CA2558659C - Genetically engineered canary poxvirus-based vaccine strain - Google Patents

Abstract

What is described is a modified vector, such as a recombinant poxvirus, particularly recombinant canary poxvirus, having enhanced safety. The modified recombinant virus has nonessential virus-encoded genetic functions inactivated therein so that virus has attenuated virulence.
In one embodiment, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor.
In another embodiment, the genetic functions are inactivated by insertional inactivation of an open reading frame encoding a virulence factor. What is also described is a vaccine containing the modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the vaccine has an increased level of safety compared to known recombinant virus vaccines.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DENIANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

CECI EST LE TOME DE _2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

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GENETICALLY ENGINEERED CANARY POXVIRUS-BASED VACCINE STRAIN
This is a divisional application of Canadian patent application Serial No. 2,105,277, filed on March 9, 1992.

FIELD OF THE INVENTION

The present invention relates to a modified poxvirus and to methods of making and using the same. More in particular, the invention relates to improved vectors for the insertion and expression of foreign genes for use as safe immunization vehicles to protect against a variety of pathogens.

Several publications are referenced in this application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned. These publications relate to the art to which this invention pertains.

The subject matter of this divisional application is directed to a recombinant canary poxvirus comprising exogenous DNA from a non-poxvirus source, vaccines comprising the virus, and methods involving use of the virus.

The subject matter of the parent application was restricted to vaccinia virus. However, it should be understood that the expression "the invention" and the like encompass the subject matter of both the parent and this divisional application.

la BACKGROUND OF THE INVENTION

Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini iet 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 the vaccinia virus described in U.S. Patent Nos. 4,769,330, 4,772,848 and 4,603,112.

7'- ,3-25 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. HoIDologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs.
A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
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 ~2/15672 PCT/US92/. 16 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 praperties. 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-NZ'O 92/1 ? PCT/US92/01906 ' -5-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 sequenced (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.

92/15672 PC"T/US92 -06 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 'JVO 92/ '2 PCT/US92/01906 region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,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 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 N1L 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 ideritified as host range genes, K1L (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).
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 the virus causing a productive t 92/15672 PCT/US92/ 36 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 FPV as a vaccine vector in poultry 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).
The use of live attenuated vectored vaccines present a number of potential advantages. The vaccines are inexpensive to produce and a number of poultry pathogens can potentially be incorporated into one vector. The immunogen is presented to the immune system in an authentic manner such that both humoral and cell mediated responses can be invoked. Because the disease agent is not replicating, side effects of vaccination are minimal and the continual re-introduction of the disease agent into the environment is eliminated.
It can be appreciated that provision of a novel vaccine strains having enhanced safety would be a highly desirable advance over the current state of technology. For instance, so as to provide safer vaccines or safer products from the expression of a gene or genes by a virus.
OBJECTS OF THE INVENTION
It is therefore an 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 vaccine having an increased level of safety compared to known recombinant poxvirus vaccines.

WO 92/1 2 PC'T/US92/01906 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 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.
STATEMENT OF THE INVENTION
In one 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.
In another aspect, the present invention relates to a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine including a carrier and a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The virus used in the vaccine according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus.
In yet another aspect, the present invention relates to an immunogenic composition containing a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The modified recombinant virus includes, within a non-essential-region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein derived from a pathogen wherein the composition, when administered to a host, is capable of ~
inducing an immunological response specific to the protein encoded by the pathogen.

In a further aspect, the present invention relates to a method for expressing a gene product in a cell cultured 5 in vitro by introducing into the cell a modified recombinant virus having attenuated virulence and enhanced safety.

In a still further aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that 10 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. In particular, the genetic functions are inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host restricted 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 - K1L, and 14L (by the terminology reported in Goebel et al., 1990a,b). In this respect, the open reading frame comprises a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range gene region or a large subunit, ribonucleotide reductase.
According to one aspect of the present invention, there is provided a recombinant virus, comprising exogenous DNA from a non-poxvirus source and encoding an antigenic protein of the source inserted by recombination in a nonessential region of a canarypox virus genome, wherein the non-poxvirus source is rabies virus, Hepatitis B virus, Japanese encephalitis virus, yellow fever virus, Dengue s 10a virus, measles virus, pseudorabies virus, Epstein-Barr virus, herpes simplex virus, human immunodeficiency virus, simian immunodeficiency virus, equine herpes virus, bovine herpes virus, bovine viral diarrhea virus, human cytomegalovirus, canine parvovirus, equine influenza virus, feline leukemia virus, feline herpes virus, Hantaan virus, C. tetani, avian influenza virus, mumps virus, or Newcastle Disease virus, and wherein said canarypox virus is attenuated.

The invention also relates to a rabies virus recombinant canarypox virus which is vCP65.

The invention also relates to a human immunodeficiency virus recombinant canarypox virus which is vCP95, vCP112, vCP60 or vCP61.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, 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 schematically shows a method for the construction of plasmid pSD460 for deletion of thymidine kinase gene and generation of recombinant vaccinia virus vP410;

FIG. 2 schematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus vP553;

FIG. 3 schematically shows a method for the construction of plasmid pMP494A for deletion of ATI region and generation of recombinant vaccinia virus vP618;
FIG. 4 schematically shows a method for the construction of plasmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus vP723;
FIG. 5 schematically shows a method for the construction of plasmid pMPCSKlA for deletion of gene cluster [C7L - K1L] and generation of recombinant vaccinia virus vP804;
FIG. 6 schematically shows a method for the construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant vaccinia virus vP866 (NYVAC);
FIG. 7 schematically shows 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. 8 is a map of the EBV coding regions inserted into EBV Triple.l plasmid;
FIG. 9 shows the DNA sequence (SEQ ID NO:213) of the synthetic spsAg gene and modified synthetic vaccinia virus H6 early/late promoter with the predicted amino acid sequence (SEQ ID N0:214);
FIG. 10 schematically shows a method for the construction of recombinant vaccinia virus vP856;
FIG. 11 shows the DNA sequence (SEQ ID NO:215) of the u promoter/lpsAg gene with the predicted amino acid sequence (SEQ ID NO:216);
FIG. 12 schematically shows a method for the construction of recombinant vaccinia virus vP896;
FIG. 13 shows the DNA sequence (SEQ ID NO:87) of the 13L promoter/S12/core gene with the predicted amino acid sequence (SEQ ID NO:217);
FIG. 14 schematically shows a method for the construction of recombinant vaccinia virus vP919;

92/15672 PCT/US92, 06 FIG. 15 shows the DNA sequence (SEQ ID NO:218) of the EPV 42 kDa promoter/1psAg gene with the predicted amino acid sequence (SEQ ID NO:219);
FIG. 16 shows the DNA sequence (SEQ ID NO:217) of a canarypox PvuII fragment containing the C5 ORF.
FIG. 17 schematically shows a method for the construction of recombinant canarypox virus vCP65 (ALVAC-RG);
FIG. 18 is a schematic of the JEV coding regions inserted in the vaccinia viruses vP555, vP825, vP908, vP923, vP857 and vP864;
FIG. 19 is a schematic of the YF coding regions inserted in the vaccinia viruses vP766, vP764, vP869, vP729 and vP725;
FIG. 20 is a schematic of the DEN coding regions inserted in the vaccinia viruses vP867, vP962 and vP955;
FIG. 21 shows the nucleotide sequence (SEQ ID NO:221) of a 3661 base pair fragment of TROVAC DNA containing the F8 ORF;
FIG. 22 shows the DNA sequence (SEQ ID NO:222) of a 2356 base pair fragment of TROVAC DNA containing the F7 ORF;
FIG. 23 shows the nucleotide sequence of EIV HA
(Al/Prague/56) (SEQ ID NO:279);
FIG. 24 shows the nucleotide sequence of EIV HA
(A2/Fontainebleu/79) (SEQ ID NO:284);
FIG. 25 shows the nucleotide sequence of EIV HA
(A2/Suffolk/89) (SEQ ID NO:300);
FIG. 26 shows the nucleotide sequence of FeLV-B
Envelope Gene (SEQ ID NO:310);
FIG. 27 shows the nucleotide sequence of FeLV-A aag and partial gol genes (SEQ ID NO:324);
FIG. 28 shows the nucleotide sequence of the FHV-1 CO
strain gD homolog gene (SEQ ID NO:290);
FIG. 29 shows the consensus F nucleotide sequence (mumps) represented by pURF3 (SEQ ID NO:370);
FIG. 30 shows the consensus HN nucleotide sequence (mumps) represented by piJRHN5 (SEQ ID NO:371);
FIG. 31 shows the cytotoxic responses of spleen cells of mice and immunized with vaccinia virus or canarypox virus wO 92/1#46, PCT/US92/01906 -13-vectors (NYVAC, ALVAC) or with vaccinia virus or canarypox virus recombinants expressing HIV III B env (vP911, vCP112);
FIG. 32 shows the sensitivity of the cytotoxic effector cells from the spleens of mice immunized with vCP112 to antibodies against cytotoxic T lymphocyte cell surface antigens Thy 1.2 and Lyt 2.2;
FIG. 33 shows the specificity of cytotoxic T lymphocyte antigen receptor for the HIV III B hypervariable V3 loop'of' gp120, but not for the V3 loop of HIV MN or SF2;
FIG. 34 shows the antibody responses to HIV III B gp120 of mice immunized with vectors (NYVAC, ALVAC) or with vaccinia virus recombinant vP911 or canarypox recombinant vCP112 expressing HIV-1 env (inverted triangle indicates time of administration of second inoculation);
FIG. 35 shows graph of rabies neutralizing antibody titers (RFFIT, IU/ml), booster effect of HDC and vCP65 (105=5 TCID50) in volunteers previously immunized with either the same or the alternate vaccine (vaccines given at days 0, 28 and 180, antibody titers measured at days 0, 7, 28, 35, 56, 173, 187 and 208);
FIG. 36 shows JEV cDNA sequences contained in vP908, vP555, vP923 and vP829;
FIG. 37 shows NEUT and HAI activities observed in swine immunized on days 0 and 28 with vP908, vP923, vP866 and PBS
(arrows indicated days of inoculation);
FIG. 38 shows time course of viremia detected in individual pigs of each group immunized with PBS, vP866, vP908 or vP923 and then challenged with the B-2358/84 strain of JEV;
Fig 39 shows schematically the ORFs deleted to generate NYVAC;
DETAILED DESCRIPTION OF THE INVENTION
To develop a new vaccinia vaccine strain, NYVAC
(vP866), the Copenhagen vaccine strain of vaccinia virus was modified by the deletion of six nonessential regions of the genome encoding known or potential virulence-factors. The sequential deletions are detailed below. All designations of vaccinia restriction fragments, open reading frames and nucleotide positions are based on the terminology reported in Goebel et al., 1990a,b.
The deletion loci were also engineered as recipient loci for the insertion of foreign genes.
The regions deleted in NYVAC are listed below. Also listed are the abbreviations and open reading frame designations for the deleted regions (Goebel et al., 1990a,b) and the designation of the vaccinia recombinant (vP) containing all deletions through the deletion specified:
(1) thymidine kinase gene (TK; J2R) vP410;
(2) hemorrhagic region (u; B13R + B14R) vP553;
(3) A type inclusion body region (ATI; A26L) vP618;
(4) hemagglutinin gene (HA; A56R) vP723;
(5) host range gene region (C7L - K1L) vP804; and (6) large subunit, ribonucleotide reductase (14L) vP866 (NYVAC).

DNA Clonina 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 Research Laboratories, Gaithersburg, MD, New England Biolabs, Beverly, MA; and Boehringer Mannheim Biochemicals, Indianapolis, IN. Klenow fragment of E. coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as specified by the various suppliers.
Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenase (Tabor et al., 1987) as previously described (Guo et al., 1989). DNA
amplification by polymerase chain reaction (PCR) for sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp*
DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler.
*Trade-mark NVO 92/' 72 PCT/CJS92/01906 Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides.
Cells, Virus, and Transfection. The origins and conditions of cultivation of the Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989). Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et al., 1989).
A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.
Example 1- CONSTRUCTION OF PLASMID p8D460 FOR
DELETION OF THYMIDINE KINASE GENE (J2R) Referring now to FIG. 1, plasmid pSD406 contains vaccinia HindIII J (pos. 83359 - 88377) cloned into pUCB.
pSD406 was cut with HindIil and PvuII, and the 1.7 kb fragment from the left side of HindIII J cloned into pUCB
cut with HindIII/SmaI, forming pSD447. pSD447 contains the entire gene for J2R (pos. 83855 - 84385). The initiation codon is contained within an NlaIII site and the termination codon is contained within an SSDI site. Direction of transcription is indicated by an arrow in FIG. 1.
To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI
fragment was isolated from pSD447, then digested with N1aIII
and a 0.5 kb HindIII/N1aIII fragment isolated. Annealed synthetic oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:1/SEQ
ID NO:2) SmaI
MPSYN43 5' TAATTAACTAGCTACCCGGG 3' MPSYN44 3' GTACATTAATTGATCGATGGGCCCTTAA 5' N1aIII EcoRI
were ligated with the 0.5 kb HindIII/N1aIII fragment into pUC18 vector plasmid cut with HindIII/EcoRI, generating plasmid pSD449.
To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with SsDI (partial) within vaccinia sequences and HindIII at ~ 92/15672 PCT/US92/ 06 the pUC/vaccinia junction, and a 2.9 kb vector fragment isolated. This vector fragment was ligated with annealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID N0:3/SEQ
ID NO:4) HindIII SmaI
MPSYN45 5' AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA
MPSYN46 3' AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT
NotI SstiI
ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5' MPSYN46 generating pSD459.
To combine the left and right flanking arms into one plasmid, a 0.5 kb indIII/SmaI fragment was isolated from pSD449 and ligated with pSD459 vector plasmid cut with indIII/SmaI, generating plasmid pSD460. pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2. 32P labelled probe was synthesized by primer extension using MPSYN45 (SEQ ID
NO:3) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:5) (5' TTAGTTAATTAGGCGGCCGC 3') as primer. Recombinant virus vP410 was identified by plaque hybridization.
Example 2 - CONSTRUCTION OF PLABMID p8D486 FOR
DELETION OF HEMORRHAGIC REGION (B13R + B14R) Referring now to FIG. 2, plasmid pSD419 contains vaccinia SalI G (pos. 160,744-173,351) cloned into pUC8.
pSD422 contains the contiguous vaccinia SalI fragment to the right, SalI 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. 2.
To remove unwanted sequences from pSD419, sequences to the left of the NcoI site (pos. 172,253) were removed by digestion of pSD419 with NcoI/SmaI 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 Hpal at the WO 92/1 " ,2 PCT/US92/01906 termination codon of B14R and by digestion with NruI 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb HincII vector fragment isolated from pSD476, generating plasmid pSD477. The location of the partial deletion of the vaccinia u region in pSD477 is indicated by a triangle. The remaining B13R coding sequences in pSD477 were removed by digestion with C1aI/HpaI, and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID NO:6/SEQ ID NO:7) C1aI BamHI HpaI
SD22mer 5' CGATTACTATGAAGGATCCGTT 3' SD20mer 3' TAATGATACTTCCTAGGCAA 5' generating pSD479. pSD479 contains an initiation codon (underlined) followed by a BamHI site. To place E. coli Beta-galactosidase in the B13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactosidase gene (Shapira et al., 1983) was inserted into the BamHI site of pSD479, generating pSD479BG. pSD479BG was used as donor plasmid for recombination with vaccinia virus vP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque in the presence of chromogenic substrate X-gal. In vP533 the B13R-B14R region is deleted and is replaced by Beta-galactosidase.
To remove Beta-galactosidase sequences from vP533, plasmid pSD486, a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized. First the ClaI/HpaI vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:8/SEQ
ID NO:9) ClaI SacI Xhol IioaI
SD42mer 5' CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3' SD40mer 3' TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5' BalII SmaI ]LamHI
generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia junction was destroyed by digestion of pSD478 with EcoRI followed by blunt ending with Kienow fragment of E. coli polymerase and ligation, generating plasmid ') 92/15672 PCT/US9: 906 pSD478E-. pSD478E- was digested with BamHI and HpaI and ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ ID NO:10/SEQ ID NO:11) $amHI EcoRI HpaI
HEM5 5' GATCCGAATTCTAGCT 3' HEM6 3' GCTTAAGATCGA 5' generating plasmid pSD486. pSD486 was used as donor plasmid for recombination with recombinant vaccinia virus vP533, generating vP553, which was isolated as a clear plaque in the presence of X-gal.
Example 3 - CONSTRUCTION OF PLABMID pMP494e FOR DELETION OF ATI REGION (A26L) Referring now to FIG. 3, pSD414 contains SalI B cloned into pUC8. To remove unwanted DNA sequences to the left of the A26L region, pSD414 was cut with XbaI within vaccinia sequences (pos. 137,079) and with HindIII 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 was cut with NdeI (partial) slightly upstream from the A26L ORF (pos. 139,004) and with HpaI (pos. 137,889) slightly downstream from the A26L ORF. The 5.2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:12/SEQ ID N0:13) NdeI
ATI3 5' TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT
ATI4 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA
BqlII EcoRI HpaI
TATATAAATAGATCTGAATTCGTT 3' ATI3 ATATATTTATCTAGACTTAAGCAA 5' ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region containing the restriction sites BalII, EcoRI and HpaI, as indicated above.
The resulting plasmid was designated pSD485. Since the BqlII and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted BqlII and EcoRI sites were removed from plasmid pSD483 (described above) by digestion with BqlII

WO 92/1! ? PCT/US92/01906 (pos. 140,136) and with coRI at the pUC/vaccinia junction, followed by blunt ending with Klenow fragment of E. coli polymerase and ligation. The resulting plasmid was designated pSD489. The 1.8 kb C aI (pos. 137,198)/EcoRV
(pos. 139,048) fragment from pSD489 containing the A26L ORF
was replaced with the corresponding 0.7 kb polylinker-containing C1aI/EcoRV fragment from pSD485, generating pSD492. The BalII and EcoRI sites in the polylinker region of pSD492 are unique.
A 3.3 kb BalII 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 Bcr1II site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus vP553. Recombinant vaccinia virus, vP581, containing Beta-galactosidase in the A26L deletion region, was isolated as a blue plaque in the presence of X-gal.
To generate a plasmid for the removal of Beta-galactosidase sequences from vaccinia recombinant virus vP581, the polylinker region of plasmid pSD492 was deleted by mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN177 (SEQ ID N0:14) (5' AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3').
In the resulting plasmid, pMP494A, vaccinia DNA encompassing positions [137,889 - 138,937), including the entire A26L ORF
is deleted. Recombination between the pMP494A and the Beta-galactosidase containing vaccinia recombinant, vP581, resulted in vaccinia deletion mutant vP618, which was isolated as a clear plaque in the presence of X-gal.
Example 4 - CONSTRUCTION OF PLASMID p8D467 FOR
DELETION OF REMAGGLIITININ GENE (A56R) Referring now to FIG. 4, vaccinia SalI G restriction fragment (pos. 160,744-173,351) crosses the HindIII A/B
junction (pos. 162,539). pSD419 contains vaccinia SalI G
cloned into pUC8. The direction of transcription for the hemagglutinin (HA) gene is indicated by an arrow in FIG. 4.
Vaccinia sequences derived from indIII B were removed by digestion of pSD419 with HindIII within vaccinia sequences ' 92/ l 5672 PCT/US92. )06 and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to the right. A56R coding sequences were removed by cutting pSD456 with RsaI (partial;
pos. 161,090) upstream from A56R coding sequences, and with EaqI (pos. 162,054) near the end of the gene. The 3.6 kb RsaI/Ea~I vector fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID NO:15), MPSYN62 (SEQ ID NO:16), MPSYN60 (SEQ ID
NO:17), and MPSYN 61 (SEQ ID NO:18) RsaI
MPSYN59 5' ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT-MPSYN62 3' TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA-MPSYN59 AGTTGATAGAACAAAATACATAATTT 3' MPSYN62 TCAACTATCT 5' MPSYN60 5' TGTAAAAATAAATCACTTTTTATA-MPSYN61 3' TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT-BctlII SmaI PstI EaaI
MPSYN60 CTAAGATCTCCCGGGCTGCAGC 3' MPSYN61 GATTCTAGAGGGCCCGACGTCGCCGG 5' reconstructing the DNA sequences upstream from the A56R ORF
and replacing the A56R ORF with a polylinker region as indicated above. The resulting plasmid is pSD466. The vaccinia deletion in pSD466 encompasses positions [161,185-162,053]. The site of the deletion in pSD466 is indicated by a triangle in FIG. 4.
A 3.2 kb BcrlII/BamHI (partial) cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) was inserted into the BalII
site of pSD466, forming pSD466KBG. Plasmid pSD466KBG was used in recombination with rescuing virus vP618.
Recombinant vaccinia virus, vP708, containing Beta-galactosidase in the A56R deletion, was isolated as a blue plaque in the presence of X-gal.
Beta-galactosidase sequences were deleted from vP708 using donor plasmid pSD467. pSD467 is identical to pSD466, except that EcoRI, SmaI and BamHI sites were removed from WO 92/1 " '2 PCT/US92/01906 the pUC/vaccinia junction by digestion of pSD466 with EcoRI/BamHI followed by blunt ending with Klenow fragment of E. coli polymerase and ligation. Recombination between vP708 and pSD467 resulted in recombinant vaccinia deletion mutant, vP723, which was isolated as a clear plaque in the presence of X-gal.
EYamgle 5 - CONSTRUCTION OF PLAS1riID pKPC81C10 FOR DELETION OF OPEN READING FRAMES jC7L-K1L]
Referring now to FIG. 5, the following vaccinia clones were utilized in the construction of pMPCSKlA. pSD420 is SalI H cloned into pUC8. pSD435 is MnI F cloned into pUC18. pSD435 was cut with St?hI and religated, forming pSD451. In pSD451, DNA sequences to the left of the SphI
site (pos. 27,416) in indIII M are removed (Perkus et al., 1990). pSD409 is indIII M cloned into pUCB.
To provide a substrate for the deletion of the [C7L-K1L] gene cluster from vaccinia, E. coli Beta-galactosidase was first inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminate the Bg1II site in pSD409, the plasmid was cut with BalII in vaccinia sequences (pos. 28,212) and with DamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphI site (pos. 27,416). M2L coding sequences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide BciII
MPSYN82 (SEQ ID NO:19) 5' TTTCTGTATATTTGCACCAATTTAGATCTT-ACTCAAAATATGTAACAATA 3' The resulting plasmid, pMP409D, contains a unique Bc1II site inserted into the M2L deletion locus as indicated above. A
3.2 kb BamHI (partial)/BalII cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with BalII. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination with rescuing vaccinia virus vP723. Recombinant vaccinia virus, vP784, containing Beta-galactosidase inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal.

