NUCLEOTIDE AND AMINO ACID SEQUENCES OF CANINE HERPESVIRUS gB, gC and gD AND USES THEREFOR

Abstract: Disclosed and claimed are nucleotides for genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues. These genes encode polypeptides of 879, 459 and 345 amino acids, respectively, which are also disclosed and claimed. The genes are useful as DNA probes or, for preparing PCR primers. The polypeptides are useful in antigenic, immunological or vaccine compositions. The nucleotides can be expressed in any suitable vector system, allowing for production of the polypeptides. Additionally, the vector system containing any or any combination of the nucleotides can be employed in an antigenic, immunological or vaccine composition, such as a poxvirus vector system, e.g., a CHV-vaccinia or avipox virus recombinant, as can the products from expression, i.e., the gB, gC and gD glycoproteins. Antibodies elicited by the glycoproteins or from expression of the vector containing the nucleotide (s) are also useful. Methods for making and using the composition are also disclosed and claimed. Also, specific canarypox-CHV gB, gC and gD recombinants vCP 320, vCP322 and vCP294 and methods for making and using them are also disclosed and claimed.

Description: NUCLEOTIDE AND AMINO ACID SEQUENCES OF CANINE HERPESVIRUS gB, gC and gD AND USES THEREFOR FIELD OF THE INVENTION This invention relates to canine herpesvirus (CHV) , nucleotides or isolated nucleic acids encoding the CHV gB, gC and gD glycoproteins, and the amino acid sequences thereof, vectors, such as a recombinant poxvirus, e.g., vaccinia and avipox virus recombinants, containing the CHV gB, gC and/or gD coding or expressing the same, glycoproteins therefrom, vaccines, immunological or antigenic compositions from the nucleotide (such as from vectors, for instance, recombinant poxvirus, e.g., vaccinia or avipox virus recombinants containing the CHV, gB, gC and/or gD coding and expressing glycoprotein(s) therefrom) , or, from the glycoproteins, for instance, from expression of the nucleotides in a vector system, and, to methods employing the nucleotides, glycoproteins, and compositions. Several publications are cited in the following text, with full citation of each set forth in the section headed References. The publications cited throughout the text are hereby incorporated herein by reference. BACKGROUND OF THE INVENTION Canine herpesvirus (CHV) causes a fatal, hemorrhagic disease in neonatal puppies and a self-limiting, usually subclinical, upper respiratory tract infection in adult dogs (Appel, 1987) . Little is known about the genomic structure of CHV. The genome has not been mapped and no nucleotide sequence has been published. In particular, genes encoding immunologically pertinent proteins have not been identified. Herpesvirus glycoproteins mediate essential viral functions such as cellular attachment and penetration, cell to cell spread of the virus and, importantly, determine the pathogenicity profile of infection. Herpesvirus glycoproteins are critical components in the interaction with the host immune system (Spear, 1985a; Spear 1985b) . Herpesvirus glycoproteins are antigens recognized by both the humoral and cellular immune systems and, have been shown to evoke protective immune responses in vaccinated hosts ( achsman et al., 1987; Marchioli et al., 1987; Eberle et al., 1980; Papp-Vid et al., 1979). During a herpesvirus infection, the majority of the immune response is directed against viral envelope glycoproteins. These antigens have been shown to elicit both humoral and cellular immune responses. Several reports have indicated that in other herpesvirus systems immunization with the herpesvirus gB, gC and/or gD glycoproteins can induce a protective immune response. The well characterized glycoproteins of herpes simplex virus include gB, gC, gD, gE, gG, gH, gl, gJ, gK, gL and gM (Spear, 1985a; Spear 1985b; Ackermann et al., 1986; Frink et al. 1983; Frame et al., 1986; Longnecker et al., 1987; Richman et al. , 1986; Swain et al., 1985; Zezulak, 1984; Roizman and Sears, 1990; Hutchinson et al., 1992a; Hutchinson et al., 1992b; Baines and Roizman, 1993). A number of studies have indicated the importance of herpes simplex virus glycoproteins in eliciting immune responses. Hence, it has been reported that gB and gD can elicit important immune responses (Ber an et al., 1983; Cantin et al., 1987; Cremer et al., 1985; Lasky et al., 1984; Martin et al., 1987a; Martin et al., 1987b; Paoletti et al., 1984; Perkus et al., 1985; Rooney et al., 1988; Wachsman et al., 1987; Zarling et al., 1986a; Zarling et al., 1986b). gC can stimulate class I restricted cytotoxic lymphocytes (Glorioso et al., 1985; Rosenthal et al., 1987) whereas gD can stimulate class II cytotoxic T cell responses (Martin et al., 1987a; Martin et al,. 1987b; Wachsman et al., 1987; Zarling et al., 1986a; Zarling 1986b). gG was shown to be a target for complement-dependent antibody directed virus neutralization (Sullivan et al., 1987; Sullivan et al., 1988) . A number of glycoproteins from other herpesviruses have also been shown to elicit important immune responses. Both subtypes of equine herpesvirus (EHV) express six abundant glycoproteins (Allen et al., 1986; Allen et al., 1987) . The genomic portions of the DNA sequences encoding gp2, gplO, gpl3, gpl4, gpl7/l8, and gp21/22a have been determined using lambda gtll expression vectors and monoclonal antibodies (Allen et al., 1987). Glycoproteins gpl3 and gpl4 were located in the same locations within the L component of the genome to which the gC and gB homologs, respectively, of herpes simplex virus map (Allen et al., 1987) . The envelope glycoproteins are the principal immunogens of herpesviruses involved in eliciting both humoral and cellular host immune responses (Ben-Porat et al., 1986; Cantin et al., 1987; Glorioso et al., 1984; Wachsman et al., 1988; Wachsman et al., 1989) and so are of the highest interest for those attempting to design vaccines. Recently, the nucleotide sequence of the Kentucky T431 strain of the EHV-1 transcriptional unit encoding gpl3 has been reported (Allen et al., 1988). The glycoprotein was shown to be homologous to the herpes simplex virus (HSV) gC- 1 and gC-2, to the pseudorabies virus (PRV) gill and the varicella-zoster virus (VZV) gpV (Allen et al., 1988). EHV- l gpl3 is thus the structural homolog of the herpesvirus gC- like glycoproteins. The nucleotide sequence of EHV-l gpl4 (Whalley et al., 1989; Riggio et al., 1989) has recently been reported. Analysis of the predicted amino acid sequence of gpl4 glycoprotein revealed significant homology to the corresponding glycoprotein of HSV, gB. Monoclonal antibodies directed against some EHV-l glycoproteins have been shown to be neutralizing (Sinclair et al., 1989). Passive immunization experiments demonstrated that monoclonal antibodies directed against gpl3 or gpl4 (Shimizu et al., 1989) or against gpl3, gpl4 or gpl7/iδ (Stokes et al., 1989) could protect hamsters against a lethal challenge. Other gB and gC glycoprotein analogs are also involved in protection against diseases caused by alphaherpesviruses (Cantin et al., 1987; Cranage et al., 1986; Glorioso et al., 1984). Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujesky's disease. The PRV genome consists of a 90 x IO 6 dalton double stranded DNA (Rubenstein et al., 1975) separated by inverted repeat sequences into unique long (U L ) or unique short (U s ) segments (Stevely, 1977; Ben-Porat et al., 1979). The PRV genome encodes approximately 100 polypeptides whose expression is regulated in a cascade-like fashion similar to other herpesviruses (Ben-Porat et al., 1985; Hampl et al., 1984). PRV glycoprotein gp50 is the Herpes simplex virus type 1 (HSV-1) gD analog (Wathen et al., 1984). The DNA open reading frame encodes 402 amino acids (Petrovskiε et al., 1986) . The mature glycosylated form (50-60 kDa) contains O- linked carbohydrate without N-linked glycosylation (Petrovskis et al., 1986). Swine serum is highly reactive with PRV gp50, suggesting its importance as an immunogen. Monoclonal antibodies to gp50 neutralize PRV in vitro with or without complement (Wathen et al., 1984; Wathen 1985; Eloit et al., 1988) and passively protect mice (Marchioli et al., 1988; Wathen et al., 1985; Eloit et al., 1988) and swine (Marchioli et al., 1988). Vaccinia virus recombinants expressing PRV gp50 induced serum neutralizing antibodies and protected both mice and swine against lethal PRV challenge (Kost et al., 1989; Marchioli et al., 1987; Ishii et al. , 1988) . PRV gill is the HSV-1 gC analog (Robbins et al., 1986). Functional replacement of PRV gill by HSVgC was not observed (Whealy et al., 1989). Although PRV gill is nonessential for replication in vitro (Wathen et al., 1986; Robbins et al., 1986), the mature glycosylated form (98 kDa) is an abundant constituent of the PRV envelope. Anti-gpIII monoclonal antibodies neutralize the virus in vitro with or without complement (Hampl et al., 1984; Eloit et al., 1988; Wathen et al., 1986) and can passively protect mice and swine (Marchioli et al., 1988). The PRV glycoprotein gill can protect mice and swine from lethal PRV challenge after immunization with a Cro/glll fusion protein expressed in E . coli (Robbins, A., R. Watson, L. Enquiεt, European Patent application 0162738A1) or when expressed in a vaccinia recombinant (Panicali, D. , L. Gritz, G. Mazzara, European Patent application 0261940A2) . PRV gpll is the HSV-l gB homolog (Robbins et al., 1987) . Monoclonal antibodies directed against PRV gpll have been shown to neutralize the virus in vitro (Ben-Porat et al., 1986) with or without complement (Wittmann et al., 1989) . Moreover, passive immunization studies demonstrated that neutralizing monoclonal antibodies partially protected swine (Marchioli et al., 1988). Immunization with NYVAC (highly attenuated vaccinia virus)-based recombinants expressing pseudorabies virus (PRV) gll (gB) or gp50 (gD) has been shown to protect swine against a virulent PRV challenge (Brockmeier et al., 1993). Furthermore, vaccinia recombinants expressing PRV gll and gp50, or gll, gill (gC) and gp50 have been shown to elicit a higher level of protection than recombinants expressing gll or gp50 alone, suggesting a potential synergistic effect with these glycoproteins (Riviere et al., 1992). The herpes simplex virus type l (HSVl) genome encodes at least eleven antigenically distinct glycoproteins: gB, gC, gD, gE, gG, gH, gl, gJ, gK, gL and gM (Roizman et al., 1990) . Mice immunized with purified HSVl gB, gC or gD are protected against lethal HSVl challenge (Chan, 1983). Mice have also been protected against lethal HSVl or HSV2 challenge by passive immunization with antibodies to total HSVl (Davis et al., 1979) or HSV2 (Oakes et al., 1978) virus and with antibodies to the individual HSV2 gB, gC, gD or gE glycoproteins (Balachandran et al., 1982). Vaccinia virus vectors expressing HSVl gB (McLaughlin- Taylor et al., 1988) and HSVl gC (Rosenthal et al., 1987) have been shown to induce cytotoxic T-cell responses. In addition, it has been shown that mice immunized with recombinant vaccinia virus expressing either HSVl gB (Cantin et al., 1987), HSVl gC (Weir et al., 1989) or HSVl gD (Paoletti et al., 1984) are protected against a lethal challenge of HSVl. A recombinant vaccinia virus expressing HSVl gD has also been shown to be protective against HSV2 in a guinea pig model system (Wachsman et al., 1987). Bovine herpesvirus 1 (BHV1) specifies more than 30 structural polypeptides, 11 of which are glycosylated (Misra et al., 1981). Three of these glycoproteins, gl, gill and gIV, have been characterized and found to be homologous to the herpes simplex virus (HSV) glycoproteins gB, gC and gD (Lawrence et al., 1986; Zamb, 1987). Immunization with purified bovine herpesvirus type 1 (BHV1) gl (gB) , gill (gC) and/or gIV (gD) has been shown to protect cattle against a BHVl /Paste rella haemolytica challenge (Babiuk et al., 1987) . Feline herpesvirus type-1 (FHV-1) has been shown to contain at least 23 different proteins (Meas et al., 1984; Fargeaud et al., 1984). Of these, at least five are glycosylated (Fargeaud et al., 1984; Compton, 1989) with reported molecular masses ranging from 120 kDa to 60 kDa. The FHV-l glycoproteins have been shown to be immunogenic (Meas et al., 1984; Compton, 1989). Like several other alphaherpesviruses, FHV-l appears to have a homolog of glycoprotein B (gB) of HSV-1 (Maeda et al., 1992). The FHV-l gB glycoprotein is a 134 kDa complex which is dissociated with B-mercaptoethanol into two glycoproteins of 66 kDa and 60 kDa. The FHV-l DNA genome is approximately 134 Kb in size (Rota et al., 1986). Epstein Barr Virus (EBV) , a human B lymphotropic herpesvirus, is a member of the genus lymphocryptovirus which belongs to the subfamily gamroaherpesvirus (Roizman et al. , 1990) . Since the EBV genome was completely sequenced (Baer et al., 1984) as the genomes of VZV (Davison et al., 1986), HSVl (McGeoch et al., 1988), MCHV (Chee et al. , 1990) and EHVl (Telford et al., 1992) numerous homologies between these different herpesviruses have been described (Kieff et al., 1990). Human cytomegalovirus (HCMV) is a member of the betaherpesvirinae subfamily (family Herpesviridae) . Three immunologically distinct families of glycoproteins associated with the HCMV envelope have been described (Gretch et al., 1988): gCI (gp55 and gp93-130) ; gCII (gp47- 52) ; and gCIII (gp85-pl45) . The gene coding for gCI is homologous to HSVl gB. In addition, immunization with a fowlpox recombinant expressing Marek's disease virus (MDV) gB has been shown to protect chickens against a virulent MDV challenge (Nazarian et al., 1992) . The results of these studies indicate that an immune response against gB, gC and/or gD glycoproteins can protect target species animals against a herpesvirus challenge and, that the provision of nucleotides for CHV gB, gC and gD glycoproteins is a valuable advance over the current state of the art as it allows for the provision of the glycoproteins and, antigenic, immunological or vaccine compositions from the vector systems or from the glycoproteins. Further, the glycoproteins from expression of the nucleotides can be used to elicit antibodies which can be further used in antibody binding diagnostic assays, kits or tests for ascertaining the presence or absence in a sample such as sera of the glycoprotein(s) and therefore the presence or absence of CHV or of an immune or antigenic response (to either CHV or to the glycoproteins) . Thus, many utilities flow from the provision of the nucleotides for CHV gB, gC and gD glycoproteins. Various vector systems exist for the expression of exogenous DNA, such as the phage, e.g., lambda, and E. coli systems (Allen et al., 1987; Robbins, EPA 0162738A1; Panicali, EPA 0261940A2, each of which is expressly incorporated herein by reference) . 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 et al., 1987). Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Patent Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587, and 5,179,993, the disclosures of which are 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. Additional strategies have recently been reported for generating recombinant vaccinia virus. 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. Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983) . With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a). The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988). A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology. A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems. The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK" vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts by a variety of routes. It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK " virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK + virus (Stanberry et al., 1985) . It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974). Attenuation of TK" vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK" recombinant vaccinia generated equivalent anti- vaccinia neutralizing antibodies as compared with the parental TK + vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK" and TK + recombinant vaccinia virus (WR strain) , significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a). Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989). Both the small (Slabaugh et al., 1988) and large (Schmitt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990). The vaccinia virus hemagglutinin gene (HA) has been mapped and εequenced (Shida, 1986) . The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA" vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987). Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation. The u_ gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u_ region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b) . Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959) . The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., I990a,b). A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al. , 1988b) . Specific genes within the deleted region include the virokine NIL and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of NIL reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like NIL into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms. The left end of the vaccinia genome includes two genes which have been identified as host range genes, KIL (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990). Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920"s by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982b) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition. FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990) . Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993 a,b; Taylor et al., 1992; 1991b; 1988b) . Thus, heretofore, the nucleotide and amino acid sequences for the CHV gB, gC and gD glycoproteins, have not been taught or suggested and, providing these sequences would be of great value. Further, vaccine, antigenic or immunological compositions from the nucleotides for the CHV gB, gC and gD glycoproteins (such as from vector systems containing such nucleotides) as well as from the glycoproteins themselves (such as from expression by the vector systems) have not heretofore been taught or suggested and, these nucleotides, vector systems, glycoproteins and compositions would be of great value. OBJECTS AND SUMMARY OF THE INVENTION It is therefore an object of the invention to provide nucleotides or isolated nucleic acids coding for CHV gB, gC and gD. It is a further object of the invention to provide vectors containing nucleotides or isolated nucleic acids coding for CHV gB, gC and/or gD. It is another object of the invention to provide CHV gB, gC and/or gD glycoproteins, especially from expression of nucleotides or isolated nucleic acids therefor in a vector system. It is an additional object of the invention to provide antigenic, vaccine or immunological compositions from the CHV gB, gC and/or gD nucleotides or isolated nucleic acids or a vector containing them or, from the glycoproteins themselves, such as by way of expression by the vector. It is yet another object of this invention to provide modified recombinant viruses, which viruses have enhanced safety, and to provide a method of making such recombinant viruses. It is an additional object of this invention to provide a recombinant poxvirus antigenic, vaccine or immunological composition having an increased level of safety compared to known recombinant poxvirus antigenic, vaccine or immunological compositions. It is a further object of this invention to provide a modified vector for expressing a gene product in a host, wherein the vector is modified so that it has attenuated virulence in the host. It is another object of this invention to provide a method for expressing a gene product, such as CHV gB, gC and/or gD, in a cell cultured in vitro using a modified recombinant virus or modified vector having an increased level of safety. These and other objects and advantages of the present invention will become more readily apparent after consideration of the following. The present invention involves the elucidation of the CHV gB, gC and gD nucleotides, glycoproteins therefrom and, antigenic, vaccine or immunological compositions employing the nucleotide sequences and the glycoproteins. Accordingly, the present invention provides a nucleotide or isolated nucleic acid coding for canine herpesvirus gB glycoprotein. The present invention provides a nucleotide or isolated nucleic acid coding for canine herpesvirus gC glycoprotein. The present invention provides a nucleotide or isolated nucleic acid coding for canine herpesvirus gD glycoprotein. The nucleotides are preferably DNA. The nucleotides or isolated nucleic acids preferably have the DNA sequences as set forth in Figs. 1, 4 and 7. The present invention also provides canine herpesvirus glycoprotein gB. The present invention provides canine herpesvirus glycoprotein gC. The present invention provides canine herpesvirus glycoprotein gD. The present invention further provides a vector containing the nucleotide or isolated nucleic acid for canine herpesvirus gB, gC and/or gD. Preferably the vector is a recombinant poxvirus such as a recombinant vaccinia or avipox virus, more preferably the vaccinia or avipox virus is attenuated such as NYVAC, ALVAC or TROVAC. Thus, in one preferred aspect, the present invention relates to a modified recombinant virus having inactivated virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The functions can be non-essential, or associated with virulence. The virus is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes a CHV antigenic protein, e.g., CHV gC, gB, and gD or any combination thereof. In a still further preferred aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the virus has attenuated virulence, and wherein the modified recombinant virus further contains DNA from a heterologous source in a nonessential region of the virus genome. The DNA can code for a CHV gB, gC and gD, or any combination thereof. In particular, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host 95/26751 -18 - reεtricted viruses. The virus used according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. Advantageously, the open reading frame is selected from the group consisting of J2R, B13R + B14R, A26L, A56R, C7L - KIL, and I4L (by the terminology reported in Goebel et al., 1990a,b) ; and, the combination thereof. In this respect, the open reading frame comprises a thymidine kinase gene, a hemorrhagic region, an A type incluεion body region, a hemagglutinin gene, a hoεt range gene region or a large εubunit, ribonucleotide reductase; or, the combination thereof. The modified Copenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia et al., 1992) . The present invention still further provides an antigenic, vaccine or immunological composition for inducing an antigenic or immunological reεponεe in a hoεt, εuch as a canine, comprising a suitable vector containing the nucleotide(ε) or isolated nucleic acid(ε) for canine herpeεviruε gB, gC and/or gD and a suitable carrier; or, canine herpesvirus gB, gC and/or gD glycoprotein(s) , such as from expression thereof in a vector containing the nucleotide(ε) of the invention, and a suitable carrier. The present invention yet further provides methods employing the inventive nucleotide(s) or isolated nucleic acid(s), glycoprotein(s) , composition(s) . Thus, the invention provideε a method for preparing canine herpesvirus gB, gC and/or gD comprising inserting the nucleotide(ε) or iεolated nucleic acid(ε) therefor into a suitable vector, cultivating the vector, and, collecting the glycoprotein from the vector. The vector can be a poxvirus, such aε vaccinia or avipox virus, a phage such as lambda, or E. coli or any other suitable virus or bacterial vector. The cultivating can be infecting cells susceptible to viral infection by the virus vector or, growing colonies of the bacterial vector system, such as by plate or broth methods. And, collecting can be by separating the glycoprotein(ε) from the viral-infected cells or from the bacterial cells. Thus, in a preferred aεpect, the present invention relates to a method for expressing a gene product in a cell cultured in vitro by introducing into the cell a modified recombinant virus having attenuated virulence and enhanced safety. