Note: Descriptions are shown in the official language in which they were submitted.
CA 02221570 1997-12-05
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Title: Neutr~ ;ng conforma~; n~l epitopes of Ch; ck~n
anemia virus .
BRIEF DESCRIPTION OF T~ lNv~NllON
The present invention provides the formation and
characterization of the ..~Ll-li~;ng conforma~;~Al
epitope of the CAV protein VPl, which is required for
eliciting a protective Lmmune ~e~u~l~e in vA~c;nAted
animals.
In par~ l1 Ar, the production of ll~uL~ ;ng
an~; h~; ~5 againt CAV is descr; h~ . In a diagnostic test,
based on these produced neutrAl;~;ng ant;ho~;~s, CAV
(part;~l~s) are detected.
Various vA~c;n~ ve~Lo~ A;nct CAV infections are
~;cclnsed~ All these vACc;n~c are less pa~h~en;~ than
CAV. All these vA~;n~ vector are shown to ~ u~e the
required neutr~ ;n~ confol~t;onAl epitope of CAV.
One vector provides a subunit vA~c;n~, based on a
recombinant baculovirus expressing both VPl and VP2 in the
same cell. A second type of vA~c;n~ is a live virus
vector, comprising a Marek's disease vector stably
h~rhoring the coA;ng se~n~c for VPl and VP2. The third
CAV vA~C;ne comprises genet;~lly attenuated CAV str~;ns
with a reduced cytopathogenic effect.
BACRGROUND OF THE lNv~NllON
Chicken An- ~A virus (CAV) is a sm~ll virus of a
unique type with a particle diameter of 23 to 25 nm and a
genome consisting of a circular c; n~l e-stranded
(m;nus-strand) DNA (Gelder blom et al., 1989, No~hnrn et
al., 1991 and Todd et ~l., 1990). This DNA mult.;rl;~5 in
infected cells via a circular ~ hle-stranded replicative
intermediate, which was L~l.Lly clon~ and fully
se~-~n~e~. The CAV genome is 2319 nucleotides long
(Not~h~rn and De Boer, 1990). DNA analysis of CAV strains
isolated in different continents revealed only m;nor
5UBSTITUTE SHEET (RULE 26)
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differences among the various isolates (Meehan et al.,
1992; accession number M81223, Claessens et al., 1991;
D10068, Kato et al., 1995; D31965 and Pallister et al.,
1994 Soine et al. 1993 and 1994; L14767). Recently, on the
basis of the genome structure, CAV has been placed into
the novel virus family of Circoviridae. However, CAV is
not related to the other members of this virus family
consisting of ~n;m~l single-stranded circular-DNA viruses,
such as porcine circovirus and psittacine
beak-and-feather-disease virus (Noteborn and Koch, 1995).
The CAV genome contains one evident promoter/enhancer
region (Noteborn et al. 1994) which regulates the CAV
transcription. The major transcript from the CAV genome is
an unspliced polycistronic mRNA of about 2100 nucleotides
encoding three proteins of 51.6 kDa (VPl~, 24.0 kDa (VP2)
and 13.3 kDa (VP3 or apoptin (Noteborn and Koch, 1995,
Noteborn et al., 1992 and Phenix et al., 1994). All three
predicted CAV proteins are synthesi zed in CAV-infected
cells (Noteborn and Koch, 1995). Tmmlln;zation with
(recombinant) VPl and VP2 synchronously synthesized in the
same cells elicits a protective response and can be used
as subunit vaccine against chicken infectious anemia (Koch
et al., 1995 and Noteborn and Koch, 1994c).
CAV causes clinical and subclinical disease in
chickens, and is recognized as an important avian pathogen
worldwide (McIlroy et al., 1992 and McNulty et al., 1991).
Day-old chicks are especially susceptible to CAV
infections. In these ~n;m~l S lethargy, anorexia and anemia
are observed from 10 days after inoculation with CAV.
After infection mortality may increase to a m~; mllm of
50%. With increasing age the resistance also increases
(Jeurissen et al., 1992). The hematocrit values of chicks
that had been infected with CAV at an age of 1-3 days are
decreased. CAV infections of 1-21 days old chicks result
in a depletion of in partlcular the thymus cortex.
However, in older chickens CAV can subclinically multiply.
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CAV infection in older-chickens can be determined by the
occurence of seroconversion.
The depletion of the cortical thymocytes is
considered to cause immunodeficiency resulting in enhanced
concurrent infections and to vaccination failures
(Noteborn and Koch, 1995). The depletion of thymocytes and
most likely also of erythroblatoid cells occur via
CAV-induced apoptosis (Jeurissen et al., 1992a). The
CAV-encoded protein apoptin is the main inducer of this
phenomenon (Noteborn et al., 1994).
Maternal antibodies have been found to give an
important protection against CAV infection. Vielitz et al.
(1991) reported that hens exposed to CAV from large
amounts of antibodies, which are passively transferred to
the offspring per ovum. These antibodies protect against
chicken infectious-anaemia-associated disease.
CAV-neutralizing antibodies were detected in yolk of eggs
produced by hens that had been inoculated with lysates of
cells that had been co-infected with CAV-recombinant
baculovirus and produced all three CAV proteins, or mainly
VP1 and VP2. Specific clinical signs did not develop in
CAV-challenged progeny that hatched from these eggs (Koch
et al., 1995).
Vielitz and Landgraf (1988) have developed a vaccine
against infectious anaemia, which is based on CAV
propagated in chicken embryos. Currently, this is the only
commercially available vaccine. The offspring of
vaccinated breeders are protected againt infectious
anaemia. Vaccination is possible because when maternal
immunity vanished, the birds are susceptible to virus
infection without developing the disease. Obviously, the
use of live vaccines based on non-attenuated virus harbors
risks. Experimental infection of 3-week-old chickens
resulted in a comprehensive decrease of the functions of
the immune system (McConnell et al., 1993, and 1993a) in
the absence of disease. In line with this finding, McIlroy
(1992) have provided evidence that CAV also causes
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considerable economic losses in the absence of disease:
the subclinical disease negatively affected feed
conversion and average body weight and increased
medication, both of which result in a considerable
financial burden. So far, non-pathogenic CAV has not been
isolated.
