Note: Descriptions are shown in the official language in which they were submitted.
CA 022~3229 1998-10-26
WO97/41236 PCT~B97/00444
POLYNUCLEOTIDE VACCINE AGAINST CANINE
DISTEMPER
Technical Field
The invention concerns polynucleotide vac-
cines against the canine distemper virus (CDV), methods
of preparation of the polynucleotides and the vaccines
comprising them, and the use of the polynucleotides as
o vaccines for prophylactic immunization of animals suscep-
tible to canine distemper.
Background Art
Canine distemper is a highly infectious,
acute or subacute, febrile viral disease of dogs and
other carnivores, which occurs world-wide. Some dogs show
primarily respiratory signs, others intestinal signs and
at least 30~ of the animals develop neurological symp-
toms. All experimentally infected dogs have histopa-
thological lesions in the central nervous system. The
mortality rate ranges between 30 and 80%. In a minority
of cases, dogs that have recovered continue to harbour
the virus in brain cells where it replicates slowly and
eventually produces old dog encephalitis. The situation
is analogous to that of subacute sclerosing panencephali-
tis in the corresponding human infection, measles. Dogs
surviving distemper have life-long immunity to re-
infection. Immunization is recommended for the control of
distemper in dogs, using attenuated live virus vaccines
at the age of 8 weeks and again at 12 to 16 weeks. Annual
re-vaccination is recommended.
- The importance of effective vaccines against
morbillivirus infections is emphasized by recent reports
- 35 on the discovery of new members of this virus group, af-
fecting both terrestrial and marine mammals (Kennedy et
al. 1988; Domingo et al. 1991). There have been several
.
CA 022~3229 1998-10-26
WO97/41236 PCT~B97/00444
outbreaks of canine distemper among lions of the Ser-
engeti and lions, tigers and leopards in American zoos
(Appel et al. 1994; Leary, 1994). It was surprising, that
these big cats are susceptible to CDV. Furthermore, in
Australia a disease of horses, acute equine respiratory
syndrome (AERS) occurred and it was shown, that the AERS
virus belongs to the genus morbillivirus of the
paramyxoviridae (Murray, 1994). This virus not only in-
fects horses but is also transmissible to man. Morbil-
o liviruses thus seem to have expanded their host range.Increasing incidence of canine distemper has also been
noted in Japan, Finland, Italy and Switzerland despite
vaccination. The tested virus lsolates were different
from vaccine stralns, in terms of reactivity with anti-
bodies raised against the vaccine strains (Mori et al.1994). In Germany and Switzerland CDV infections among
wild carnivores have been reported, and mustelids may be
a hidden reservoir of CDV (Alldinger et al. 1994). Recent
experiments demonstrated CDV-RNA in bone tissues of hu-
mans with a chronic bone illness characterized by exces-
sive bone resorption, new bone formation and deformity,
the so-called Paget's disease (Gordon et al. 1992).
Therefore, CDV has been suggested to be involved in the
pathogenesis of Paget's disease. It is well known that
CDV can infect bone cells of its natural host ~Gordon et
al. 1992; Mee et al. 1992). Moreover, bone lesions were
observed in young dogs with experimental and spontaneous
distemper (Baumgartner et al. 1995). In addition to acute
infections, two members of the morbilliviruses, measles
virus and canine distemper virus, also produce a persis-
tent infection.
Canine distemper is caused by CDV, a member
of the genus morbillivirus (family paramyxoviridae). CDV
is closely related to the viruses of measles and
rinderpest.
The canine distemper virions (Fig. 1) are
enveloped and contain a negative-strand RNA genome of
CA 022~3229 1998-10-26
WO97141236 PCT~B97/00444
15'616 nucleotides which has been entirely sequenced for
the cell culture adapted Onderstepoort (OP-CDV) strain
(Sidhu et al , 1993, and references therein). The viral
genome encodes 6 proteins: the nucleocapsid (N) protein,
the phosphoprotein (P), the matrix (M) protein, the fu-
sion (F) protein, the hemagglutinine (H) protein, and the
large (L) protein. The genes are arranged in the genomic
RNA in the order (3'-5'): N, P, M, F, H, and L and each
protein is translated from a unique mRNA transcribed from
the negative strand RNA template.
The currently used vaccines against canine
distemper have a number of drawbacks. They may induce im-
munosuppression (M. Vandevelde, University of Berne,
pers. comm.) or neurological disorders (cited in Ham-
burger et al., 1991). Even cases of vaccine-induced dis-
temper have been reported (C. Green, University of Geor-
gia; R. Higgins, University of Davis; R. Maes, University
of Michigan, pers. comm.~. Furthermore, these vaccines
are not particularly satisfactory in terms of efficacy
since cases of canine distemper in vaccinated dogs are
not rare. Thus, of 84 dogs with diagnosed neurologic dis-
temper, 32 had complete, and 21 partial vaccine coverage
(Tipold et al., 1994). The incomplete protection provided
by the vaccine strains is most likely the consequence of
changes occurring in the virus upon cell culture adapta-
tion. Such changes are demonstrated by the fact that af-
ter adaptation to cell lines the virus quickly loses its
ability to cause disease (Bittle et al., 1962) and that
loss of virulence is associated with structural altera-
tions in the viral nucleocapsid protein (Hamburger etal., 1991). Similarly, the observation that radiolabelled
hybridization probes derived from tissue cuiture-adapted
virus failed to detect viral nucleic acids in the brain
of animals infected with virulent virus is an indication
that the vaccine and virulent strains differ markedly
(Mitchell et al., 1987). In view of these differences it
is not surprising that immunity induced by vaccine
~ ~ , ,,
CA 022~3229 1998-10-26
W097/41236 PCT~B97/00444
strains is not able to provide complete protection
against virulent virus.
Since the first report of protection of mice
against challenge with influenza virus following intra-
muscular injection of DNA (Ulmer et al., 1993) it hasbeen recognized that injection of naked nucleic acids en-
coding vaccine antigens represents a potent novel avenue
in vaccine development (review: Montgomery et al., 1994).
The advantages of nucleic acid vaccines are obvious. Such
0 vaccines should be safe, since no live organisms are
used. Furthermore, plasmid DNA is easy and cheap to pro-
duce and is stable even in adverse climatic conditions
which makes DNA vaccines particularly attractive for de-
veloping countries. An additional advantage is that new
plasmids can be constructed and tested in a relatively
short time which is important for designing vaccines
against pathogens for which the protective antigens have
not yet been identified. Perhaps the most attractive fea-
ture of nucleic acid vaccines is that they induce both
antibody and cell-mediated immune responses (Ulmer et
al., 1993).
Several methods for delivering DNA are cur-
rently available (review: Montgomery et al., 1994). The
most convenient method is direct injection into muscle
tissue (Wolff et al., 1992).