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes. an antigenic protein, e.g., CHV gB, gC and gD, or any combination thereof. Likewise, the invention provides a method for inoculating or for stimulating an antigenic or immunological response in a hoεt such as a canine against canine herpesviruε compriεing adminiεtering the inventive antigenic, vaccine or immunological compoεition to the hoεt, e.g., canine. Additionally, the invention includeε an antibody elicited by the expreεεion of the inventive nucleotide(ε) . The antibody can be generated into a monoclonal antibody by known techniqueε and, the antibody or the monoclonal antibody can be employed in a binding diagnoεtic aεεay, test or kit to determine the presence or absence of CHV gB, gC and/or gD in a sample such as sera and therefore the presence or absence of CHV or, of an antibody or immune responεe to CHV or to glycoproteinε thereof. These and other embodiments within the present invention are deεcribed or are obvious from the following detailed description. BRIEF DESCRIPTION OF DRAWINGS The following detailed deεcription, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: FIG. 1 showε the nucleotide sequence and predicted amino acid sequence of the CHV gB homologue (SEQ ID N0S:1, 2); FIG. 2 shows the hydropathicity analysis of the CHV gB homologue; FIGS. 3A and 3B εhow the amino acid homology of 8 gB homologueε (SEQ ID NOS:2-9); FIG. 4 εhowε the nucleotide εequence and predicted amino acid εequence of the CHV gC homologue and 0RF2 (SEQ ID NOS:10-11); FIG. 5 εhows the hydropathicity analysiε of the CHV gC homologue; FIG. 6 εhowε the amino acid homology of 4 gC homologues (SEQ ID NOS:11-14) ; FIG. 7 shows the nucleotide εequence and predicted amino acid εequence of the CHV gD homologue; SEQ ID NOS:15- 16); FIG. 8 εhows the hydropathicity analysis of the CHV gD homologue; FIG. 9 showε amino acid homology of 4 gD homologueε (SEQ ID NOS:16-20) ; FIG. 10 schematically εhowε a method for the conεtruction of plaεmid pSD460 for deletion of thymidine kinaεe gene and generation of recombinant vaccinia viruε vP410; FIG. 11 εchematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus VP553; FIG. 12 εchematically showε a method for the conεtruction of plaεmid pMP494Δ for deletion of ATI region and generation of recombinant vaccinia viruε VP618; FIG. 13 εchematically εhowε a method for the conεtruction of plaεmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus vP723; FIG. 14 schematically showε a method for the conεtruction of plaεmid pMPCKlΔ for deletion of gene cluεter [C7L - KIL] and generation of recombinant vaccinia viruε vP804; FIG. 15 schematically showε a method for the conεtruction of plaεmid pSD548 for deletion of large εubunit, ribonucleotide reductaεe and generation of recombinant vaccinia viruε vP866 (NYVAC) ; FIG. 16 εchematically εhows a method for the construction of plasmid pRW842 for insertion of rabies glycoprotein G gene into the TK deletion locus and generation of recombinant vaccinia virus vP879; FIG. 17 shows the DNA εequence (SEQ ID NO:59) of a canarypox PvuII fragment containing the C5 ORF. FIGS. 18A and 18B εchematically show a method for the construction of recombinant canarypox viruε vCP65 (ALVAC- RG); FIG. 19 εhowε schematically the ORFs deleted to generate NYVAC; FIG. 20 shows the nucleotide sequence (SEQ ID NO:69) of a fragment of TROVAC DNA containing an F8 ORF; FIG. 21 showε the DNA εeguence (SEQ ID NO:72) of a 2356 baεe pair fragment of TROVAC DNA containing the F7 ORF; FIGS. 22A to 22D εhow graphε of rabieε neutralizing antibody titerε (RFFIT, IU/ml) , booεter effect of HDC and VCP65 (IO 5,5 TCID50) in volunteerε previouεly immunized with either the same or the alternate vaccine (vaccineε given at dayε 0, 28 and 180, antibody titerε meaεured at days 0, 7, 28, 35, 56, 173, 187 and 208); FIG. 23 shows the nucleotide sequence of the I3L- promoted CHV gB gene contained in pCHV37 and VCP320; FIG. 24 shows the nucleotide sequence of the ALVAC C6 flanking arms; FIG. 25 shows the immunoprecipitation analysis of vCP320-infected cells (Lysateε from 35 S-labeled mock- infected cellε (lane A) , ALVAC-infected cells (lane B) , vCP320-infected cells (lane C) and CHV-infected cellε (lane D) were immunoprecipitated with a CHV gB-specific monoclonal antibody, 1125B2 (obtained from Rhone Merieux, Lyon, France), and resolved on an SDS-polyacrylamide gel. Molecular weight standards are resolved in lane E) ; FIG. 26 shows the nucleotide εequence of the H6- promoted CHV gC gene contained in pCHV40 and VCP322; FIG. 27 εhowε the immunoprecipitation analyεiε of vCP322-infected cellε (Lysates from 35 S-labeled mock- infected cells (lane A), ALVAC-infected cells (lane B) , vCP322-infected cells (lane C) and CHV-infected cellε (lane D) were immunoprecipitated with a CHV gC-specific monoclonal antibody, 2011A9 (obtained from Rhone Merieux, Lyon, France) , and reεolved on an SDS-polyacrylamide gel. Molecular weight εtandards are resolved in lane E) ; FIG. 28 εhowε the nucleotide εequence of the H6- promoted CHV gD gene contained in pCHV26 and VCP294; and FIG. 29 εhows the immunoprecipitation analysis of vCP294-infected cells (Lysates from 35 S-labeled mock- infected cells (lane A) , ALVAC-infected cells (lane B) , vCP294-infected cells (lane C) and CHV-infected cells (lane D) were immunoprecipitated with a CHV gD-specific monoclonal antibody, 208D11 (obtained from Rhone Merieux, Lyon, France), and reεolved on an SDS-polyacrylamide gel. Molecular weight εtandards are resolved in lane E) . DETAILED DESCRIPTION This invention provideε nucleotideε coding for the CHV gB, gC and gD genes. These genes encode polypeptides of 879, 459 and 345 amino acidε, reεpectively. Comparison of the predicted amino acid sequence of these glycoproteins with the gB, gC and gD amino acid εequenceε of other herpeεviruses indicates that CHV is an alpha-herpesvirus; a concluεion that iε conεistent with the previous classification of this viruε according to biological propertieε. This analysiε alεo revealed that the homology among gB homologues is greater than the homology among gC or gD homologueε, εuggeεting that the εtructural and functional conεtraintε on gB may be greater than thoεe on gC or gD. Alignment of homologouε gB, gC and gD polypeptideε revealed that the vaεt majority of cysteine residueε are perfectly conserved. These results suggest that these cysteine residues, due to their ability to form disulfide bonds, are important in maintaining the structural and functional integrity of the gB, gC and gD glycoproteins. In fact, in HSVl gD, it has been εhown that cyεteine 1 forms a disulfide bond with cysteine 5, cysteine 2 forms a disulfide bond with cyεteine 6 and cysteine 3 forms a disulfide bond with cysteine 4 (Long et al., 1992). Furthermore, it has been shown that a mutation of any of these residues haε a profound effect on the conformation, proceεεing and function of the reεulting glycoprotein (Wilcox et al., 1988; Long et al., 1990). Therefore, the conεervation of cyεteine reεidueε in the glycoproteins of the invention may also have εtructural significance. The high degree of homology among the gC, gD and, in particular, gB homologues alεo εuggeεts that these glycoproteins have common functions. In fact, it has been shown that the BHV1 gB homologue can rescue a gB" PRV viruε, indicating that theεe 2 glycoproteinε are functionally equivalent (Kopp & Mettenleiter, 1992) . Alignment of the gB, gC and gD amino acid sequences alεo revealed that potential N-linked glycoεylation sites are somewhat conserved. N-linked glycosylation is thought to play a role in a variety of functions, such as maintenance of protein conformation and protection against proteolytic degradation. The biological significance of N- linked carbohydrateε on herpesvirus glycoproteins, however, iε not completely underεtood. For example, tunicamycin treatment of HSVl infected cellε haε been εhown to inhibit the production of infectiouε virionε (Pizer et al., 1980). In addition, endoglycosidase treatment of HSVl virions haε been shown to decrease infectivity (Kuhn et al., 1988). On the other hand, N-linked glycosylation of HSVl gD does not appear to be absolutely essential, since mutagenesis of the glycosylation εites on this glycoprotein does not affect infectivity (Sodora et al., 1991). Therefore, although the glycoεylation siteε on the gB, gC and gD glycoproteinε are relatively well conεerved, proper glycoεylation of each of theεe polypeptideε may not be absolutely essential. The G+C content of herpesviruses varies from 33%-75% (Roizman, 1982) . It haε been εuggeεted that thiε extenεive variability is due to a nonselective mutational bias based on the presence (or absence) of virally encoded or induced enzymeε involved in nucleotide metaboliεm (Honess, 1984). For example, VZV and herpesvirus saimiri (HVS) both have relatively low G+C contentε (46% and 46%, reεpectively) and both encode an enzyme, thymidylate εynthetase, which is involved in TTP εyntheεiε (Daviεon & Scott, 1986; Honess et al., 1986). HSVl, HCMV and EBV, on the other hand, have relatively high G+C contents (68%, 57% and 60%, respectively) and do not appear to encode a thymidylate εynthetaεe (Honeεε et al., 1986). CHV has been determined by DNA density analyεiε to have the lowest G+C content of any herpesviruε, 33% (Plummer et al., 1969; Roizman, 1982); a value which is consiεtent with the relatively low G+C content of the nucleotideε of the invention (29%) . Without wiεhing it to be bound by the theory that CHV doeε not encode an enzyme involved in nucleotide metaboliεm, from the preεent invention the ORF located immediately downεtream from the CHV gC gene iε not homologouε to VZV thymidylate εynthetaεe. Therefore, if CHV containε a thymidylate εynthetaεe gene, it iε not found at the εame genomic location aε VZV. Newborn pupε expoεed to CHV uεually die without forming CHV-εpecific neutralizing antibodieε. Alεo, the maternal antibodieε or treatment with immune εerum from seropositive dogε can protect pupε from a fatal CHV infection (Carmichael, 1970). Therefore, εerum neutralizing antibodieε can protect pupε againεt a fatal CHV infection. Likewiεe, εerum neutralizing antibodieε can protect adult dogε from the self-limiting subclinical, upper respiratory tract infection. Three CHV glycoproteinε, gpl45/112, gp80 and gp47, are known to elicit CHV neutralizing antibodieε (Xuan et al., 1991) . The geneε encoding theεe glycoproteinε have not been identified. Without wishing to be bound by any one theory, it is possible, however, that these antigens are encoded by the gB, gC and gD genes of this invention. Since several reports have indicated that an immune responεe against gB, gC and/or gD can provide protection of target species animalε against a herpesvirus challenge (Babiuk et al., 1987; Nazarian et al., 1992; Riviere et al., 1992; Brockmeier et al., 1993), the CHV gB, gC and gD genes of this invention provide efficacious CHV glycoproteins, immunological or vaccine compositionε and methodε of uεing the same. In particular, the nucleotides of this invention can be inserted into any suitable vector system for expresεion. For inεtance, the nucleotide(ε) can be inεerted into any εuitable bacterial vector εystem such as the E. coli syεtem, employing known methods (εee, e.g., Robbins, EPA 0162738A1; Panicali, EPA 0261940A2). The nucleotide(s) can be inεerted into any suitable phage or viral vector syεtem εuch aε lambda, poxvirus, herpesvirus (see Roizman, U.S. Patent No. 4,769,331, incorporated herein by reference), baculovirus, polio virus (see Kitson et al., J. Virol. 65, 3068-3075, 1991, incorporated herein by reference) , and adenovirus (see Grunhaus et al., 1992, "Adenoviruε aε cloning vectors," Seminars in Virology (Vol. 3) p. 237-52, 1993; Ballay et al., EMBO Journal, vol. 4, p. 3861-65; Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen. Virol. 70, 429- 434, each of which is incorporated herein by reference) syεtemε employing known methodε. The preferred vector εystem iε a poxviruε vector system, especially an avipox vaccinia virus system wherein recombination is as in U.S. Patents Noε. 4,769,330, 4,772,848, 4,603,112, 5,100,587 and 5,179,993. However, an attenuated poxviruε system is even more preferred. To develop a new vaccinia vaccine strain, NYVAC (vP866) , the Copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonesεential regionε of the genome encoding known or potential virulence factorε. The sequential deletionε are detailed below. All deεignationε 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 deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designationε for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified: (1) thymidine kinase gene (TK; J2R) VP410; (2) hemorrhagic region (u; B13R + B14R) VP553; (3) A type incluεion body region (ATI; A26L) vP618; (4) hemagglutinin gene (HA; A56R) VP723; (5) hoεt range gene region (C7L - KIL) VP804; and (6) large εubunit, ribonucleotide reductaεe (I4L) VP866 (NYVAC) . NYVAC iε a genetically engineered vaccinia virus strain that waε generated by the εpecific deletion of eighteen open reading frames encoding gene products asεociated with virulence and hoεt range. NYVAC iε highly attenuated by a number of criteria including i) decreaεed virulence after intracerebral inoculation in newborn mice, ii) inocuity in genetically (2vu + /E u+ ) or chemically (cyclophoεphamide) immunocoropromiεed mice, iii) failure to cause disseminated infection in immunocompromised mice, iv) lack of significant induration and ulceration on rabbit skin, v) rapid clearance from the site of inoculation, and vi) greatly reduced replication competency on a number of tisεue culture cell lineε including thoεe of human origin. Nevertheless, NYVAC based vectorε induce excellent reεponses to extrinεic immunogenε and provided protective immunity. TROVAC refers to an attenuated fowlpox that was a plague-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chickε. ALVAC iε an attenuated canarypox viruε-baεed vector that waε a plaque-cloned derivative of the licenεed canarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has some general properties which are the same as some general properties of Kanapox. ALVAC-baεed recombinant viruεeε expreεεing extrinεic immunogens have alεo been demonεtrated efficaciouε aε vaccine vectorε (Tartaglia et al., 1993 a,b) . This avipox vector is reεtricted to avian species for productive replication. On human cell cultures, canarypox virus replication is aborted early in the viral replication cycle prior to viral DNA εyntheεiε. Nevertheless, when engineered to expresε extrinsic immunogens, authentic expreεεion and proceεεing iε observed in vitro in mammalian cells and inoculation into numerous mammalian species induces antibody and cellular immune responses to the extrinsic immunogen and provides protection against challenge with the cognate pathogen (Taylor et al., 1992; Taylor et al., 1991). Recent Phase I clinical trials in both Europe and the United States of a canarypox/rabies glycoprotein recombinant (ALVAC-RG) demonstrated that the experimental vaccine was well tolerated and induced protective levels of rabiesviruε neutralizing antibody titerε (Cadoz et al., 1992; Frieε et al., 1992). Additionally, peripheral blood mononuclear cells (PBMCs) derived from the ALVAC-RG vaccinates demonstrated significant levels of lymphocyte proliferation when εtimulated with purified rabieε viruε (Frieε et al., 1992). NYVAC, ALVAC and TROVAC have also been recognized as unique among all poxviruseε in that the National Inεtitutes of Health ("NIH") (U.S. Public Health Service), Recombinant DNA Advisory Committee, which isεueε guidelineε for the phyεical containment of genetic material εuch as viruseε and vectors, i.e., guidelines for safety procedures for the uεe of such viruses and vectors which are based upon the pathogenicity of the particular virus or vector, granted a reduction in phyεical containment level: from BSL2 to BSL1. No other poxviruε has a BSL1 physical containment level. Even the Copenhagen strain of vaccinia virus - the common smallpox vaccine - has a higher phyεical containment level; namely, BSL2. Accordingly, the art haε recognized that NYVAC, ALVAC and TROVAC have a lower pathogenicity than any other poxvirus. Clearly based on the attenuation profileε of the NYVAC, ALVAC, and TROVAC vectorε and their demonεtrated ability to elicit both humoral and cellular immunological reεponεes to extrinsic immunogens (Tartaglia et al., 1993a,b; Taylor et al., 1992; Konishi et al., 1992) εuch recombinant viruses offer a distinct advantage over previouεly deεcribed vaccinia-baεed recombinant viruεeε. After growing the bacteria or infecting cellε with the recombinant virus, the glycoprotein(s) are collected by known techniqueε such aε chromatography (see Robbins, EPA 0162738A1; Panicali, EPA 0261940A2) . The collected glycoprotein(s) can then be employed in a vaccine, antigenic or immunological compoεition which also contains a suitable carrier. Accordingly, the inventive nucleotides are quite useful. Alternatively, the viral vector syεtem, eεpecially the preferred poxviruε vector εyεtem, can be employed in a vaccine, antigenic or immunological compoεition which also contains a suitable carrier. The CHV recombinant poxvirus in the compoεition expreεεes the CHV glycoprotein in vivo after administration or inoculation. The antigenic, immunological or vaccine composition of the invention (either containing glycoprotein(s) expreεεed from a vector εystem containing the inventive nucleotide(s) or containing a suitable vector system such as the CHV recombinant poxviruε) is administered to pups in the εame faεhion aε maternal antibodieε or immune εerum from εeropositive dogs (Carmichael, 1970) . Seronegative dogε are administered the composition in the same fashion aε other antigenic, vaccine or immunological compositions are administered. One skilled in the veterinary arts can determine dosage from this discloεure without undue experimentation, taking into conεideration εuch factorε aε the age, weight, breed, sex and general health of the particular dog or pup. Additionally, the inventive recombinant poxvirus and the expresεion products therefrom εtimulate an immune or antibody reεponεe in animals. From those antibodies, by techniques well-known in the art, monoclonal antibodies can be prepared and, those monoclonal antibodies or the antigens expresεed from the inventive nucleotideε, can be employed in well known antibody binding aεεays, diagnostic kits or teεtε to determine the preεence or abεence of particular CHV gB, gC and/or gD antigen(ε) or antibodies thereto and therefrom the presence or absence of the virus or, to determine whether an immune response to the virus or antigen(s) has simply been stimulated. Monoclonal antibodieε are immunoglobulinε produced by hybridoma cellε. A monoclonal antibody reactε with a εingle antigenic determinant and provides greater specificity than a conventional, εerum-derived antibody. Furthermore, εcreening a large number of monoclonal antibodieε makeε it poεεible to select an individual antibody with desired specificity, avidity and isotype. Hybridoma cell lineε provide a constant, inexpensive source of chemically identical antibodies and preparationε of εuch antibodieε can be eaεily εtandardized. Methodε for producing monoclonal antibodieε are well known to those of ordinary skill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265, iεεued Apr. 1, 1989, incorporated herein by reference. Uses of monoclonal antibodies are known. One εuch uεe iε in diagnostic methods, e.g., David, G. and Greene, H. , U.S. Pat. No. 4,376,110, iεεued Mar. 8, 1983, incorporated herein by reference. Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, e.g. Milstein, C. , 1980, Scientific American 243:66, 70, incorporated herein by reference. Additionally, the inventive nucleotideε can be used as probeε to aεcertain the preεence of CHV DNA in sampleε, aε well as in the generation of PCR primers for replicating or cloning CHV DNA. Methods for using DNA as probes or for preparing PCR primers are known in the art. Thus, the inventive nucleotides and expresεion products of the inventive nucleotides (and therefore the nucleotides) are quite useful. The following non-limiting Examples are given by way of illustration only and are not to be considered a limitation of thiε invention. EXAMPLES METHODS AND MATERIALS: Preparation of genomic CHV DNA. CHV (obtained from L. Carmichael, Cornell Univerεity) was propagated on Madin- Darby canine kidney (MDCK) cells (ATCC CCL34) . Viral DNA was isolated by εtandard methodology (Tartaglia et al., 1990) . DNA hybridization. CHV genomic DNA was digested with restriction enzymes, run on agarose gelε and tranεferred to Gene-Screen membraneε (New England Nuclear) under conditionε recommended by the manufacturers. Hybridizations were performed at 44°C, 53°C or 59°C in IM NaCl, 1% SDS and 10% dextran sulfate. The hybridization probe included a 1800 bp BamHI-Xbal fragment, containing an internal εegment of the feline herpesvirus (FHV) gB gene, a 950 bp BamHI-EcoRI fragment, containing the 3'-end of the FHV gC gene and a 970 bp BamHI-Hindlll fragment, containing the 3'-end of the FHV gD gene (Audonnet, unpublished resultε) . Cloninσ and DNA εequencinσ. CHV genomic fragmentε were εubcloned into pBlueεcriptSK (Stratagene) . Plaεmid DNA waε prepared and manipulated using εtandard techniqueε. Nucleotide sequencing was performed on double-stranded plaεmid templateε, uεing the modified T7 enzyme, Sequenaεe (U.S. Biochemical Corporation), and εtandard protocolε recommended by the manufacturer. M13 forward and reverεe primers were uεed to obtain initial εequence, and cuεtom primers, prepared with a Biosearch 8700 or an Applied Bioεystems 380B oligonucleotide εyntheεizer, were uεed for subsequent reactions. DNA and amino acid sequence analyses. DNA and amino acid εequence analyseε were performed with PC/GENE (IntelliGeneticε, Incorporated), ALIGN Pluε (Scientific and Educational Software) and IBI-Puεtell (International Biotechnologieε, Incorporated) εoftware packages. Homology searches were conducted on the SWISS-PROT (Releaεe 20 or 23) (IntelliGeneticε, Incorporated) databaεe, uεing the FASTA program (Pearson & Lipman, 1988) . DNA Cloning and Synthesis. Plasmids were constructed, screened and grown by standard procedures (Maniatis et al., 1982; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from Bethesda Reεearch Laboratorieε, Gaitherεburg, MD, New England Biolabε, Beverly, MA; and Boehringer Mannheim Biochemicalε, Indianapoliε, IN. Klenow fragment of E. coli polymeraεe waε obtained from Boehringer Mannheim Biochemicalε. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabε. The reagents were used as specified by the various suppliers. Synthetic oligodeoxyribonucleotides were prepared on a Bioεearch 8750 or Applied Bioεyεtems 380B DNA syntheεizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenaεe (Tabor et al., 1987) aε previouεly deεcribed (Guo et al., 1989). DNA amplification by polymeraεe chain reaction (PCR) for εequence verification (Engelke et al., 1988) waε performed uεing custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler. Exceεs DNA sequences were deleted from plasmids by restriction endonucleaεe digeεtion followed by limited digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides. Cells. Viruε. and Transfection. The originε and conditionε of cultivation of the Copenhagen strain of vaccinia virus has been previouεly deεcribed (Guo et al. , 95/26751 r -32- 1989) . Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are aε previouεly deεcribed (Piccini et al., 1987). The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus and NYVAC has been previously described (Guo et al., 1989; Tartaglia et al., 1992) . Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filterε and εcreening for B-galactoεidase activity are aε previously described (Panicali et al., 1982; Perkuε et al., 1989). The parental canarypox virus (Rentschler εtrain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 εerial paεsages on chick embryo fibroblaεtε. A master viral seed was subjected to four succeεsive plaque purifications under agar and one plaque clone was amplified through five additional passageε after which the εtock virus was used as the parental virus in in vitro recombination tests. The plaque purified canarypox isolate is designated ALVAC. The strain of fowlpox virus (FPV) designated FP-1 has been described previously (Taylor et al., 1988a). It is an attenuated vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France as a fowlpox scale from a chicken. The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on chicken embryo fibroblast cells. The virus was subjected to four succesεive plaque purificationε. One plaque iεolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, established. NYVAC, ALVAC and TROVAC viral vectors and their derivatives were propagated as described previously (Piccini et al., 1987; Taylor et al., 1988a,b) . Vero cells and chick embryo fibroblasts (CEF) were propagated aε deεcribed previouεly (Taylor et al., 1988a,b) . Examole 1 - IDENTIFICATION AND SEQUENCING OF THE CHV αB GENE Hybridization of CHV genomic DNA at relatively low εtringency with a radiolabelled probe containing the feline herpeεviruε (FHV) gB, gC and gD geneε (Audonnet, unpubliεhed reεults) identified one complimentary sequence. A 6 kb Xbal fragment containing this sequence was cloned and the nucleotide sequence of the hybridizing region was determined. The sequence of the nucleotide coding the CHV gB gene is shown in Fig. 1 together with the predicted amino acid expresεion (gB glycoprotein) therefrom. The putative tranεmembrane regionε and potential TATA, CAAT and polyadenylation εignal εequenceε are underlined. Nucleotideε and predicted amino acid reεidueε are numbered to the right of the sequence. An open reading frame (ORF) starting at position 201 and ending at position 2840 waε identified. The tranεlation product (predicted) of thiε ORF is 879 amino acids long. Comparison of this amino acid εequence with the SWISS-PROT (Release 20) database revealed significant homology with the gB glycoprotein of numerous herpesviruses. Additional analyseε revealed that the CHV gene product (predicted) waε more homologouε to the gB glycoprotein of alpha- herpeεviruεeε, εuch aε herpeε εimplex viruε type l (HSVl) , than beta- or gamma-herpeεviruses, εuch aε human cytomegaloviruε (HCMV) or Epεtein-Barr viruε (EBV) . Theεe analyεes and the results thereof are shown in Table 1 below. These results indicate that CHV should be classified as an alpha-herpesvirus; a concluεion that is consiεtent with the previouε classification of this viruε according to biological properties (Carmichael et al., 1965; Roizman, 1982). Table 1. HOMOLOGY BETWEEN THE PREDICTED AMINO ACID SEQUENCES OF 10 HERPESVIRUS αB GLYCOPROTEINS FHV EHV1 PRV BHV1 VZV MDV HSVl HCMV EBV CHV 78 FHV EHV1 PRV BHV1 VZV MDV HSVl HCMV Values in Table 1 were obtained using the ALIGN Plus program and are expressed as percent homology. The entire gB amino acid sequence was uεed. The alignment parameterε were: mismatch penalty •= 2, open gap penalty = 4, extended gap penalty «•= 1. References: FHV (Maeda et al., 1992), EHV1 (Whalley et al., 1989), PRV (Robbinε et al., 1987), BHV1 (Whitbeck et al., 1988), VZV (Keller et al., 1986), MDV (Roεε et al., 1989), HSVl (Bzik et al., 1984), HCMV (Kouzarides et al., 1987) and EBV (Pellett et al., 1985). Example 2 - ANALYSIS OF THE CHV σB NUCLEOTIDE SEQUENCE The 5'- and 3'-noncoding regions of the CHV gB gene contain numerous RNA polymerase II regulatory sequence motifs, εuch aε TATA box, CAAT box and polyadenylation εignal sequences (Corden et al., 1980; Proudfoot & Brownlee, 1976) (Fig. 1) . Potential TATA box sequenceε are found at poεitionε 34, 36, 119 and 148, approximately 165 bp, 160 bp, 80 bp and 50 bp upεtream from the CHV gB initiation codon. Potential CAAT box εequenceε (ATTG) are found at positions 89, 97 and 165, approximately 110 bp, 100 bp and 35 bp upstream from the gB initiation codon. Potential polyadenylation εignal sequences (AATAAA) are found at positionε 2839 and 2961, approximately 0 bp and 120 bp downεtream from the CHV gB termination codon. The nucleotide εequence εurrounding the initiation codon haε been εhown to affect the efficiency of tranεlation initiation (Kozak, 1986) . In particular, the sequence, [A/G]NNATGG r has been found to be moεt efficient. Therefore, relative to Kozak , ε ruleε, the nucleotide sequence εurrounding the CHV gB initiation codon (AGTATGT) iε favorable at poεition -3, but not at poεition +4 (Fig. 1) . The fact that the CHV gB gene doeε not follow Kozak'ε ruleε iε not unuεual. The FHV (Maeda et al., 1992), PRV (Robbinε et al., 1987), varicella-zoεter viruε (VZV) (Keller et al., 1986), MDV (Roεε et al., 1989) and HSVl (Bzik et al., 1984) gB genes alεo contain a pyrimidine at position +4. Example 3 - ANALYSIS OF THE PREDICTED CHV gB AMINO ACID SEQUENCE The deduced amino acid sequence of the CHV gB homologue is presented in Fig. 1. ' Hydropathicity analyεiε of thiε amino acid εequence iε εhown in Fig. 2. The profile waε obtained with the PC/GENE SOAP program, using the method of Kyte & Doolittle (1982) and an interval of 13 amino acids. The vertical axis representε relative hydropathicity, where poεitive valueε are hydrophobic and negative values are hydrophilic. The horizontal axis representε the amino acid number of the CHV gB homologue. Hydropathicity analyεiε of thiε amino acid sequence revealed the presence of 2 prominent hydrophobic peaks. The firεt peak, located at the N-terminus, without wishing to be bound by any one theory, represents a potential signal sequence. N-terminal signal sequenceε initiate tranεport across the endoplasmic reticulum membrane and can be critical for the proper post-tranεlational modification and targeting of glycoproteinε (Blobel, 1980) . Signal sequences vary in length from about 15-30 residueε and uεually conεiεt of a baεic N-terminal region, a central hydrophobic region and a short, relatively polar C-terminal region. In addition, the cleavage site usually conforms to the -3,-1 rule, where the residue at position -1 is small (Ala, Ser, Gly, Cys, Thr or Gin) and the residue at poεition -3 is not aromatic (Phe, His, Tyr or Trp) , charged (Aεp, Glu, Lys or Arg) or large and polar (Asn or Gin) , and reεidueε -3 through +1 are not Pro (von Heijne, 1986). Although analyεiε with PSIGNAL, a PC/GENE program deεigned to detect eukaryotic εignal εequenceε, doeε not identify the N- terminal end of CHV gB aε a potential εignal εequence, thiε region doeε have elements consiεtent with typical εignal sequences; namely a hydrophobic core (residues 2-17) and a relatively polar C-terminal region (Fig. 1) . The fact that PSIGNAL doeε not detect a εignal εequence in the N-terminal region of CHV gB iε not unique. Thiε algorithm alεo doeε not detect a signal sequence in the N-terminal region of the VZV gB homologue. The second, very broad, hydrophobic peak(s) (Fig. 2), with predicted membrane-εpanning segments between amino acid residueε 725 and 741 and 746-750 and 766-772 (uεing the method of Klein et al. (1985)), without wishing to be bound by any one theory, functions as a membrane anchor region. It has been hypothesized that the