IE68404B1 - Measles virus recombinant poxvirus vaccine - 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

MEASLES VIRUS RECOMBINANT POXVIRUS VACCINE CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of copending application Serial No. 07/621,614 filed November 5 20, 1990.

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, which virus expresses gene products of a Morbillivirus gene, and to vaccines which provide protective immunity against Morbillivirus infections.

Several publications are referenced in this application by arabic numerals within 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 which 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 between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini etal., 1987).

Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the 30 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 E. coli plasmid construct into which 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 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 with 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 between 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 between 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 within 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., 1960). 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), fowlpox 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., 1990) 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 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 Kilt 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 al., 1988b). This recombinant virus is also described in PCT Publication No. WO89/03429. 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 controlled by the use of live attenuated vaccines (Fenner et al., 1987; Preblud et al., 1988). immunization is β 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 within the vaccinated animals. These deleterious effects are most notable when the CDV vaccine strain is co-inoculated with canine adenovirus 1 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 means of overcoming the documented inhibitory effect that maternal antibody has on vaccination with presently utilized •Ts 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 harboring NV 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 Morbillivirus recombinant poxvirus, and of vaccines which provide protective immunity against Morbillivirus infections, would be a highly desirable advance over the current state of technology.

OBJECTS OP THE INVENTION It is therefore an object of this invention to provide recombinant poxviruses, which viruses express gene products of Morbilliviruses, and. to provide a method of making such recombinant poxviruses.

It is an additional object of this invention to provide for the cloning and expression of Morbillivirus coding sequences, particularly measles virus coding sequences, in a poxvirus vector, particularly vaccinia virus vectors.

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

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

According to a first aspect of the present invention there is provided a recombinant poxvirus comprising in a nonessential region of its genome exogenous Morbillivirus DNA coding for an antigen characterised in that 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 a second aspect of the present invention there is provided a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, the vaccine comprising a carrier and a recombinant poxvirus according to the first aspect of the present invention.

The present Invention therefore relates to a recombinant poxvirus as just defined which contains therein a DNA sequence from Morbillivirus in a nonessential region of the poxvirus genome.

The poxvirus is a vaccinia virus. The Morbillivirus is advantageously measles virus.

According to the present invention, the recombinant poxvirus expresses gene products of the foreign Morbillivirus gene. In particular, the foreign DNA 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.

The present invention also relates to a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine including a carrier and a recombinant poxvirus according to the present invention which contains, in a 2Q nonessential region thereof, DNA from Morbillivirus, particularly measles virus. Advantageously, the DNA codes for and expresses a measles virus glycoprotein, particularly measles virus hemagglutinin glycoprotein and measles virus fusion glycoprotein. A plurality of measles virus glycoproteins advantageously are co-expressed in the host. The poxvirus used in the vaccine according to the present invention is a vaccinia virus.

