CA2253229A1 - Polynucleotide vaccine against canine distemper - Google Patents

Abstract

Disclosed are polynucleotide vaccines against the canine distemper virus, methods of preparation of the polynucleotides and the vaccines, and the use of the polynucleotides and the vaccines for prophylactic immunization of mammals susceptive to canine distemper.

Description

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 CA 022~3229 1998-10-26 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;

... . .... . . . ... ... .

CA 022~3229 l998-l0-26 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;

CA 022~3229 1998-10-26 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.

CA 022~3229 1998-10-26 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) CA 022~3229 1998-10-26 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) CA 022~3229 1998-10-26 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.

CA 022~3229 1998-10-26 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 ~ . . , ~

CA 022~3229 1998-10-26 WO97/41236 PCT~B97/00444 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

CA 022~3229 1998-10-26 WO97/41236 PCTnB97/00444 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

CA 022~3229 1998-10-26 WO97/41236 PCT~B97/00444 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;

CA 022~3229 1998-10-26 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 CA 022~3229 1998-10-26 WO97/41236 PCT~B97/00444 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 CA 022~3229 1998-10-26 WO97/41236 PCT~B97/00444 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 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.

W O 97/41236 PCT~B97/00444 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 CA 022~3229 1998-10-26 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 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 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.

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W O 97/41236 PCT~B97/00444 SEQUENCE LISTING

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(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 (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 (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 (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 Cc~ lAAG AGCCTCACAT TATTCAAGAG GACTCGGGAC CAACCCCCAC TTGCCTCGGG 120 GGTAATCCCA AGCATCAACT ~lllGCGG TCTTACATTT GCATCCAGAG GAGCAAGTTT 420 AC~l~rC GGGGAATTCA GAATGAACAA AATATGGCTT GATATTGTTA GAAACAGAAT 720 TCAACA&ATG GGTGAAACAG CACCGTACAT GGTTATTCTG GA M ATTCTG TTCAGAACAA 1020 ATTTAGTGCA GGATCCTACC CAll~l~lG GAGTTATGCT ATGGGAGTTG GTGTTGAACT 1080 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) GA~l~l~lCC CAAGGAGCGG GATCCCGGCT CAAAAGGCGG CAATCCAATG CAACCAACTC 420 A~G~1~lCAG TGCACCTGGT TAGTCCTATG GTGCATTGGA ATAGCCAGTC I~l~ ~ 480 CCTACAGTCA GTAGGGTCAG GTAGGAGACA AAGGCGTTTT GCAGGAGTGG IG~llGCAGG 780 GAGATTAGGG TTA~AACTGC TTAGGTATTA TACCGAGTTG TTGTCAATAT TTGGCCCGAG1080 TTTACGTGAT CCTATTTCAG CCGAGATATC AATTCAAGCA CTGAGTTATG ~l~ll~GG&G1140 AGAAATTCAT AAGATACTTG AGAAGTTGGG ATAIl~lG~A AATGATATGA TTGCAATTTT1200 .CTTAAGTATC TCATACCCAA CTTTATCAGA AGTCAAGGGG GTCATAGTCC ACAGACTGGA1320 GGGTGACACT TCAI~.l~lG CTCGGACCTT G~l~l~l~6G ACGATGGGCA ACAAGTTTAT1560 TCCCATATTA ATATGTACAG CC~lGG~lll ~ lG~lG ATTTACTGCT GTAAAAGACG1980 ATTGTCAGGC TTGAAATCTA TAAATCCCCC CCAATTTTCT TCA~AAGCTA TCAAACTACA2160 CA 022~3229 l998-l0-26 WO97/41236 PCT~B97/00444 ~ (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) 35 GACCACCCTA Tl~ ,L11 GTCCTTCTCA TCCTACTGGT TGGAATCATG GCCTTGCTTG 180 TCAAACAATT TATCCTTCAA AAGACAAACT TCTTCAATCC GAACAGGGAG TTcGACTTCC 420 CACTCTCCGG AGGCAGAGGT GACATATTCC CACCATACAG ATGCAGTGGA GcTAcTAcTT 600 CA 022~3229 1998-10-26 GGTTCATCAA ACG~b~lG AATGACATGC CATTACTCCA GACAACCAAC TATATGGTCC 840 C~lLG~l~l AGATGAGAGC ACCGTATTGT TATATCATGA CAGCGATGGT TCACAAGATG 960 GTATTCTAGT GGTGACGCTG GGAATA~TTG GGGCAACACC TATGGATCAA GTTGAAGAGG 1020 GAGACGGTAT GGATTATTAT GAAAGCCCAC ~l~lG~ACTC CGGATGGCTT ACCATTCCCC 1380 TAATCCCCCA IG~ CACA TTTGCGCCCA GGGAATCAAG TGGAAATTGT TATTTACCTA 1500 TTCAAACATC CCAGATTATG GATAAAGATG TCCTTACTGA GTCCAATTTA b-~blGllGC 1560 ~ lATTA TGTTTATGAC CCAATCCGGG CGATTTCTTA TACGTACCCA TTTAGACTAA 1680