92/15672 PCT/US92 )06 A plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUCB cut with maI, indIII and blunt ended with Klenow fragment of E. coli polymerase. The left flanking arm consisting of vaccinia HindIII C sequences was obtained by digestion of pSD420 with XbaI (pos. 18,628) followed by blunt ending with Klenow fragment of E. coli polymerase and digestion with BalII (pos. 19,706). The right flanking arm consisting of vaccinia HindIII K
sequences was obtained by digestion of pSD451 with BalII
(pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP581CK is deleted for vaccinia sequences between the BalII site (pos. 19,706) in HindIII C and the BcxlII site (pos. 29,062) in HindIII K. The site of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 5.
To remove excess DNA at the vaccinia deletion junction, plasmid pMP581CK, was cut at the NcoI sites within vaccinia sequences (pos. 18,811; 19,655), treated with Bal-31 exonuclease and subjected to mutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233 (SEQ ID NO:20) 5'-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3'.
The resulting plasmid, pMPCSKlA, is deleted for vaccinia sequences positions 18,805-29,108, encompassing 12 vaccinia open reading frames [C7L - K1L3. Recombination between pMPCSKld and the Beta-galactosidase containing vaccinia recombinant, vP784, resulted in vaccinia deletion mutant, vP804, which was isolated as a clear plaque in the presence of X-gal.
Example 6 - CONSTRUCTION OF PLASKID p8D548 FOR DELETION OF
LARGE BIIBIINIT, RIBONUCLEOTIDE REDUCTASE (14L) Referring now to FIG. 6, plasmid pSD405 contains vaccinia HindIII I (pos. 63,875-70,367) cloned in pUCB.
pSD405 was digested with EcoRV within vaccinia sequences (pos. 67,933) and with SmaI at the pUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 was used as the source of all the vaccinia restriction fragarents used in the construction of pSD548.
The vaccinia 14L gene extends from position 67,371-65,059. Direction of transcription for 14L is indicated by WO 92/' 72 PCT/US92/01906 ' -23-an arrow in FIG. 6. To obtain a vector plasmid fragment deleted for a portion of the 14L coding sequences, pSD518 was digested with BamHI (pos. 65,381) and HDaI (pos. 67,001) and blunt ended using Klenow fragment of E. coli polymerase.
This 4.8 kb vector fragment was ligated with a 3.2 kb SmaI
cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990), resulting in plasmid pSD524KBG. pSD524KBG was used as donor plasmid for recombination with vaccinia virus vP804.
Recombinant vaccinia virus, vP855, containing Beta-galactosidase in a partial deletion of the 14L gene, was isolated as a blue plaque in the presence of X-gal.
To delete Beta-galactosidase and the remainder of the 14L ORF from vP855, deletion plasmid pSD548 was constructed.
The left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and presented schematically in FIG. 6.
To construct a vector plasmid to accept the left vaccinia flanking arm, pUCB was cut with BamHI/ coRI and ligated with annealed synthetic oligonucleotides 518A1/518A2 (SEQ ID NO:21/SEQ ID NO:22) BamHI RsaI
518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT
518A2 3' GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA
Balli EcoRI
TTGAGAATAAAAAGATCTTAGG 3' 518A1 AACTCTTATTTTTCTAGAATCCTTAA 5' 518A2 forming plasmid pSD531. pSD531 was cut with RsaI (partial) and BamHI and a 2.7 kb vector fragment isolated. pSD518 was cut with BalII (pos. 64,459)/ RsaI (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 14L coding sequences.
To construct a vector plasmid to accept the right vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and 92/15672 PCT/US92 )06 ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID NO:23/SEQ ID NO:24) BamHI BalII SmaI
518B1 5' GATCCAGATCTCCCGGGAAP.AAAATTATTTAACTTTTCATTAATAG-518B2 3' GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATC-~saI EcoRI
GGATTTGACGTATGTAGCGTACTAGG 3' 518B1 CCTAAACTGCATACTACGCATGATCCTTAA 5' 518B2 forming plasmid pSD532. pSD532 was cut with RsaI
(partial)/EcoRI and a 2.7 kb vector fragment isolated.
pSD518 was cut with RsaI within vaccinia sequences (pos.
67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two fragments were ligated together, forming pSD538, which contains the complete vaccinia flanking arm to the right of 14L coding sequences.
The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BalII fragment from pSD538 and ligated into pSD537 vector plasmid cut with EcoRI/BalII. In the resulting plasmid, pSD539, the 14L ORF (pos. 65,047-67,386) is replaced by a polylinker region, which is flanked by 0.6 kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right, all in a pUC background. The site of deletion within vaccinia sequences is indicated by a triangle in FIG. 6. 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 14L deletion cassette was moved from pSD539 into pRC11, a pUC derivative from which all Beta-galactosidase sequences have been removed and replaced with a polylinker region (Colinas et al., 1990). pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment isolated. This fragment was ligated into pRC11 cut with EcoRI/PstI (2.35 kb), forming pSD548.
Recombination between pSD548 and the Beta-galactosidase containing vaccinia recombinant, vP855, resulted in vaccinia deletion mutant vP866, which was isolated as a clear plaque in the presence of X-gal.
DNA from recombinant vaccinia virus vP866 was analyzed by restriction digests followed by electrophoresis on an agarose gel. The restriction patterns were as expected.
Polymerase chain reactions (PCR) (Engelke et al., 1988) WO 92/1 " '2 PCT/US92/01906 using vP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR generated fragments around the areas of the deletion junctions confirmed that the junctions were as expected. Recombinant vaccinia virus vP866, containing the six engineered deletions as described above, was designated vaccinia vaccine strain "NYVAC."
Example 7 - 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 inserted into TK deletion plasmid pSD513. pSD513 is identical to plasmid pSD460 (FIG. 1) except for the presence of a polylinker region.
Referring now to FIG. 7, the polylinker region was inserted by cutting pSD460 with SmaI and ligating the plasmid vector with annealed synthetic oligonucleotides VQ1A/VQ1B (SEQ ID NO:25/SEQ ID NO:26) SmaI Bg1II XhoI PstI NarI BamHI
VQ1A 5' GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3' VQ1B 3' CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5' to form vector plasmid pSD513. pSD513 was cut with SmaI and ligated with a SmaI ended 1.8 kb cassette 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 was used as donor plasmid for recombination with NYVAC rescuing virus (vP866). Recombinant vaccinia virus vP879 was identified by plaque hybridization using 32P-labelled DNA probe to rabies glycoprotein G coding sequences.
The modified recombinant viruses of the present invention provide advantages as recombinant vaccine vectors.
The attenuated virulence of the vector advantageously reduces the opportunity for the possibility 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.

92/15672 PCT/US92. >06 The modified recombinant viruses are also advantageously used in a method for expressing a gene product in a cell cultured in vitro by introducing into the cell the modified recombinant virus having foreign DNA which codes for and expresses gene products in the cell.
Example 8- CONSTRUCTION OF TROVAC-NDV EZPR$SSING THE
FUSION AND HB1rtAGGLIITININ-NEURAMINIDASE
GLYCOPROTEINS OF NEWCASTLE DISEASE VIRUS

A fowlpox virus (FPV) vector expressing both F and HN
genes of the virulent NDV strain Texas was 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 chicks protects 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-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 scab 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 successive plaque purifications. One plaque isolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, established. The stock virus used in the in vitro recombination test to produce TROVAC-NDV had been subjected to.twelve passages in primary CEF cells from the plaque isolate.
Construction of a Cassette 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 pUC18 to form pCE13. The vaccinia virus H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) was inserted into pCE13 by digesting pCE13 with SalI, filling in the sticky ends with WO 92/ 1= "2 PCT/US92/01906 Klenow fragment of E. coli DNA polymerase and digesting with HindIII. A HindIII - EcoRV fragment containing the H6 promoter sequence was then inserted into pCE13 to form pCE38. A perfect 5' end was generated by digesting pCE38 with KpnI and NruI and inserting the annealed and kinased oligonucleotides CE75 (SEQ ID NO:27) and CE76 (SEQ ID NO:28) to generate pCE47.
CE75:
CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGATCCCGGTAC
CE76:
CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGATATCG.
In order to remove non-coding sequence from the 3' end of the NDV-F a SmaI to PstI fragment from pCE13 was inserted into the SmaI and PstI sites of pUC18 to form pCE23. The non-coding sequences were removed by sequential digestion of pCE23 with SacI, BamHI, Exonuclease III, SI nuclease and EcoRI. The annealed and kinased oligonucleotides CE42 (SEQ
ID NO:29) and CE43 (SEQ ID NO:30) were then inserted to form pCE29.
CE42: AATTCGAGCTCCCCGGG
CE43: CCCGGGGAGCTCG
The 3' end of the NDV-F sequence was then inserted into plasmid pCE20 already containing the 5' end of NDV-F by cloning a~stI - SacI fragment from pCE29 into the PstI and SacI sites of pCE20 to form pCE32. Generation of pCE20 has previously been described in Taylor et al., 1990.
In order to align the H6 promoter and NDV-F 5' sequences contained in pCE47 with the 3' NDV-F sequences contained in pCE32, a indIII -PstI fragment of pCE47 was inserted into the HindIII and PstI sites of pCE32 to form pCE49. The H6 promoted NDV-F sequences were then transferred to the de-ORFed F8 locus (described below) by cloning a HindIII - NruI fragment from pCE49 into the HindIII and SmaI sites of pJCA002 (described below) to form pCE54. Transcription stop signals were inserted into pCE54 by digesting pCE54 with SacI, partially digesting with BamHI
and inserting the annealed and kinased oligonucleotides CE166 (SEQ ID NO:31) and CE167 (SEQ ID NO:32) to generate pCE58.

92/15672 PCT/US92, )06 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 oligonucleotides CE182 (SEQ ID NO:33) and CE183 (SEQ ID
NO:34) as primers.
CE182: CTTAACTCAGCTGACTATCC
CE183: TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC
The PCR fragment was digested with PvuII and HpaI and cloned into pCE58 that had been digested with HpaI and partially digested with PvuII. The resulting plasmid was designated pCE64. Translation stop signals were inserted by cloning a HindIII - HpaI fragment which contains the complete H6 promoter and F coding sequence from pCE64 into the HindIII
and HpaI sites of pRW846 to generate pCE71, the final cassette for NDV-F. Plasmid pRW846 is essentially equivalent to plasmid pJCA002 (described below) but containing the H6 promoter and transcription and translation stop signals. Digestion of pRW846 with HindIII and HDaI
eliminates the H6 promoter but leaves the stop signals intact.
Construction of Cassette for NDV-HN. Construction of plasmid pRW802 was previously described in Edbauer et al., 1990. This plasmid contains the NDV-HN sequences linked to the 3' end of the vaccinia virus H6 promoter in a pUC9 vector. A HindIII - EcoRV fragment encompassing the 5' end of the vaccinia virus H6 promoter was inserted into the HindIII and EcoRV sites of pRW802 to form pRW830. A perfect 3' end for NDV-HN was obtained by inserting the annealed and kinased oligonucleotides CE162 (SEQ ID NO:35) and CE163 (SEQ ID NO:36) into the EcoRI site of pRW830 to form pCE59, the final cassette for NDV-HN.
CE162:
AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATTTAATTTTTAT
AAGCTTG
CE163:
AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTAGTAAAGGAAC
GATCCTG

WO 92 t '2 PCT/1JS92/01906 Construction of FPV Insertion Vector. Plasmid pRW731-15 contains a 10kb PvuII - PvuII fragment cloned from genomic DNA. The nucleotide sequence was determined on both strands for a 3660 bp PvuII - EcoRV fragment. The limits of an open reading frame designated here as F8 were determined.
Plasmid pRW761 is a sub-clone of pRW731-15 containing a 2430 bp EcoRV - coRV fragment. The F8 ORF was entirely contained between an XbaI site and an SSDI site in pRW761.
In order to create an insertion plasmid which on recombination with TROVAC genomic DNA would eliminate the F8 ORF, the following steps were followed. Plasmid pRW761 was completely digested with XbaI and partially digested with SSDI. A 3700 bp XbaI - SsPI band was isolated from the gel and ligated with the annealed double-stranded oligonucleotides JCA017 (SEQ ID N0:37) and JCA018 (SEQ ID
N0:38) .
JCA017:5' CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGATCCTTATACGG
GGAATAAT
JCA018:5' ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATATTAAAAAACATAA
AGTGT
The plasmid resulting from this ligation was designated pJCA002.
Construction of Double Insertion Vector for NDV F and HN. The H6 promoted NDV-HN sequence was inserted into the H6 promoted NDV-F cassette by cloning a HindIII fragment from pCE59 that had been filled in with Klenow fragment of E. coli DNA polymerase into the HpaI site of pCE71 to form pCE80. Plasmid pCE80 was completely digested with NdeI and partially digested with BalII to generate an NdeI - E_qlII
4760 bp fragment containing the NDV F and HN genes both driven by the H6 promoter and linked to F8 flanking arms.
Plasmid pJCA021 was obtained by inserting a 4900 bp PvuII -F}indII fragment from pRW731-15 into the SmaI and HindII
sites of pBSSK+. Plasmid pJCA021 was then digested with NdeI and BglII and ligated to the 4760 bp NdeI - BglII
fragment of pCE80 to form pJCA024. Plasmid pJCA024 therefore contains the NDV-F and HN genes inserted in = ~ 92/15672 PCT/US92 )06 opposite orientation with 3' ends adjacent between FPV
flanking arms. Both genes are linked to the vaccinia virus H6 promoter. The right flanking arm adjacent to the NDV-F
sequence 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 was 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 plaques were selected on the basis of hybridization to specific NDV-F and HN radiolabelled probes and subjected to five sequential rounds of plaque purification until a pure population was achieved. One representative plaque was then amplified and the resulting TROVAC recombinant was designated TROVAC-NDV (vFP96).
Immunofluorescence. Indirect immunofluorescence was performed as described (Taylor et al., 1990) using a polyclonal anti-NDV serum and, as mono-specific reagents, sera produced in rabbits against vaccinia virus recombinants expressing NDV-F or NDV-HN.
Immunoprec' itation. Immunoprecipitation reactions were performed as described (Taylor et al., 1990) using a polyclonal anti-NDV serum obtained from SPAFAS Inc., Storrs, CN.
The stock virus was screened by in situ plaque hybridization to confirm that the F8 ORF was deleted. The correct insertion of the NDV genes into the TROVAC genome and the deletion of the F8 ORF was also confirmed by Southern blot hybridization.
In NDV-infected cells, the F glycoprotein is anchored in the membrane via a hydrophobic transmembrane region near the carboxyl terminus and requires post-translational cleavage of a precursor, Fo, into two disulfide linked polypeptides F1 and F2. Cleavage of FO 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 site is therefore critical in determining viral virulence. It has ',WO 92/1 '2 PCT/US92/01906 been determined that amino acids at the cleavage site in the NDV-F sequence inserted into FPV to form recombinant vFP29 had the sequence Arg - Arg - Gln - Arg - Arg (SEQ ID NO:39) (Taylor et al., 1990) which conforms to the sequence found to be a requirement for virulent NDV strains (Chambers et al., 1986; Espion et al., 1987; Le et al., 1988; McGinnes and Morrison, 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 expression of F and HN genes in TROVAC-NDV was analyzed to confirm that the gene products were authentically processed and presented. Indirect-immunofluorescence using a polyclonal anti-NDV chicken serum confirmed that immunoreactive proteins were presented on the infected cell surface. To determine that both proteins were presented on the plasma membrane, mono-specific rabbit sera were produced against vaccinia recombinants expressing either the F or HN glycoproteins. Indirect immunofluorescence using these sera confirmed the surface presentation of both proteins.
Immunoprecipitation experiments were performed by using (35S) methionine labeled lysates of CEF cells infected with parental and recombinant viruses. The expected values of apparent molecular weights of the glycolysated forms of F1 and F2 are 54.7 and 10.3 kDa respectively (Chambers et al., 1986). In the immunoprecipitation experiments using 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 single recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV.
No NDV specific products were detected from uninfected and parental TROVAC infected CEF cells.
In CEF cells, the F and HN glycoproteins are appropriately presented on the infected cell surface where 92/15672 PCT/US92, )06 they are recognized by NDV immune serum.
Immunoprecipitation analysis indic _ed that the Fo protein is authentically cleaved to the F1 and F2 components required in virulent strains. Similarly, the HN
glycoprotein was authentically processed in CEF cells infected with recombinant TROVAC-NDV.
Previous reports (Taylor et al., 1990; Edbauer et al., 1990; Boursnell et al., 1990a,b,c; Ogawa et al., 1990) would indicate that expression of either HN or F alone is sufficient to elicit protective immunity against NDV
challenge. Work on other paramyxoviruses has indicated, however, that antibody to both proteins 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 suggested that vaccine failures with killed measles virus vaccines were due to inactivation of the fusion component (Norrby et al., 1975). Since both NDV glycoproteins have been shown to be responsible for eliciting virus neutralizing antibody (Avery et al., 1979) and both glycoproteins, when expressed individually in a fowipox vector are able to induce a protective immune response, it can be appreciated that the most efficacious NDV vaccine should express both glycoproteins.
Esample 9 - CONSTRUCTION OF NY9AC-KV RECOMBINANT
EYPRESBING1riEA8LE8 FUSION AND HEMAGGLIITININ
GLYCOPROTEINS

cDNA copies of the sequences encoding the HA and F
proteins of measles virus MV (Edmonston strain) were inserted into NYVAC to create a double recombinant designated NYVAC-MV. The recombinant authentically expressed both measles glycoproteins on the surface of infected cells. Immunoprecipitation analysis demonstrated correct processing of both F and HA glycoproteins. The recombinant was also shown to induce syncytia formation.
Cells and Viruses. The rescuing virus used in the production of NYVAC-MV was the modified Copenhagen strain of vaccinia virus designated NYVAC. All viruses were grown and titered on Vero cell monolayers.

1?VO 92/1 '2 PC7T/US92/01906 Plasmid Construction. Plasmid pSPM2LHA (Taylor et al., 1991c) contains the entire measles HA gene linked in a precise ATG to ATG configuration with the vaccinia virus H6 promoter which has been previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989). A 1.8kpb coRV/SmaI fragment containing the 3' most 24 bp of the H6 promoter fused in a precise ATG:ATG configuration with the HA gene lacking the 3' most 26 bp was isolated from pSPM2LHA. This fragment was used to replace the 1.8 kbp EcoRV/SmaI fragment of pSPMHHAll (Taylor et al., 1991c) to generate pRW803. Plasmid pRW803 contains the en- tire H6 promoter linked precisely to the entire measles HA gene.
In the confirmation of previous constructs with the measles HA gene it was noted that the sequence for codon 18(CCC) was deleted as compared to the published sequence (Alkhatib et al., 1986). The CCC sequence was replaced by oligonucleotide mutagenesis via the Kunkel method (Kunkel, 1985) using oligonucleotide RW117 (SEQ ID NO:40) (5'GACTATCCTACTTCCCTTgGGATGGGGGTTATCTTTGTA-3').

Single stranded template was derived from plasmid pRW819 which contains the H6/HA cassette from pRW803 in pIBI25 (International Biotechnologies, Inc., New Haven, CT). The mutagenized plasmid containing the inserted (CCC) to encode for a proline residue at codon 18 was designated pRW820.
The sequence between the KindIII and XbaI sites of pRW820 was confirmed by nucleotide sequence analysis. The indIII
site is situated at the 5' border of the H6 promoter while the XbaI site is located 230 bp downstream from the initiation codon of the HA gene. A 1.6 kbp Xbal/ECoRI
fragment from pRW803, containing the HA coding sequences downstream from the XbaI (above) and including the termination codon, was used to replace the equivalent fragment of pRW820 resulting in the generation of pRW837.
The mutagenized expression cassette contained within pRW837 was derived by digestion with indIII and coRI, blunt-ended using the Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs, and inserted into the Sm~I site of pSD513 to yield pRW843. Plasmid pSD513 was derived from 92/15672 PCT/LS92. )06 plasmid pSD460 by the addition of polylinker sequences.
Plasmid pSD460 was derived to enable deletion of the thymidine kinase gene from vaccinia virus.
To insert the measles virus F gene into the HA
insertion plasmid, manipulations were performed on pSPHMF7.
Plasmid pSPHMF7 (Taylor et al., 1991c) contains the measles F gene juxtaposed 3' to the previously described vaccinia virus H6 promoter. In order to attain a perfect ATG for ATG
configuration and remove intervening sequences between the 3' end of the promoter and the ATG of the measles F gene oligonucleotide directed mutagenesis was performed using oligonucleotide SPMAD (SEQ ID NO:41).
SPMAD: 5'- TATCCGTTAAGTTTGTATCGTAATGGGTCTCAAGGTGAACGTCT-3' The resultant plasmid was designated pSPMF75M20.
The plasmid pSPMF75M20 which contains the measles F
gene now linked in a precise ATG for ATG configuration with the H6 promoter was digested with NruI and EagI. The resulting 1.7 kbp blunt ended fragment containing the 3' most 27 bp of the H6 promoter and the entire fusion gene was isolated and inserted into an intermediate plasmid pRW823 which had been digested with NruI and XbaI and blunt ended.
The resultant plasmid pRW841 contains the H6 promoter linked to the measles F gene in the pIBI25 plasmid vector (International Biotechnologies, Inc., New Haven, CT). The H6/measles F cassette was excised from pRW841 by digestion with SmaI and the resulting 1.8 kb fragment was inserted into pRW843 (containing the measles HA gene). Plasmid pRW843 was first digested with NotI and blunt-ended with Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs. The resulting plasmid, pRW857, therefore contains the measles virus F and HA genes linked in a tail to tail configuration. Both genes are linked to the vaccinia virus H6 promoter.
Develoument of NYVAC-MV. Plasmid pRW857 was transfected into NYVAC infected Vero cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of in situ plaque hybridization to specific MV F and HA radiolabelled probes WO 92/1 2 PC.'T/US92/01906 and subjected to 6 sequential rounds of plaque purification until a pure population was achieved. One representative plaque was then amplified and the resulting recombinant was designated NYVAC-MV (vP913).
Immunofluorescence. Indirect immunofluorescence was performed as previously described (Taylor et al., 1990).
Mono-specific reagents used were sera generated by inoculation of rabbits with canarypox recombinants expressing either the measles F or HA genes.
Immunoprecioitation. Immunoprecipitation reactions were performed as previously described (Taylor et al., 1990) using a guinea-pig anti measles serum (Whittaker M.A.
.Bioproducts, Walkersville, MD).
Cell Fusion Experiments. Vero cell monolayers in 60mm dishes were inoculated at a multiplicity of 1 pfu per cell with parental or recombinant viruses. After 1 h absorption at 37 C the inoculum was removed, the overlay medium replaced and the dishes inoculated overnight at 37 C. At 20 h post-infection, dishes were examined.
In order to determine that the expression products of both measles virus F and HA genes were presented on the infected cell surface, indirect immunofluorescence analysis was performed using mono-specific sera generated in rabbits against canarypox recombinants expressing either the measles F or HA genes. The results indicated that both F and HA
gene products were expressed on the infected cell surface, as demonstrated by strong surface fluorescence with both mono-specific sera. No background staining was evident with either sera on cells inoculated with the parental NYVAC
strain, nor was there cross-reactive staining when mono-specific sera were tested against vaccinia single recombinants expressing either the HA or F gene.
In order to demonstrate that the proteins expressed by NYVAC-MV were immunoreactive with measles virus specific sera and were authentically processed in the infected cell, immunoprecipitation analysis was performed. Vero cell monolayers were inoculated at a multiplicity of 10 pfu/cell of parental or recombinant viruses in the presence of 35S-methionine. Immunoprecipitation analysis revealed a HA

