -) or AcBTV 10-3 expressed VP3 ( - - ) (continuous lines) and sheep antisera to
BTV-10 (e), BTV-17 (O), and normal rabbit serum (a). (B) ELISA using AcBTV 10.6 expressed NS1 and sheep antisera to 5 USA
serotypes of BTV.
anti-BTV-17 (lanes 3 and 9). Strong positive reactions were detected with all antisera. Weak reactions were also detected with VP6 using EHDV-1
C
I
2
3
4
5
6
7
8
9
10
NS1VP6-
Fig. 4. Western analyses of VP6 and NS1 using different
anfisem. Lane 1:anfi-BTV-10 rabble (immunized with crude
virus); lane 2: anti-BTV-lO rabbit (immunized with purified
virus); lane 3:anti-BTV.17 rabbit; lanes 4 and 5: normal sheep
serum; lane 6:anfi-BTV-10 sheep serum; lane 7:anfi*BTV-11
sheep serum; lane 8:anti-BTV-13 sheep serum; lane 9: antiBTV-17 sheep serum; lane 10: anti.EFIDV-I sheep serum; lane
11:anti-EHDV-2 sheep serum; C: Coomassie blue-stained
strip.
and 2 antisera (lanes 10 and 11), but not with
NS1.
Polyclonal antisera raised against each of these
recombinant proteins can also recognise the viral
antigens. Antisera raised against recombinant VF7
of BTV-10 can recognise not only BTV-2, -10, -11,
-13 and -17, but also EHDV-1 and EHDV-2 in a
Western blot analysis. These results indicated that
using the expressed antigens or using the antisera
raised with these antigens, it is possible to develop
diagnostic reagents either for detection of antibodies from infected sera or viral antigens from infected samples.
3.3. Vaccination of sheep with bluetongue-AcNPV
recombinants and protection against lethal infection
BTV is highly capable of reassorting the RNA
segments between different serotypes. Therefore,
the whole virus vaccines might play a significant
role in the generation of endemic strains [10,13].
Sin,~ t~,~neticengineering techniques allow safe,
large-~ale production of subunit vaccines, we have
tested the efficacy of these baculovirus-expressed
antigens for development as subunit vaccines.
The protective capabilities of these recombinant proteins against virus infection in sheep were
228
performed in collaborations with the scientists in
The Veterinary Research Institute, Onderstepoort,
South Africa. Twenty-four Merino sheep that
originated from a BTV-free region of the North
Eastern Cape of South Africa were used for vaccination trials. Prior to vaccination, the BTV-susceptible status of the sheep was determined by
analysing the sera in immunodiffusion tests, followed by confirmation with an ELISA test.
Three groups of 4 sheep each were injected
subcutaneously with various doses of the recombinant VP2 protein (AcBTV-10.2), the major neutralization outer capsid protein of BTV-10 [14-16].
To determine whether the second outer capsid
protein, VP5, can also induce the neutralizing
antibody, a group of 4 sheep were injected with a
mixture of crude infected cell lysates containing
VP2 (100 pg) and VP5 (25 /~g). In addition, to
determine whether BTV-directed non-structural
proteins or virion core proteins have any role in
immunity and protection, 4 sheep were also inocnlated with mixtures of recombinant virus-derived
proteins (VP2, VP5) and also 4 core proteins, 2
major (VP3, VP7) and 2 minor (VP1, VP6). In
addition, the sheep were inoculated with 3 nonstructural proteins NS1, NS2 and NS3. The control group of 4 sheep received only saline. To
evaluate whether the adjuvants are essential for
induction of neutralizing antibodies in sheep, 2
sheep in each group received vaccines with adjuvants.
To assess the ability of the recombinant antigens to induce neutralizing antibody, sera were
collected between 25-74 days post-immunization
and were tested for neutralizing antibody against
BTV-10 by the plaque reduction neutralization
test. As shown in Table 1, all the immunized sheep
produced BTV-10 neutrafizing antibody (NA). The
VP2 recombinants with or without adjuvant were
able to induce NA, although the higher the amount
of VP2 used, the better was the titre of NA.
However, significant differences are observed when
adjuvant was included either with a high dose of
VP2 alone, or with a low dose of VP2 when mixed
with VP5 or with the mixture of all other antigens.