Claims (18)

1. A nucleic acid construct comprising one or more canine distemper virus gene of virulent canine distemper virus, wherein said nucleic acid construct is capable of inducing the expression of an antigenic canine distemper 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 resultant uptake of the nucleic acid construct by the cells which express the encoded canine distemper virus gene.

2. A nucleic acid construct according to Claim 1, wherein the canine distemper virus gene encodes the nucleocapsid (N) protein, the phosphoprotein (P), the matrix (M) protein, the fusion (F) protein, the hemagglutinin (H) protein, or the large (L) protein.

3. A nucleic acid construct according to Claim 1 or 2, wherein the canine distemper virus gene encodes the nucleocapsid (N) protein, the fusion (F) protein, or the hemagglutinin (H) protein.

4. A DNA construct according to anyone of Claims 1 to 3, which is the plasmid N/CMV5 or N/pCI, which encode the nucleocapsid (N) protein, the plasmid H/CMVS or H/pCI, which encode the hemagglutinin (H) protein, or the plasmid F/CMV5 or F/pCI which encode the fusion (F) protein of canine distemper virus strain A75/17.

5. A polynucleotide vaccine comprising an effective amount of a DNA or RNA construct according to anyone of Claims 1 to 4 and a physiologically acceptable carrier.

6. A polynucleotide vaccine according to Claim 5 which induces neutralizing antibodies against canine distemper virus, canine distemper virus specific cytotoxic lymphocytes, or protective immune responses upon introduction of said vaccine into animal tissue in vivo, wherein the animal is a mammal, carnivor, in particular a dog, or a human.

7. A polynucleotide vaccine according to Claim 5 or 6 comprising one or more of the plasmids selected from N/CMV5 or N/pCI, which encode the nucleocapsid (N) 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 canine distemper virus strain A75/17 and a vaccine carrier.

8. A polynucleotide vaccine according to any-one of Claims 5 to 7 additionally comprising further components to form a multivalent vaccine.

9. A method for protecting an animal susceptible to canine distemper infection against disease by canine distemper virus which comprises immunization of said animal with a prophylactically effective amount of a polynucleotide vaccine of anyone of claims 5 to 8.

10. A method according to Claim 9, wherein the animal is a mammal, such as a carnivor, in particular a dog.

11. A method according to Claim 9 or 10, wherein at least one polynucleotide is administered directly into the animal tissue in vivo.

12. A method according to Claims anyone of 9 to 11, wherein the polynucleotide is administered either in naked form in a physiologically acceptable solution, or contained in a liposome, or in a mixture with an adjuvant or a transfection facilitating agent.

13. A method for using a canine distemper virus gene of virulent canine distemper virus to induce 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 sequences which, when introduced into a living tissue, direct the transcription initiation and subsequent translation of the gene, and c) introducing the gene into a living tissue of an animal suceptible to canine distemper.

14. A method according to Claim 13, which comprises multiple introduction of the canine distemper virus gene for boosting the immune response.

15. A method according to Claim 13 or 14, wherein the canine distemper virus gene encodes the nucleocapsid (N) protein, the hemagglutinin (H) protein, or the fusion (F) protein of canine distemper virus strain A75/17.

16. A method according to anyone of Claims 13 to 15, wherein the canine distemper virus gene product for immunization is selected from the plasmids F/CMV5 or F/pCI, which encode the fusion (F) protein, H/CMV5 or H/pCI, which encode the hemagglutinin (H) protein, or N/CMV5 or N/pCI which encode the nucleocapsid protein of canine distemper virus strain A75/17.

17. A composition of nucleic acid constructs encoding canine distemper genes of virulent canine distemper virus from more then one canine distemper strain.

18. The use of an isolated canine distemper virus gene of virulent canine distemper virus operatively linked to one or more control sequences for the preparation of a vaccine for use in immunization against disease by canine distemper virus.