transmembrane domain of HSVl gB, as well as other gB homologueε, transverses the membrane 3 times (Pellett et al., 1985). Hydropathicity analysiε of CHV gB revealε the preεence of at leaεt 2 diεtinct hydrophobic peakε. Therefore, CHV gB and HSVl gB have similar transmembrane structureε. Alignment of the CHV gB amino acid εequence with εimilar εequenceε from other herpeεviruεeε revealed extenεive homology throughout the entire εequence, with the exception of the N-terminuε, a region surrounding the putative cleavage εite (see below) and a region near the C- terminus. Figs. 3A and 3B show the amino acid homology of 8 gB homologues. The amino acid εequences of the CHV, FHV, EHV1, PRV, HSVl, VZV, HCMV and EBV gB homologues (for references from which the εequences were obtained, see text below Table 1) were aligned using the PC/GENE CLUSTAL program. Gaps, indicated by daεheε, were introduced to maximize homology. Aligned residues which are identical in all 8 εequenceε are indicated by an aεteriεk (*) . Aligned residues which are identical in the majority of εequences are indicated by a period (.). Conserved cysteine residueε are boxed. Potential N-linked glycosylation sites are shaded. Putative proteolytic cleavage εiteε are underlined. Thiε alignment alεo revealed that the vast majority of cysteine reεidueε are perfectly conεerved. For example, CHV gB contains 11 cysteine residues, 10 of which are perfectly conserved in all alpha-, beta- and gamma-herpesviruεes. In fact, the only cysteine residue in CHV gB that is not conserved is found near the N-terminus and may be located in the putative signal sequence. These resultε εhow that the gB glycoproteinε have relatively εimilar tertiary εtructureε. Alignment of the gB amino acid εequenceε also revealed that the potential N-linked glycosylation sites are relatively well conserved (Figs. 3A and 3B) . N-linked oligoεaccharideε can be added to Asn residues that have the εequence Asn-X-Ser or Asn-X-Thr, where X is not Pro (Bauεe, 1983) . CHV gB containε 13 potential N-linked glycoεylation εiteε. Three of theεe εiteε, however, are εituated in the putative cytoplasmic domain and, therefore, may not be glycosylated. The location of the potential N-linked glycoεylation siteε is relatively well conserved in the majority of gB glycoproteins (Figs. 3A and 3B) . The gB glycoprotein of most herpesviruses is cleaved internally during maturation, with the subεequent peptides being held together by disulfide bonds. The VZV gB homologue (gpll) , for example, is cleaved between Arg and Ser residueε, resulting in 2 glycoproteins of approximately 60 kd (Keller et al., 1986). The gB glycoproteins of FHV (Maeda et al., 1992), equine herpesvirus type 1 (EHV1) (Whalley et al., 19989), PRV (Robbins et al., 1987), BHV1 (Whitbeck et al., 1988), MDV (Ross et al., 1989) and HCMV (Kouzarides et al., 1987) are alεo cleaved. Furthermore, a sequence, Arg-X-Arg-Arg/Lys—Ser/Ala, similar to the εequence at the VZV cleavage site, Arg-Thr-Arg-Arg—Ser, is preεent at virtually the εame location in each of these gB glycoproteinε. Converεely, thiε εequence iε not found in the HSVl (Bzik et al., 1984) and EBV (Pellett et al., 1985) gB glycoproteins, which are not cleaved. The significance of this cleavage event iε unknown. It doeε not appear, however, to be eεεential for replication, in vitro , since εtrainε of BHV1 (Blewett & Miεra, 1991) and HCMV (Spaete et al., 1990) that have been mutated at the cleavage εite, and therefore encode an uncleaved gB glycoprotein, are εtill infectiouε. Without wiεhing to be bound by the theory that CHV gB is cleaved internally, proteolytically, the sequence, Arg-Lyε-Arg-Arg—Ser, iε preεent at the εame location in CHV aε in VZV, FHV, EHV1, PRV, BHV1, MDV and HCMV. Example 4 - IDENTIFICATION AND SEQUENCING OF THE CHV σC GENE CHV genomic fragmentε were randomly cloned into pBlueεcriptSK. The nucleotide εequence of the termini of theεe fragmentε waε determined and the predicted amino acid εequence of potential ORFε were analyzed for homology againεt the SWISS-PROT (Releaεe 20) amino acid databaεe. Uεing this methodology, a 12 kb Xbal fragment encoding an ORF with homology to herpesviruε gC glycoproteins was identified. The nucleotide εequence of thiε ORF iε preεented in Fig. 4. Fig. 4 εhowε the nucleotide εequence and predicted amino acid εequence of the CHV gC homologue and ORF2. The putative tranεmembrane region and potential TATA, CAAT and polyadenylation εignal εequenceε are underlined. Nucleotideε and predicted amino acid reεidueε are numbered to the right of the sequence. The putative CHV gC gene starts at position 201 and ends at poεition 1580. The predicted tranεlation product iε 459 amino acidε long. Comparison of this amino acid sequence with the sequence of gC glycoproteins from other herpesviruses is shown in Table 2, below, and revealed extensive homology, indicating that this ORF encodes the CHV gC homologue (Table 2) . Table 2. HOMOLOGY BETWEEN THE PREDICTED AMINO ACID SEQUENCES OF 9 HERPESVIRUS oC GLYCOPROTEINS FHV EHV1 EHV4 PRV BHV1 VZV MDV HSVl CHV 44 32 FHV 32 EHV1 EHV4 PRV BHV1 VZV MDV Valueε in Table 2 were obtained uεing the ALIGN Pluε program and are expreεεed aε percent homology. The entire gC amino acid εequence waε uεed. See Table 1 for alignment parameters. References: FHV (Audonnet, unpublished resultε) , EHV1 (Allen & Coogle, 1988), EHV4 (Nicolεon & Onions, 1990), PRV (Robbins et al., 1986), BHVi (Fitzpatrick et al., 1989), VZV (Daviεon & Scott, 1986), MDV (Ihara et al., 1989) and HSVl (McGeoch et al., 1988). Example 5 - ANALYSIS OF THE CHV σC NUCLEOTIDE SEQUENCE Potential TATA box εequenceε (TATA) are found at poεitions 22 and 81, approximately 180 bp and 120 bp upstream from the CHV gC initiation codon (Fig. 4) . An additional TATA sequence is found at poεition 175. Due to itε proximity to the gC initiation codon, however, thiε sequence may not be a potential TATA box sequence. Potential CAAT box sequenceε (CAAT and ATTG) are found at poεitionε 13, 59 and 119, approximately 190 bp, 140 bp and 80 bp upεtream from the gC initiation codon. A potential polyadenylation signal sequence (AATAAA) is found at poεition 1744, approximately 165 bp downεtream from the CHV gC termination codon and 45 bp within ORF2 (see below) . Other potential polyadenylation signal-like εequenceε are also found in the gC 3 , -noncoding region. Like the CHV gB gene, the nucleotide sequence surrounding the CHV gC initiation codon (AAAATGA) is favorable with respect to Kozak , s ruleε at position -3, but not at position +4 (Fig. 4). The FHV (Audonnet, unpublished resultε) , EHV1 (Allen & Coogle, 1988) and VZV (Daviεon & Scott, 1986) gC geneε alεo contain an unfavorable nucleotide at poεition +4. Example 6 - ANALYSIS OF THE CHV gC AMINO ACID SEQUENCE PREDICTED The deduced amino acid εequence of the CHV gC homologue iε preεented in Fig. 4. Fig. 5 εhows the hydropathicity analysiε of the CHV gC homologue. The profile waε obtained with the PC/GENE SOAP program, uεing the method of Kyte & Doolittle (1982) and an interval of 13 amino acidε. The vertical axiε repreεentε relative hydropathicity, where poεitive valueε are hydrophobic and negative valueε are hydrophilic. The horizontal axis representε the amino acid number of the CHV gC homologue. Hydropathicity analysis of the predicted CHV gC amino acid sequence revealed the presence of 2 prominent hydrophobic peaks (Fig. 5) . The first peak, located at the N-terminuε, without wiεhing to be bound by any one theory, repreεentε a potential εignal εequence. Although analyεiε with PSIGNAL doeε not identify the N-terminal end of thiε polypeptide aε a potential εignal sequence, this region does have a basic N-terminal region, a hydrophobic core (residues 6-20) and a relatively polar C-terminal region (Fig. 4) . The second hydrophobic peak, with a predicted membrane- spanning segment between reεidueε 424-433 and 449-456 (uεing the method of Klein et al. (1985)), without wishing to be bound by any one theory, functions aε a membrane anchor region. Fig. 6 εhowε the amino acid homology of 4 gC homologueε. The amino acid sequences of the CHV, FHV, EHV1 and HSVl gC homologues (for references see Table 2) were aligned using the PC/GENE CLUSTAL program. Gaps, indicated by dashes, were introduced to maximize homology. Aligned residues which are identical in all 4 sequences are indicated by an aεteriεk (*) . Aligned residueε which are identical in the majority of εequences are indicated by a period (.). Conserved cysteine residueε are boxed. Potential N-linked glycosylation siteε are shaded. Alignment of the CHV gC amino acid sequence with homologous sequences from other herpesviruεeε revealed a moderate level of homology throughout the entire sequence, with the exception of the N-terminus (Fig. 6) . This alignment alεo revealed that the majority of cyεteine reεidueε are perfectly conserved. For example, CHV gC contains 10 cysteine residueε, 8 of which are perfectly conserved in all alpha-herpesviruεeε. In fact, the only cysteine residueε in CHV gC that are not conεerved are located in the putative tranεmembrane or intracellular domainε. These resultε show that the gC glycoproteins have relatively similar tertiary structureε. Alignment with other gC εequenceε alεo revealed the relative conεervation of potential N-linked glycoεylation sites. Example 7 - IDENTIFICATION AND SEQUENCING OF ORF2 Nucleotide sequence analysiε of the region downεtream from the CHV gC gene revealed the presence of a second ORF (Fig. 4) . This ORF (ORF2) starts at position 1699 and endε at poεition 2226. The predicted translation product is 175 amino acidε long. Table 3, below, shows the comparison of this amino acid sequence with the SWISS-PROT (Release 23) database revealed significant homology with the ORFs located downstream from other alpha-herpesviruε gC geneε. The homology scores for the ORF2 homologues showε that in CHV, FHV, EHV1, equine herpeεviruε type 4 (EHV4) , MDV, herpesvirus of turkey (HVT) and posεibly HSVl, the ORF located downstream from the gC gene represents a highly divergent, but evolutionarily related, gene family. Conversely, the ORF (gene 13) located next to the VZV gC gene does not exhibit significant homology with any of the other comparably positioned ORFs. Furthermore, gene 13 iε oriented on the genome in the oppoεite direction relative to all the other ORF2-like geneε (Davison & Scott, 1986) . These resultε are conεiεtent with the propoεed functions of the proteins encoded by theεe.2 groups of genes; VZV gene 13 encodes a thymidylate synthetase (Daviεon and Scott, 1986) , whereaε the HSVl ORF2-like gene (UL45) encodeε a putative virion protein (Telford et al., 1992). Therefore, the ORFε located next to the gC gene in CHV, FHV, EHV1, EHV4, MDV, HVT and poεεibly HSVl encode proteins that are structurally and functionally unrelated to the protein encoded downstream from the VZV gC homologue. Valueε in Table 3 were obtained uεing the FASTA and RDF2 programε (Pearεon & Lipman, 1988) . A ktup of l waε used. Values in parentheεeε repreεent the number of εtandard deviationε between the FASTA εcore and the mean of the scores obtained from 100 randomly permutated versionε of the potentially related sequence. References: FHV (Audonnet, unpublished results), EHVl (Telford et al., 1992), EHV4 (Nicolεon & Onions, 1990), MDV (Ihara et al., 1989), HVT (Kato et al., 1989), HSVl (McGeoch et al., 1988) and VZV (Davison & Scott, 1986) . Example 8 - ANALYSIS OF THE CHV ORF2 NUCLEOTIDE SEQUENCE Potential TATA box sequences (TATA) are found at positions 1604, 1606, 1635 and 1662, approximately 95, 93, 65 and 35 bp upstream from the ORF2 initiation codon and approximately 24, 26, 55 and 80 bp downstream from the gC gene termination codon (Fig. 4) . A potential CAAT box sequence (CAAT) is found at position 1584, approximately 115 bp upstream from the initiation codon. Potential polyadenylation εignal εequences (AATAAA) are found at overlapping positionε 2225, 2229, 2234 and 2238, approximately 0-15 bp downstream from the 0RF2 termination codon. The nucleotide sequence surrounding the 0RF2 initiation codon (AATATGGϊ is favorable with respect to Kozak's rules at positionε -3 and +4. Example 9 - IDENTIFICATION AND SEQUENCING OF THE CHV σD GENE ; Employing the same methodology used to map the CHV gC homologue, a 7 kb PstI fragment encoding an ORF with homology to herpesvirus gD glycoproteins was identified. Fig. 7 shows the nucleotide sequence and predicted amino acid sequence of the CHV gD homologue. The putative εignal εequence, tranεmembrane region and potential polyadenylation εignal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence. The CHV gD gene startε at poεition 201 and ends at position 1238. The translation product (predicted) is 345 amino acids long. Table 4, below, provides comparison of this amino acid sequence with the sequence of other gD glycoproteins and, revealed extenεive homology, indicating that thiε ORF encodeε the CHV gD homologue. Table 4. Homology between the predicted amino acid εequenceε of 6 herpeεviruε gD glvcoproteinε FHV EHV1 PRV BHV1 HSVl CHV 45 35 27 34 21 FHV 31 30 34 24 EHV1 26 27 21 PRV 37 27 BHV1 24 Valueε in Table 4 were obtained uεing the ALIGN Pluε program and are expressed as percent homology. The entire gD amino acid sequence was used. See Table 1 for alignment parameters. References: FHV (Audonnet, unpublished resultε) , EHV1 (Flowerε et al., 1991), PRV (Petrovskis et al., 1986), BHV1 (Tikoo et al., 1990) and HSVl (Lasky & Dowbenko, 1984) . Example 10 - ANALYSIS OF THE CHV αD NUCLEOTIDE SEQUENCE No TATA or CAAT/ATTG sequences were identified immediately upstream from the CHV gD gene (Fig. 7) . Numerous potential TATA box-like εequenceε, however, were found. Potential polyadenylation εignal sequences (AATAAA) were found at positions 1260 and 1287, approximately 25 bp and 50 bp downstream from the CHV gD termination codon. Like the CHV gB and gC genes, the nucleotide sequence εurrounding the CHV gD initiation codon (AAAATGA) iε favorable with reεpect to Kozak'ε ruleε at poεition -3, but not at poεition +4 (Fig. 7) . The FHV (Audonnet, unpublished reεultε) , EHV1 (Audonnet et al., 1990; Flowerε et al., 1991), PRV (Petrovεkiε et al., 1986) and BHV1 (Tikoo et al., 1990) gD geneε alεo contain an unfavorable nucleotide at poεition +4. Exam p le 11 - ANALYSIS OF THE PREDICTED CHV gD AMINO ACID SEQUENCE The deduced amino acid εequence of the CHV gD homologue iε preεented in Fig. 7. Fig. 8 εhows the hydropathicity analyεiε of the CHV gD homologue. The profile waε obtained with the PC/GENE SOAP program, uεing the method of Kyte & Doolittle (1982) and an interval of 11 amino acidε. The vertical axiε repreεentε relative hydropathicity, where poεitive valueε are hydrophobic and negative valueε are hydrophilic. The horizontal axiε represents the amino acid number of the CHV gD homologue. Hydropathicity analysis of the predicted CHV gD amino acid sequence revealed the presence of 2 prominent hydrophobic peakε (Fig 8) . The firεt peak, located at the N-terminuε, without wiεhing to be bound by any one theory, represents a potential εignal εequence. In fact, PSIGNAL identified a potential cleavage site between positions 16 and 17. The second hydrophobic peak, with a predicted membrane-spanning segment between residues 304-311 and 327- 332 (using the method of Klein et al. (1985)), without wiεhing to be bound by any one theory, functions as a membrane anchor region. Fig. 9 showε amino acid homology of 4 gD homologueε. The amino acid εequenceε of the CHV, FHV, EHV1 and HSVl gD homologueε (for referenceε εee Table 4) were aligned uεing the PC/GENE CLUSTAL program. Gapε, indicated by daεheε, were introduced to maximize homology. Aligned reεidueε which are identical in all 4 εequenceε are indicated by an aεteriεk (*) . Aligned reεidueε which are identical in the majority of εequenceε are indicated by a period (.). Conserved cysteine residues are boxed. Potential N-linked glycosylation sites are shaded. Alignment of the CHV gD amino acid sequence with homologous εequenceε from other herpesviruseε revealed a moderate level of homology throughout the entire sequence, with the exception of the N- terminuε (Fig. 9) . Thiε alignment alεo revealed that the vast majority of cysteine reεidues are perfectly conserved. For example, CHV gD contains 6 cysteine residues, all of which are perfectly conserved in all alpha-herpeεviruses. These results show that the gD glycoproteins have relatively similar tertiary structureε. Thiε alignment alεo revealed that the potential N-linked glycoεylation εiteε are well conserved. Without wishing to be bound by any theory that the CHV gD glycosylation siteε are utilized, it iε known that all of the potential HSVl gD glycoεylation εiteε are used (Sodora et al., 1991). Example 12 - GENOMIC ORGANIZATION The gB, gC and gD genes were not mapped to specific locations on the CHV genome. Nucleotide sequence analyses of the regions flanking these genes, however, indicateε that the genomic organization of CHV iε εimilar to other alpha- herpeεviruses. For example, the ORF located immediately upstream from the CHV gB gene has homology with gene 30 of VZV (Davison & Scott, 1986) and UL28 of HSVl (McGeoch et al., 1988), both of which are located immediately upstream from the gB homologue in those viruses. 0RF2, located immediately downstream from the CHV gC gene, has homology with the ORFε located immediately downεtream from the gC homologue in FHV (Audonnet, unpublished resultε) , EHV1 (Telford et al., 1992), EHV4 (Nicolεon & Onionε, 1990), HVT (Kato et al., 1988) and perhapε HSVl (McGeoch et al., 1988. Additionally, the ORF located immediately downεtream from the CHV gD gene haε homology to the gl gene of EHV1 (Audonnet et al., 1990) and the gp63 gene of PRV (Petrovεkiε et al., 1986), both of which are located immediately downεtream from the gD homologue in thoεe viruεeε (data not εhown) . Exam p le 13 - CONSTRUCTION OF PLASMID pSD460 FOR DELETION OF THYMIDINE KINASE GENE (J2R) Referring now to FIG. 10, plaεmid pSD406 containε vaccinia Hindlll J (poε. 83359 - 88377) cloned into pUC8. pSD406 waε cut with Hindlll and PvuII. and the 1.7 kb fragment from the left εide of Hindlll J cloned into pUC8 cut with Hindlll/Smal. forming pSD447. pSD447 containε the entire gene for J2R (poε. 83855 - 84385). The initiation codon iε contained within an Nlalll εite and the termination codon is contained within an Sspl εite. Direction of tranεcription iε indicated by an arrow in FIG. 10. 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 iεolated. Annealed synthetic oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:21/SEQ ID NO:22) Smal MPSYN 3 5' TAATTAACTAGCTACCCGGG 3' MPSYN44 3' GTACATTAATTGATCGATGGGCCCTTAA 5' Nlalll EcoRI were ligated with the 0.5 kb flindlll/fflalll fragment into pUClδ vector plasmid cut with Hindlll/EcoRI. generating plasmid pSD449. To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with Sεpl (partial) within vaccinia εequenceε and Hindlll at the pUC/vaccinia junction, and a 2.9 kb vector fragment isolated. Thiε vector fragment was ligated with annealed synthetic oligonucleotideε MPSYN45/MPSYN46 (SEQ ID NO:23/SEQ ID NO:24) Hindlll Smal MPSYN45 5' AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA MPSYN46 3' AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT NotI Sεpl ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5' MPSYN46 generating pSD459. To combine the left and right flanking armε into one plaεmid, a 0.5 kb Hindlll/Smal fragment waε iεolated from pSD449 and ligated with pSD459 vector plaεmid cut with Hindlll/Smal. generating plaεmid pSD460. pSD460 waε uεed as donor plaεmid for recombination with wild type parental vaccinia viruε Copenhagen εtrain VC-2. 3 P labelled probe waε synthesized by primer extension using MPSYN45 (SEQ ID NO:23) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:25) (5' TTAGTTAATTAGGCGGCCGC 3') as primer. Recombinant virus vP410 was identified by plaque hybridization. Example 14 - CONSTRUCTION OF PLASMID pSD486 FOR DELETION OF HEMORRHAGIC REGION (B13R + B14RΪ Referring now to FIG. 11, plasmid pSD419 containε 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, B13R - B14R (pos. 172,549 - 173,552), pSD419 was used as the source for the left flanking arm and pSD422 was used as the source of the right flanking arm. The direction of transcription for the u region is indicated by an arrow in FIG. 11. To remove unwanted εequences from pSD419, sequences to the left of the Ncol site (pos. 172,253) were removed by digestion of pSD419 with Ncol/Smal followed by blunt ending with Klenow fragment of E. coli polymerase and ligation generating plasmid pSD476. A vaccinia right flanking arm was obtained by digestion of pSD422 with Hoal at the de¬ termination codon of B14R and by digestion with Nrul 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb Hindi vector fragment iεolated 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 εequenceε in pSD477 were removed by digeεtion with Clal/Hpal. and the reεulting vector fragment waε ligated with annealed εynthetic oligonucleotideε SD22mer/SD20mer (SEQ ID NO:26/SEQ ID NO:27) £lal lamHI Hoal SD22mer 5' CGATTACTATGAAGGATCCGTT 3' SD20mer 3' TAATGATACTTCCTAGGCAA 5' generating pSD479. pSD479 containε an initiation codon (underlined) followed by a BamHI εite. To place E. coli Beta-galactoεidaεe in the B13-B14 (u) deletion locuε under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactoεidaεe gene (Shapira et al., 1983) waε inεerted into the BamHI εite of pSD479, generating pSD479BG. pSD479BG waε uεed aε donor plasmid for recombination with vaccinia virus VP410. Recombinant vaccinia viruε VP533 waε iεolated aε a blue plaque in the preεence of chromogenic substrate X-gal. In VP533 the B13R- B14R region is deleted and is replaced by Beta- galactosidase. To remove Beta-galactosidase εequences from vP533, plasmid pSD486, a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized. First the Clal/Hpal vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:28/SEQ ID NO:29) Clal Sad Xhol Hpal SD42mer 5 * CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3 ' SD40mer 3 ' TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5 ' Bαlll Smal BamHI generating plaεmid 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 plaεmid pSD478E~ pSD478E" waε digeεted with BamHI and Hpal and ligated with annealed εynthetic oligonucleotides HEM5/HEM6 (SEQ ID NO:30/SEQ ID NO:31) BamHI EcoRI Hoal HEM5 5' GATCCGAATTCTAGCT 3' HEM6 3' GCTTAAGATCGA 5' generating plasmid pSD486. pSD486 was used aε donor plaεmid for recombination with recombinant vaccinia viruε VP533, generating VP553, which waε iεolated aε a clear plaque in the presence of X-gal. Exam p le IS - CONSTRUCTION OF PLASMID pMP 94Δ FOR DELETION OF ATI REGION (A26L) Referring now to FIG. 12, pSD414 contains Sail B cloned into pUC8. To remove unwanted DNA sequenceε to the left of the A26L region, pSD414 waε cut with Xbal within vaccinia sequences (pos. 137,079) and with Hindlll at the pUC/vaccinia junction, then blunt ended with Klenow fragment of E. coli polymerase and ligated, resulting in plasmid pSD483. To remove unwanted vaccinia DNA sequences to the right of the A26L region, pSD483 was cut with EcoRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484. To remove the A26L coding region, pSD484 waε 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 waε isolated and ligated with annealed εynthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:32/SEQ ID NO:33) Ndel ATI3 5' TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT ATI4 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA SailI EcoRI Hpal TATATAAATAGATCTGAATTCGTT 3' ATI3 ATATATTTAT C TA G A C TTAA GC AA 5 . ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region containing the restriction sites Bglll. EcoRI and Hpal. as indicated above. The reεulting plasmid was designated pSD485. Since the Bglll and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted Bglll and EcoRI sites were removed from plaεmid pSD483 (deεcribed above) by digeεtion with Balll (poε. 140,136) and with EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenow fragment of E. coli polymeraεe and ligation. The reεulting plaεmid waε deεignated pSD489. The 1.8 kb £lal (poε. 137,198)/ficoRV (poε. 139,048) fragment from pSD489 containing the A26L ORF waε replaced with the correεponding 0.7 kb polylinker- containing Clal/EcoRV fragment from pSD485, generating pSD492. The Bglll and EcoRI sites in the polylinker region of pSD492 are unique. A 3.3 kb Bglll 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) was inserted into the Bglll εite of pSD492, forming pSD493KBG. Plaεmid pSD493KBG waε uεed in recombination with reεcuing viruε VP553. Recombinant vaccinia viruε, VP581, containing Beta-galactoεidase in the A26L deletion region, was iεolated aε a blue plaque in the preεence of X-gal. To generate a plaεmid for the removal of Beta- galactoεidase εeguenceε from vaccinia recombinant viruε VP581, the polylinker region of plaεmid pSD492 waε deleted by mutageneεiε (Mandecki, 1986) uεing synthetic oligonucleotide MPSYN177 (SEQ ID NO:34) (5' AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3'). In the resulting plaεmid, pMP494Δ, vaccinia DNA encompaεsing poεitionε [137,889 - 138,937], including the entire A26L ORF iε deleted. Recombination between the pMP494Δ and the Beta- galactosidase containing vaccinia recombinant, VP581, resulted in vaccinia deletion mutant vP618, which was isolated aε a clear plaque in the preεence of X-gal. Example 16 - CONSTRUCTION OF PLASMID pSD 67 FOR DELETION OF HEMAGGLUTININ GENE (A56R) Referring now to FIG. 13, vaccinia Sail G restriction fragment (poε. 160,744-173,351) croεεeε the Hindlll A/B junction (poε. 162,539). pSD419 containε vaccinia Sail G cloned into pUC8. The direction of tranεcription for the hemagglutinin (HA) gene iε indicated by an arrow in FIG. 13. Vaccinia εequenceε derived from Hindlll B were removed by digeεtion of pSD4l9 with Hindlll within vaccinia εequenceε and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, containε the HA gene, A56R, flanked by 0.4 kb of vaccinia εequenceε to the left and 0.4 kb of vaccinia εequenceε to the right. A56R coding sequenceε were removed by cutting pSD456 with Rεal (partial; poε. 161,090) upεtream from A56R coding sequenceε, and with EaqI (poε. 162,054) near the end of the gene. The 3.6 kb Rεal/Eagl vector fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:35), MPSYN62 (SEQ ID NO:36), MPSYN60 (SEQ ID NO:37), and MPSYN61 (SEQ ID N0:38) Real MPSYN59 5' ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT- MPSYN62 3' TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA- MPSYN59 AGTTGATAGAACAAAATACATAATTT 3' MPSYN62 TCAACTATCT 5' MPSYN60 5' TGTAAAAATAAATCACTTTTTATA- MPSYH61 3' TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT- MPSYN60 3' MPSYN61 5' reconstructing the DNA sequenceε upεtream from the A56R ORF and replacing the A56R ORF with a polylinker region aε indicated above. The reεulting plaεmid iε pSD466. The vaccinia deletion in pSD466 encompaεεeε poεitionε [161,185- 162,053]. The εite of the deletion in pSD466 iε indicated by a triangle in FIG. 13. A 3.2 kb Bglll/BamHI (partial) caεεette 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 inεerted into the Bglll site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination with rescuing virus VP618. Recombinant vaccinia virus, vP708, containing Beta- galactoεidase in the A56R deletion, waε isolated as a blue plaque in the preεence of X-gal. Beta-galactoεidaεe εequenceε were deleted from VP708 uεing donor plasmid pSD467. pSD467 iε identical to pSD466, except that EcoRI. Smal and BamHI εiteε were removed from the pUC/vaccinia junction by digeεtion of pSD466 with EcoRI/BamHI followed by blunt ending with Klenow fragment of E. coli polymeraεe and ligation. Recombination between VP708 and pSD467 reεulted in recombinant vaccinia deletion mutant, vP723, which waε isolated as a clear plaque in the preεence of X-gal. Example 17 - CONSTRUCTION OF PLASMID pMPCSKlΔ FOR DELETION OF OPEN READING FRAMES TC7L-K1L1 Referring now to FIG. 14, the following vaccinia clones were utilized in the construction of pMPCSKlΔ. 