IE71643B1 - A recombinant poxviral vaccine for canine distemper - Google Patents

Abstract

A recombinant poxvirus containing therein DNA from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper, is described. The viral vector may be canary poxvirus or vaccinia virus with the following open reading frames deleted: a thymidine kinase gene, a haemorrhagic gene, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region and a large subunit ribonucleotide reductase gene. The Morbillivirus DNA may be the measles virus glycoproteins haemagglutinin and/or fusion glycoproteins.

Description

A RECOMBINANT POXVIRAL VACCINE FOR CANINE DISTEMPER This patent application is a divisional of Patent Application No. 3960/91.

FIELD OF THE INVENTION The present invention relates to a modified poxvirus and to methods of making and using the same. More in particular, the invention relates to recombinant poxvirus, vhich virus expresses gene products of a Morbillivirus gene, and to vaccines vhich provide protective immunity against Morbillivirus infections.

Several publications are referenced in this application by arabic numerals vithin parentheses. Full citation to these references is found at the end of the specification immediately preceding the claims. These references describe the state-of-the-art to vhich this invention pertains.

BACKGROUND OF THE INVENTION Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination betveen pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).

Specifically, the recombinant poxviruses are constructed in tvo steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Patent No. 4,603,112, the disclosure of which patent is incorporated herein by reference.

First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an £. coli plasmid construct into vhich DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter**gene 643 linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA ^sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).

Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along vith the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term foreign DNA designates exogenous DNA*, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.

Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA.

In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.

Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination betveen viral genes may .occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.

However, recombination can also take place betveen sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence vithin the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable.by the presence of that genetic marker - »· or gene in the recombinant viral genome.

Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.

Canine distemper virus (CDV) and measles virus (MV) are members of the Morbillivirus subgroup of the family Paramyxovirus genus (Diallo, 1990; Kingsbury et al., 1978). The viruses contain a non-segmented single-stranded RNA genome of negative polarity. Canine distemper is a highly infectious febrile disease of dogs and other carnivores.

The mortality rate is high; ranging between 30 and 80 percent. Dogs surviving often have permanent central nervous system damage (Fenner, et al., 1987). Similarly, measles virus causes an acute infectious febrile disease characterized by a generalized macropapular eruption. The disease mainly affects children.

The characteristics of Morbilliviruses have recently been reviewed by Norrby and Oxman (1990) and Diallo (1990) . As reported for other Paramyxoviruses (Avery and Niven, 1979; Merz et al., 1980) two structural proteins are Crucial for the induction of a protective immune response. These are the membrane glycoprotein hemagglutinin (HA), which is responsible for hemagglutination and attachment of the virus to the host cell, and the fusion glycoprotein (F), which causes membrane fusion between the virus and the infected cell or between the infected and adjacent uninfected cells (Graves et al., 1978). The order of genes in the MV genome has been deduced by Richardson et al. (1985) and Dowling et al. (1986). The nucleotide sequence of the MVHA gene and MVF gene has been determined by Alkhatib and Briedis (1986) and Richardson et al. (1986), respectively.

CDV and MV are structurally similar and share a close serological relationship. Immunoprecipitation studies have shown that antiserum to MV will precipitate all CDV proteins (P, NP, F, HA and M). By contrast, antiserum to CDV will precipitate all MV proteins except the HA glycoprotein (Hall et al., 1980; Orvell et al., 1980; Stephenson, et al., 1979). In light of this close serological relationship, it has previously been demonstrated that vaccination with MV will elicit protection against CDV challenge in dogs (Gillespie et al., 1960; Moura et al., 1961; Warren et al., i960). Neutralizing antibodies against CDV have been reported in human anti-MV sera (Adams et al., 1957; Imagawa et al., 1960; Karzon, 1955; Karzon, 1962) but neutralizing antibodies against MV have not been found in anti-CDV sera from dogs (Delay et al., 1965; Karzon, 1962; Roberts, 1965).

MV HA and F genes have been expressed in several viral vectors including vaccinia virus (Drillien et al., 1988; Wild et al., 1991), fovlpox virus (Spehner et al., 1990; Wild et al., 1990), adenovirus (Alkhatib et al., 1990) and baculovirus (Vialard et al., 1990). In these studies, authentic MV proteins were expressed which were functional in hemagglutination (Vialard et al., Ϊ990) hemolysis (Alkhatib et al., 1990; Vialard et al., 1990) or cell fusion (Alkhatib et al., 1990; Vialard et al., 1990; Wild et al., 1991) assays. When inserted into a vaccinia virus vector, the expression of either the HA or the F protein was capable of eliciting a protective immune response in mice against MV encephalitis (Drillien et al., 1988). Similarly, expression of the F protein in a fowlpox virus vector elicited protective immunity against MV encephalitis in mice (Wild et al., 1990). No protection studies were reported with other vectors.

European Patent Application No. 0 314 569 relates to the expression of an MV gene in fowlpox.

Perkus et al. (1990.) recently described the definition of two unique host range genes in vaccinia virus These genes encode -host range functions which permit vaccinia virus replication on various cell substrates in 5 vitro. The genes encode host range functions for vaccinia virus replication on human cells as well as cells of rabbit and porcine origin. Definition of these genes provides for the development of a vaccinia virus vector, which, while still expressing foreign genes of interest, would be severely restricted in its ability to replicate in defined cells. This would greatly enhance the safety features of vaccinia virus recombinants.

An attenuated vector has been developed by the sequential deletion of six non-essential regions from the Copenhagen strain of vaccinia virus. These regions are known to encode proteins that may have a role in viral virulence. The regions deleted are the tk gene, the hemorrhagic gene, the A-type inclusion gene, the hemagglutinin gene and the gene encoding the large subunit of the ribonucleotide reductase as well as the C7L through K1L sequences defined previously (Perkus et al., 1990). The sequences and genomic locations of these genes in the Copenhagen strain of vaccinia virus have been defined previously (Goebel et al., 1990 a,b) . The resulting attenuated vaccinia strain is designated as NYVAC.

The technology of generating vaccinia virus recombinants has recently been extended to other members of the poxvirus family which have a more restricted host range. The avipoxvirus, fowlpox, has been engineered as a recombinant virus expressing the rabies G gene (Taylor et 25 al., 1988b). This recombinant virus is also described in PCT Publication No. N089/03429. On inoculation of the recombinant into a number of non-avian species an immune . response to rabies is elicited which in mice, cats and dogs is protective against a lethal rabies challenge.

Both canine distemper and measles are currently 3q controlled by the use of live attenuated vaccines (Fenner et al., 1987; Preblud et al., 1988). Immunization is recommended for control of CDV using a live attenuated vaccine at eight weeks of age and again at 12 to 16 weeks of age. Although immunity to CDV is life-long, because of the highly infectious nature of the agent and the severity of the disease, annual revaccination is usually recommended.

One problem with the current policy of continual revaccination is that CDV immune mothers pass neutralizing antibody to offspring in the colostrum. It is difficult to ascertain when these antibody levels will wane such that pups can be vaccinated. This leaves a window when pups may be susceptible to CDV infection. Use of a recombinant vaccine expressing only the measles virus glycoproteins may provide a means to overcome the inhibitory effects of maternal antibody and allow vaccination of newborns. In fact, it has been demonstrated that CDV-specific antibodies in pups that suckled CDV immune mothers did not prevent the development of MV-specific antibodies when inoculated with a MV vaccine (Baker et al., 1966).

Other limitations of the commonly used modified live CDV vaccines have been previously documented (Tizard, 1990) and are linked to the ability of these vaccine strains to replicate vithin the vaccinated animals. These deleterious effects are most notable when the CDV vaccine strain is co-inoculated with canine adenovirus l and 2 into dogs resulting in immunosuppression, thrombocytopenia, and encephalitis (Bestetti et al., 1978;'Hartley, 1974; Phillips et al., 1989). The modified live CDV vaccines have also been shown to induce distemper in other animal species including foxes, Kinkajous, ferrets, and the panda (Bush et al., 1976; Carpenter et al., 1976; Kazacos et al., 1981). Therefore, the use of a recombinant CDV vaccine candidate would eliminate the continual introduction of modified live CDV into the environment and potential vaccine-associated and vaccine-induced complications which have arisen with the use of the conventional CDV vaccines.

The use of poxvirus vectors may also provide a Beans of overcoming the documented inhibitory effect that maternal antibody has on vaccination with presently utilized live attenuated CDV strains in dogs. Pups born to mothers previously immunized at a young age with a poxvirus recombinant may avoid the interference of CDV-specific maternal antibody. Additionally, the ability of both vaccinia virus and canarypox virus vectors harbouring MV HA and F genes to elicit these responses and the lack of serological cross-reactivity between the two poxviruses provides a further advantage in that one vector could be utilized early in the pup’s life and the other later, to boost CDV-specific immunity. This would eliminate the release of live attenuated CDV strains into the environment, an event linked to the occurrence of vaccine-induced and vaccine-associated complications (Tizard, 1990).

It can thus be appreciated that provision of a Morbilliviros recombinant poxvirus, and of vaccines which provide protective immunity against Morbilliviros infections, would be a highly desirable advance over the current state of technology.

It is an object of this invention to provide a vaccine which is capable of eliciting Morbilliviros neutralizing antibodies, hemagglutination-inhibiting antibodies and protective immunity against Morbilliviros infection and a lethal Morbilliviros challenge, for providing cross-protection of dogs against canine distemper, particularly using a measles virus recombinant poxvirus vaccine.

It is also an object of this invention to provide recombinant poxviruses for use as the vaccine according to the present invention, which viruses express gene products of Morbilliviros, and to provide a method of making such recombinant poxviruses.

It is also an object of this invention to provide for the cloning and expression of Morbilliviros coding sequences, particularly measles virus coding sequences, in a poxvirus vector, particularly vaccinia virus vectors, for use as the vaccine according to the present invention.

These and other objects and advantages of the present invention will become more readily apparent after consideration of the following.

According to the present invention there is provided a recombinant poxvirus containing therein exogenous DNA from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper, wherein the exogenous Morbillivirus DNA codes for an antigen and the poxvirus is a modified vaccinia virus having at least the following open reading frames deleted therefrom: a thymidine kinase gene, a haemorrhagic gene region, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region, and a large subunit, ribonucleotide reductase gene.

According to the present invention there is also provided use of a recombinant poxvirus containing therein exogenous DNA from Morbillivirus in a nonessential region of the poxvirus genome for the preparation of a medicament for protecting a dog against canine distemper, wherein the exogenous Morbillivirus DNA codes for an antigen and the poxvirus is a modified vaccinia virus having at least the following open reading frames deleted therefrom: a thymidine kinase gene, a haemorrhagic gene region, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region, and a large subunit, ribonucleotide reductase gene.

Therefore, the present invention relates to a vaccine for inducing an immunological response in a host animal - i.e. a dog - inoculated with the vaccine, said vaccine including a carrier and a recombinant poxvirus as defined above containing, in a nonessential region thereof, DNA from Morbillivirus, particularly measles virus. Advantageously, the DNA codes for an expresses a measles virus glycoprotein, particularly measles virus hemagglutinin glycoprotein and measles virus fusion glycoprotein. A plurality of measles virus glycoproteins advantageously are coexpressed in the host. The poxvirus used in the vaccine according to the present invention is a vaccinia virus.

For use as the vaccine according to the present invention, the present invention provides a recombinant poxvirus containing therein a ONA sequence from Morbillivirus in a nonessential region of the poxvirus senome. The poxvirus is a vaccinia virus. The Morbillivirus is advantageously measles virus. For use as the vaccine according to the present invention, the recombinant poxvirus expresses gene products of the foreign Morbillivirus gene. In particular, the foreign ONA codes for a measles virus glycoprotein, advantageously measles virus hemagglutinin glycoprotein and measles virus fusion glycoprotein. Advantageously, a plurality of measles virus glycoproteins are co-expressed in the host by the recombinant poxvirus.