~ 92/15672 PCT/US9: 906 glycoprotein of approximately 76 kDa and the cleaved fusion products F1 and F2 with molecular weights of 44 kDa and 23 kDa, respectively. No measles specific products were detected in uninfected Vero cells or Vero cells infected with the parental NYVAC virus.
A characteristic of MV cytopathology is the formation of syncytia which arise by fusion of infected cells with surrounding infected or uninfected cells followed by migration of the nuclei toward the center of the syncytium (Norrby et al., 1982). This has been shown to be an important method of viral spread, which for Paramyxoviruses, can occur in the presence of HA-specific virus neutralizing antibody (Merz et al., 1980). In order to determine that the MV proteins expressed in vaccinia virus were functionally active, Vero cell monolayers were inoculated with NYVAC and NYVAC-MV and observed for cytopathic effects.
Strong cell fusing activity was evident in NYVAC-MV infected Vero cells at approximately 18 hours post infection. No cell fusing activity was evident in cells infected with parental NYVAC.
Example 10 - CONSTRUCTION OF NYVAC RECOMBINANTS EXPRESSING
GLYCOPROTEINS OF PSEUDORABIES VIRUS

It has been demonstrated that vaccinia virus recombinants expressing the PRV gpII, gpIII, and gp50 glycoproteins either individually or in combination provide efficacious vaccine candidates, in that, they protect swine from a virulent challenge with live PRV. Considering the inability of the NYVAC vector to productively replicate in porcine cell cultures and the inherent safety of the vector due to the deletion of known potential virulence genes, NYVAC-based recombinants containing the PRV gpII, gpIII, and gp50 either alone or in various combinations have been generated. These recombinants were generated to provide efficacious vaccine candidates against PRV that were safe .or swine and eliminated or severely limited transmission to the environment.
Viruses and Cells. Manipulations of NYVAC and molecular cloning were performed by standard techniques (Piccini et al., 1987; Maniatis et al., 1982). Cultivation of NYVAC and NYVAC-based recombinants was as previously described (Piccini et al., 1987).
Cloning of the PRV qplI, apIII, and =50 Genes. The growth of PRV, extraction of PRV genomic DNA, and the identification of the PRV gpII, gpIII, and gp5O genes have been described.
Cloning and Expression of the Pseudorabies Virus (PRV) Genes into NYVAC (vP866). The NYVAC deletion mutant lacking a region encompassing the human and porcine host range genes (C7L and K1L), vP866, was the basic vector used to insert the PRV genes. This vector also lacks the vaccinia virus tk gene, hemagglutinin gene, hemorrhagic gene, ribonucleotide reductase (large subunit) gene, and A-type inclusion gene.
Importantly, vP866 does not replicate efficiently, if at all=, on human or pig kidney (LLC-PK1) cells. PRV genes gpII, gpIII, and gp50, which are homologous to the herpes simplex virus gB (Robbins et al., 1987), gC (Robbins et al., 1986b), and gD (Wathen and Wathen, 1984), respectively, were inserted into vP866 as outlined below.
Insertion of the PRV qpII Gene into the Hemagglutinin Locus of vP866. The DNA sequence encoding the PRV gpII gene resides in the DamHI fragment 1 of the PRV genome (Mettenleiter et al., 1986).
The plasmid designated pPR9.25, containing the PRV
BamHI fragment 1 inserted into the BamHI site of pBR322 was digested with NcoI. The resultant restriction fragments were fractionated on a 0.8% agarose gel and a 6.2 kb NcoI
DNA fragment was purified using Geneclean (Bio101, Inc., LaJolla, CA) and subsequently inserted into the NcoI site of pBR328 (Boehringer Mannheim Biochemicals, Indianapolis, IN) treated with CiAP. The resulting plasmid, pPR2.15, was digested with SphI and fractionated on an agarose gel. The 2.7 kb and 1.8 kb fragments were purified and inserted into the ,SDhI site of pUC18 to create pPR1 and pPR2, respectively.
The 1060 bp PRV SphI/dheI fragment from-pPR1 was isolated from an agarose gel and inserted into the BamHI/SphI sites of pIBI25 with annealed oligonucleotides MRSYN1 (SEQ ID NO:42) (5'-GATCCATTCCATGGTTG-31) and MRSYN2 92/15672 PCT/US92 )06 (SEQ ID NO:43) (5'-TAGCAACCATGGAATG-3') to generate pPR6.
pPR6 was digested with HindIII and ApaI. The ApaI site is located 32 bp downstream from the ATG initiation codon of PRV gpII. This 3920 bp fragment was ligated to annealed oligonucleotides MRSYN3 (SEQ ID NO:44) (5'-AGCTTGATATCCGTTAAGTTTGTATCGTAATGCCCGCTGGTGGCGGTCTTTGGCGCGGGC
C-3' ) and MRSYN4 (SEQ ID NO:45) (5'-CGCGCCAAAGACCGCCAACCAGCGGGATTACGATACAAACTTAACGGATATCA-3') to generate pPR9. These annealed oligonucleotides provide the DNA sequences specifying the vaccinia virus H6 promoter from the EcoRV site through the ATG, followed by the PRV gpII
coding sequences. The plasmid pPR9 was digested with BamHI
and NheI and treated with Calf Intestinal Alkaline Phosphatase (CiAP), and ligated to annealed oligonucleotides MRSYN7 (SEQ ID NO:46) (5'-CCCAGATCTCCTTG-3' ) and NII2SYN8 (SEQ
ID NO:47) (5'-GTACGGGTCTAGAGGAACCTAG-3') and a 1640 bp SDhI/dheI fragment obtained from pPRi generating plasmid pPR12.
The 1030 bp HindII/ERhI fragment from pPR2 was isolated from an agarose gel and inserted into a HincII/SphI pUC18 vector. The resulting plasmid, pPR10, was digested with HindIII and NaeI and treated with CiAP. The aeI site is located 44 bp upstream of the termination codon (TAG).
Annealed oligonucleotides MRSYN9 (SEQ ID NO:48) (5'-GGCACT
ACCAGCGCCTCGAGAGCGAGGACCCCGACGCCCTGTAGAATTTTTATCGGCCGA-3') and MRSYN10 (SEQ ID NO:49) (5'-AGCTTCGGCCGATAAAAATTCTA
CAGGGCGTCGGGGTCCTCGCTCTCGAGGCGTAGTGCC-3') were ligated to the 3720 bp NaeI/HindIII fragment of pPR10 to yield plasmid pPRll. A 770 bp SphI/ incII fragment from pPR2 was purified from an agarose gel and inserted using the BamHI/SphI
phosphorylated linker MIItSYN7 (SEQ ID NO : 4 6) and IrIl2SYN8 (SEQ
ID NO:47) into the BamHI/HincII sites of CiAP treated pPRll to generate pPR13. Plasmid pPR12 digested with EcoRI and SphI was ligated using MRSYN19 (SEQ ID N0:50) (5'-AGCTTCTGC
AGCCATGGCGATCGG-3') and MRSYN20 (SEQ ID NO:51) (5'-AATTCCG
ATCGCCATGGCTGCAGA-3') to a 990 bp HindIII/SphI fragment from pPR13 to yield plasmid pPR15. Plasmid, pPRl5, was digested with HindIII/EcoRV to yield a 2780 bp fragment. This fragment was inserted into pTP15 (Guo et al., 1989) which was digested with XmaIII and coRV to generate pPR18. In pPR18, the PRVgpII is linked with the vaccinia virus H6 promoter in a HA deletion plasmid. pPR18 was used in in vitro recombination experiments with vP866 as the rescue virus to generate vP881.
Insertion of the PRV gpIII gene into the TK Locus of NYVAC. The sequences encoding the PRV gpIII gene map to the BamHI 2 and 9 fragments of the PRV genome (Robbins et al., 1986b). Plasmids pPR9.9 and pPR7.35 contain the PRV DamHI
fragments 2 and 9, respectively, inserted into the BamHI
site of pBR322. An SphI/BamHI fragment containing the 5' end of the PRV gpIII gene was isolated from pPR9.9. An NcoI/BamHI fragment containing the remainder of the gpIII
gene was isolated from pPR7.35. The entire PRV gpIII gene was assembled by the ligation of these two fragments into pIBI25 to yield pPR17.
The PRV gpIII gene was manipulated to be expressed under the control of the early vaccinia virus hemorrhagic promoter, located in the IindIiI B region (Goebel et al., 1990a,b). Using site-directed mutagenesis, an siI site was introduced by changing the sequence CGC (bases 192-194) in PRV gpIII to ATG and an CbaI site was introduced by changing the sequence GTCACGT to TTCTAGA (bases 1632-1638). To perform the mutagenesis reactions, single-stranded DNA was generated from plasmid pPR17 using the helper phage R408 (Stratagene, LaJolla, CA). The site-directed mutagenesis was done using MRSYN5 (SEQ ID NO:52) (5'-GCGAGCGAGGCCATGC
ATCGTGCGAATGGCCCC-3') and MRSYN6 (SEQ ID NO:53) (5'-GGGGG
GACGCGCGGGTCTAGAAGGCCCCGCCTGGCGG-3') and selection on E.
coli dut- una_ strain. CJ236 (International Biotechnologies, Inc., New Haven, CT). Mutagenesis was performed according to the protocols of Kunkel (1985).
These mutations resulted in the generation of plasmid pPR28.
Plasmid pPR28 was digested with NsiI and XbaI and treated with Mung bean nuclease. A 1440 bp fragment was purified and inserted into a BalII/HpaI digested pSD478VC
after treatment with Mung bean nuclease and calf-intestine alkaline phosphatase. The resultant plasmid was designated as pPR24.

The plasmid pPR24 was digested with g=B1 and DraI to liberate a 1500 bp blunt-ended fragment containing the u promoter and PRV gpIII gene. This fragment was ligated into SmaI digested pSD513VC to yield pPRVIIIVCTK. In vitro recombination experiments were performed with pPRVIIIVCTK
and vP866 as the rescue virus to generate vP883. In vP883, the vaccinia tk coding sequences are replaced by the PRV
gpIII gene inserted in a right to left orientation, with respect to the genome, under the control of the 120 bp vaccinia u promoter element.
Insertion of the PRV an50 Gene into the ATI Locus of NYVAC. DNA encoding the gene for the PRV glycoprotein gp5O
is located on the amHI fragment 7 of the PRV genome (Petrovskis et al., 1986a,b). Plasmid pPR7.1 contains the PRV BamHI fragment 7 inserted into the &_mliI site of pBR322.
A Stul/N eI subfragment of pPR7.1 containing the entire gp50 gene was subcloned into pIBI25 to yield plasmid #856.
The coding sequences for PRV gp5O were placed under the control of the early/intermediate vaccinia promoter, 13L
(Schmitt and Stunnenberg, 1988; Vos and Stunnenberg, 1988).
This promoter element has been used previously to express foreign genes in vaccinia recombinants (Perkus et al., 1985;
Bucher et al., 1989). DNA corresponding to promoter sequences upstream from the 13L open reading frame (Schmitt and Stunnenberg, 1988) was derived by PCR (Saiki et al., 1988) using synthetic oligonucleotides P50PPBAM (SEQ ID
NO:54) (5'-ATCATCGGATCCGGTGGTTTGCCATTCCG-3') and P50PPATG
(SEQ ID NO:55) (5'-GATTAAACCTAAATAATTG-3') and pMPIVC, a subclone of the Copenhagen HindIII I region, as template.
The resulting 126 bp fragment was digested with BamHI to generate a$gmHI cohesive end at the 5' end of the promoter sequence. The 3' end remained blunt-ended.
The PRV gp5O coding region was excised from plasmid 1856. Plasmid #856 was initially digested with figjI, which cuts 7 bp upstream from the ATG and results in a 3' overhang. The 3' overhang was blunt-ended with T4 DNA
polymerase in the presence of 2mM dNTPs. The resulting DNA
was partially digested with W II, and a 1.3kb blunt/ $alII
fragment containing the PRV gp50 gene was isolated.

WO 92/" '2 PCT/US92/01906 The 126 bp 13L promoter fragment (DamHI/blunt) and the 1.3kb gp50 gene containing fragment (blunt/=II) were ligated into pBS-SK (Stratagene, La Jolla, CA) digested with BamHI. The resultant plasmid was designated as pBSPRV50I3.
The expression cassette containing the 13L promoter linked to the PRV gp50 gene was excised by a BamH1/partial SmaI
digestion. A 1.4 kb fragment containing the 13L
promoter/PRV gp50 gene was isolated and blunt-ended using the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs.
The 1.4 kb blunt-ended fragment containing the 13L
promoter/PRV gp50 gene was inserted into the ATI insertion plasmid pSD541. Flanking arms for the ATI region were generated by PCR using subclones of the Copenhagen HindIII A
region as template. Oligonucleotides MPSYN267 (SEQ ID
NO:56) (5'-GGGCTGAAGCTTGCGGCCGCTCATTAGACAAGCGAATGAGGGAC-3') and MPSYN268 (SEQ ID NO:57) (5'-AGATCTCCCGGGCTCGAGTAATTAATTAATTTTTATTACACCAGAAAGACGGCTTGAGAT
C-3') were used to derive the 420 bp vaccinia arm to the right of the ATI deletion. Synthetic oligonucleotides MPSYN269 (SEQ ID NO:58) (5'-TAATTACTGAGCCCGGGAGATATAATTTAATTTAATTTATATAACTCATTTTTTCCCC-3') and MPSYN270 (SEQ ID NO:59) (5'-TATCTCGAATTCCCGCGGCTTTAAATGGACGGAACTCTTTTCCC-3') were used to derive the 420 bp vaccinia arm to the left of the deletion. The left and right arms were fused together by PCR and are separated by a polylinker region specifying restriction sites for BalII, SmaI, and XhoI. The PCR-generated fragment was digested with Hin_dIII and EcoRI to yield sticky ends, and ligated into pUC8 digested with HindIII and EcoRI to generate pSD541.
The pSD541 plasmid containing the 13L/PRV gp5O gene was designated as pATIgp5O. This plasmid was used in in vitro recombination experiments to generate vP900. vP900 contains the PRV gp50 gene in place of the ATI gene.
Generation of Double and Triple PRV Recombinants in NYVAC. In vitro recombination experiments were performed to generate NYVAC-based recombinants containing multiple PRV
genes. in vitro recombination experiments using the donor plasmid, pATIP50, were performed with vP881, vP883, and vP915 to generate vP912, vP916, and vP925, respectively (Table 1). Experiments were done with plasmid pPR18 and vP883 as rescue virus to yield vP915 (Table 1).
Immunoprecipitation from NYVAC/PRV Recombinant Infected Cells. Vero cells were infected at an m.o.i. of 10 pfu per cell with the individual recombinant viruses, with the NYVAC
parent virus, or were mock infected. After a 1 hr absorption period, the inoculum was removed and infected cells were overlaid with methionine-free media containing 35S-methionine (20uCi/ml). All samples were harvested at 8 hr post infection. For samples analyzed with the sheep anti-gpII sera, cells were harvested by centrifugation and were dissociated with RIPA buffer (1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 0.O1M methionine, 5mM EDTA, 5mM 2-mercapto-ethanol, im/ml BSA, and 100 u/ml aprotinin).
Samples analyzed with sheep anti-gpIII and a monoclonal specific for gp50 were lysed in 1X Buffer A(1$ NP40, 10mM
Tris (pH7.4), 150mM NaCl, 1mM EDTA, 0.0% Na Azide, 0.2mg/ml PMSF). All sera was preadsorbed with vP866 infected Vero cells and all lysates were preadsorbed with normal sera (sheep or mouse) and protein A-sepharose linked to the secondary antibody.
Lysates from the infected cells were analyzed for PRV
gpII expression using a sheep anti-gpII serum. This primary antiserum was incubated with protein A-sepharose conjugated with rabbit anti-sheep IgG (Boehringer-Mannheim). After an overnight incubation at 4 C, samples were washed 4 times with 1 x RIPA buffer, 2 times with LiCl-urea (0.2M LiCl, 2M
urea, lOmM Tris, ph 8.0). Precipitates were harvested by micro centrifugation. Precipitated protein was dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH 6.8), 4% (SDS), 20% glycerol, 10% 2 mercapto-ethanol) and boiling for 5 min. Proteins were fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M Na-Salicylate for fluorography.
Lysates were analyzed for PRV gpIII expression using a sheep-anti gpIII sera. This primary antisera was incubated with Protein A-Sepharose conjugated with rabbit anti-sheep IgG (Boehringer-Mannhein). After an overnight incubation at 40C, samples were washed 4 times with 1X Buffer A and 2 times with the LiCl-urea buffer. Precipitates were treated.
and analyzed by flurography as described above.
Lysates were analyzed for PRV gp50 expression using monoclonal antibody, 22M4 (provided by Rhone Merieux, Lyon, France). This primary antibody was incubated with Protein A-Sepharose conjugated with goat anti-mouse IgG and IgM
(Boehringer-Mannheim). The precipitates were recovered and analyzed as described above for the PRV gpIII
immunoprecipitations.
Expression of the PRV Glycoproteins in Cells Infected with the NYVAC/PRV Recombinants. The PRV gpII, gpIII, and gp50 products are typical glycoproteins associated with membranous structures in PRV infected cells. Anti-gpII, anti-gpIII and anti-gp50 specific monoclonal antibodies followed by fluorescein-conjugated goat anti-mouse IgG were used to analyze the PRV glycoprotein expression on the surface of recombinant infected Vero cells. Surface expression of neither gpII, gpIII, nor gp50 was detectable on the surface of mock infected cells or cells infected with the NYVAC (vP866) parent virus. PRV gpII expression was observed on the surface of vP881, vP912, vP915, and vP925 infected cells. PRV gpIII surface expression was observed in vP883, vP915, vP916, and vP925 infected cells. PRV gp50 surface expression was observed in vP900, vP912, vP916, and vP925 infected cells. In summary, the surface expression of the particular PRV glycoproteins was only detectable in cells infected with NYVAC/PRV recombinants containing the appropriate PRV gene(s).
Immunoprecipitation of PRV Glycoproteins from Cells Infected with the NYVAC/PRV Recombinants. The authenticity of the expressed PRV gpII, qpIII, and gp50 glycoproteins in Vero cells infected with the NYVAC/PRV recombinants was analyzed by immunoprecipitation. The PRV gpII gene product represents one of the major glycoproteins encoded in PRV-infected cells. The mature protein consists of a complex of glycoproteins linked by disulfide bonds (Hampl et al., 1984;

Lukacs et al., 1985). Under reducing conditions, three species are resolved from this complex. These spec--es (IIa-Iic) migrate with apparent size of 120 kDa, 74-67 kDa, and 58 kDa, respectively, on an SDS-polyacrylamide gel (Hampl et al., 1984).
In immunoprecipitation analyses using the anti-PRVgpII
specific serum, no PRV-specific protein species were precipitated from mock infected cells or cells infected with the NYVAC (vP866) parent virus. PRV gpII was also not detectable in cells infected with the non-gpII containing NYVAC/PRV recombinants vP916, vP883, and vP900. It is evident that PRV gpII was expressed in all the NYVAC/PRV
recombinants which harbor the PRV gpII gene. These are vP925, vP912, vP915 and vP881. Lysates from Vero cells infected with the PRV gpII containing recombinants all contained protein species consistent with the proper expression and processing of gpII to gpIIa (120 kDa), gpIIb (74-67 kDa), and gIIc (58 kDa). Two additional protein species of 45 kDa and 10 kDa were specifically precipitated with the anti-gpII serum. These protein species appear to emerge by an aberrant proteolytic processing of PRV gpII at late times in recombinant infected cells.
The PRV gpIII product is another major PRV
glycoprotein. The.gpIII exists as a monomer not complexed with other viral proteins that migrates with an apparent molecular weight of 92 kDa (Hampi et al., 1984; Robbins et al., 1986b). In immunoprecipitation analyses from NYVAC/PRV
recombinant infected cells using antisera specific for gpIII, no anti-gpIII specific protein species were present in lysates from mock infected cells, nonrecombinant infected cells, or cells infected with NYVAC/PRV recombinants not containing gpIII (vP912, vP881, and vP900, respectively).
Lysates from vP925, vP915, vP916, and vP883 infected cells all contained the 92 kDa PRV gpIII gene product.
The mature PRV gp50 gene product is approximately 50 to 60 kDa (Petrovskis et al., 1986a; Wathen et al., 1984), that most likely contains 0-linked carbohydrate (Petrovskis et al., 1986b). In immunoprecipitations from lysates of cells infected with the NYVAC/PRV recombinants using antisera WO 92/. '2 PC'T/US92/01906 specific to the PRV gp50 gene product, gp50 was not present in lysates from mock infected cells, nonrecombinant infected cells, and cells infected with the recombinants not containing the gp50 gene (vP915, vP881, and vP883, respectively). Lysates from cells infected with recombinant NYVAC viruses containing the PRV gp50 gene (vP925, vP912, vP916, and vP900, respectively) all expressed a 50-60 kDa protein species which was specifically precipitated with the anti-PRV gp50 serum.
Table 1. NYVAC Recombinants Expressing PRV glycoproteins gpII, gpIII and gp50 Recombinant Parent Donor Plasmid PRV Glycoprotein vP881 vP866 pPR18 gpII
vP883 vP866 pPRVIIIVCTK gpIII
vP900 vP866 pATIgp50 gp50 vP912 vP881 pATIgp5O gpII, gp50 vP915 vP883 pPR18 gpII, gpIII
vP916 vP883 pATIgp5O gpIII, gp50 vP925 vP915 pATIgp50 gpII, gpIII, gp50 Example 11 - CONSTRUCTION OF NYVAC RECOKBINANTB EXPRESSING
THE qp340, gB and gH GENES OF EPSTEIN-BARR
VIRUS

A NYVAC donor plasmid containing the EBV gp340, gB, and gH genes was constructed. This donor plasmid was used to generate two recombinants: vP941 and vP944.
Restriction enzymes were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD), New England BioLabs, Inc. (Beverly, MA) or Boehringer-Mannheim (Indianapolis, IN). T4 DNA ligase and DNA polymerase I
Klenow fragment were obtained from New England BioLabs, Inc.
Standard recombinant DNA techniques were used (Maniatis et al., 1982) with minor modifications for cloning, screening and plasmid purification. Nucleic acid sequences were confirmed using standard dideoxychain-termination reactions (Sanger, 1977) on alkaline-denatured double-stranded plasmid templates. M13mp18 phage, pIBI24 and pIBI25 plasmids were obtained from International Biotechnologies, Inc., CT.
Cell Lines and Virus Strains. NYVAC was used as a rescue virus to generate recombinants. All vaccinia virus stocks were produced in Vero (ATCC CCL81) cells in Eagles MEM medium supplemented with 5-10% newborn calf serum (Flow Laboratories, Mclean, VI).
Oligonucleotide-Directed Mutagenesis. The uracil-substituted single-stranded DNA template used for the mutagenesis reactions was from CJ236 transformed cells. The mutations were achieved by using the protocol of Kunkel et al. (1987). The various oligonucleotides were synthesized using standard chemistries (Biosearch 8700, San Rafael, CA;
Applied Biosystems 380B, Foster City, CA) Construction of Vaccinia Virus Recombinants.
Procedures for transfection of recombinant donor plasmids into tissue culture cells infected with a rescuing vaccinia virus and identification of recombinants by in situ hybridization on nitrocellulose filters were as previously described (Panicali et al., 1982; Piccini et al., 1987).
Modifications and Expression in Vaccinia Recombinants of EBV Genes gp340, gB, and gH. The gp340 gene corresponds to the open reading frame BLLFla of the complete EBV
sequence (Baer et al., 1984). The gp220 gene derives from the gp340 mRNA by an internal splicing event (open reading frame BLLFlb). The gp340 and gp220 genes were isolated from cDNA clones (plasmids pMLPgp340 and pMLPgp220, respectively) provided by Dr. Perricaudet (Centre de Recherche sur le Cancer-IRSG1 7 rue Guy Mocquet, 94802 Villejuif, France).
The 2100 bp XmaI/C1aI fragment of pMLPgp220 was inserted into maI/C1aI M13 mp18, and the resulting plasmid was called mp18gp220. By in vitro mutagenesis using the oligonucleotides CM4 and CM5 the 5' and 3' extremities of gp220 gene were modified for expression under the control of the vaccinia H6 promoter. The plasmid containing the modified gp220 gene was called mp18gp220(4+5). The nucleotide composition of CM4 (SEQ ID N0:60) and CM5 (SEQ ID
NO:61) were as follows:
CM4: TAAAGTCAATAAATTTTTATTGCGGCCGCTACCGAGCTCGAATTCG
NotI -CM5: GCTTGCATGCCTGCAGATATCCGTTAAGTTTGTATCGTAATGGAGGCAGCCTTGC
EcoRV Met ti'O 92/1. 2 PCT/US92/01906 The 2300 bp NarI/ coRV fragment of mp18gp220(4+5) was cloned into the NarI/EcoRV plasmid SP131NotI. SP131NotI
contains the complete H6 vaccinia promoter as previously defined (Taylor et al., 1988a, b). The resulting plasmid was called SP131gp220.
The 2360 bp ScaI/XhoI fragment of pMLPgp340 was cloned into the ScaI/XhoI SP131gp220 plasmid. The resulting plasmid was called SP131gp340.
The 2800 bp NotI/NotI fragment of SP131gp340 was cloned into the SmaI digested vaccinia donor plasmid pSD486. The resulting plasmid was called 486H6340.
The EBV gB gene corresponds to the open reading frame BALF4 of the complete EBV sequence (Baer et al., 1984). A
3500 bp EcoRI/XmnI fragment was isolated from the EBV BamHI
A fragment and cloned into the HincII/EcoRI plasmid pIBI25.
The resulting plasmid was called p25gB.
By in vitro mutagenesis, using the oligonucleotides EBVM5 (SEQ ID NO:62) and EBVM3 (SEQ ID NO:63), the EBV gB
gene was adapted for expression under the control of the vaccinia H6 promoter. The nucleotide composition of EBVM5 (SEQ ID NO:62) and EBVM3 (SEQ ID NO:63) were as follows:
EBVM5:
CCCTACGCCGAGTCATTACGATACAAACTTAACGGATATCAGAGTCGTACGTAGG
EBVM3: CTGGAAACACTTGGGAATTCAAGCTTCATAAAAAGGGTTATAGAAGAGTCC
The resulting plasmid was called p25gB(5+3).
The 2600 bp EcoRV/EcoRI fragment of p25gB(5+3) was cloned into the EcoRV/EcoRI Sp131 plasmid. The resulting plasmid was called SP131gB.
The EBV gH gene corresponds to the BXLF2 open reading frame of the complete EBV sequence (Baer et al., 1984). The complete BXLF2 open reading frame is contained in two BamHI
EBV fragments: BamHI X and BamHI T. The complete BXLF2 open reading frame was reconstituted by cloning the 830 bp SmaI/BamHI fragment of EBV DAmHI T fragment into the SmaI/BamHI pIBI24 plasmid; the resulting plasmid was called 24gH5. The 1850 bp BamHI/HindIII fragment of EBV BamHI X
fragment was cloned into the $amHI/HindIII 24gH5 plasmid.
The resulting plasmid was called 24gH.