Disclosure of the invention
Object of the presented invention is to pro-
duce novel nucleic acid vaccines against canine distemper
which lack the drawbacks of hitherto vaccines against
this disease. In particular, said vaccine is a polynu-
cleotide vaccine containing virulent canine distemper vi-
rus genes which are important for eliciting neutralizing
antibodies, and which are essential for cell-mediated im-
munity. These genes are to be inserted into expression
plasmids which after delivery to living tissues produce
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WO97/41236 PCT~B97/00444
an immunizing effect. It is believed that a nucleic acid
vaccine containing genes of virulent distemper virus has
significant advantages~in terms of efficacy over conven-
tional attenuated vaccine strains which differ markedly
from virulent virus. Furthermore, no reversion to viru-
lence, which has been demonstrated for distemper virus
vaccine strains (Appel,1978) and which may result in dis-
temper outbreaks in vaccinated animals is possible (Bush
et al., 1976; Carpenter et al., 1976; Hartley et al.,
o 1974). In addition, the inclusion of different genes in
combination in the nucleic acid vaccine will generate
both a humoral and a cellular immune response. A further
advantage of a nucleic acid vaccine against canine dis-
temper is that such a vaccine, in contrast to conven-
tional live vaccine strains, will not induce immunesup-
pression. This is particularly important when the canine
distemper vaccine is administered together with other
components in a multivalent vaccine. In this situation,
immunesuppression of the host renders other live vaccine
components more virulent, possibly resulting in disease
induced by these vaccine strains. Immunesuppression by
canine distemper vaccine strains also reduces the immmllne
response to inactivated components contained in a multi-
valent vaccine. A nucleic acid vaccine against canine
distemper will not have these undesirable side effects.
Thus, the inventive vaccine is im many aspects superior
to hitherto known vaccines.
Brief Description of the Sequence Listings
30 and the Figures:
SEQU ID NO 1 shows the primer sequence corre-
sponding to the leader of CDV strain A75/17;
SEQU ID NO 2 shows the primer sequence corre-
3s sponding to the end of the N gene of CDV strain A75/17;
... . .... . . . ... ... .
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W O 97/41236 PCT~B97/00444
SEQU ID NO 3 shows the primer sequence corre-
sponding to the M gene at position M 116 of strain OP-
CDV;
SEQU ID NO 4 shows the primer sequence corre-
s sponding to the F gene at position F 1092 of strain OP-
CDV;
SEQU ID NO 5 shows the primer sequence corre-
sponding to the F gene at position F 177 of strain OP-
CDV;
lo SEQU ID NO 6 shows the primer sequence corre-
sponding to the F gene at position F 2058 of strain OP-
CDV;
SEQU ID NO 7 shows the primer sequence corre-
sponding to the F gene at position F 2002 of strain OP-
CDV;
SEQU ID NO 8 shows the primer sequence corre-
sponding to the H gene at position H 716 of strain OP-
CDV;
SEQU ID NO 9 shows the primer sequence corre-
sponding to the H gene at position H 675 of strain OP-
CDV;
SEQU ID NO 10 shows the primer sequence cor-
responding to the L gene at position L 78 of strain OP-
CDV;
2s SEQU ID NO 11 shows the primer sequence for
generating the 5' end of the N gene with a Kpn I restric-
tion site;
SEQU ID NO 12 shows the primer sequence for
generating the 3' end of the N gene with a Sal I restric-
tion site;
SEQU ID NO 13 shows the primer sequence F1
corresponding to the F gene of strain OP-CDV at position
1 with a Mlu I restriction site;
SEQU ID NO 14 shows the primer sequence F2
corresponding to the F gene of strain OP-CDV at position
2033;
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WO97/41236 PCTAB97/00444
SEQU ID NO 15 shows the primer sequence F3 -
corresponding to the F gene of strain OP-CDV at position
2014;
SEQU ID NO 16 shows the primer sequence F4
corresponding to the F gene of strain OP-CDV at position
2095 with a Sal I restriction site;
SEQU ID NO 17 shows the primer sequence H1
corresponding to the H gene of strain OP-CDV at position
18 with a Kpn I restriction site;
SEQU ID NO 18 shows the primer sequence H2
corresponding to the H gene of strain OP-CDV at position
705;
SEQU ID NO 19 shows the primer sequence H3
corresponding to the H gene of strain OP-CDV at position
684;
SEQU ID NO 20 shows the primer sequence H4
corresponding to the H gene of strain OP-CDV at position
1835 with a Sal I restriction site;
SEQU ID NO 21 shows the sequence correspond-
ing to the N gene of virulent CDV strain A75/17. Position1 corresponds to 5' end of the N mRNA. The translation
initiation (ATG) and termination (TAA) codons are under-
lined;
SEQU ID NO 22 shows the sequence correspond-
ing to the F gene of virulent CDV strain A75/17. Position
1 corresponds to 5' end of the F mRNA.
SEQU ID NO 23 shows the sequence correspond-
ing to the H gene of virulent CDV strain A75/17. Position
1 corresponds to 5' end of the H mRNA.
Figure 1 shows a schematic representation of
the CDV particle. The location of the viral M, H, F, N, P
and L proteins are indicated.
Figure 2 shows the expression plasmid H/CMV5
- 35 for the CDV H gene of strain A75/17.
Figure 3 shows the expression plasmid H/pCI
for the CDV H gene of strain A75/17.
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WO97/41236 PCTnB97100444
Figure 4 shows the expression plasmid N/CMV5
for the CDV N gene of strain A75/17.
Figure 5 shows the expression plasmid N/pCI
for the CDV N gene of strain A75/17.
Figure 6 shows the expression plasmid F/CMV5
for the CDV F gene of strain A75/17.
Figure 7 shows the expression plasmid F/pCI
for the CDV F gene of strain A75/17.
Figure 8 shows CTL assays of mice immunized
o with plasmid N/pCI or empty vector after 2nd immuniza-
tion.
Figure 9 shows CTL assays of mice immunized
with plasmid N/pCI or empty vector after 3rd immuniza-
tion.
Figure 10 shows anti-N antibody titers of
dogs immunized with standard vaccine or with plasmid
N/pCI.
Modes for Carrying out the Invention
In one embodiment the invention concerns a
nucleic acid construct comprising a canine distemper vi-
rus gene, wherein said nucleic acid construct is capable
of inducing the expression of an antigenic canine distem-
per virus gene product which induces a canine distemper
virus specific immune response upon introduction of said
nucleic acid construct into animal tissue in vivo and re-
sultant uptake of the nucleic acid construct by the cells
which express the encoded canine distemper virus gene.
The nucleic acid construct is a DNA or RNA
construct, preferably a DNA construct.
The invention concerns in particular a nu-
cleic acid construct, wherein the canine distemper virus
gene encodes the nucleocapsid (N) protein, the phos-
phoprotein (P), the matrix (M) protein, the fusion (F)
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WO97/41236 PCT~B97/00444
protein, the hemagglutinin (H) protein, or the large (L)
protein.
The nucleic acid construct is in particular
such, wherein the canine distemper virus gene encodes the
~ s nucleocapsid (N) protein, the fusion (F) protein, or the
hemagglutinin (H) protein.