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; ο FIG. 2 schematically shows a method for the construction of plasmid pSPMFVC used to derive recombinant vaccinia virus VP4S5 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 pRW8io used to derive recombinant canarypox viruses VCP50 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 pRH852 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; 1 FIG. 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; FIG. 14 schematically shows a method for the 5 construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant vaccinia virus vP866 (NYVAC, ; and FIG. 15 schematically shows a method for the construction of plasmid pRW857 used to derive recombinant NYVAC -|0 virus vP913 co-expressing the MV hemagglutinin and fusion genes. DETAILED DESCRIPTION OF THE INVENTION 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, 14 and 16 are only for information purposes. 2 Example 1 - GENERATION OF VACCINIA VIRUS RECOMBINANTS CONTAINING THE MEASLES HEMAGGLUTININ GENE The rescuing virus used in the production of both recombinants was the Copenhagen strain of vaccinia virus from 5 ' 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 10 al., 1986; Taylor et al., 1988a,b) was constructed by annealing four overlapping oligonucleotides, H6SYN A-D. The resultant H6 sequence is as follows: Vaccinia Virus H6 Promoter (SEQ ID NO:1/SEQ ID NO:2): HindiII · AGCTTCTTTATTCTATACTTAAAAAGTGAAAATAAATACAAAGGTTCTTGAGG GTTGT AGAAATAAGATATGAATTTTTCACTTTTATTTATGTTTCCAAGAACTCCCAACA 2 0 GTTAAATTGAAAGCGAGAAATAATCATAAATTATTTCATTATCGCGATATCCGTT AAGTT CAATTTAACTTTCGCTCTTTATTAGTATTTAATAAAGTAATAGCGCTATAGGCAA TTCAA 3 TGTATCGTAC-3' ACATAGCATGAGCT-5' Xhoi Referring now to Figure 1, the annealed H6SYN oligonucleotides were ligated into pMP2LVC digested with Xhol/HindHI to yield plasmid pSP131. The plasmid pMP2LVC 10 contains the leftmost 0.4kbp of the vaccinia virus (Copenhagen strain) HindHI K region within pUCl8. The construction of pMP2LVC was performed as follows: a 0.4kbp HindHI/Sail fragment from the HindHI K region was 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 pUCl8 which had been digested with Pvull. The resulting plasmid was designated pMP2VC. The plasmid pHP2VC 20 was linearized with Ssol. Synthetic oligonucleotides MPSYN52 (SEQ ID NO:3) (5 *-ATTATTTTTATAAGCTTGGATCCCTCGAGGGTACCCCCGGGGAGCTCGAATTCT-3') and MPSYN53 (SEQ ID NO:4) (5*25 AGAATTCGAGCTCCCCGGGGGTACCCTCGAGGGATCCAAGCTTATAAAAATAAT-3 *) were annealed and inserted into the leftmost of the two SspI sites located within 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*35 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 ligated to a 1.8kbp Xhol/Smal fragment from pMH22 containing 4 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 with mung bean nuclease. Insertion into this vector yielded plasmid pSPMHA41. 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 with vaccinia virus vP458 as the rescue virus to generate recombinant vP557. VP458 contains the £. 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 31 end of the measles fusion gene, a vaccinia virus early transcription termination signal (Yuen et al., 1987) and Eagl and Hindlll ends were ligated to a lkbp Sall/HaelH fragment from pCRF2 (obtained from C. Richardson, National Research Council of Canada (Biotechnology Institute), Montreal, Canada H3A 1A1) and pUC8 digested with Sail and Hindlll. The resulting plasmid pMF3PRl4 contains the 3' end of the lkbp fragment of the measles fusion gene.

-Annealed oligonucleotides 5PA (SEQ ID NO:10) (5'GGGATGGGTCTCAAGGTGAACGTCTCTGCCATATTC-3 ·) and 5PB (SEQ ID NO:11) (5'-ATGGCAGAGACGTTCACCTTGAGACCCATCCC-3'), containing a 5* Smal site and a 3' BstXI site, were ligated to a 820bp gstXI/S&lI fragment from pCRF2 and pUC8 digested with Smal and Sail. The resultant plasmid pSPMF5P16 contains the 51 portion of the measles fusion gene. The 820bp Smal/Sail fragment from pSPMF5P16 and the lkbp Sail/Eagl fragment from PMF3PR14 were ligated into pTPl5 digested with Smal and Eagl. The plasmid pTP15 (Guo et al., 1989) contains the vaccinia virus early/late H6 promoter flanked by sequences from the HA locus of the vaccinia virus (Copenhagen strain) genome. The resultant plasmid containing the measles fusion gene juxtaposed 3' to the H6 promoter within the HA insertion plasmid was designated pSPHMF7.

Oligonucleotide directed mutagenesis was performed on pSPHMF7. Initially an in vitro mutagenesis reaction (Mandecki, 1982) was performed to create a precise ATG:ATG I 6 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-TTGTATGGTAATGGGTCTCAAGGTGAACGTCT’ 3'). This resulted in the generation of pSPMF75M20.

Subsequently, the Bglll site at the 5' end of the H6. promoter was removed using oligonucleotide SPBGLD (SEQ ID NO:13) (5'-AATAAATCACTTTTTATACTAATTCTTTATTCTATACTTLO AAAAAGT-3’) according to a known procedure (Mandecki, 1982). The resultant plasmid was designated pSPMFVC. This plasmid was used in in vitro recombination experiments with vaccinia 15 virus vP410 as rescue virus to generate vP455.