A better understanding of the present invention will be had by referring to the accompanying drawings, in which: Figure 1 schematically shows a method for the construction of plasmid pSPM2LHAVC used to derive recombinant vaccinia virus vP557 expressing the MV hemagglutinin gene; FIG. 2 schematically shows a method for the construction of plasmid pSPMFVC used to derive recombinant vaccinia virus vP455 expressing the MV fusion gene; FIG. 3 schematically shows a method for the construction of plasmid pRW843 used to derive recombinant vaccinia virus vP756 expressing the MV hemagglutinin gene; FIG. 4 schematically shows a method for the construction of plasmid pRW850 used to derive recombinant vaccinia virus vP800 expressing the MV fusion gene; FIG. 5 schematically shows a method for the construction of plasmid pRW800 used to derive recombinant canarypox virus VCP40 expressing the MV fusion gene; FIG. 6 schematically shows a method for the construction of plasmid pRW810 used to derive recombinant canarypox viruses yCP50 expressing the MV hemagglutinin gene and vCP57 co-expressing the MV fusion and hemagglutinin genes; FIG. 7 schematically shows a method for the construction of plasmid pRW852 used to derive recombinant canarypox virus VCP85 expressing the MV hemagglutinin gene; FIG. 8 schematically shows a method for the construction of plasmid pRW853A used to derive recombinant canarypox virus VCP82 co-expressing the MV hemagglutinin and fusion genes; FIG. 9 schematically shows a method for the construction of plasmid pSD460 for deletion of thymidine kinase gene and generation of recombinant vaccinia virus VP410; FIG. 10 schematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus vP553; FIG. 11 schematically shows a method for the construction of plasmid ρΜΡ494Δ for deletion of ATI region and generation of recombinant vaccinia virus VP618; FIG. 12 schematically shows a method for the construction of plasmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus VP723; I 1 Figure 13 schematically shows a method for the construction of plasmid pMPCSKla for deletion of gene cluster [C7L - K1L] and generation of recombinant vaccinia virus vP804; Figure 14 schematically shows a method for the construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant vaccinia virus vP866 (NYVAC); and Figure 15 schematically shows a method for the construction of plasmid pRW857 used 10 to derive recombinant NYVAC virus vP913 co-expressing the MV hemagglutinin and fusion genes.

A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

In the following examples, Examples 1-14 contain general background information for preparing and testing recombinant poxvirus vaccines such as the measles virus recombinant poxvirus vaccine of the present invention, which is illustrated in Examples 15 and 16. In particular, the reference to and the information relating to canary poxviruses in Examples 9, 10, 11, 12, 13 and 14 are only for information purposes. ’ 2 Example 1 - GENER^TIONIIF-VACCINTA VIBlJS.RECQMBIKAiNTS CONTAINING THE MEASLES HEMAGGLUTININ GENE The rescuing vims used in the production of both recombinants was the Copenhagen strain of vaccinia virus from which the thymidine kinase gene had been deleted. All viruses were grown and titered on VERO cell monolayers.

The early/late vaccinia virus H6 promoter (Rosel et al., 1986; Taylor et al., 1988a,b) was constructed by annealing four overlapping oligonucleotides, H6SYN AD. The resultant H6 sequence is as follows: Vaccinia Vims H6 Promoter (SEQ ID NOtl/SEQ ID NO:2): Hindlll S’AGCTTCnTAITCrATACTTAAAAAGTGAAAATAAATACAAAGGTTCrTGAGG GTTGT AGAAATAAGATATGAATmT G4CTTTTATTTATGTTTCCAAGAACTCCCAACA GTTAAATIGAAAGCGAGAAATAATCATAAATTATrrCATTATCGCGATATCCGTT AAGTT CAATTTAACrTTCGCTCTTTATTAGTATTTAATAAAGTAATAGCGCTATAGGCAA TTCAA 3 TGTATCGTAC-3' ACATAGCATGAGCT-5' ZhPl Referring now to Figure 1, the annealed H6SYN 5 oligonucleotides were ligated into pMP2LVC digested vith Xhol/Hindlll to yield plasmid pSP131. The plasmid pMP2LVC contains the leftmost 0.4kbp of the vaccinia virus (Copenhagen strain) Hindlll X region within pUC18. The 10 construction of pMP2LVC was performed as follows: a 0.4kbp Hindlll/Sail fragment from the Hindlll K region vas isolated and blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. This fragment was inserted into pUC18 which had been digested with PvuII. The resulting plasmid was designated pMP2VC. The plasmid pMP2VC was linearized with Sspl. Synthetic oligonucleotides MPSYN52 (SEQ ID NO:3) (5'-ATTATTTTTATAAGCTTGGATCCCTCGAGGGTACCCCCGGGGAGCTCGAATTCT-3 ·) and MPSYN53 (SEQ ID NO:4) (5'20 AGAATTCGAGCTCCCCGGGGGTACCCTCGAGGGATCCAAGCTTATAAAAATAAT-3 ·) were annealed and inserted into the leftmost of the two Sspl sites located vithin the vaccinia virus sequences. The resultant plasmid pMP2LVC contains a multiple cloning region in the intergenic region between the K1L and K2L open reading frames.

-Annealed oligonucleotides 3P1 (SEQ ID NO:5) (5’GGGAAG-ATGGAACCAATCGCAGATAG-3') and 3P2 (SEQ ID NO:6) (5'AATTCTATCTG-CGATTGGGGTTCCATCTTCCC-3') containing the extreme 3* sequences of the HA gene and a sticky EcoRI end were 30 ligated to a l.8kbp Xhol/Smal fragment from pHH22 containing the remainder of the HA gene and pSP131 digested with Xhol and EcoRI. The resultant plasmid was designated pSPMHAll. The plasmid pMH22 was derived from a full length cDNA clone of the measles HA gene by creating a Xhol site at the ATG initiation codon (Alkhatib et al., 1986).

A 1.9kbp Hindlll/EcoRI fragment from pSPMHAll, containing the measles HA gene, was isolated and blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. The isolated fragment was inserted into pMP409DVC (Guo et al., 1989) digested with Belli and blunt-ended by. treatment vith mung bean nuclease. Insertion into this vector yielded plasmid pSPMHA4l. The Xhol site between the H6 promoter and the initiation codon of the HA gene was removed by oligonucleotide directed double strand break mutagenesis (Mandecki, 1982) using oligonucleotide HAXHOD (SEQ ID NO:7) (5'ATATCCGTTAAGTTTGTATCGTAATGTCACCACAACGAGACCGGAT-3 ·) . Plasmid PSPM2LHAVC was generated by this procedure. Insertion plasmid pSPM2LHAVC was used in in vitro recombination experiments vith vaccinia virus vP458 as the rescue virus to generate recombinant vP557. vP458 contains the E. coli lac Z gene in the M2L insertion site of vP410. This vaccinia virus recombinant contains the measles HA gene in the M2L locus of the genome, replacing the lac Z gene. example 2 - GENERATION OF VACCINIA VIRUS RECOMBINANTS CONTAINING THE MEASLES FUSION GENE Referring now to Figure 2, annealed oligonucleotides 3PA (SEQ ID NO:8) (5·CCTAAAGCCTGATCTTACGGGAACATCAAAATCCTAT1 5 GTAAGGTCGCTCTGATTTTTATCGGCCGA-3') and 3PB (SEQ ID NO:9) (5*· AGCTTCGGCCGATAAAAATCAGAGCGACCTTACATAGGATTTTGATGTTCCCGTAAGATCAGGCTTTAGG-3') containing the 3' end of the measles fusion gene, a vaccinia virus early transcription termination signal (Yuen et al., 1987) and EaqI and HindlH ends were ligated to a lkbp Sall/Haelll fragment from pCRF2 (obtained from C. Richardson, National Research Council of Canada (Biotechnology Institute), Montreal, Canada H3A 1A1) and pUC8 digested with Sail and HindlH. The resulting plasmid pMF3PR14 contains the 3* end of the lkbp fragment of the measles fusion gene.

Annealed oligonucleotides 5PA (SEQ ID NO: 10) (5 Oligonucleotide directed mutagenesis was performed on pSPHMF7. Initially an in vitro mutagenesis reaction (Handecki, 1982) was performed to create a precise ATG:ATG linkage of the H6 promoter with the measles fusion gene by removing the Smal site using the oligonucleotide SPMAD (SEQ . > ID NO : 12 ) ( 5»-TATCCGTTAAGT-TTGTATGGTAATGGGTCTCAAGGTGAACGTCT3*). This resulted in the generation of pSPMF75M20. Subsequently, the Balll site at the 5' end of the H6 promoter was removed using oligonucleotide SPBGLD (SEQ ID NO: 13 ) ( 5 · -AATAAATCACTTTTTATACTAATTCTTTATTCTATACTTAAAAAGT-3’) according to a known procedure (Mandecki, 1982). The resultant plasmid was designated pSPMFVC. This plasmid vas used in in vitro recombination experiments with vaccinia virus vP410 as rescue virus to generate vP455. r Example 3 - IMMUNOPRECIPITATION ANALYSIS In order to determine that recombinants VP455 and vP557 expressed authentic proteins, immunoprecipitation experiments were performed essentially as described (Taylor et al., 1990). Briefly, VERO cell monolayers were infected at 10 pfu per cell with either parental or recombinant viruses in the presence of 35S-methionine. The fusion protein was specifically precipitated from the infected cell lysate using a rabbit antiserum directed against a carboxy terminal fusion peptide. The hemagglutinin protein was specifically precipitated from the infected cell lysate using a polyclonal monospecific anti-hemagglutinin serum.

With respect to immunoprecipitation using a fusion specific serum, no radiolabelled products were detected in uninfected VERO cells, parentally infected VERO cells, or cells infected vith the HA recombinant vP557. In cells infected with the fusion recombinant vP455, the fusion precursor Fo with a molecular weight of approximately 60 kd I ? and the two cleavage products F, and F2 with molecular weights of 44 kd and 23 kd were detected. Similarly, with respect to immunoprecipitation of the glycosylated form of the HA protein with a molecular weight of approximately 7577 kd, no products were detected in uninfected VERO cells, parental infected cells, or VERO cells infected with vP455.

In addition, immunofluorescence studies indicated that both proteins were expressed on the infected cell surface. example 4 - CELL FUSION EXPERIMENTS A characteristic of Morbillivirus cytopathogenicity is the formation of syncytia which arise by fusion of infected cells with surrounding uninfected cells followed hy migration of the nuclei toward the center of the syncytium (Norrhy et al., 1982). This has been shown to be an important method of viral spread, which for Paramyxoviruses can occur in the presence of hemagglutinin specific antibody (Merz et al., 1980). This ability has been assigned by analogy with other Paramyxoviruses to the amino terminus of the FI peptide (Choppin et al., 1981; Novick et al., 1988; Paterson et al., 1987).

In order to determine that the measles proteins expressed in vaccinia virus were functionally active, VERO cell monolayers were inoculated with parental or recombinant viruses VP455 and vP557, respectively, at 1 pfu per cell. After 1 h absorption at 37"C the inoculum was removed, the overlay medium replaced, and the dishes incubated overnight at 37*C. At 18 h post-infection, plates were examined with a microscope and photographed. No cell fusing activity was evident in VERO cells inoculated with parental virus, VP455 or VP557. However, when vP455 and vP557 were co-inoculated, efficient cell fusing activity was observed.

This result has recently been confirmed by Wild et al. (1991) who determined that syncytium formation in a variety of cell lines infected with measles/vaccinia virus recombinants required expression of both fusion and hemagglutinin genes. The result, however, is in contrast to a previous report (Alkhatib, 1990) which described cell fusion in 293 cells infected with high multiplicities of an adenovirus recombinant expressing the measles fusion protein. Similarly, it has been reported (Vialard et al., 1990) that cell fusion was observed in insect cells infected with a baculovirus recombinant expressing the measles fusion protein but only when incubated at pH 5.8. In neither case was the fusion activity enhanced by co-infection with the appropriate recombinant expressing the measles hemagglutinin protein. Variables vhich may be involved in the fusion process are cell type (Giraudon et al., 1984), pH of medium (Vialard et al., 1990) and level of expression of the fusion protein (Norrby et al., 1982).

Example 5 - SEROLOGICAL TESTS The technique for virus neutralizing (VN) antibody testing was previously described in detail (Appel et al., 1973). Testing for CDV-VN antibody titers was made in VERO cells with the adapted Onderstepoort strain of CDV. Testing for MV-VN antibody titers was made in VERO cells with the adapted Edmonston strain of MV. The results of the serological tests are shown in Table 1. 9 Dogs immunized as described in Example 6 with either the vaccinia parental virus or vP455 expressing the measles fusion protein did not develop neutralizing antibody to MV. Dogs immunized with either VP557 expressing the HA protein or co-inoculated with both recombinants vP455 and VP557 did develop neutralizing antibodies after one inoculation. Levels of antibody were equivalent to those induced by inoculation with the attenuated Edmonston strain of MV.