The recombinant CAV proteins synthesized by means of
the baculovirus expression system can be used as a subunit
vaccine. The recombinant CAV proteins VPI and VP2 have
been proven to protect chicks by maternal immlln-ty (Koch
et al, 1995). Since the baculovirus vector is an
insect-specific virus, known to be non-pathogenic for
vertebrates, it can be cultured and supplied to chicken
without undue risks ~Vlak and Keus, 1990).
In general, live-virus vaccines induce a better
immune response and are less expensive than sub-unit
vaccines. Therefore, knowledge about the immunogenicity of
the various CAV proteins can be used to construct
attenuated CAV or other recombinant-virus vectors, such as
avian herpes viruses (Nakamura et al., 1992, Morgan et
al., 1993) or fowlpox virus (Nazerian et al., 1992, Boyle
and Heine, 1993). These recombinant viruses should express
the CAV protein VPl and VP2, in addition to their own
proteins, and may be applicable as vaccine for both layer
breeders and broiler breeders to protect their offspring
against infectious anaemia. In addition, such vectors may
be used to protect maternally immune broilers against
subclinical disease.
DETAILED DESCRIPTION OF THE INVENTION.
The present invention relates to production and
analyses of the neutralizing conformational epitope
structure of chicken anemia virus (CAV).
Furthermore, the production of neutralizing
monoclonal antibodies directed against CAV is disclosed.
It is shown that these neutralizing antibodies are
CA 02221~70 1997-12-o~
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directed against a conformational epitope of CAV protein
VPl.
Also a diagnostic test kit, based on these
neutralizing antibodies, for the detection of CAV will be
described. This invention provides evidence for the
me~h~nism by which the neutralizing antibodies neutralize
CAV particles.
For the formation of the neutralizing conformational
epitope of VPl, the synthesis of VP2 within the same cell
is required. Recombinant baculovirus expressing only VPl
in insect cells only does not react with neutralizing
antibodies directed against CAV, but these neutralizing
antibodies will react when VPl and VP2 are synthesized in
one cell.
In purified CAV capsids, however, only VPl is
present. This invention discloses that during the
synthesis of VPl, and most likely during its complex
formation resulting in the CAV capsids, VP2 binds
temporarily to VPl. Denaturation of the CAV capsids
results in the destruction of the neutralizing epitope,
indicating that the neutralizing epitope is an
conformational one.
Besides, the invention relates to vaccines and
compositions for preventing or treating virus infections
in poultry, in particular infections with CAV.
In particular, the invention relates to vaccines that
are less pathogenic than the CAV itself but are still
capable of producing the neutralizing conformational
epitope. Vaccination of chickens with these type of
vaccines will lead to the generation of neutralizing
antibodies and thus protect the ~ n; m~ and their progeny
against CAV infections.
The invention relates to a baculovirus vector which
contains separately on its genome the coding sequences for
VPl or VP2. This recombinant baculovirus is able to
synthesize VPl and VP2 in the same cell, resulting in the
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formation of the neutralizing conformational epitope of
CAV.
The invention also relates to the construction of a
Marek's~disease virus (MDV) vectors cont~in;ng CAV
sequences encoding VP1, VP2 proteins. In particular, this
recombinant MDV vector synthesizes the recombinant CAV
proteins in such a way that the neutralizing
conformational epitope of VP1 is properly made.
Furth~rmnre, the invention describes the formation of
various attenuated CAV strains, which reveals a reduced
cytopathogenic effect in chicken T cells in comparison to
a a wild-type-derived CAV strain. The attenuated CAV
strains were made by introducing point mutations in a
cloned CAV DNA genome, in particular a sequence within the
promoter/enhancer region has been mutated. The attenuated
CAV mutant strains are able to produce the neutralizing
conformational epitope.
Processes for the prophylaxis or control of CAV
infections, in particular in chickens, and processes for
the preparation of recombinant parts of CAV comprising
sequences, and processes for the preparation of vaccine
are also subjects of the invention.
Thus, reiterating the invention provides a
neutralizing antibody or an antibody fragment or
derivative thereof reacting with a conformational epitope
of viral protein 1 (VP1) of chicken anaemia virus (CAV)
recognized by a monoclonal antibody designated as 132-1,
132-2 or 132-3, as produced by the hybridoma's deposited
under no.'s xxxxxx, yyyyyy, zzzzzz at the Insitut Pasteur,
Paris, France. A workable experiment for det~rmining
whether an antibody is an antibody according to the
invention is to see if it cross-reacts (or competes) with
an antibody from the deposited hybridomas. Fragments and
derivatives of antibodies are well known in the art and
hardly need further explanation for the person skilled in
the art.
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The invention also provides a conformational
neutralizing epitope of viral protein 1 of chicken anaemia
virus recognized by an antibody as disclosed above. The
epitope may be part of a larger molecule, it may for
5 instance be bound to a carrier or the epitope may be
& repeated (at intervals) in a polypeptide chain. Preferably
the epitope is part of a larger part of VPl, because such
a molecule will have the right conformation more easily.
As described the conformational epitope will only be
10 present on VPl if it is produced in one cell, preferably
from one vector together with VP2. Therefore the.invention
provides a method for producing a viral protein 1
comprising a conformational epitope as disclosed a~ove,
comprising the expression of said viral protein 1 in one
15 cell together with viral protein 2 of said CAV whereby the
genetic information encoding VP1 and the genetic
information encoding VP2 are separately present on one
recombinant vector.
The invention also includes vectors for use in a
20 method just described comprising as two separate coding
sequences the genetic information encoding VP1 and the
genetic information encoding VP2. Preferably such a vector
is based on Marek's disease virus vector so that a vaccine
can be produced giving protection against two pathogens.
A very safe and efficient expression system for the
viral proteins according to the invention are vectors
which are based on a baculo virus vector. As explained
before Baculovirus is generally regarded as safe for use
in vertebrates since it cannot infect them.
The invention also provides another way of arriving
at vaccines or vaccine components which do have the very
important neutralizing comformational epitope, but have
reduced pathogenicity. (Attenuated viruses for instance.)