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 sequenceε to the left of the Sohl εite (pos. 27,416) in Hindlll M are removed (Perkus et al., 1990). pSD409 is Hindlll M cloned into pUC8. To provide a subεtrate for the deletion of the [C7L- K1L] gene cluεter from vaccinia, E. coli Beta-galactoεidaεe waε first inserted into the vaccinia M2L deletion locuε (Guo et al., 1990) aε followε. To eliminate the Bglll εite in pSD409, the plaεmid waε cut with Bglll in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plaεmid pMP409B. pMP409B was cut at the unique SphI site (pos. 27,416). M2L coding sequences were removed by mutagenesiε (Guo et al., 1990; Mandecki, 1986) uεing εynthetic oligonucleotide BolII MPSYN82 (SEQ ID NO:39) 5' TTTCTGTATATTTGCACCAATTTAGATCTT- ACTCAAAATATGTAACAATA 3' The reεulting plaεmid, pMP409D, containε a unique B^III εite inserted into the M2L deletion locus aε indicated above. A 3.2 kb BamHI (partial) /Bglll caεεette containing the E. coli Beta-galactoεidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) waε inserted into pMP409D cut with Bglll. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plaεmid for recombination with reεcuing vaccinia viruε VP723. Recombinant vaccinia viruε, VP784, containing Beta- galactoεidaεe inεerted into the M2L deletion locuε, waε isolated aε a blue plaque in the preεence of X-gal. A plasmid deleted for vaccinia genes [C7L-K1L] waε assembled in pUC8 cut with Smal. Hindlll and blunt ended with Klenow fragment of E. coli polymerase. The left flanking arm consiεting of vaccinia Hindlll C εequences was obtained by digestion of pSD420 with Xbal (poε. 18,628) followed by blunt ending with Klenow fragment of E. coli polymerase and digestion with BolII (pos. 19,706). The right flanking arm consisting of vaccinia &indIII K sequenceε waε obtained by digestion of pSD451 with Bglll (poε. 29,062) and EcoRV (poε. 29,778). The reεulting plaεmid, pMP581CK iε deleted for vaccinia εequenceε between the Ig ll εite (poε. 19,706) in Hindlll C and the Bglll εite (poε. 29,062) in Hindlll K. The εite of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 14. To remove exceεε DNA at the vaccinia deletion junction, plaεmid pMP581CK, waε cut at the Ncol εiteε within vaccinia sequences (pόs. 18,811; 19,655), treated with Bal-31 exonucleaεe and εubjected to mutageneεiε (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:40) 5'- TGTCATTTAACACTATACTCATATTAAT AAAAATAATATTTATT-3' . The resulting plasmid, pMPCSKlΔ, is deleted for vaccinia sequences positions 18,805-29,108, encompasεing 12 vaccinia open reading frameε [C7L - KIL]. Recombination between pMPCSKlΔ and the Beta-galactoεidaεe containing vaccinia recombinant, VP784, reεulted in vaccinia deletion mutant, vP804, which waε iεolated aε a clear plaque in the preεence of X-gal. Example 18 - CONSTRUCTION OF PLASMID pED5 8 FOR DELETION OF LARGE SUBUNIT, RIBONUCLEOTIDE REDUCTASE (I4L) Referring now to FIG. 15, plaεmid pSD405 containε vaccinia Hindlll I (poε. 63,875-70,367) cloned in pUC8. pSD405 waε digeεted with EcoRV within vaccinia sequences (pos. 67,933) and with Smal at the pUC/vaccinia junction, and ligated, forming plaεmid pSD518. pSD518 waε uεed aε the εource of all the vaccinia reεtriction fragmentε uεed in the conεtruction of pSD548. The vaccinia I4L gene extendε from poεition 67,371- 65,059. Direction of tranεcription for I4L iε indicated by an arrow in FIG. 15. To obtain a vector plaεmid fragment deleted for a portion of the I4L coding εequenceε, pSD518 waε digested with BamHI (pos. 65,381) and Hpal (pos. 67,001) and blunt ended using Klenow fragment of E. coli polymerase. This 4.8 kb vector fragment waε ligated with a 3.2 kb Smal caεεette containing the E. coli Beta-galactoεidaεe gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkuε et al., 1990), reεulting in plaεmid pSD524KBG. pSD524KBG waε uεed aε donor plaεmid for recombination with vaccinia viruε VP804. Recombinant vaccinia viruε, vP855, containing Beta- galactoεidaεe in a partial deletion of the I4L gene, waε iεolated aε a blue plaque in the preεence of X-gal. To delete Beta-galactoεidase and the remainder of the I4L ORF from vP855, deletion plasmid pSD548 was constructed. The left and right vaccinia flanking arms were aεsembled separately in pUC8 as detailed below and presented εchematically in FIG. 15. To conεtruct a vector plaεmid to accept the left vaccinia flanking arm, pUC8 waε cut with BamHI/EcoRI and ligated with annealed εynthetic oligonucleotideε 518A1/518A2 (SEQ ID N0:41/SEQ ID NO:42) BamHI Real 518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT 518A2 3' GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA Bσlll EcoRI TTGAGAATAAAAAGATCTTAGG 3' 5 8A1 AACTCTTATTTTTCTAGAATCCTTAA 5' 518A2 forming plaεmid pSD531. pSD53l waε cut with Rsal (partial) and BamHI and a 2.7 kb vector fragment isolated. pSD518 was cut with Bailf (pos. 64,459)/ Esal (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 sequenceε. 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:43/SEQ ID NO:44) BamHI Bαlll Smal 518B1 5' GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAG- 518B2 3' GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATC- Real EcoRI GGATTTGACGTATGTAGCGTACTAGG 3' 518B1 CCTAAACTGCATACTACGCATGATCCTTAA 5' 518B2 forming plasmid pSD532. pSD532 was cut with Rsal (partial) /EcoRI and a 2.7 kb vector fragment iεolated. pSD518 was cut with Rsal within vaccinia sequenceε (poε. 67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment iεolated. The two fragmentε were ligated together, forming pSD538, which containε the complete vaccinia flanking arm to the right of I4L coding εequences. The right vaccinia flanking arm waε iεolated aε a 0.6 kb EcoRI/Bglll fragment from pSD538 and ligated into pSD537 vector plaεmid cut with EcoRI/Bglll. In the reεulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) iε replaced by a polylinker region, which iε 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. 15. To avoid possible recombination of Beta-galactosidase sequences in the pUC-derived portion of pSD539 with Beta- galactosidase sequences in recombinant vaccinia virus vP855, the vaccinia I4L deletion cassette was moved from pSD539 into pRCll, a pUC derivative from which all Beta- galactosidase sequences have been removed and replaced with a polylinker region (Colinaε et al., 1990). pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment iεolated. Thiε fragment waε ligated into pRCll cut with EcoRI/PstI (2.35 kb) , forming pSD548. Recombination between pSD548 and the Beta-galactoεidase containing vaccinia recombinant, VP855, resulted in vaccinia deletion mutant VP866, which waε iεolated aε a clear plaque in the preεence of X-gal. DNA from recombinant vaccinia viruε VP866 waε analyzed by restriction digests followed by electrophoreεiε on an agaroεe gel. The reεtriction patternε were as expected. Polymerase chain reactions (PCR) (Engelke et al., 1988) using VP866 aε template and primerε flanking the six deletion loci detailed above produced DNA fragments of the expected εizeε. Sequence analyεiε of the PCR generated fragmentε around the areas of the deletion junctions confirmed that the junctions were aε expected. Recombinant vaccinia viruε VP866, containing the εix engineered deletionε aε deεcribed above, waε designated vaccinia vaccine εtrain ' • NYVAC. H Example 19 - INSERTION OF A RABIES GLYCOPROTEIN G GENE INTO NYVAC The gene encoding rabies glycoprotein G under the control of the vaccinia H6 promoter (Taylor et al., 1988a,b) was inεerted into TK deletion plaεmid pSD513. pSD513 iε identical to plaεmid pSD460 (FIG. 10) except for the preεence of a polylinker region. Referring now to FIG. 16, the polylinker region waε inεerted by cutting pSD460 with Smal and ligating the plasmid vector with annealed synthetic oligonucleotides VQ1A/VQ1B (SEQ ID NO:45/SEQ ID NO:46) Smal Bglll Xhol PstI Narl BamHI VQ1A 5' GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3' VQ1B 3' CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5' to form vector plasmid pSD513. pSD513 was cut with Smal and ligated with a Smal ended 1.8 kb caεsette containing the gene encoding the rabies glycoprotein G gene under the control of the vaccinia H6 promoter (Taylor et al., 1988a,b) . The resulting plasmid was designated pRW842. pRW842 waε uεed aε donor plaεmid for recombination with NYVAC reεcuing viruε (vP866) . Recombinant vaccinia virus VP879 waε identified by plaque hybridization uεing 32 P- labelled DNA probe to rabieε glycoprotein G coding sequenceε. The modified recombinant viruεeε of the preεent invention provide advantageε aε recombinant vaccine vectorε. The attenuated virulence of the vector advantageouεly reduceε the opportunity for the poεsibility of a runaway infection due to vaccination in the vaccinated individual and also diminishes transmission from vaccinated to unvaccinated individuals or contamination of the environment. The modified recombinant viruseε are alεo advantageously used in a method for expresεing a gene product in a cell cultured in vitro by introducing into the cell the modified recombinant viruε having foreign DNA which codeε for and expreεεeε gene productε in the cell. Exam p le 20 - CONSTRUCTION OF TROVAC-NDV EXPRESSING THE FUSION AND HEMAGGLUTININ-NEURAMINIDASE GLYCOPROTEINS OF NEWCASTLE DISEASE VIRUS Thiε example deεcribeε the development of TROVAC, a fowlpox virus vector and, of a fowlpox Newcastle Disease Virus recombinant deεignated TROVAC-NDV and itε safety and efficacy. A fowlpox virus (FPV) vector expresεing both F and HN geneε of the virulent NDV strain Texas waε constructed. The recombinant produced was designated TROVAC-NDV. TROVAC-NDV expresses authentically processed NDV glycoproteins in avian cells infected with the recombinant virus and inoculation of day old chickε protectε against subsequent virulent NDV challenge. Cells and Viruses. The Texas strain of NDV is a velogenic strain. Preparation of cDNA clones of the F and HN genes has been previously described (Taylor et al., 1990; Edbauer et al., 1990). The strain of FPV designated FP-l haε been deεcribed previously (Taylor et al., 1988a). It iε a vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France as 95/26751 -58- a fowlpox εcab from a chicken. The virus was attenuated by approximately 50 εerial paεεages in chicken embryonated eggs followed by 25 passageε on chicken embryo fibroblast cells. The viruε waε εubjected to four εucceεsive plaque purifications. One plaque isolate was further amplified in primary CEF cellε and a εtock viruε, deεignated aε TROVAC, eεtabliεhed. The εtock virus used in the in vitro recombination teεt to produce TROVAC-NDV had been εubjected to twelve paεεages in primary CEF cells from the plaque iεolate. Construction of a Casεette for NDV-F. A 1.8 kbp BamHI fragment containing all but 22 nucleotides from the 5' end of the F protein coding sequence was excised from pNDV81 (Taylor et al., 1990) and inserted at the BamHI site of pUClδ to form pCE13. The vaccinia virus H6 promoter previouεly described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) waε inεerted into pCE13 by digeεting pCE13 with Sail, filling in the sticky ends with Klenow fragment of E. coli DNA polymerase and digeεting with HiDdlll. A Hindlll - EcoRV fragment containing the H6 promoter εeguence waε then inεerted into pCE13 to form pCE38. A perfect 5' end waε generated by digeεting pCE38 with Konl and Nrul and inεerting the annealed and kinased oligonucleotides CE75 (SEQ ID NO:47) and CE76 (SEQ ID NO:48) to generate pCE47. CE75: CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGATCCCGGTAC CE76: CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGATATCG. In order to remove non-coding sequence from the 3' end of the NDV-F a Smal to PstI fragment from pCE13 was inserted into the Smal and PstI sites of pUC18 to form pCE23. The non-coding sequences were removed by sequential digestion of pCE 3 with Sad. BamHI. Exonuclease III, SI nuclease and EcoRI. The annealed and kinased oligonucleotides CE42 (SEQ ID NO: 9) and CE43 (SEQ ID NO:50) were then inserted to form pCE29. CE42: AATTCGAGCTCCCCGGG CE43: CCCGGGGAGCTCG The 3' end of the NDV-F εequence waε then inεerted into plaεmid pCE20 already containing the 5' end of NDV-F by cloning a Pεtl - Sad fragment from pCE29 into the Pεtl and Sad εiteε of pCE20 to form pCE32. Generation of pCE20 has previously been deεcribed in Taylor et al., 1990. In order to align the H6 promoter and NDV-F 5' sequenceε contained in pCE47 with the 3' NDV-F sequences contained in pCE32, a Hindlll - Pεtl fragment of pCE47 waε inεerted into the Hindlll and Pεtl εiteε of pCE32 to form pCE49. The H6 promoted NDV-F εequenceε were then transferred to the de-ORFed F8 locus (described below) by cloning a Hindlll - Nrul fragment from pCE49 into the fiindlll and Smal εiteε of pJCA002 (described below) to form pCE54. Transcription εtop εignalε were inserted into pCE54 by digesting pCE54 with Sad, partially digesting with BamHI and inserting the annealed and kinased oligonucleotides CE166 (SEQ ID N0:51) and CE167 (SEQ ID NO:52) to generate pCE58. CE166: CTTTTTATAAAAAGTTAACTACGTAG CE167: GATCCTACGTAGTTAACTTTTTATAAAAAGAGCT A perfect 3' end for NDV-F was obtained by using the polymerase chain reaction (PCR) with pCE54 as template and oligonucleotideε CE182 (SEQ ID NO:53) and CE183 (SEQ ID NO:54) aε primerε. CE182: CTTAACTCAGCTGACTATCC CE183: TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC The PCR fragment waε digeεted with PvuII and Hpal and cloned into pCE58 that had been digeεted with Hpal and partially digeεted with PvuII. The reεulting plaεmid waε deεignated pCE64. Translation stop signalε were inεerted by cloning a Hindlll - Hoal fragment which contains the complete H6 promoter and F coding sequence from pCE64 into the Hindlll and Hpal sites of pRW846 to generate pCE71, the final cassette for NDV-F. Plasmid pRW846 iε eεεentially equivalent to plaεmid pJCA002 (deεcribed below) but containing the H6 promoter and tranεcription and tranεlation stop signals. Digestion of pRW846 with Hindlll and Hoal eliminateε the H6 promoter but leaves the εtop εignalε intact. Construction of Caεsette for NDV-HN. Construction of plaεmid pRW802 waε previouεly deεcribed in Edbauer et al., 1990. Thiε plaεmid containε the NDV-HN εequenceε linked to the 3' end of the vaccinia virus H6 promoter in a pUC9 vector. A Hindlll - EcoRV fragment encompaεεing the 5' end of the vaccinia viruε H6 promoter waε inεerted into the Hindlll and EcoRV sites of pRW802 to form pRW830. A perfect 3' end for NDV-HN was obtained by inεerting the annealed and kinaεed oligonucleotideε CE162 (SEQ ID NO:55) and CE163 (SEQ ID NO:56) into the EcoRI εite of pRW830 to form pCE59, the final caεεette for NDV-HN. CE162: AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATTTAATTTTTAT AAGCTTG CE163: AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTAGTAAAGGAAC GATCCTG Conεtruction of FPV Inεertion Vector. Plaεmid pRW731- 15 containε a lOkb PvuII - PvuII fragment cloned from genomic DNA. The nucleotide εequence waε determined on both strands for a 3660 bp PvuII - EcoRV fragment. The limits of an open reading frame designated here aε F8 were determined. Plaεmid pRW761 iε a sub-clone of pRW731-15 containing a 2430 bp EcoRV - EcoRV fragment. The F8 ORF was entirely contained between an XJbal site and an Sεpl εite in pRW761. In order to create an inεertion plaεmid which on recombination with TROVAC genomic DNA would eliminate the F8 ORF, the following εteps were followed. Plasmid pRW761 waε completely digeεted with Xbal and partially digeεted with Sspl. A 3700 bp Xbal - Sspl band was iεolated from the gel and ligated with the annealed double-stranded oligonucleotides JCA017 (SEQ ID NO:57) and JCA018 (SEQ ID N0:58). JCA017 : 5 ' C TAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGATCCTTATACGG GGAATAAT JCA018:5' ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATATTAAAAAACATAA AGTGT The plasmid reεulting from thiε ligation waε deεignated pJCA002. Conεtruction of Double Inεertion Vector for NDV F and HN. The H6 promoted NDV-HN sequence was inserted into the H6 promoted NDV-F caεsette by cloning a Hindlll fragment from pCE59 that had been filled in with Klenow fragment of E. coli DNA polymerase into the Hpal εite of pCE7l to form pCE80. Plaεmid pCE80 waε completely digeεted with Ndel and partially digeεted with Bglll to generate an Ndel - Bglll 4760 bp fragment containing the NDV F and HN geneε both driven by the H6 promoter and linked to F8 flanking arms. Plasmid pJCA021 waε obtained by inεerting a 4900 bp PvuII - flindll fragment from pRW731-15 into the Smal and Hindi! sites of pBSSK+. Plasmid pJCA021 was then digested with Ndel and. BolII and ligated to the 4760 bp Ndel - Bglll fragment of pCE80 to form pJCA024. Plasmid pJCA024 therefore contains the NDV-F and HN genes inεerted in opposite orientation with 3' ends adjacent between FPV flanking arms. Both genes are linked to the vaccinia viruε H6 promoter. The right flanking arm adjacent to the NDV-F εequence consists of 2350 bp of FPV sequence. The left flanking arm adjacent to the NDV-HN sequence consists of 1700 bp of FPV sequence. Development of TROVAC-NDV. Plasmid pJCA024 waε transfected into TROVAC infected primary CEF cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaqueε were εelected on the baεis of hybridization to specific NDV-F and HN radiolabelled probes and εubjected to five sequential rounds of plaque purification until a pure population was achieved. One representative plaque waε then amplified and the resulting TROVAC recombinant was designated TROVAC-NDV (vFP96) . Immunofluorescence. Indirect immunofluoreεcence waε performed aε deεcribed (Taylor et al., 1990) uεing a polyclonal anti-NDV εerum and, as mono-εpecific reagentε, sera produced in rabbits against vaccinia virus recombinants expreεεing NDV-F or NDV-HN. Immunoprecipitation. Immunoprecipitation reactionε were performed aε deεcribed (Taylor et al., 1990) uεing a polyclonal anti-NDV εerum obtained from SPAFAS Inc., Storrε, CT. The εtock viruε was screened by in situ plaque hybridization to confirm that the F8 ORF was deleted. The correct insertion of the NDV geneε into the TROVAC genome and the deletion of the F8 ORF waε alεo confirmed by Southern blot hybridization. In NDV-infected cellε, the F glycoprotein iε anchored in the membrane via a hydrophobic tranεmembrane region near the carboxyl terminuε and reguireε poεt-tranεlational cleavage of a precurεor, F 0 , into two diεulfide linked polypeptides T-, and F . Cleavage of F 0 is important in determining the pathogenicity of a given NDV strain (Homma and Ohuchi, 1973; Nagai et al., 1976; Nagai et al., 1980), and the sequence of amino acids at the cleavage εite iε therefore critical in determining viral virulence. It haε been determined that amino acidε at the cleavage εite in the NDV-F εequence inεerted into FPV to form recombinant VFP29 had the sequence Arg - Arg - Gin - Arg - Arg (SEQ ID NO:39) (Taylor et al., 1990) which conforms to the sequence found to be a requirement for virulent NDV strainε (Chamberε et al., 1986; Eεpion et al., 1987; Le et al., 1988; McGinneε and Morriεon, 1986; Toyoda et al., 1987). The HN glycoprotein synthesized in cells infected with virulent strains of NDV is an uncleaved glycoprotein of 74 kDa. Extremely avirulent strains such as Ulster and Queensland encode an HN precursor (HNo) which requires cleavage for activation (Garten et al., 1980). The expresεion of F and HN genes in TROVAC-NDV was analyzed to confirm that the gene products were authentically procesεed and preεented. Indirect- immunofluoreεcence uεing a polyclonal anti-NDV chicken εerum confirmed that immunoreactive proteinε were preεented on the infected cell εurface. To determine that both proteinε were preεented on the plaεma membrane, mono-εpecific rabbit εera were produced againεt vaccinia recombinantε expreεεing either the F or HN glycoproteinε. Indirect immunofluoreεcence using these εera confirmed the surface presentation of both proteins. Immunoprecipitation experiments were performed by using ( 35 S) methionine labeled lysateε of CEF cellε infected with parental and recombinant viruεeε. The expected valueε of apparent molecular weightε of the glycoεylated formε of F 2 and F 2 are 54.7 and 10.3 kDa reεpectively (Chamberε et al., 1986) . In the immunoprecipitation experimentε uεing a polyclonal anti-NDV serum, fusion specific products of the appropriate size were detected from the NDV-F single recombinant VFP29 (Taylor et al., 1990) and the TROVAC-NDV double recombinant vFP96. The HN glycoprotein of appropriate size was also detected from the NDV-HN εingle recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV. No NDV εpecific productε were detected from uninfected and parental TROVAC infected CEF cellε. In CEF cellε, the F and HN glycoproteinε are appropriately preεented on the infected cell εurface where they are recognized by NDV immune εerum. Immunoprecipitation analysis indicated that the F 0 protein iε authentically cleaved to the F and F componentε required in virulent strains. Similarly, the HN glycoprotein was authentically procesεed in CEF cells infected with recombinant TROVAC-NDV. Previous reports (Taylor et al., 1990; Edbauer et al., 1990; Bourεnell et al., 1990a,b,c; Ogawa et al., 1990) would indicate that expression of either HN or F alone iε sufficient to elicit protective immunity againεt NDV challenge. Work on other paramyxoviruεes haε indicated, however, that antibody to both proteinε may be required for full protective immunity. It has been demonstrated that SV5 virus could spread in tissue culture in the presence of antibody to the HN glycoprotein but not to the F glycoprotein (Merz et al., 1980). In addition, it has been εuggeέted that vaccine failures with killed measles viruε vaccineε were due to inactivation of the fuεion component (Norrby et al., 1975). Since both NDV glycoproteinε have been εhown to be reεponεible for eliciting viruε neutralizing antibody (Avery et al., 1979) and both glycoproteinε, when expreεεed individually in a fowlpox vector are able to induce a protective immune reεponse, it can be appreciated that the most efficacious NDV vaccine should express both glycoproteins. Example 21 - CONSTRUCTION OF ALVAC RECOMBINANTS EXPRESSING RABIES VIRUS GLYCOPROTEIN G Thiε example deεcribeε the development of ALVAC, a canarypox viruε vector and, of a canarypox-rabieε recombinant deεignated aε ALVAC-RG (vCP65) and itε safety and efficacy. Cells and Viruseε. The parental canarypox virus (Rentεchler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial pasεageε on chick embryo fibroblasts. A maεter viral seed was subjected to four successive plaque purifications under agar and one plaque clone waε amplified through five additional paεεages after which the εtock virus waε uεed aε the parental viruε in in vitro recombination teεtε. The plaque purified canarypox isolate is designated ALVAC. Construction of a Canarypox Inεertion Vector. An 880 bp canarypox PvuII fragment waε cloned between the PvuII sites of pUC9 to form pRW764.5. The sequence of this fragment is εhown in FIG. 17 (SEQ ID NO:59) between positions 1372 and 2251. The limits of an open reading frame designated as C5 were defined. It was determined that the open reading frame was initiated at position 166 within the fragment and terminated at position 487. The C5 deletion was made without interruption of open reading frames. Bases from position 167 through poεition 455 were replaced with the sequence (SEQ ID NO:60) GCTTCCCGGGAATTCTAGCTAGCTAGTTT. This replacement εequence contains Hindlll. Smal and EcoRI insertion siteε followed by tranεlation εtopε and a tranεcription termination εignal recognized by vaccinia viruε RNA polymeraεe (Yuen et al., 1987) . Deletion of the C5 ORF waε performed aε deεcribed below. Plaεmid pRW764.5 waε partially cut with Rsal and the linear product was isolated. The Rsal linear fragment was recut with Bglll and the pRW764.5 fragment now with a Rsal to fialll deletion from position 156 to position 462 waε iεolated and uεed aε a vector for the following εynthetic oligonucleotideε: RW145 (SEQ ID NO:61) : ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA RW146 (SEQ ID NO:62) : GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW 764.5 Rsal and Bglll vector described above. The resulting plasmid is designated pRW831. Construction of Inεertion Vector Containing the Rabieε G Gene. Conεtruction of pRW838 iε illuεtrated below. Oligonucleotides A through E, which overlap the translation initiation codon of the H6 promoter with the ATG of rabies G, were cloned into pUC9 aε pRW737. Oligonucleotides A through E contain the H6 promoter, starting at Nrul. through the Hindlll site of rabies G followed by Bglll. Sequences of oligonucleotides A. through E ((SEQ ID NO:63)-(SEQ ID NO:67)) are: A (SEQ ID NO:63): CTGAAATTATTTCATTATCGCGATATCCGTTAA GTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT B (SEQ ID NO:64): CATTACGATACAAACTTAACGGATATCGCGATAA TGAAATAATTTCAG C (SEQ ID NO:65): ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAA TTCCCTATTTACACGATCCCAGACAAGCTTAGATCTCAG D (SEQ ID NO:66): CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATA GGGAATTTCCCAAAACA E (SEQ ID NO:67): CAACGGAAAAACCAGAAGGGGTACAAACAGGAGA GCCTGAGGAAC The diagram of annealed oligonucleotides A through E is as follows: A c B E Oligonucleotides A through E were kinaεed, annealed (95°C for 5 minuteε, then cooled to room temperature) , and inεerted between the PvuII εites of pUC9. The resulting plasmid, pRW737, was cut with Hindlll and Bglll and used as a vector for the 1.6 kbp Hindlll-BgJLII fragment of ptgl55PRO (Kieny et al., 1984) generating pRW739. The ptgl55PR0 Hindlll εite iε 86 bp downεtream of the rabieε G tranεlation initiation codon. Bglll iε downεtream of the rabieε G translation stop codon in ptgl55PR0. pRW739 was partially cut with Nrul. completely cut with Bglll. and a 1.7 kbp Nrul-Bqlll fragment, containing the 3' end of the H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) through the entire rabies G gene, was inεerted between the Nrul and BamHI εites of pRW824. The resulting plasmid iε deεignated pRW832. Inεertion into pRW824 added the H6 promoter 5' of Nrul. The pRW824 sequence of BamHI followed by Smal iε (SEQ ID NO:68): GGATCCCCGGG. pRW824 is a plasmid that contains a nonpertinent gene linked preciεely to the vaccinia viruε H6 promoter. Digeεtion with Nrul and BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832 Smal fragment, containing H6 promoted rabies G, was inserted into the Smal of pRW831, to form plaεmid pRW838. Development of ALVAC-RG. Plaεmid pRW838 waε tranεfected into ALVAC infected primary CEF cellε by uεing the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of hybridization to a specific rabies G probe and subjected to 6 sequential rounds of plaque purification until a pure population was achieved. One representative plaque waε then amplified and the resulting ALVAC recombinant was designated ALVAC-RG (VCP65) (see alεo FIGS. 18A and 18B) . • The correct insertion of the rabies G gene into the ALVAC genome without subεequent mutation waε confirmed by sequence analysiε. jmjptipnfluoreεcence. During the final εtageε of aεsembly of mature rabies viruε particleε, the glycoprotein component iε tranεported from the golgi apparatus to the plasma membrane where it accumulates with the carboxy terminuε extending into the cytoplaεm and the bulk of the protein on the external εurface of the cell membrane. In order to confirm that the rabieε glycoprotein expreεsed in ALVAC-RG was correctly presented, immunofluoreεcence waε performed on primary CEF cells infected with ALVAC or ALVAC- RG. Immunofluoreεcence waε performed aε previouεly deεcribed (Taylor et al., 1990) uεing a rabieε G monoclonal antibody. Strong surface fluorescence was detected on CEF cells infected with ALVAC-RG but not with the parental ALVAC. Immunoprecipitation. Preformed monolayers of primary CEF, Vero (a line of African Green monkey kidney cells ATCC # CCL81) and MRC-5 cells (a fibroblast-like cell line derived from normal human fetal lung tiεsue ATCC # CCL171) were inoculated at 10 pfu per cell with parental virus ALVAC and recombinant virus ALVAC-RG in the presence of radiolabelled 35 S-methionine and treated as previously deεcribed (Taylor et al., 1990). Immunoprecipitation reactionε were performed uεing a rabies G specific monoclonal antibody. Efficient expression of a rabies specific glycoprotein with a molecular weight of approximately 67 kDa was detected with the recombinant ALVAC-RG. No rabies specific products were detected in uninfected cellε or cellε infected with the parental ALVAC virus. Sequential Pasεaginq Experiment. In εtudieε with ALVAC viruε in a range of non-avian species no proliferative infection or overt diseaεe waε obεerved (Taylor et al., 1991b) . However, in order to eεtabliεh that neither the parental nor recombinant virus could be adapted to grow in non-avian cellε, a εequential paεεaging experiment was performed. The two viruεeε, ALVAC and ALVAC-RG, were inoculated in 10 εequential blind passages in three cell lineε: (1) Primary chick embryo fibroblaεt (CEF) cellε produced from 11 day old white leghorn embryoε; (2) Vero cellε - a continuous line of African Green monkey kidney cells (ATCC # CCL81) ; and (3) MRC-5 cellε - a diploid cell line derived from human fetal lung tissue (ATCC # CCL171) . The initial inoculation waε performed at an m.o.i. of 0.1 pfu per cell uεing three 60mm diεheε of each cell line containing 2 X IO 6 cellε per diεh. One diεh waε inoculated in the preεence of 40μg/ml of Cytoεine arabinoεide (Ara C) , an inhibitor of DNA replication. After an abεorption period of 1 hour at 37°C, the inoculum waε removed and the monolayer washed to remove unabεorbed viruε. At thiε time the medium waε replaced with 5ml of EMEM + 2% NBCS on two dishes (sampleε to and t7) and 5ml of EMEM + 2% NBCS containing 40 μg/ml Ara C on the third (εample t7A) . Sample to waε frozen at -70°C to provide an indication of the residual input viruε. Sampleε t7 and t7A were incubated at 37 β C for 7 dayε, after which time the contentε were harvested and the cells disrupted by indirect sonication. One ml of εample t7 of each cell line waε inoculated undiluted onto three diεheε of the εame cell line (to provide εampleε to, t7 and t7A) and onto one diεh of primary CEF cellε. Sampleε to, t7 and t7A were treated aε for paεsage one. The additional inoculation on CEF cells waε included to provide an amplification εtep for more εenεitive detection of viruε which might be preεent in the non-avian cellε. This procedure was repeated for 10 (CEF and MRC-5) or 8 (Vero) εeguential blind paεεageε. Sampleε were then frozen and thawed three timeε and assayed by titration on primary CEF onolayers. 