Example 3 - IMMPNOPRECIPITATION 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 25 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 with the HA recombinant vP557. In cells infected with the fusion recombinant vP455, the fusion precursor Fo with a molecular weight of approximately 60 kd 7 and the two cleavage products F1 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 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 hemagglutinin specific antibody (Merz et al., 1980). This ability has been assigned by analogy with other Paramyxoviruses to the k amino terminus of the Fl 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 37eC 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 8 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 which 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 NV. The results of the serological tests are shown in Table 1.

I 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 NV. 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.

Tabla 1 Measles virus neutralizing antibody titers in response to vaccination Days past vaccination Immunization Dog No. 0’ 7 14 21b 28 35e Vacc. 4/1 4/2 <1.0 <1.0 <1.0 <1.0 <1.0 <1.0 <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 & 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 Λ5 © a) Time of first immunization b) Time of second immunization (first immunization with MV) c) Time of challenge d) Titer expressed as log10 of last antibody dilution showing complete neutralization of infectivity in a raicrotiter neutralization test as described by Appel et al. (1973) .

V 1 Example 6 - 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 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 two injections three weeks apart. The first group received vaccinia virus only. The 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 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 Rockbom strain of CDV subcutaneously two weeks before challenge with virulent CDV. Two control dogs remained uninoculated before challenge.

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 were 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 of dogs tovirulent CBY No. of days after inoculation with virulent CDV Immunization Dog Number Deoression Height Loss Elevated Bodv TemD." Lympho- neniab Virus Isolation6 Death Vacc. 4/1 4-10d 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 — 4/6 ND ND ND ND ND — VP455 & VP557 4/7 ND ND 7 7 7 M 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 — None 4/18 6,14-17d 7-17 5,7 13-17 10 17 4/19 4-10d 3-10 4,5,7 3,7,10 3-10 10 2o a) Above 39.5°C b) Less than 2xlO3 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 3 Non-immunized control dogs and dogs vaccinated with parental vaccinia virus developed clinical signs of severe disease and were euthanized when dehydration was evident. Both dogs immunized with vP455 showed 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 with both recombinants showed minimal signs of infection and survived challenge. Dogs inoculated with either attenuated Edmonston strain of MV 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 with the HA gene lacking the 3'-most 26bp was isolated from pSPM2LHAVC. This fragment was used to replace the 1.8kbp EcoRV/Smal fragment of pSPMHAll to generate pRN803. Plasmid pRW803 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) was 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 pRW819 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 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.Skbp 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 pRW820 resulting in the generation of pRW837.

The mutagenized expression cassette contained within pRW837 was derived by digestion with Hindlll and EcoRI. blunt-ended using the Klenow fragment of E. 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 by immunoprecipitation analysis to correctly express a hemagglutinin glycoprotein of approximately 75kd.

Referring now to Figure 4, a second vaccinia virus recombinant (vP800) harboring the measles fusion gene in the ATI locus of the genome was generated using insertion plasmid pRW850. To construct pRW850, the following manipulations were performed. The plasmid pSPHF75N20 containing the measles fusion gene linked in a precise ATG:ATG configuration with the H6 promotet was digested with Nrul and Eacrl. 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 with 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 was derived from pRW841 by digestion with Smal and the resulting l.Skbp fragment was inserted into pSD494VC digested with Smal to yield pRW850. The plasmid pRW850 was 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 VP455 Two rabbits were inoculated intradermally at 5 sites with a total of lxio8 pfu of recombinant VP455 expressing the measles fusion protein. Both rabbits were boosted with 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 1x10® 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 Aniwal Guinea Pig 1 ! N.D. N.D. N.D.-.8b 1.3-1.3 1.3-1.5 1.3 N.T*5 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 c) Not tested Example 9 GENERATION OP MEASLES VIRUS RECOMBINANT RYPOX 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 l.Skbp blunt-ended Belli/Eaal fragment from pSPMF75M20 containing the H6 promoted measles F gene was inserted into the blunt-ended EcoRI site of pRW764.2. Plasmid pRW764.2 contains a 3.4kbp Pyull 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 was used in recombination experiments with canarypox as the rescuing virus to generate VCP40.