This is achieved by providing a method for providing
a viral protein 1 comprising a neutralizing conformational
epitope according to the invention, comprising expressing
at least a functional part of VP1 and VP2 from genes
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encoding them, at least one of which genes is under
control of a regulatory sequence derived from the CAV
sequence upstream of the transcription intitiation site
which regulatory sequence is modified to reduce its
efficiency. In this way recomnbinant viruses can be
produced which do not replicate as fast and therefor are
less pathogenic ~i.e. attenuated).
Such virus particles are also part of the invention.
As disclosed hereinbefore the modification is preferably
in the promoter/enhancer region and most preferably the
modification is in the 12 base pair insert in the promoter
enhancer region.
Recombinant virus particles obtainable by any method
according to the invention are also part of the invention,
as are nucleic acids for use in any method according to
the invention, for instance vectors comprising a gene
encoding at least a functional part of VPl and a gene
encoding a functional part of VP2, at least one of the
genes being inder control of a regulatory sequence which
is modified to reduce its efficiency.
The antibodies and epitopes according to the
invention are also useful in a diagnostic test kit for
detecting or det~rmining the presence of CAV or antibodies
to CAV in a sample
Furthermore vaccines for the treatment or prophylaxis
of CAV associated disease comprising an antibody or an
epitope according to the invention together with a
suitable adjuvans and/or a suitable vehicle for
administration are also provided herewith as are vaccines
for the treatment or prophylaxis of CAV associated disease
comprising recombinant virus particles according to the
invention together with a suitable adjuvans and/or a
suitable vehicle for a~min;ctration.
The invention will be explained in more detail on the
basis of the following experimental part. This is only for
the purpose of illustration and should not be lnterpreted
as a limitation of the scope of protection.
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EXPERIMENTAL
Purification of CAV particles to elicit neut~alizing
mo~oclonal antiho~ies aga;n.~t CAV
For the production of neutralizing monoclonal
antibodies against CAV, mice were injected with purified
CAV particles.
The supernatant of a 1-liter culture of CAV-infected
MDCC-MSB1 cells was fourty times concent~ated by means of
a MILLITAN 300-kDa filter (Millipore, USA). The
supernatant was dialyzed against lOmM Tris(ph 8.7)-lmM
EDTA (TE) buffer. Subsequently, sodium dodecyl sulphate
(SDS) to an end-concentration of 0.5% was added to the
CAV-capsid suspension and incubated for 30 minutes at
37 C. Finally, the CAV capsids were pelleted on a 30%
sucrose cushion. The pellet cont~;n;ng the CAV capsids was
resuspended in 1 ml TE buffer. Mice were twice injected
with 100 ~ul CAV-capsid suspension.
In-vitro neutralization test.
The supernatants of the candidate monoclonal
antibodies were diluted 1:2 and then a two-fold dilution
series was made. The diluted supernatants were incubated
for 1 hour with 104-105 TCID50 CAV-Cux-1 (Von Bulow et
al., 1983, Von Bulow, 1985). Approximately one hundred
thousand cells of the T cell line MDCC-MSB1 transformed by
Marek's disease virus were infected with this mixture of
diluted supernatant and virus (Yuasa, 1983, Yuasa et al.
1983).
Tmmllnofluorescence and immunoperoxidase assay
Cells were fixed with 80~ acetone and used for
immunofluorescence assays with CAV-specific monoclonal
antibodies and goat anti-mouse IgC conjugated with
fluorescein, as decribed by Noteborn et al. (1990).
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WO96/40931 PCT~L~6,0~230
Recombinant-Vpl/VP2-MDV-infected CEF were used for
immunoperoxidase stai ning with CAV-pecific monoclonal
antibodies, as described by Jeurissen et al. (1988).
5 EnzymP-l;nked ;mml~nosorh~n~ assay f~TISA~
Microtiter wells (Greiner~ FRG) were coated with the
CAV-specific neutralizing monoclonal antibody 132.1, which
was 1:10,000 diluted in 50 mM sodiumbicarbonate pH 9.6.
Wash the wells three times with tapped water cont~;n;ng
0.05% Tween 80. Saturate the wells with 100 ~ul
phosphate-buffered saline cont~;n;ng 4% horse serum, 51
gram/liter NaCl, and 0.05% Tween 80 (saturation buffer)
for 30 minutes at 37 C. Wash the wells three times with
tap water containing 0.05% Tween 80. Next, 50 u~l of
non-diluted chicken serum and 50 ~ul of thirty times
concentrated supernatant containing CAV particles, or 50
~ul of a lysate of insect cells containing recombinant VP1
and VP2 proteins, were mixed, added per well and incubated
for 1 hour at 37 C. Wash the wells three times with tap
water containing 0.05% Tween 80. Add per well 100 u~l a
1:2000 fold dilution of a peroxidase-labeled rabbit an
ti-mouse immunoglobulin G conjugate in saturation buffer.
Incubate for 1 hour at 37 C. Wash again three times with
tap water containing 0.05% Tween 80. Add 100 ~1 of a
standard solution of tetramethylbenzidine, sodiumacetate
and hydrogenperoxidase to the wells and incubate for 10
minutes at room temperature. The reactlons are blocked
with 10~ H2SO4. The various wells are ~m; ned at 450 nm,
as standard.
Baculovirus ,~nd i~ect cells.
The recombinant baculovirus AcRP23-lacZ (Bishop,
1992) was obtained from Dr. R. Possee, NERC Institute of
Virology, Oxford, England, and the genomic DNA was
purified as described by Summers and Smith (1987).
Spodoptera frugiperda (Sf9) cell were obtained from the
American Tissue Culture Collection (no. CRL 1711).
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11
Baculovirus stocks were grown in confluent monolayers and
suspension cultures in TC-100 medium (GIBCO/BRL)
containing 10% fetal calf serum, as described by Summers
and Smith (19~7).
Clon; nq of CAV DNA.
All CAV DNA sequences are originally derived from the
plasmid DNA pIc-20H/CAV-EcoRI (Noteborn and De Boer,
1990). All cloning steps with plasmid DNA were in
principle carried out according to the methods described
by Maniatis et al. (1982).