") 92/15672 PCT/US9: 906 By in vitro mutagenesis using the oligonucleotides HM5, HM4, and HM3 the EBV gH gene was modified to be expressed under the control of the vaccinia B13R hemorrhagic promoter (Goebel et al., 1990a,b). The oligonucleotide HM4 was used to modify a sequence corresponding to a vaccinia early transcription termination signal. The nucleotide compositions of HM5 (SEQ ID NO:64), HM4 (SEQ ID NO:65), and HM3 (SEQ ID NO:66) were as follows:
HM5: ACACAGAGCAACTGCAGATCTCCCGATTTCCCCTCT
HM4: GGGCAAAGCCACAAAATATGCAGGATTTCTGCG
HM3: GCCAGGGTTTTCCCAGATCTGATAAAAACGACGGCCAGTG
The resulting plasmid containing the modified gH was called 24gH(5+4+3).
The vaccinia hemorrhagic promoter does not appear to be a strong promoter when compared with other pox promoters.
The EBV gH gene has been placed under the control of the 42 kDa entomopox promoter. This was achieved by using the polymerase chain reaction (PCR), specific oligonucleotides 42gH (SEQ ID NO:67) and BAMgH (SEQ ID NO:68) and the plasmid 24gH(5+4+3) as template.
42gH: GGGTCAAAATTGAAAATATATAATTACAATATAAAATGCAGTTGCTCTGTGTT
Met BAM-H: ATGGATCCTTCAGAGACAG (The first A residue corresponds to position 292 of the gH coding sequence) The PCR reaction was processed in a Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) with 36 cycles at 94 C for 1 minute, 42 C for 1.5 minutes, and 72 C for 3 minutes, and a final extension step at 72 C for 5 minutes. The PCR product was purified, digested with BamHI and cloned into the 4550 bp SmaI/BamHI fragment of 24gH(5+4+3). The resulting plasmid was called 24BXLF2.42K.
Insertion of EBV qp340, gB, and gH Genes into the Vaccinia Donor Plasmid pSD542 and Isolation of vP941 and vP944. The vaccinia donor plasmid pSD542 is a derivative of pSD460 with an expanded polylinker region; it is used to recombine foreign genes into the vaccinia TK locus.

WO 92/1 '2 PCT/US92/01906 The 2820 bp BamHI/BalII fragment of 486H6340 plasmid was cloned into the BamHI/BalII pSD542 plasmid. The resulting plasmid was called 542.340.
The 2150 bp SmaI/BalII fragment of 24BXLF2.42K plasmid was cloned into the SmaI/BalII 542.340 plasmid. The resulting plasmid was called 542.340gH.
The 2700 bp HindIII/HindIII fragment of SP13lgB plasmid was cloned into the Bg1II 542.340gHplasmid. The resulting plasmid was called EBV Triple.l. A map of the EBV coding regions inserted into EBV Triple.1 plasmid is presented in FIG. 8. The direction of transcription is indicated by the arrows in FIG. S.
EBV Triple.1 plasmid was digested by NotI and transfected into Vero cells infected with NYVAC or vP919, a NYVAC based vaccinia recombinant containing three HBV genes.
The corresponding recombinant vaccinia viruses vP944 and vP941 were isolated.
Esample 12 - CONSTRUCTION OF NYVAC RECOKBINAIJTB EXPRESSING
THE qB, qC and gD GENES OF HERPES SIMPLEB

A recombinant vaccinia virus that expresses the HSV2 gB, gC and gD genes was constructed.
Cells and Viruses. HSV 2 (strain G) was propagated in VERO cells (ATCC CCL81) and purified by centrifugation on a sucrose gradient (Powell et al., 1975).
Vaccinia virus (Copenhagen) and recombinants derived therefrom were propagated in VERO cells (ATCC CCL81) as previously described (Panicali et al., 1982; Guo et al., 1989).
Isolation of the HSV2 gB Gene. A 12 kb BalII fragment, containing the HSV2 gB gene, was isolated from HSV2 genomic DNA and inserted into the BamHI site ofsite of pSD48 pUC19.
The resulting plasmid was designated pJ4.
The gB gene was then cloned between vaccinia virus flanking arms. This was accomplished by cloning the 2,700 bp SstII-SacI (partial) fragment of pJ4 into the SstII-SacI
fragment of pMP409DVC (Guo et al., 1989). This placed the gB gene between the vaccinia virus sequences flanking the M2L gene. The plasmid generated by this manipulation was designated pGB1.

An in-frame terminati-on codon was then added to the 3'-end of the gB gene. This was accomplished by cloning the oligonucleotides, GBL3 (SEQ ID NO:69) 5'-CTAATAG-3' and GBL4 (SEQ ID NO:70) 5'-GATCCTATTAGAGCT-3', into the 6,300 bp BamHI-SacI (partial) fragment of pGB1. The plasmid generated by this manipulation was designated pGB2.
The vaccinia virus H6 promoter (Taylor et al., 1988a, b; Perkus et al., 1989) was then cloned upstream of the gB
gene. This was accomplished by cloning the 370 bp BalII
fragment of pBLVH14 (Portetelle et al., 1991), containing the H6 promoter, into the BalII site of pGB2. The plasmid generated by this manipulation was designated pGB3.
The initiation codon of the H6 promoter was then aligned with the initiation codon of the gB gene. This was accomplished by cloning the oligonucleotides, GBL1 (SEQ ID
NO:71) 5'-ATCCGTTAAGTTTGTATCGTAATGCGCGGGGGGGGCTTGATTTGCGCGCTGGTCGTGGGG
GCGCTGGTGGCCGC-3' and GBL2 (SEQ ID NO:72) 5'-GGCCACCAGCGCCCCCACGACCAGCGCGCAAATCAAGCCCCCCCCGCGCATTACGATACA
AACTTAACGGAT-3', into the 6,300 bp SstII-EcoRV (partial) fragment of pGB3. The plasmid generated by this manipulation was designated pGB5.
The H6-promoted gB gene was then cloned into a different vaccinia virus donor plasmid. This was accomplished by cloning the 2,800 bp Bg1II-BamHI fragment of pGB5, containing the H6- promoted gB gene, into the Bct1II
site of pSD513VCVQ. (pSD513VCVQ is a subclone of the vaccinia virus HindIII J fragment in which the thymidine kinase (tk) gene is replaced by a polylinker region.) This placed the H6-promoted gB gene between the vaccinia virus sequences flanking the tk gene. The plasmid generated by this manipulation was designated pGB6.
Isolation of the HSV2 gC Gene. A 2,900 bp SalI
fragment, containing the HSV2 gC gene, was isolated from HSV2 genomic DNA and inserted into the SalI site of pIBI25.
The resulting plasmid was designated pGC3.
The gC gene was then cloned between vaccinia virus flanking arms. This was accomplished by cloning the 2,900 bp XhoI-BamHI fragment of pGC3 into the XhoI-BamHI site of WO 92/. 72 PCF/US92/01906 pGC2. pGC2 was generated by cloning the 370 bp $alII
fragment of pBLVH14 (Portetelle et al., 1991), containing the H6 promoter, into the BalII site of pSD486 (FIG. 2).
This placed the gC gene between the vaccinia virus sequences flanking the u gene. The plasmid generated by this manipulation was designated pGC5.
The initiation codon of the H6 promoter was then aligned with the initiation codon of the gC gene. This was accomplished by cloning the oligonucleotides, GCL1 (SEQ ID
NO:73) 5'-ATCCGTTAAGTTTGTATCGTAATGGCCCTTGGACGGGTGGGCCTAGCCGTGGGCCTGTG-3' and GCL2 (SEQ ID NO:74) 5'-AGGCCCACGGCTAGGCCCACCCGTCCAAGGGCCATTACGATACAAACTTAACGGAT-3', into the 5,400 bp NruI-SfiI fragment of pGC5. The plasmid generated by this manipulation was designated pGC10.
Extraneous 3'-noncoding sequence was then eliminated from pGC10. This was accomplished by recircularizing the E.
coli DNA polymerase I(Klenow fragment) filled-in 4,900 bp SalI-SmaI (partial) fragment of pGC10. The plasmid generated by this manipulation was designated pGC11.
Additional 3'-noncoding sequence was then eliminated from pGC11. This was accomplished by cloning the oligonucleotide, GCL3 5'-CTAGGGCC-3', into the 4,900 bp XbaI-ApaI (partial) fragment of pGC11. The plasmid generated by this manipulation was designated pGC12.
Isolation of the HSV2 gD Gene. A 7.5 kb XbaI fragment, containing the HSV2 gD gene, was isolated from HSV2 genomic DNA and inserted into the ~baI site of pIBI25. The resulting plasmid was designated pGD1.
The gD gene was then cloned downstream of the H6 promoter and between vaccinia virus flanking arms. This was accomplished by cloning the 1,500 bp DraI-PstI fragment of pGD1 into the 3,700 bp SmaI-PstI fragment of pTP15 (Guo et al., 1989). This placed the gD gene downstream of the H6 promoter and between the vaccinia virus sequences flanking the HA gene. The plasmid generated by this manipulation was designated pGD2.
The initiation codon of the H6 promoter was then aligned with the initiation codon of the gD gene. This was 92/1-55,672 PCT/US92 106 accomplished by cloning the oligonucleotides, GDL1 (SEQ ID
NO:75) 5'-ATCCGTTAAGTTTGTAfiCGTAATGGGGCGTTTGACCTCCGG-3' and GDL2 (SEQ ID NO:76) 5'-CGCCGGAGGTCAAACGCCCCATTACGATACAAACTTAACGGAT-3', into the 5,100 bp EcoRV-FhaII (partial) fragment of pGD2. The plasmid generated by this manipulation was designated pGD5.
Extraneous 3'-noncoding sequence was then eliminated.
This was accomplished by cloning the oligonucleotides, GDL3 (SEQ ID NO:77) 5'-GGCAGTACCCTGGCGGCGCTGGTCATCGGCGGTATTGCGTTTTGGGTACGCCGCCGGCGC
TCAGTGGCCCCCAAGCGCCTACGTCTCCCCCACATCCGGGATGACGACGCGCCCCCCTCG
CACCAGCCATTGTTTTACTAGCTGCA-3' and GDL4 (SEQ ID NO:78) 5'-GCTAGTAAAACAATGGCTGGTGCGAGGGGGGCGCGTCGTCATCCCGGATGTGGGGGAGAC
GTAGGCGCTTGGGGGCCACTGAGCGCCGGCGGCGTACCCAAAACGCAATACCGCCGATGA
CCAGCGCCGCCAGGGTACTGCC-3', into the 4,800 bp NaeI-PstI
fragment of pGD5. The plasmid generated by this manipulation was designated pGD7.
Additional sequence was then added upstream of the H6 promoter. This was accomplished by cloning the 150 bp Bcr1II-EcoRV fragment of pGB6 (see above) into the 4,800 bp BalII-EcoRV fragment of pGD7. The plasmid generated by this manipulation was designated pGD8.
Construction of a Vaccinia Virus Donor Plasmid Containing the HSV2 gB, aC and aD Genes. A plasmid containing the gC and gD genes was constructed. This was accomplished by cloning the 1,850 bp PstI fragment of pGC12, containing the H6-promoted gC gene, into the PstI site of pGDB. The plasmid generated by this manipulation was designated pGCD1.
A plasmid containing the gB, gC and gD genes was then constructed. This was accomplished by cloning the 2,800 bp Bg1II-BamHI fragment of pGB6, containing the H6-promoted gB
gene, into the 6,800 bp BamHI (partial) fragment of pGCD1.
The plasmid generated by this manipulation was designated pGBCD1.
Extraneous DNA was then eliminated. This was accomplished by cloning the E. coli DNA polymerase I(Klenow fragment) filled-in 6,000 bp HindIII-BamHI (partial) fragment of pGBCD1, containing the H6-promoted gB, gC and gD

'.='VO 92/1 '2 PC'I'/US92/01906 into the SmaI site of pMP831. The plasmid generated genes, by this manipulation was designated pGBCDC1.
The H6-promoted gB, gC and gD genes were then cloned between vaccinia virus flanking arms. This was accomplished by cloning the oligonucleotides, HSVL1 (SEQ ID NO:79) 5'-TCGATCTAGA-3' and HSVL2 (SEQ ID NO:80) 5'-AGCTTCTAGA-3', and the 5,700 bp HindIII-BamHI (partial) fragment of pGBCDC1, containing the H6-promoted gB, gC and gD genes, into the 3,600 bp XhoI-BalII fragment of pSD541. This placed the H6-promoted gB, gC and gD genes between the vaccinia virus sequences flanking the ATI gene. The plasmid generated by this manipulation was designated pGBCD4.
Construction of vP914. A vaccinia virus recombinant, vP914, containing the HSV2 gB, gC and gD genes, was constructed. The procedures used to construct vaccinia virus recombinants have been described previously (Panicali et al., 1982; Guo et al., 1989; Guo et al., 1990). The vaccinia virus recombinant, vP914, was generated by transfecting pGBCD4 into vP866 (NYVAC) infected cells. The HSV2 genes in this recombinant are under the transcriptional control of the vaccinia virus H6 promoter.
Immunofluorescence and ImmunoDrecipitation of vP914 Infected Cells. Immunofluorescence and immunoprecipitations were performed as previously described (Guo et al., 1989).
Rabbit antisera against HSV2 was obtained from DAKO Corp.
(code no. B116). Monoclonal antibodies against HSV2 gB
(H233) and HSV2 gD (HD1) (Meignier et al., 1987) were obtained from B. Meignier (Institut Merieux, Lyon, France).
In HSV2 infected cells, gB, gC and gD (as well as other HSV2 glycoproteins) are expressed on the cell surface.
Immunofluorescence studies with vP914 infected cells, using monoclonal antibodies specific for HSV2 gB (H233) and HSV2 gD (HD1), indicated that the HSV2 gB and gD glycoproteins produced in these cells were also expressed on the cell surf ace .
In HSV2 infected cells, gB, gC and gD have molecular weights of approximately 117 kDa, 63 kDa and 51 kDa, respectively (Marsden et al., 1978; Marsden et al., 1984;
Zweig et al., 1983). Immunoprecipitation of vP914 infected V 92/ l 5672 PC.'T/US92/ 36 cells with a gB-specific monoclonal antibody (H233) precipitated three major proteins with molecular weights of approximately 117 kDa, 110 kDa and 100 kDa, as well as other minor proteins. Immunoprecipitation with a gD-specific monoclonal antibody (HD1) precipitated a major protein with a molecular weight of approximately 51 kDa and minor proteins with molecular weights of approximately 55 kDa and 46 kDa. Additionally, immunoprecipitation of vP914 infected cells with polyclonal antisera against HSV2 precipitated a protein with a molecular weight similar to gC, 63 kDa, (as well as an 85 kDa protein) and proteins corresponding in size to gB and gD. Therefore, cells infected with vP914 .appeared to express HSV2 proteins with molecular weights similar to gB, gC and gD.
Example 13 - CONSTRUCTION OF NYVAC RECOMBINANTS EXPRESSING
HEPATITIS B VIRUS GENES

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 Research Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA) and Boehringer Mannheim Biochemicals (Indianapolis, IN). T4 DNA ligase was obtained from New England Biolabs. T4 polynucleotide kinase was obtained from Bethesda Research Laboratories. Plasmid pGEM-3Z was obtained from Promega (Madison, WI). The origin of plasmid pTHBV containing the HBV genome cloned in pBR322 has been previously described (Paoletti et al., 1984).
Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain terminating method (Sanger et al., 1977) using Sequenase (Tabor and Richardson, 1987) as previously described (Guo et al., 1989). DNA
amplification by polymerase chain reaction (PCR) for cloning and sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer WO 92/. 72 PCT/US92/01906 Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA
Thermal Cycler.
Virus and Transfection. The NYVAC strain of vaccinia virus and its intermediate ancestor, vP804 (FIG. 5), were used. Generation and processing of recombinant virus are as previously described (Panicali et al., 1982).
Immunoprecipitation. Vero cells were infected at an m.o.i. of 10 pfu per cell with recombinant vaccinia virus, with the NYVAC parent virus (vP866) or were mock infected.
After a 1 hour adsorption period, the inoculum was removed and infected cells were overlayed with methionine-free media containing 35S-methionine (20 uCi/ml). All samples were harvested at 8 hours post infection. Samples were lysed in 3x buffer A containing triton and DOC (3% NP-40, 3% triton, 31 DOC, 30 mM Tris pH 7.4, 450 mM NaCl, 3 mM EDTA, 0.03 %
NaAzide, 0.6 mg/mi PMSF) containing 50 ul aprotinin (Sigma Chemical Co., St. Louis, MO, # A6279). All lysates were precleared against normal rabbit sera linked to protein A-sepharose.
Rabbit antisera raised to HBV core antigen and to HBV
S2 peptide (aa 120-153) were obtained from R. Neurath (The Lindsley F. Kimball Research Institute of the New York Blood Center). Anti-S2 antiserum was preadsorbed with vP866 infected Vero cells. HBV proteins were immunoprecipitated using anti-core or anti-S2 antiserum and resuspended in 2x Laemmli sample buffer (Laemmli, 1970) for electrophoresis and subsequent autoradiography.
Serolocrv. Rabbits and guinea pigs were inoculated with 108 pfu recombinant vaccinia virus vP919 in sets of two by intradermal, subcutaneous or intramuscular route. Six weeks after the primary inoculation, rabbits were boosted once by the same route and dose. Seven weeks after the primary inoculation, guinea pigs were boosted once by the same route and dose. Groups of 12 mice were inoculated with 107 pfu recombinant vaccinia virus vP919 by intradermal, subcutaneous or intramuscular route. Seven weeks after the primary inoculation, mice were boosted once by the same route. Sera were collected at weekly intervals. Weekly bleedings from each group of mice were pooled. All sera ~''~ 92/15672 PCT/US92/ '06 were analyzed for antibody to HBV surface antigen using the AUSAB radioimmunoassay kit (Abbott, North Chicago, IL). All sera were analyzed for antibody to HBV core antigen using the CORAB competitive radioimmunoassay kit (Abbott) using standard techniques.
Construction of vP919. Vaccinia recombinant vP919 contains three genes from Hepatitis B Virus inserted into NYVAC vaccinia virus vector. The genes were inserted individually into three different sites of the virus. The three HBV genes encode the following protein products: (1) HBV M protein, (referred to here as small pre S antigen, or spsAg), (2) HBV L protein (referred to here as large pre S
antigen, or 1psAg) and (3) a fusion protein, (referred to here as S12/core) composed of the entire pre-S region (Si +
S2) linked onto the amino terminus of the core antigen.
Vaccinia virus does not maintain multiple copies of the same heterologous DNA sequences inserted contiguously into a single vaccinia genome (Panicali et al., 1982) Since coding sequences for the spsAg are contained within coding sequences for the 1psAg, insertion of both genes into a single vaccinia genome would be expected to lead to instability of the genome. Similarly, an S1+S2 DNA region present in a hybrid S12/core gene could undergo recombination with the equivalent S1+S2 region of 1psAg.
These potential problems were prevented in two ways. (1) The three genes were inserted into three different loci in the vaccinia genome, separated from each other by large regions of vaccinia DNA containing essential genes. Thus, any recombination between the HBV genes would lead to incomplete vaccinia genomes which would not produce viable vaccinia progeny. (2) DNAs encoding the spsAg gene and the Sl+S2 region of the S12/core hybrid gene were synthesized chemically with different codon u: ge to minimize DNA
homology with the native HBV gene encoding the 1psAg and with each other. The native HBV gene encoding the lpsAg and the synthetic gene encoding the spsAg are of the ayw subtype; the S1+S2 region for the fusion S12/core gene was synthesized to correspond to the adw2 subtype (Valenzuela et al., 1979).