Referring now to Figure 6, the l.Skbp 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/EcoRl fragment of pRW803 containing the H6 promoted measles HA gene was blunt-ended and inserted into the bluntended Belli site of plasmid pRW764.5. Plasmid pRW764.5 contains an 800bp Pvuli fragment of the canarypox genome having a unique Belli site which 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, VCP50 and vCP57 expressed authentic proteins, immunoprecipitation analysis-was performed using mono-specific sera directed against either the HA or F proteins. A correctly processed fusion polypeptide was specifically precipitated from lysates of cells infected with VCP40 and VCP57. The fusion precursor Fo with a molecular weight of approximately 60kd and the two cleavage products F, and Fz with molecular weights of approximately 44 and 23kd, respectively, were detected. No fusion specific products were detectable in uninfected CEF cells, parentally infected CEF cells or CEF cells infected with the HA recombinant vCP50. Similarly, a glycoprotein of approximately 75kd was specifically precipitated from CEF cells infected with the single HA recombinant VCP50 and double recombinant VCP57. No HA specific products were detected in uninfected cells, parentally infected cells or cells infected with fusion recombinant vCP40.

Example ii - cell fusion experiments In order to determine that the measles virus recombinants were functionally active, cell fusion assays were 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 was evident in VERO cells inoculated with parental, vCP40 of VCP50 viruses. However, when VERO cells were inoculated with the double recombinant VCP57 or when cells are efficient cell Example 12 Dogs the canarypox/HA recombinant VCP50, 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 inoculation. Neither of the two dogs inoculated with the canarypox/F recombinant vCP40 developed neutralizing co-xnfected with both vCP40 and vCPSO, fusing activity is evident.

SEROLOGICAL TESTS inoculated as described in Example 13 with 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 Mo. 0’ 7 14 216 28 35* 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/ 4 <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 & 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 CDV-Ro ..-9/15 <1.0 <1.0 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 5 induce a protective immune response against CDV challenge, ten week old specific pathogen free beagle dogs were 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 with each of the single vaccinia virus recombinants vP455 and vP557 and a combination of both. One dog was also inoculated intramuscularly with 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 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 after exposure of dogs to virulent,CDV.

No, of davs after inoculation with virulent CDV Immunization Doa No. Deoression Elevated Wt. Loss Lympho Virus Bodv TemD.* Deniab Isolation1 Canary pox 9/ 1 4-10d 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 VCP50 9/ 3 ND* 7-10 5-7 5-7 7 9/ 4 ND 7 6,7 7 ND 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/ 8 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 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 4-10d 3-10 4-6 5-10 5-10 9/14 4-10d 3-10 4-5 3-10 5-7 CDV-Ro 9/15 ND ND ND ND ND ajAbove 39.5°C.b)Less than 2x10* Lymphocytes per mm3, o)Isolated from buffy 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 with 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 with 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 following insertion plasmids were created. A l.8kbp EcoRV/EcoRI fragment from pRW837 containing the 3'-most 26bp of the H6 promoter linked precisely to the measles HA, was ligated to a 3.2kbp EcoRV/EcoRI 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 Pvull canarypox genomic fragment was inserted between the Pvull sites of pUC9. the resultant plasmid was designated pRW764.5. 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 £s^i linear fragment was recut with BolII 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 N0:16): (51GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT-3 *) Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW764.5 Rsal-BolII vector described above. The resulting plasmid is pRW831.

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 RW146) which include the restriction sites for Hindlll. Smal. and EcoRI.

The plasmid pRW838, was derived from pRW831 by the insertion of a Sjnal 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 pRW837 with 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 to yield vCP85. ALVAC is a plague 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 with recombinant VCP85.

Referring now to Figure 8, to generate a canarypox virus recombinant harboring both the MV HA and F genes the following constructs were engineered. Smal restriction sites were added to the ends of the H6 promoted measles fusion gene. To accomplish this, pRW823, which is plBI25 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 Eagl/Nrul fragment from pSPMF75. The resultant plasmid was designated as pRW841.

The 1.8kbp Smal fragment derived by digestion of pRW841 was inserted into the C5 deletion vector, pRW831.