DNA transformations were carried out in the E.coli
strain HB101. All plasmid were multiplied in large culture
under agitation, purified on CsCl gradients, and then by
filtration over Sephacryl-S500 columns or by filtrations
on QIAGEN-chromatography columns.
Conctruct;on ~n~ 5~lect;0n of rec~mh;nAnt-VPl/VP2
bacl1lovirus.
Recombinant transfer vector pAcVP1/VP2 DNA was
transfected with linearized recombinant baculovirus
AcRP23-lacZ DNA, in Sf9 cells. After homologous
recombinantion baculoviruses were obtained, which had
incorporated in the polyhedrin unit instead of the lacZ
the two CAV proteins VP1 and VP2 under regulation of the
promoter of the plO or polyhedrin gene, respectively. In
first instance, the recombinant CAV viruses were
characterized for the absence of beta-galactosidase
activity in plaques of baculovirus-infected insect cells.
Further the integration of CAV DNA sequences in the
baculovirus genome was det~r~;ned by means of a
CAV-specific DNA probe in a hybridization experiment.
Immunoprecipitation assay
Two days after infection, the cells were incubated
with Promix label (ICN, USA~ and four hours later, the
cells were lysed in ElA buffer (50 mM Tris (pH7.5), 0.1
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12
Triton-X-100, 250 mM NaCl, 50 mM NaF, and 5 mM EDTA) and
incubated with monoclonal antibody 111.1 directed against
VP2 for 2 hours at 4 C, washed with ElA buffer and
separated on a PAA-SDS gel.
Tr~n~fection of ~nv DNA ;n ~s.
For the construction of recombinant-VP1/VP2 MDV,
co-transfections with purified recombinant-VP1/VP2
MDV-transfervector and DNA, isolated from chicken embryo
fibro blasts (CEF) infected with MDV Rispens isolate (van
Vloten et al. 1972) were carried out according the method
described by Graham and Van der Eb (1973).
~ansfection of chicken T cells with mutant-CAV ge~omes.
The various mutated-CAV clones were digested with
EcoRI and the EcoRI fragmerlts cont~; n; ng the CAV (mutated)
sequences was recircularized and transfected in MDCC-MSB1
cells, by using the DEAE-dextran method as has been
described by Noteborn et al. (1991). As control, the whole
procedure was in parallel carried out for the pCAV-EcoRI
clone.
RESULTS AND DISCUSSION
The production of neutralizing monoclonal antibodies
against CAV.
Noteborn and Koch (1994) have described two types of
CAV-specific monoclonal antibodies. One type is directed
against VP2, while the other is directed against VP3. None
of these monoclonal antibodies reveal a CAV-specific
neutralizing activity. Even, more important was that none
of these monoclonal antibodies was directed against VP1.
We assumed that a neutralizing monoclonal antibody might
be directed against VPl, for the capsids contain mainly
VPl (Todd et al., 1990). Below, we describe the production
of neutralizing antibodies against CAV.
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13
For the production of neutralizing monoclonal
antibodies against CAV, mice were injected with purified
CAV particles.
As a first screening for (neutralizing) monoclonal
antibodies against CAV, microtitre wells were coated with
recombinant-VP1/VP2-baculovirus infected insect cells,
which co-synthesized both VP1 and VP2 (see below).
CAV-specific antisera with a high neutralizing titer
reacted at a dilution of 1:1000 specifically with the
recombinant VPl and/or VP2 products (see below). Several
different hybridoma cell lines producing monoclonal
antibodies, which specifically reacted with recombinant
VP1/VP2 products, were obtained.
A CAV-specific serum neutralization test, showed that
three of these monoclonal antibodies obtained, had a
neutralizing activity against CAV. These three
CAV-specific neutralizing monoclonal antibodies were
called 132.li 132.2 and 132.3.(deposited under no's
xxxx,yyyy,zzzz at the Institut Pasteur, France.
Microscopy examinations of the neutraliz;ng
monoclonal antibodies d~ ected aga;n~t CAV.
Immunofluorescence showed that the three neutralizing
monoclonal antibodies 132.1, 132.2 and 132.3 recognize
specific structures in CAV-infected MDCC-MSBl cells. None
of these monoclonal antibodies reacted with uninfected
MDCC-MSB1 cells.
Electron-microscopic analysis was carried out with
purified CAV particles incubated with neutralising
antibodies against CAV (132.1) or with monoclonal
antibodies 111.1 (against VP2) or 111.3 (against VP3).
Todd et al. (1990) have reported that purified CAV capsids
contain onlya 50-kDa protein, which is most likely VPl.
The various monoclonal antibodies were detected by
immunogold labeling. Only the neutralizing monoclonal
antibodies 132.1 were found to bind to CAV particles.
Binding of the monoclonal antibody 132.1 to a CAV particle
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14
resulted in its lysis. Furth~rmorer CAV capsids, which
were lysed due to incubation with the neutralising
monoclonal antibody 132.1, showed no binding with
monoclonal antibo dies directed against VP2 or VP3.
These results reveal the mech~n; ~m by which the
neutralizing monoclonal antibodies act: They cause the
lysis of the virus capsids, by doing so causing
non-infectious particles. Furthermore, these data show
that purified CAV particles contain (almost) only VP1.
Neutr~l;z;ng ~noclon~l anl-;hodies are directed aaain~t an
conformation~l epitope on VP1.
Pepscan analysis (Geysen et al., 1984) revealed that
none of the 3 neutralizing monoclonal antibodies reacted
significantly with one of the 12-mers derived from VP1 or
VP2, or VP3. For the sake of brevity only the data
obtained wwith monoclonal antibody 132.1 are shown for VPl
in Figure 1, and for VP2 in Figure 2. These results
indicate that the neutralizing monoclonal antibodies are
directed against a conformational epitope.
These data were confirmed by the following
experiments. Purified CAV particles, dotted on a nylon
filter under native conditions, still could react with the
neutralizing monoclo nal antibody 132.1. However, after
boiling in the presence of SDS, the CAV capsid proteins
did not bind to monoclonal antibody 132.1.
Immunoprecipitations experïments, carried under
native conditions, as described by Noteborn et al.
(1994b), with partially purified CAV particles and
monoclonal antibody 132.1, 132.2 or 132.3 showed that a
protein of about 50 kDa was precipated by these monoclonal
antibodies.