Prefered DNA constructs are the plasmids
~/CMV5 and H/pCI, which encode the hemagglutinin (H) pro-
tein, the plasmids F/CMV5 and F/pCI, which encode the fu-
lo sion (F) protein of canine distemper virus strain A75/17,and in particuiar the plasmids N/CMV5 and N/pCI, which
encode the nucleocapsid (N) protein,.
Nucleic acids coding for polypeptides of the
wild-type strain A75/17 and expression vectors ~or the
expression of such polypeptides in vivo are of particular
importance because this strain induces distemper.
The present nucleic acid constructs are in
particular expression plasmids comprising at least one
and preferably one of the canine distemper genes opera-
tively linked to a promotor and optionally to other se-
quences improving the expression of the gene, e.g. such
as an enhancer, as well as an appropriate terminator se-
quence. Expression plasmids comprising such functional
sequences necessary for expression of the gene are known
2s in the art, and are e.g. plasmids CMV5 and pCI.
In another embodiment the invention concerns
a polynucleotide vaccine comprising an effective amount
of a nucleic acid construct, e.g. a DNA or RNA construct,
and a physiologically acceptable carrier. Said vaccine
induces neutralizing antibodies against canine distemper
virus, canine distemper virus specific cytotoxic lympho-
cytes, or protective immune reponses upon introduction
thereof into animal tissue in vivo, wherein said animal
is a mammal, a human, and in particular a dog.
In particular prefered is a polynucleotide
- vaccine comprising one or more of the plasmids selected
from N/CMV5 or N/pCI, which encode the nucleocapsid (N)
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WO97/41236 PCT~B97/00444
protein, H/CMV5 or H/pCI, which encode the hemagglutinin
(H) protein, or F/CMV5 or F/pCI which encode the fusion
(F) protein of the vir-ulent canine distemper virus strain
A75/17, and a physiologically acceptable carrier.
Physiologically acceptable vaccine carriers
are known in the art and are e.g. physiologically accept-
able injectable fluids, such as buffer solutions, e.g.
phosphate-buffered saline (P~S) of appropriate pH, pref-
erably of between about 7 to about 7.4, or injectable
o liposome preparations. The vaccine may also contain an
adjuvant or a transfection facilitating agent. The vac-
cine comprises an effective, that is an immunizing amount
of a nucleic acid construct of the present invention, or
a combination of two or more constructs, e.g. in a con-
centration of about 0.01 to 100, preferably about 0.1 to1 mg /ml.
In yet another aspect of the invention one or
more inventive constructs, each of which is carrying at
least one of the canine distemper genes, are components
of a multivalent vaccine. The components of said multiva-
lent vaccine can be packed in admixed form or one or more
components can be packed separatedly from other compo-
nents but are administered either together, i.e. after
mixing, or separatedly but almost simultaneously, i.e. a
second administration directly after a first one.
In another embodiment the invention concerns
a method for protecting an animal susteptible to infec-
tion by canine distemper virus which comprises immuniza-
tion of said animal with a prophylactically effective
amount of at least one polynucleotide construct compris-
ing a gene of canine distemper virus optionally together
or simultaneously with at least one other component as a
multivalent vaccine.
A number of animals are known as being sus-
ceptible to canine distemper virus. Such animals are inparticular m~mm?.ls, such as carnivors, in particular
dogs, and also humans.
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W O 97/41236 PCT~B97/00444 11
In particular prefered is the method, wherein
the polynucleotide is administered directly into tissue,
preferably into muscle-tissue, in vivo. The polynucleo-
tide may be administered either in naked form in a
physiologically acceptable solution, or contained in a
liposome, or in a mixture with an adjuvant or a transfec-
tion facilitating agent. In particular prefered ist the
method of using a vaccine according to the present inven-
tion.
o In another embodiment the invention concerns
a method for using a canine distemper virus gene to in-
duce an immune response in vivo which comprises:
a) isolating the gene
b) linking the gene to regulatory sequences
such that the gene is operatively linked to control se-
quences which, when introduced into a living tissue, di-
rect the transcription of the gene and subse~uent trans-
lation of the mRNA, and
c) introducing the gene into a living tissue.
In particular prefered is the method, which
comprises multiple introduction of the canine distemper
gene for boosting the immune response.
In particular prefered is the method, wherein
the canine distemper gene encodes the nucleocapsid (N)
protein, the hemagglutinin (H) protein, or the fusion (F)
protein of canine distemper virus strain A75/17.
In particular prefered is the method, wherein
the canine distemper gene product for immunization is se-
lected from the plasmids F/CMV5 or F/pCI, H/MCV5 or
H/pCI, N/CMV5 or N/pCI which encode proteins of the wild
type canine distemper virus strain A75/17, or a combina-
tion of those plasmids.
In another embodiment the invention concerns
a composition of nucleic acid constructs encoding CDV
genes from more than one canine distemper virus strain.
In another embodiment the invention concerns
the use of an isolated canine distemper gene operatively
~ . . , ~
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WO97/41236 PCT~B97/00444
12
linked to one or more control sequences for the prepara-
tion of a vaccine for use in immunization against infec-
tion by CDV.
The following examples serve to further de-
scribe the invention, however, they should not be con-
strued as a limitation thereof.
Example 1: Preparation of cDNA clones from
canine distemper virus strain A75/17 (wild type) infected
primary dog brain cell cultures
a) Preparation of cytoplasmic RNA
Primary dog brain cell cultures (DBCC) were
prepared as described by Zurbriggen and Vandevelde, 1984.
DBCC were infected 10-14 days after seeding,
when confluency was reached, with the virulent canine
distemper virus strain A75/17 (Zurbriggen et al., 1993).
About 40 days after infection, RNA was pre-
pared from infected DBCC grown in 9-cm diameter cell cul-
ture petri dishes as follows: The medium was removed andreplaced by lml of ice-cold buffer A (150 mM NaCl, 1.5 mM
MgCl2, 10mM Tris, pH 7.8) The cells were scraped off with
a rubber policeman and transferred to a centrifuge tube.
The tu~e was kept on ice for 10 min and then centrifuged
for 3 min at 1000 x g. The supernatant was transferred to
a new tube. The pellet was resuspended in lml of ice-cold
buffer A and again centrifuged for 3 min at 1000 x g. The
supernatant was combined with the first. To the combined
supernatants, 2 ml of 7 M urea, 350 mM NaCl, 10 mM EDTA,
10 mM Tris pH 7.9, 1% SDS was added. The obtained mixture
was extracted with 4 ml of phenol-chloroform (1:1) and
the resulting aqueous phase treated with 3 volumes of
EtOH. The precipitated RNA was centrifuged and suspended
in 100 ~l of PBS.
b) Synthesis of cDNA
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WO97/41236 PCTnB97/00444
13
A series of overlapping cDNA clones from the
CDV genome was obtained as outlined below. The procedure
is described for generating clones containing the entire
N, F and H gene sequences. The M, P and L genes may be
isolated in the same manner using specific primers for
these genes.
c) First strand cDNA
Primers used for first strand cDNA synthesis
were selected on the basis of the pu~lished sequence of
the OP-CDV vaccine strain (Sidhu et al., 1993). They are
located in regions which are highly conserved in Morbil-
liviruses. The l0 primers used and their sequence identi-
fication numbers SEQ ID NO l to l0 are given hereinafter.