Measles virus neutralising antibody titers in response to vaccination Table 1 Immunization Dog No. Days past vaccination 0* 7 14 21® 28 35e Vacc. 4/1 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 4/2 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 VP455 4/3 . <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 4/4 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 VP557 4/5 <1.0 2.2d 2.9 2.9 3.4 3.4 4/6 <1.0 2.7 2.9 2.9 3.9 3.4 VP455 6 VP557 4/7 <1.0 2.7 3.4 3.2 3.4 3.4 4/8 <1.0 2.5 2.9 2.9 3.6 2.9 MV 4/14 <1.0 2.9 4/15 <1.0 3.2 a) Time of first immunization b) Time of second immunization (first immunization with MV) c) Time of challenge d) Titer expressed as logl0 of last antibody dilution showing complete neutralization of 20 infectivity in a microtiter neutralization test as described by Appel et al. (1973). 1 Example β - ANIMAL PROTECTION STUDIES In order to determine whether expression of the measles virus proteins in dogs inoculated with the recombinants was sufficient to induce a protective immune response against CDV challenge, fourteen 10 week old 5 specific pathogen free beagle dogs were studied. Blood samples were collected at the initiation of the experiment and repeatedly thereafter. Four groups with two dogs in each group were immunized with tvo injections three weeks apart. The first group received vaccinia virus only. The 10 second group received vaccinia virus with an insert for the F protein of measles virus (vP455). The third group received vaccinia virus with an insert for the HA antigen of MV (vP557), and the fourth group received a combination of 2 and 3. Each dog was inoculated with approximately 4 x 108 pfu of vaccinia virus in 1 ml amounts (0.6 ml subcutaneously and 0.4 ml intramuscularly). Two control dogs received 105 15 50% tissue culture infectious doses (TCIDS0) of the attenuated Edmonston strain of MV intramuscularly (1 ml amount) and two control dogs received 104 TCIDS0 of the attenuated Rockborn strain of COV subcutaneously two weeks before challenge vith virulent CDV. Two control dogs remained uninoculated before challenge. 2q All dogs were challenged by intranasal inoculation of 1 ml of tissue culture fluid containing 104 TCIDS0 of the Snyder Hill strain of virulent CDV two weeks after the last inoculation. The clinical reactions of the dogs vere monitored by daily observations and recording of body temperature and by biweekly recording of weight gain or losses. Circulating blood lymphocytes were counted before challenge and on days post challenge (dpc) 3, 5, 7 and 10.

Virus isolation from buffy coat cells by co-cultivation with dog lung macrophages (Appel et al., 1967) was attempted on dpc 3, 5, 7 and 10. Blood samples for serological tests were collected before vaccination and in weekly intervals until time of challenge, and on dpc 7, 10 and 20.

The results of challenge are shown in Table 2.

Table 2 Effects of immunization on clinical signs after exposure ofdogs tovlrulent CDV No. of days after inoculation with virulent CDV Immunization Dog Number Deoression Height Loss Elevated Bodv Temo.a Lympho- oeniab Virus Isolation6 Death Vacc. 4/1 4-10" 3-10 4,5,7,10 7-10 3-7 10 4/2 4-10d 3-10 4,5,8-10 3-10 3-7 10 VP455 4/3 4-8 7-10 4,5,7-10 5,7 5-7 — 4/4 4-6 7-10 4—6 7 5-7 — 10 VP557 4/5 ND* ND 6,7 10 7 a* 4/6 ND ND ND ND ND - VP455 & VP557 4/7 ND ND 7 7 7 4/8 ND 7 6 ND 7 - MV 4/14 ND ND ND 7 ND 15 4/15 ND 7 5 5 ND - CDV-Ro 4/16 ND ND ND ND ND «* 4/17 ND ND ND ND ND «Μ None 4/18 6,14-17" 7-17 5,7 13-17 10 17 4/19 4-10" 3-10 4,5,7 3,7,10 3-10 10 a) Above 39.5°C b) Less than 2x10s Lymphocytes per mm3 c) Isolated from buffy coat cells co-cultivated with dog lung macrophages d) Dog became dehydrated and was euthanized e) None detected Non-immunized control dogs and dogs vaccinated with parental vaccinia virus developed clinical signs of severe disease andjwere euthanized when dehydration was evident. Both dogs immunized with vP455 shoved some signs of infection with CDV including weight loss, elevated body temperature, and lymphopenia although these symptoms were of shorter duration than were seen in control dogs.

Nonetheless, both dogs survived lethal challenge with CDV. Dogs inoculated with vP557 or co-inoculated vith both recombinants showed minimal signs of infection and survived challenge. Dogs inoculated with either attenuated Edmonston strain of NV or the attenuated Rockborn strain of CDV also survived challenge with minimal signs of disease.

Example 7 - ADDITIONAL VACCINIA/MEASLES CONSTRUCTS Referring now to Figure 3, a second vaccinia virus recombinant containing the measles HA gene within the tk locus was generated (vP756) using insertion plasmid pRW843. pRW843 was constructed in the following manner. A 1.8kbp EcoRV/Smal fragment containing the 3’-most 24bp of the H6 promoter fused in a precise ATG:ATG configuration vith the HA gene lacking the 3'-most 26bp was isolated from pSBM2LHAVC. This fragment vas used to replace the 1.8kbp EcoRV/Smal fragment of pSPMHAll to generate pRN803. Plasmid pRN803 contains the entire H6 promoter linked precisely to the entire measles HA gene.

In the confirmation of previous constructs with the measles HA gene it was noted that the sequence for codon 18 (CCC) vas deleted as compared to the published sequence (Alkhatib et al., 1986). The CCC sequence was replaced by oligonucleotide mutagenesis via the Kunkel method (Kunkel, 1985) using oligonucleotide RW117 (SEQ ID NO: 14) (5« — GACTATCCTACTTCCCTTGGGATGGGGGTTATCTTTGTA-3 ·) .

Pro 18 Single stranded template was derived from plasmid pRH819 which contains the H6/HA cassette from pRW803 in pIBI25 (IBI, New Haven, CT.). The mutagenized plasmid containing the inserted (CCC) to encode for a proline residue at codon 4 was designated pRW820. The sequence between the Hindlll and Xbal sites of pRW820 was confirmed by nucleotide sequence analysis. ?The Hindlll site is situated at the 5' border of the H6 promoter while the Xbal site is located 230bp downstream from the initiation codon of the HA gene.

A l.6kbp Xbal /EcoRI fragment from pRW803, containing the HA coding sequences downstream from the Xbal and including the termination codon, was used to replace the equivalent fragment of pRH820 resulting in the generation of pRW837.

The mutagenized expression cassette contained within pRW837 was derived by digestion vith Hindlll and EcoRI. blunt-ended using the Klenow fragment of Σ. coli DNA polymerase in the presence of 2mM dNTPs, and inserted into the Smal site of PSD573VCVQ to yield pRW843. The plasmid pRW843 was used in in vitro recombination experiments with VP618 as the rescue virus to yield vP756. Parental virus vP618 is a Copenhagen strain virus from which the thymidine kinase, hemorrhagic and A-type inclusion genes have been deleted. Recombinant vP756 has been shown hy immunoprecipitation analysis to correctly express a hemagglutinin glycoprotein of approximately 75kd.

Referring nov to Figure 4, a second vaccinia virus recombinant (VP800) harboring the measles fusion gene in the ATI locus of the genome vas generated using insertion plasmid pRW850. To construct pRW850, the following manipulations were performed. The plasmid pSPMF75M20 containing the measles fusion gene linked in a precise ATG:ATG configuration with the H6 promoter vas digested with Nrul and Eagl. The 1.7kbp blunt ended fragment containing the 3’-most 28bp of the H6 promoter and the entire fusion gene was isolated and inserted into pRW823 digested vith Nrul and Xbal and blunt-ended. The resultant plasmid pRW841 contains the H6 promoter linked to the measles fusion gene in the pIBI25 plasmid vector (IBI, New Haven, CT.). The H6/measles fusion expression cassette vas derived from pRW841 by digestion vith Smal and the resulting 1.8kbp fragment vas inserted into pSD494VC digested with Sjngl to yield pRN850. The plasmid pRH850 vas used in in vitro recombination experiments with vP618 as the rescue virus to yield VP800. Recombinant vP800 has been shown by immunoprecipitation; analysis to express an authentically processed fusion glycoprotein. example 8 - ASSESSMENT OF MEASLES NEUTRALIZING ANTIBODY IN GUINEA PIGS AND RABBITS INOCULATED WITH VP4S5 Two rabbits were inoculated intradermally at 5 sites with a total of 1x10* pfu of recombinant VP455 expressing the measles fusion protein. Both rabbits were boosted vith an identical inoculation at week 12. Serial bleeds were collected, and at week 14, two weeks after the boost, the rabbits were tested for the presence of serum neutralizing antibodies.

Four guinea pigs were inoculated subcutaneously with lxlO8 pfu each of recombinant VP455. An identical booster inoculation was given at 21 days. Serial bleeds were collected.

The presence of measles virus serum neutralizing antibody was assessed using a microtiter test (Appel et al., 1973) using 10 TCIDS0 of virus per microtiter well. The results are shown in Table 3.

Table 3 Results of measles virus serum neutralizing antibodies in guinea pigs and rabbits inoculated with vP455 Week Post-Inoculation 0 2 3 4 5 7 14 Anhoal Guinea Pig • 1 N.D.· N.D. N.D.-.8b 1.3-1.3 1.3-1.5 1.3 N.Te 2 N.D. N.D. .8-1.0 .8-1.3 1.3-1.5 N.D. N.T. 3 N.D. N.D. N.D.-.8 .8-1.5 1.0-1.3 1.0 N.T. 6 N.D. N.D. .8 -.8 .8- .8 1.0-1.3 1.0 N.T. Rabbit W44 N.D. N.T. N.T. N.T. N.T. N.T. 1.5 W86 N.D. N.T. N.T. N.T. N.T. N.T. 1.5 a) Not detectable b) Results of two assays 15 c) Not tested Example 9 " GENERATION OP MEASLES VIRUS RECOMBINANT CANARYPOX VIRUS Measles/canarypox virus recombinants were developed using a similar strategy to that previously described for fowlpox virus (Taylor et al., 1988a,b).

Plasmids for insertion of the measles F and HA genes into canarypox virus were generated as follows.

Referring now to Figure 5, the 1.8kbp blunt-ended Bglll/EaqI fragment from p5PMF75M20 containing the H6 promoted measles F gene was inserted into the blunt-ended EcoRI site of pRH764.2. Plasmid pRW764.2 contains a 3.4kbp PvuII fragment from the canarypox genome having a unique EcoRI site which has been determined to be non-essential for viral replication. The resultant plasmid containing the measles F gene was designated pRW800 and vas used in recombination experiments with canarypox as the rescuing virus to generate VCP40.

Referring now to Figure 6, the l.8kbp EcoRV/Smal fragment from pSPM2LHA containing the 3'-most 28bp of the H6 promoter fused in a precise ATG:ATG configuration with HA was inserted between the EcoRV and Smal sites of pSPMHAll. The- resultant plasmid was designated pRW803. A 2kbp Hindlll/EcoRI fragment of pRH803 containing the H6 promoted measles HA gene was blunt-ended and inserted into the bluntended SsXII site of plasmid pRW764.5. Plasmid pRW764.5 contains an 800bp PvuII fragment of the canarypox genome having a unique Bglll site vhich has previously been determined to be non-essential for viral growth. This insertion created plasmid pRW810 which was used in recombination tests to generate VCP50.

Insertion of the measles F and HA sequences individually led to the development of recombinants vCP40 and vCP50, respectively. In order to create a double recombinant, the single F recombinant VCP40 was used as a rescue virus for insertion of the HA gene contained in * pRW810. This led to the development of double recombinant VCP57.

Example 10 - IMMUNOPRECIPITATION ANALYSIS In order to confirm that recombinants VCP40, vCPSO and vCP57 expressed authentic proteins, immunoprecipitation analysis· was performed using mono-specific sera directed 5 against either the HA or F proteins. A correctly processed fusion polypeptide was specifically precipitated from lysates of cells infected vith vCP40 and VCP57. The fusion precursor Fo vith a molecular weight of approximately 60kd and the two cleavage products F, and F2 with molecular weights of approximately 44 and 23kd, respectively, were detected. Ho fusion specific products were detectable in uninfected CSF cells, parentally infected CSF cells or CEF cells infected with the HA recombinant vCP50. Similarly, a glycoprotein of approximately 75kd was specifically precipitated from CEF ce'lls infected with the single HA recombinant VCP50 and double recombinant vCP57. No HA specific products vere detected in uninfected cells, parentally infected cells or cells infected with fusion recombinant vCP40.

Example 11 - CELL FUSION EXPERIMENTS In order to determine that the measles virus recombinants were functionally active, cell fusion assays vere performed. VERO cell monolayers were infected with 1 pfu per cell of CP parental or recombinant viruses and examined for cytopathic effects at 18 hours post infection. No cell fusing activity vas evident in VERO cells inoculated vith parental, vCP40 or vCPSO viruses. However, vhen VERO cells were inoculated with the double recombinant vCP57 or when cells are co-infected with both VCP40 and vCPSO, efficient cell fusing activity is evident.