In contrast, the adjuvant did not seem to have any
affect with the low dose of VP2 antigen. Very low
amounts of recombinant VP5 (ca. 25/tg) together
with VP2 antigen induced a surprisingly high titre
of neutralizing antibody (1 : 512). The presence of
other viral proteins did not appear to make any
difference to the NA titre produced. In all cases
the plaque reduction titres dropped significantly
with time.
When immunoprecipitation analysis was performed with the antisera collected from vaccinated
animals, it was clear that sheep that received VP2
alone precipitated VP2, and the sheep that received the mixture of VP2 and VPS, precipitated
both of these proteins. All major proteins of BTV,
namely VP2, VP3, VP5 and VP7 were detected in
the immunoprecipitation analysis with the serum
collected from a sheep that was immunized with a
mixture of all 9 baculovirus recombinant proteins.
To assess the ability of the recombinant viral
antigens to induce protective immunity, on day 75
(4 weeks after the 2nd booster injection) all sheep
were challenged with the lethal infectious sheep
blood containing 2 × 10 6 pfu of virulent BTV-10.
Rectal temperatures were recorded twice daily and
the sheep were carefully examined for cfinical
manifestation of bluetongue disease which was
expressed numerically by the clinical reaction index (CRI) (see Table 2). Serum and blood samples
were collected daily after the challenge for the first
I I days and from then on every 3rd day. The
post-challenge blood samples were screened for
viraemia by passaging in 10-12 day embryonated
chicken eggs and observing the fate of the embryos. Plaque reduction titres were determined on
sera taken 21 days post-challenge.
As shown in Table 3, apart from 2 sheep that
received the low dose (ca. 100 /tg) of the VP2
recombinant alone, all the sheep injected with
recombinant antigens were highly immune to the
infectious virus. They had no clinical symptoms or
signs of viraemia. All of the control sheep, on the
other hand, developed typical bluetongne disease
with a relatively high CRI. In addition, the postchallenge sera induced positive virae~rda in ECE
as well as high plaque reduction titres characteristic of a primary infection.
Data presented here indicate that recombinant
baculovirus-derived subunit vaccines can replace
the attenuated five virus vaccines currently in use.
Moreover, since the expression level of these pro-
229
teins is high (20-30 mg per 1 × 109 cellS) the
production of these 'safe' subunit vaccines can be
significantly cost-effective.
3.4. Three-dimensional structure of B T V
The three-dimensional structure of singleshelled
BTV
has been
determined
to a resolution
of 35 A using cryo-electron microscopy and image
processing mierography of unstained, unfixed virus
particles embedded in vitreous ice. This study
demonstrates that the viral core has icosahedral
symmetry with a triangulation number (T) of 13and exhibits a bristly surface. A distinctive feature
of the particle is the presence of 132 large chan-
nels at all the 5- and 6- co-ordinated positions as
indicated in the reconstructed structure (Fig. 5.)
These are about 70 ~, deep and about 70
wide at the surface. The protein mass is mainly
concentrated on the local and at strict 3-fold axes
suggesting a trimeric clustering. The capsid has a
diameter of 680 .~, and is divided into predominantly 3 layers, with an outer layer of 110 ,A
(between the radii of 230 A and 340 ~ ) which
appears to be composed of 780 molecules of only
one protein, probably the VP7 protein. The middie laver is about 90 ~, thick, between the radii
140 A and 230 .~ and probably contains the
second major protein VP3.
Table 2
V a c c i n a tioa o f sheep by oaculovirus expressed BTV antigens
Pairs o f a n i n ~
were inoculated with (v) or without ( - ) incomplete Freund's adjuvant on the day indicated.
B T V - 1 0 antigens
Adjuvant
Inoculation
(day)
Serum neutralization titers . a
against BTV-10 (days)
0
21
42
BTV-10,
VP2: ca. 50 F g
v
v
v
v
v
v
v
v
v
v
v
v
v
v
32
32
16
<4
32
32
16
4
64
32
32
16
64
32
32
16
32
16
32
16
64
8
16
8
16
8
12
8
8
4
8
8
BTV-10.
VP2: ca. 100 F g
v
v
v
v
v
v
v
v
v
v
-
> 32
> 32
32
16
64
64
32
16
32
16
16
32
16
16
16
16
16
16
8
?