Reaction mixtures for cDNA synthesis con-
tained: 24.5 ~l H2O, l0 ~l SX AMV reverse transcription
buffer, 1 ~l of a 75 ~M dNTP solution, 2,5 ~l of a 40 ~M
primer solution, 1 ~l RNAse inhibitor, 1 ~l AMV reverse
transcriptase (5 units/~l), l0 ~l of the above obtained
RNA/PBS solution. Samples were incubated for 2 h at 42~C
and then heated at 75~C for l0 min.
e) Synthesis of double stranded cDNA
Double stranded cDNA was synthesized using
polymerase chain reaction (PCR). Reaction mixtures for
amplification of a specific region of the CDV genome con-
tained both the 3' and 5' primers (see SEQ ID NOs). Syn-
thesis was performed in a volume of l00 ~l and contained
the following: 77.4 ~l H2O, l0 ~l l0X Taq buffer, l.l ~l
of a solution containing all 4 dNTPs at 20 ~M each, 0.5
~1 of a 40 ~M primer solution, 1 ~l of Taq polymerase
(0.5 units/~l) and l0 ~l of first strand cDNA, heated to
75~C for l0 min and then cooled on ice. PCR reactions
were performed for 30 cycles under standard conditions.
~ f) Cloning of cDNA
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WO97/41236 PCT~B97/00444
14
PCR amplified cDNA was cloned into the pCR II
vector (Invitrogen) using standard conditions (Samb~ook
et al., 1989).
g) Assembly of contiguous genes
The procedure described above for producing
cDNA clones resulted in the isolation of the complete N
gene.
For the F and H genes, a series of overlap-
ping clones was obtained. To assemble these genes into
contiguous DNA segments, recombinant PCR (Ho et al.,
1989) was used.
Example 2: Preparation of the N Gene
lS Appropriate 5' and 3' ends for insertion of
the N gene into expression plasmids were generated by
PCR. The following primers were used:
Nl, SEQ ID NO ll: 5' GGG GTA CCT CAG GGT TCA
GAC CTA CCA 3', for generating the 5' end of the gene;
and
N2, SEQ ID NO 12: 5' GCG TCG ACG ACT GAT GTA
ACA CTG GTC T 3', for generating the 3' end.
This created KpnI and SalI sites at the 5'
and 3' ends, respectively. PCR reactions were performed
under standard conditions.
Example 3: Preparation of the F Gene
The primers Fl-F4 used in this experiment
were designed according to partial sequences of the
A75/17. However, the positions of the underlined nucleo-
tides correspond to the positions of the of the OP-CDV
genes according to Barrett et al., 1987. The primers were
synthesized with a nucleic acid synthesizer machine.
Fl, SEQ ID NO 13: 5' CGA CGC GTA GGG TCC AGG
ACG TAG CA 3', position l;
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WO97/41236 PCT~B97/00444
F2, SEQ ID NO 14: 5' CAG GTT TAA ATG TCG GAT
CG 3', position 2033;
F3, SEQ ID-NO 15: 5' CGA TCC GAC ATT TAA ACC
TG 3', position 2014;
F4, SEQ ID NO 16: 5' GCGTCG ACA AGA CGT GTG
ACC AGA GTG 3', position 2095.
The F gene was isolated as 3 overlapping
clones. First, the 5' portion of the gene was assembled.
lo A first cDNA clone containing parts of the M and F genes
was cleaved with SacI in the vector DNA and with HindIII
at position 687 in the F gene and the fragment of 2035 bp
was isolated. A second cDNA clone, containing most of the
F gene coding sequences in reverse orientatio~ with re-
spect to the first clone, was also c~eaved with HindIIIand SacI. The 1405 bp fragment was isolated. Both frag-
ments were ligated into the pBluescript (Stratagene, La
Jolla, CA) plasmid cleaved with SacI. To add the 3' end
of the F gene, and to generate correct 5' and 3' ends for
cloning into expression plasmids, PCR was used. The 5'
portion of the gene was amplified by PCR using primers F1
(5' CGA CGC GTA GGG TCC AGG ACG TAG CA 3') and F2 (5' CAG
GTT TAA ATG TCG GAT CG 3') and the DNA fragment was puri-
fied by gel electrophoresis on an agarose gel. Similarly,
the 3' portion of the gene was amplified by PCR with
primers F3 (5' CGA TCC GAC ATT TAA ACC TG 3') and F4 (5'
GCGTCG ACA AGA CGT GTG ACC AGA GTG 3') and purified. Fi-
nally, the two parts of the gene were assembled by recom-
binant PCR using the gel purified 5' and 3' portions of
the gene and primers F1 and F4. This allowed to synthe-
size the entire F gene as 1 contiguous DNA segment with
Mlu I and Sal I sites at the 5' and 3' ends, respec-
tively, for cloning into expression plasmids.
Example 4: Preparation of the H Gene
The primers H1-H4 used in this experiment
were designed according to partial sequences of the
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16
A75/17 genome. However, the positions of the underlined
nucleotides correspond to the positions of the OP-CDV
genes according to Curran et al., 1991. The primers were
synthesized with a nucleic acid synthesizer machine.
H1, SEQ ID NO 17: 5' GCG GTA CCA CAA TGC TCT
CCT ACC AG 3', position 18;
H2, SEQ ID NO 18: 5' CAT ACA CTC CGT CTG AGA
TAG C 3', position 705;
H3, SEQ ID NO l9: 5' GCT ATC TCA GAC GGA GTG
TAT G 3', position 684;
H4, SEQ ID NO 20: 5' GCG TCG ACT TAA CGG TTA
CAT GAG AAT CT 3', position 1835:
The H gene coding sequences were cloned as 2
overlapping cDNA clones. The gene was assembled by PCR
technology. First, the 5' portion of the gene was ampli-
fied by PCR using primers H1 (5' GCG GTA CCA CAA TGC TCT
CCT ACC AG 3') and H2 (5' CAT ACA CTC CGT CTG AGA TAG C
3') and the resulting DNA fragment was isolated. The 3'
portion of the gene was amplified with primers H3 (5' GCT
ATC TCA GAC GGA GTG TAT G 3') and H4 (5' GCG TCG ACT TAA
CGG TTA CAT GAG AAT CT 3') and the DNA fragment was also
isolated. The two portions of the gene were fused in a
recombinant PCR reaction containing both DNA fragments
and primers H1 and H4. This resulted in the synthesis of
a DNA fragment containing the entire H gene coding se-
quences with a KpnI site at the ~' end and a SalI site at
the 3' end for cloning into expression plasmids.