5/26751 -69- Viruε yield in each εample waε then determined by plaque titration on CEF monolayers under agarose. Summarized reεultε of the experiment are εhown in Tableε 5 and 6. The reεultε indicate that both the parental ALVAC and the recombinant ALVAC-RG are capable of εuεtained replication on CEF monolayerε with no loεε of titer. In Vero cells, levels of virus fell below the level of detection after 2 paεεageε for ALVAC and 1 passage for ALVAC-RG. In MRC-5 cells, a similar result waε evident, and no viruε waε detected after 1 passage. Although the results for only four passages are shown in Tables 5 and 6 the series was continued for 8 (Vero) and 10 (MRC-5) passages with no detectable adaptation of either virus to growth in the non-avian cells. In passage 1 relatively high levels of viruε were present in the t7 sample in MRC-5 and Vero cells. However this level of virus was equivalent to that εeen in the to sample and the t7A sample incubated in the presence of Cytosine arabinoside in which no viral replication can occur. This demonstrated that the levels of viruε εeen at 7 days in non-avian cells repreεented reεidual viruε and not newly replicated viruε. In order to make the aεεay more sensitive, a portion of the 7 day harvest from each cell line was inoculated onto a permissive CEF monolayer and harvested at cytopathic effect (CPE) or at 7 dayε if no CPE was evident. The results of this experiment are shown in Table 7. Even after amplification through a permisεive cell line, viruε was only detected in MRC-5 and Vero cells for two additional passages. These resultε indicated that under the conditionε used, there was no adaptation of either virus to growth in Vero or MRC-5 cells. Inoculation of Macaqueε. Four HIV seropositive macaques were initially inoculated with ALVAC-RG as described in Table 8. After 100 days these animals were re- inoculated to determine a booεter effect, and an additional εeven animalε were inoculated with a range of doses. Blood was drawn at appropriate intervals and εera analyzed, after heat inactivation at 56°C for 30 minuteε, for the preεence of anti-rabieε antibody uεing the Rapid Fluoreεcent Focus Inhibition Asεay (Smith et al., 1973). Inoculation of Chimpanzees. Two adult male chimpanzees (50 to 65 kg weight range) were inoculated intramuscularly or subcutaneously with l X IO 7 pfu of vCP65. Animals were monitored for reactionε and bled at regular intervalε for analyεiε for the preεence of anti-rabieε antibody with the RFFI teεt (Smith et al., 1973). Animalε were re-inoculated with an equivalent doεe 13 weekε after the initial inoculation. Inoculation of Mice. Groupε of mice were inoculated with 50 to 100 μl of a range of dilutionε of different batcheε of VCP65. Mice were inoculated in the footpad. On day 14, mice were challenged by intracranial inoculation of from 15 to 43 mouεe LD 50 of the virulent CVS εtrain of rabieε viruε. Survival of mice waε monitored and a protective doεe 50% . (PD 50 ) calculated at 28 dayε poεt- inoculation. Inoculation of Dogs and Cats. Ten beagle dogs, 5 monthε old, and 10 catε, 4 monthε old, were inoculated εubcutaneouεly with either 6.7 or 7.7 log 10 TCID 50 of ALVAC- RG. Four dogε and four catε were not inoculated. Animalε were bled at 14 and 28 dayε post-inoculation and anti-rabieε antibody aεεeeεed in an RFFI test. The animals receiving 6.7 log 10 TCID 50 of ALVAC-RG were challenged at 29 days poεt-vaccination with 3.7 log 10 mouεe LD 50 (dogε) or 4.3 lo*3ιo • mouse *-- j * D 50 (catε) of the NYGS rabieε viruε challenge εtrain. Inoculation of Squirrel Monkeys. Three groups of four squirrel monkeys (Saimiri εciureus) were inoculated with one of three viruses (a) ALVAC, the parental canarypox virus, (b) ALVAC-RG, the recombinant expreεεing the rabieε G glycoprotein or (c) VCP37, a canarypox recombinant expressing the envelope glycoprotein of feline leukemia virus. Inoculationε were performed under ketamine anaeεtheεia. Each animal received at the εame time: (1) 20 μl inεtilled on the εurface of the right eye without scarification; (2) 100 μl aε εeveral dropletε in the mouth; (3) 100 μl in each of two intradermal injection εiteε in the shaven skin of the external face of the right arm; and (4) 100 μl in the anterior muεcle of the right thigh. Four monkeyε were inoculated with each viruε, two with a total of 5.0 log 10 pfu and two with a total of 7.0 log 10 pfu. Animalε were bled at regular intervalε and εera analyzed for the presence of antirabies antibody uεing an RFFI teεt (Smith et al., 1973). Animalε were monitored daily for reactionε to vaccination. Six monthε after the initial inoculation the four monkeyε receiving ALVAC-RG, two monkeyε initially receiving VCP37, and two monkeyε initially receiving ALVAC, aε well aε one naive monkey were inoculated with 6.5 log 10 pfu of ALVAC-RG εubcutaneouεly. Sera were monitored for the presence of rabies neutralizing antibody in an RFFI test (Smith et al., 1973). Inoculation of Human Cell Lines with ALVAC-RG. In order to determine whether efficient expresεion of a foreign gene could be obtained in non-avian cellε in which the viruε does not productively replicate, five cell types, one avian and four non-avian, were analyzed for viruε yield, expreεεion of the foreign rabieε G gene and viral εpecific DNA accumulation. The cellε inoculated were: (a) Vero, African Green monkey kidney cellε, ATCC # CCL81; (b) MRC-5, human embryonic lung, ATCC # CCL 171; (c) WISH human amnion, ATCC ≠ CCL 25; (d) Detroit-532, human foreskin. Downs'ε syndrome, ATCC ≠ CCL 54; and (e) Primary CEF cells. Chicken embryo fibroblast cellε produced from 11 day old white leghorn embryos were included as a poεitive control. All inoculationε were performed on preformed monolayers of 2 X IO 6 cells as diεcuεsed below. Methods for DNA analysis. Three dishes of each cell line were inoculated at 5 pfu/cell of the virus under test, allowing one extra dish of each cell line un-inoculated. One dish waε incubated in the presence of 40 μg/ml of cytosine arabinoεide (Ara C) . After an adεorption period of 60 minuteε at 37°C, the inoculum waε removed and the monolayer waεhed twice to remove unadsorbed virus. Medium (with or without Ara C) was then replaced. Cellε from one dish (without Ara C) were harvested aε a time zero sample. The remaining diεhes were incubated at 37°C for 72 hours, at which time the cells were harvested and used to analyze DNA accumulation. Each εample of 2 X IO 6 cellε waε reεuεpended in 0.5 ml phoεphate buffered saline (PBS) containing 40 mM EDTA and incubated for 5 minuteε at 37 β C. An equal volume of 1.5% agarose prewarmed at 42°C and containing 120 mM EDTA was added to the cell εuεpenεion and gently mixed. The εuspenεion waε transferred to an agarose plug mold and allowed to harden for at leaεt 15 min. The agaroεe plugε were then removed and incubated for 12-16 hourε at 50°C in a volume of lyεiε buffer (1% εarkoεyl, 100 μg/ml proteinaεe K, 10 mM Triε HCl pH 7.5, 200 mM EDTA) that completely coverε the plug. The lyεiε buffer waε then replaced with 5.0 ml εterile 0.5 X TBE (44.5 mM Tris-borate, 44.5 mM boric acid, 0.5 mM EDTA) and equilibrated at 4°C for 6 hours with 3 changeε of TBE buffer. The viral DNA within the plug was fractionated from cellular RNA and DNA using a pulse field electrophoresiε εystem. Electrophoresiε waε performed for 20 hourε at 180 V with a ramp of 50-90 sec at 15 β C in 0.5 X TBE. The DNA was run with lambda DNA molecular weight standardε. After electrophoreεiε the viral DNA band was visualized by staining with ethidium bromide. The DNA was then transferred to a nitrocellulose membrane and probed with a radiolabelled probe prepared from purified ALVAC genomic DNA. B. Eεtimation of viruε yield. Dishes were inoculated exactly as described above, with the exception that input multiplicity was 0.1 pfu/cell. At 72 hourε poεt infection, cellε were lysed by three successive cycles of freezing and thawing. Virus yield waε assessed by plaque titration on CEF monolayers. C. Analyεiε of expression of Rabies G gene. Diεheε were inoculated with recombinant or parental viruε at a multiplicity of 10 pfu/cell, allowing an additional diεh aε an uninfected viruε control. After a one hour abεorption period, the medium was removed and replaced with methionine free medium. After a 30 minute period, this medium waε replaced with methionine-free medium containing 25 uCi/ml of 35 S- Methionine. Infected cellε were labelled overnight (approximately 16 hourε) , then lyεed by the addition of buffer A lyεiε buffer. Immunoprecipitation waε performed aε previouεly deεcribed (Taylor et al., 1990) using a rabieε G specific monoclonal antibody. Resultε: Eεtimation of Viral Yield. The reεultε of titration for yield at 72 hourε after inoculation at 0.1 pfu per cell are shown in Table 9. The resultε indicate that while a productive infection can be attained in the avian cellε, no increaεe in viruε yield can be detected by thiε method in the four non-avian cell εyεtemε. Analvεiε of Viral DNA Accumulation. In order to determine whether the block to productive viral replication in the non-avian cells occurred before or after DNA replication, DNA from the cell lysates was fractionated by electrophoresis, transferred to nitrocellulose and probed for the presence of viral specific DNA. DNA from uninfected CEF cells, ALVAC-RG infected CEF cells at time zero, ALVAC- RG infected CEF cells at 72 hourε poεt-infection and ALVAC- RG infected CEF cellε at 72 hourε poεt-infection in the presence of 40 μg/ml of cytosine arabinoside all showed some background activity, probably due to contaminating CEF cellular DNA in the radiolabelled ALVAC DNA probe preparation. However, ALVAC-RG infected CEF cellε at 72 hourε poεt-infection exhibited a εtrong band in the region of approximately 350 kbp repreεenting ALVAC-εpecific viral DNA accumulation. No εuch band iε detectable when the culture iε incubated in the preεence of the DNA εyntheεiε inhibitor, cytosine arabinoside. Equivalent samples produced in Vero cells εhowed a very faint band at approximately 350 kbp in the ALVAC-RG infected Vero cellε at time zero. Thiε level repreεented reεidual viruε. The intenεity of the band waε amplified at 72 hourε poεt- infection indicating that εome level of viral εpecific DNA replication had occurred in Vero cellε which had not reεulted in an increaεe in viral progeny. Equivalent εampleε produced in MRC-5 cellε indicated that no viral εpecific DNA accumulation waε detected under theεe conditionε in thiε cell line. Thiε experiment waε then extended to include additional human cell lineε, εpecifically WISH and Detroit-532 cellε. ALVAC infected CEF cellε served as a positive control. No viral εpecific DNA accumulation waε detected in either WISH or Detroit cellε inoculated with ALVAC-RG. It εhould be noted that the limitε of detection of thiε method have not been fully aεcertained and viral DNA accumulation may be occurring, but at a level below the sensitivity of the method. Other experiments in which viral DNA replication was meaεured by 3 H-thymidine incorporation εupport the reεultε obtained with Vero and MRC-5 cellε. Analyεiε of Rabieε Gene Expreεsion. To determine if any viral gene expreεεion, particularly that of the inεerted foreign gene, waε occurring in the human cell lineε even in the abεence of viral DNA replication, immunoprecipitation experimentε were performed on 35 S-methionine labelled lyεates of avian and non-avian cells infected with ALVAC and ALVAC-RG. The results of immunoprecipitation using a rabieε G εpecific monoclonal antibody illuεtrated specific immunoprecipitation of a 67 kDa glycoprotein in CEF, Vero and MRC-5, WISH and Detroit cells infected with ALVAC-RG. No εuch εpecific rabieε gene products were detected in any of the uninfected and parentally infected cell lysateε. The reεultε of this experiment indicated that in the human cell lines analyzed, although the ALVAC-RG recombinant waε able to initiate an infection and expreεε a foreign gene product under the tranεcriptional control of the H6 early/late vaccinia viruε promoter, the replication did not proceed through DNA replication, nor waε there any detectable viral progeny produced. In the Vero cellε, although some level of ALVAC-RG specific DNA accumulation waε observed, no viral progeny was detected by these methods. These resultε would indicate that in the human cell lineε analyzed the block to viral replication occurε prior to the onεet of DNA replication, while in Vero cells, the block occurε following the onεet of viral DNA replication. In order to determine whether the rabieε glycoprotein expressed in ALVAC-RG was immunogenic, a number of animal species were tested by inoculation of the recombinant. The efficacy of current rabies vaccineε iε evaluated in a mouse model syεtem. A similar test was therefore performed uεing ALVAC-RG. Nine different preparationε of viruε (including one vaccine batch (J) produced after 10 serial tissue culture passages of the seed virus) with infectiouε titers ranging from 6.7 to 8.4 log 10 TCID 50 per ml were εerially diluted and 50 to 100 μl of dilutionε inoculated into the footpad of four to six week old mice. Mice were challenged 14 days later by the intracranial route with 300 μl of the CVS strain of rabies viruε containing from 15 to 43 mouεe LD 50 aε determined by lethality titration in a control group of mice. Potency, expreεεed aε the PD 50 (Protective doεe 50%) , waε calculated at 14 dayε post-challenge. The resultε of the experiment are shown in Table 10. The resultε indicated that ALVAC-RG waε conεistently able to protect mice against rabies virus challenge with a PD 50 value ranging from 3.33 to 4.56 with a mean value of 3.73 (STD 0.48). As an extension of this study, male mice were inoculated intracranially with 50 μl of virus containing 6.0 log 10 TCID 50 of ALVAC-RG or with an equivalent volume of an uninfected cell εuspenεion. Mice were εacrificed on dayε l, 3 and 6 poεt-inoculation and their brains removed, fixed and sectioned. Histopathological examination εhowed no evidence for neurovirulence of ALVAC-RG in mice. In order to evaluate the safety and efficacy of ALVAC- RG for dogs and catε, a group of 14, 5 month old beagleε and 14, 4 month old catε were analyzed. Four animalε in each εpecieε were not vaccinated. Five animalε received 6.7 log 10 TCID 50 εubcutaneouεly and five animalε received 7.7 log 10 TCID 50 by the εame route. Animalε were bled for analyεiε for anti-rabieε antibody. Animalε receiving no inoculation or 6.7 log 10 TCID 50 of ALVAC-RG were challenged at 29 dayε poεt-vaccination with 3.7 log 10 mouεe LD 50 (dogs, in the temporal muscle) or 4.3 log 10 mouεe LD 50 (catε, in the neck) of the NYGS rabieε viruε challenge strain. The resultε of the experiment are εhown in Table 11. No adverεe reactionε to inoculation were seen in either cats or dogε with either doεe of inoculum viruε. Four of 5 dogε immunized with 6.7 log 10 TCID 50 had antibody titers on day 14 post-vaccination and all dogs had titers at 29 days. All dogε were protected from a challenge which killed three out of four controlε. In catε, three of five catε receiving 6.7 log 10 TCID 50 had εpecific antibody titerε on day 14 and all catε were poεitive on day 29 although the mean antibody titer waε low at 2.9 IU. Three of five catε survived a challenge which killed all controls. All cats immunized with 7.7 log 10 TCID 50 had antibody titers on day 14 and at day 29 the Geometric Mean Titer waε calculated aε 8.1 International Unitε. The immune reεponεe of εquirrel monkeyε (Saimiri εciureus) to inoculation with ALVAC, ALVAC-RG and an unrelated canarypox viruε recombinant waε examined. Groupε of monkeyε were inoculated aε deεcribed above and sera analyzed for the presence of rabieε εpecific antibody. Apart from minor typical skin reactions to inoculation by the intradermal route, no adverse reactivity was seen in any of the monkeys. Small amounts of residual viruε were iεolated from εkin lesions after intradermal inoculation on days two and four post-inoculation only. All specimens were negative on day seven and later. There was no local reaction to intra-muεcular injection. All four monkeys inoculated with ALVAC-RG developed anti-rabies serum neutralizing antibodies as measured in an RFFI test. Approximately six months after the initial inoculation all monkeys and one additional naive monkey were re-inoculated by the subcutaneous route on the external face of the left thigh with 6.5 log 10 TCID 50 of ALVAC-RG. Sera were analyzed for the presence of anti-rabies antibody. The results are shown in Table 12. Four of the five monkeys naive to rabies developed a serological reεponεe by seven days post-inoculation with ALVAC-RG. All five monkeys had detectable antibody by 11 dayε poεt-inoculation. Of the four monkeyε with previouε expoεure to the rabieε glycoprotein, all εhowed a significant increase in serum neutralization titer between days 3 and 7 post-vaccination. The resultε indicate that vaccination of εquirrel monkeyε with ALVAC-RG doeε not produce adverεe side-effects and a primary neutralizing antibody response can be induced. An amnanestic reεponεe iε also induced on re-vaccination. Prior exposure to ALVAC or to a canarypox recombinant expresεing an unrelated foreign gene doeε not interfere with induction of an anti-rabieε immune reεponse upon re-vaccination. The immunological response of HIV-2 seropositive macaques to inoculation with ALVAC-RG was assessed. Animals were inoculated aε described above and the presence of anti- rabies serum neutralizing antibody asεeεsed in an RFFI test. The results, shown in Table 13, indicated that HIV-2 positive animals inoculated by the subcutaneouε route developed anti-rabies antibody by 11 days after one inoculation. An anamnestic responεe waε detected after a booster inoculation given approximately three monthε after the first inoculation. No reεponεe was detected in animals receiving the recombinant by the oral route. In addition, a series of six animals were inoculated with decreaεing doεes of ALVAC-RG given by either the intra-muscular or subcutaneous routes. Five of the six animals inoculated responded by 14 days post-vaccination with no εignificant difference in antibody titer. Two chimpanzeeε with prior expoεure to HIV were inoculated with 7.0 log 10 pfu of ALVAC-RG by the εubcutaneouε or intra-muscular route. At 3 months post- inoculationε both animalε were re-vaccinated in an identical faεhion. The reεultε are εhbwn in Table 14. No adverεe reactivity to inoculation was noted by either intramuscular or subcutaneous routes. Both chimpanzees responded to primary inoculation by 14 days and a strongly rising reεponεe waε detected following re- vaccination. Table 5. Sequential Passage of ALVAC in Avian and non-Avian Cells. CEF Vero MRC-5 3.0 2.6 1.4 0.4 1.2 0.4 0.4 N.D. 0.4 N.D. N.D. N.D. 0.4 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. This sample was harvested at zero time and represents the residual input virus. The titer is expressed as loς 1Q pfu per ml. This sample was harvested at 7 days poet-infection. This sample was inoculated in the presence of 40 μg/ml of Cytoβine arabinoside and harvested at 7 days post infection. Not detectable Table 6. Sequential Passage of ALVAC-RG in Avian and non-Avian Cells ss∑ e o MRC-5 This sample was harvested at zero time and represents the residual input virus. The titer is expressed as log 1Q pfu per ml. b: This sample was harvested at 7 dayε post-infection. c: This sample was inoculated in the presence of 40 μg/ml of Cytosine arabinoside and harvested at 7 days post-infection. Not detectable. Table 7. Amplification of residual virus by passage in CEF cells CEF Vero MRC-5 a) ALVAC 3 7.5 4.1 4.9 4 7.5 N.D. N.D. 5 7.1 N.D. N.D. b) ALVAC-RG 3 7.2 5.0 5.1 4 7.2 N.D. N.D. 5 7.2 N.D. N.D. a: Pass 2 represents the amplification in CEF cells of the 7 day sample from Pass 1. b: Titer expressed as log 1fJ pfu per ml c: Not Detectable Table 8. Schedule of inoculation of rheβus macaques with ALVAC-RG (VCP65) Animal Inoculation 176L Primary: 1 X 10 8 pfu of VCP65 orally in TANG Secondary: 1 X IO 7 pfu of vCP65 plus 1 X IO 7 pfu of vCP82 β by SC route 185 L Primary: 1 X IO 8 pfu of vCP65 orally in Tang Secondary: 1 X IO 7 pfu of vCP65 plus 1 X IO 7 pfu of vCP82 by SC route 177 L Primary: 5 X IO 7 pfu SC of vCP65 by SC route Secondary: 1 X IO 7 pfu of vCP65 plus 1 X IO 7 pfu of vCP82 by SC route 186L Primary: 5 X IO 7 pfu of VCP65 by SC route Secondary: 1 X IO 7 pfu of VCP65 plus 1 X IO 7 pfu of vCP82 by SC route 178L Primary: 1 X IO 7 pfu of vCP65 by SC route 182L Primary: 1 X IO 7 pfu of vCP65 by IM route 179L Primary: 1 X 10° pfu of vCP65 by SC route 183L Primary: 1 X 10 6 pfu of vCP65 by IM route 180L Primary: 1 X 10 6 pfu of vCP65 by SC route 184L Primary: 1 X 10 5 pfu of VCP65 by IM route 187L Primary 1 X IO 7 pfu of VCP65 orally vCP82 is a canarypox virus recombinant expressing the measles virus fusion and hemagglutinin genes. Table 9. Analysis of yield in avian and non-avian cells inoculated with ALVAC-RG Sample Time Cell Type to t72 t72A c Expt 1 CEF 3.3* 7.4 1.7 Vero 3.0 1.4 1.7 MRC-5 3.4 2.0 1.7 Expt 2 CEF 2.9 7.5 <1.7 WISH 3.3 2.2 2.0 Detroit-532 2.8 1.7 <1.7 a: Titer expressed aβ log 10 pfu per ml b: Culture incubated in the presence of 40 μg/ml of Cytosine arabinoβide Table 10. Potency of ALVAC-RG as tested in mice Test Challenge Dose* PD 50 Initial aeed 43 4.56 Primary seed 23 3.34 Vaccine Batch H 23 4.52 Vaccine Batch I 23 3.33 Vaccine Batch K 15 3.64 Vaccine Batch L 15 4.03 Vaccine Batch M 15 3.32 Vaccine Batch N 15 3.39 Vaccine Batch J 23 3.42 a: Expressed as mouse LD 50 b : Expressed as log 1fJ TCID 50 Table 11. Efficacy of ALVAC-RG in dogs and cats Doαs Cats Doβe Antibody 8 Survival' Antibody Survival 6.7 11.9 5/5 2.9 3/5 7.7 10.1 N.T. 8.1 N.T. Antibody at day 29 post inoculation expressed as the geometric mean titer in International Units. Expressed as a ratio of survivors over animals challenged Table 12. Anti-rabies βerological response of Squirrel monkeys inoculated with canarypox recombinants 10 15 0 a: As determined by RFFI teat on days indicated and expressed in International Units b: Day-196 represents serum from day 28 after primary vaccination c: Animals received 5.0 log 1fJ TCID-;,, Of ALVAC 20 d: Animals received 5.0 l g ή TCIDςή of vCP37 e: Animals received 5.0 log 1fJ TCID 50 of ALVAC-RG f: Animals received 7 log 1fJ TCID 5 „ of ALVAC-RG 9' Not tested. Table 13. Inoculation of rhesus macaques with ALVAC-RG a Days post- Route of Primary Inoculation Inoculation _or/Tanα_ ^ SC_ ^ SC_ _SC_ ^ IM_ ^ SC ^ _IM_ _SC_ IM ^ _OR I76L P 185L 177L 186L 178L 182L 179L 183L 180L 184L Tβ7L b -84 -9 10 3 6 11 19 35 15 59 75 99 c I 2 ∞ 6 I 20 15 29 55 57 16 128 128 16 16 25 a! See Table 9 for schedule of inoculations. b: Animals 176L and 185L received 8.0 log 10 pfu by the oral route in 5 ml Tang. Animal 187 received 7.0 log, n pfu by oral route not in Tang. c: Day of re-vaccination for animals 176L, I85L, I77 and 186 by S.C. route, and primary vaccination for animals I78 , 1821., 179L, 183L, I80 , 1R4L and 1871, d: Titers expressed as reciprocal of last dilution showing inhibition of fluorescence in an RFFI test. Table 14. Inoculation of chimpanzeeβ with ALVAC-RG Weeks post- Animal 431 Animal 457 Inoculation I.M. S.C. 0 <8 β <8 1 <8 <8 2 8 32 4 16 32 8 16 32 12 b /0 16 8 13/1 128 128 15/3 256 512 20/8 64 128 26/12 32 128 a: Titer expressed as reciprocal of last dilution showing inhibition of fluorescence in an RFFI test b: Day of re-inoculation Example 22 - IMMUNIZATION OF HUMANS USING CANARYPOX EXPRESSING RABIES GLYCOPROTEIN (ALVAC-RG: VCP65) ALVAC-RG (vCP65) waε generated aε deεcribed in Example 21 and FIGS. 18A and 18B. For εcaling-up and vaccine manufacturing ALVAC-RG (vCP65) waε grown in primary CEF derived from εpecified pathogen free eggε. Cellε were infected at a multiplicity of 0.01 and incubated at 37°C for three dayε. The vaccine viruε suspenεion waε obtained by ultraεonic diεruption in serum free medium of the infected cellε; cell debris were then removed by centrifugation and filtration. The resulting clarified suspension was supplemented with lyophilization stabilizer (mixture of amino-acids) , diεpenεed in εingle doεe vialε and freeze dried. Three batches of decreasing titer were prepared by ten-fold serial dilutions of the virus suεpenεion in a mixture of εerum free medium and lyophilization εtabilizer, prior to lyophilization. Quality control teεtε were applied to the cell subεtrateε, media and viruε seeds and final product with emphasiε on the εearch for adventitiouε agentε and innocuity in laboratory rodentε. No undeεirable trait waε found. Preclinical data. Studieε in vitro indicated that VERO or MRC-5 cellε do not εupport the growth of ALVAC-RG (vCP65) ; a series of eight (VERO) and 10 (MRC) blind serial paεεageε caused no detectable