12
6
8
8
4
<4
<4
<4
BTV-10,
VP2: ca. 200 F g
-
v
v
-
-
v
v
-
v
v
v
v
v
v
-
> 32
> 32
> 32
> 32
128
64
128
512
32
16
64
128
32
16
64
128
32
16
32
128
16
16
32
64
BTV-10,
VP2: ca, 50 F g
VP$: ca. 25/~g
v
v
v
v
v
v
v
v
v
v
v
v
v
v
< 4
< 4
> 32
32
< 4
4
128
64
16
16
512
128
8
8
256
128
8
8
128
64
IITV-10, V P l ,
VP2, VP3,
VPS, VP6, VPT,
NS1, NS2, N S 3
v
v
v
v
v
v
v
v
v
v
v
v
v
v
8
16
> 32
> 32
> 4
4
128
64
8
8
256
128
8
8
256
128
Saline
v
v
v
v
v
v
v
v
v
v
v
v
v
v
< 4
<4
<4
<4
<4
<4
<4
<4
<4
<4
25
a Reciprocal o f dilution that caused a 507o plaque reduction
<4
<4
42
48
8
50
52
60
?
67
<4
74
<4
16
8
32
64
8
8
16
32
8
8
128
32
4
?
128
32
4
4
96
24
8
8
128
64
16
24
64
64
16
24
64
48
16
12
16
24
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
230
Fig.5. Modelof BTVcoresas determinedby cryoelectronmicroscopy
3.5. Core protein morphology
Little is known about the functions, interactions or stoichiometry of the 5 core proteins.
Appreciation of such factors is obviously important if we are to understand the morphology of
a complex virus such as BTV. Our cryoelectron
microscopy studies suggest that the core of BTV
consists of a nucleoprotein centre surrounded by 2
distinct protein layers, each of which is composed
of a single polypeptide species. Immunogold analysis indicates that VP7 is the principal component
of the outermost layer of core [17]. We were
therefore interested to see if we could synthesize
BTV core particles from 2 major core proteins,
VP7 and VP3. To this end the segment 3 and
segment 7 genes were inserted into a novel baculovirus dual expression transfer vector (Fig. 6).
Such vectors incorporate 2 copies of the polyhedrin promoter and transcription termination sequences [18] with a unique enzyme restriction site
located downstream of each promoter to allow for
the insertion of 2 foreign genes. The promoters are
present in opposite orientations to minimize the
possibility of homologous sequence recombination
and excision of one or other of the foreign genes.
Recombinant baculoviruses were prepared by the
established procedure of co-transfecting S. frugiperda cells with the dual expression plasmid DNA
and wild-type AcNPV DNA.
Electron micrographs of S. frugiperda cells infected with the recombinant baculovirus showed
large aggregates of foreign material in the cytoplasm which, under high magnification, appeared
to consist of spherical particles.
This expressed material was isolated by lysing
the cells with NP40 and then purified on a discontinuous sucrose gradient. When examined under
the electron microscope the material was found to
consist of empty core-like particles whose size and
appearance were indistinguishable from authentic
BTV core particles prepared from BTV-infected
BHK cells (Fig.7).
The synthesis of empty BTV core particles from
the major core proteins VP3 and VP7 reveals some
important aspects of BTV morphology. It can be
concluded that their formation is not dependent
on the presence of the 3 minor core proteins, nor
the BTV dsRNA. In addition, the 3 BTV nonstructural proteins (NS1, NS2, NS3) are not required to assist or direct the assembly of the VP3
and VP7 proteins to form stoichiometrically-correct empty particles.
The genes encoding the outer coat proteins of
the virus (VP2, VP5) have also been similarly
inserted into a dual expression vector and used
together with the recombinant that expressed the
major inner core proteins (VP3, VP7) to co-infect
insect cells. Particles were purified from ~.he in-
BTV-SOSellmerA7
Pill
231
polytm4fInptomotofs
~
i
o
~
B
81mH
IITV-17Ieiment3
n
Bem~4!
i
AATAAA
mill
OemHIcut
Pafthl|PIUcut~ 5 a l
Electr°elul"'TV-17~33
~l~nedlge.... Ltgelion
I cut
Kfenow
El"~n
e...............
a'
IIlmHI
8amN
OamHI
~
B01I •
81mHt
Plft~lOamHIcut
Ete¢~f o e l u l e ~ m
e,e9O T V - 1 7 . 3
i]iii .......
BglHcut
mn
. .......
.....
AATAAA
Fig. 6. Preparation of a dual expression vector, pAcVC3. BTV-10.7.BTV-17.3.
232
Table 3
Protection of sheep by baculovirus expressed BTV antigens.