Example 5: Cloning into eukaryotic expression
plasmids
The recombinant PCR products were purified by
gel electrophoresis on an agarose gel. The ends were ren-
dered blunt by Klenov polymerase and the fragments werecloned into the EcoRV site of the plasmid pBluescript
(Stratagene, La Jolla, CA) and amplified. The inserts
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WO97/41236 PCT~B97/00444
17
were isolated from plasmids containing the F gene by di-
gestion with MluI and SalI and from plasmids harboring
the N and H genes by KpnI and SalI.
The fragments were then cloned either into
the plasmid pCI (Promega) or into plasmid pCMV-5
(Andersson et al., 1989). The obtained expression plas-
mids F/CMV5, F/pCI, H/CMV5, HCPI, N/MCV5 and N/pCI were
purified according to standard methods and are shown in
Figures 2 to 7.
Example 6: Preparation of vaccines
Vaccines are prepared by dissolvin~ one or
more of the obtained expression plasmids in sterilized
PBS of pH 7.4 in a concentration of l mg/ml. The vaccine
solution may be freshly prepared just before use or
filled under sterile conditions in vials of appropriate
size.
Example 7: Antibody response in mice immu-
nized with N/pCI
The immune response following intramuscular
injection of plasmid N/pCI was tested in mice. Two inde-
pendent experiments were performed. In the first one
2s (Table l, Experiment No. I), 5 Balb-c mice were injected
with plasmid N/pCI purified by the Qiagen procedure
(Qiagen Inc, Chatsworth, CA, USA) according to the in-
structions of the supplier. Five mice were injected with
empty vector DNA purified in the same manner. As a fur-
ther control, 5 animals were injected with PBS alone. Inthe second experiment (Experiment No. II) 5 mice were in-
jected with plasmid pCI/N purified by cesium chloride
gradient centrifugation (Sambrook et al., 1989) and 5
mice with empty vector DNA purified by the same proce-
dure. In both experiments each animal was injected withlO0 ~g of DNA in PBS at a concentration of l mg/ml, re-
ceiving 50 ~g in each quadriceps muscle per inoculation.
CA 022~3229 1998-10-26
W O 97/41236 PCT~B97/00444
18
A total of 4 inoculations were performed at biweekly in-
tervals. Two weeks after the last injection the animals
were sacrificed and the serum was collected.
Antibody titers were determined by ELISA us-
ing serially diluted mouse sera. Maxisorp ELISA plates(Nunc, Roskilde, Denmark) were coated with 50 ng of re-
combinant N protein per well in carbonate/bicarbonate
buffer (15 mM Na2CO3, 35 mM NaHCO3, 0,02% NaN3, pH 9.6) at
4~C for 16 hours. After 3 washes with TBS-T (137 mM NaCl,
o 2.68 mM KCl, 24.7 mM Tris, 0.05 % Tween-20; pH 7.5) the
plates were blocked at room temperature for 60 min with
PBS-T/LM (PBS containing 0.05% Tween-20 and 2% low fat
milk powder. The plates were subsequently washed 3 times
with TBS-~ before adding 50 ~l of the mouse sera diluted
in PBS-T/LM. After incubation at 37~C for 60 min. and 3
washes with TBS-T, horseradish peroxidase-labelled goat
anti-mouse IgG (Sigma, St. Louis, MO, USA ), diluted
1000-fold in PBS-T/LM was added as the secondary anti-
body. The plates were incubated at 37~C for 60 min and
then washed 3 times with TBS-T. Finally, 50 ul of a solu-
tion of 1 mg/ml of 1,2 phenylene-diamine in 0.1 M Na-
citrate, pH 5.0, containing 0.001 volumes of 30% H2O2 was
added per well. The reaction was stopped with 50 ~l of 4
M H2SO4 per well, and the optical density was read at a
wave length of 490 nm in a Microplate reader 3550 (Bio-
Rad Laboratories, Hercules, CA, USA).
The results (Table 1) show that in contrast
to control animals, all animals injected with plasmid
N/pCI had significant anti-N antibody titers of up to
1:25'600. Intramuscular injection of plasmid N/pCI thus
induces a good immune response, demonstrating the useful-
ness of the proposed vaccine for protecting animals
against canine distemper.
CA 02253229 1998-10-26
W O 97/41236 PCT~B97/00444
19
Table 1: Anti-N antibody titers in mice injected with rlq~ N/pCI
Experiment No.Treatment Exp.-MouseNo. Titer
I- 1 ~ 1: 50
I-2 < 1: 50
PBS I- 3 < 1: 50
I-4 < 1: 50
I-5 <1 :50
.. . . ..
I-6 1 :200
I- 7 1: 200
pCI I- 8 1: 200
I-9 1 :200
I- 10 1 : 200
I-11 1: 800
I-12 1: 1600
N/pCl I-13 1: 3200
I- 14 1: 6400
I- 15 1 : 25600
II-1 <1 :50
II-2 <I :50
II pCI II- 3 < 1: 50
II-4 < 1: 50
II-5 < 1: 50
II- 6 l: 3200
II- 7 1: 1600
II N/pCl II- 8 l: 200
II- 9 l: 12800
II-10 1: 3200
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WO97/41236 PCT~B97/00444
Example 8: CTL response in mice immunized
with N/pCI
Groups of 4 mice were immunized by either 1,
2, or 3 intramuscular injections at 21-day intervals with
a total of 100 ~g of plasmid N/pCI. Control animals were
~njected with empty vector. Twelve days after the first,
second, or third injection the mice were sacrificed and
the spleen was removed. Splenocytes were isolated using a
cell strainer and resuspended in DMEM supplemented with
5% heat-inactivated fetal calf serum, 100 ~g/ml penicil-
lin, 100 U/ml streptomycin, 0.05 mM ~-mercaptoethanol, 10
mM HEPES, and non-essential amino acids. The cells were
then stimulated by incubation with a synthetic 9 amlno
acid peptide (YPALGLHEF~ which has been shown to repre-
sent a CTL epitope in the measles virus N protein
(Beauverger et al., 1993) and which is conserved in CDV
strains Onderstepoort and A75/17. The peptide was used at
a concentration of 10 ~M. After 5-7 days the cells were
counted in Trypan blue and adjusted to 2 x 106 viable
cells/ml. The cells were then diluted into microtiter
plates to yield effector to target cell ratios ranging
from 100:1 to 0.1:1.
P 815 mastocytoma cells were used as targets
2s for the CTL assay. Briefly, 106 cells were incubated for
1 hour at room temperature with the CTL peptide at a fi-
nal concentration of 1 ~M. Control cells were incubated
in the absence of the peptide. After incubation, the
cells were centrifuged and resuspended in 100 ~l of me-
dium. Then, 100-150 ~Ci of 51Cr was added and the cells
were incubated for 1 hour at 37~C with occasional shak-
ing. The cells were then washed extensively before adding
2 x 103 target cells per well of effector cells. Target
and effector cells were incubated 37~C for 4-5 hours. The
plates were then centrifuged and from each well 100 111 of
medium was removed and the radioactivity was counted in a
gamma counter. The radioactivity released by control
CA 022~3229 1998-10-26
WO97/41236 PCT~B97/00444
21
cells incubated without the CTL peptide was subtracted
from the value obtained from cells incubated with t~e
peptide. The resulting value was used to calculate per-
centage specific lysis.
s No CTL response was observed after a single
immunization (not shown). Importantly, however, after 2
and 3 injections of plasmid N/pCI all mice showed high
CTL activity. In contrast, control mice immunized with
the empty vector showed very little CTL activlty (Fig.
lC 8). Fig. ~ represents CTL assay of mice immunized with
plasmid N/pCI or with empty plasmid. Per cent specific
lysis was obtained by subtracting the value of non spe-
cific lysis of target cells incubated with effector cells
in the absence of the CTL peptide. Each curve represents
the values obtained with splenocytes from one mouse.