It can be concluded that the presented results
indicate that the neutralizing monoclo nal antibodies are
directed against an conformational epitope of VP1.
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~nzyme-linked ;mm~lnosorbens assay (~TISA) based on 2
neutralizina antibody againct CAV.
We have developed a complex-trapping-blocking
~ ~CTB)-ELISA. One can use enriched CAV particles derived
from CAV-infected MDCC-MSB-1 cells or recombinant VPl/VP2
proteins synthesized by means of the above described
baculovirus system.
Serum from CAV-infected chickens contains antibodies
which will block all epitopes on the CAV capsids.or
recombinant VP1/VP2. This means that the CAV capsid or
recombinant VPl/VP2 will not bind to the coated monoclonal
antibody 132.1. Negative serum, howe ver, will allow
binding of CAV capsids or recombinant VPl/VP2 to the
coated 132.1. A signal smaller than 0.5 of the signal
detected with a negative control serum will be ~m; ned as
positive.
The detection level of our CTB-ELISA are titers of 24
to 25 a det~rm;ned in a serum neutralization test, which
is very sensitive. More than 400 sera were analyzed.
Comparison to the serum neutralization test revealed that
96.5% of the positive sera within the serum neutrali
zation test were positive within the CTB-ELISA, and 98.3%
of the negative sera within the serum neutralization test
were negative within the CTB-ELISA.
Construction of a recom~;n~nt-VPl/VP2 transfer vector.
The coding sequences for the CAV proteins VPl and VP2
were cloned into the baculovirus transfer vector pAcUW51
(cat. no: 21205P), which was co~-rcially obtained from
PharMingen, San Diego, USA. This vector is shown in Figure
3 and contains the polyhe drin flanking region, within
their midst the baculovirus polyhdrin promoter and the plO
promo ter and for both transcription units, the required
3'-non-coding transcrioptional sequences including the
polyadenylation signals. The transfer vector contains
prokaryotic sequences for multiplication in bacteria.
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16
The plasmid pET-16b-VP2 (Noteborn et al., dates not
publ;~h~) contains CAV DNA sequences of positions 380 to
1512. This CAV DNA fragment contains the coding region for
VP2 flanked by 484 bp 3'-non-coding CAV DNA sequences. 106
bp downstream of the start codon for VP2 the start codon
for VP3 is found in another reading frame. The plasmid
pET-16b-VP2 was treated with the restriction enzymes NdeI
and NheI, and the sticky ellds were filled by means of
Klenow polymerase. A 0.8 kb CAV DNA fragment was isolated.
The plasmid pAcUW51 was linearized with BamH1, the sticky
ends filled by means of Klenow polymerase and finally
treated with alkaline phosphatase ~CIP). The 0.8 kB CAV
DNA fragment was ligated at the linearized pAcUW51 DNA.
The orientation of VP2 in pAcUW51 was determined by
restriction enzyme analysis. This construct was called
pUW-VP2.
The pl~-cm;~ pET-16b-VP1 (Noteborn et al., data not
publ;qhe~) contains CAV DN~ sequences of positions 853 to
2319. The CAV DNA insertion contains the complete coding
region for the protein VPl flanked by 117 bp 3'-non-coding
CAV DNA sequences. The plasmid pET-16b-VPl was treated
with the restriction enzymes NdeI and EcoRI, and the
sticky ends were filled by means of Klenow polymerase. A
1.45 kb CAV DNA fragment was isolated. The plasmid pUW-VP2
was linearized by EcoRI, the sticky ends filled by means
of Klenow polymerase and finally treated by CIP. The 1.45
kb CAV DNA fragment was ligated at the linearized pUW-VP2.
The orientation of VP1 opposite of the plO promoter unit
was determined by restriction-enzyme analysis, and the
final construct pAcVPl/VP2 is shown in Figure 3.
Construction of rec~mhin~nt-VPl/VP2 baclllovirus.
The open reading frames encoding VPl and VP2 were
separately cloned into a single baculovirus
transfervector, viz pAcUW51. The CAV sequences encoding
VPl are under the regulation of the baculovirus plO
promoter and VP2 under the regulation of the baculovirus
CA 0222l~70 l997-l2-0~
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17
poyhedrin promoter. Transfection of Sf9 insect cells
occurred with 'naked' baculovirus DNA and transfervector
DNA. After homologous recombinantion baculovirues were
obtained which had incorporated the VP1/VP2 expression
unit, integrated in the polyhedrin region of the
recombinant baculovirus, instead of the lacZ unit. The
recombinant baculoviruses were characterized for the
absence of beta-galactosidase activity and integration of
CAV DNA sequences.
Expression of the CAV prote; n~ VP1 and VP2 ; n Sf9 c~lls.
The simultaneous expression of the CAV proteins VP1
and VP2 in Sf9 cells infected with recombinant-VP1/VP2
baculovirus was analyzed by Coomassie-brilliant blue
staining and protein labeling with Promix (ICN, USA) or
3H-leucine (Amersham, UK) and PAA-SDS gel
electrophoresis.
In the PCT/NL94/00168 it was shown that lysates of
insect cells infected with recombi nant-VP1 baculovirus
contained a CAV-specific protein of 52 kD and expression
of VP2 by recombinant-VP2 baculovirus in infected insect
cells produced a major specific product of 30 kDa.
Infection of insect cells with recombinant-VP1/VP2
baculovirus resulted in the synthesis of both the
CAV-specific proteins of 52 kD and 30 kD. Both
CAV-specific products could be detected as radioactively
labeled protein band or Coomassie-brilliant blue stained
protein band. The latter result indicates that both
products are produced in relatively high levels in recombi
nant-VPl/VP2-baculovirus-infected insect cells. Sf9 cells
infected with a recombinant-lacZ baculovirus did not
contain these CAV-specific proteins.
Thus we have obtained evid~nce that inoculation in
hens of crude lysates of recombinant-VP1/VP2-infected Sf9
- 35 cells are able to induce neutralizing antibodies directed
against CAV.
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18
The role OI' VP2 for the formation of a conformational
neutr~lizing epitope of VPl.