Example 12 - SEROLOGICAL TESTS Dogs inoculated as described in Example 13 vith the canarypox/HA recombinant vCPSO, vaccinia/HA recombinant vP557, the canarypox/HA/F double recombinant VCP57 or coinoculated with vP455 and vP557 developed significant serum neutralizing antibody to measles virus after one 30 inoculation. Neither of the two dogs inoculated with the canarypox/f recombinant vCP40 developed neutralizing antibody after one or two inoculations. The results of the serological tests are shown in Table 4.

In addition, guinea pigs inoculated with the vCP40 recombinant did develop low but reproducible levels of serum neutralizing antibody.

Table 4 Measles virus neutralizing antibody titers (in log10) Days post vaccination Immunization Dog No. 0' 7 14 23? 28 35c Canary pox virus 9/ 1 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 (CPV) 9/ 2 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 VCP50 9/ 3 <1.0 2.7d 2.9 3.2 4.4 ’ 4.1 9/ « <1.0 1.7 2.7 2.7 3.9 3.9 VCP40 9/ 5 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 9/ 6 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 VCP57 9/ 7 <1.0 2.0 2.7 2.5 3.9 3.6 9/ 8 <1.0 1.0 2.2 2.0 3.6 3.4 VP455 9/ 9 <1.0 <1.0 <1.0 1.0 1.0 1.0 VP557 9/10 <1.0 2.9 2.5 3.2 3.4 3.4 VP455 6 VP557 9/11 <1.0 1.3 2.9 2.9 2.9 2.9 MV 9/12 <1.0 2.5 2.5 Control 9/13 <1.0 9/14 <1.0 CPV-Ro. 2215 <1,Q..... ,. JLUS a) Time of first immunization. b) Time of second immunization. (First immunization with MV and CDV-Ro,. c) Time of challenge. d) Assessment of serum neutralization titers assessed in known manner (Appel et al., 1973). 1 Example 13 - ANIMAL PROTECTION STUDIES In order to determine whether non-replicating canarypox vectors expressing measles virus proteins would induce a protective immune response against CDV challenge, ten week old specific pathogen free beagle dogs were 5 inoculated with canarypox parental and recombinant viruses.

Two dogs were inoculated simultaneously with two subcutaneous injections of 1x10 8 pfu of each recombinant at three week intervals. For comparison, one dog was inoculated in the same regimen vith each of the single 1θ vaccinia virus recombinants vP455 and vP557 and a combination of both. One dog was also inoculated intramuscularly vith one dose of 10s TCIDS0 of the attenuated Edmonston strain of MV. One dog was inoculated subcutaneously with one. dose of 104 TCIDS0 of the attenuated Rockborn strain of CDV. Dogs were challenged two weeks after the final inoculation via intranasal inoculation with 15 a lethal dose of 104 TCIDS0 of the virulent Snyder Hill strain of CDV. Clinical reactions of dogs were monitored daily. The results are shown in Table 5.

Table 5 Effects of Immunization on clinical signs afterexposure of dogs to virulent CDV Wo. of davs after Inoculation with virulent CDV Immunization Doa No. Deoression Elevated Wt. Loss Lympho virus Bodv Tern».® oeniab Isolation1 Canary pox 9/ 1 4-10- 3-10 4,5,7,8 5-10 3-10 virus (CPV) 9/ 2 4-10- 7-10 4,5,7,8 3-10 3-10 vCPSO 9/ 3 ND· 7-10 5-7 5-7 7 9/ 4 ND 7 6,7 7 ND 10 VCP40 9/ 5 4-10 3-10 4 5-10 5-7 9/ 6 4-6 3-10 4-6 5 5-7 VCP57 9/ 7 ND 7-10 5,6 5 ND 9/ β ND 7-10 4-7,10 7-10 5-7 VP455 9/ 9 6-8 7-10 4,5,8,9 5-10 3-10 15 VP557 9/10 ND 3-10 ND ND ND VP455 & VP557 9/11 ND 3-10 ND 5-7 ND MV 9/12 ND 7-10 ND 5 5 None 9/13 4-10- 3-10 4-6 5-10 5-10 20 9/14 4-10- 3-10 4-5 3-10 5-7 CDV-Ro 9/15 ND ND ND ND ND ajAbove 39.5®C.bjLees than 2x10* Lymphocytes per mm3, ojIsolated from huffy coat cells co-cultivated with dog lung macrophages in known manner (Appel et al., 1967). d, Dog became dehydrated and was euthanized, e) None detected.

No adverse reactions to vaccination were noticed in any of the dogs during the course of the experiment. The two dogs immunized with parental canarypox virus and two ·» non-immunized control dogs showed severe disease after challenge with virulent CDV. All four dogs became depressed, showed elevated body temperature, weight loss, lymphopenia and severe dehydration. Dogs immunized vith CDV-Rockborn developed serum neutralizing antibodies against CDV but not against MV prior to challenge and survived challenge, symptom free. Dogs immunized with attenuated MV developed serum neutralizing antibodies to MV but not CDV prior to challenge, and survived challenge with mild signs of infection. Dogs inoculated with vCP50, VCP57, VP557 or co-inoculated with VP455 and VP557 developed significant serum neutralizing antibody to MV after one inoculation and « survived challenge vith only minor signs of infection. Example 14 - ADDITIONAL CANARYPOX/MEASLES CONSTRUCTS Referring now to Figure 7, to generate a canarypox virus recombinant expressing the MV HA gene the folloving insertion plasmids were created. A 1.8kbp EcoRVZEcoRI fragment from pRW837 containing the 3'-most 26bp of the H6 promoter linked precisely to the measles HA, was ligated to a 3.2kbp EcpRV/£gpRI fragment from pRW838. The pRW838 derived fragment includes the 5' portion of the H6 promoter and C5 locus flanking arms. Plasmids pRW838 and pRW831 (see below) were derived as follows.

An 880 bp PvuII canarypox genomic fragment was inserted between the PvuII sites of pUC9. the resultant plasmid was designated pRH764.5. The nucleotide sequence of the 880 bp canarypox fragment was determined using the modified T7 enzyme Sequenase™ Kit (United States Biochemical, Cleveland, OH) according to manufacturer's specifications. Sequence reactions utilized custom synthesized primers (17-18 mers) prepared with the Biosearch 8700 (San Rafael, CA) or Applied Biosystems 3800 (Foster City, CA). This enabled the definition of the C5 open reading frame.

To specifically delete the C5 open reading frame, PRW764.5 was partially cut with Rsal and the linear product was isolated. The Rsal linear fragment was recut with Belli and the pRW764.5 fragment with a RsaI-ΒαΙΙΙ deletion from position 156 to position 462 was isolated and used as a vector for the following synthetic oligonucleotides: RW145 (SEQ ID NO:15): (5'-ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA-3') RW146 (SEQ ID NO:16): (5·GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT-3') Oligonucleotides RW145 and RW146 vere annealed and inserted into the pRW764.5 RsaI-ΒαΙΙΙ vector described above. The resulting plasmid is pRH83l.

This C5 deletion plasmid was constructed without interruption of other canarypox virus open reading frames. The C5 coding sequence was replaced with the above annealed oligonucleotides (RW145 and RN146) which include the restriction sites for Hindlll. Smal. and EcoRI.

The plasmid pRW838, was derived from pRW831 by the insertion of a Smal fragment containing the Rabies G gene (Taylor et al., 1988b) juxtaposed 3' to the vaccinia virus H6 promoter. Ligation of the 1.8 kbp EcoRV/EcoRI fragment from pRN837 vith the 3.2 kbp EcoRV/EcoRI fragment from pRW838 led to the construction of plasmid pRW852. Plasmid pRW852 was used in recombination experiments with a canarypox isolate designated ALVAC td yield vCP85. ALVAC is a plaque cloned isolate of canarypox virus (CPV) derived from the Rentschler strain, a highly attenuated strain of CPV used for vaccination of canaries. Replication of ALVAC and derived recombinants is restricted to avian species. Immunoprecipitation analysis has confirmed that a protein of approximately 75kd recognized by a rabbit anti-HA serum is expressed in CEF cells infected vith recombinant vCP85.

Referring now to Figure 8, to generate a canarypox virus recombinant harboring both the NV HA and F genes the following constructs vere engineered. Smal restriction sites were added to the ends of the H6 promoted measles fusion gene. To accomplish this, pRW823, vhich is pIBI25 containing the vaccinia virus H6 promoter, was digested downstream of the promoter sequence at the Xbal site. The ends were blunted with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. The bluntended DNA was subsequently digested with Nrul to liberate a 3.0kbp fragment containing the 5’-most lOObp of the H6 promoter. This fragment was isolated and ligated to a 1.7kbp blunt-ended EaqI/Nrul fragment from pSPMF75. The resultant plasmid was designated as pRW841.

The l.8kbp Smal fragment derived by digestion of pRW84l was inserted into the C5 deletion vector, pRW83l.

The plasmid pRW851 was linearized at the EcoRI site situated 3 * to the fusion gene and was blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. The plasmid^ pRW837, containing the measles HA gene juxtaposed 3’ to the H6 promoter sequences, was digested with HindlH and EcoRI and blunt-ended vith the Klenow fragment. The resultant 1.8kbp fragment was isolated and inserted into pRH85l that had been linearized with EcpRl and blunt-ended. The resultant plasmid, which contains both genes in a tail to tail configuration, vas designated pRW853A and was utilized in in vitro recombination experiments with canarypox (ALVAC) as the rescue virus to generate vCP82 also designated ALVAC-MV. Expression analysis using immunoprecipitation and immunofluorescence confirmed that in cells infected with recombinant VCP82 authentically processed HA and F proteins vere expressed.

The recombinant vas also functional for cell fusing activity.

Results of serological analysis of sera of rabbits and guinea pigs inoculated with ALVAC-MV (vCP821 Four guinea pigs were inoculated by the subcutaneous route with ALVAC-MV (vCP82). Two animals (026 and 027} each received 1x10s pfu and two aniaals (028 and 029} each received lxio7 pfu. At 28 days, animals were re-inoculated vith an identical dose. Two rabbits were inoculated with lxlO8 pfu of ALVAC-MV (vCP82) by the subcutaneous route. At 28 days, animals were re-inoculated with an identical dose. Serial bleeds of these animals were analyzed for measles virus neutralizing activity using either a microtiter .neutralization test described by Appel and Robson (1973) or a plague reduction neutralization test described by Albrecht et al. (1981). In addition, sera were 5 analyzed for the presence of antibody capable of blocking measles virus induced cell-cell fusion in an anti-fusion assay performed as described in Merz et al. (1980).

The results of analysis for the presence of measles virus serum neutralizing antibody are shown in Tables 6 and 7. Both guinea pigs (026 and 027) receiving 1x10s pfu of ALVAC-MV sero-converted after a single inoculation and sera showed an antibody rise after the booster inoculation. One animal (029) receiving lxio7 pfu also sero-converted after one inoculation. The fourth animal (028) did not show a detectable response after one inoculation but did achieve equivalent titers after the second inoculation.

Rabbit sera were also analyzed using a plague reduction neutralization method. The results are shown in Table 7. Both animals sero-converted after one inoculation. Sera of rabbit 063 was tested by both the micro-titer neutralization test and the plague reduction neutralization test. Titers achieved were similar using both methods. Zt has been reported that a minimal serum neutralizing titer of 1.2 to 1.9 in vaccinated children is required for protection from disease (Lennon and Black, 1986; Black et al., 1984). Using this criteria, all animals, except the one guinea-pig vhich did not sero-convert until the second inoculation showed a protective level of antibody after one inoculation.

Table 6 Serological analysis of sera of guinea pigs inoculated with ALVAC-MV (vCP82): Analysis performed by microtiter serum * · neutralization assay.

Animal Days post-inoculation 0 14 21 28e 42 48 56 Guinea pigs 026d - N.T.* 1.25b 1.49 2.45 2.68 2.92 027 - N.T. 1.97 1.49 2.68 2.45 2.21 028* - N.T. - - 1.73 2.45 1.97 029 N.T. 0.8 1.49 2.45 2.45 2.45 a) Not tested. b) Titer expressed as logw of reciprocal of last dilution showing complete neutralization of cytopathic effect. c) Animals boosted at 28 days post-inoculation. d) Animals 026 and 027 received 1x10s pfu. e) Animals 028 and 029 received lxlO7 pfu.