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 Hindlll and EcoRI and blunt-ended with the Klenow fragment. The resultant l.Skbp fragment was isolated and inserted into pRW85l that had been linearized with EcoRI and blunt-ended. The resultant plasmid, which contains both genes in a tail to tail configuration, was 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 were expressed.

The recombinant was also functional for cell fusing activity.

Results of serological analysis of sera of rabbits and guinea pics 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 1x10® pfu and two animals (028 and 029) each received lxio7 pfu. At 28 days, animals were re-inoculated with an identical dose. Two rabbits were inoculated with 1X10® 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 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 1x10* pfu of ALVAC-MV sero-converted after a single inoculation and sera showed an antibody rise after the booster inoculation. One animal (029) receiving lxlO7 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. It 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 which did not sero-convert until the second inoculation showed a protective level of antibody after one inoculation. v Table β 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 28c 42 48 56 Guinea pigs 02 6d - 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 028e - 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 log1Q of reciprocal of last dilution showing complete neutralization of cytopathic effect.

I c) Animals boosted at 28 days post-inoculation. d) Animals 026 and 027 received lxio8 pfu. e) Animals 028 and 029 received lxio7 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 reduction method 063 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 logw of reciprocal of last dilution showing a 50% reduction in plague number as compared to pre-inoculation serum. b) Animals boosted at 28 days post-inoculation. c) Titer expressed as logw of reciprocal of last dilution showing complete neutralization of cytophatic effect.

Previous studies have shown that an inactivated vaccine was associated with poor protective efficacy and an enhanced measles disease 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. 0 Table 8 Analysis of sera of guinea pigs and rabbits inoculated with ALVAC-MV for anti-fusion activity Animal Designation Immunogen Anti-Fusion Titer Pre-inoc. Post-Vacc.

Guinea-pig 026 027 ALVAC-MV ALVAC-MV - 2.4*' b 1.2 Rabbit 063 ALVAC-MV — 1.8C 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 with 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 with 4.8 log10 TCID5o, and five rabbits were inoculated with .8 log10 TCIDS0. All animals were re-inoculated at 28 days with an equivalent dose. Animals were 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 showed 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 was 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. Four of five animals sero-converted 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. 3 Table 9 Serological response of mice to inoculation with ALVAC-MV (VCP82) Anti-canarvpox response ELISA TITER Week post-inoculation Mouse Group 0 2 4 5 6 8 1* -0.0098 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 log10 titer of 1 i.e. 1:10 dilution. Titer expressed as logv of reciprocal of highest dilution showing inhibition of hemagglutination.

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 0.038’ 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 a) Optical density in an ELISA assay on serum at a 1:3200 dilution. b) 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 ii Serological response of rabbits to inoculation with ALVAC-MV (VCP82) Anti-canarvoox 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 response HI TITER Rabbit 0 Week post-inoculation 8 2 4 5 6 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 dilution of 1:1600. b) 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): 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 shown in Table 12. All monkeys were 10 inoculated with one dose of 5.8 log10 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 was 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 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. 7 Table 12 Inoculation of squirrel monkeys with ALVAC-MV (vCP82,: Immune response in the face of pre-existing ALVAC immunity.

Monkey # Previous Immunity Anti-Measles HI response to Poxviruses Primary® 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. 10 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. 15 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) Animals received 5.8 log10 pfu by s.c. route. b) Animals 39, 42, 52 and 53 were boosted with an identical dose 15 weeks after the first inoculation. 8 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 (g; 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).

DNA Cloning and Synthesis Plasmids were 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. Klenow fragment of E. coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as specified by the various suppliers.