As reported in PCT/NL94/00168 and by Koch et al.
(1995), simultaneous synthesis and not simply mixing of
5 recombinant CAV proteins VPl and VP2 is required to obtain
a neutralizing and protective immune response. These data
suggest that VP2 is a non-structural protein that at some
stage of infection is required for virus assembly and/or
the correct conformation of VP1, which result(s) in the
10 formation of the neutralizing epitope(s). One explanation
of the requirement of VP2 might be that it acts ~s a
scaffold protein that is necessary during the assembly of
the virion but absent in the final product (Leibowitz and
Horwitz, 1975). Examples of scaffold proteins are the IVa2
15 and 39kDa proteins of adenovirus (D'Halluin et al., 1978;
Persson et al., 1979). These proteins act as scaffolds for
the formation of the so-called light capsid, but are
L~..oved in the next step. VP2 might function in a s;m;1;~r
way during the formation of CAV virions. However, at this
20 stage, we cannot entirely exclude that (very) small
amounts of VP2 that remained undetected in electroblots of
purified CAV preparations (Todd et al., 1990) or in
electron microscopic photographs of lysed CAV particles
incubated with immunogold-labeled VP2-specific monoclonal
25 antibodies, as described above, associate with VP1 and
form conformational neutralizing epitopes. Recently, weak
evidence for the presence of VP2 in gradient-purified CAV
was reported (Buchholz, 1994).
In the following experiments, evidence is provided
30 that the neutralizing epitope of VP1 is only (optimal)
present, when VP2 is simultaneously synthesized. Insect
cells were infected with recombinant-CAV baculoviruses
expressing VP1, VP2 (see PCT/NL94/00168) or both VP1 plus
VP2. The infected Sf9 cells were harvested 3 or 4 days
35 after infection and used for immunofluorescence tests with
the CAV-specific neutralizing monoclonal antibody 132.1.
The cells containing only the CAV-specific protein VP2 did
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19
not react at all with the monoclonal antibody 132.1. Cells
cont~;n; ng only VPl revealed a very poor
immunofluorescence signal after incubation with monoclonal
antibody 132.1. However, insect cells infected with
recombinant-VP1/VP2 baculovirus expressing both VP1 and
VP2 bound very strongly to the neutralising monoclonal
132.1. PAA-SDS gel electrophoresis of in parallel
radioactive-labeled lysates of insect cells expressing
VP1, VP2 or VP1 plus VP2, revealed that VP1 is expressed
at the same level when expressed only or simultaneously
with VP2.
VP1 and VP2 interact temporarily with each other.
The neutralizing epitope of VP1 is only formed when
VP2 is present, and therefore is a conformationally
distinct epitope. This implies that VP1 and VP2 associate
with each other during a short time period. By means of
;mmllnoprecipitations under very mild conditions, we have
~m; ned whether VP1 could associate with VP2. Sf9 insect
cells were infected with recombinant baculoviruses, which
synthesized VP1, VP2, or VP1 plus VP2.
The results clearly reveal that monoclonal antibody
111.1 precipates VP2 when VP2 is synthesized alone or in
the presence of VP1. In the case that besides VP2, VP1 was
expres sed also, VP1 co-precipitated to a small extent
with VP2. The monoclonal antibody 111.1 did not detectably
precipitate VP1, when VP1 was synthesized in the absence
of VP2. These data indicate that VP1 and VP2 are (to a
relatively small amount) associated to each other. During
this association event, VP1 might obtain its conformation
resulting in the neutralizing epitope.
Basis for the development of vaccines aaa;nst CAV
infections
The above presented results show that for the
induction of neutralizing antibodies against CAV, VP1 is
required which has a specific conformation. In a
CA 02221~70 1997-12-0~
WO 96/40931 PCT/NL96/00230
baculovirus expression system, this correct VP1
conformation is only possible, when VP1 plus VP2 or VPl
plUS VP2 plus VP3 are simultaneously synthesized.
Denaturation of VP1 with e.g. sodium-dodecyl-sulphate
result also in the destruction of it neutralizing epitope,
indicating that the VPl neutralizing epitope is an
conformational one.
The recombinant CAV products, VP1 plus VP2 or VP1
plus VP2 plus VP3, which will be used for vaccination of
laying-hens, can be synthesized by means of the
baculovirus system. The CAV proteins can also be
synthesized by means of other expression systems, such as
yeast cells, via (retro)- viral infection or gene
amplification (CHO-dhfr system) in m~mm~l ian cell systems.
In principle, the expression of fragments of VP1 (in
combination with VP2 or VP2 and VP3) may be sufficient for
the induction of a protective immune response. The fact
that 12-mers of VP1 can not react with neutralizing
antibodies against CAV indicates that larger VPl fragments
20 are needed for getting the correct VPl conformation to
form the neutralizing epitope. However, one should take
into account that minor amino-acid mutations or a few
amino-acid deletions might not influence the formation of
the neutralizing epitope of VPl.
The fact that 2 or 3 proteins encoded by the CAV open
reading frames can induce a protective immune respons is
also applicable to the development of living virus
vectors. The coding sequences for VPl plus VP2 or VPl plus
VP2 plus VP3 are then cloned into living virus vectors.
It is also possible that one of the CAV proteins,
VPl, VP2 or VP3, separately, but then within the context
of a living virus vector, is also suitable for the
induction of a protective immune response against CAV
infections. The expression of fragments of one of the abo
ve-mentioned CAV proteins by living virus vectors may be
sufficient for the induction of a protective immune
response.
CA 0222l~70 l997-l2-0~
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21
The fact that VP3 causes apoptosis in, i.e. chicken
mononuclear cells (PCT/NL94/00168i Noteborn et al., 1994a
favors to construct living virus vectors not expres sing
VP3. The replication of i.e. Marek_s disease virus might
be negatively influenced by VP3-induced apoptosis.
Alternatively, one can produce attenuated CAV
expressing VP1 with the required conformation needed for
eliciting a neutralizing and protective immune response
agnaist CAV infections.
Co~truction of a rec~mh;n~nt-~nV-VP1/VP2 trAncfçr vector.