Table 7 Serological analysis of sera of rabbits inoculated with ALVAC-MV (VCP82) Animal Days post-inoculation 0 14 21 28b 42 56 Plague 063 reduction method 1.9’ 2.8 1.6 2.2 2.2 064 - 2.2 2.5 2.8 3.1 2.8 Microtiter neutralization method 063 1.5C 1.7 1.5 1.7 a) Titer expressed as log10 of reciprocal of last dilution showing a 50% reduction in plague number as compared to pre-inoculation serum.

Animals boosted at 28 days post-inoculation.

Titer expressed as log10 of reciprocal of last dilution showing complete neutralization of cytophatic effect. b) c, Previous studies have shown that an inactivated vaccine was associated with poor protective efficacy and an enhanced measles dipease on re-exposure to the virus. Recipients of the inactivated vaccine demonstrated an absence of antibody to the fusion protein and it was proposed that the inactivation process had rendered the protein non-immunogenic (Norrby and Gollmar, 1975; Norrby et al., 1975). In addition, it has been shown for other paramyxoviruses that antibody to the F protein is able to inhibit cell to cell spread of virus in tissue culture while antibody to the hemagglutinin component is not (Merz et al., 1980).

It was therefore significant to demonstrate that animals inoculated with ALVAC-MV (VCP82) were able to induce antibody to the F component which was capable of blocking cell to cell transmission of measles virus. The results of this anti-fusion assay are shown in Table 8. Anti-fusion activity was evident in sera of both guinea-pigs and rabbits inoculated with ALVAC-MV (vCP82). The sera analyzed was taken two or three weeks after the boost inoculation. No anti-fusion activity could be detected in sera of rabbits inoculated with ALVAC parental virus.

Table 8 Analysis of sera of guinea pigs and rabbits inoculated with ALVAC-MV for anti-fpsion activity Animal Designation Immunogen Anti-Fusion Titer Pre-inoc. Post-Vacc. Guinea-pig 026 ALVAC-MV - 2.4®' b 027 ALVAC-MV - 1.2 Rabbit 063 ALVAC-MV - 1.8e 064 ALVAC-MV - 1.8 Rabbit W121 ALVAC - - W123 .· ALVAC * a) Guinea pig sera tested at 7 weeks post-vaccination. b) Titer expressed as log10 of reciprocal of highest dilution showing complete inhibition of measles virus induced cell fusing activity, c) Rabbit sera test at 6 weeks post-vaccination. 1 In further tests to demonstrate the presence of antibody to both the MV hemagglutinin and MV fusion proteins in sera of animals inoculated vith ALVAC-MV, immunoprecipitation experiments were performed. Sera of rabbits inoculated with ALVAC-MV was shown to specifically precipitate both the hemagglutinin and fusion proteins from radiolabelled lysates of Vero cells infected with Edmonston strain MV.

In a similar study, groups of guinea pigs, rabbits and mice were inoculated by the intra muscular route with ALVAC-MV, and their serological response to measles virus monitored using the hemagglutination-inhibition (HI) test. The serological response to canarypox virus was monitored by ELISA assay. In this study, five guinea pigs were inoculated with 5.5 logw TCIDS0, thirty mice were inoculated vith 4.8 log10 TCID50, and five rabbits vere inoculated with 5.8 log10 TCIDjg. All animals were re-inoculated at 28 days vith an equivalent dose. Animals vere bled at regular intervals and their response to measles virus assessed in an HI assay. The limit of detection in the HI assay corresponds to a log10 titer of 1 and it is considered that sero-positive (protected) children have a serum titer in the range of 1.6 to 2.8. The results of analysis are shown in Tables 9, 10 and 11.

Sera of mice were analyzed in groups of 5 animals (Table 9). All animals shoved a primary response to canarypox virus which was boosted after the second inoculation. The mice did not show a response to MV after one inoculation. Three of the six groups showed titers within the protective range at 8 weeks post-inoculation. Similarly, all guinea-pigs (Table 10) showed a response to canarypox virus after one inoculation which vas boosted after the second inoculation. Four of five animals developed anti-HI titers after one inoculation, one of these being in the protective range. One week after the second inoculation, the titers of all animals were in the protective range. These titers were maintained through 8 weeks post-inoculation when the experiment was concluded.

All rabbits (Table 11) inoculated with ALVAC-MV (vCP82) responded serologically to canarypox inoculation. Pour of five animals sero-cohverted to measles virus after one inoculation (one in the protective range). Serum titers of all animals were in the protective range one week after the second inoculation.

Table 9 Serological response of mice to inoculation with ALVAC-MV (VCP82) Anti-canarvpox response ELISA TITER 0 2 Week post-inoculation Mouse Group 4 5 6 8 1* —0.009° 0.364 0.193 1.821 1.616 1.123 2 -0.026 0.047 0.240 1.739 1.963 1.986 3 -0.006 0.148 0.641 1.860 1.861 1.947 4 -0.005 0.130 0.451 1.506 1.937 1.124 5 0.687 0.542 Mean -0.012 0.275 • 0.413 1.732 1.844 1.395 Anti-measles resoonse HI TITER Week post-inoculation Mouse Group 0 2 4 5 6 8 1 c <1 <1 <1 1 1 2 <1 <1 <1 1 1.6 1.6 3 <1 <1 1 1 2.2 2.2 4 <1 <1 <1 1.6 1 1.8 5 <1 <1 <1 1.3 1.8 1.2 Mean - - 1 1.2 1.5 1.5 a) . Groups of five mice were exsanguinated and sera pooled. b) optical density in an ELISA assay on sera at dilution of 1:800 c) Limit of detection in HI test corresponds to a logw titer of 1 i.e. 1:10 dilution. Titer expressed as logv of reciprocal of highest dilution showing inhibition of hemagglutination. 4 Table 10 Serological response of guinea-pigs to inoculation with ALVAC-MV (VCP82, ' Anti-canarvpox response ELISA TITER Guinea-pig Week post-inoculation 6 8 0 2 4 5 1 0i038a 0.045 0.111 .771 1.970 1.856 2 0.010 0.072 0.234 1.768 1.786 1.785 3 -0.011 0.426 0.529 1.567 1.586 1.700 4 0.016 0.045 0.076 1.583 1.696 1.635 5 -0.020 0.012 0.050 1.583 1.859 1.847 Anti-measles response HI TITER Week post- inoculation Guinea pig 0 2 4 5 6 8 1 b 1.18 1.90 3.11 3.41 3.11 2 <1 <1 1.00 2.20 2.20 2.08 3 <1 <1 1.18 2.51 2.68 2.98 4 <1 <1 <1 1.60 1.90 1.90 5 <1 <1 1.30 1.90 2.20 2.20 b) a) Optical density in an ELISA assay on serum at a 1:3200 dilution.

Limit of detection in HI test corresponds to a log10 titer of 1 i.e. 1:10 dilution. Titer expressed as in legend to Table 9.

Table 11 Serological response of rabbits to inoculation with ALVAC-MV (VCP82) Anti-canarvpox response ELISA TITER Rabbit 0 Week post-inoculation 8 2 4 5 6 1* —0.009* 0.085 0.113 1.953 1.754 1.249 2 -0.002 0.065 0.068 0.717 0.567 0.353 3 -0.003 0.090 0.079 0.921 0.692 0.481 4 —0.005 0.034 0.068 1.558 1.324 1.076 5 -0.003 0.072 0.092 1.785 1.226 0.710 Anti-measles resDonse HI TITER Week post-; inoculation Rabbit 0 2 4 5 6 8 1 b <1 1.00 2.81 2.51 2.20 2 <1 <1 <1 2.20 1.90 1.60 3 <1 <1 1.30 2.81 2.51 2.38 4 <1 1.30 1.60 3.11 3.11 2.51 5 <1 1.00 1.30 2.68 2.38 1.90 a) Optical density in an ELISA assay on sera at a b) dilution of 1:1600.

Limit of detection in HI test corresponds to a log10 titer of 1 i.e. 1:10 dilution. Titer expressed as in legend to Table 9.

Results of serological analysis of sera of squirrel monkeys inoculated with ALVAC-MV (vCP82)s Influence of prior exposure to poxvirus on induction of a measles virus specific immune response Nine squirrel monkeys (Saimiri sciureus) were inoculated with ALVAC-MV (VCP82). All monkeys were naive to measles virus. Seven of the monkeys had prior exposure to vaccinia virus and/or canarypox virus. The previous immunization history is shovn in Table 12. All monkeys were inoculated with one dose of 5.8 logw pfu by the subcutaneous route. Four of the animals (#39, 42, 53 and 58} were re-inoculated with an equivalent dose fifteen weeks after the primary inoculation. Anti-measles antibody vas measured in the HI test. The results are shown in Table 12.

After the first .inoculation, two of the nine monkeys showed a low response to inoculation with ALVAC-MV. After the second inoculation, the four monkeys re-inoculated all 15 sero-converted with significant antibody titers in the range required for protective immunity. The titers achieved were equivalent whether the monkey had prior exposure to vaccinia virus and ALVAC or no prior poxvirus exposure. . 47 Table 12 Inoculation of squirrel monkeys with ALVAC-MV (VCP82): Immune response in the face of pre-existing ALVAC immunity.

Monkey # Previous Immunity to Poxviruses Anti-Measles Primary® HI response Boost* 36 W, ALVAC <1 N.B. 37 W, ALVAC-RG <1 N.B. 39 W, ALVAC-RG, CP-FeLV 1 2.2. 40 W, CP-FeLV <1 N.B. 42 None .. <1 2.2. 52 ALVAC <1 N.B. 53 ALVAC-RG, ALVAC-RG <1 1.6. 56 CP-FeLV <1 N.B. 58 None 1 2.2. W: Vaccinia virus, Copenhagen strain ALVAC-RG: ALVAC recombinant expressing rabies G gene CP-FeLV: Canarypox recombinant expressing FeLV env gene NB: Not boosted a) b) Animals received 5.8 log10 pfu by S.C. route.

Animals 39, 42, 52 and 53 were boosted with an identical dose 15 weeks after the first inoculation.

Example 15 - ATTENUATED VACCINIA VACCINE STRAIN NYVAC To develop a new vaccinia vaccine strain, the Copenhagen vaccine Strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence factors. The sequential deletions are detailed below. All designations of vaccinia restriction fragments, open reading frames and nucleotide positions are based on the terminology reported in Goebel et al. (1990a,b).

The deletion loci were also engineered as recipient loci for the insertion of foreign genes.

The regions sequentially deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designations for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified: (1) thymidine kinase gene (TK; J2R) vP410; (2) hemorrhagic region (u; B13R + B14R) VP553; (3) A type inclusion body region (ATI; A26L) vP618; (4) hemagglutinin gene (HA; A56R) vP723; (5) host range gene region (C7L - K1L) vP804; and (6) large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC).

DMA Cloning and Synthesis Plasmids vere constructed, screened and grown by standard procedures (Maniatis et al., 1986; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from GIBCO/BRL, Gaithersburg, MD, New England Biolabs, Beverly, MA; and Boehringer Mannheim Biochemicals, Indianapolis, IN. Klenov fragment of B. coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents vere used as specified by the various suppliers.

Synthetic oligodeoxyribonucleotides vere prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA 9 sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously;described (Guo et al., 1989). DNA amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) was performed 5 using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler. Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited 10 digestion by BAL-31 exonuclease and mutagenesis (Handecki, 1986) using synthetic oligonucleotides.

Cells. Virus, and Transfection The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989). Generation of recombinant virus by recombination, in situ hybridization of 15 nitrocellulose filters and screening for Beta-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et al., 1989).

Construction of Plasmid PSD460 for Deletion of Thymidine Kinase Gene (J2R) Referring now to FIG. 9, plasmid pSD406 contains 2q vaccinia Hindlll J (pos. 83359 - 88377) cloned into pUC8. pSD406 was cut with Hindlll and PvuII. and the 1.7 kb fragment from the left side of Hindlll J cloned into pUC8 cut with Hindlll/Smal, forming pSD447. pSD447 contains the entire gene for J2R (pos. 83855 - 84385). The initiation codon is contained vithin an Nlalll site and the termination codon is contained within an SspI site. Direction of transcription is indicated by an arrow in FIG. 9.

To obtain .a left flanking arm, a 0.8 kb Hindlll /EcoRI fragment was isolated from pSD447, then digested with Elalll and a 0.5 kb Hindlll/Nlalll fragment isolated. Annealed synthetic oligonucleotides • MPSYN43/MPSYN44 (SEQ ID NO:17/SEQ ID NO: 18) Smal MPSYN43 5' TAATTAACTAGCTACCCGGG 3' MPSYN44 3’ GTACATTAATTGATCGATGGGCCCTTAA 5’ Nlalll > EcoRI were ligated with the 0.5 kb Hindlll/Nlalll fragment into pUC18 vector plasmid cut with HindlH/EcoRI. generating plasmid pSD449.