Cassettes containing-the three individual HBV genes under the control of poxvirus promoters were assembled in different vaccinia donor plasmids and inserted sequentially into vaccinia virus as detailed below.
The synthetic version of the gene encoding the HBV
spsAg was synthesized using vaccinia favored codons with the following deviations. (1) The T5NT early transcription terminator TTTTTCT occurring in amino acids 19 through 21 of the sAg (HBV S protein) was modified to TTCTTTC, and codon utilization was adjusted to prevent the generation of other T5NT termination signals (Yuen et al., 1987). (2) To avoid possible aberrant translation products, codon usage was adjusted to prevent the generation of any out of frame ATG
initiation codons in either direction. The synthetic spsAg gene was linked precisely to the modified synthetic vaccinia virus H6 early/late promoter (Perkus et al., 1989). The complete sequence of promoter and gene is given in FIG. 9.
Amino acid sequence is based on the sequence in plasmid pTHBV, which differs from the published ayw sequence (Galibert et al., 1979) at two amino acid positions in the S2 region: Galibert, aa 31 thr; aa 36 leu; pTHBV, aa 31 ala;
aa 36 pro.
Plasmid pGJ15 contains the H6 promoter/synthetic spsAg gene in the vaccinia ATI insertion locus (Perkus et al., 1990). pGJ15 was constructed by assembling portions of the synthetic spsAg gene in pGEM-3Z, then transferring the assembled gene to insertion plasmid pMP494H, a derivative of pSD492 which contains the synthetic H6 promoter in the ATI
deletion locus.
Referring now to FIG. 10, the synthetic HBV spsAg was assembled in three parts. Plasmids pGJ5, pGJ3, and pGJ7 were generated from 6, 5, and 8 pairs of complementary oligonucleotides respectively as follows. Complementary oligonucleotide pairs synthesized with standard chemistries were kinased under standard conditions followed by heating at 65 C and allowed to cool slowly to room temperature to effect annealing. Aliquots of the annealed pairs comprising each fragment were combined with appropriately digested pGEM-3Z (Promega) and ligated under standard conditions.

92/15672 PCT/US92i 06 Fragment SX (indicated with a solid box), bounded by SphI
and XbaI restriction sites, was ligated to pGEM-3Z vector plasmid digested with those enzymes creating plasmid pGJ5.
Vector plasmid sequences are indicated with open regions.
Similarly, fragments XB (diagonal cross-hatch) and BH
(horizontal cross-hatch), were assembled in plasmid pGEM-3Z
digested with either XbaI and BamHI, or LamHI and HindIII, respectively, generating plasmids pGJ3 and pGJ7. The integrity of the insert in each plasmid was verified by determination of the DNA sequence.
Synthetic HBV gene fragments were isolated by digestion of the plasmids pGJ5, pGJ3 and pGJ7 with the appropriate restriction enzymes flanking the SX, XB and BH gene segments and subsequently ligated to pGEM-3Z digested with SphI and HindIII generating plasmid pGJ9 which contains the contiguous HBV synthetic spsAg sequence. Oligonucleotides H6LINK (SEQ ID NO:81) (5'-CTCGCGATATCCGTTAAGTTTGTATCGTAATGCAGTGG-3') and H6LINK2 (SEQ ID NO:82) (5'-AATTCCACTGCATTACGATACAAACTTAACGGATATCGCGAGGTAC-31) containing the 3' 28 bp of the H6 promoter (diagonal hatch) appended to the synthetic spsAg at the initiating methionine through th.e coRI site 9 bp downstream from the first codon, were ligated to pGJ9 digested with KDnI (5' to the SQhI site within the multiple cloning region derived from pGEM-3Z) and with coRI, generating plasmid pGJ12.. A NruI/HnaI fragment was isolated from pGJ12 and ligated to similarly digested pMP494H, generating plasmid pGJ15. pMP494H is an ATI
insertion plasmid containing the vaccinia H6 promoter in the ATI deletion region. pGJ15 contains the H6 promoter-driven HBV synthetic spsAg gene flanked by vaccinia sequences (stippled) surrounding the ATI locus.
pGJ15 was used as donor plasmid for recombination with vaccinia recombinant vP804, generating recombinant vaccinia virus vP856. vP804 contains the NYVAC deletions for the TK, HA, u and [C7L - K11]. Recombinant virus vP856 contains the above deletions with the insertion of the HBV synthetic spsAg gene replacing the ATI region. Progeny virus recombinant described below containing an insert in the 14L

WO 92/. '2 PCT/US92/01906 region will be equivalent to NYVAC in terms of deletions (TK, HA, ATI, 14L, u, [C7L - K1L]).
The gene encoding the HBV 1psAg was derived from plasmid pTHBV. In addition to the amino acid changes in the S2 region referred to above, pTHBV differs from the published ayw subtype at one amino acid position in the S1 region: Galibert et al., 1979, aa 90 ser; pTHBV aa 90 thr.
The early translational termination signal in sAg referred to above was modified from TTTTTCT to TTCTTCT. The entire ipsAg gene was placed under the control of the 105 bp cowpox u promoter (Pickup et al., 1986) The entire sequence of the u promoter/1psAg gene cassette is given in FIG. 11.
Plasmid pMP550ulps contains the u promoter/ipsAg gene in the vaccinia 14L deletion locus. The construction of pMP550ulps is presented schematically in FIG. 12. The 14L
deletion in pMP550ulps is equivalent to the 14L deletion in NYVAC.
Referring now to FIG. 12(A), by PCR using synthetic oligonucleotide primers MPSYN322 (SEQ ID NO:83), MPSYN323 (SEQ ID NO:84) and template plasmid pBScow, the 5' end of the HBV ipsAg gene was added to the cowpox u promoter (orientation indicated by an arrow) generating pMPuSl. (The dark box indicates the u promoter and the striped box indicates HBV sequences.) pSD550 is a vaccinia insertion plasmid for the 14L deletion region. (The triangle indicates the site of deletion and the open box indicates vaccinia sequences.) A SnaBI/BamHI fragment containing the u promoter/HBV junction was isolated and inserted into pSD550 cut with SmaI/BamHI, forming pMP550u.
Referring now to FIG. 12(B), a 1.1 kb raI fragment containing the entire HBV 1psAg was isolated from pTHBV and inserted into pUC8, generating pMPBS. Translation initiating codon and stop codon are indicated. (*) indicates the site of T5NT transcriptional termination signal (Yuen et al., 1987). The transcriptional termination signal was removed from pMP8S by PCR mutagenesis as indicated, generating pMP8ST. A 1.1 kb BamHI (partial) fragment containing the bulk of the 1psAg gene was isolated from pMP8ST and inserted into plasmid pMP550u cut with hamHI, generating pMP550ulps. pMP550ulps was used for recombination with vaccinia recombinant vP856, generating vP896. Synthetic oligonucleotide sequences are as follows:
Bcx1II C1aI
MPSYN322 (SEQ ID NO:83) 5' CCCAGATCTATCGATTGCCATGGGGCAGA 3' BamHI
MPSYN323 (SEQ ID NO:84) 5' TCTGAAGGCTGGATCCAACT 3' XhoI
MPSYN330 (SEQ ID NO:85) 5' CAATCTTCTCGAGGATT 3' incII
MPSYN331 (SEQ ID NO:86) 5' AACAAGAAGAACCCCGCC 3' The HBV initiation codon in MPSYN322 (SEQ ID NO:83) is underlined, the mutated base in MPSYN331 (SEQ ID NO:86) is underlined and restriction sites are indicated.
pMP550ulps was used as donor plasmid for recombination with rescuing virus vP856 described above to generate recombinant virus vP896. vP896 contains both the genes for HBV spsAg and HBV 1psAg in a NYVAC background (deletion of TK, HA, ATI, 14L, u, [C7L - K1L]). To generate a recombinant containing only the HBV 1psAg gene for purposes of comparison with multivalent HBV vaccinia recombinants, pMP550ulps was also used in recombination with vP866 (NYVAC), generating recombinant virus vP897.
The third HBV gene inserted into vaccinia virus encodes a fusion protein. Synthetic DNA specifying the HBV Sl and S2 regions was cloned onto the 5' end of the gene specifying the HBV core antigen. Synthetic DNA was designed to encode the S1 + S2 regions of the adw subtype (Valenzuela et al., 1979), starting with the met at aa position 12 (equivalent to position 1 of the ayw subtype) (Galibert et al., 1979).
Total translation region of S1 + S2 is 163 codons. To prevent unwanted intramolecular recombination among HBV
genes in a multivalent HBV vaccinia recombinant virus, codon utilization was adjusted to minimize DNA homology of the synthetic Si + S2 region with the native ayw Si + S2 region present in pTHBV and as well as the synthetic S2 region in pGJ15.
The entire gene encoding the core antigen was obtained from pTHBV. The amino acid sequence of the core antigen WO 92/' '2 PCT/L,'S92/01906 encoded by pTHBV agrees with the published ayw sequence (Galibert et al., 1979). The hepatitis fusion gene encoding S12/core was placed under the control of the vaccinia 13L
early/intermediate promoter (Vos et al., 1988; Goebel et al., 1990b positions 64,973 - 65,074). The entire sequence of the 13L promoter/S12/core gene cassette is given in FIG.
13 (SEQ ID NO:87).
Plasmid pMP544I3S12C contains the 13L
promoter/S1+S2/core gene in the HA deletion locus (Guo et al., 1989). The construction of pMP544I3S12C is presented schematically in FIG. 14.
Referring now to FIG. 14, plasmid pMPCA-B contains a 1 kb HhaI fragment from pTHBV inserted into the SmaI site of pUC9. pMP9CA-B contains the entire coding sequences for the HBV core antigen, as well as flanking HBV DNA upstream and downstream from the gene. pMP9CA-B was cut with BalII
(partial) 30 bp upstream from the 3' end of the gene and with EcoRI in the polylinker region at the HBV/pUC junction.
The 3.4 kb vector fragment containing the bulk of the HBV
gene was isolated and ligated with annealed synthetic oligonucleotides MPSYN275/MPSYN276, (SEQ ID NO:88/SEQ ID
NO:89) BalII
MPSYN275 (SEQ ID NO:88) 5'GATCTCAATCTCGGGAATCTCAATGTTAGAT-SmaI
AACTAATTTTTATCCCGGGT 3' MPSYN276 (SEQ ID NO:89) 3' AGTTAGAGCCCTTAGAGTTACA-ATCTATTGATTAAAAATAGGGCCCATTAA 5' generating pMP9CA-C. Restriction sites are indicated, the translational stop codon is underlined and the early vaccinia transcriptional terminator is overlined.
pMP9CA-C contains the entire coding sequence for the HBV core antigen, and was used as the source for the bulk of the gene as indicated above.
The synthetic S1+S2 region was assembled in five double stranded sections A through E as indicated above using synthetic oligonucleotides, MPSYN290 through MPSYN308 (SEQ
ID NO:90)-(SEQ ID NO:99), as set out below.
Oligonucleotides ranged in size from 46mer through 71mer, 92/ 1-5672 PCr/US92, 06 with 4 to 8 bp sticky ends. 5' ends of oligonucleotides which were at internal positions within a section were kinased before annealing of the section. Sequence of synthetic oligonucleotides used to construct sections A
through E are given below. Only the coding strand is shown.
Relevant restriction sites are noted. Initiation codons for S1 (section A), S2 (section C) and core (section E) are underlined.
Section A, MPSYN290-294 (SEQ ID NO:90)-(SEQ ID NO:92) HindIII RsaI (13L) (S1) MPSYN290 (SEQ ID NO:90) 5'AGCTTGTACAATTATTTAGGTTTAATCATGGGAA

CGAACCTATCTGTT 3' MPSYN292 (SEQ ID NO:91) 5'CCCAACCCACTTGGATTTTTTCCTGATCATCAGT
TAGACCCTGCTTTC 3' MPSYN294 (SEQ ID NO:92) 5'GGAGCCAACTCAAACAATCCTGACTGGGATTT
PstI
TAACCCCGTCAAAGACGATTGGCCTGCA 3' Section B, MPSYN296-299 Ps I
MPSYN296 (SEQ ID NO:93) 5'GCCAACCAAGTAGGTGTGGGAGCTTTCGGACC-AAGGCTCACTCCTCCACACGGCGGT 3' MPSYN298 (SEQ ID NO:94) 5'ATATTAGGTTGGTCTCCACAAGCTCAAGG-incII EcoRI
CATATTGACCACAGTGTCAACCCG 3' Section C, MPSYN300-303 indIII HincII
MPSYN300 (SEQ ID NO:95) 5' AGCTTGTCAACAATTCCTCCACCAGCCTCT-ACTAATCGGCAGTCTGGT 3' MPSYN302 (SEQ ID NO:96) 5' AGACAGCCAACTCCCATCTCTCCTCCTCTA-(S2) coRI
AGAGACAGTCACCCACAAGCTATGCAGTGG 3' Section D, MPSYN304-305 HindIII EcoRI
MPSYN304 (SEQ ID NO:97) 5' AGCTTGGGAATTCAACTGCTTTTCACCAG-PstI
ACACTTCAAGACCCTAGAGTCAGGGGTCTATATCTTCCTGCA 3' Section E, MPSYN306-308 PstI
MPSYN306 (SEQ ID NO:98) 5'GGTGGATCTAGTTCTGGAACTGTAAACCCAGCT-CCGAATATTGCCAGTCACATCTC 3' MPSYN308 (SEQ ID NO:99) 5' GTCTATCTCCGCGAGGACTGGAGACCCAGTGAC
( core ) TagI
GAACATGGACAT 3' The vaccinia 13L promoter was synthesized using pMP1, a subclone of HindIII I, as template and synthetic .oligonucleotides MPSYN310 (SEQ ID NO:100), MPSYN311 (SEQ ID
NO:101) as PCR primers. Restriction sites are indicated.
MPSYN310 (SEQ ID NO:100) 5' HindIII Smai CCCCCCAAGCTTCCCGGGCTACATCATGCAGTGGTTAAAC 3' RsaI
MPSYN311 (SEQ ID NO:101) 5' ACTTTGTAATATAATGAT 3' The 13 promoter/HBV S1+S2/core expression cassette was assembled in pUCB and pUC9 in steps, using the intermediate plasmid clones detailed above, resulting in pMP9I3S12core.
Restriction sites are indicated only where relevant.
Plasmid pMP9I3S12core was digested with SmaI and a 1.2 kb fragment containing the entire promoter/gene cassette was isolated. Vaccinia HA deletion plasmid pSD544 was cut with SmaI and ligated with the 1.2 kb fragment, producing plasmid pMP544I3S12C.
pMP544I3S12C was used as donor plasmid for recombination with vaccinia recombinant vP896 described above to generate recombinant vaccinia virus vP919. vP919 contains all three HBV inserts: spsAg, 1psAg and S12/core fusion in the NYVAC background. The sequence of all HBV
insertions in vP919 was confirmed by polymerase chain reaction (PCR) using vP919 as template, followed by dideoxy sequencing of PCR generated material. In addition, pMP544I3S12C was used in recombination with vP804 described above to generate recombinant vaccinia virus vP858 containing only the HBV S12/core fusion. pMP544I3S12C was also used in recombination with recombinant vaccinia virus vP856 to generate recombinant vaccinia virus vP891. vP891 contains two HBV gene insertions, spsAg and S12/core.
Expression of HBV Proteins by vP919. To assay for the various HBV proteins synthesized by the triple HBV
recombinant, metabolically labelled lysates from cells infected with vP919 and appropriate vaccinia recombinants containing single and double HBV gene insertions were subjected to immunoprecipitation and analyzed by SDS-polyacrylamide gel electrophoresis followed by radioautography. Proteins in uninfected cells and cells infected with vP866 (NYVAC), vP856 (spsAg), vP896 (spsAg +
1psAg) or vP919 (spsAg, 1psAg, S12/core) were immunoprecipitated using rabbit anti-S2 antiserum. Proteins in additional uninfected cells and additional cells infected with vP919 (spsAg, lpsAg, S12/core), vP858 (S12/core) or vP866 (NYVAC) were immunoprecipitated using rabbit anti-core antiserum. Anti-S2 serum precipitates a major protein of 33 kDa from vaccinia single recombinant vP856 containing the gene for spsAg. This corresponds to the expected size for the singly glycosylated form of HBV spsAg. A protein 36 kDa, corresponding to the expected size for the doubly glycosylated form of spsAg is precipitated in lesser amount.
Anti-S2 serum precipitates the same proteins from vaccinia double recombinant vP896, containing the genes for spsAg and 1psAg. In addition, two larger proteins of 38 and 41 kDa are precipitated, which correspond well to the expected sizes of 1psAg (39 kDa unglycosylated and 42 kDa glycosylated). All proteins precipitated by anti-S2 serum from vP856 and vP896 are also precipitated from vaccinia HBV
triple recombinant vP919.
The predicted size for the HBV S12/core fusion protein is 38 kDa. Rabbit anti-core antiserum precipitated a protein of the predicted size as well as a variety of smaller proteins from vP858, the vaccinia single recombinant containing the HBV fusion gene S12/core. The most abundant protein precipitated from vP858 by anti-core serum had a size of 27 kDa. This corresponds in size to the translation product which would be predicted if translation of the !N'O 92/1' '2 PCT/US92/01906 fusion protein gene began at the second (S2) ATG. The 29 kDa protein precipitated from vP858 may be the glycosylated form of the 27 kDa protein. A smaller protein of 20 kDa, corresponding in size,to the translation product for core protein alone, was also precipitated from vP858 in lesser amounts. Vaccinia recombinant vP919, containing all three HBV genes (spsAg, 1psAg and S12/core fusion), gave an identical pattern to that observed with vP858 following immunoprecipitation with anti-core antiserum. The 27 kDa and 29 kDa proteins precipitated from vP858 and vP919 by anti-core antiserum were, as expected, also precipitated from vP919 by anti-S2 antiserum.
Antibody Response to vP919. To test for serological response to HBV proteins produced by vP919, the virus was inoculated into rabbits, guinea pigs and mice. Rabbits and guinea pigs were inoculated with 108 pfu recombinant vaccinia virus vP919 in sets of two by intradermal, subcutaneous or intramuscular route. Six weeks after the primary inoculation, rabbits were boosted once by the same route and dose. Seven weeks after the primary inoculation, guinea pigs were boosted once by the same route and dose.
Groups of 12 mice were inoculated with 107 pfu recombinant vaccinia virus vP919 by intradermal, subcutaneous or intramuscular route. Seven weeks after the primary inoculation, mice were boosted once by the same route. Sera were collected at weekly intervals. Weekly bleedings from each group of mice were pooled. All sera were analyzed for antibody to HBV surface antigen using the AUSAB
radioimmunoassay kit (Abbott). All sera were analyzed for antibody to HBV core antigen using the CORAB competitive radioimmunoassay kit (Abbott). Assays were performed using standard techniques. The results of these analyses are presented in Tables 2 (rabbits), 3 (guinea pigs) and 4 (mice).
Summarizing the results presented in Table 2, all six rabbits exhibited an anti-core antibody response following a single inoculation with vP919. In five of the six rabbits, the anti-core antibody response was boosted by a second inoculation of vP919. Four of six rabbits exhibited an anti sAg response following a single inoculation of vP919. These four rabbits, plus one additional rabbit, showed an increase in the anti sAg response following the second inoculation.
Summarizing the results presented in Table 3, one guinea pig exhibited an anti-core response following an initial inoculation with vP919; following the boost at 7 weeks, a total of three guinea pigs showed an anti-core response. One of these animals showed an anti-sAg antibody response in week eight only.
Summarizing the results presented in Table 4, all three groups of mice showed anti-core antibody responses at various times after inoculation with vP919; two of the three groups also showed anti-sAg responses.
AUSRIA Assay. Expression of particulate HBV surface antigen from cells infected with HBV-containing vaccinia recombinants was assayed using the commercially available AUSRIA 11-125 kit (Abbott Laboratories, North Chicago, IL).
Dishes containing 2 x 106 Vero cells were infected in triplicate with recombinant vaccinia virus at 2 pfu/cell.
After 24 h, culture medium was removed, cells were washed with 2 ml PBS and the wash combined with the medium and centrifuged at 1000 rpm for 10 min. The supernatant was designated the medium fraction. The cell fraction was prepared by adding 2 ml PBS to the dish, scraping off the cells and combining with the cell pellet from above. The final volume of both medium and cell fractions were adjusted to 4 ml with PBS. Cell fractions were sonicated for 2 min before assay. Cell fractions and medium fractions were assayed for the presence of HBV surface antigen at a 1:5 dilution using the AUSRIA kit. Samples below the cutoff value of 2.1 x the negative control supplied in the kit were considered negative. output virus of cell and medium fractions from all dishes were titered on Vero cells.
Results are shown in Table 5.
Construction of Vaccinia Recombinants Expressing the HBV 1psAcx under the Control of the EPV 42 kDa Promoter.
Vaccinia recombinant vP919 contains three distinct HBV genes under the control of three different poxvirus promoters which function at early times post infection. To compare WO 92/1 '2 PCT/US92/01906 the relative strength of various poxvirus promoters expressing a foreign gene at early times post infection in the same vaccinia background, a sandwich ELISA assay was developed, utilizing the rabies glycoprotein G gene as the test gene. Using this test system, the vaccinia H6 promoter and the vaccinia 13L promoter were found to be stronger promoters than the cowpox u promoter. In vP919 the H6 promoter directs expression of the HBV spsAg, the 13L
promoter directs expression of the HBV S12/core fusion, and the u promoter directs expression of the HBV 1psAg. The relatively weak u promoter was purposely selected for expression of HBV 1psAg, since it has been shown that coexpression of 1psAg interferes with particle formation and secretion of sAg or spsAg (Ou et al., 1987; Cheng et al., 1986; McLachlan et al., 1987; Chisari et al., 1986).
The AUSRIA radioimmunoassay kit was used to measure the in vitro production of particles containing sAg or spsAg by recombinant vaccinia virus expressing HBV genes.
Preliminary investigation showed that AUSRIA-reactive particle formation and secretion occurred in vP856 (containing spsAg), vP896 (containing spsAg + lpsAg) and vP919 (containing spsAg + 1psAg + S12/core). In vP896 and vP919, the relative levels of secretion of AUSRIA-reactive particles were lower than that observed with vP856.
To determine whether formation and secretion of AUSRIA-reactive particles could be observed in the presence of higher levels of lpsAg expression, the 1psAg gene was placed under the control of the entomopox (EPV) 42 kDa promoter.
By the comparative ELISA test described above, the EPV 42 kDa promoter in a vaccinia recombinant virus directed the expression of a foreign gene at a level equivalent to that observed with the vaccinia H6 promoter or the vaccinia 13L
promoter.
Plasmid pMP55oulps contains the 1psAg gene under the control of the cowpox u promoter in the vaccinia 14L
deletion locus (FIG. 12). The cowpox u promoter present in plasmid pMP550ulps was replaced by the EPV 42 kDa promoter as follows: Complementary oligonucleotides MPSYN371-374 were kinased at the internal 5' ends (MPSYN372; MPSYN373), 92/15672 PCT/L,'S92. -06 annealed, and cloned into pUCB cut with EcoRI/BamHI, forming plasmid pMP371/374. MPSYN371 (SEQ ID NO:102), MPSYN373 (SEQ
ID NO:103) MPSYN372 (SEQ ID NO:104), and MPSYN374 (SEQ ID
NO:105).
EcoRI Bg1II
MPSYN371 5' AATTCAGATCTCAAAATTGAAAATATATAATTACAATA
TAAAATGGGGC 3' MPSYN373 3' GTCTAGAGTTTTAACTTTTATATATTAATGTTATAT

TTTACCCCGTCTT 5' MPSYN372 5' AGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACC
BamHI
ACCAGTTG 3' MPSYN374 3' AGAAAGGTGGTCGTTAGGAGACCCTAAGAAAGGGCTGGTGGTC
AACCTAG 5' contain a 31 bp EPV 42 kDa promoter element, followed by HBV
S1 region (ATG underlined) to the DamHI site. Following DNA
sequence confirmation, the insert was isolated from pMP371/374 by digestion with BamHI/BalII, and used to replace the corresponding u promoter/HBV sequence in pMP550ulps as follows: pMP550ulps was digested with BamHI
(partial)/Bg1II, and the appropriate 5 kb vector fragment isolated and ligated with the BamHI/BalII fragment from pMP371/374. In the resulting plasmid, pMP550E311ps, the HBV
1psAg is under the control of the EPV 42 kDa promoter. The entire sequence of the EPV 42 kDa promoter/1psAg gene cassette is given in FIG. 15.
pMP550E311ps was used as donor plasmid with vaccinia recombinant vP856, containing the spsAg gene, to generate the double HBV recombinant vaccinia virus vP932. vP932 was used as rescuing virus with donor plasmid pMP544I3S12C
containing the S12/core fusion to generate a triple HBV
recombinant vaccinia virus vP975. To generate a vaccinia recombinant containing only the EPV 42 kDa promoter/1psAg, pMP550E311ps was used as donor plasmid with vP866, generating recombinant vaccinia virus vP930.
Secretion of HBV Surface Antigen In Vitro by Recombinant Vaccinia Viruses. Dishes containing Vero cells WO 92/1 2 PCT/L'S92/01906 were infected in triplicate with NYVAC (vP866) or recombinant vaccinia virus expressing HBV spsAg and/or HBV
1psAg. The relative amounts of HBV surface antigen particles associated with infected cells were compared with the amounts secreted into the medium using the AUSRIA 11-125 kit (Table 5). The presence of HBV surface antigen in the medium was not due to lysis of infected cells because more than 99.8 t of viral infectivity remained cell associated.
Volumes of cell pellets and medium were equalized to allow for direct comparison. In cells infected with recombinant vaccinia virus vP856, expressing the spsAg, 42% of the AUSRIA reactive surface antigen was secreted into the medium. Coexpression of 1psAg under the control of the relatively weak u promoter (vP896) did not dramatically change the amount of cell associated AUSRIA reactive material,.but decreased the relative amount of secreted material to 24% of the total. Coexpression of 1psAg under the control of the relatively strong EPV 42 kDa promoter (vP932) lowered the relative amount of secreted material to 6% of the total. As with vP896, coexpression of 1psAg with spsAg in vP932 did not lower the amount of AUSRIA reactive cell associated material. Interestingly, expression of lpsAg alone under the control of the EPV 42 kDa promoter (vP930) resulted in the production of a level of cell associated AUSRIA reactive material significantly above background for the assay, whereas expression of 1psAg under the control of the u promoter (vP897) did not. This is most likely due to the higher levels of spsAg or sAg produced in vP930 infected cells due to initiation at internal (S2 or S) initiation codons.