Solid line: mice immunized with plasmid N/pCI; broken
line: mice immunized with empty vector. The effector (E)
to target (T) cell ratio is indicated.
Example 9: Immunization of dogs with N/pCI
Beagle dogs of 6 weeks of age were used for
immunization experiments. Five control animals (Fig. 9,
dogs l-5) received intramuscular injections of a commer-
cially available multivalent vaccine (standard vaccine)
containing inactivated canine adenovirus, parainflunza
virus, parvovirus, leptospira and live CDV Onderstepoort
strain. Ten dogs (dogs 6-15) were injected into one quad-
riceps muscle with l00 ~lg of plasmid N/pCI. Standard vac-
cine lacking the CDV component was injected into the
other quadriceps. A total of 3 injections were performed
at 2-week intervals. Before the first, and 2 weeks after
each injection (I-III) blood samples were drawn and anti-
N antibody levels were determined by ELISA using recombi-
- nant CDV N protein as antigen as described for ELISA as-
says in mice. With standard vaccine, anti N antibody
CA 022~3229 1998-10-26
W O 97/41236 PCT~B97/00444
22
titers were already elevated with respect to the pre-
iIlunune serum after the first vaccination and then reached
a plateau. With plasmid N/pCI, in most animals the titers
were low after the first and second injection. However,
s after the third injection, the titers increased and in
some animals reached values similar to those obtained
with standard vaccine.
The results obtained are visualized in Figure
9. Titers were determined 2 weeks after the first (I),
second (II), or third (III) immun1zation and are repre-
sented as the highest serum dilution in which the OD
value measured in the ELISA assay was at least twice as
high as the value of the corresponding pre-immune serum
at the same dilution.
A toxicity test was performed according to
the description of the European Pharmacopoeia. Five
healthy mice and two healthy guinea pigs were injected
with the polynucleotide vaccine as described above. The
20 animals were observed for 7 days. None of the animals
showed local or systemic reactions.
REEERENCES
2s
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W097/41236 PCT~B97/00~4
23
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WO97/41236 PCTAB97/00444
24
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WO97/41236 PCT~B97/00444
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Zurbriggen, A. and Vandevelde, M. (1984):
Morphological and immunocytochemical characterisation of
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Wittek, Riccardo
(B) STREET: Ch. de la mésange lA
(C) CITY: Vufflens-la-Ville
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP): 1302
(i) APPLICANT:
(A) NAME: Zurbriggen, Andreas
(B) STREET: Muhlestrasse 158
(C) CITY: Munche~buchsee
(E) COUNTRY: Switzerland
(F) POSTAL CODE (ZIP): 3053
(ii) TITLE OF INVENTION: Polynucleotide
Vaccine against Canine Distemper
(iii) NUMBER OF SEQUENCES: 23
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release
#1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
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CA 022~3229 1998-10-26
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(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11
5' GGG GTA CCT CAG GGT TCA GAC CTA CCA 3'
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 022~3229 1998-10-26
W 0 97/41236 PCTnB97/00444
34
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12
5' GCG TCG ACG ACT GAT GTA ACA CTG GTC T 3'
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
. (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D)- DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13
5' CGA CGC GTA GGG TCC AGG ACG TAG CA 3'
(2) INFORMATION FOR SEQ ID NO:14:
CA 022~3229 1998-10-26
WO97/41236 PCT~B97/0~4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleotides
~B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14
5' CAG GTT TAA ATG TCG GAT CG 3'
(2) INFORMATION FOR SEQ ID NO:l5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
~D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
.. . . . . .
CA 022~3229 1998-10-26
WO97/41236 PCTAB97/00444
36
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15
5' CGA TCC GAC ATT TAA ACC TG 3'
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: slngle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
2s (G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16
5' GCG TCG ACA AGA CGT GTG ACC AGA GTG 3'
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
4 5 (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
. . ..
CA 022~3229 1998-10-26
WO97/41236 PCT~B97/00444
37
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