To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with SspI (partial) within vaccinia sequences and HindlH at the pUC/vaccinia junction, and a 2.9 kb vector fragment isolated. This vector fragment was ligated with annealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID NO;19/SEQ ID NO:20) HindlH Smal HPSYN45 5* AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA MPSYN46 3· AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT Notl SSPI ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5 * MPSYN46 generating pSD459.

To combine the left and right flanking arms into one plasmid, a 0.5 kb Hindlll/Smal fragment was isolated from PSD449 and ligated vith pSD459 vector plasmid cut with Hindlll/Smal. generating plasmid pSD460. pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2. KP labeled probe was synthesized by primer extension using MPSYN45 (SEQ ID NO: 19) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:21) (5«-TTAGTTAATTAGGCGGCCGC-3 ·) as primer. Recombinant virus VP410 was identified by plaque hybridization.

Construction of Plasmid PSD486 for Deletion of Hemorrhagic Region (B13R + B14R1 Referring now to FIG. 10, plasmid pSD419 contains vaccinia Sail G (pos. 160,744-173,351) cloned into pUC8. pSD422 contains the contiguous vaccinia Sail fragment to the right. Sail J (pos. 173,351-182,746) cloned into pUC8. To construct a plasmid deleted for the hemorrhagic region, u, 1 B13R - B14R (pos. 172,549 - 173,552), pSD419 was used as the source for the left flanking am and pSD422 was used as the source of the right; flanking am. The direction of transcription for the u region is indicated by an arrow in FIG. 10.

To remove unwanted sequences from pSD419, sequences to the left of the Ncol site (pos. 172,253) were removed by digestion of pSD419 with Ncol/Smal followed by blunt ending vith Klenow fragment of E. coli polymerase and ligation generating plasmid pSD476. A vaccinia right flanking arm 10 vas obtained by digestion of pSD422 with Hpal at the termination codon of B14R and by digestion with Nrul 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb Hindi vector fragment isolated from pSD476, generating plasmid pSD477. The location of the partial deletion of the vaccinia u region in pSD477 is indicated by a triangle. The remaining B13R coding sequences in pSD477 15 were removed by digestion vith Clal/Hpal. and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID NO:22/SEQ ID NO:23) Clal BamHI Hpal SD22mer 5* CGATTACTATGAAGGATCCGTT 3* SD20mer 3' TAATGATACTTCCTAGGCAA 5' 2Q generating pSD479. pSD479 contains an initiation codon (underlined) followed by a BamHI site. To place E. coli Beta-galactosidase in the B13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactosidase gene (Shapira et al., 1983} was inserted into the BamHI site of pSD479, generating pSD479BG. pSD479BG was used as donor plasmid for - 25 recombination with vaccinia virus VP410. Recombinant vaccinia virus VP533 was isolated as a blue plaque in the presence of chromogenic substrate X-gal. In vP533 the B13RB14R region is deleted and is replaced by Betagalactosidase .

To remove Beta-galactosidase sequences from VP533, plasmid pSD486, a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized. First the Clal/Hpal vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides 5042mer/SD4Omer (SEQ ID NO:24/SEQ ID NO:25) Clal Sacl Xhol Hpal SD42mer 5’ CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3* SD40mer 3* TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 51 Belli Smal BamHI generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia junction was destroyed by digestion of pSD478 with EcoRI followed by blunt ending with Klenow fragment of E. coli polymerase and ligation, generating plasmid pSD478E~ . pSD478E~ was digested with BamHI and Hpal and ligated vith annealed synthetic oligonucleotides HEM5/HEM6 (SEQ ID NO:26/SEQ ID NO:27) HEM5 HEM6 BamHI EcoRI iHoal 5' GATCCGAATTCTAGCT 3' 31 GCTTAAGATCGA 51 generating plasmid pSD486. pSD486 was used as donor plasmid for recombination vith recombinant vaccinia virus VP533, generating vP553, vhich was isolated as a clear plague in the presence of X-gal.

Construction of Plasmid ΡΚΡ494Δ for Deletion of ATI Region (A2SL) Referring now to FIG. 11, pSD414 contains Sail B cloned into pDC8. To remove unwanted DNA sequences to the left of the A26L region, pSD414 was cut with Xbal vithin vaccinia sequences (pos. 137,079) and with Hindlll at the pUC/vaccinia junction, then blunt ended with Klenow fragment of E. coli polymerase and ligated, resulting in plasmid pSD483. To remove unwanted vaccinia DNA sequences to the right of the A26L region, pSD483 vas cut with £ssRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding region, pSD484 was cut with lidel (partial) slightly upstream from the A26L ORF (pos. 139,004) and vith HES.I (pos. 137,889) slightly downstream from the A26L ORF. The 5.2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:28/SEQ ID NO:29) Ndel.

ATI 3 5’ TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT ATI 4 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA Ballf’gssRI Hpal TATATAAATAGATCTGAATTCGTT 3' ATI3 5 ATATATTTATCTAGACTTAAGCAA 5' ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF vith a short polylinker region containing the restriction sites BgXII, £coRI and Hsal, as indicated above. The resulting plasmid vas designated pSO485. Since the IQ Belli and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted Balll and EcoRI sites vere removed from plasmid pSD483 (described above) by digestion vith Balll (pos. 140,136) and vith EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenov fragment of E. coli polymerase and ligation.* The resulting plasmid was designated pSD489. The 1.8 kb Clal (pos. 137.198) /EcoRV (pos. 139,048) fragment from pSD489 containing the A26L ORF was replaced with the corresponding 0.7 kb polylinkercontaining Clal/EcoRV fragment from pSO485, generating pSD492. The Balll and EcoRI sites in the polylinker region of pSD492 are unique.

A 3.3 kb Balll cassette containing the E. coli Betagalactosidase gene (Shapira et al., 1983) under the control 20 of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990} vas inserted into the Balll site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination vith rescuing virus vP553. Recombinant vaccinia virus, vP581, containing Beta-galactosidase in the A26L deletion region, was isolated as a blue plaque in the presence of X-gal.

To generate a plasmid for the removal of Betagalactosidase sequences from vaccinia recombinant virus vP58l, the polylinker region of plasmid pSD492 was deleted by mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN177 (SEQ ID NO:30) (5‘AAAATGGGCGTGGATTGTTAACTTTATATA-ACTTATTTTTTGAATATAC—3 ·) . In the resulting plasmid, ρΜΡ494Δ, vaccinia DNA encompassing positions [137,889 - 138,937], including the entire A26L ORF is deleted. Recombination between the ρΜΡ494Δ and the Betagalactosidase containing vaccinia recombinant, vP581, resulted in vaccinia deletion mutant vP618, which was isolated as a clear plague in the presence of X-gal.

Construction of Plasmid PSD467 for Deletion of Hemagglutinin Gene Referring now to FIG. 12, vaccinia Sail G restriction fragment (pos. 160,744-173,351) crosses the Hindlll A/B junction (pos. 162,539). pSD4l9 contains vaccinia Sail G cloned into pUC8. The direction of transcription for the hemagglutinin (HA) gene is indicated by an arrow in FIG. 12. Vaccinia sequences derived from Hindlll B were removed by digestion of pSD419 with Hindlll within vaccinia sequences and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, contains the HA gene, A56R, flanked hy 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to the right. A56R coding sequences were removed hy cutting PSO456 with Rsal (partial; pos. 161,090) upstream from A56R coding sequences, and with Eaol (pos. 162,054) near the end of the gene. The 3.6 kb Rsal/Eaal vector fragment from PSD456 vas isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:31), MPSY62 (SEQ ID NO:32), HPSYN60 (SEQ ID NO:33), and MPSYN 61 (SEQ ID NO:34) Rsal MPSYN59 5’ ACACGAATGATTTTCTAAAGTATTTGGAAAGTnTATAGGTAGTTGATAGAMPSYN62 3* TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCATCAACTATCT 5’ MPSYN59 -ACAAAATACATAATTT MPSYN60 5* TGTAAAAATAAATCACTTTTTATACTAAGATCTMPSYN61 3* TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATATGATTCTAGASmal Pstl £gg| MPSYN60 -CCCGGGCTGCAGC 3’ MPSYN61 -GGGCCCGACGTCGCCGG 5’ reconstructing the DNA sequences upstream from the A56R ORF and replacing the A56R ORF vith a polylinker region as indicated above. The resulting plasmid is pSD466. The vaccinia deletion in pSD466 encompasses positions [161,185162,053]. The site of the deletion in pSD466 is indicated by a triangle in FIG. 12.

A 3.2 kb BolII/BamHl (partial) cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983} under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) was inserted into the BolII site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination vith rescuing virus VP618.

Recombinant vaccinia virus, vP708, containing Betagalactosidase in the A56R deletion, vas isolated as a blue plaque in the presence of X-gal.

Beta-galactosidase sequences were deleted from VP708 using donor plasmid pSD467. pSD467 is identical to pSD466, except that EcoRI. Smal. and BamHl sites vere removed from the pUC/vaccinia junction by digestion of pSD466 with EcoRI/BamHl followed by blunt ending with Klenow fragment of B. coli polymerase and ligation. Recombination between VP708 and pSD467 resulted in recombinant vaccinia deletion mutant, VP723, vhich was isolated as a clear plague in the presence of X-gal.

Construction of Plasmid PKPCSKlA for Deletion of Open Reading Frames TC7L-K1L1 Referring now to FIG. 13, the following vaccinia clones were utilized in the construction of pMPCSKlA. pSD420 is Sail H cloned into pUC8. pSD435 is Konl F cloned into pUC18. pSD435 was cut with Sohl and religated, forming pSD451. In pSD451, DNA sequences to the left of the Sohl site (pos. 27,416) in Hindlll M are removed (Perkus et al., 25 1990). pSD409 is Hindlll M cloned into pUC8.

To provide a substrate for the deletion of the [C7LKll>] gene cluster from vaccinia, E. coli Beta-galactosidase was first inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminate the Belli site in pSD409, the plasmid was cut with BolII in vaccinia seguences (pos. 28,212) and with BamHl at the pUC/vaccinia junction, then ligated to form plasmid pHP409B. pMP409B was cut at the unique Sohl site (pos. 27,416). M2L coding seguences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide ;· BolII MPSYN82 (SEQ ID NO: 35) 5* TTTCTGTATATTTGCACCAATTTAGATCTTACTCAAAA TATGTAACAATA 3· The resulting plasmid, pMP409D, contains a unique BolII site inserted into the M2L deletion locus as indicated above. A 3.2 kb BamHI (partial)/figlll cassette containing the E„ coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with BolII. The resulting plasmid, pMP409DBG (Guo et al.r 1990), was used as donor plasmid for recombination vith rescuing vaccinia virus vP723. Recombinant vaccinia virus, VP784, containing Betagalactosidase inserted'into the M2L deletion locus, vas isolated as a blue plaque in the presence of X-gal.

* A plasmid deleted for vaccinia genes (C7L-K1L) vas assembled in pUC8 cut vith Smal. Hindlll and blunt ended with Klenow fragment of E. coli polymerase. The left flanking arm consisting of vaccinia Hindlll C sequences was obtained by digestion of pSD420 vith Xbal (pos. 18,628) followed by blunt ending with Klenov fragment of E. coli polymerase and digestion vith BolII (pos. 19,706). The right flanking arm consisting of vaccinia Hindlll K sequences was obtained by digestion of pSD45l with BolII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP581CK is deleted for vaccinia sequences between the BolII site (pos. 19,706) in Hindlll C and the BolII site (pos. 29,062) in Hindlll K. The site of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 13.

To remove excess DNA at the vaccinia deletion junction, plasmid pMP58lCK, was cut at the figgl sites within vaccinia sequences (pos. 18,811; 19,655), treated with Bal31 exonuclease and subjected to mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:36) 5’TGTCATTTAACACTA57 TACTCATATTAATAAAAATAATATTTATT-3'. The resulting plasmid, pMPCSKlA, is deleted for vaccinia sequences positions 18,805—29,108, encompassing 12 vaccinia open reading frames » [C7L - K1L]. Recombination between pMPCSKlA and the Beta5 galactosidase containing vaccinia recombinant, vP784, resulted in vaccinia deletion mutant, vP804, which was isolated as a clear plague in the presence of X-gal. Construction of Plasmid PSD548 for Deletion of Large Subunit. Ribonucleotide Reductase (I4L) Referring now to FIG. 14, plasmid pSD4O5 contains 10 vaccinia Hindlll I (pos. 63,875-70,367) cloned in pUC8. pSD405 was digested with EcoRV within vaccinia sequences (pos. 67,933) and with Smal at the pUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 was used as the source of all the vaccinia restriction fragments used in the construction of pSD548.