The coding sequences for the CAV proteins VP1 and VP2
were cloned into the Marek's disease virus (MDV)
transfervector pMD-US10-SV (Koch, unpublished data). The
transfervector contains the SV40 early promoter, which are
flanked by sequences of the MDV US10 region, and
prokaryotic sequences for multiplication in bacteria.
A schematic representa tion of the transfer vector is
given in Figure 4.
The CAV coding sequences from position nt 368 to nt
2319 were inserted in the pMD-US10-SV transfervector under
the regulation of the Sv40 ealy promoter. This recombinant
transfervector was checked by restriction enzyme digestion
and is called pMD-US10-SV-VP1/VP2.
By introducing a point-mutation at position 549 {T
was changed into an A) an extra stopcodon has been
introduced in the gene encoding VP3 (Noteborn, unpublished
data). Therefore, the inserted CAV sequences will only
express VP1 and vP2. Indirect-immunofluorescence analysis
of chicken embryo fibroblasts (CEF) transfected with the
vector pMD-US10-SV-VP1/VP2 proved that VP1 and VP2 were
indeed expressed, whereas VP3 was not.
Construction of rec~mhin~nt-VP1/VP2 ~nv.
Using the Ca-phosphate transfection method,
recombinant transfer vector pMD-US10-SV-VP1/VP2 DNA was
transfected with MDV (Rispens isolate) DNA,in CEF cells.
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22
After homologous recombination, recombinant MDVs were
obtained, which had incor porated the CAV coding sequences
in the the USlO region ~Nakamura et al., 1992). The
recombinant-VPl/VP2 MDVs were characterized for the
presence of the expression of VP2. By in-parallel plaque
purification and ;mmllno-peroxidase analyis using a
monoclonal antibody directed against VP2, several
100%-purified recombinant-VPl~VP2 MDV independent strains
were obtained.
Char~cter;~at;on of recnmhin~nt-VPl~VP2 ~nv.
The correct integration of the CAV-DNA sequences in
the MDV genome was determined by means of PCR and
restriction-enzyme analysis. By subsequent passaging of
the various purified recombinant-VPl/VP2 MDVs, it was
shown that they remained stable. No recombinants were
obtained with (partial) loss of the VPl/VP2 expresssion
unit.
The simultaneous expression of the CAV proteins VPl
and VP2 in CEF cells, which were infected with the
recombinant-VPl/VP2 MDV, was proven by indirect
immunofluorescence. The monoclonal antibodies
CVI-CAV-111.1 (111.1), directed against VP2 and
CVI-CAV-132.1 (132.1), directed against VP1 were used.
The monoclonal antibody 132.1 is directed against a
neutralizing epitope of VP1. The fact that the
neutralizing antibody 132.1 can recognize recombinant-MDV
expressed VP1, co-synthesized with VP2, implies that the
required neutralizing epitope is present on MDV-expressed
VP1. Therefore, vaccination of chickens with
recombinant-VP1/VP2 MDV will result in the induction of a
neutralizing/protective antibody response. Besides the
synthesis of the CAV VP1 and VP2 proteins, the
recombinant-VP1/VP2 MDV will synthesize the MDV proteins
required for the induction of a protective immune response
against MDV infections.
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WO 96/40931 . PCT/NL'~6/00'~0
23
Pro~uc~;on of attenuated CAV based on the cloned CAV
genome pCAV/EcoRI.
The CAV-EcoRI clone is used for the production of
~ viable infectious CAV particles. To that end, the complete
CAV insert, the 2,319-bp EcoRI fragment has to be isolated
from the bacterial sequences and recircularized by DNA
polymerase treatment. Subsequently, e.g. MDCC-MSB1 cells
are transfected with the recircularized EcoRI fragments
and after several days wild-type CAV will be produced.
One can introduce mutational changes in the CAV
sequences of the CAV-EcoRI clone and isolate,
recircularize and transfect in MDCC-MSBl cells. Instead of
wild-type CAV, in principle one can produce mutant CAV and
if the mutant is less pathogenic, attenuated CAV. The
complete strategy for the production of attenuated CAV is
shown in Figure 5.
Constrllct;o~ of CAV gen~mes co~tA;ninq mlltate~
promoter/~nh~ncer reaions.
A remarkable feature of the CAV promoter/enhancer
region is a sequence of 4 or 5 near-perfect 21-bp repeats,
interrupted by an insert of 12-bp (Noteborn et al., 1991).
This region is involved in the regulation of the CAV
transcription (Noteborn et al., 1994b).
For the production of attenuated CAV, we have
introduced mutational changes in the 12-bp
insert/direct-repeat region of the CAV-EcoR1 clone
(Noteborn et al., 1991). The mutated promoter/enhancer
region of the wild-type (wt) and the various CAV mutants
are shown in Fig. 6. The Nae-mutant contains no
12-bp/direct-repeat region sequences at all. Tnstead of
these, a NaeI site (5'-GCCGGC-3') has been introduced,
which is given as 'N' in figure 6.
All other mutants contain 4 original direct repeats
instead of 5, as is the case for the CAV-EcoRI clone. The
various CAV mutants, called '6b'p, '12bp', '24bp' contain
changed 12-bp inserts. The mutant 'wt in Nae' and of
CA 02221~70 1997-12-0~
WO96/40931 PCT~L96/00230
24
course the original CAV-EcoRI clone, called 'wt', contain
the original 12-bp insert sequences 5'-AAGAGGCGTTCC-3'.
The CAV mutants '6bp', '12bp', '18bp', '24bp' and 'wt
in Nae' contain all additional sequences flanking the
12-bp insert/direct-repeat region. At the 5'-site of this
region, the linker 5'-GCCCATGT-3', and at the 3'-site the
linker 5'-GATCCGCC-3' has ~een introduced.
Mtltate~ CAV a~nomes resl~lts in the production of
attenuated infectious CAV.
To detPrmine whether the mutated CAV genomes could
produce live CAV particles, firstly the synthesis of VP3
was ~mined. At several timepoints after transfection, a
part of the various transfected MDCC-MSBl-cell cultures
were aceton-fixed and analysed by indirect
immllnofluorescence using a monoclonal antibody directed
against VP3. In parallel, 1 ml of each culture was added
to 9 ml of fresh RPMI-medium, which was supplemented with
10~ fetal bovine serum.