s (A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17
5' GCG GTA CCA CAA TGC TCT CCT ACC AG 3'
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
~o (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
3s (A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18
5' CAT ACA CTC CGT CTG AGA TAG C 3'
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
, . ~ , .. .. .
CA 022~3229 1998-10-26
WO97/41236 PCTnB97/00444
38
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19
5' GCT ATC TCA GAC GGA GTG TAT G 3'
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 nucleotides
(B) TYPE: deoxyoligonucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(C) SYNTHETIC: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20
5' GCG TCG ACT TAA CGG TTA CAT GAG AAT CT 3'
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
CA 02253229 l998-l0-26
WO 97/41236 PCT ~ 97/00444
39
(A) LENGTH: 1678 nucleotides
(B) TYPE: cDNA
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
~o (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: CANINE DISTEMPER VI-
RUS, STRAIN CDV A75/17
(D) DEVELOPMENTAL STAGE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE: N/CMV5 OR N/PCI
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21
AGGGTCAATG ATCCTACCTT AGAGAACAAG GTCAGGGTTC AGACCTACCA ATATGGCTAG 60
Cc~ lAAG AGCCTCACAT TATTCAAGAG GACTCGGGAC CAACCCCCAC TTGCCTCGGG 120
CTCCGGAGGA GCAATCAGAG GGATAAAGCA TGTCATTATA GTCCTAATCC CGGGTGACTC 180
AAGCATTGTT ACAAGATCTC GACTATTGGA TAGACTTGTT AGATTGGTCG GTGATCCGGA 240
AATCAACGGG CCTAAATTAA CTGGGATTTT AATCAGTATC CTCTCCTTGT TCGTGGAATC 300
CCCTGGACAG TTGATCCAGA GGATCATAGA CGACCCTGAT ATAAGCATCA AGTTAGTAGA 360
GGTAATCCCA AGCATCAACT ~lllGCGG TCTTACATTT GCATCCAGAG GAGCAAGTTT 420
GGATTCTGAG GCAGATGAGT TCTTCAM AT TGTAGACGAA GGGTCGAAAG CTCAAGGACA 480
ATTAGGCTGG TTGGAGAATA AGGATATTGT AGACATAGAA GTTGATGATG CTGAGCAATT 540
CAATATATTG CTAGCTTCCA TCTTGGCCCA AATTTGGATC CTGCTAGCTA AAGCGGTGAC 600
TGCTCCTGAT ACTGCAGCCG ACTCGGAGAT GAGAAGGTGG ATTAAGTATA CCCAACAGAG 660
AC~l~rC GGGGAATTCA GAATGAACAA AATATGGCTT GATATTGTTA GAAACAGAAT 720
TGCTGAGGAC TTATCTTTGA GGCGGTTCAT GGTGGCACTC ATCTTGGATA TCAAACGATC 780
CCCAGGGAAC AAGCCTAGAA TTGCTGAAAT GATTTGTGAT ATAGATAACT ACATTGTGGA 840
AGCTGGATTA GCTAGTTTCA TCTTAACTAT CAAATTTGGC ATTGAAACTA TGTATCCGGC 900
TCTTGGGTTG CATGAGTTTT CCGGAGAGTT AACAACTATT GAATCCCTTA TGATGCTATA 960
TCAACA&ATG GGTGAAACAG CACCGTACAT GGTTATTCTG GA M ATTCTG TTCAGAACAA 1020
ATTTAGTGCA GGATCCTACC CAll~l~lG GAGTTATGCT ATGGGAGTTG GTGTTGAACT 1080
TGAAAACTCC ATGGGAGGGT TAAATTTCGG TAGATCCTAC TTTGATCCAG CTTATTTCAG 1140
GCTCGGGCAA GAAATGGTTA GAAGATCTGC CGGCAAAGTA AGCTCTGCAC TTGCCGCCGA 1200
GCTTGGCATC ACCAAGGAAG AGGCTCAACT AGTGTCAGAA ATAGCATCCA AGACAACGGA 1260
GGACCGGACG ATTCGCGCTG CTGGTCCCAA GCAATCTCAA ATCACTTTTC TGCACTCAGA 1320
AAGATCCGAA GTCACTAATC AACAACCCCC AACCATCAAC AAGAGGTCCG AAAACCAAGG 1380
AGGAGACAAA TACCCCATCC ACTTCAGTGA TGAACGGTTT CCAGGGTATA CCCCAGATGT 1440
CAACAGCTCC GAATGGAGTG AATCACGCTA TGATACCCAA ACTATTCAAG ATGATGGAAA 1500
CGACGATGAC CGGAAATCGA TGGAAGCAAT CGCCAAGATG AGAATGCTTA CTAAGATGCT 1560
CAGTCAACCT GGGACCAGTG AAGAGAGTTC TCCTGTCTAT AATGATAGAG AGCTACTCAA 1620
TTAAATATTC AAGACCAGTG TTACATCAGT CAACGATTCT CCTTCTAAAC TCATTATA 1678
55 (2) INFORMATION FOR SEQUENCE ID NO: 22
(i) SEQUENCE CHARACTE~ISTICS:
(A) LENGTH: 2198
(B) TYPE: nucleic acid
(C) STRANDENESS: single
(D) TOPOLOGY: linear
, ., ., .. . . . ~ . , ,, .. ,, , "
CA 022~3229 l998-l0-26
WO97/41236 PCT~B97/00444
(ii) MOLECULAR TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "deoxynucleotlde"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi~ ORIGINAL SOURCE:
(A) ORGANISM: Canine distemper virus
(B) STRAIN: A75/17
(viii~ POSITION IN GENOME:
(~) MAP POSITION: F gene position is 5'end of F
mRNA
(C) UNITS: bp
(ix) FEATURE:
(A) NAME/KEY: mRNA
(B) LOCATION: complement (12198)
AGGGTCCAGG ACGTAGCAAG CCTACAGGCC AACCAAGTCC ACCAACTCTA GGCCGGGCAG 60
GAACCCCCAC GAACAGACAA GCCCCATGCA CAACAAAATC CCCAAAAGGT CCAACACCCG 120
AAAACACACC CAACAAGACC TCCCCCCACA ACACAGCACC AAATCCGCCG AGACCAAGAC 180
CTCCCAAGCA CGACACAGCA CAACATCGGC TCGGCGATCC ACGCACCATG GTCCTCTAAC 240
ATCGGACAGG CCCATCCACT ACATCATGAA CAGGATCAGG TCCTGCAAGC AAGCCAGCCA 300
CAGATCGGAT AACATCCCGG CTCACGGAGA CCATGAGGGC ACCATCCATC ACACACCAGG 360
GA~l~l~lCC CAAGGAGCGG GATCCCGGCT CAAAAGGCGG CAATCCAATG CAACCAACTC 420
A~G~1~lCAG TGCACCTGGT TAGTCCTATG GTGCATTGGA ATAGCCAGTC I~l~ ~ 480
TTCTAAGGCT CAGATACATT GGAATAATTT GTCAACTATT GGGATTATCG GGACTGACAG 540
TGTCCATTAT AAGATCATGA CTAGACCCAG TCACCAGTAC TTGGTCATAA AACTAATGCC 600
TAATGTTTCA CTTATAGATA ATTGTACCAA AGCAGAATTA GGTGAGTATG AGAAATTATT 660
AAATTCAGTC CTCGAGCCAA TCAATCAAGC TTTGACTCTA ATGACCAAGA ATGTGAAGCC 720