adaptation of the virus to grow in these non avian lines. Analyseε of human cell lineε (MRC-5, WISH, Detroit 532, HEL, HNK or EBV-tranεformed ly phoblaεtoid cellε) infected or inoculated with ALVAC-RG (vCP65) showed no accumulation of viruε εpecific DNA suggesting that in these cells the block in replication occurs prior to DNA synthesis, significantly, however, the expression of the rabies virus glycoprotein gene in all cell lines tested indicating that the abortive step in the canarypox replication cycle occurs prior to viral DNA replication. The εafety and efficacy of ALVAC-RG (VCP65) were documented in a εerieε of experimentε in animalε. A number of species including canaries, chickens, ducks, geese, laboratory rodents (suckling and adult mice) , hamsterε, guinea-pigε, rabbitε, cats and dogs, εguirrel monkeyε, rhesus macaques and chimpanzees, were inoculated with doses ranging from 10 5 to IO 8 pfu. A variety of routes were uεed, moεt commonly εubcutaneouε, intra uεcular and intradermal but alεo oral (monkeyε and mice) and intracerebral (mice) . In canaries, ALVAC-RG (VCP65) caused a • •take" leεion at the εite of εcarification with no indication of diεeaεe or death. Intradermal inoculation of rabbitε reεulted in a typical poxviruε inoculation reaction which did not εpread and healed in εeven to ten dayε. There waε no adverεe side effects due to canarypox in any of the animal teεtε. Immunogenicity waε documented by the development of anti- rabies antibodies following inoculation of ALVAC-RG (vCP65) in rodents, dogs, cats, and primates, as measured by Rapid Fluorescent Focus Inhibition Test (RFFIT) . Protection was also demonstrated by rabies virus challenge experiments in mice, dogs, and cats immunized with ALVAC-RG (vCP65) . Volunteers. Twenty-five healthy adults aged 20-45 with no previous hiεtory of rabieε immunization were enrolled. Their health status was aεεessed by complete medical hiεtories, physical examinations, hematological and blood chemiεtry analyses. Exclusion criteria included pregnancy, allergies, immune depresεion of any kind, chronic debilitating diεeaεe, cancer, injection of immune globinε in the past three months, and seropoεitivity to human immunodeficiency virus (HIV) or to hepatitis B viruε εurface antigen. Study deεiσn. Participantε were randomly allocated to receive either standard Human Diploid Cell Rabies Vaccine (HDC) batch no E0751 (Pasteur Merieux Serums & Vaccine, Lyon, France) or the study vaccine ALVAC-RG (VCP65) . The trial was deεignated aε a dose escalation study. Three batches of experimental ALVAC-RG (vCP65) vaccine were uεed εeguentially in three groupε of volunteers (Groups A, B and C) with two week intervals between each step. The concentration of the three batches was IO 3 ' 5 , IO 4*5 , IO 5,5 Tisεue Culture Infectiouε Doεe (TCID 50 ) per doεe, reεpectively. Each volunteer received two doεeε of the εame vaccine subcutaneously in the deltoid region at an interval of four weeks. The nature of the injected vaccine was not known by the participants at the time of the firεt injection but waε known by the inveεtigator. In order to minimize the riεk of immediate hypersensitivity at the time of the second injection, the volunteers of Group B allocated to the medium doεe of experimental vaccine were injected 1 h previouεly with the lower doεe and thoεe allocated to the higher doεe (Group C) received εucceεsively the lower and the medium doεe at hourly intervals. Six months later, the recipientε of the higheεt doεage of ALVAC-RG (VCP65) (Group C) and HDC vaccine were offered a third doεe of vaccine; they were then randomized to receive either the εame vaccine aε previously or the alternate vaccine. As a result, four groups were formed corresponding to the following immunization scheme: 1. HDC, HDC - HDC; 2. HDC, HDC - ALVAC-RG (VCP65) ; 3. ALVAC-RG (VCP65) , ALVAC-RG (VCP65) - HDC; 4. ALVAC-RG (VCP65) , ALVAC-RG (VCP65) , ALVAC-RG (VCP65) . Monitoring of Side Effects. All subjects were monitored for 1 h after injection and re-examined every day for the next five days. They were aεked to record local and systemic reactions for the next three weeks and were questioned by telephone two timeε a week. Laboratory Inveεtiσatorε. Blood specimenε were obtained before enrollment and two, four and six days after each injection. Analysis included complete blood cell count, liver enzymes and creatine kinase assays. Antibodv aεεayε. Antibody aεεayε were performed seven days prior to the first injection and at days 7, 28, 35, 56, 173, 187 and 208 of the study. The levels of neutralizing antibodieε to rabies were determined using the Rapid Fluorescent Focus Inhibition test (RFFIT) (Smith & Yaeger, In Laboratory Techniques on Rabies) . Canarypox antibodies were meaεured by direct ELISA. The antigen, a εuεpenεion of purified canarypox viruε disrupted with 0.1% Triton X100, was coated in microplateε. Fixed dilutionε of the εera were reacted for two hourε at room temperature and reacting antibodieε were revealed with a peroxidaεe labelled anti-human IgG goat εerum. The reεultε are expreεsed aε the optical denεity read at 490nm. Analvεiε. Twenty-five εubjectε were enrolled and completed the εtudy. There were 10 maleε and 15 femaleε and the mean age waε 31.9 (21 to 48). All but three subjects had evidence of previous smallpox vaccination; the three remaining subjects had no typical scar and vaccination hiεtory. Three εubjectε received each of the lower doεeε of experimental vaccine (10 3-5 and IO 4 ' 5 TCID 50 ) , nine εubjectε received 10 5 * 5 TCID 50 and ten received the HDC vaccine. Safety (Table 14). During the primary εeries of immunization, fever greater than 37.7°c was noted within 24 hours after injection in one HDC recipient (37.8 β C) and in one VCP65 10 5#s TCID 50 recipient (38 β C) . No other εyεtemic reaction attributable to vaccination waε observed in any participant. Local reactions were noted in 9/10 recipientε of HDC vaccine injected subcutaneously and in 0/3, 1/3 and 9/9 recipients of VCP65 10 3 * 5 , IO 4*5 , 10 5 ' 5 TCID 50 , respectively. Tendernesε waε the moεt common symptoms and was always mild. Other local symptoms included rednesε and induration which were also mild and transient. All symptoms usually subsided within 24 hours and never lasted more than 72 hours. There was no significant change in blood cell counts, liver enzymes or creatine kinaεe valueε. Immune Reεponεes: Neutralizing Antibodies to Rabies (Table 16) . Twenty eight days after the first injection all the HDC recipients had protective titerε (≥0.5 IU/ml) . By contraεt none in groupε A and B (IO 3,5 and IO 4,5 TCID 50 ) and only 2/9 in group C (IO 5 ' 5 TCID 50 ) ALVAC-RG (VCP65) recipientε reached thiε protective titer. At day 56 (i.e. 28 dayε after the εecond injection) protective titers were achieved in 0/3 of Group A, 2/3 of Group B and 9/9 of Group C recipients of ALVAC-RG (vCP65) vaccine and persisted in all 10 HDC recipients. At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and 11.5 IU/ml in groupε A, B, C and HDC respectively. At day 180, the rabies antibody titers had subεtantially decreased in all subjectε but remained above the minimum protective titer of 0.5 IU/ml in 5/10 HCD recipientε and in 5/9 ALVAC-RG (VCP65) recipientε; the geometric mean titers were 0.51 and 0.45 IU/ml in groups HCD and C, respectively. Antibodies to the Canarypox viruε (Table 17) . The pre- immune titerε obεerved varied widely with titerε varying from 0.22 to 1.23 O.D. unitε deεpite the abεence of any previouε contact with canary birdε in thoεe εubjectε with the higheεt titerε. When defined aε a greater than two-fold increase between preimmunization and post second injection titers, a seroconversion was obtained in 1/3 subjectε in group B and in 9/9 εubjectε in group C whereaε no εubject seroconverted in groupε A or HDC. Booster Injection. The vaccine was similarly well tolerated six months later, at the time of the booster injection: fever was noted in 2/9 HDC booster recipients and in 1/10 ALVAC-RG (vCP65) booster recipients. Local reactions were present in 5/9 recipients of HDC booster and in 6/10 recipients of the ALVAC-RG (vCP65) booster. Obεervationε. FIGS. 22A-22D εhowε graphε of rabieε neutralizing antibody titerε (Rapid Fluoreεcent Focuε Inhibition Test or RFFIT, IU/ml) : Booster effect of HDC and VCP65 (IO 5,5 TCID 50 ) in volunteers previously immunized with either the same or the alternate vaccine. Vaccineε were given at dayε 0, 28 and 180. Antibody titerε were measured at days 0, 7, 28, 35, 56, 173, and 187 and 208. As εhown in FIGS. 22A-22D, the booεter doεe given reεulted in a further increaεe in rabieε antibody titerε in every subject whatever the immunization scheme. However, the ALVAC-RG (vCP65) booster globally elicited lower immune reεponses than the HDC booεter and the ALVAC-RG (VCP65) , ALVAC-RG (VCP65) - ALVAC-RG (VCP65) group had εignificantly lower titerε than the three other groupε. Similarly, the ALVAC-RG (vCP65) booεter injection reεulted in an increaεe in canarypox antibody titers in 3/5 εubjectε who had previouεly received the HDC vaccine and in all five subjects previously immunized with ALVAC-RG (vCP65) . In general, none of the local εide effectε from adminiεtration of VCP65 waε indicative of a local replication of the viruε. In particular, leεionε of the skin such as those observed after injection of vaccine were absent. In spite of the apparent absence of replication of the viruε, the injection reεulted in the volunteerε generating εignificant amountε of antibodieε to both the canarypox vector and to the expressed rabies glycoprotein. Rabies neutralizing antibodieε were aεεayed with the Rapid Fluoreεcent Focuε Inhibition Teεt (RFFIT) which iε known to correlate well with the εero neutralization test in mice. Of 9 recipients of 10 5 * 5 TCID 50 , five had low level responseε after the firεt doεe. Protective titerε of rabieε antibodieε were obtained after the εecond injection in all recipientε of the higheεt doεe teεted and even in 2 of the 3 recipientε of the medium dose. In this study, both vaccines were given εubcutaneously as usually recommended for live vaccines, but not for the inactivated HDC vaccine. This route of injection was selected as it best allowed a careful examination of the injection εite, but thiε could explain the late appearance of antibodieε in HDC recipientε: indeed, none of the HDC recipientε had an antibody increase at day 7, whereas, in moεt εtudieε where HDC vaccine iε give intramuεcularly a εignificant proportion of εubjectε do (Klietmann et al., Int'l Green Cross - Geneva, 1981; Kuwert et al., Int'l Green Crosε - Geneva, 1981). However, thiε invention iε not neceεεarily limited to the εubcutaneouε route of adminiεtration. The GMT (geometric mean titerε) of rabieε neutralizing antibodieε waε lower with the inveεtigational vaccine than with the HDC control vaccine, but εtill well above the minimum titer required for protection. The clear doεe effect response obtained with the three dosageε uεed in thiε study suggest that a higher dosage might induce a stronger responεe. Certainly from thiε diεcloεure the εkilled artiεan can εelect an appropriate doεage for a given patient. The ability to booεt the antibody response is another important reεult of thiε Example; indeed, an increaεe in rabieε antibody titerε waε obtained in every εubject after the 6 month doεe whatever the immunization εcheme, εhowing that preexiεting immunity elicited by either the canarypox vector or the rabieε glycoprotein had no blocking effect on the booεter with the recombinant vaccine candidate or the conventional HDC rabieε vaccine. Thiε contraεtε findingε of otherε with vaccinia recombinantε in humanε that immune reεponεe may be blocked by pre-exiεting immunity (Cooney et al., Lancet 1991, 337:567-72; Etinger et al., Vaccine 9:470- 72, 1991). Thuε, thiε Example clearly demonεtrateε that a non- replicating poxviruε can serve as an immunizing vector in animals or humans, with all of the advantages that replicating agents confer on the immune reεponεe, but without the safety problem created by a fully permisεive virus. TABLE 15: Reactions in the 5 days following vaccination 0.007 student t test 95/26751 -98- TABLE 17: Canarypox antibodies: ELISA Geometric Mean Titers* optical density at 1/25 dilution ExamPle 23 - COMPARISON OF THE LD 50 OF ALVAC AND NYVAC WITH VARIOUS VACCINIA VIRUS STRAINS Mice. Male outbred Swiεε Webster mice were purchased from Taconic Farms (Germantown, NY) and maintained on mouse chow and water ad libitum until use at 3 weekε of age ("normal" mice) . Newborn outbred Swiεε Webεter mice were of both sexes and were obtained following timed pregnancies performed by Taconic Farms. All newborn mice used were delivered within a two day period. Viruseε. ALVAC waε derived by plague purification of a canarypox viruε population and was prepared in primary chick embryo fibroblast cellε (CEF) . Following purification by centrifugation over sucrose density gradients, ALVAC was enumerated for plaque forming units in CEF cells. The WR(L) variant of vaccinia virus was derived by selection of large plaque phenotypes of WR (Panicali et al., 1981). The Wyeth New York State Board of Health vaccine εtrain of vaccinia viruε waε obtained from Pharmaceuticalε Calf Lymph Type vaccine Dryvax, control number 302001B. Copenhagen strain vaccinia virus VC-2 was obtained from Inεtitut Merieux, France. Vaccinia viruε εtrain NYVAC waε derived from Copenhagen VC-2. All vaccinia viruε strains except the Wyeth strain were cultivated in Vero African green monkey kidney cellε, purified by εucroεe gradient denεity centrifugation and enumerated for plaque forming unitε on Vero cellε. The Wyeth εtrain waε grown in CEF cellε and enumerated in CEF cells. Inoculations. Groupε of 10 normal mice were inoculated intracranially (ic) with 0.05 ml of one of εeveral dilutionε of viruε prepared by 10-fold serially diluting the εtock preparationε in sterile phosphate-buffered saline. In some inεtances, undiluted stock virus preparation was used for inoculation. Groups of 10 newborn mice, 1 to 2 days old, were inoculated ic εimilarly to the normal mice except that an injection volume of 0.03 ml was used. All mice were observed daily for mortality for a period of 14 dayε (newborn mice) or 21 days (normal mice) after inoculation. Mice found dead the morning following inoculation were excluded due to potential death by trauma. The lethal dose required to produce mortality for 50% of the experimental population (LD 50 ) was determined by the proportional method of Reed and Muench. Comparison of the LD 50 of ALVAC and NYVAC with Various Vaccinia Virus Strains for Normal. Young Outbred Mice bv the ic Route. In young, normal mice, the virulence of NYVAC and ALVAC were several orders of magnitude lower than the other vaccinia virus strainε teεted (Table 18) . NYVAC and ALVAC were found to be over 3,000 timeε leεε virulent in normal mice than the Wyeth strain; over 12,500 times lesε virulent than the parental VC-2 εtrain; and over 63,000,000 times lesε virulent than the WR(L) variant. Theεe reεultε would suggest that NYVAC is highly attenuated compared to other vaccinia εtrainε, and that ALVAC iε generally nonvirulent for young mice when adminiεtered intracranially, although both may cause mortality in mice at extremely high doseε (3.85X10 8 PFUs, ALVAC and 3xl0 8 PFUs, NYVAC) by an undetermined mechanism by this route of inoculation. Comparison of the LD 50 of ALVAC and NYVAC with Variouε Vaccinia Virus Strains for Newborn Outbred Mice bv the ic Route. The relative virulence of 5 poxvirus strains for normal, newborn mice was teεted by titration in an intracranial (ic) challenge model εyεtem (Table 19) . With mortality aε the endpoint, LD 50 valueε indicated that ALVAC iε over 100,000 timeε less virulent than the Wyeth vaccine strain of vaccinia virus; over 200,000 times less virulent than the Copenhagen VC-2 εtrain of vaccinia virus; and over 25,000,000 times lesε virulent than the WR-L variant of vaccinia virus. Nonethelesε, at the higheεt doεe teεted, 6.3X10 7 PFUs, 100% mortality resulted. Mortality rates of 33.3% were observed at 6.3xl0 6 PFUs. The cauεe of death, while not actually determined, waε not likely of toxicological or traumatic nature since the mean survival time (MST) of mice of the higheεt doεage group (approximately 6.3 LD 50 ) was 6.7 ± 1.5 days. When compared to WR(L) at a challenge dose of 5 LD 50 , wherein MST is 4.8 ± 0.6 dayε, the MST of ALVAC challenged mice waε significantly longer (P=0.001). Relative to NYVAC, Wyeth waε found to be over 15,000 timeε more virulent; VC-2, greater than 35,000 timeε more virulent; and WR(L) , over 3,000,000 timeε more virulent. Similar to ALVAC, the two higheεt doεeε of NYVAC, 6x10 8 and 6xl0 7 PFUs, caused 100% mortality. However, the MST of mice challenged with the higheεt dose, corresponding to 380 LD 50 , was only 2 dayε (9 deathε on day 2 and 1 on day 4) . In contraεt, all mice challenged with the highest dose of WR-L, equivalent to 500 LD 50 , εurvived to day 4. 95/26751 -102- Table 18. Calculated 50% Lethal Dose for mice by various vaccinia virus εtrains and for canarypox viruε (ALVAC) by the ic route. Table 19. Calculated 50% Lethal Doεe for newborn mice by variouε vaccinia viruε εtrainε and for canarypox viruε (ALVAC) by the ic route. POXVIRUS CALCULATED STRAIN LD 50 (PFUε) ExamPle 24 - EVALUATION OF NYVAC (VP866) AND NYVAC-RG (V-P879) Immunoprecipitations♦ Preformed onolayers of avian or non-avian cellε were inoculated with 10 pfu per cell of parental NYVAC (VP866) or NYVAC-RG (VP879) viruε. The inoculation waε performed in EMEM free of methionine and supplemented with 2% dialyzed fetal bovine serum. After a one hour incubation, the inoculum was removed and the medium replaced with EMEM (methionine free) containing 20 μCi/ml of 35 S-methionine. After an overnight incubation of approximately 16 hourε, cellε were lyεed by the addition of Buffer A (1% Nonidet P-40, 10 mM Triε pH7.4, 150 mM NaCl, l mM EDTA, 0.01% εodium azide, 500 unitε per ml of aprotinin, and 0.02% phenyl methyl sulfonyl fluoride). Immunoprecipitation was performed using a rabies glycoprotein specific monoclonal antibody designated 24-3F10 supplied by Dr. C. Trimarchi, Griffith Laboratories, New York State Department of Health, Albany, New York, and a rat anti-mouse conjugate obtained from Boehringer Mannheim Corporation (Cat. #605-500) . Protein A Sepharose CL-48 obtained from Pharmacia LKB Biotechnology Inc., Piεcataway, New Jersey, was used as a support matrix. I munoprecipitateε were fractionated on 10% polyacrylamide gelε according to the method of Dreyfuεε et. al. (1984). Gelε were fixed, treated for fluorography with IM Na- εalicylate for one hour, and expoεed to Kodak XAR-2 film to viεualize the immunoprecipitated protein species. Sources of Animalε. New Zealand White rabbits were obtained from Hare-Marland (Hewitt, New Jersey) . Three week old male Swiss Webster outbred mice, timed pregnant female Swiss Webster outbred mice, and four week old Swiss Webster nude (nu + nu + ) mice were obtained from Taconic Farms, Inc. (Germantown, New York) . All animals were maintained according to NIH guidelines. All animal protocols were approved by the institutional IACUC. When deemed necesεary, mice which were obviously terminally ill were euthanized. 95/26751 -104- Evaluation of Lesions in Rabbits. Each of two rabbits was inoculated intradermally at multiple sites with 0.1 ml of PBS containing IO 4 , 10 5 , IO 6 , IO 7 , or IO 8 pfu of each test virus or with PBS alone. The rabbitε were observed daily from day 4 until lesion reεolution. Indurationε and ulcerationε were meaεured and recorded. Viruε Recovery from Inoculation Siteε. A εingle rabbit waε inoculated intradermally at multiple εiteε of 0/1 ml of PBS containing io 6 , 10 7 , or IO 8 pfu of each teεt viruε or with PBS alone. After 11 days, the rabbit was euthanized and skin biopsy specimenε taken from each of the inoculation εites were aεeptically prepared by mechanical diεruption and indirect εonication for viruε recovery. Infectiouε viruε waε aεsayed by plaque titration on CEF monolayerε. Virulence in Mice. Groupε of ten mice, or five in the nude mice experiment, were inoculated ip with one of εeveral dilutionε of viruε in 0.5 ml of sterile PBS. Reference is also made to Example 23. Cvclophospha ide (CY) Treatment. Mice were injected by the ip route with 4 mg (0.02 ml) of CY (SIGMA) on day -2, followed by viruε injection on day 0. On the following days post infection, mice were injected ip with CY: 4 mg on day 1; 2 mg on days 4, 7 and 11; 3 mg on days 14, 18, 21, 25 and 28. Immunosuppresεion waε indirectly monitored by enumerating white blood cellε with a Coulter Counter on day 11. The average white blood cell count waε 13,500 cellε per μl for untreated mice (n=4) and 4,220 cellε per μl for CY- treated control mice (n=5) . Calculation of LD 50 . The lethal doεe required to produce 50% mortality (LD 50 ) waε determined by the proportional method of Reed and Muench (Reed and Muench 1938) . Potency Testing of NYVAC-RG in Mice. Four to six week old mice were inoculated in the footpad with 50 to 100 μl of a range of dilutions (2.0 - 8.0 log 10 tissue culture infective dose 50% (TCID S0 )) of either W-RG (Kieny et al., 1984), ALVAC-RG (Taylor et al., 1991b), or the NYVAC-RG. Each group consiεted of eight mice. At 14 dayε post- vaccination, the mice were challenged by intracranial inoculation with 15 LD 50 of the rabies virus CVS εtrain (0.03 ml). On day 28, εurviving mice were counted and protective doeε 50% (PD 50 ) calculated. Derivation of NYVAC (vP866) . The NYVAC εtrain of vaccinia virus was generated from VC-2, a plaque cloned isolate of the COPENHAGEN vaccine εtrain. To generate NYVAC from VC-2, eighteen vaccinia ORFε, including a number of viral functionε aεεociated with virulence, were preciεely deleted in a εerieε of sequential manipulations as described earlier in this disclosure. These deletions were constructed in a manner designed to prevent the appearance of novel unwanted open reading frames. FIG. 19 εchematically depictε the ORFε deleted to generate NYVAC. At the top of FIG. 19 iε depicted the Hindlll reεtriction map of the vaccinia viruε genome (VC-2 plaque iεolate, COPENHAGEN εtrain) . Expanded are the εix regionε of VC-2 that were sequentially deleted in the generation of NYVAC. The deletions were described earlier in this discloεure (Examples 13 through 18) . Below such deletion locus is listed the ORFs which were deleted from that locuε, along with the functionε or ho ologieε and molecular weight of their gene products. Replication Studies of NYVAC and ALVAC on Human Tiεεue Cell Lines. In order to determine the level of replication of NYVAC strain of vaccinia virus (vP866) in cells of human origin, six cell lines were inoculated at an input multiplicity of 0.1 pfu per cell under liquid culture and incubated for 72 hours. The COPENHAGEN parental clone (VC- 2) was inoculated in parallel. Primary chick embryo fibroblast (CEF) cells (obtained from 10-11 day old embryonated eggs of SPF origin, Spafas, Inc., Storrs, CT) were included to represent a permissive cell substrate for all viruses. Cultures were analyzed on the basis of two criteria: the occurrence of productive viral replication and expression of an extrinsic antigen. The replication potential of NYVAC in a number of human derived cellε are εhown in Table 20. Both VC-2 and NYVAC are capable of productive replication in CEF cellε, although NYVAC with εlightly reduced yieldε. VC-2 iε alεo capable of productive replication in the εix human derived cell lines teεted with comparable yieldε except in the EBV tranεformed ly phoblaεtoid cell line JT-1 (human lymphoblaεtoid cell line transformed with Epstein-Barr viruε, εee Rickinεon et al., 1984). In contract, NYVAC iε highly attenuated in itε ability to productively replicate in any of the human derived cell lines tested. Small increases of infectious virus above reεidual viruε levelε were obtained from NYVAC- infected MRC-5 (ATCC #CCL171, human embryonic lung origin) , DETROIT 532 (ATCC #CCL54, human foreεkin. Downs Syndrome), HEL 299 (ATCC #CCL137, human embryonic lung cells) and HNK (human neonatal kidney cells, Whittiker Bioproductε, Inc. Walkerεville, MD, Cat #70-151) cellε. Replication on theεe cell lineε waε εignificantly reduced when compared to viruε yieldε obtained from NYVAC-infected CEF cellε or with parental VC-2 (Table 20) . It εhould be noted that the yieldε at 24 hourε in CEF cellε for both NYVAC and VC-2 iε equivalent to the 72-hour yield. Allowing the human cell line cultureε to incubate an additional 48 hourε (another two viral growth cycleε) may, therefore, have amplified the relative viruε yield obtained. Consistent with the low levels of virus yields obtained in the human-derived cell lines, MRC-5 and DETROIT 532, detectable but reduced levelε of NYVAC-εpecific DNA accumulation were noted. The level of DNA accumulation in the MRC-5 and DETROIT 532 NYVAC-infected cell lineε relative to that obεerved in NYVAC-infected CEF cellε paralleled the relative viruε yieldε. NYVAC-εpecific viral DNA accumulation waε not obεerved in any of the other human- derived cellε. An equivalent experiment was also performed uεing the avipox viruε, ALVAC. The reεults of virus replication are also shown in Table 20. No progeny virus waε detectable in any of the human cell lines consistent with the host range restriction of canarypox virus to avian species. Alεo conεiεtent with a lack of productive replication of ALVAC in theεe human-derived cells is the observation that no ALVAC- εpecific DNA accumulation was detectable in any of the human-derived cell lineε. Expreεεion of Rabieε Glycoprotein by NYVAC-RG (vP879) in Human Cellε. In order to determine whether efficient expression of a foreign gene could be obtained in the absence of significant levels of productive viral replication, the εame cell lines were inoculated with the NYVAC recombinant expressing the rabies virus glycoprotein (VP879, Example 19) in the presence of 35 S-methionine. Immunoprecipitation of the rabies glycoprotein waε performed from the radiolabelled culture lyεate uεing a monoclonal antibody εpecific for the rabieε glycoprotein. Immunoprecipitation of a 67kDa protein was detected consistent with a fully glycosylated form of the rabies glycoprotein. No serologically crossreactive product waε detected in uninfected or parental NYVAC infected cell lyεateε. Equivalent reεultε were obtained with all other human cellε analyzed. Inoculationε on the Rabbit Skin. The induction and nature of εkin leεionε on rabbitε following intradermal (id) inoculationε haε been previouεly uεed aε a measure of pathogenicity of vaccinia virus strains (Buller et al., 1988; Child et al., 1990; Fenner, 1958, Flexner et al., 1987; Ghendon and Chernos 1964). Therefore, the nature of leεionε aεsociated with id inoculations with the vaccinia strains WR (ATCC #VR119 plaque purified on CV-1 cells, ATCC #CCL70, and a plaque isolate designated L variant, ATCC #VR2035 selected, as described in Panicali et al., 1981)), WYETH (ATCC #VR325 marketed as DRYVAC by Wyeth Laboratories, Marietta, PA), COPENHAGEN (VC-2), and NYVAC was evaluated by inoculation of two rabbits (A069 and A128) . The two rabbits displayed different overall sensitivities to the viruseε, with rabbit A128 diεplaying leεε severe reactions than rabbit A069. In rabbit A128, leεions were relatively εmall and reεolved by 27 days post-inoculation. On rabbit A069, lesionε were intense, especially for the WR inoculation εiteε, and reεolved only after 49 dayε. Intenεity of the leεionε was also dependent on the location of the inoculation εites relative to the lymph drainage network. In particular, all siteε located above the backεpine displayed more intense lesionε and required longer timeε to reεolve the leεionε located on the flankε. All leεionε were meaεured daily