Synthetic oligodeoxyribonucleotides were 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 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 digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 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 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 vaccinia Eindlll 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 within an Nlalll site and the termination codon is contained within an Ssol 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 Nlalll and a 0.5 kb Hindlll/Nlalll fragment isolated. Annealed synthetic oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:17/SEQ ID NO:18) Smal HPSYN43 5' TAATTAACTAGCTACCCGGG 3* MPSYN44 3· GTACATTAATTGATCGATGGGCCCTTAA 5' Elgin EcoRI were ligated with the 0.5 kb Hindlll/Nlalll fragment into pUC18 vector plasmid cut with Hlndlll/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 Hindlll 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) Hindlll SffiAl MPSYN45 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 with 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. 32P labeled probe was synthesized by primer extension using MPSYN45 (SEQ ID NO :19) as template and the complementary 2 Omer 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 CB13R + B14R? Referring now to FIG. 10, plasmid pSD4l9 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 £ 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 NcoI/Smal followed by blunt ending with Klenow fragment of E. coli polymerase and ligation generating plasmid pSD476. A vaccinia right flanking am was obtained by digestion of pSD422 with Hpal at the temination 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 were removed by digestion with clal/Hpal. and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ZD NO:22/SEQ ID NO:23) Clal BamHI Hpal SD22mer 5' CGATTACTATGAAGGATCCGTT 3* SD20mer 3' TAATGATACTTCCTAGGCAA 5* 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 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/Hoal vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:24/SEQ ID NO:25) CIbI Sacl Xhol Hoal SD42mer 5' CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3' SD40mer 3' TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5' Bglll 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 Hoa! and ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ ID NO:26/SEQ ID NO:27) BamHi EcoRI Hoal HEM5 5' GATCCGAATTCTAGCT, 3’ HEM6 3' GCTTAAGATCGA 5' generating, plasmid pSD486. pSD486 was used as donor plasmid for recombination with recombinant vaccinia virus vP533, generating vP553, which was isolated as a clear plague in the presence of X-gal.

Construction of Plasmid ΡΜΡ494Δ for Deletion of ATI Region 1A26.D Referring now to FIG. 11, pSD4l4 contains Sail B cloned into pUC8. To remove unwanted DNA seguences to the left of the A26L region, pSD4l4 was cut with Xbal within vaccinia seguences (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 seguences to the right of the A26L region, pSD483 was cut with £coRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding region, pSD484 was cut with Ndel (partial) slightly upstream from the A26L ORF (pos. 139,004) and with Hoal (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) 3 Ndel ATI3 5’ TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT ATI4 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA fiSlII EcoRI Hpal TATATAAATAGATCTGAATTCGTT 3 ' ATI 3 ATATATTTATCTAGACTTAAGCAA 5 * ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region containing the restriction sites BolII. EcoRI and Hpal. as indicated above. The resulting plasmid was designated pSD485. Since the Belli and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted BolII and EcoRI sites were removed from plasmid pSD483 (described above) by digestion with BolII (pos. 140,136) and with EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenow 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 pSO489 containing the A26L ORF was replaced with the corresponding 0.7 kb polylinkercontaining Clal/EcoRV fragment from pSD485, generating pSD492. The BolII and EcoRI sites in the polylinker region of pSD492 are unique.

A 3.3 kb Ball I cassette containing the E. coli Betagalactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990) was inserted into the BolII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus vP553. Recombinant vaccinia virus, vP58l, 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 vP581, 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 (A56R1 Referring now to FIG. 12, vaccinia Sail G restriction fragment (pos. 160,744-173,351) crosses the Hindlll A/B junction (pos. 162,539). pSD419 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 by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to the right. A56R coding sequences were removed by cutting pSD456 with Rsal (partial; pos. 161,090) upstream from A56R coding sequences, and with Eaal (pos. 162,054) near the end of the gene. The 3.6 kb Rsal/Eaal vector fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:31), MPSY62 (SEQ ID NO:32), MPSYN60 (SEQ ID NO:33), and MPSYN 61 (SEQ ID NO:34) Rsal MPSYN59 5’ ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGTAGTTGATAGAMPSYN62 3* TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCATCAACTATCT 5’ MPSYN59 -ACAAAATACATAATTT’ Bglll MPSYN60 5’ TGTAAAAATAAATCACTTTTTATACTAAGATCTMPSYN61 3’ TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATATGATTCTAGASmal EsJl Eagl MPSYN60 -CCCGGGCTGCAGC 3’ MPSYN61 -GGGCCCGACGTCGCCGG 5’ reconstructing the DNA sequences upstream from the A56R ORF and replacing the A56R ORF with 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 Belli/BamHI (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 Belli site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination with rescuing virus VP618.

Recombinant vaccinia virus, VP708, containing Betagalactosidase in the A56R deletion, was isolated as a blue plaque in the presence of X-gal.