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~' 92/15672 PCT/US92/ )06 Example 14 - CONSTRUCTION OF NYVAC R$COliBINANTB EXPRESSING
HEPATITIS B VIRUS AND EPSTEIN BARR VIRUS
Since Epstein Barr Virus (EBV) and Hepatitis B Virus (HBV) are endemic over similar geographical areas, including Africa, it would be advantageous to produce a recombinant vaccinia virus expressing immunogens for both pathogens. To this end, vP941, a recombinant vaccinia virus containing three EBV genes and three HBV genes in a NYVAC background was generated.
ImmunoDrecipitation of HBV Proteins. Metabolic labelling and immunoprecipitation of HBV proteins were as described for vP919 in Example 13 with the following modifications. Infections with recombinant vaccinia virus, parental NYVAC virus (vP866) and mock infections were performed on RK-13 cells, rather than Vero cells. Both anti-S2 and anti-core antisera were preadsorbed with vP866 infected RK-13 cells.
Generation of Recombinant Vaccinia Virus vP941.
Plasmid EBV Triple.1, the donor plasmid containing three EBV
genes which was used to generate the vaccinia virus recombinant EBV triplet vP944, was used in recombination with vP919, the vaccinia virus recombinant HBV triplet, as rescuing virus. The resulting virus, vP941, was identified by 32P-labelled EBV DNA. Like vP944, vP941 contained EBV
genes gH under the control of the Entomopox virus 42 kDa promoter, gB under the control of the vaccinia H6 promoter and gp340 under the control of the vaccinia H6 promoter, all inserted in the vaccinia TK deletion locus. Like vP919, vP941 contained the synthetic HBV spsAg under the control of the vaccinia H6 promoter inserted into the ATI deletion locus, the HBV 1psAg under the control of the cowpox u promoter inserted into the 14L deletion locus, and the HBV
S12/core fusion gene under the control of the 13L promoter inserted into the HA deletion locus. The integrity of the genome of recombinant vaccinia virus vP941 was confirmed by restriction analysis of the DNA.

Expression of HBV Proteins by vP941. To assay for the various HBV proteins synthesized by sextuplet HBV/EBV
vaccinia recombinant vP941, metabolically labelled proteins synthesized in RK-13 cells infected with vP941 and appropriate single, double and triple HBV recombinants were subjected to immunoprecipitation. Proteins in uninfected cells and cells infected with vP866 (NYVAC), vP856 (spsAg), vP896 (spsAg + 1psAg), vP919 (spsAg + 1psAg + S12/core), or vP941 were immunoprecipitated using rabbit anti-S2 antiserum. Proteins in additional uninfected cells and additional cells infected with vP941, vP919, vP858 (S12/core), or vP866 were immunoprecipitated using anti-core antiserum.
Anti-S2 serum precipitates two proteins of 33 kDa and 36 kDa from vaccinia single recombinant vP856 containing the gene for spsAg. These correspond to the expected sizes for the singly and doubly glycosylated forms of HBV spsAg.
Anti-S2 serum precipitates the same proteins from vaccinia double recombinant vP896, containing the genes for spsAg and 1psAg. In addition, a protein of 42 kDa, corresponding to the singly glycosylated form of 1psAg is precipitated, as well as larger proteins of 45 kDa and 48 kDa. The 39 kDa protein corresponding to the nonglycosylated form of 1psAg is precipitated in minor amounts compared to the glycosylated forms. All proteins precipitated by anti-S2 serum from vP856 and vP896 are also precipitated from HBV
triple recombinant vP919 and the HBV/EBV sextuplet, vP941.
In the radioautogram, HBV proteins are immunoprecipitated by anti-S2 serum from RK-13 cells infected with vaccinia recombinants. When HBV proteins were immunoprecipitated from Vero cells infected with the same vaccinia recombinants (vP856, vP896 and vP919) the same proteins were observed but in different relative amounts. In general, both spsAg and 1psAg expressed by these recombinant vaccinia virus seems to be more fully glycosylated in RR-13 cells than in Vero cells.

~ 92/15672 PCT/US92i 06 As was seen with Vero cells infected with vP858, the most abundant protein precipitated by anti-core serum from RK-13 cells infected with vP858 has a size of 27 kDa. This corresponds to the size of the translation product which would be predicted if translation of the S12/core fusion gene began at the second (S2) ATG. Unlike the situation observed following vP858 infection of Vero cells, vP858 infection of RK-13 cells followed by immunoprecipitation with anti-core serum does not result in a visible band corresponding in size to the 38 kDa expected for the complete S12/core translation product. All proteins precipitated by anti-core serum from HBV single recombinant vP858 are also precipitated from HBV triple recombinant vP919 and HBV/EBV sextuplet vP941.
EBample 15 - CONSTRUCTION OF ALVAC RECOMBINANTS EXPRESSING
RABIES VIRUS GLYCOPROTEIN G

This example describes the development of a canarypox-rabies recombinant designated as ALVAC-RG (vCP65) and its safety and efficacy.
Cells and Viruses. The parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries. The vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts. A master viral seed was subjected to four successive plaque purifications under agar and one plaque clone was amplified through five additional passages after which the stock virus was used as the parental virus in in vitro recombination tests. The plaque purified canarypox isolate is designated ALVAC.
Construction of a Canarypox Insertion Vector. An 880 bp canarypox PvuII fragment was cloned between the PvuII
sites of pUC9 to form pRW764.5. The sequence of this fragment is shown in FIG. 16 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 WO 92/1 '2 PCT/US92/01906 terminated at position 487. The C5 deletion was made without interruption of open reading frames. Bases from position 167 through position 455 were replaced with the sequence (SEQ ID NO:106) GCTTCCCGGGAATTCTAGCTAGCTAGTTT.
This replacement sequence contains HindIII, SmaI and EcoRI
insertion sites followed by translation stops and a transcription termination signal recognized by vaccinia virus RNA polymerase (Yuen et al., 1987). Deletion of the C5 ORF was performed as described below. Plasmid pRW764.5 was partially cut with RsaI and the linear product was isolated. The RsaI linear fragment was recut with BalII and the pRW764.5 fragment now with a RsaI to BalII deletion from position 156 to position 462 was isolated and used as a vector for the following synthetic oligonucleotides:
RW145 (SEQ ID NO:107):
ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA
RW146 (SEQ ID NO:108):
GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT
Oligonucleotides RW145 and RW146 were annealed and inserted into the pRW 764.5 RsaI and BalII vector described above.
The resulting plasmid is designated pRW831.
Construction of Insertion Vector Containing the Rabies G Gene. Construction of pRW838 is illustrated 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 as pRW737. Oligonucleotides A
through E contain the H6 promoter, starting at NruI, through the HindIII site of rabies G followed by BalII. Sequences of oligonucleotides A through E (SEQ ID NO:109)-(SEQ ID NO.
113) are:
A (SEQ ID NO:109): CTGAAATTATTTCATTATCGCGATATCCGTTAA
GTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT
B (SEQ ID NO:110): CATTACGATACAAACTTAACGGATATCGCGATAA
TGAAATAATTTCAG

92/15672 PCT/l,'S92, -06 C (SEQ ID NO:111): ACCCCTTCTGGTTTTTCCGTTGTGTTTT
GGGAAATTCCCTATTTACACGATCCCAGACA
AGCTTAGATCTCAG
D (SEQ ID NO:112): CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATA
GGGAATTTCCCAAAACA
E (SEQ ID NO:113): CAACGGAAAAACCAGAAGGGGTACAAACAGGAGA
GCCTGAGGAAC
The diagram of annealed oligonucleotides A through E is as follows:
A C
-------------------------- ------------------------------------------ --------!---------- ---------------B E D
Oligonucleotides A through E were kinased, annealed (95 C for 5 minutes, then cooled to room temperature), and inserted between the vuII sites of pUC9. The resulting plasmid, pRW737, was cut with indIII and BglII and used as a vector for the 1.6 kbp indIII-BqlII fragment of ptgl55PRO
(Kieny et al., 1984) generating pRW739. The ptgl55PRO
indIII site is 86 bp downstream of the rabies G translation initiation codon. Bc1II is downstream of the rabies G
translation stop codon in ptgl55PRO. pRW739 was partially cut with NruI, completely cut with B~II, and a 1.7 kbp ruI-= II 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 inserted between the NruI and BAMHI sites of pRW824. The resulting plasmid is designated pRW832.
Insertion into pRW824 added the H6 promoter 5' of NruI. The pRW824 sequence of BamHI followed by maI is: GGATCCCCGGG.
pRW824 is a plasmid that contains a nonpertinent gene linked precisely to the vaccinia virus H6 promoter. Digestion with JruI and BamHI completely excised this nonpertinent gene.
The 1.8 kbp pRW832 SmaI fragment, containing H6 promoted rabies G, was inserted into the SmaI of pRW831, to form plasmid pRW838.

WO 92/1_ 2 PCT/US92/01906 Development of ALVAC-RG. Plasmid pRW838 was transfected into ALVAC infected primary CEF cells by using 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 was then amplified and the resulting ALVAC recombinant was designated ALVAC-RG
(vCP65). The correct insertion of the rabies G gene into the ALVAC genome without subsequent mutation was confirmed by sequence analysis.
Immunofluorescence. During the final stages of assembly of mature rabies virus particles, the glycoprotein component is transported from the golgi apparatus to the plasma membrane where it accumulates with the carboxy terminus extending into the cytoplasm and the bulk of the protein on the external surface of the cell membrane. In order to confirm that the rabies glycoprotein expressed in ALVAC-RG was correctly presented, immunofluorescence was performed on primary CEF cells infected with ALVAC or ALVAC-RG. Immunofluorescence was performed as previously described (Taylor et al., 1990) using a rabies 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 tissue ATCC # CCL171) were inoculated at 10 pfu per cell with parental virus ALVAC
and recombinant virus ALVAC-RG in the presence of radiolabelled 35S-methionine and treated as previously described (Taylor et al., 1990). Immunoprecipitation reactions were performed using a rabies G specific monoclonal antibody. Efficient expression of a rabies 92/15672 PCT/US92, )06 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 cells or cells infected with the parental ALVAC
virus.
Sequential Passaging Experiment. In studies with ALVAC
virus in a range of non-avian species no proliferative infection or overt disease was observed (Taylor et al., 1991b). However, in order to establish that neither the parental nor recombinant virus could be adapted to grow in non-avian cells, a sequential passaging experiment was performed.
The two viruses, ALVAC and ALVAC-RG, were inoculated in sequential blind passages in three cell lines:
(1) Primary chick embryo fibroblast (CEF) cells produced from 11 day old white leghorn embryos;
(2) Vero cells - a continuous line of African Green monkey kidney cells (ATCC #' CCL81); and (3) MRC-5 cells - a diploid cell line derived from human fetal lung tissue (ATCC # CCL171).
The initial inoculation was performed at an m.o.i. of 0.1 pfu per cell using three 60mm dishes of each cell line containing 2 X 106 cells per dish. One dish was inoculated in the presence of 40 g/ml of Cytosine arabinoside (Ara C), an inhibitor of DNA replication. After an absorption period of 1 hour at 37 C, the inoculum was removed and the monolayer washed to remove unabsorbed virus. At this time the medium was replaced with 5m1 of EMEM + 2% NBCS on two dishes (samples tO and t7) and 5m1 of EMEM + 2% NBCS
containing 40 g/ml Ara C on the third (sample t7A). Sample t0 was frozen at -70 C to provide an indication of the residual input virus. Samples t7 and t7A were incubated at 37 C for 7 days, after which time the contents were harvested and the cells disrupted by indirect sonication.
One ml of sample t7 of each cell line was inoculated undiluted onto three dishes of the same cell line (to provide samples to, t7 and t7A) and onto one dish of primary CEF cells. Samples tO, t7 and t7A were treated as for passage one. The additional inoculation on CEF cells was included to provide an amplification step for more sensitive detection of virus which might be present in the non-avian cells.
This procedure was repeated for 10 (CEF and MRC-5) or 8 (Vero) sequential blind passages. Samples were then frozen and thawed three times and assayed by titration on primary CEF monolayers.
Virus yield in each sample was then determined by plaque titration on CEF monolayers under agarose.
Summarized results of the experiment are shown in Tables 6 and 7.
The results indicate that both the parental ALVAC and the recombinant ALVAC-RG are capable of sustained replication on CEF monolayers with no loss of titer. In Vero cells, levels of virus fell below the level of detection after 2 passages for ALVAC and 1 passage for ALVAC-RG. In MRC-5 cells, a similar result was evident, and no virus was detected after 1 passage. Although the results for only four passages are shown in Tables 6 and 7 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 virus were present in the t7 sample in MRC-5 and Vero cells. However this level of virus was equivalent to that seen 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 virus seen at 7 days in non-avian cells represented residual virus and not newly replicated virus.
In order to make the assay 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 F 92/15672 PCT/US92/ )06 (CPE) or at 7 days if no CPE was evident. The results of this experiment are shown in Table 8. Even after amplification through a permissive cell line, virus was only detected in MRC-5 and Vero cells for two additional passages. These results indicated that under the conditions used, there was no adaptation of either'virus to growth in Vero or MRC-5 cells.
Inoculation of Macaques. Four HIV seropositive macaques were initially inoculated with ALVAC-RG as described in Table 9. After 100 days these animals were re-.inoculated to determine a booster effect, and an additional seven animals were inoculated with a range of doses. Blood was drawn at appropriate intervals and sera analyzed, after heat inactivation at 56 C for 30 minutes, for the presence of anti-rabies antibody using the Rapid Fluorescent Focus Inhibition Assay (Smith et al., 1973).
Inoculation of Chimpanzees. Two adult male chimpanzees (50 to 65 kg weight range) were inoculated intramuscularly or subcutaneously with 1 X 107 pfu of vCP65. Animals were monitored for reactions and bled at regular intervals for analysis for the presence of anti-rabies antibody with the RFFI test (Smith et al., 1973). Animals were re-inoculated with an equivalent dose 13 weeks after the initial inoculation.
Inoculation of Mice. Groups of mice were inoculated with 50 to 100 l of a range of dilutions of different batches of vCP65. Mice were inoculated in the footpad. On day 14, mice were challenged by intracranial inoculation of from 15 to 43 mouse LD50 of the virulent CVS strain of rabies virus. Survival of mice was monitored and a protective dose 50% (PD50) calculated at 28 days post-inoculation.
inoculation of Dogs and Cats. Ten beagle dogs, 5 months old, and 10 cats, 4 months old, were"inoculated subcutaneously with either 6.7 or 7.7 loglc) TCID50 of ALVAC-RG. Four dogs and four cats were not inoculated. Animals V1'O 92/1 '2 PC.T/US92/01906 were bled at 14 and 28 days post-inoculation and anti-rabies antibody assessed in an RFFI test. The animals receiving 6.7 loglo TCID50 of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 loglo mouse LD50 (dogs) or 4.3 loglo mouse LD50 (cats) of the NYGS rabies virus challenge strain.
Inoculation of Squirrel Monkeys. Three groups of four squirrel monkeys (Saimiri sciureus) were inoculated with one of three viruses (a) ALVAC, the parental canarypox virus, (b) ALVAC-RG, the recombinant expressing the rabies G
glycoprotein or (c) vCP37, a canarypox recombinant expressing the envelope glycoprotein of feline leukemia virus. Inoculations were performed under ketamine anaesthesia. Each animal received at the same time: (1) 20 l instilled on the surface of the right eye without scarification; (2) 100 l as several droplets in the mouth;
(3) 100 l in each of two intradermal injection sites in the shaven skin of the external face of the right arm; and (4) 100 l in the anterior muscle of the right thigh.
Four monkeys were inoculated with each virus, two with a total of 5.0 loglo pfu and two with a total of 7.0 loglo pfu. Animals were bled at regular intervals and sera analyzed for the presence of antirabies antibody using an RFFI test (Smith et al., 1973). Animals were monitored daily for reactions to vaccination. Six months after the initial inoculation the four monkeys receiving ALVAC-RG, two monkeys initially receiving vCP37, and two monkeys initially receiving ALVAC, as well as one naive monkey were inoculated with 6.5 loglo pfu of ALVAC-RG subcutaneously. 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 expression of a foreign gene could be obtained in non-avian cells in which the virus does not productively replicate, five cell types, one avian and four non-avian, were analyzed for virus yield, 92/15672 PCT/US92, )06 expression of the foreign rabies G gene and viral specific DNA accumulation. The cells inoculated were:
(a) Vero, African Green monkey kidney cells, ATCC #
CCL81;
(b) MRC-5, human embryonic lung, ATCC # CCL 171;
(c) WISH human amnion, ATCC # CCL 25;
(d) Detroit-532, human foreskin, Downs's syndrome, ATCC # CCL 54; and (e) Primary CEF cells.
Chicken embryo fibroblast cells produced from 11 day old white leghorn embryos were included as a positive control. All inoculations were performed on preformed monolayers of 2 X 106 cells as discussed below.
A. 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 was incubated in the presence of 40 g/ml of cytosine arabinoside (Ara C). After an adsorption period of 60 minutes at 37 C, the inoculum was removed and the monolayer washed twice to remove unadsorbed virus.
Medium (with or without Ara C) was then replaced.
Cells from one dish (without Ara C) were harvested as a time zero sample. The remaining dishes were incubated at 37 C for 72 hours, at which time the cells were harvested and used to analyze DNA accumulation. Each sample of 2 X 106 cells was resuspended in 0.5 ml phosphate buffered saline (PBS) containing 40 mM EDTA
and incubated for 5 minutes at 37 C. An equal volume of 1.5% agarose prewarmed at 42 C and containing 120 mM
EDTA was added to the cell suspension and gently mixed.
The suspension was transferred to an agarose plug mold and allowed to harden for at least 15 min. The agarose plugs were then removed and incubated for 12-16 hours at 50 C in a volume of lysis buffer (1% sarkosyl, 100 g/ml proteinase K, 10 mM Tris HC1 pH 7.5, 200 mM EDTA) WO 92/1 '2 PCT/US92/01906 that completely covers the plug. The lysis buffer was then replaced with 5.0 ml sterile 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 changes of TBE
buffer. The viral DNA within the plug was fractionated from cellular RNA and DNA using'a pulse field electrophoresis system. Electrophoresis was performed for 20 hours at 180 V with a ramp of 50-90 sec at 15 C
in 0.5 X TBE. The DNA was run with lambda DNA
molecular weight standards. After electrophoresis 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. Estimation of virus yield.
Dishes were inoculated exactly as described above, with the exception that input multiplicity was 0.1 pfu/cell.
At 72 hours post infection, cells were lysed by three successive cycles of freezing and thawing. Virus yield was assessed by plaque titration on CEF monolayers.
C. Analysis of expression of Rabies G gene.
Dishes were inoculated with recombinant or parental virus at a multiplicity of 10 pfu/cell, allowing an additional dish as an uninfected virus control. After a one hour absorption period, the medium was removed and replaced with methionine free medium. After a 30 minute period, this medium was replaced with methionine-free medium containing 25 uCi/ml of 35S-Methionine. Infected cells were labelled overnight (approximately 16 hours), then lysed by the addition of buffer A lysis buffer. Immunoprecipitation was performed as previously described (Taylor et al., 1990) using a rabies G specific monoclonal antibody.
Results: Estimation of Viral Yield. The results of titration for yield at 72 hours after inoculation at 0.1 pfu per cell are shown in Table 10. The results indicate that )92/ 15672 PCT/US92. )06 while a productive infection can be attained in the avian cells, no increase in virus yield can be detected by this method in the four non-avian cell systems.
Analysis 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 hours post-infection and ALVAC-RG infected CEF cells at 72 hours post-infection in the presence of 40 g/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 cells at 72 hours post-infection exhibited a strong band in the region of approximately 350 kbp representing ALVAC-specific viral DNA accumulation. No such band is detectable when the culture is incubated in the presence of the DNA synthesis inhibitor, cytosine arabinoside. Equivalent samples produced in Vero cells showed a very faint band at approximately 350 kbp in the ALVAC-RG infected Vero cells at time zero. This level represented residual virus. The intensity of the band was amplified at 72 hours post-infection indicating that some level of viral specific DNA
replication had occurred in Vero cells which had not resulted in an increase in viral progeny. Equivalent samples produced in MRC-5 cells indicated that no viral specific DNA accumulation was detected under these conditions in this cell line. This experiment was then extended to include additional human cell lines, specifically WISH and Detroit-532 cells. ALVAC infected CEF
cells served as a positive control. No viral specific DNA
accumulation was detected in either WISH or Detroit cells inoculated with ALVAC-RG. It should be noted that the WO 92/1~,u72 PCT/US92/01906 limits of detection of this method have not been fully ascertained 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 measured by 3H-thymidine incorporation support the results obtained with Vero and MRC-5 cells.
Analysis of Rabies Gene Enression. To determine if any viral gene expression, particularly that of the inserted foreign gene, was occurring in the human cell lines even in the absence of viral DNA replication, immunoprecipitation experiments were performed on 35S-methionine labelled lysates of avian and non-avian cells infected with ALVAC and ALVAC-RG. The results of immunoprecipitation using a rabies G specific monoclonal antibody illustrated specific immunoprecipitation of a 67 kDa glycoprotein in CEF, Vero and MtC-5, WISH and Detroit cells infected with ALVAC-RG.
No such specific rabies gene products were detected in any of the uninfected and parentally infected cell lysates.
The results of this experiment indicated that in the human cell lines analyzed, although the ALVAC-RG recombinant was able to initiate an infection and express a foreign gene product under the transcriptional control of the H6 early/late vaccinia virus promoter, the replication did not proceed through DNA replication, nor was there any detectable viral progeny produced. In the Vero cells, although some level of ALVAC-RG specific DNA accumulation was observed, no viral progeny was detected by these methods. These results would indicate that in the human cell lines analyzed the block to viral replication occurs prior to the onset of DNA replication, while in Vero cells, the block occurs following the onset of viral DNA
replication.
In order to determine whether the rabies glycoprotein expressed in ALVAC-RG was immunogenic, a number of animal species were tested by inoculation of the recombinant. The efficacy of current rabies vaccines is evaluated in a mouse ) 92/15672 PCT/US92. .906 model system. A similar test was therefore performed using ALVAC-RG. Nine different preparations of virus (including one vaccine batch (J) produced after 10 serial tissue culture passages of the seed virus) with infectious titers ranging from 6.7 to 8.4 loglo TCID50 per ml were serially diluted and 50 to 100 l of dilutions inoculated into the footpad of four to six week old mice. Mice were challenged 14 days later by the intracranial route with 300 l of the CVS strain of rabies virus containing from 15 to 43 mouse LD50 as determined by lethality titration in a control group of mice. Potency, expressed as the PD50 (Protective dose 50%), was calculated at 14 days post-challenge. The results of the experiment are shown in Table 11. The results indicated that ALVAC-RG was consistently able to protect mice against rabies virus challenge with a PD50 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 l of virus containing 6.0 logio TCID50 of ALVAC-RG or with an equivalent volume of an uninfected cell suspension. Mice were sacrificed on days 1, 3 and 6 post-inoculation and their brains removed, fixed and sectioned. Histopathological examination showed no evidence for neurovirulence of ALVAC-RG in mice.
In order to evaluate the safety and efficacy of ALVAC-RG for dogs and cats, a group of 14, 5 month old beagles and 14, 4 month old cats were analyzed. Four animals in each species were not vaccinated. Five animals received 6.7 logla TCID50 subcutaneously and five animals received 7.7 logio TCID50 by the same route. Animals were bled for analysis for anti-rabies antibody. Animals receiving no inoculation or 6.7 loglo TCID50 of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 log10 mouse LD50 (dogs, in the temporal muscle) or 4.3 loglo mouse LD50 (cats, in the neck) of the NYGS rabies virus challenge strain. The results of the experiment are shown in Table 12.