Approximately 15% and 90% of the cultured MDCC-MSB1
cells, transfected with 'wt' recircularized CAV genome
DNA, contained VP3 after 6 days and 11 days (the cells
were one time passaged after transfection), respectively.
The CAV mutant genome 'Nae', lacking the complete
12-bp insert/direct-repeat region, was shown not to
produce infectious virus particles. Six days after
transfection, at most 2~ of the cells produced VP3, which
is due to transient expression of the 'Nae' DNA. A simil~r
percentage is obtained with expression plasmid pRSV-VP3
30 (Noteborn et al., 1994b) encoding VP3 only, and which are
known not to replicate in eukaryotic cells. After 3 weeks
and several passages, no VP3 was present anymore in the
Nae-DNA transfected MDCC-MSB1 cell cultures.
At various time-intervals after transfection, the
percentage of VP3-positive cells of the MDCC-MSB1
cultures, transfected with the mutant-DNA genomes '6bp',
12bp', 18bp, 24bp and 'wt in Nae' was significantly lower
CA 0222l~70 l997-l2-0~
WO96/40931 PCT~L96/00230
compared with cultures, transfected with 'wt'-DNA genome.
These data imply that the various CAV mutants replicate
less fast than the wild-type CAV. Especially, the mutants
'6bp' and '18bp' were reduced in their replication rate.
Most likely, the mutational changes of the 12-bp insert
results in the lower virus spead of the mutated CAV. The
results of these experiments are shown in Figure 7.
Subsequently, we have infected fresh MDCC-MSB1
cultures with supernatants of cells tran~fected with all
10 ~mi ned mutated CAV-DNA genomes. All supernatants, except
the one derived from 'Nae'-DNA-transfected cells~ were
proven to contain infectious virus parti cles. For these
cultures, infected with the 'mutated-virus' containing
supernatants, it could be shown by indirect
immunofluorescence, that cells were present containing
VP3.
CAV mllt~nts h~ve ~ re~uce~ cvtopa~hoq~n;c effect.
In parallel with the above described detection of VP3
synthesis, the transfected cells wçre analysed whether
they had become apoptotic. CAV is known to induce
apoptosis, 2-3 days after infection. To that end, the
cells were stained with propidium iodide, which stains
'normal' DNA strongly, but apoptotic DNA weakly and/or
irregularly. Already, 11 days after transfection almost
all MDCC-MSB1 cells transfected with 'wt' DNA were shown
to be apoptotic, whereas the majority of the cells,
transfected with the various mutated CAV-DNA genome were
not. The capacity for inducing apoptosis was shown to be
mostly reduced for the CAV mutants '6bp' and '18bp'.
DNA ~lys;~ of att~nl~ted CAV.
By polymerase-chain reaction and sequence analysis of
the 12-bp insert/direct-repeat region and flanking
sequences, it was shown that the original mutations were
not changed for the various CAV mutants '6bp', '12bp',
'18bp', '24bp' and 'wt in Nae'. These results indicate
CA 02221~70 1997-12-0~
wos6/4o93l PCT~L96/00230
26
that the various CAV mutants are stable, at least after
culturing in MDCC-MSBl for about l month and that indeed
these CAV mutants are viable and causing a reduced
cytopathogenic effect in cultured chicken T cells.
Southern blot analysis of DNA isolated from MDCC-MSBl
cultures, infected with the various CAV mutants, revealed
that all viable CAV mutants produced all CAV DNAs, as was
described for wild-type CA~. The double-stranded
replication intermediate and sing le-stranded DNA were
detected for both wild-type as mutated CAV.
Attenuated CAV synthesizes the CAV-specific neutral;zin~
epitope
By indirect-immunofluorescence, using the
neutralizing monoclonal antibody CVI-CAV 132.l and a
monoclonal antibody directed against VP2, viz CVI-CAV
lll.2 and VP3, viz CVI-CAV-85.l, it was shown that all
mutated CAV g~nnm~S were able to synthesize the CAV
proteins VPl, VP2 and VP3. Even more important is that the
neutralizing monoclo nal antibody 132.l reacted with
cells, infected with the various CAV mutants. This finding
indicates that the mutated CAVs will produce the required
neutralizing CAV-specific epitope, whcih has to be
recognized by the immune system of vaccinated chickens as
CAV for eliciting a protecvctive immune response.
It can be concluded that based on the CAV clone
pCAV-EcoRI, mutated CAV genomes can be made, resulting in
the production of viable mutated CAV particles. These CAV
mutants replicate in cultured chicken T cells less fast
than wild-type CAV. The cytopa thogenic effect caused by
these CAV mutants is also reduced compared to wild-type
CAV. The CAV mutants are all able to produce all CAV
proteins resulting in the required neutrali zing epitope
on VPl.
Therefore, these CAV mutants are attenuated versions
of CAV in the field.
CA 02221~70 1997-12-0~
WO 96/40931 PCT/NL96/00230
27
Description of the Figllres
Figure 1 shows the pepscan analysis of the
neutralizing monoclonal antibodies of type 132.1 with
- peptides (12-mers~ derived from VP1.
Figure 2 shows the pepscan analysis of the
- neutralizing monoclonal antibodies of type 132.1 with
peptides (12-mers) derived from VP2.
Figure 3 shows the diagrammatic representation of the
recombinant transfer pUW-VP1/VP2.
Figure 4 shows the diagrammatic representation of the
recombinant transfer vector pMD-US10-SV-VP1/VP2.,
Figure 5 shows the strategy for the production of
attenuated CA~-mutants, based on the plasmid pCAV/EcoR1
containing 2319 bp of wild-type CAV.
Figure 6 shows the schematic structure of the CAV
promoter/enhancer region of the various mutated and
wild-type CAV strains.
Figure 7 shows the VP3-expression rates obtained for
the various mutant-CAVs and wild-type CAV after
tranfection of mutant or wild-type circularized DNA. The
VP3-expression rate are given as percentage of
VP3-positive MDCC-MSB1 cells.
Figure 8 gives the sequence of CAV.
CA 0222l~70 l997-l2-0~
WO96/40s31 PCT~L96/00230
28
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