Beta-galactosidase sequences were deleted from vP708 using donor plasmid pSD4C7. pSD467 is identical to pSD466, except that EcoRI, Smal and BamHI sites were removed from the pUC/vaccinia junction by digestion of pSD466 with EcoRI/BamHI followed by blunt ending with Klenow fragment of E. coli polymerase and ligation. Recombination between VP708 and pSD467 resulted in recombinant vaccinia deletion mutant, vP723, which was isolated as a clear plaque in the presence of X-gal.

Construction of Plasmid pMPCSKlA for Deletion of Open Reading Frames rC7L-K!L1 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 Kpnl F cloned into pUC18. pSD435 was cut with SphI and religated, forming pSD451. In pSD451, DNA sequences to the left of the SphI site (pos. 27,416) in HindHI M are removed (Perkus et al., 1990). pSD409 is HindHI M cloned into pUC8.

To provide a substrate for the deletion of the [C7LK1L] 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 JgglH site in pSD409, the plasmid was cut with Belli in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique Sohl site (pos. 27,416). M2L coding sequences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide BolII MPSYN82 (SEQ ID NO:35) 5’ TTTCTGTATATTTGCACCAATTTAGATCTTACTCAAAA TATGTAACAATA 31 The resulting plasmid, pMP409D, contains a unique BolII site inserted into the M2L deletion locus as indicated above. A 3.2 kb BamHI (partial)/BolII cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the θ control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with BolII. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination with rescuing vaccinia virus vP723. Recombinant vaccinia virus, vP784, containing Betagalactosidase inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal.

A plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUC8 cut with Smal. Hindlll and blunt ended with Klenow fragment of E. coli polymerase. The left 20 flanking arm consisting of vaccinia Hindlll C sequences was obtained by digestion of pSD420 with Xbal (pos. 18,628) followed by blunt ending with Klenow fragment of Έ. coli polymerase and digestion with BolII (pos. 19,706). The right flanking arm consisting of vaccinia Hindlll K 25 sequences was obtained by digestion of pSD451 with BolII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP58lCK is deleted for vaccinia sequences between the BolII site (pos. 19,706) in Hindlll C and the Ball I 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 pMP581CK, was cut at the Ncol sites within vaccinia sequences (pos. 18,811; 19,655), treated With Bal35 exonuclease and subjected to mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:36) 5·TGTCATTTAACACTA5 7 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 plaque in the presence of X-gal. Construction of Plasmid PSD548 for Deletion of Large Subunit. Ribonucleotide Reductase (I4L) 10 Referring now to FIG. 14, plasmid pSD405 contains vaccinia Hindlll I (pos. 63,875-70,367) cloned in pUC8. pSD405 was digested with EcoRV within vaccinia sequences (pos. 67,933) and with Smai 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. 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 Hoal (pos. 67,001) and blunt ended using Klenow fragment of E. coli polymerase. This 4.8 kb vector fragment was ligated with a 3.2 kb Smai 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 with vaccinia virus vP804. Recombinant vaccinia virus, vP855, containing Beta30 galactosidase in a partial deletion of the I4L gene, was 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 31 GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA fiSlII EcoRI TTGAGAATAAAAAGATCTTAGG 31 518A1 AACTCTTATTTTTCTAGAATCCTTAA 5' 518A2 forming plasmid pSD531. pSD53l was cut with Rsal (partial) and BamHi and a 2.7 kb vector fragment isolated. pSD518 was cut with Bglll (pos. 64,459)/ Rsal (pos. 64,994) and a 0.5 kb fragment isolated. The two fragments were ligated together, forming pSD537, which contains the complete vaccinia flanking arm left of the I4L coding seguences.

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) gamHI Bglll Smal 518B1 5· GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAGGGATTT 518B2 31 GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATCCCTAAA Rsal EcoRI GACGTATGTAGCGTACTAGG 3 * 518B1 CTGCATACTACGCATGATCCTTAA 5' 518B2 forming plasmid pSD532. pSD532 was cut with Rsal c (partial)/BggRI and a 2.7 kb vector fragment isolated. pSD518 was cut with Rsal within vaccinia seguences (pos. 67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two fragments were ligated together, forming pSD538, which contains the complete vaccinia flanking arm to the right of I4L coding seguences.