WO 92/1 '2 PCT/US92/01906 No adverse reactions to inoculation were seen in either cats or dogs with either dose of inoculum virus. Four of 5 dogs immunized with 6.7 loglo TCID50 had antibody titers on day 14 post-vaccination and all dogs had titers at 29 days.
All dogs were protected from a challenge which killed three out of four controls. In cats, three of five cats receiving 6.7 loglo TCID50 had specific antibody titers on day 14 and all cats were positive on day 29 although the mean antibody titer was low at 2.9 IU. Three of five cats survived a challenge which killed all controls. All cats immunized with 7.7 loglo TCID50 had antibody titers on day 14 and at day 29 the Geometric Mean Titer was calculated as 8.1 International Units.
The immune response of squirrel monkeys (Saimiri sciureus) to inoculation with ALVAC, ALVAC-RG and an unrelated canarypox virus recombinant was examined. Groups of monkeys were inoculated as described above and sera analyzed for the presence of rabies specific 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 virus were isolated from skin 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-muscular 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 loglo TCID50 of ALVAC-RG. Sera were analyzed for the presence of anti-rabies antibody. The results are shown in Table 13.
Four of the five monkeys naive to rabies developed a serological response by seven days post-inoculation with ALVAC-RG. All five monkeys had detectable antibody by 11 )92/15672 PCT/US92. 906 days post-inoculation. Of the four monkeys with previous exposure to the rabies glycoprotein, all showed a significant increase in serum neutralization titer between days 3 and 7 post-vaccination. The results indicate that vaccination of squirrel monkeys with ALVAC-RG does not produce adverse side-effects and a primary neutralizing antibody response can be induced. An amnanestic response is also induced on re-vaccination. Prior exposure to ALVAC or to a canarypox recombinant expressing an unrelated foreign gene does not interfere with induction of an anti-rabies immune response upon re-vaccination.
The immunological response of HIV-2 seropositive macaques to inoculation with ALVAC-RG was assessed. Animals were inoculated as described above and the presence of anti-rabies serum neutralizing antibody assessed in an RFFI test.
The results, shown in Table 14, indicated that HIV-2 positive animals inoculated by the subcutaneous route developed anti-rabies antibody by 11 days after one inoculation. An anamnestic response was detected after a booster inoculation given approximately three months after the first inoculation. No response was detected in animals receiving the recombinant by the oral route. In addition, a series of six animals were inoculated with decreasing doses 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 significant difference in antibody titer.
Two chimpanzees with prior exposure to HIV were inoculated with 7.0 loglo pfu of ALVAC-RG by the subcutaneous or intra-muscular route. At 3 months post-inoculations both animals were re-vaccinated in an identical fashion. The results are shown in Table 15.
No adverse reactivity to inoculation was noted by either intramuscular or subcutaneous routes. Both chimpanzees responded to primary inoculation by 14 days and WO 92/1 :,v 12 PCT/US92/01906 a strongly rising response was detected following re-vaccination.

92/15672 PC"T/US92, 406 Table 6. Sequential Passage of ALVAC in Avian and non-Avian Cells.

CEF Vero MRC-5 Pass 1 Sample toa 2.4 3.0 2.6 t7b 7.0 1.4 0.4 t7Ac 1.2 1.2 0.4 Pass 2 Sample to 5.0 0.4 N.D.d t7 7.3 0.4 N.D.
t7A 3.9 N.D. N.D.
Pass 3 Sample to 5.4 0.4 N.D.
t7 7.4 N.D. N.D.
t7A 3.8 N.D. N.D.
Pass 4 Sample to 5.2 N.D. N.D.
t7 7.1 N.D. N.D.
t7A 3.9 N.D. N.D.
-----------------------------------------------------------a: This sample was harvested at zero time and represents the residual input virus. The titer is expressed as loglopfu per ml.

b: This sample was harvested at 7 days post-infection.

c: This sample was inoculated in the presence of 40 g/ml of Cytosine arabinoside and harvested at 7 days post infection.

d: Not detectable WO 92/1. 2 PCT/US92/01906 Table 7. Sequential Passage of ALVAC-RG in Avian and non-Avian Cells CEF Vero MRC-5 Pass 1 Sample t0a 3.0 2.9 2.9 t7b 7.1 1.0 1.4 t7Ac 1.8 1.4 1.2 Pass 2 Sample tO 5.1 0.4 0.4 t7 7.1 N.D.d N.D.
t7A 3.8 N.D. N.D.
Pass 3 Sample tO 5.1 0.4 N.D.
t7 7.2 N.D. N.D.
t7A 3.6 N.D. N.D.
Pass 4 Sample tO 5.1 N.D. N.D.
t7 7.0 N.D. N.D.
t7A 4.0 N.D. N.D
------------------------------------------------------------a: This sample was harvested at zero time and represents the residual input virus. The titer is expressed as loglopfu per ml.

b: This sample was harvested at 7 days post-infection.

c: This sample was inoculated in the presence of 40 g/ml of Cytosine arabinoside and harvested at 7 days post-infection.

d: Not detectable.

92/15672 PC.'T/US92, )O6 Table 8. Amplification of residual virus by passage in CEF
cells CEF Vero MRC-5 a) ALVAC

Pass 2a 7.Ob 6.0 5.2 3 7.5 4.1 4.9 4 7.5 N.D.c N.D.

7.1 N.D. N.D.
b) ALVAC-RG

Pass 2a 7.2 5.5 5.5 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 loglo pfu per ml c: Not Detectable Table 9. Schedule of inoculation of rhesus macaques with ALVAC-RG (vCP65) Animal Inoculation 176L Primary: 1 X 108 pfu of vCP65 orally in TANG
Secondary: 1 X 107 pfu of vCP65 plus 1 X 107 pfu of vCP82a by SC route 185 L Primary: 1 X 108 pfu of vCP65 orally in Tang Secondary: 1 X 107 pfu of vCP65 plus 1 X 107 pfu of vCP82 by SC route 177 L Primary: 5 X 107 pfu SC of vCP65 by SC route Secondary: 1 X 107 pfu of vCP65 plus 1 X 107 pfu of vCP82 by SC route 186L Primary: 5 X 107 pfu of vCP65 by SC route Secondary: 1 X 107 pfu of vCP65 plus 1 X 107 pfu of vCP82 by SC route 178L Primary: 1 X 107 pfu of vCP65 by SC route 182L Primary: 1 X 107 pfu of vCP65 by IM route 179L Primary: 1 X 106 pfu of vCP65 by SC route 183L Primary: 1 X 106 pfu of vCP65 by IM route 180L Primary: 1 X 106 pfu of vCP65 by SC route 184L Primary: i X 105 pfu of vCP65 by IM route 187L Primary 1 X 107 pfu of vCP65 orally a: vCP82 is a canarypox virus recombinant expressing the measles virus fusion and hemagglutinin genes.

V 92/15672 PCT/US92/k J6 Table 10. Analysis of yield in avian and non-avian cells inoculated with ALVAC-RG
Sample Time Cell Type t0 t72 t72Ab Expt 1 CEF 3.3a 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 as log10 pfu per ml b: Culture incubated in the presence of 40 g/ml of Cytosine arabinoside WO 92/1 _ 2 PCT/US92/01906 Table 11. Potency of ALVAC-RG as tested in mice Test Challenge Dosea PD50b Initial seed 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 LD50 b: Expressed as loglo TCID50 92/15672 PCT/US92it .406 Table 12. Efficacy of ALVAC-RG in dogs and cats Dogs Cats Dose Antibodya Survivalb Antibody Survival 6.7 11.9 5/5 2.9 3/5 7.7 10.1 N.T. 8.1 N.T.
a: Antibody at day 29 post inoculation expressed as the geometric mean titer in International Units.
b: Expressed as a ratio of survivors over animals challenged CA 02558659 1992-03-09 ~

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Weeks post- Animal 431 Animal 457 Inoculation I.M. S.C.
0 <8a <8 1 <8 <8 12b/0 16 8 a: Titer expressed as reciprocal of last dilution showing inhibition of fluorescence in an RFFI test b: Day of re-inoculation )92/15672 PCT/US921 - 906 Example 16 - CONSTRUCTION OF NYVAC RECOMBINANTS EXPRESSING
FLAVIVIRIIS PROTEINS

This example describes the construction of NYVAC donor plasmids containing genes from Japanese encephalitis virus (JEV), yellow fever virus (YF) and Dengue type 1, the isolation of the corresponding NYVAC Flavivirus recombinants and the ability of vaccinia recombinants expressing portions of the genomes of JEV or YF to protect mice against lethal challenge with the homologous virus.
Cell Lines and Virus Strains. A thymidine kinase mutant of the Copenhagen strain of vaccinia virus vP410 (Guo et al., 1989) was used to generate recombinants vP825, vP829, vP857 and vP864 (see below). The generation of vP555 has previously been described (Mason et al., 1991).
Biosynthetic studies were performed using HeLa cells grown at 37 C in Eagle's minimal essential medium supplemented with FBS and antibiotics. The JEV virus used in all in vitro experiments was a clarified culture fluid prepared from C6/36 cells infected with a passage 55 suckling mouse brain suspension of the Nakayama strain of JEV (Mason, 1989). Animal challenge experiments were performed using the highly pathogenic P3 strain of JEV (see below).
Cloning of JEV Genes Into a Vaccinia Virus Donor Plasmid. The JEV cDNAs used to construct the JEV-vaccinia recombinant viruses were derived from the Nakayama strain of JEV (McAda et al., 1987).
Plasmid pDr20 containing JEV cDNA (nucleotides -28 to 1000) in the SmaI and EcoRI sites of pUC18, was digested with BamHI and coRI and the JEV cDNA insert cloned into pIBI25 (International Biotechnologies, Inc., New Haven, CT) generating plasmid JEV18. JEV18 was digested with ApaI
within the JE sequence (nucleotide 23) and h~oI within pIBI25 and ligated to annealed oligonucleotides J90 (SEQ ID
NO:114) and J91 (SEQ ID NO:115) (containing an ioI sticky end, naI site, and JE nucleotides 1 to 23) generating plasmid JEV19. JEV19 was digested with XhoI within pIBI25 and AccI within JE sequences (nucleotide 602) and the resulting 613 bp fragment was cloned into the XhoI and AccI
fragment of JEV2 (Mason et al., 1991) containing the plasmid WO 92/1. 2 PCT/US92/01906 origin and JEV cDNA encoding the carboxy-terminal 40% prM
and amino-terminal two thirds of E(nucleotides 602 to 2124), generating plasmid JEV20 containing JE sequences from the ATG of C through the SacI site (nucleotide 2124) found in the last third of E.
The SmaI-SacI fragment from JEVB (a plasmid analogous to JEVL Mason et al., 1991) in which TTTTTGT nucleotides 1304 to 1310 were changed to TCTTTGT), containing JE
sequences from the last third of E through the first two amino acids of NS2B (nucleotides 2124 to 4126), the plasmid origin and vaccinia sequences, was ligated to the purified SmaI-SacI insert from JEV20 yielding JEV22-1. The 6 bp corresponding to the unique SmaI site used to construct JEV22-1 were removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating JEV24 in which the H6 promoter immediately preceded the ATG start codon.
Plasmid JEV7 (Mason et al., 1991) was digested with SphI within JE sequences (nucleotide 2180) and HindIII
within IBI24. Ligation to annealed oligonucleotides J94 and J95 [containing a SphI sticky end, translation stop, a vaccinia early transcription termination signal (TTTTTAT;
Yuen et al., 1987) a translation stop, an EaeI site and a HindIII sticky end] generated plasmid JEV25 which contains JE cDNA extending from the SacI site (nucleotide 2124) in the last third of E through the carboxy-terminus of E. The SacI-EaaI fragment from JEV25 was ligated to the SacI-EacrI
fragment of JEV8 (containing JE cDNA encoding 15 aa C, prM
and amino-terminal two thirds of E nucleotides 337 to 2124, the plasmid origin and vaccinia sequences) yielding plasmid JEV26. A unique SmaI site preceding the ATG start codon was removed as described above, creating JEV27 in which the H6 promoter immediately preceded the ATG start codon.
Oligonucleotides J96, J97, J98 and J99 (containing JE
nucleotides 2243 to 2380 with an SphI sticky end) were kinased, annealed and ligated to SmaI-SDhI digested and alkaline phosphatase treated pIBI25 generating plasmid JEV28. JEV28 was digested with HpaI within the JE sequence (nucleotide 2301) and with HindIII within the pIBI25 1 92/15672 PCT/US92, .906 sequence and alkaline phosphatase treated. Ligation to the HpaI-HindIII fragment from JEV1 or HDaI-HindIII fragment from JEV7 (Mason et al., 1991) yielded JEV29 (containing a Smal site followed by JE cDNA encoding 30 aa E, NS1, NS2A
nucleotides 2293 to 4126) and JEV30 (containing a Smal site followed by JE cDNA encoding 30 aa E, NS1, NS2A, NS2B
nucleotides 2293 to 4512).
The SmaI-EagI fragment from JEV29 was ligated to SmaI-EaaI digested pTP15 (Mason et al., 1991) yielding JEV31.
The 6 bp corresponding to the unique maI site used to produce JEV31 were removed as described above creating JEV33 in which the H6 promoter immediately preceded the ATG start codon.
The SmaI-EaaI fragment from JEV30 was ligated to SmaI-EaaI digested pTP15 yielding JEV32. The 6 bp corresponding to the unique SmaI site used to produce JEV32 were removed as described above creating 7EV34 in which the H6 promoter immediately preceded the ATG start codon. Oligonucleotides J90 (SEQ ID NO:114), J91 (SF.,~ ID NO:115), J94 (SEQ ID
NO:116), J95 (SEQ ID NO:117), J96 and J97 (SEQ ID NO:118), and J99 and J98 (SEQ ID NO:119) are as follows:
J90 5'-TCGAG CCCGGG atg ACTAAAAAACCAGGA GGGCC-3' J91 3'- C GGGCCC TAC TGATTTTTTGGTCCT C -5' XhoI SmaI ApaI

J94 5'- C T tga tttttat tga CGGCCG A -3' J95 3'-GTACG A ACT AAAAATA ACT GCCGGC TTCGA-5' SDhI Eaql indIII
J96+J97 5'-GGG atg GGCGTTAACGCACGAGACCGATCAATTGCTTTGGCC
J99+J98 3'-CCC TAC CCGCAATTGCGTGCTCTGGCTAGTTAACGAAACCGG
TTCTTAGCCACAGGAGGTGTGCTCGTGTTCTTAGCGACCAATGT GCATG-3' AAGAATCGGTGTCCTCCACACGAGCACAAGAATCGCTGGTTACA C -5' SDhI
Construction of Vaccinia Virus JEV Recombinants.
Plasmids JEV24, JEV27, JEV33 and JEV34 were transfected into vP410 infected cells to generate the vaccinia recombinants vP825, vP829, vP857 and vP864 respectively (FIG. 18).
In Vitro Virus Infection and Radiolabeling. HeLa cell monolayers were prepared in 35 mm diameter dishes and infected with vaccinia viruses (m.o.i. of 2 pfu per cell) or JEV (m.o.i. of 5 pfu per cell) before radiolabeling. Cells WO 92/1. 2 PCT/US92/01906 were pulse labeled with medium containing 35S-Met and chased for 6 hr in the presence of excess unlabeled Met exactly as described by Mason et al. (1991).
Radioimmunoprecipitations, Polyacrylamide Gel Electrophoresis, and Endoglycosidase Treatment.
Radiolabeled cell lysates and culture fluids were harvested and the viral proteins were immunoprecipitated, digested with endoglycosidases, and separated in SDS-containing polyacrylamide gels (SDS-PAGE) exactly as described by Mason (1989).
Animal Protection Experiments. Mouse protection experiments were performed exactly as described by Mason et al. (1991). Briefly, groups of 3-week-old mice were immunized by intraperitoneal (ip) injection with 107 pfu of vaccinia virus recombinants, and 3 weeks later sera were collected from selected mice. Mice were then either re-inoculated with the recombinant virus or challenged with 1.3 x 103 LD50 by intraperitoneal injection with a suspension of suckling mouse brain infected with the P3 strain of JEV.
Three weeks later, the boosted animals were rebled and challenged with 4.9 x 105 LD50 of the P3 strain of JEV.
Following challenge, mice were observed at daily intervals for three weeks and lethal-dose titrations were performed in each challenge experiment using litter-mates of the experimental animals. In addition, sera were collected from all surviving animals 4 weeks after challenge.
Evaluation of Immune Response to the Recombinant Vaccinia Viruses. Sera were tested for their ability to precipitate JEV proteins from detergent-treated cell lysates or culture fluids obtained from 35S-Met-labeled JEV-infected cells exactly as described by Mason et al. (1991).
Hemagglutination inhibition (HAI) and neutralization (NEUT) tests were performed as described by Mason et al. (1991) except carboxymethylcellulose was used in the overlay medium for the NEUT test.
Structure of Recombinant Vaccinia Viruses. Four different vaccinia recombinants (in the HA locus) were constructed that expressed portions of the JEV coding region extending from C through NS2B. The JEV cDNA sequences 1 92/ l5672 PCT/L!S92,,, , 906 contained in these recombinant viruses are shown in FIG. 18.
In all four recombinant viruses the sense strand of the JEV
cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from=
naturally occuring JEV Met codons located at the 5' ends of the viral cDNA sequences.
Recombinant vP825 encoded the capsid protein, structural protein precursor prM, the structural glycoprotein E, the nonstructural glycoprotein NS1, and the nonstructural protein NS2A (McAda et al., 1987).
Recombinant vP829 encoded the putative 15 aa signal sequence preceding the amino-terminus of prM, as well as prM, and E
(McAda et al., 1987). Recombinant vP857 contained a cDNA
encoding the 30 aa hydrophobic carboxy-terminus of E, followed by NS1 and NS2A. Recombinant vP864 contained a cDNA encoding the same proteins as vP857 with the addition of NS2B. In recombinants vP825 and vP829 a potential vaccinia virus early transcription termination signal in E
(TTTTTGT; nucleotides 1304-1310) was modified to TCTTTGT
without altering the aa sequence. This change was made in an attempt to increase the level of expression of E since this sequence has been shown to increase transcription termination in in vitro transcription assays (Yuen et al., 1987).
E and DrM Are Correctly Processed When Expressed By Recombinant Vaccinia Viruses. Pulse-chase experiments demonstrate that proteins identical in size to E were synthesized in cells infected with all recombinant vaccinia viruses containing the E gene (Table 16). In the case of cells infected with JEV, vP555 and vP829, an E protein that migrated slower in SDS-PAGE was also detected in the culture fluid harvested from the infected cells (Table 16). This extracellular form of E produced by JEV- and vP555-infected cells contained mature N-linked glycans (Mason, 1989; Mason et al., 1991), as confirmed for the extracellular forms of E
produced by vP829-infected cells. Interestingly, vP825, which contained the C coding region in addition to prM and E
specified the synthesis of E in a form that is not released into the extracellular fluid (Table 16).

V-'O 92/1. 2 PCT/US92/01906 Immunoprecipitations prepared from radiolabeled recombinant vaccinia-infected cells using a MAb specific for M (and prM) revealed that prM was synthesized in cells infected with vP555, vP825, and vP829, and M was detected in the culture fluid of cells infected with vP555 or vP829 (Table 16).
The extracellular fluid harvested from cells infected with vP555 and vP829 contained an HA activity that was not detected in the culture fluid of cells infected with vP410, vP825, vP857 or vP864. This HA appeared similar to the HA
produced in JEV infected cells based on its inhibition by anti-JEV antibodies and its pH optimum (Mason et al., 1991).
Sucrose density gradients were prepared with culture fluids from cells infected with vaccinia virus JEV recombinants vP829, vP825, vP857 and vP864. Analysis of the gradients identified a peak of HA activity in the vP829 infected sample that co-migrated with the peak of slowly-sedimenting hemagglutinin (SHA) found in the JEV culture fluids (data not shown). This result indicated that vP829 infected cells produce extracellular particles similar to the empty viral envelopes containing E and M observed in the culture fluids harvested from vP555 infected cells (Table 16 and Mason et al., 1991).
NS1 Is Correctly Processed and Secreted When Expressed Bv Recombinant Vaccinia Virus. The results of pulse-chase experiments demonstrated that proteins identical in size to authentic NS1 and NS1' were synthesized in cells infected with vP555, vP825, vP857 and vP864. NS1 produced by vP555-infected cells was released into the culture fluid of infected cells in a higher molecular weight form. NS1 was also released into the culture fluid of cells infected with vP857 and vP864, whereas NS1 was not released from cells infected with vP825 (Table 16). Comparison of the synthesis of NS1 from recombinant vaccinia viruses containing either the NS2A (vP857) or both the NS2A and NS2B coding regions showed that the presence or absence of the NS2B coding region had no affect on NS1 expression, consistent with previous data showing that only the NS2A gene is needed for the authentic processing of NS1 (Falgout et al., 1989; Mason et al., 1991).