CCTACAGTCA GTAGGGTCAG GTAGGAGACA AAGGCGTTTT GCAGGAGTGG IG~llGCAGG 780
TGCAGCTTTA GGAGTAGCCA CAGCTGCACA AATCACTGCA GGGATAGCTT TACATCAATC 840
CAACCTCAAT GCTCAAGCAA TCCAATCTCT GAGAACTAGC CTTGAACAGT CCAACAAGGC 900
TATAGAAGAA ATTAGGGAGG CAACCCAGGA AACCGTCATT GCCGTTCAGG GAGTTCAGGA 960
TTACGTCAAT AATGAACTCG TCCCTGCTAT GCAACATATG TCGTGTGAAT TAGTTGGGCA1020
GAGATTAGGG TTA~AACTGC TTAGGTATTA TACCGAGTTG TTGTCAATAT TTGGCCCGAG1080
TTTACGTGAT CCTATTTCAG CCGAGATATC AATTCAAGCA CTGAGTTATG ~l~ll~GG&G1140
AGAAATTCAT AAGATACTTG AGAAGTTGGG ATAIl~lG~A AATGATATGA TTGCAATTTT1200
GGAGAGTCGG GGGATAAAAA CAAAAATAAC CCATGTTGAT CTCCCCGGGA AACTCATCAT1260
.CTTAAGTATC TCATACCCAA CTTTATCAGA AGTCAAGGGG GTCATAGTCC ACAGACTGGA1320
AGCAGTTTCT TATAATATAG GGTCACAGGA GTGGTACACC ACTGTCTCGA GGTATGTTGC1380
AACTAATGGT TACTTAATAT CTAATTTTGA TGAGTCACCC TGTGTATTCG TCTCAGAATC1440
AGCCATTTGT AGCCAGAACT CCCTATACCC CATGAGCCCG CTTCTACAAC AATGCATTAG1500
GGGTGACACT TCAI~.l~lG CTCGGACCTT G~l~l~l~6G ACGATGGGCA ACAAGTTTAT1560
TCTGTCAAAA GGTAATATCG TCGCAAATTG TGCTTCTATA CTGTGTAAGT GTTATAGCAC1620
AGGCACAATT ATCAATCAGA GTCCTGATAA ATTGCTGACA TTTATTGCCT CCGGTACCTG1680
CCCACTGGTT GAGATAGATG GTGTAACTAT CCAGGTTGGA GGGAGGCAAT ACCCTGATAT1740
GGTATACGAA AGCAAAGTTG CCTTAGGCCC TGCTATATCA CTTGAGAGGT TAGATGTAGG1800
TACAAATTTA GGGAACGCCC TTAAGAAACT GGATGATGCT AAGGTACTGA TAGACTCCTC1860
TAACCAGATC CTTGAGACGG TTAGGCGCTC TTCCTTTAAT TTTGGCAGTC TTCTCAGCGT1920
TCCCATATTA ATATGTACAG CC~lGG~lll ~ lG~lG ATTTACTGCT GTAAAAGACG1980
CTACCAACAG ACACTCAAGC AGAATGCTAA GGTCGATCCG ACATTTAAAC CTGATTTGAC2040
- TGGAACTTCG AAATCCTATG TAAGATCACT CTAAAGCACT CTGGTCACAC GTCTTACCCG2100
ATTGTCAGGC TTGAAATCTA TAAATCCCCC CCAATTTTCT TCA~AAGCTA TCAAACTACA2160
ACAAATAGTG GAGAGGACTG ACTACGATTA TCGTAATT 2198
CA 022~3229 l998-l0-26
WO97/41236 PCT~B97/00444
41
~ (2)INFORMATION FOR SEQUENCE ID NO: 23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1969
(B) TYPE: nucleic acid
(C) STRANDENESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: other nucleic ~cid
(A) DESCRIPTION: /desc =
"deoxynucleotide"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Canine distemper virus
(B) STRAIN: A75/17
(viii) POSITION IN GENOME:
(B) MAP POSITION: H gene position is
25 5'end of H mRNA
(C) UNITS: bp
(ix) FEATURE:
(A) NAME/KEY: mRNA
(B) LOCATION: complement (1... 1969)
AGGGCTCAGG TAGTCCAACA ATGCTCTCCT ACCAGGACAA GGTGAGTGCC TTCTATAAGG 60
ATAATGCAAG AGCTAATTCA TCCAAGCTAT CCTTAGTGAC AGAAGAGCAA GGGGGCAGGA 120
35 GACCACCCTA Tl~ ,L11 GTCCTTCTCA TCCTACTGGT TGGAATCATG GCCTTGCTTG 180
CTATCACTGG AGTTCGATTT CACCAAGTAT CAACTAGCAA TATGGAATTT AGCAGATTGC 240
TGAAAGAGGA TATGGAGAAA TCAGAGGCCG TACATCACCA AGTCATAGAT GTCTTGACAC 300
CGCTCTTCAA AATTATTGGA GATGAGATTG GGTTACGGTT GCCACAAAAA CTAAACGAGA 360
TCAAACAATT TATCCTTCAA AAGACAAACT TCTTCAATCC GAACAGGGAG TTcGACTTCC 420
- 40 GCGATCTCCA CTGGTGCATT AACCCACCTA GTAAGATCAA AGTGAATTTT ACTAATTACT 480
GCGATACAAT TGGGATCAGA AAATCTATTG CATCGGCAGC AAATCCTATC CTTTTATCAG 590
CACTCTCCGG AGGCAGAGGT GACATATTCC CACCATACAG ATGCAGTGGA GcTAcTAcTT 600
CAGTAGGCAG AGTTTTCCCC CTATCAGTAT CATTGTCCAT GTCTTTGATC TCAAGAACAT 660
CAGAGATAAT CAATATGCTA ACCGCTATCT CAGACGGAGT GTATGGTAAA ACTTATTTGC 720
CA 022~3229 1998-10-26
WO 97/41236 PCT/IB97/00444
42
TAGTTCATGA TTATATTGAA GGGGGGTTCG ACACGCAAAA GATTCGAGTC TTTGAGATAG 780
GGTTCATCAA ACG~b~lG AATGACATGC CATTACTCCA GACAACCAAC TATATGGTCC 840
TCCCGGAGAA TTCCAAAGCC AAGGTATGTA CTATAGCGGT GGGCGAGTTG ACACTGGCTT 900
C~lLG~l~l AGATGAGAGC ACCGTATTGT TATATCATGA CAGCGATGGT TCACAAGATG 960
GTATTCTAGT GGTGACGCTG GGAATA~TTG GGGCAACACC TATGGATCAA GTTGAAGAGG 1020
TGATACCTGT TGCTCACCCA TCAGTAGAAA AAATACATAT AACAAATCAC CGTGGGTTCA 1080
TAAAAGATTC AATAGCAACC TGGATGGTGC CTGCATTGGT ATCTGAGAAA CAAGAGGAAC 1140
AAAAAAATTG TCTGGAGTCG GCTTGTCAAA GAAAATCCTA CCCTATGTGC AACCAAACGT 1200
CATGGGAACC CTTTGGAGGA GGACAGTTGC CATCTTATGG GCGGTTGACA TTACCTCTAG 1260
ATCCAAGCAT TGACCTTCAA CTTAACATCT CGTTTACATA CGGTCCGGCT ATACTGAATG 1320
GAGACGGTAT GGATTATTAT GAAAGCCCAC ~l~lG~ACTC CGGATGGCTT ACCATTCCCC 1380
CCAAGAACGG AACAGTCCTT GGATTGATAA ACAAAGCAAG TAGAGGAGAC CAATCCACTG 1440
TAATCCCCCA IG~ CACA TTTGCGCCCA GGGAATCAAG TGGAAATTGT TATTTACCTA 1500
TTCAAACATC CCAGATTATG GATAAAGATG TCCTTACTGA GTCCAATTTA b-~blGllGC 1560
CTACACAGAA TTTTAGATAT GTCATAGCAA CATATGATAT ATCCCGGGGC GATCATGCGA 1620
~ lATTA TGTTTATGAC CCAATCCGGG CGATTTCTTA TACGTACCCA TTTAGACTAA 1680
CTACCAAGGG TAGACCTGAT TTCCTAAGGA TTGAATGTTT TGTGTGGGAT GACGATTTGT 1740
GGTGTCACCA ATTTTACCGA TTCGAGGCTG ACAGCACCAA CTCTACAACC AGTGTTGAGA 1800
ATTTAGTCCG TATAAGATTC TCATGTAATC GTTCAAAACC TTGACAGTAT GATGATACAC 1860
ATTTCAATTG GACTTAGGTA TGATGACTGT GGTGAGAAAT TCCTTACCGA CGATTGAATT 1920
AAACCATCTC CAGCATTATA AAAAAACTAA GGATCCAGGA TCCTTTTAG 1969