The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/Bglll fragment from pSD538 and ligated into pSD537 vector plasmid cut with EcoRI/Bglll. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a polylinker 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 pUC-derived portion of pSD539 with Betagalactosidase sequences in recombinant vaccinia virus vP855, the vaccinia I4L deletion cassette was moved from pSD539 into pRCll, a pUC derivative from which all Betagalactosidase sequences have been removed and replaced with a polylinker region (Colinas et al., 1990). pSD539 was cut with EcoRI/Pstl and the 1.2 kb fragment isolated. This fragment was 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 was 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 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 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 BEMAGGLUTININ GLYCOPROTEINS cDNA copies of the sequences encoding the HA and F proteins of measles virus MV (Edmonston strain) were 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 pSPH2LHA contains the entire measles HA gene linked in a precise ATG to ATG configuration with the vaccinia virus H6 promoter which has been previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989). A 1.8kpb IcoRV/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/Smal 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 pSD5l3VCVQ 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 sequences between the 31 end of the promoter and the ATG of the measles F gene oligonucleotide directed mutagenesis was performed using oligonucleotide SPMAD (SEQ ID NO:41).

SPMAD: 5 ’ -TATCCGTTAAGTTTGTATCGTAATGGGTCTCAAGGTGAACGTCT-3 ' The resultant plasmid was designated pSPMF75M20.

The plasmid pSPMF75M20 which contains the measles F gene now linked in a precise ATG for ATG configuration with the H6 promoter was digested with Nrul and Eagl. The resulting 1.7 kbp blunt ended fragment containing the 3' 1 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 pRW84l contains the H6 promoter linked to the measles F gene in the pIBI25 plasmid vector (IBI, New Haven, CT). The H6/measles F cassette was excised from pRW841 by digestion with Smal and the resulting 1.8 kb fragment was inserted into pRW843 (containing the measles HA gene). Plasmid pRW843 was first digested with Not! and blunt-ended witli 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 pRW857 was 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 plaque purification until a pure population was achieved. One representative plaque was then amplified and the resulting recombinant was designated NYVAC-MV (VP913). Tmamwofiuoreseence ' 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.

Tmamnoorecipitation 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 of l 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 was performed using mono-specific sera In order to demonstrate that the proteins expressed by NYVAC-MV were 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 in the presence of 35Smethionine. Immunoprecipitation analysis revealed a HA glycoprotein of approximately 76 kDa and the cleaved fusion products Ft and F2 with molecular weights of 44 kDa and 23 kDa, respectively. No measles specific products were detected in uninfected Vero cells or Vero cells infected with the parental NYVAC virus.

A characteristic of MV cytopathology is the formation of syncytia which 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 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. No4cell fusing activity was evident in cells infected with parental NYVAC.

Results of serological analysis of sera of rabbits inoculated with NYVAC-MV (vP913) In this study, two rabbits were inoculated with 1x10* 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 (vP913) into rabbits similarly induces a level of measles virus neutralizing antibody which would be protective. 4 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* A116 <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 pre-inoculation serum. c) Animals were re-inoculated at 28 days.

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Claims (10)

1. A recombinant poxvirus comprising in a nonessential region of its genome exogenous Morbillivirus DNA coding for an antigen characterised in that 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 according to claim 1 wherein the exogenous Morbillivirus DNA is exogenous measles virus DNA.

3. A recombinant poxvirus 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 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 according to any one of claims 2 to 4 wherein the exogenous Morbillivirus DNA is from the Edmonston strain of measles virus.

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

7. A recombinant poxvirus according to any one of the preceding claims wherein the exogenous Morbillivirus DNA further includes a promoter for expressing the antigen.

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

9. A recombinant pox virus according to any of the preceding claims wherein the deleted open reading frames comprise C7L-K1L, J2R, B13R+B14R, A26L, A56R and I4L. 5 10. A recombinant pox virus according to claim 9 wherein the poxvirus is the NYVAC vaccinia virus. 11. The recombinant poxvirus vP913.

10. 12. A vaccine for inducing an immunological response in a host animal inoculated I with the vaccine, the vaccine comprising a carrier and a recombinant poxvirus according to any one of the preceding claims.