from day 4 to the diεappearance of the laεt leεion, and the meanε of maximum leεion εize and dayε to reεolution were calculated (Table 21) . No local reactions were observed from εites injected with the control PBS. Ulcerative leεionε were obεerved at εiteε injected with WR, VC-2 and WYETH vaccinia viruε εtrainε. Significantly, no induration or ulcerative leεionε were obεerved at εiteε of inoculation with NYVAC. Perεiεtence of Infectious Virus at the Site of Inoculation. To asεeεε the relative perεiεtence of theεe viruεeε at the εite of inoculation, a rabbit waε inoculated intradermally at multiple εiteε with 0.1 ml PBS containing IO 6 , IO 7 or IO 8 pfu of VC-2, WR, WYETH or NYVAC. For each viruε, the IO 7 pfu doεe waε located above the backεpine, flanked by the 10 6 and IO 8 doεeε. Siteε of inoculation were obεerved daily for 11 dayε. WR elicited the moεt intenεe reεponεe, followed by VC-2 and WYETH (Table 22) . Ulceration waε firεt obεerved at day 9 for WR and WYETH and day 10 for VC-2. Sites inoculated with NYVAC or control PBS displayed no induration or ulceration. At day 11 after inoculation, skin samples from the siteε of inoculation were exciεed, mechanically disrupted, and virus was titrated on CEF cells. The results are shown in Table 22. In no case was more viruε recovered at thiε timepoint than waε administered. Recovery of vaccinia strain, WR, was approximately IO 6 pfu of virus at each site irrespective of amount of virus administered. Recovery of vaccinia strains WYETH and VC-2 was IO 3 to IO 4 pfu regardless of amount administered. No infectiouε viruε waε recovered from εites inoculated with NYVAC. Inoculation of Genetically or Chemically Immune Deficient Mice. Intraperitoneal inoculation of high doεeε of NYVAC (5 X IO 8 pfu) or ALVAC (IO 9 pfu) into nude mice caused no deaths, no lesions, and no apparent disease through the 100 day observation period. In contrast, mice inoculated with WR (10 3 to IO 4 pfu) , WYETH (5 x IO 7 or 5 x IO 8 pfu) or VC-2 (IO 4 to IO 9 pfu) displayed disεeminated lesions typical of poxviruses first on the toes, then on the tail, followed by severe orchitis in some animals. In mice infected with WR or WYETH, the appearance of disεeminated leεionε generally led to eventual death, whereas most mice infected with VC-2 eventually recovered. Calculated LD 50 values are given in Table 23. In particular, mice inoculated with VC-2 began to display lesionε on their toeε (red papuleε) and 1 to 2 dayε later on the tail. Theεe leεionε occurred between 11 and 13 days post-inoculation (pi) in mice given the highest doses (10 9 , 10 8 , 10 7 and IO 6 pfu), on day 16 pi in mice given IO 5 pfu and on day 21 pi in mice given IO 4 pfu. No lesionε were obεerved in mice inoculated with IO 3 and 10 2 pfu during the 100 day obεervation period. Orchitiε waε noticed on day 23 pi in mice given IO 9 and IO 8 pfu, and approximately 7 dayε later in the other groupε (10 7 to 10 4 pfu) . Orchitiε waε especially intense in the IO 9 and IO 8 pfu groups and, although receding, was observed until the end of the 100 day observation period. Some pox-like lesionε were noticed on the skin of a few mice, occurring around 30-35 days pi. Most pox lesions healed normally between 60-90 days pi. Only one mouse died in the group inoculated with IO 9 pfu (Day 34 pi) and one mouse died in the group inoculated with IO 8 pfu (Day 94 pi) . No other deaths were observed in the VC-2 inoculated mice. Mice inoculated with IO 4 pfu of the WR strain of vaccinia started to display pox lesionε on Day 17 pi. Theεe lesions appeared identical to the lesionε displayed by the VC-2 injected mice (εwollen toes, tail) . Mice inoculated with IO 3 pfu of the WR strain did not develop lesions until 34 days pi. Orchitis waε noticed only in the mice inoculated with the higheεt doεe of WR (IO 4 pfu) . During the latter εtageε of the obεervation period, leεionε appeared around the mouth and the mice εtopped eating. All mice inoculated with IO 4 pfu of WR died or were euthanized when deemed neceεεary between 21 dayε and 31 dayε pi. Four out of the 5 mice injected with IO 3 pfu of WR died or were euthanized when deemed neceεεary between 35 dayε and 57 dayε pi. No deathε were obεerved in mice inoculated with lower doses of WR (1 to 100 pfu) . Mice inoculated with the WYETH strain of vaccinia viruε at higher doses 5 x IO 7 and 5 x IO 8 pfu) showed leεionε on toeε and tailε, developed orchitiε, and died. Mice injected with 5 x IO 6 pfu or leεε of WYETH εhowed no εignε of diεeaεe or leεionε. Aε εhown in Table 23, CY-treated mice provided a more senεitive model for aεεaying poxviruε virulence than did nude mice. LD 50 valueε for the WR, WYETH, and VC-2 vaccinia viruε εtrainε were εignificantly lower in thiε model εyεtem than in the nude mouse model. Additionally, lesionε developed in mice injected with WYETH, WR and VC-2 vaccinia viruses, as noted below, with higher doseε of each virus resulting in more rapid formation of lesionε. Aε waε εeen with nude mice, CY-treated mice injected with NYVAC or ALVAC did not develop leεionε. However, unlike nude mice, some deaths were observed in CY-treated mice challenged with NYVAC or ALVAC, regardlesε of the doεe. Theεe random incidenceε are εuεpect aε to the cauεe of death. Mice injected with all doεeε of WYETH (9.5 x IO 4 to 9.5 x IO 8 pfu) displayed pox lesionε on their tail and/or on their toes between 7 and 15 days pi. In addition, the tails and toes were swollen. Evolution of lesionε on the tail waε typical of pox leεionε with formation of a papule, ulceration and finally formation of a εcab. Mice inoculated with all doses of VC-2 (1.65 x 10 s to 1.65 x IO 9 ) also -Ill- developed pox lesionε on their tailε and/or their toeε analogouε to thoεe of WYETH injected mice. Theεe leεions were observed between 7-12 days post inoculation. No leεionε were obεerved on mice injected with lower doεeε of WR viruε, although deathε occurred in theεe groupε. Potency Teεting of NYVAC-RG. In order to determine that attenuation of the COPENHAGEN εtrain of vaccinia viruε had been effected without εignificantly altering the ability of the reεulting NYVAC εtrain to be a uεeful vector, comparative potency teεtε were performed. In order to monitor the immunogenic potential of the vector during the εequential genetic manipulationε performed to attenuate the viruε, a rabieεviruε glycoprotein waε uεed aε a reporter extrinεic antigen. The protective efficacy of the vectorε expreεεing the rabieε glycoprotein gene waε evaluated in the εtandard NIH mouse potency test for rabieε (Seligmann, 1973) . Table 24 demonεtrateε that the PD 50 valueε obtained with the highly attenuated NYVAC vector are identical to thoεe obtained using a COPENHAGEN-based recombinant containing the rabieε glycoprotein gene in the £k locuε (Kieny et al., 1984) and εimilar to PD 50 valueε obtained with ALVAC-RG, a canarypox baεed vector reεtricted to replication to avian species. Observationε. NYVAC, deleted of known virulence geneε and having reεtricted in vitro growth characteriεticε, waε analyzed in animal model εyεtemε to aεεeεε its attenuation characteristicε. These studies were performed in comparison with the neurovirulent vaccinia virus laboratory εtrain, WR, two vaccinia viruε vaccine εtrainε, WYETH (New York City Board of Health) and COPENHAGEN (VC-2), aε well aε with a canarypox viruε εtrain, ALVAC (See also Example 23). Together, these viruses provided a spectrum of relative pathogenic potentials in the mouse challenge model and the rabbit skin model, with WR being the most virulent strain, WYETH and COPENHAGEN (VC-2) providing previously utilized attenuated vaccine strains with documented characteristicε, and ALVAC providing an example of a poxviruε whoεe 5/26751 -112- replication iε restricted to avian species. Results from these in vivo analyseε clearly demonstrate the highly attenuated properties of NYVAC relative to the vaccinia virus εtrainε, WR, WYETH and COPENHAGEN (VC-2) (Tableε 18- 24). Significantly, the LD 50 valueε for NYVAC were comparable to thoεe obεerved with the avian host restricted avipoxvirus, ALVAC. Deaths due to NYVAC, aε well aε ALVAC, were obεerved only when extremely high doεeε of viruε were adminiεtered via the intracranial route (Example 23, Tableε 18, 19, 23). It haε not yet been eεtabliεhed whether theεe deathε were due to nonεpecific conεeguenceε of inoculation of a high protein maεε. Reεultε from analyεes in immunocompromised mouεe modelε (nude and CY-treated) also demonstrate the relatively high attenuation characteriεticε of NYVAC, aε compared to WR, WYETH and COPENHAGEN strains (Tables 21 and 22) . Significantly, no evidence of diεεeminated vaccinia infection or vaccinial disease was observed in NYVAC-inoculated animals or ALVAC-inoculated animals over the observation period. The deletion of multiple virulence-associated genes in NYVAC showε a εynergiεtic effect with reεpect to pathogenicity. Another meaεure of the innocuity of NYVAC waε provided by the intradermal adminiεtration on rabbit εkin (Tableε 21 and 22) . Considering the resultε with ALVAC, a viruε unable to replicate in nonavian species, the ability to replicate at the site of inoculation is not the εole correlate with reactivity, since intradermal inoculation of ALVAC caused areas of induration in a dose dependent manner. Therefore, it iε likely that factorε other than the replicative capacity of the viruε contribute to the formation of the leεionε. Deletion of geneε in NYVAC preventε leεion occurrence. Together, the reεultε in this Example and in foregoing Examples, including Example 23, demonεtrate the highly attenuated nature of NYVAC relative to WR, and the previouεly utilized vaccinia viruε vaccine strains, WYETH and COPENHAGEN. In fact, the pathogenic profile of NYVAC, in the animal model εyεte s teεted, was εi ilar to that of ALVAC, a poxviruε known to productively replicate only in avian εpecieε. The apparently reεtricted capacity of NYVAC to productively replicate on cellε derived from humanε (Table 20) and other εpecieε, including the mouse, swine, dog and horse, provides a considerable barrier that limits or prevents potential tranεmission to unvaccinated contacts or to the general environment in addition to providing a vector with reduced probability of disεemination within the vaccinated individual. Significantly, NYVAC-baεed vaccine candidates have been εhown to be efficaciouε. NYVAC reco binantε expreεsing foreign gene products from a number of pathogens have elicited immunological responεeε towards the foreign gene products in several animal species, including primates. In particular, a NYVAC-based recombinant expreεεing the rabieε glycoprotein waε able to protect mice againεt a lethal rabieε challenge. The potency of the NYVAC-baεed rabieε glycoprotein recombinant was comparable to the PD 50 value for a COPENHAGEN-based recombinant containing the rabies glycoprotein in the tk locuε (Table 24). NYVAC-baεed recombinantε have also been shown to elicit measleε viruε neutralizing antibodieε in rabbitε and protection againεt pεeudorabieε virus and Japaneεe encephalitiε viruε challenge in swine. The highly attenuated NYVAC strain confers safety advantages with human and veterinary applicationε (Tartaglia et al., 1990). Furthermore, the use of NYVAC as a general laboratory expression vector syεtem may greatly reduce the biological hazardε associated with using vaccinia virus. By the following criteria, the results of this Example and the Examples herein, including Example 23, show NYVAC to be highly attenuated: a) no detectable induration or ulceration at site of inoculation (rabbit skin) ; b) rapid clearance of infectious virus from intradermal site of inoculation (rabbit skin) ; c) absence of testicular inflammation (nude mice) ; d) greatly reduced virulence (intracranial challenge, both three-week old and newborn mice) ; e) greatly reduced pathogenicity and failure to disεeminate in immunodeficient subjects (nude and cyclophosphamide treated mice) ; and f) dramatically reduced ability to replicate on a variety of human tiεsue culture cellε. Yet, in εpite of being highly attenuated, NYVAC, aε a vector, retains the ability to induce strong immune responεeε to extrinεic antigenε. Table 20. Replication of COPENHAGEN (VC-2), NYVAC and ALVAC in avian or human derived cell lines a: Yield of NYVAC at 72 hours post-infection expressed as a percentage of yield of VAC-2 after 72 hours on the same cell line. b: Titer expressed as LOG 50 pfu per ml. c: Sample was incubated in the presence of 40μg/ml. of cytosine arabinoside. d: Not determined. *: ATCC #CCL25 Human amnionic cells. Table 21. Induration and ulceration at the εite of intradermal inoculation of the rabbit εkin a pfu of indicated vaccinia viruε in 0.1 ml PBS inoculated intradermally into one εite. b mean maximum εize of leεionε (mm 2 ) c mean time after inoculation for complete healing of lesion. no lesionε diεcernable. Table 22. Persiεtence of poxviruses at the site of intradermal inoculation a: expreεsed aε log 10 pfu. Table 23. Virulence studieε in immunocompromiεed mice a: Calculated 50% lethal dose (pfu) for nude or cyclophosphamide treated mice by the indicated vaccinia viruεeε and for ALVAC by intraperitoneal route. b: 5 out of 10 mice died at the higheεt doεe of 5 x IO 8 pfu. Table 24. Comparative efficacy of NYVAC-RG and ALVAC-RG in mice Recombinant J P D≥al W-RG 3.74 ALVAC-RG 3.86 NYVAC-RG 3.70 a: Four to εix week old mice were inoculated in the footpad with 50-100μl of a range of dilutionε (2.0 - 8.0 log 10 tissue culture infection dose 50% (TCID 50 ) of either the W-RG (Kieny et al., 1984), ALVAC-RG (VCP65) or NYVAC-RG (VP879) . At day 14, mice of each group were challenged by intracranial inoculation of 30μl of a live CVS εtrain rabieε viruε corresponding to 15 lethal dose 50% (LD 50 ) per mouse. At day 28, surviving mice were counted and a protective dose 50% (PD 50 ) waε calculated. ExamDle 25 - CONSTRUCTION OF TROVAC RECOMBINANTS EXPRESSING THE HEMAGGLUTININ GLYCOPROTEINS OF AVIAN INFLUENZA VIRUSES This Example deεcribeε the development of fowlpox viruε recombinantε expreεsing the hemagglutinin genes of three serotypeε of avian influenza virus. Cells and Viruses. Plasmidε containing cDNA clones of the H4, H5 and H7 hemagglutinin genes were obtained from Dr. Robert Webεter, St. Jude Children'ε Reεearch Hoεpital, Memphiε, Tenneεsee. The strain of FPV designated FP-1 has been described previously (Taylor et al., 1988a, b) . It is a vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France aε a fowlpox εcab from a chicken. The virus was attenuated by approximately 50 serial pasεageε in chicken embryonated eggε followed by 25 paεεageε on chick embryo fibroblaεt (CEF) cellε. Thiε viruε waε obtained in September 1980 by Rhone Merieux, Lyon, France, and a master viral seed established. The virus was received by Virogenetics in September 1989, where it waε subjected to four succeεεive plaque purifications. One plaque isolate was further amplified in primary CEF cellε and a εtock viruε, deεignated aε TROVAC, waε eεtablished. The stock virus used in the in vitro recombination test to produce TROVAC-AIH5 (vFP89) and TR0VAC-AIH4 (vFP92) had been further amplified though 8 passages in primary CEF cells. The stock virus used to produce TR0VAC-AIH7 (vFPlOO) had been further amplified through 12 passages in primary CEF cells. Construction of Fowlpox Insertion Plaεmid at F8 Locuε. Plaεmid pRW731.15 containε a 10 kbp PvuII-PvuII fragment cloned from TROVAC genomic DNA. The nucleotide sequence waε determined on both strands for a 3659 bp £yuII-£≤oRV fragment. This sequence is shown in FIG. 20 (SEQ ID NO:69). The limits of an open reading frame designated in this laboratory as F8 were determined within this sequence. The open reading frame is initiated at position 495 and terminates at position 1887. A deletion was made from position 779 to position 1926, as described below. Plaε id pRW761 iε a εub-clone of pRW731. 15 containing a 2430 bp EcoRV-EcoRV fragment . Plaεmid pRW761 waε completely digested with Xbal and partially digested with Sspl . A 3700 bp Xbal-Sεol band waε iεolated and ligated with the annealed double-εtranded oligonucleotideε JCA017 (SEQ ID NO: 57) and JCA018 (SEQ ID NO: 58) . JCA017 ( SEQ ID NO: 57 ) 5 ' CTAGACACTTTATGTTTTTTAATATCCGGTCTT AAAAGCTTCCCGGGGATCCTTATACGGGGAATAAT 3' JCA018 (SEQ ID NO:58) 5' ATTATTCCCCGTATAAGGATCCCCCGGGAA GCTTTTAAGACCGGATATTAAAAAACATAAAGTGT 3' The plaεmid reεulting from thiε ligation was designated pJCA002. Plasmid pJCA004 contains a non-pertinent gene linked to the vaccinia viruε H6 promoter in plaεmid pJCA002. The sequence of the vaccinia virus H6 promoter haε been previouεly deεcribed (Taylor et al., 1988a, b; Guo et al. 1989; Perkuε et al., 1989). Plaεmid pJCA004 waε digeεted with EcoRV and BamHI which deletes the non-pertinent gene and a portion of the 3' end of the H6 promoter. Annealed oligonucleotides RW178 (SEQ ID NO:70) and RW179 (SEQ ID NO:71) were cut with EcoRV and BamHI and inserted between the EcoRV and BamHI siteε of JCA004 to form pRW846. R 178 (SEQ ID NO:70): 5' TCATTATCGCGATATCCGTGTTAACTAGCTA GCTAATTTTTATTCCCGGGATCCTTATCA 3' RW179 (SEQ ID NO:71): 5' GTATAAGGATCCCGGGAATAAAAATTAGCT AGCTAGTTAACACGGATATCGCGATAATGA 3' Plaεmid pRW846 therefore containε the H6 promoter 5' of EcoRV in the de-ORFed F8 locuε. The Hindi εite 3*' of the H6 promoter in pRW846 is followed by translation stop codons, a transcriptional stop sequence recognized by vaccinia virus early promoters (Yuen et al., 1987) and a Smal site. Construction of Fowlpox Inεertion Plaεmid at F7 Locuε. The original F7 non-de-ORFed insertion plasmid, pRW731.13, contained a 5.5 kb FP genomic PvuII fragment in the PvuII site of pUC9. The insertion site was a unique Hindi site within these sequences. The nucleotide sequence shown in FIG. 21 (SEQ ID NO:72) was determined for a 2356 bp region encompassing the unique fiincll site. Analysis of this sequence revealed that the unique Hindi εite (FIG. 21, underlined) waε εituated within an ORF encoding a polypeptide of 90 amino acidε. The ORF beginε with an ATG at poεition 1531 and terminateε at position 898 (positionε marked by arrowε in FIG. 21) . The armε for the de-ORFed inεertion plaεmid were derived by PCR uεing pRW731.13 aε template. A 596 bp arm (deεignated aε HB) correεponding to the region upεtream from the ORF waε amplified with oligonucleotideε F73PH2 (SEQ ID NO:73) (5'-GACAATCTAAGTCCTATATTAGAC-3') and F73PB (SEQ ID NO:74) (5'-GGATTTTTAGGTAGACAC-3') . A 270 bp arm (deεignated aε EH) correεponding to the region downstream from the ORF was amplified using oligonucleotides F75PE (SEQ ID NO:75) (5 , -TCATCGTCTTCATCATCG-3') and F73PH1 (SEQ ID N0:76) (5'- GTCTTAAACTTATTGTAAGGGTATACCTG-3') . Fragment EH was digested with EcoRV to generate a 126 bp fragment. The EcoRV site is at the 3"-end and the 5'-end was formed, by PCR, to contain the 3' end of a Hindi site. This fragment was inserted into pBS-SK (Stratagene, La Jolla, CA) digested with Hindi to form plasmid pF7Dl. The εequence waε confirmed by dideoxynucleotide sequence analyεiε. The plaεmid pF7Dl waε linearized with Apal. blunt-ended using T4 DNA polymerase, and ligated to the 596 bp HB fragment. The resultant plasmid waε deεignated aε pF7D2. The entire sequence and orientation were confirmed by nucleotide sequence analysis. The plasmid pF7D2 was digested with EcoRV and Bglll to generate a 600 bp fragment. This fragment was inserted into pBS-SK that was digested with Apal. blunt-ended with T4 DNA polymerase, and subsequently digested with BamHI. The resultant plasmid was designated as pF7D3. Thiε plasmid contains an HB arm of 404 bp and a EH arm of 126 bp. The plasmid pF7D3 was linearized with Xhol and blunt- ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. Thiε linearized plasmid was ligated with annealed oligonucleotideε F7MCSB (SEQ ID NO:77) (5'-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTTTTTATTAGTTT AGTTAGTC-3 ' ) and F7MCSA (SEQ ID NO:78) (5'-GACTAACTAACTAATA AAAAACCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAATCGTT-3') . Thiε was performed to inεert a multiple cloning region containing the reεtriction εiteε for Hindlll. Pεtl and Smal between the EH and HB arms. The resultant plasmid was deεignated aε pF7DO. Conεtruction of Insertion Plasmid for the H4 Hemagglutinin at the F8 Locus. A cDNA copy encoding the avian influenza H4 derived from A/Ty/Min/833/80 was obtained from Dr. R. Webεter in plaεmid pTM4H833. The plasmid was digested with HLndlll and Nrul and blunt-ended using the Klenow fragment of DNA polymeraεe in the preεence of dNTPε. The blunt-ended 2.5 kbp Hindlll-Nrul fragment containing the H4 coding region waε inεerted into the Hindi εite of pIBI25 (International Biotechnologieε, Inc., New Haven, CT) . The reεulting plaεmid pRW828 waε partially cut with Banll. the linear product iεolated and recut with fiindlll. Plaεmid pRW828 now with a 100 bp Hindlll-Banll deletion waε used as a vector for the synthetic oligonucleotides RW152 (SEQ ID NO:79) and RW153 (SEQ ID N0:80). These oligonucleotides repreεent the 3" portion of the H6 promoter from the £c_oRV εite and align the ATG of the promoter with the ATG of the H4 CDNA. RW152 (SEQ ID NO:79): 5' GCACGGAACAAAGCTTATCGCGATATCCGTTA AGTTTGTATCGTAATGCTATCAATCACGATTCTGTTCCTGCTCATAGC AGAGGGCTCATCTCAGAAT 3' RW153 (SEQ ID NO:80): 5' ATTCTGAGATGAGCCCTCTGCTATGAGCAGGA ACAGAATCGTGATTGATAGCATTACGATACAAACTTAACGGATATCGC GATAAGCTTTGTTCCGTGC 3' The oligonucleotideε were annealed, cut with Banll and Hindlll and inεerted into the Hindlll-Banll deleted pRW828 vector described above. The resulting plasmid pRW844 was cut with EcoRV and Dral and the 1.7 kbp fragment containing the 3" H6 promoted H4 coding sequence was inserted between the EcoRV and Hindi sites of pRW846 (described previously) forming plasmid pRW848. Plasmid pRW848 therefore contains the H4 coding sequence linked to the vaccinia viruε H6 promoter in the de-ORFed F8 locuε of fowlpox virus. Conεtruction of Insertion Plasmid for H5 Hemagglutinin at the F8 Locus. A cDNA clone of avian influenza H5 derived from A/Turkey/Ireland/1378/83 waε received in plaεmid pTH29 from Dr. R. Webεter. Synthetic oligonucleotideε RW10 (SEQ ID NO:81) through RW13 (SEQ ID NO:84) were designed to overlap the translation initiation codon of the previously described vaccinia virus H6 promoter with the ATG of the H5 gene. The sequence continues through the 5" Sail εite of the H5 gene and beginε again at the 3' H5 Dral εite containing the H5 stop codon. RW10 (SEQ ID NO:81): 5' GAAAAATTTAAAGTCGACCTGTTTTGTTGAGT TGTTTGCGTGGTAACCAATGCAAATCTGGTC ACT 3' RW11 (SEQ ID NO:82): 5' TCTAGCAAGACTGACTATTGCAAAAAGAAGCA CTATTTCCTCCATTACGATACAAACTTAACG GAT 3' R 12 (SEQ ID NO:83): 5' ATCCGTTAAGTTTGTATCGTAATGGAGGAAA TAGTGCTTCTTTTTGCAATAGTCAGTCTTGCTAGAAGTGACCAGATTT GCATTGGT 3' RW13 (SEQ ID NO:84): 5' TACCACGCAAACAACTCAACAAAACAGGTCG ACTTTAAATTTTTCTGCA 3' The oligonucleotides were annealed at 95°C for three minutes followed by slow cooling at room temperature. This resultε in the following double εtrand εtructure with the indicated endε. EcoRV PstI RW12 R 13 RWΪ3 1 RWΪϋ Cloning of oligonucleotideε between the EcoRV and Pεtl sites of pRW742B resulted in ρRW744. Plasmid pRW742B contains the vaccinia virus H6 promoter linked to a non¬ pertinent gene inserted at the Hindi site of pRW731.15 described previously. Digestion with PstI and EcoRV eliminates the non-pertinent gene and the 3'-end of the H6 promoter. Plasmid pRW744 now contains the 3' portion of the H6 promoter overlapping the ATG of avian influenza H5. The plasmid also contains the H5 sequence through the 5' Sail site and the 3' sequence from the H5 stop codon (containing a Dral εite). Uεe of the Dral εite removes the H5 3' non- coding end. The oligonucleotides add a transcription termination signal recognized by early vaccinia virus RNA polymeraεe (Yuen et al., 1987). To complete the H6 promoted H5 conεtruct, the H5 coding region waε iεolated as a 1.6 kpb Sall-Dral fragment from pTH29. Plasmid pRW744 was partially digested with Dral f the linear fragment iεolated, recut with Sail and the plaεmid now with eight bases deleted between Sail and Dral waε uεed aε a vector for the 1.6 kpb pTH29 Sail and β£al fragment. The resulting plasmid pRW759 was cut with EcoRV and Dral. The 1.7 kbp PRW759 EcoRV-Dral fragment containing the 3' H6 promoter and the H5 gene was inεerted between the EcoRV and fiincll εiteε of pRW846 (previouεly deεcribed) . The reεulting plaεmid pRW849 contains the H6 promoted avian influenza virus H5 gene in the de-ORFed F8 locus. Construction of Insertion Vector for H7 Hemagglutinin at the F7 Locus. Plaεmid pCVH71 containing the H7 hemagglutinin from A/CK/VIC/1/85 waε received from Dr. R. Webεter. An EcoRI-BamHI fragment containing the H7 gene waε blunt-ended with the Klenow fragment of DNA polymeraεe and inεerted into the Hindi εite of pIBI25 aε PRW827. Synthetic oligonucleotideε RW165 (SEQ ID NO:85) and RW166 (SEQ ID NO:86) were annealed, cut with Hindi and Stvl and inserted between the EcoRV and Stvl siteε of pRW827 to generate pRW845. R 165 (SEQ ID NO:85): 5' GTACAGGTCGACAAGCTTCCCGGGTATCGCG ATATCCGTTAAGTTTGTATCGTAATGAATACTCAAATTCTAATACTCA CTCTTGTGGCAGCCATTCACACAAATGCAGACAAAATCTGCCTTGGAC ATCAT 3' RW166 (SEQ ID NO:86): 5' ATGATGTCCAAGGCAGATTTTGTCTGCATTTG TGTGAATGGCTGCCACAAGAGTGAGTATTAGAATTTGAGTATTCATTA CGATACAAACTTAACGGATATCGCGATACCCGGGAAGCTTGTCGACCT GTAC 3' Oligonucleotideε RW165 (SEQ ID NO:85) and RW166 (SEQ ID NO:86) link the 3' portion of the H6 promoter to the H7 gene. The 3' non-coding end of the H7 gene waε removed by isolating the linear product of an ApaLI digeεtion of pRW845, recutting it with EcoRI. iεolating the largeεt fragment and annealing with εynthetic oligonucleotideε RW227 (SEQ ID NO:87) and RW228 (SEQ ID NO:88). The resulting plasmid was pRW854. R 227 (SEQ ID NO:87): 5' ATAACATGCGGTGCACCATTTGTATAT AAGTTAACGAATTCCAAGTCAAGC 3' RW228 (SEQ ID NO:88): 5' GCTTGACTTGGAATTCGTTAACTTATA TACAAATGGTGCACCGCATGTTAT 3' The stop codon of H7 in PRW854 iε followed by an Hoal εite. The intermediate H6 promoted H7 conεtruct in the de-ORFed F7 locus (described below) was generated by moving the pRW854 EcoRV-Hoal fragment into pRW858 which had been cut with EcoRV and blunt-ended at its PstI site. Plasmid pRW858 (described below) containε the H6 promoter in an F7 de-ORFed inεertion plaεmid. The plaεmid pRW858 was constructed by insertion of an 850 bp Smal/Hoal fragment, containing the H6 promoter linked to a non-pertinent gene, into the Smal site of pF7DO described previously. The non-pertinent εequences were excised by digestion of pRW858 with EcoRV (site 24 bp upstream of the 3'-end of the H6 promoter) and PstI. The 3.5 kb resultant fragment waε iεolated and blunt-ended uεing the Klenow fragment of the E. coli DNA polymeraεe in the preεence of 2mM dNTPε. This blunt-ended fragment was ligated to a 1700 bp EcoRV/Hpal fragment derived from pRW854 (described previously) . Thiε EcoRV/Hpal fragment containε the entire AIV HA (H7) gene juxtapoεed 3' to the 3'-moεt 24 bp of the W H6 promoter. The reεultant plaεmid waε designated pRW861. The 126 bp EH arm (defined previously) waε lengthened in pRW861 to increase the recombination frequency with genomic TROVAC DNA. To accomplish this, a 575 bp AccI/SnaBI fragment was derived from pRW 731.13 (defined previously). The fragment was isolated and inserted between the AccI and Nael sites of pRW861. The resultant plasmid, containing an EH arm of 725 bp and a HB arm of 404 bp flanking the AIV H7 gene, was deεignated aε pRW869. Plasmid pRW869 therefore consists of the H7 coding sequence linked at its 5' end to the vaccinia virus H6 promoter. The left flanking arm conεiεtε of 404 bp of TROVAC εequence and the right flanking arm of 725 bp of TROVAC εequence which directε inεertion to the de-ORFed F7 locuε. pevelopment of TRQVAC-Avian Influenza Viruε Recombinantε. Inεertion plaεmidε containing the avian influenza viruε HA coding εequenceε were individually tranεfected into TROVAC infected primary CEF cellε by uεing the calcium phoεphate precipitation method previouεly described (Panicali et al., 1982; Piccini et al., 1987). Positive plaqueε were εelected on the baεiε of hybridization to HA εpecific radiolabelled probes and subjected to εequential roundε of plaque purification until a pure population waε achieved. One repreεentative plaque was then amplified to produce a stock virus. Plasmid pRW849 waε uεed in an in vitro recombination teεt to produce recombinant TR0VAC-AIH5 (vFP89) expreεεing the H5 hemagglutinin. Plaεmid pRW848 waε uεed to produce recombinant TR0VAC-AIH4 (VFP92) expreεεing the H4 hemagglutinin. Plaεmid pRW869 waε uεed to produce recombinant TR0VAC-AIH7 (vFPlOO) expreεεing the H7 hemagglutinin. Immunofluoreεcence. In influenza viruε infected cells, the HA molecule is εynthesized and glycosylated aε a precursor molecule at the rough endoplasmic reticulum. During passage to the plasma membrane it undergoeε extenεive poεt-tranεlational modification culminating in proteolytic cleavage into the diεulphide linked HA 2 and HA 2 εubunitε and inεertion into the hoεt cell membrane where it iε subsequently incorporated into mature viral envelopes. To determine whether the HA molecules produced in cellε infected with the TROVAC-AIV recombinant viruεeε were expreεsed on the cell surface, immunofluoreεcence studies were performed. Indirect immunofluoreεcence waε performed aε deεcribed (Taylor et al., 1990). Surface expreεsion of the H5 hemagglutinin in TR0VAC-AIH5, H4 hemagglutinin in TROVAC-AIH4 and H7 hemagglutinin in TR0VAC-AIH7 was confirmed by indirect immunofluoreεcence. Expreεsion of the H5 hemagglutinin was detected using a pool of monoclonal antibodieε εpecific for the H5HA. Expresεion of the H4HA waε analyzed uεing a goat monoεpecific anti-H4 εerum. Expresεion of the H7HA waε analyzed uεing a H7 εpecific monoclonal antibody preparation. Immunoprecipitation. It haε been determined that the sequence at and around the cleavage site of the hemagglutinin molecule plays an important role in determining viral virulence since cleavage of the hemagglutinin polypeptide is necesεary for viruε particleε to be infectiouε. The hemagglutinin proteinε of the virulent H5 and H7 viruεeε poεεeεε more than one baεic amino acid at the carboxy terminuε of HA1. It iε thought that thiε allowε cellular proteaεes which recognize a serieε of baεic amino acidε to cleave the hemagglutinin and allow the infectiouε viruε to εpread both in vitro and in vivo . The hemagglutinin moleculeέ of H4 avirulent εtrainε are not cleaved in tiεεue culture unleεε exogenouε trypεin iε added. In order to determine that the hemagglutinin molecules expreεsed by the TROVAC recombinantε were authentically proceεsed, immunoprecipitation experiments were performed aε deεcribed (Taylor et al., 1990) uεing the εpecific reagentε deεcribed above. Immunoprecipitation analyεiε of the H5 hemagglutinin expressed by TROVAC-AIH5 (vFP89) showed that the glycoprotein is evident as the two cleavage productε HA-, and HA 2 with approximate molecular weightε of 44 and 23 kDa, respectively. No such proteins were precipitated from uninfected cellε or cellε infected with parental TROVAC. Similarly immunoprecipitation analyεiε of the hemagglutinin expressed by TR0VAC-AIH7 (vFPlOO) showed specific precipitation of the HA 2 cleavage product. The H χ cleavage product was not recognized. No proteins were specifically precipitated from uninfected CEF cells or TROVAC infected CEF cells. In contrast, immunoprecipitation analysiε of the expression product of TROVAC-AIH4 <vFP92) showed expresεion of only the precursor protein HA Q . This iε in agreement with the lack of cleavage of the hemagglutinins of avirulent εubtypeε in tiεεue culture. No H4 εpecific proteinε were detected in uninfected CEF cells or cells infected with TROVAC. Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidaεe activity are aε previouεly deεcribed (Panicali et al., 1982; Perkuε et al., 1989). Example 26 - CHV gB, gC AND gD NUCLEOTIDES IN VECTOR SYSTEM, EXPRESSION THEREFROM AND USE OF VECTOR SYSTEM AND EXPRESSION PRODUCT Expreεεion of the CHV gB glycoprotein iε accompliεhed by putting the CHV gB homolog gene under the control of the vaccinia viruε I3L promoter. Expreεεion of the CHV gC glycoprotein iε accompliεhed by putting the CHV gC homolog gene under the control of the vaccinia viruε H6 promoter. Expreεεion of the CHV gD glycoprotein iε accompliεhed by putting the CHV gD homolog gene under the control of the entomopox viruε 42K gene promoter. The gB and gC coding iε in the ATI locuε and, the gD coding iε in the HA locuε. Generation of Donor Plaεmid. The CHV gB coding εequence iε PCR-derived. The CHV gB fragment iε fuεed to a PCR-derived fragment containing the I3L promoter element in a plaεmid containing the caεεette I3L-CHV gB in the ATI deorfed locuε. The CHV gC coding iε PCR-derived and iε fuεed in the HA deorfed locuε in a plaεmid. A donor plaεmid iε uεed to inεert the I3L-CHV gB — H6- CHV gC double conεtruction in the NYVAC ATI deorfed locuε. In vitro recombination iε performed on Vero cells using the donor plasmid and vP866 (NYVAC) as the reεcuing viruε. Standard protocolε were uεed to identify and purify the recombinant viruε (Piccini et al., 1987). The NYVAC-baεed recombinant containing the CHV gB and gC genes in the ATI deorfed locus is designated NYVAC-CHVgBgC. Generation of Donor Plasmid. The CHV gD coding sequence iε fuεed to the 42K promoter and a resulting plasmid therefrom generated for insertion with the NYVAC HA deorfed locus. In vitro recombination is performed on Vero cells using the CHV gD-42K donor plasmid and recombinant vaccinia virus NYVAC-CHVgBgC (NYVAC background) aε the rescuing virus. This iε performed with εtandard procedures (Piccini et al., 1987) . The NYVAC-based recombinant containing the CHV gB and gC geneε in the ATI deorfed locuε and the CHV gD gene in the HA deorfed locus is designated NYVAC-CHVgBgCgD. Generation of ALVAC donor plasmid. A plaεmid donor plaεmid to inεert the I3L-CHV gB — H6-CHV gC — 42K-CHV gD triple conεtruction in the ALVAC C3 deorfed locuε iε constructed from the above plasmidε. In vitro recombination is performed on primary chick embryo fibroblasts using the donor plasmid and CPpp (ALVAC) as the rescuing virus. Standard procedures are followed to identify and purify the generated recombinant (Piccini et al., 1987). The ALVAC-based recombinant containε the CHV gB, gC and gD geneε in the C3 deorfed locuε and is designated ALVAC-CHVgBgCgD. Analysiε confirms expresεion of the glycoproteinε by the recombinantε and, the glycoproteinε are substantially within the predicted sequences. Example 27 - GENERATION OF VCP320; AN ALVAC RECOMBINANT EXPRESSING CHV σB VCP320, an ALVAC recombinant expreεsing CHV gB, was generated by the following procedure. A 6 kb Xbal fragment, containing the CHV gB gene, waε iεolated from genomic CHV DNA and cloned into the Xbal εite of pBSK+. The plaεmid generated by this manipulation is called pCHV2. The CHV gB gene was then cloned between canarypox flanking arms. This was accomplished by cloning the 3,700 bp Sad-EcoRV fragment of pCHV2, containing the CHV gB gene, into the 5,800 bp Sacl-Nael fragment of pBHVC16. (pBHVC16 contains a copy of the BHV1 gC gene cloned between C5 flanking arms.) The plaεmid generated by thiε manipulation iε called pCHV14. Extraneous 3'-noncoding sequence was then eliminated. This was accomplished by cloning a 210 bp Smal-Clal-digested PCR fragment, containing the 3'-end of the gB gene, into the 5,500 bp partial Sroal-Clal fragment of pCHV14. (Thiε PCR fragment was generated from the plasmid, pCHV2, with the primers, CHVP39 (SEQ ID NO: 89; 5'-TAAGAATGGTAATTCT-3') and CHVP40 (SEQ ID NO: 90; 5'-TTCCCGGGTTAAACTTTACTTTCATTTTC- 3').) The plaεmid generated by thiε manipulation iε called pCHV15. The I3L promoter waε then cloned upεtream from the gB initiation codon. In addition, 3 T 5 NT early tranεcription termination εignal sequences located in the 5'-end of the gB gene were modified. This was accomplished by cloning a 140 bp S^al-S^ll-digested PCR fragment, containing the I3L promoter and the T 5 NT-modified 5'-end of the gB gene, into the 6,300 bp Seal-Sail fragment of pCHVl5. (This PCR fragment waε generated from the plaεmid, pCHV2, with the primerε, CHVP42 (SEQ ID NO: 91; 5'- TTGTCGACTGAGATAAAGTGAAAATATATATCATTATATTACAAAGTACAATTATTTAGG TTTAATCATGTTTTCATTGTATCTATAT-3') and CHVP78 (SEQ ID NO: 92; 5'-TTAGTACTTTCCGGTGTTGTTGGATCACATATTATTAAAGTATAAATAATAAAGAA- 3') .) The plaεmid generated by thiε manipulation iε called PCHV27. An error in the εequence flanking the Seal-Sail fragment of pCHV27 waε then corrected. Thiε waε accompliεhed by cloning the 180 bp Seal-Sail fragment of pCHV27, containing the I3L promoter and the T 5 NT-modified 5'-end of the gB gene, into the 6,300 bp Seal-Sail fragment of pCHVlS. The plasmid generated by this manipulation is called pCHV28. An early transcription termination signal εequence near the 3'-end of the CHV gB gene waε then modified. Thiε waε accompliεhed by cloning a 330 bp Spel-Aεp718-digeεted PCR fragment, containing the T 5 NT-modified region of the CHV gB gene, into the 5,450 bp Spel-Aεp7l8 fragment of pCHV28. (Thiε PCR fragment waε generated from a 150 bp PCR fragment, a 280 bp PCR fragment and the primerε, CHVP89 (SEQ ID NO: 93; 5'-TGGAATGAAGTTATGAAACT-3') and CHVP92 (SEQ ID NO: 94; 5'-TGCACTGATCATTTCAATTTC-3') . The 150 bp PCR fragment waε generated from the plaεmid, pCHV2, with the primerε, CHVP89 (SEQ ID NO: 93; 5'-TGGAATGAAGTTATGAAACT-3') and CHVP90 (SEQ IS NO: 95; 5'-TGGAATTTTGAATGAAAACACTAGAACC-3') . The 280 bp PCR fragment was generated from the plasmid, pCHV2, with the primers, CHVP91 (SEQ ID NO: 96; 5'- TTCTAGTGTTTTCATTCAAAATTCCAT-3') and CHVP92 (SEQ ID NO: 94; 5'-TGCACTGATCATTTCAATTTC-3') .) The plaεmid generated by thiε manipulation iε called pCHV31. An early tranεcription termination εignal εequence in the middle of the CHV gB gene waε then modified. Thiε waε accompliεhed by cloning a 480 bp BamHI-BsaBI-digested PCR fragment, containing the T 5 NT-modified region of the gB gene, into the 5,000 bp BamHI-BsaBI fragment of pCHV31. (Thiε PCR fragment waε generated from a 380 bp PCR fragment, a 210 bp PCR fragment and the primerε, CHVP87 (SEQ ID NO: 97; 5'-CCTTCAAAGTTTAATACACC-3') and CHVP94 (SEQ ID NO: 98; 5'-TATGGCTTCACGTTTGGCAC-3') . The 380 bp PCR fragment waε generated from the plaεmid, pCHV2, with the primerε, CHVP93 (SEQ ID NO: 99; 5'- CACCGGGGATATAATTCATATGTCCCCTTTCTTTGGATTACGAGATGGT-3') and CHVP94 (SEQ ID NO: 98; 5'-TATGGCTTCACGTTTGGCAC-3') . The 210 bp PCR fragment waε generated from the plaεmid, pCHV2, with the primerε, CHVP87 (SEQ ID NO: 97; 5'-CCTTCAAAGTTTAATACACC- 3') and CHVP88 (SEQ ID NO: 100; 5'- CCATCTCGTAATCCAAAGAAAGGGGACATATGAAT-3') .) The plaεmid generated by thiε manipulation iε called pCHV32. A portion of the gB gene removed in the previouε manipulation waε then cloned back into pCHV32. Thiε waε accompliεhed by cloning the 2,000 bp partial BεaBI-Pεtl fragment of pCHV31, containing the 3'-end of the gB gene removed in the previouε manipulation, into the 5,450 bp BεaBI-PstI fragment of pCHV32. The plaεmid generated by this manipulation is called pCHV36. The I3L-promoted gB gene was then cloned between C6 flanking arms. This was accomplished by cloning the 2,750 bp Sall-Smal fragment of pCHV36, containing the I3L-promoted gB gene, into the 4,350 bp Sall-Smal fragment of pHIV34. (pHIV34 contains a copy of the H6-promoted HIV2 gpl20 (+ TM) gene cloned between C6 flanking arms.) The plasmid generated by thiε manipulation iε called pCHV37. The DNA εequence of the I3L-promoted gB gene in pCHV37 (SEQ ID NO: 101) iε εhown in Fig. 23. The DNA εequence of the ALVAC C6 flanking armε (SEQ ID NO: 102) iε εhown in Fig. 24. pCHV37 waε uεed in in vitro recombination experimentε with ALVAC aε the reεcuing viruε to yield VCP320. Immunoprecipitation analyεiε waε performed to determine whether VCP320 expreεεeε CHV gB. MDCK cell monolayerε were either mock infected or infected with the parental viruε (ALVAC) (m.o.i. = 15 PFU/cell) , VCP320 (m.o.i. = 15 PFU/cell) or CHV (m.o.i. = 10 PFU/cell). Following an hour adεorption period, the inoculum waε removed and the cellε were overlayed with 2 mlε of modified Eagle'ε medium (minuε cyεteine) containing 2% dialyzed fetal bovine serum and [ 35 S]-cysteine (50 μCi/ml) . The lysateε were harveεted at 18 hrε poεt-infection in 1 ml 3X buffer A (450 mM NaCl, 3% NP-40, 30 mM Triε (pH = 7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/ml PMSF) and analyzed for CHV gB expreεεion uεing a 1:100 dilution of a gB-εpecific monoclonal antibody, 1125B2 (obtained from Dr. Michel Riviere, Rhone Merieux, Lyon, France) . Lyεateε, precleared with normal mouεe εera and a goat anti-mouεe-protein A-εepharoεe complex, were incubated overnight at 4°C with a monoclonal antibody-goat anti-mouεe- protein A-sepharose complex and waεhed 4X with IX buffer A and 2X with a LiCl 2 /urea buffer. Precipitated proteinε were diεεociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Triε (pH = 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteinε were fractionated on an SDS-polyacrylamide gel, fixed and treated with 1 M Na-εalicylate for fluorography. Proteinε of the appropriate εize were precipitated from CHV- infected cells (lane D) and vCP320-infected cells (lane C) , but were not precipitated from mock-infected cellε (lane A) or ALVAC-infected cellε (lane B) (Fig. 25) . Theεe reεultε indicate that VCP320 expresseε CHV gB. Examole 28 - GENERATION OF VCP322; AN ALVAC RECOMBINANT EXPRESSING CHV C VCP322, an ALVAC recombinant expreεεing CHV gC, was generated by the following procedure. A 2.2 kb Ejc_gRI fragment, containing the CHV gC gene, was isolated from genomic CHV DNA and cloned into the EcoRI site of PVQH6CP3LSA. (pVQH6CP3LSA contains a copy of the H6 promoter cloned between C3 flanking armε.) Thiε manipulation poεitionε the gC gene downεtream from the H6 promoter and between C3 flanking armε. The plasmid generated by this manipulation is called pCHV17. Extraneous 3'-noncoding sequence waε then eliminated and 3 T 5 NT early tranεcription termination εignal εequences located near the 3'-end of the gC gene were modified. This was accomplished by cloning the oligonucleotides, CHVL66 (SEQ ID NO: 103; 5'- CGATGTTAATAAGTATTACCACAATAATTGGTGGAGCCATTTTCGTTATAGTATTGATTT TCATAACAGCTTTATGTTTCTATTGTTCAAAAAATAATAAGATCTAACTGCA-3') and CHVL67 (SEQ ID NO: 104; 5'- GTTAGATCTTATTATTTTTTGAACAATAGAAACATAAAGCTGTTATGAAAATCAATACTA TAACGAAAATGGCTCCACCAATTATTGTGGTAATACTTATTAACAT-3') , into the 8,400 bp partial Clal-Pεtl fragment of pCHVl7. The plaεmid generated by thiε manipulation iε called pCHV20. The initiation codon of the gC gene waε then aligned with the initiation codon of the H6 promoter. In addition, 2 early tranεcription termination εignal εequenceε were modified. This was accompliεhed by cloning a 740 bp Nrul- BεrGI-digeεted PCR fragment, containing the 3'-end of the H6 promoter and the 5'-end of the T 5 NT-modified gC gene, into the 7,900 bp ffruI-BεrGI fragment of pCHV20. (Thiε PCR fragment was generated from a 500 bp PCR fragment, a 300 bp PCR fragment and the oligonucleotides, CHVP96 (SEQ ID NO: 105; 5'-CGTAGATTCCAATGGAAAGT-3') and CHVP97 (SEQ ID NO: 106; 5'-TTTTCGCGATATCCGTTAAGT-3') . The 500 bp PCR fragment waε generated from the plaεmid, pCHV13, with the oligonucleotideε, CHVP68 (SEQ ID NO: 107; 5'- TTTTCGCGATATCCGTTAAGTTTGTATCGTAATGAGTTTTAAAAATTTCTATCTAATATA TGTAATTATAATTTTCATAAACTCGATAATAAC-3') and CHVP69 (SEQ ID NO: 108; 5'-TTTGTATACCTAATAAGAAATCATTATAAAAGT-3') . The 300 bp PCR fragment was generated from the plaεmid, pCHVl3, with the oligonucleotideε, CHVP95 (SEQ ID NO: 109; 5'- CTTTTATAATGATTTCTTATTAGGTATACAAAATC-3') and CHVP96 (SEQ ID NO: 105; 5'-CGTAGATTCCAATGGAAAGT-3') . pCHV13 was obtained by cloning the 2.2 kb EcoRI CHV genomic fragment, containing the gC gene, into the EcoRI εite of pBSK+.) The plaεmid generated by thiε manipulation iε called pCHV38. The H6-promoted gC gene was then cloned between C6 flanking arms. This was accomplished by cloning the 1,400 bp NruI-PstI fragment of pCHV38, containing the H6-promoted gC gene, and the oligonucleotide, CHVL98 (SEQ ID NO:110; 5'- AATTTGCA-3') , into the 4,500 bp Nrul-EcoRI fragment of pHIV34. (pHIV34 containε a copy of the H6-promoted HIV2 gpl20 (+ TM) gene cloned between C6 flanking armε.) The plaεmid generated by thiε manipulation iε called pCHV40. The DNA εequence of the H6-promoted gC gene in pCHV40 (SEQ ID NO: 111) iε shown in Fig. 26. The DNA sequence of the ALVAC C6 flanking arms (SEQ ID NO: 102) is shown in Fig. 24. pCHV40 was uεed in in vitro recombination experiments with ALVAC as the rescuing virus to yield VCP322,. Immunoprecipitation analyεiε waε performed to determine whether VCP322 expreεses CHV gC. MDCK cell monolayerε were either mock infected or infected with the parental viruε (ALVAC) (m.o.i. = 15 PFU/cell), VCP322 (m.o.i. = 15 PFU/cell) or CHV (m.o.i. = 10 PFU/cell). Following an hour adsorption period, the inoculum was removed and the cellε were overlayed with 2 mlε of modified Eagle'ε medium (minuε cyεteine) containing 2% dialyzed fetal bovine serum and [ 35 S]-cysteine (50 μCi/ml) . The lysateε were harveεted at 18 hrε poεt-infection in 1 ml 3X buffer A (450 mM NaCl, 3% NP-40, 30 mM Triε (pH - 7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/ml PMSF) and analyzed for CHV gC expreεεion uεing a 1:100 dilution of a gC-specific monoclonal antibody, 2011A9 (obtained from Dr. Michel Riviere, Rhone Merieux, Lyon, France) . Lyεates, precleared with normal mouse sera and a goat anti-mouse-protein A-sepharoεe complex, were incubated overnight at 4°C with a monoclonal antibody-goat anti-mouεe- protein A-εepharoεe complex and waεhed 4X with IX buffer A and 2X with a LiCl 2 /urea buffer. Precipitated proteinε were dissociated from the immune complexes by the addition of 2X Laemmli'ε buffer (125 mM Triε (pH = 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 in. Proteinε were fractionated on an SDS-polyacrylamide gel, fixed and treated with 1 M Na-εalicylate for fluorography. Proteinε of the appropriate size were precipitated from CHV- infected cells (lane D) and vCP322-infected cellε (lane C) , but were not precipitated from mock-infected cells (lane A) or ALVAC-infected cells (lane B) (Fig. 27) . These resultε indicate that VCP322 expreεεeε CHV gC. Exam p le 29 - GENERATION OF VCP29 ; AN ALVAC RECOMBINANT EXPRESSING CHV CD VCP294, an ALVAC recombinant expreεεing CHV gD, waε generated by the following procedure. A 7 kb pεtl fragment, containing the CHV gD gene, waε iεolated from genomic CHV DNA and cloned into the Pεtl εite of pBSK+. The plasmid generated by this manipulation iε called pCHVll. The CHV gD gene waε then cloned between canarypox flanking armε. Thiε waε accompliεhed by cloning the 1,475 bp Pεtl-SnaBI fragment of pCHVll, containing the CHV gD gene, into the 5,600 bp Pεtl-Smal fragment of pHIV34. (pHIV34 containε a copy of the H6-promoted HIV2 gpl20 (+ TM) gene cloned between C6 flanking armε.) Thiε placeε the CHV gD gene between C6 flanking armε and downεtream from the H6 promoter. The plasmid generated by this manipulation iε called pCHVlδ. The initiation codon of the H6 promoter waε then aligned with the initiation codon of the CHV gD gene. Thiε waε accomplished by cloning the oligonucleotideε, CHVL81 (SEQ ID NO: 112; 5'- CGATATCCGTTAAGTTTGTATCGTAATGATTAAACTTCTATTTATCTTATTTTATTTTAA CCCAATAA-3') and CHVL82 (SEQ ID NO: 113; 5'- TTGGGTTAAAATAAAATAAGATAAATAGAAGTTTAATCATTACGATACAAACTTAACGGA TATCG-3'), into the 5,600 bp NruI-PflMI fragment of pCHV18. The plaεmid generated by thiε manipulation iε called pCHV21. Three early tranεcription termination εignal sequences at the 3'-end of the CHVgD gene were then modified. This was accompliεhed by cloning, the 1,400 bp BglII-Aεp718 fragment of pCHV22, containing the "T 5 NT-modified" 3'-end of the CHV gD gene and C3 flanking arm, into the 3,700 bp BglII-Aεp718 fragment of pCHV21. (pCHV22 waε generated by cloning a 430 bp Bglll-EcoRl-digested PCR fragment, containing the "T 5 NT-modified" 3'-end of the CHV gD gene, into the 3,900 bp BolII-EcoRI fragment of pHIV43 (pHIV43 contains a copy of the H6-promoted HIV1 gpl20-murine IL-2 fusion gene cloned between C3 flanking arms) . (This PCR fragment was generated from the plasmid, pCHVlδ, with the oligonucleotides, CHVP79 (SEQ ID NO: 114; 5'- TTGAATTCCTAAACATTTGTTGTTAATTTTTTATAATTATTATATATTTTTTGTC TTTTATAAACAAAGAAT-3') and CHVP80 (SEQ ID NO: 115; 5'- TTAGATCTGTAGGAGCATCAAAAGTTGACGATGAACTTTTCTATCTAAATAGAGC TGGTCCCCAAACCCTGCTTAAATATTATGTTATTAAAGATTTCTAT-3') .) ) The plasmid generated by thiε manipulation is called pCHV24. The M T 5 NT-modified" central region of the CHV gD gene was then cloned into pCHV24. Thiε waε accompliεhed by cloning a 240 bp Bglll-digeεted PCR fragment, containing the "T 5 NT-modified" central region of the CHV gD gene, into the Bglll εite of pCHV24. (Thiε PCR fragment waε generated from the plasmid, pCHVlδ, with the oligonucleotides, CHVP83 (SEQ ID NO: 116; 5'- TTAGATCTAGATTCCTTACACCATTCCATAAAAGTTGGTTCAAATTTATCTTCTTTAGAG AAATAACAAGTTTCTCGTGGTAATTGAACCATAAAATCAGTATAGAAAAC-3') and CHVP84 (SEQ ID NO: 117; 5'-TATTTTGATTGTGATCC-3') .) The plaεmid generated by thiε manipulation iε called pCHV25. The C3 flanking arm waε then replaced with the C6 flanking arm. This was accomplished by cloning the 1,160 bp EcoRI-Asp718 fragment of pCHV21, containing the C6 flanking arm, into the 4,100 bp EcoRI-Asp718 fragment of pCHV25. The plasmid generated by this manipulation is called pCHV26. The DNA sequence of the H6-promoted gD gene in pCHV26 (SEQ ID NO: 118) iε εhown in Fig. 28. The DNA sequence of the ALVAC C6 flanking arms (SEQ ID NO: 102) is εhown in Fig. 24. pCHV26 was used in in vitro recombination experiments with ALVAC aε the reεcuing virus to yield VCP294. Immunoprecipitation analysiε was performed to determine whether VCP294 expresεeε CHV gD. MDCK cell monolayerε were either mock infected or infected with the parental viruε (ALVAC) (m.o.i. = 15 PFU/cell), VCP294 (m.o.i. = 15 PFU/cell) or CHV (m.o.i. ■ 10 PFU/cell). Following an hour adεorption period, the inoculum waε removed and the cellε were overlayed with 2 mlε of modified Eagle'ε medium (minus cysteine) containing 2% dialyzed fetal bovine serum and [ 35 S]-cysteine (50 μCi/ml) . The lysateε were harvested at 18 hrs poεt-infection in 1 ml 3X buffer A (450 mM NaCl, 3% NP-40, 30 mM Triε (pH = 7.4), 3 mM EDTA, 0.03% Na-Azide and 0.6 mg/ml PMSF) and analyzed for CHV gD expreεεion uεing a 1:100 dilution of a gD-εpecific monoclonal antibody, 208D11 (obtained from Dr. Michel Riviere, Rhone Merieux, Lyon, France). Lysates, precleared with normal mouεe sera and a goat anti-mouse-protein A-sepharoεe complex, were incubated overnight at 4°C with a monoclonal antibody-goat anti-mouse- protein A-sepharose complex and waεhed 4X with IX buffer A and 2X with a LiCl 2 /urea buffer. Precipitated proteinε were dissociated from the immune complexes by the addition of 2X Laemmli'ε buffer (125 mM Triε (pH = 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were fractionated on an SDS-polyacrylamide gel, fixed and treated with 1 M Na-salicylate for fluorography. Proteins of similar size were precipitated from CHV-infected cells (lane D) and vCP294-infected cells (lane C) , but were not precipitated from mock-infected cells (lane A) or ALVAC- infected cells (lane B) (Fig. 29). These reεultε indicate that VCP294 expresses CHV gD. Having thus described in detail preferred embodiments of the preεent invention, it iε to be underεtood that the invention defined by the appended claimε iε not to be limited by particular details set forth in the above deεcription aε many apparent variations thereof are possible without departing from the spirit or scope thereof. 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Claims: WHAT IS CLAIMED IS: 1. An iεolated nucleic acid coding for canine herpeεviruε gB, gC or gD glycoprotein. 2. The iεolated nucleic acid of claim 1 which haε a sequence of any one of SEQ ID NOS: 1, 10 and 15. 3. The iεolated nucleic acid of claim 1 which iε DNA. 4. The iεolated nucleic acid of claim 1 coding for canine herpeεviruε gB glycoprotein. 5. The iεolated nucleic acid of claim 1 coding for canine herεpeviruε gC glycoprotein. 6. The iεolated nucleic acid of claim 1 coding for canine herpeεviruε gD glycoprotein. 7. A vector containing the iεolated nucleic acid of claim 3. 8. The vector of claim 7 wherein the vector iε a poxviruε. 9. The vector of claim 8 wherein the poxviruε iε an avipox viruε or a vaccinia viruε. 10. The vector of claim 7 which iε a modified recombinant viruε, εaid modified recombinant viruε having virus-encoded genetic functions inactivated therein εo that the viruε haε attenuated virulence, yet retained efficacy; said virus further comprising exogenous DNA in a nonessential region of the virus genome, εaid exogenouε DNA encoding at leaεt one of canine herpeεviruε gB, gC and gD. 11. The vector of claim 10 wherein said virus iε a poxviruε. 12. The vector of claim 11 wherein the poxviruε iε a vaccinia viruε. 13. The vector of claim 12 wherein the genetic functionε are inactivated by deleting at leaεt one open reading frame. 14. The vector of claim 13 wherein the deleted genetic functionε include a C7L-K1L open reading frame, or, host range functions. 15. The vector of claim 14 wherein at leaεt one additional open reading frame is deleted; and, the additional open reading frame iε εelected from the group conεiεting of: J2R, B13R + B14R, A26L, A56R, and I4L. 16. The vector of claim 14 wherein at leaεt one additional open reading frame iε deleted; and, the additional open reading frame iε εelected from the group conεiεting of: a thymidine kinaεe gene, a hemorrhagic region, an A type incluεion body region, a hemagglutinin gene, and a large εubunit, ribonucleotide reductaεe. 17. The vector of claim 15 wherein J2R, B13R + B14R, A26L, A56R, C7L - KIL and I4L are deleted from the viruε. 18. The vector of claim 16 wherein a thymidine kinaεe gene, a hemorrhagic region, an A type incluεion body region, a hemagglutinin gene, a hoεt range region, and a large εubunit, ribonucleotide reductaεe are deleted from the viruε. 19. The vector of claim 17 which iε a NYVAC recombinant viruε. 20. The vector of claim 18 which iε a NYVAC recombinant viruε. 21. The vector of claim 7 which iε a modified recombinant avipox viruε which iε modified εo that it haε attenuated virulence in a hoεt; and, which containε exogenouε DNA in a noneεεential region of the viruε genome, said exogenous DNA encoding at least one of canine herpeεviruε gB, gC and gD. 22. The vector of claim 21 wherein εaid viruε iε a canarypox viruε. 23. The vector of claim 22 wherein the canarypox viruε iε a Rentεchler vaccine strain which was attenuated through more than 200 serial paεεageε on chick embryo fibroblaεtε, a master seed therefrom was subjected to four successive plaque purifications under agar, from which a plaque clone was amplified through five additional paεεageε. 24. The vector of claim 23 which is an ALVAC recombinant virus. 25. The vector of claim 24 which iε VCP320, VCP322 or, VCP294. 26. A compoεition for inducing an antigenic or immunological reεponεe compriεing a vector aε claimed in any one of claimε 7, 10, 19, 21 or 24 in admixture with a εuitable carrier. 27. A method for expreεεing a gene product in a cell cultured in vitro compriεing introducing into the cell a vector as claimed in any one of claims 7, 10, 19, 21 or 24. 28. Canine herpesviruε glycoprotein gB, gC or gD iεolated from in vitro expreεεion of a vector aε claimed in any one of claimε 7, 10, 19, 21 or 24.

Inventors: LIMBACH KEITH JEFFREY, PAOLETTI ENZO, LIMBACH, KEITH, JEFFREY, PAOLETTI, ENZO
Applicants: VIROGENETICS CORP, VIROGENETICS CORPORATION
Application year: 1995
Main classification: A61K39
IPC codes: A61K39, A61P31, C07H21, C07K14, C12N15, C12P21