Animals were challenged on day 74. Clinical reaction index
(CRI) was obtained by addk~gthe following 3 scores (a + b + c):
(a) the fever scores are cumulative totals of fever readings
above 40°C on days 3-14 after challenge (maximum score
12); (b) the lesion score. Lesions of the mouth, nose and feet
where each scored on a scale of 0-4 and added together
(maximmn score 12); (c) the death score. - 4 points if death
occurred g~thin 14 days post-challeoge.
Antigens
Serum neutral
titers against
BTV-10 (21
days postchallenge)
Clinical Viremia
reaction (days
index
postchallenge)
BTV-10
VP2: ca. 100 pg
160
640
40
320
0.0
1.4
0.0
3.1
0.0
4.6
0.0
9.
BTV-10,
VP2: ca. 100 pg
VP5: ca. 25 pg
40
40
120
60
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BTV-IO,
VP2:100 pg-500 pg
40
< 20
< 20
80
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BTV-10,
VP2: 500pg-lmg
80
40
80
< 20
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
BTV-10 VPl0
VP2, VP3,
VP5, VP6, VP7,
NS1,
NS2, NS3
20
20
< 20
20
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
> 640
640
640
> 640
7.4
5.0
4.6
5.1
4.9
4.10
4.9
4.10
Saline
Fig. 7. Lower microg~ph consists of BTV cores derived from
BTV virions. Top micrograph shows core made by co-expression of VP3 and VP7.
fected cells (Fig. 8) a n d s h o w n to c o n t a i n cores
(C) a n d larger p a r t i c l e s (P) s i m i l a r in d i a m e t e r to
virions.
By p r o t e i n a n a l y s e s the p u r i f i e d m a t e r i a l obt a i n e d from in fected cells w a s f o u n d to h a v e all 4
s t r u c t u r a l p r o t e i n s o f the virus (VP2, VP3, VP5
a n d VP7).
Fig, 8. Co-expression of VP2, VP3, VP5 and VP7 by recombinant baculoviruses forms cores (C) and vlrus-like particles (P).
233
4. DISCUSSION
For virus diagnosis, antigens are required that
represent the etiologic agent. Traditionally such
antigens are derived from virus-infected tissues
a n d other materials (e.g., in vitro cultures) and in
the majority of cases the materials work well.
However, o n occasion such reagents are not satisfactory, for instance due to low antigen production, reactivity, or the presence of materials or
reactions that complicate interpretation of the results. However, products of expression of c D N A
clones representing the requisite antigens can
eliminate many of these problems.
Similarly, there is always a need for safe vaccines that can protect animals from infection and
disease. For virus infections, such vaccines may be
live, attenuated viruses, or they may come from
killed virus preparations. As in virus diagnostics,
bioteclmology offers new types of products for
vaccine development.
Recombinant gene expression has as its particular goal, the synthesis of proteins that represent
the authentic products in terms of size, modification a n d other post-translational processing events.
The proteins may be required for basic research
(structure-function analyses), or for practical purposes (diagnostic or therapeutic reagents, vaccines,
etc.). It is quite clear from our studies that the
baculovirus expression system has tremendous
potential, b o t h for development of diagnostic reagents and for vaccine development.
In addition it can be anticipated from the results obtained with the multiple expression vectors
that it will now be possible to study the molecular
basis for the foru~ation of morphological structures (viral and subviral) representing multiple
proteins as in BTV. This can be undertaken by a
variety of procedures including X-ray crystallography, site-directed mutagenesis and deletion analyses.
The value of the baculovirus-expressed BTVlike structures needs to be investigated further.
particularly with regard to their use as vaccines.
There is every reason to believe that it should be
possible to make vaccine chimaeras representing
different BTV serotypes (e.g., involving the expression of several BTV VP2 genes), as well as
chimaeras containing protein sequences representing other pathogens (e.g., chimaeric genes involving VP2 and or VP5 and or VP3 and or VP7
sequences and selected sequences of viral, bacterial, fungal, or protozoan pathogens).
ACKNOWLEDGEMENTS
I would like to thank the scientific staff of the
Veterinary Research Institute of Onderstepoort, S.
Africa, especially Dr. A.A. Van Dijk and B.J.
Erasmus who have performed all the experiments
for testing the vaccine in sheep. I am also grateful
to Dr. B.V. Prasad, Baylor College of Medicine,
TX, U.S.A., for the analyses of BTV core particles
and for the photograph of the three-dimensional
structures. In part this work was supported by
EEC contract BAP-0120 U.K. and by N I H G r a n t
AL-126879.
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