The vaccinia I4L gene extends from position 67,37165,059. Direction of transcription for I4L is indicated by an arrow in FIG. 14. To obtain a vector plasmid fragment deleted for a portion of the I4L coding sequences, pSD518 was digested with BamHI (pos. 65,381) and Hpal (pos. 67,001) and blunt ended using Klenow fragment of E. coli polymerase. This 4.8 kb vector fragment vas ligated with a 3.2 kb Smal 20 cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990), resulting in plasmid pSD524KBG. pSD524KBG was used as donor plasmid for recombination vith vaccinia virus vP804. Recombinant vaccinia virus, vP855, containing Betagalactosidase in a partial deletion of the I4L gene, vas isolated as a blue plaque in the presence of X-gal.

To delete Beta-galactosidase and the remainder of the I4L ORF from VP855, deletion plasmid pSD548 was constructed. The left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and presented schematically in FIG. 14.

To construct a vector plasmid to accept the left vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518A1/518A2 (SEQ ID NO:37/SEQ ID NO:38) BamHI Rsal 518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT 518A2 3 * GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA Bglll EcoRI TTGAGAATAAAAAGATCTTAGG 3 ’ 518A1 AACTCTTATTTTTCTAGAATCCTTAA 5* 518A2 forming plasmid pSD53l. pSD531 vas cut vith Rsal (partial) and BamHI and a 2.7 kb vector fragment isolated. pSD5l8 was cut vith BolII (pos. 64,459)/ Rsal (pos. 64,994) and a 0.5 kb fragment isolated. The two fragments were ligated together, forming pSD537, vhich contains the complete vaccinia flanking arm left of the I4L coding sequences.

To construct a vector plasmid to accept the right • · vaccinia flanking arm, *pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID NO:39/SEQ ID NO:40) BamHI figlll Smal 518B1 5' GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAGGGATTT 518B2 3' GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATCCCTAAA Rsal EcoRI GACGTATGTAGCGTACTAGG 31 518B1 CTGCATACTACGCATGATCCTTAA 5 * 518B2 forming plasmid pSD532. pSD532 was cut with Rsal (partial) /EcoRI and a 2.7 kb vector fragment isolated. pSD518 was cut with Rsal within vaccinia sequences (pos. 67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kh fragment isolated. The tvo fragments were ligated together, forming pSD538, vhich contains the complete vaccinia flanking arm to the right of I4L coding sequences.

The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BolII fragment from pSD538 and ligated into pSD537 vector plasmid cut vith EcoRI/BolII. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a poly linker region, which is flanked by 0.6 kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right, all in a pUC background. The site of deletion within vaccinia sequences is indicated by a triangle in FIG. 14.

To avoid possible recombination of Beta-galactosidase sequences in the pVC-derived portion of pSD539 with Betagalactosidase sequences in recombinant vaccinia virus VP855, the vaccinia I4L deletion cassette vas moved from pSD539 5 into pRCll, a pUC derivative from which all Betagalactosidase sequences have been removed and replaced vith a polylinker region (Colinas et al., 1990). pSD539 was cut with EcoRI/Pstl and the 1.2 kb fragment isolated. This fragment vas ligated into pRCll cut with EcoRI/Pstl (2.35 kb), forming pSD548. Recombination between pSD548 and the Beta-galactosidase containing vaccinia recombinant, vP855, resulted in vaccinia deletion mutant VP866, which vas isolated as a clear plaque in the presence of X-gal.

DNA from recombinant vaccinia virus VP866 was analyzed by restriction digests followed by electrophoresis on an agarose gel. The restriction patterns were as 15 expected. Polymerase chain reactions (PCR) (Engelke et al., 1988) using VP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected. Recombinant 20 vaccinia virus VP866, containing the six engineered deletions as described above, was designated vaccinia vaccine strain NYVAC.

Example 16 - CONSTRUCTION OF NYVAC-MV RECOMBINANT EXPRESSING MEASLES FUSION AND HEMAGGLUTININ GLYCOPROTEINS cDNA copies of the sequences encoding the HA and F proteins of measles virus NV (Edmonston strain) vere inserted into NYVAC to create a double recombinant designated NYVAC-MV (VP913). The recombinant authentically expressed both measles glycoproteins on the surface of infected cells. Immunoprecipitation analysis demonstrated correct processing of both F and HA glycoproteins. The recombinant was also shown to induce syncytia formation.

Cells and Viruses The rescuing virus used in the production of NYVACMV was the modified Copenhagen strain of vaccinia virus designated NYVAC. All viruses were grown and titered on Vero cell monolayers.

Plasmid Construction Referring now to Fig. 15 and Taylor et al. (1991), plasmid pSPM2LHA contains the entire measles HA gene linked in a precise ATG to ATG configuration with the vaccinia virus H6 promoter vhich has been previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989). A 1.8kpb EcoRV/Smal fragment containing the 3' most 24 bp of the H6 promoter fused in a precise ATG;ATG configuration with the HA gene lacking the 3' most 26 bp was isolated from pSPM2LHA.· This fragment was used to replace the 1.8 kbp EcoRV/Sraal fragment of pSPMHAll (Taylor et al., 1991) to generate pRW803. Plasmid pRW803 contains the entire H6 promoter linked precisely to the entire measles HA gene.

Plasmid pSD513VCVQ was derived from plasmid pSD460 by the addition of polylinker sequences. Plasmid pSD460 was derived to enable deletion of the thymidine kinase gene from vaccinia virus (FIG. 9).

To insert the measles virus F gene into the HA insertion plasmid, manipulations were performed on pSPHMF7. Plasmid pSPHMF7 (Taylor et al., 1991) contains the-measles F gene juxtaposed 3' to the previously described vaccinia virus H6 promoter. In order to attain a perfect ATG for ATG configuration and remove intervening seguences between the 31 end of the promoter and the ATG of the measles F gene oligonucleotide directed mutagenesis vas performed using oligonucleotide SPMAD (SEQ ID NO:41).

SPMAD : 51 -TATCCGTTAAGTTTGTATCGTAATGGGTCTCAAGGTGAACGTCT-3 ’ The resultant plasmid was designated pSPMF75M20.

The plasmid pSPMF75M20 which contains the measles F gene nov linked in a precise ATG for ATG configuration with the H6 promoter vas digested vith Nrul and Eagl. The resulting 1.7 kbp blunt ended fragment containing the 3* 1 • 25 most 27 bp of the H6 promoter and the entire fusion gene was isolated and inserted into an intermediate plasmid pRW823 which had been digested with Nrul and Xbal and blunt ended. The resultant plasmid pRW841 contains the H6 promoter linked to the measles F gene in the pIBI25 plasmid vector (IBI, New Haven, CT). The H6/measles F cassette vas excised from pRW84l by digestion with Smal and the resulting 1.8 kb fragment vas inserted into pRW843 (containing the measles HA gene). Plasmid pRW843 was first digested with Notl and blunt-ended with Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs. The resulting plasmid, pRW857, therefore contains the measles virus F and HA genes linked in a tail to tail configuration. Both genes are linked to the vaccinia virus H6 promoter.

Development of NYVAC-MV.

Plasmid pRH857 vas .transfected into NYVAC (VP866) infected Vero cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plagues were selected on the basis of in situ plague hybridization to specific MV F and HA radiolabeled probes and subjected to 6 sequential rounds of plague purification until a pure population vas achieved. One representative plague was then amplified and the resulting recombinant was designated NYVAC-MV (vP913). Tianniwofiuoreseence Indirect immunofluorescence was performed as previously described (Taylor et al., 1990). Mono-specific reagents used were sera generated by inoculation of rabbits with canarypox recombinants expressing either the measles F or HA genes.

Immunoprecipitation Immunoprecipitation reactions were performed as previously described (Taylor et al., 1990) using a guineapig anti measles serum (Whittaker M.A. Bioproducts, Walkersville, MD).

Cell Fusion Experiments Vero cell monolayers in 60mm dishes were inoculated at a multiplicity .df 1 pfu per cell with parental or recombinant viruses. After 1 h absorption at 37°C the inoculum was removed, the overlay medium replaced and the dishes inoculated overnight at 37°C. At 20 h postinfection, dishes were examined.

In order to determine that the expression products of both measles virus F and HA genes were presented on the infected cell surface, indirect immunofluorescence analysis vas performed using mono-specific sera generated in rabbits against canarypox recombinants expressing either the measles F or HA genes. The results indicated that both F and HA gene products vere expressed on the infected cell surface, as demonstrated by strong surface fluorescence vith both mono-specific sera. No background staining was evident vith either sera on cells inoculated with the parental NYVAC strain, nor vas there cross-reactive staining when monospecific sera vere tested against vaccinia single recombinants expressing either the HA or F gene.

In order to demonstrate that the proteins expressed by NYVAC-MV vere immunoreactive with measles virus specific sera and were authentically processed in the infected cell, immunoprecipitation analysis was performed. Vero cell monolayers were inoculated at a multiplicity of 10 pfu/cell of parental or recombinant viruses ih the presence of 35Smethionine. Immunoprecipitation analysis revealed a HA glycoprotein of approximately 76 kDa and the cleaved fusion products F, and F2 vith molecular weights of 44 kDa and 23 kDa, respectively. No measles specific products vere detected in uninfected Vero cells or Vero cells infected with the parental NYVAC virus.

A characteristic of MV cytopathology is the formation of syncytia vhich arise by fusion of infected cells with surrounding infected or uninfected cells followed by migration of the nuclei toward the center of the syncytium (Norrby et al., 1982). This has been shown to be an important method of viral spread, which for Paramyxoviruses, can occur in the presence of HA-specific virus neutralizing antibody (Merz et al., 1980). In order to determine that the MV proteins expressed in vaccinia virus were functionally active, Vero cell monolayers were 5 inoculated with NYVAC and NYVAC-MV and observed for cytopathic effects. Strong cell fusing activity was evident in NYVAC-MV infected Vero cells at approximately 18 hours post infection. No.cell fusing activity was evident in cells infected with parental NYVAC.

Results of serological analysis of sera of rabbits 10 inoculated with NYVAC-MV (VP913) In this study, two rabbits were inoculated with lxio8 pfu of NYVAC-MV (vP913) by the subcutaneous route. At 28 days, animals were boosted with an equivalent dose. Serial bleeds were analyzed for MV neutralizing activity using the plaque reduction method. The results are shown in Table 13.

The results^indicate that neither rabbit responded to the initial inoculation of NYVAC-MV. However, the sharply rising response after the second inoculation indicates that the animals were primed. Both animals achieved neutralizing antibody titers in the protective range.

The in vivo analysis of immunogenicity of ALVAC-MV (VCP82) shown in Example 14 indicates that on inoculation of a range of species, the recombinant is able to induce a serological response which is measurable in standard serological tests. The titers achieved are in the range required for protection from disease. Inoculation of NYVACMV (vP9l3) into rabbits similarly induces a level of measles virus neutralizing antibody which would be protective.

Table 13 Anti-measles neutralizing antibody titers (log10) in sera of rabbits inoculated with NYVAC-MV (vP913) Animal Titer at weeks post-inoculation WO W2 W4C W5 W6 W7 Rabbit® All 6 <1 <1 <1 2.8b 2.2 2.2 A117 <1 <1 <1 1.9 1.9 1.9 a) Rabbits received 8.0 log10 pfu of NYVAC-MV (vP913) by S.C. route. b) Titer expressed as log10 of reciprocal of last dilution showing a 50% reduction in plague number as compared to prerinoculation serum. c) Animals were re-inoculated at 28 days.

REFERENCES 1. Adams, J.M., and D.T. Imagawa, Proc. Soc. Exper.

Biol. Med. 96, 240-244 (1957). · 2. Albrecht, P., K. Herman, and G.R. Burns, J. Virol. Methods 3, 251-260 (1981). 3. Alkhatib, G., and D. Briedis, Virology 150, 479-490 (1986). 4. Alkhatib, G., C. Richardson, and S-H. Shen, Virology 175, 262-270 (1990).

. Appel, M.J.G., and O.R. Jones, Proc. Soc. Exp. Biol. Med'. 126, 571-574 (1967). 6. Appel, M.J.G., and D.S. Robson, Am. J. Vet. Res. 34, 1459-1463 (1973). 7. Avery, R.J., and J. Niven, Infect. Iramun. 26, 795801 (1979). 8. Baker, J.A., B.E. Sheffy, D.S. Robson, J. Gilmartin, Cornell Vet (USA, 56, 588-594 (1966). 9. Bertholet, C., R. Drillien, and R. Hittek, Proc. Natl. Acad. Sci. USA 82, 2096-2100 (1985).

. Bestetti, G., R. Fatzer, and R. Frankhauser, Acta Neuropathol. 43, 69-75 (1978). 11. Black, F.L., L.L. Berman, M. Libel, C.A. Reichelt, F. de P. Pinheiro, A.T. da Rosa, F. Figuera, and E.S. Gonzales, Bull, W.H.O. 62, 315-319 (1984). 12. Bush, M., R.J. Montali, D. Brownstein, A.E. James, Jr., and M.J.G. Appel, J. Am. Vet. Med. Assoc. 169, 959-960 (1976). 13. Carpenter, J.W., M.J.G. Appel, R.C. Erickson, and M.N. Novilla, J. Am. Vet. Med. Assoc. 169, 961-964 (1976). 14. Choppin, P.W., C.D. Richardson, D.C. Merz, W.W.

Hall, and A. Scheid, J. Infect. Dis. 143, 352-363 (1981).

. Clewell, D.B., J. Bacteriol. 110, 667-676 (1972). 16. Clewell, D.B., and D.R. Helinski, Proc. Natl. Acad. Sci. USA 62, 1159-1166 (1969). 17. Colinas, R.J., R.C. Condit, and E. Paoletti, Virus Research 18, 49-70 (1990). 18. DeLay, P.D., S.S. Stone, D.T. Karzon, S. Katz, and J. Enders, Am. J. Vet. Res. 26, 1359-1373 (1965). 19. Dialloj A., Vet. Micro. 23, 155-163 (1990).

. Dowling, P.C., B.M. Blumberg, J. Menonna, j.E. Adamus, P. Cook, J.C. Crowley, D. Kolakofsky, and S.D. Cook, J. Gen. Virol. 67, 1987-1992 1 (1986). 21. Drillien, R., D. Spehner, A. Kirn, P. Giraudon, R. Buckland, F. Wild, and J.P. Lecocg, Proc. Natl.

Acad. Sci. USA 85, 1252-1256 (1988). 22. Engelke, D.R., P.A. Hoener, and F.S. Collins, Proc. Natl. Acad. Sci. USA 85, 544-548 (1988). 23. Fenner, F., P.A. Bachmann, E.P.J. Gibbs, F.A.

Murphy, M.J. Studdert, and D.O. White, In Veterinary Virology, ed. F. Fenner, (Academic Press, Inc., Nev York) pp. 485-503 (1987). 24. Gillespie,.-J.H., and D.T. Karzon, Proc. Soc. Exp. Biol Med. 105, 547-551 (1960).

. Giraudon, P., Ch. Gerald, and T.F. Wild, Intervirology 21, 110-120 (1984). 26. Goebel, S.J., G.P. Johnson, M.E. Perkus, S.W. Davis, J.P. Winslow, and £. Paoletti, Virology 179, 247-266 (1990a). 27. Goebel, S.J., G.P. Johnson, M.E. Perkus, S.W. Davis, J.P. Winslow, and E. Paoletti, Virology 179, 517-563 (1990b). 28. Graves, M.C., S.M. Silver, and P.W. Choppin, Virology 86, 254-263 (1978). 29. Guo, P., S. Goebel, S. Davis, M.E. Perkus, B. Languet, P. Desmettre, G. Allen, and E. Paoletti, J. Virol. 63, 4189-4198 (1989).

. Guo, P., S. Goebel, S. Davis, M.E. Perkus, J.

Taylor, E. Norton, G. Allen, B. Languet, P. Desmettre, and E. Paoletti, J. Virol. 64, 2399-2406 (1990). 31. Hall, W.W., R.A. Lamb, and P.W. Choppin, Virology 100, 433-449 (1980). 32. Hartley, W.J., Vet. Path. 11, 301-312 (1974). 33. Imagava, D.T., P. Goret, and J.M. Adams, Proc. Natl. Acad. Sci. USA 46, 1119-1123 (1960). 34. Karzon, D.T., Pediatrics 16, 809-818 (1955).

. Karzon, D.T., Annals of the N.Y. Academy of Sci. 101, 527-539 (1962). 36. Kazacos, K.;R., H.L. Thacker, H.L. Shivaprasad, and P.P. Burger, J. Am. Vet. Med. Assoc. 179, 1166-1169 (1981). 37. Kingsbury, D.W., M.A. Bratt, P.W. Choppin, R.P. Hanson, T. Hosaka, V. ter Meulen, E. Norrby, W. Plowright, R. Rott, and W.H. Wunner, Intervirology 10, 137-152 (1978). 38. Kunkel, T.A., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985). 39. Lennon, J.L., and F.L. Black, J. Ped. 108, 671-676 (1986). 40. Mandecki, W., Proc. Natl. Acad. Sci. USA 83, 7177-7181 (1982). 41. Mandecki, W., Proc. Natl. Acad. Sci. USA 83, 7177-7181 (1986). 42. Maniatis, T., E.F. Fritsch, and J. Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory, NY 545 pages (1982). 43. Maniatis, T., E.F. Fritsch, and J. Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory, NY 545 pages (1986). 44. Merz, D.C., A. Schied, and P. Choppin, J. Exp. Med. 151, 275-288 (1980). 45. Moura, R.A., and J. Warren, J. Bact. 82, 702-705 (1961). 46. Norrby, E., and Y. Gollmar, Infect. Immun. ll, 231239 (1975). 47. Norrby, E., G. Enders-Ruckle, and V. ter Meulen, J. Infect. Dis. 132, 262-269 (1975). 48. Norrby, E., S.N. Chen, T. Togashi, H. Shesberadaran, and K.P. Johnson, Archives of Virology 71, 1-11 (1982). 49. Norrby, E., and M.N. Oxman, In Fields Virology 2nd Ed., B.N. Fields and D.M. Knipe, eds. (Raven Press, NY) pp. 1013-1044 (1990). 50. Novick, S.L. and D. Hoekstra, Proc. Natl. Acad. Sci. USA 85, 7433-7437 (1988). 51. Orvell, c., and E. Norrby, J. Gen. Virol. 50, 231245 (1980).

Panicali, 0., and E. Paoletti, Proc. Natl. Acad.

Sci. USA 79, 4927-4931 (1982).

Paterson, &.G., and R.A. Lamb, Cell 48, 441-452 (1987).

Perkus, M.E., A. Piccini, B.R. Lipinskas, and E. Paoletti, Science 229, 981-984 (1985).

Perkus, M.E., K. Limbach, and E. Paoletti, J. Virol. 63, 3829-3836 (1989).

Perkus, M.E., S.J. Goebel, s.W. Davis, G.P Johnson, K. Limbach, E.K. Norton, and E. Paoletti, Virology 179, 276-286 (1990).

Phillips, T.R., J.L. Jensen, M.J. Rubino, W.C. Yang, and R.D. Schultz, Can. J. Vet. Res. 53, 154-160 (1989).

Piccini, A., M.E. Perkus, and E. Paoletti, In Methods in Enzymology, Vol. 153, eds. Wu, R., and Grossman, L., (Academic Press) pp. 545-563 (1987).

Preblud, S.R., and S.L. Katz, In Vaccines, eds. S.A. Plotkin and E.A. Mortimer, (W.B. Saunders Co.) pp. 182-222 (1988).

Richardson, C.D., A. Berkovich, S. Rozenblatt, and W. Bellini, J. Virol. 54, 186-193 (1985).

Richardson, C., D. Hull, P. Greer, K. Hasel, A. Berkovich, G. Englund, W. Bellini, B. Rima, and R. Lazzarini, Virology 155, 508-523 (1986).

Roberts, J.A., J. Immunol. 94, 622-628 (1965).

Rosel, J.L., P.L. Earl, J.P. Weir, and B. Moss, J. Virol. 60, 436-449 (1986).

Sanger, F., S. Nicklen, and A.R. Coulson, Proc.

Natl. Acad. sci. USA 74, 5463-5467 (1977).

Shapira, S.K., J. Chou, F.V. Richaud, and M.J. Casadaban, Gene 25, 71-82 (1983).

Spehner, D., R. Drillien, and J.P. Lecocq, J. Virol. 64, 527-533 (1990).

Stephenson, J.R. and V. ter Meulen, Proc. Nat. Acad. Sci. USA 76, 6601-6605 (1979).

Tabor, S., and C.C. Richardson, Proc. Natl. Acad. Sci. USA 84, 4767-4771 (1987).

Taylor, J., R. Weinberg, Y. Kawaoka, R.G. Webster, and E. Paoletti, Vaccine 6, 504-508 (1988a). 6» 70. Taylor, J., R. Weinberg, B. Languet, P. Desmettre, and E. Paoletti, Vaccine 6, 497-503 (1988b). 71. Taylor, J.., $C. Edbauer, A. Rey-Senelonge, J.F. Bouquet, E. Norton, S. Goebel, P. Desmettre, and E. Paoletti, J. Virol. 64, 1441-1450 (1990). 72. Taylor, J., S. Pincus, J. Tartaglia, C. Richardson, G. Alkhatib, D. Briedis, M. Appel, E. Norton, and E Paoletti, J. Virol. 65, 4263-4272 (1991). 73. Tizard, I., J. Am. Vet. Med. Assoc. 196, 1851-1858 (1990). 10 74. Vialard, J., M. Lalumiere, T. Vernet, D. Briedis, G Alkhatib, D. Henning, D. Levin, and C. Richardson, J. Virol. 64, 37-50 (1990). 75. Warren, J., M.K. Nadel, E. Slater, and S.J. Millian, Amer. J. Vet. Res. 21, 111-119 (1960). 76. Wild, T.F., E. Malvoisin, and R. Buckland, J. Gen. Virol. 72, 439-442 (1991). 15 77. Wild, F., P. Giraudon, D. Spehner, R. Drillien, and J-P. Lecocq, Vaccine 8, 441-442 (1990). 78. Yuen, L., and B. Moss, Proc. Natl. Acad. Sci. USA 84, 6417-6421 (1987).

Claims (13)

1. A recombinant poxvirus containing therein exogenous DNA from Morbillivirus in a nonessential region of the poxvirus genome, for use in protecting a dog against canine distemper, wherein the exogenous Morbillivirus DNA codes for an antigen and the poxvirus is a modified vaccinia virus having at least the following open reading frames deleted therefrom: a thymidine kinase gene, a haemorrhagic gene region, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region, and a large subunit, ribonucleotide reductase gene.

2. A recombinant poxvirus for the use according to claim 1 wherein the exogenous Morbillivirus DNA is exogenous measles virus DNA.

3. A recombinant poxvirus for the use according to claim 2 wherein the exogenous Morbillivirus DNA codes for a measles virus glycoprotein, a measles virus haemagglutinin glycoprotein, or a measles virus fusion glycoprotein.

4. A recombinant poxvirus for the use according to claim 3 wherein the exogenous Morbillivirus DNA codes for two measles virus glycoproteins, preferably a haemagglutinin glycoprotein and a fusion glycoprotein.

5. A recombinant poxvirus for the use according to any one of claims 1 to 4 wherein the exogenous Morbillivirus DNA is from the Edmonston strain of measles virus.

6. A recombinant poxvirus for the use according to any one of claims I to 5 wherein the exogenous Morbillivirus DNA is introduced into the poxvirus by recombination.

7. A recombinant poxvirus for the use according to any one of claims 1 to 6 wherein the exogenous Morbillivirus DNA further indudes a promoter for expressing the antigen.

8. A recombinant poxvirus for the use according to claim 7 wherein the promoter is an H6 promoter.

9. -A recombinant poxvirus for the use according to any one of claims 1 to 8 wherein the deleted open reading frames comprise C7L-K1L, J2R, B13R+B14R, A26L, A56L, A56R and I4L.

10. A recombinant poxvirus for the use according to claim 9 wherein the poxvirus is the NYVAC vaccinia virus.

11. A recombinant poxvirus for the use according to claim 1 wherein the recombinant poxvirus is vP913.

12. A recombinant poxvirus for the use according to any one of the preceding claims when admixed with a vaccine carrier.

13. Use of a recombinant poxvirus containing therein exogenous DNA from Morbillivinis in a nonessential region of the poxvirus genome for the preparation of a medicament for protecting a dog against canine distemper, wherein the exogenous Morbillivinis DNA codes for an antigen and the poxvirus is a modified vaccinia virus having at least the following open reading frames deleted therefrom: a thymidine kinase gene, a haemorrhagic gene region, an A type inclusion body gene region, a haemagglutinin gene, a host range gene region, and a large subunit, ribonucleotide reductase gene.