) 92/15672 PCT/US92. .906 Recombinant Vaccinia Viruses Induced Immune Responses To JEV Antigens. Pre-challenge sera pooled from selected animals in each group were tested for their ability to immunoprecipitate radiolabeled E and NS1. The results of these studies (Table 16) demonstrated that: (1) the magnitude of immune response induced to E was vP829>vP555>vP825, (2) all viruses encoding NS1 and NS2A
induced antibodies to NS1, and (3) all immune responses were increased by a second inoculation with the recombinant viruses. Analysis of the neutralization and HAI data for the sera collected from these animals (Table 17) confirmed the results of the immunoprecipitation analyses, showing that the immune response to E as demonstrated by RIP
correlated well with these other serological tests (Table 11).
Vaccination With the Recombinant Viruses Provided Protection From Lethal JEV Infection. All of the recombinant-vaccinia viruses were able to provide mice with some protection from lethal infection by the peripherally pathogenic P3 strain of JEV (Huang, 1982) (Table 17). These studies confirmed the protective potential of vP555 (Mason et al., 1991) and demonstrated similar protection in animals inoculated with vP825 and vP829. Recombinant viruses vP857 and vP864 which induced strong immune responses to NS1 showed much lower levels of protection, but mice inoculated with these recombinants were still significantly protected when compared to mice inoculated with the control virus, vP410 (Table 17).
Post-Challencte Immune Responses Document the Level of JEV Replication. In order to obtain a better understanding of the mechanism of protection from lethal challenge in animals inoculated with these recombinant viruses, the ability of antibodies in post-challenge sera to recognize JEV antigens was evaluated. For these studies antigen present in lysates of radiolabeled JEV-infected cells was utilized, and the response to the NS3 protein which induces high levels of antibodies in hyperimmunized mice (Mason et al., 1987a) was examined. The results of these studies (Table 18) correlates with the survival data in that groups WO 92/1'-, 2 PCT/US92/01906 of animals vaccinated with recombinant viruses that induced high levels of protection (vP829, vP555, and vP825) showed low post-challenge responses to NS3, whereas the sera from survivors of groups vaccinated with recombinants that expressed NS1 alone (vP857 and vP864) showed much higher post-challenge responses to NS3.

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WO 92/1: 2 PCT/L,'S92/01906 Table 17. Protection of mice and immune response following single or double inoculations with recombinant vaccinia virus expressing JEV proteins Immunizing yirusa Protectionb vP555 vP829 vP825 vP857 vP864 single inoculation 7/10 10/10 8/10 0/10 1/10 double inoculation 10/10 9/10 9/10 5/10 6/10 Neut titerc single inoculation 1:20 1:160 1:10 <1:10 <1:10 double inoculation 1:320 1:2560 1:320 <1:10 <1:10 HAI titerd single inoculation 1:20 1:40 1:10 <1:10 <1:10 double inoculation 1:80 1:160 1:40 <1:10 <1:10 a Groups of 20 mice were inoculated by ip route with 107 pfu of the indicated vaccinia virus JEV recombinant.
Sera were collected after three weeks. At this time, 10 mice per group were challenged with JEV as indicated in the text (single inoculation). The remaining 10 mice in each group were boosted with the same vaccinia virus JEtI recombinant (double inoculation). After three weeks, sera were collected and the mice were challenged with JEV. All mice were observed for 21 days post challenge.

b Protection is expressed as number os mice surviving at 21 days post challenge/total.

c Neutralization titer is expressed as the reciprocal of the highest dilution that gives 90% JEV plaque reduction.

d HAI titer is expressed as the reciprocal of the highest dilution that gives measurable inhibition of hemagglutination of red blood cells.

.O 92/156'2 PCT/L,'S9. .01906 Table 18. Post challenge immune response following single or double inoculation with recombinant vaccinia virus expressing JEV proteins.

Immunizing Virus vP555 vP829 vP825 vP857 vP864 single ++ + ++ -a ++++
double +/-b - - ++ +++
+ NS3 antibodies present in post-challenge sera a No surviving mice b Very low level NS3 antibodies present in post-challenge sera 21 day post challenge sera from mice surviving JEV challenge following single or double inoculation with vaccinia virus JEV recombinants (Table 17) were analyzed for the presence of antibodies to JEV NS3.

WO 92/, 12 PCi'/US92/01906 Cloning of JEV Genes Into a Vaccinia (NYVAC) Donor Plasmid. Plasmid pMP2VCL (containing a polylinker region within vaccinia sequences upstream of the K1L host range gene) was digested within the polylinker with indIII and XhoI and ligated to annealed oligonucleotides SPHPRHA A
through D generating SP126 containing a HindIII site, H6 promoter -124 through -1 (Perkus et al., 1989) and XhoI, KDnI, SmaI, SacI and EcoRI sites.
Plasmid pSD544VC (containing vaccinia sequences surrounding the site of the HA gene which was replaced with a polylinker region and translation termination codons in six reading frames) was digested with XhoI within the polylinker, filled in with the Klenow fragment of DNA
polymerase I and treated with alkaline phosphatase. SP126 was digested with indIII, treated with Klenow and the H6 promoter isolated by digestion with SmaI. Ligation of the H6 promoter fragment to pSD544VC generated SPHA-H6 which contained the H6 promoter in the polylinker region (in the direction of HA transcription).
Plasmid'JEVL14VC was digested with EcoRV in the H6 promoter and SacI in JEV sequences (nucleotide 2124) and a 1789 bp fragment isolated. JEVL14VC (Mason et al., 1991) was digested with Ec1XI at the EagI site following the T5NT, filled in with the Klenow fragment of DNA polymerase I and digested with SacI in JEV sequences (nucleotide 2124) generating a 2005 bp fragment. The 1789 bp coRV-SacI and 2005 bp (SacI-filled ~c1XI) fragments were ligated to EcoRV
(within H6) and SmaI digested (within polylinker) and alkaline phosphatase treated SPHA-H6 generating JEV35.
JEV35 was transfected into vP866 (NYVAC) infected cells to generate the vaccinia recombinant vP908 (FIG. 18).
JEV35 was digested with SacI (within JE sequences nucleotide 2124) and Ec1XI (after T5NT) a 5497 bp fragment isolated and ligated to a SacI (JEV nucleotide 2124) to ~ca I
fragment of JEV25 (containing the remaining two thirds of E, translation stop and T5NT) generating JEV36. JEV36 was transfected into vP866 (NYVAC) infected cells to generate the vaccinia recombinant vP923 (FIG. 18).

92/156'2 PC'T/US92, 906 SPHPRHA A through D Oligonucleotides SPHPRHA:A+B (SEQ ID
NO:120) and SPHPRHA:C+D (SEQ ID NO:121) are as follows 'ndIII
5'- AGCTTCTTTATTCTATACTTAA.AAAGTGAAAATAAATACAAAGGTTCTTGA
3'- AGAAATAAGATATGAATTTTTCACTTTTATTTATGTTTCCAAGAACT

EcoRV
GGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATTATTTCATTATCGCGATATCCG
CCCAACACAATTTAACTTTCGCTCTTTATTAGTATTTAATAAAGTAATAGCGCTATAGGC
TTAAGTTTGTATCGTAC -3' AATTCAAACATAGCATGAGCT -5' XhoI
Construction of Plasmids ContainincL YF Genes. The YF
17D cDNA clones used to construct the YF vaccinia recombinant viruses (clone 10III and clone 28111), were obtained from Charles Rice (Washington University School of Medicine, St. Louis, MO). All nucleotide coordinates are derived from the sequence data presented in Rice et al., 1985.
Plasmid YFO containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NS1 (nucleotides 537-3266) was derived by cloning an AvaI to siI fragment of YF cDNA (nucleotides 537-1659) and an NsiI to KonI fragment of YF cDNA (nucleotides 1660-3266) into Aval and KpnI
digested IBI25 (International Biotechnologies, Inc., New Haven, CT). Plasmid YF1 containing YF cDNA encoding C and amino-terminal 20% prM (nucleotides 119-536) was derived by cloning a RsaI to AvaI fragment of YF cDNA (nucleotides 166-536) and annealed oligonucleotides SP46 and SP47 (containing a disabled HindIII sticky end, XhoI and C1aI sites and YF
nucleotides 119-165) into AvaI and indIII digested IBI25.
Plasmid YF3 containing YF cDNA encoding the carboxy-terminal 60% of E and amino-terminal 25% of NS1 was generated by cloning an AvaI to BamHI fragment of YF cDNA (nucleotides 1604-2725) into aRaI and Ba I digested IBI25. Plasmid YF8 containing YF cDNA encoding the carboxy-terminal 20% NS1 NS2a, NS2B and amino-terminal 20% NS3 was derived by cloning a KDnI to Xbal fragment of YF cDNA (nucleotides 3267-4940) into KDnI and XbaI digested IBI25. Plasmid YF9 containing YF cDNA encoding the carboxy-terminal 60% NS2B and amino-WO 92/1 2 PCf/US92/01906 terminal 20% NS3 was generated by cloning a SacI to XbaI
fragment of YF cDNA (nucleotides 4339-4940) into SacI and XbaI digested IBI25. Plasmid YF13 containing YF cDNA
encoding the carboxy-terminal 25% of C, prM and amino-terminal 40% of E was derived by cloning a~alI to ApaI
fragment of YF cDNA (nucleotides 384-1603) into ADaI and SmaI digested IBI25.
Oligonucleotide-directed mutagenesis (Kunkel, 1985) was used to change the following potential vaccinia virus early transcription termination signals (Yuen et al., 1987) (1) 49 aa from the amino-terminus of the C gene in YF1 (TTTTTCT
nucleotides 263-269 and TTTTTGT nucleotides 269-275) to (SEQ
ID N0:122) TTCTTCTTCTTGT creating plasmid YF1B, (2) in the E
gene in YF3 (nucleotides 1886-1893 TTTTTTGT to TTCTTTGT 189 aa from the carboxy-terminus and nucleotides 2429-2435 TTTTTGT to TTCTTGT 8 aa from the carboxy-terminus) creating plasmids YF3B and YF3C, respectively. A PstI to BamHI
fragment from YF3C (nucleotides 1965-2725) was exchanged for the corresponding fragment of YF3B generating YF4 containing YF cDNA encoding the carboxy-terminal 60% E and amino-terminal 25% NS1 (nucleotides 1604-2725) with both mutagenized transcription termination signals. An ApaI to BamHI fragment from YF4 (nucleotides 1604-2725) was substituted for the equivalent region in YFO creating plasmid YF6 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NS1 (nucleotides 537-3266) with both mutagenized transcription termination signals.
Plasmid YF6 was digested with EcoRV within the IBI25 sequences and AvaI at nucleotide 537 and ligated to an EcoRV
to AvaI fragment from YF1B ( coRV within IBI25 to AvaI at nucleotide 536) generating YF2 containing YF cDNA encoding C
through the amino-terminal 80% of NS1 (nucleotides 119-3266) with an XhoI and C1aI site at 119 and four mutagenized transcription termination signals.
Oligonucleotide-directed mutagenesis described above was used (1) to insert XhoI and ClaI sites preceding the ATG
17 aa from the carboxy-terminus of E (nucleotides 2402-2404) in plasmid YF3C creating YF5, (2) to insert X}oI and C1aI
sites preceding the ATG 19 aa from the carboxy-terminus of 92/15672 PCT/US92, _4b6 prM (nucleotides 917-919) in plasmid YF13 creating YF14, (3) to insert an Xhol site preceding the ATG 23 aa from the carboxy-terminus of E (nucleotides 2384-2386) in plasmid YF3C creating plasmid YF25, (4) and to insert an Xhol site and ATG (nucleotide 419) in plasmid YF1 21 aa from the carboxy-terminus of C generating YF45.
An ADaI to BamHI fragment from YF5 (nucleotides 1604-2725) was exchanged for the corresponding region of YFO
creating YF7 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NS1 (nucleotides 537-3266) with XhoI and C1aI sites at 2402 (17 aa from the carboxy-terminus of E) and a mutagenized transcription termination signal at 2429-2435 (8 aa from the carboxy-terminus of E). The ApaI to BamHI fragment from YF25 (nucleotides 1604-2725) was exchanged for the corresponding region of YFO generating YF26 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NS1 (nucleotides 537-3266) with an XhoI site at nucleotide 2384 (23 aa from the carboxy-terminus of E) and mutagenized transcription termination signal at 2428-2435 (8 aa from the carboxy-terminus of E).
An AvaI to AiDaI fragment from YF14 (nucleotides 537-1603) was substituted for the corresponding region in YF6 generating YF15 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NS1 (nucleotides 537-3266) with XhoI and C ai sites at nucleotide 917 (19 aa from the carboxy-terminus of prM) and two mutagenized transcription termination signals. YF6 was digested within IBI25 with EcoRV and within YF at nucleotide 537 with vaI
and ligated to an EcoRV (within IBI25) to ~v I fragment of YF45 generating YF46 containing YF cDNA encoding C through the amino-terminal 80% NS1 (nucleotides 119-3266) with an 'XhoI site at 419 (21 aa from the carboxy-terminus of C) and two transcription termination signals removed.
Oligonucleotide-directed mutagenesis described above was used to insert a SmaI site at the carboxy-terminus of NS2B (nucleotide 4569) in plasmid YF9 creating YF11, and to insert a SmaI site at the carboxy-terminus of NS2A
(nucleotide 4180) in plasmid YF8 creating YF10. A SacI to WO 92/1: 2 PCT/US92/01906 XbaI fragment from YF11 (nucleotides 4339-4940) and As~718 to acI fragment from YF8 (nucleotides 3262-4338) were ligated to Asp718 and CbaI digested IBI25 creating YF12 containing YF cDNA encoding the carboxy-terminal 20% NS1, NS2A, NS2B and amino-terminal 20% NS2B (nucleotides 3262-4940) with a SmaI site after the carboxy-terminus of NS2B
(nucleotide 4569).
Cloning of YF Genes into a pHES System Vaccinia Virus Donor Plasmid. Prior to insertion of YF cloning sequences into a NYVAC donor plasmid, YF coding sequences were inserted into vaccinia plasmid pHES4 (Perkus et al., 1989).
A KDnI to SmaI fragment from YF12 encoding carboxy-terminal '20% NS1, NS2A and NS2B (nucleotides 3267-4569), XhoI to KDnI
fragment from YF15 encoding 19 aa prM, E and amino-terminal 80% NS1 (nucleotides 917-3266) and ~ioI-SmaI digested pHES4 were ligated generating YF23. An XhoI to DamHI fragment from YF26 encoding 23 aa E, amino-terminal 25% NS1 (nucleotides 2384-2725) was ligated to an XhoI to BamHI
fragment from YF23 (containing the carboxy-terminal 75% NS1, NS2A and NS2B, the origin of replication and vaccinia sequences) generating YF28.
XhoI-Smal digested pHES4 was ligated to a purified XhoI
to DnI fragment from YF7 encoding 17 aa E and amino-terminal 80% NS1 (nucleotides 2402-3266) plus a KpnI to SmaI
fragment from YF10 encoding the carboxy-terminal 20% NS1 and NS2A (nucleotides 3267-4180) creating YF18. An h~oI to BamHI fragment from YF2 encoding C, prM, E and amino-terminal 25% NS1 (nucleotides 119-2725) was ligated to a XhoI to BamHI fragment of YF18 (containing the carboxy-terminal 75% NS1 and NS2A, the origin of replication and vaccinia sequences) generating YF19. The same XhoI to BamHI
fragment from YF2 was ligated to a h~oI to BamHI fragment from YF28 (containing the carboxy-terminal 75% NS1 and NS2A, the origin of replication and vaccinia sequences) generating YF20. AXhQI to DamHl fragment from YF46 encoding 21 aa C, prM, E and amino-terminal 25% NS1 (nucleotides 419-2725) was ligated to the XhoI to LamHI fragment from YF18 generating YF47. Oligonucleotide SP46 (SEQ ID N0:123) AND SP47 (SEQ ID
NO:124) are as follows:

92/15672 PCT/US92/ . 406 indIII
SP46 5'- AGCTT CTCGAGCATCGATTACT atg TCTGGTCGTAAAGCTCAGGG
SP47 3'- A GAGCTCGTAGCTAATGA TAC AGACCAGCATTTCGAGTCCC
AAAAACCCTGGGCGTCAATATGGT -3' TTTTTGGGACCCGCAGTTATACCA -5' Construction of Recombinant YF Vaccinia Viruses. Five different vaccinia virus recombinants that expressed portions of the YF coding region extending from C through NS2B were constructed utilizing a host range selection system (Perkus et al., 1989). Plasmids YF18, YF23, YF20, YF19 and YF47 were transfected into vP293 infected cells to generate the vaccinia recombinants vP725, vP729, vP764, vP766 and vP869. The YF cDNA sequences contained in these recombinants are shown in FIG. 19. In all five recombinant viruses the sense strand of YF cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from Met codons located at the 5' ends of the viral cDNA sequences (FIG. 19).
Recombinant vP725 encoded the putative 17-aa signal sequence preceding the N terminus of the nonstructural protein NS1 and the nonstructural proteins NS1 and NS2A
(Rice et al., 1985). Recombinant vP729 encoded the putative 19-aa signal sequence preceding the N terminus of E, E, NS1, NS2A and NS2B (Rice et al., 1985). Recombinant vP764 encoded C, prM, E, NS1, NS2A and NS2B (Rice et al., 1985).
Recombinant vP766 encoded C, prM, E, NS1 and NS2A (Rice et al., 1985). Recombinant vP869 encoded the putative 21-aa signal sequence preceding the N terminus of the prM
structural protein precursor as well as prM, E, NS1 and NS2A
(Rice et al., 1985).
Protection From Lethal YF Challenge. vP869 secreted an HA activity not found in the culture fluid of cells infected with any of the other recombinants. This HA appeared similar to the HA produced in YF infected cells based on its inhibition by anti-YF antibodies and pH optimum.
Three-week-old mice were inoculated intraperitoneally with 107 pfu vP869, vP764 or YF-17D and challenged three weeks later with 100 LD50 of French neurotropic strain of YF. vP869 provided significant protection (Table 19) whereas vP764 offered no better protection than a control vaccinia virus lacking YF genes (vP457).

Table 19. Protection of mice by recombinant vaccinia viruses from YF challenge Immunizing Virus Survival/total vP457 2/10 vP764 2/10 vP869 9/10 Cloning of YF Genes Into a NYVAC Donor Plasmid. A XhoI
to SmaI fragment from YF47 (nucleotides 419-4180) containing YF cDNA encoding 21 amino acids C, prM, E, NS1, NS2A (with nucleotide 2962 missing in NS1) was ligated to XhoI-SmaI
digested SPHA-H6 (HA region donor plasmid) generating YF48.
YF48 was digested with SacI (nucleotide 2490) and partially digested with AsD718 (nucleotide 3262) and a 6700 bp fragment isolated (containing the plasmid origin of replication, vaccinia sequences, 21 amino acids C, prM, E, amino-terminal 3.5% NS1, carboxy-terminal 23% NS1, NS2A) and ligated to a SacI-Asp718 fragment from YF18 (containing the remainder of NS1 with the base present at 2962) generating YF51. The 6 bp corresponding to the unique XhoI site in YF51 were removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating plasmid YF50 encoding YF 21 amino acids C, prM, E, NS1, NS2A in the HA locus donor plasmid. Donor plasmid YF50 was transfected into vP866 (NYVAC) infected cells to generate vaccinia recombinant vP984.
The 6 bp corresponding to the unique XhoI site in YF48 were removed using oligonucleotide-directed double-strand break mutagenesis creating YF49. Oligonucleotide-directed mutagenesis (Kunkel, 1985) was used to insert a SmaI site at the carboxy-terminus of E (nucleotide 2452) in YF4 creating YF16. An ApaI-SmaI fragment of YF49 (containing the plasmid origin of replication, vaccinia sequences and YF cDNA
encoding 21 amino acid C, prM, and amino-terminal 43% E) was ligated to an ApaI-SmaI fragment from YF16 (nucleotides ) 92/15672 PCT/US91. 4906 1604-2452 containing the carboxy-terminal 57% E) generating YF53 containing 21 amino acids of C, prM, E in the HA locus.
Donor plasmid YF53 was transfected into vP913 (NYVAC-MV) infected cells to generate the vaccinia recombinant vP997.
Cloning of Denctue Type 1 Into a Vaccinia Virus Donor Plasmid. Plasmid DEN1 containing DEN cDNA encoding the carboxy-terminal 84% NS1 and amino-terminal 45% NS2A
(nucleotides 2559-3745, Mason et al., 1987b) was derived by cloning an coRI-XbaI fragment of DEN cDNA (nucleotides 2559-3740) and annealed oligonucleotides DEN1 (SEQ ID
NO:125) and DEN2 (SEQ ID NO:126) (containing a~baI sticky end, translation termination codon, T5AT vaccinia virus early transcription termination signal (Yuen et al. 1987), EaQI site and HindIII sticky end) into HindIII- coRI
digested pUCB. An EcoRI- indIII fragment from DEN1 (nucleotides 2559-3745) and acI-EcoRI fragment of DEN cDNA
encoding the carboxy-terminal 36% of E and amino-terminal 16% NS1 (nucleotides 1447-2559, Mason et al., 1987b) were ligated to HindIII -SacI digested IBI24 (International Biotechnologies, Inc., New Haven, CT) generating DEN3 encoding the carboxy-terminal 64% E through amino-terminal 45% NS2A with a base missing in NS1 (nucleotide 2467).
HindIII-XbaI digested IBI24 was ligated to annealed oligonucleotides DEN9 (SEQ ID NO:127) and DEN10 (SEQ ID
NO:128) [containing a HindIII sticky end, SmaI site, DEN
nucleotides 377-428 (Mason et al., 1987b) and XbaI sticky end] generating SPD910. SPD910 was digested with SacI
(within IBI24) and AvaI (within DEN at nucleotide 423) and ligated to an vaI-SacI fragment of DEN cDNA (nucleotides 424-1447 Mason et al., 1987) generating DEN4 encoding the carboxy-terminal 11 aa C, prM and amino-terminal 36% E.
Plasmid DEN6 containing DEN cDNA encoding the carboxy-terminal 64% E and amino-terminal 18% NS1 (nucleotides 1447-2579 with nucleotide 2467 present Mason et al., 1987b) was derived by cloning a acI-X oI fragment of DEN cDNA into IBI25 (International Biotechnologies, Inc., New Haven, CT).
Plasmid DEN15 containing DEN cDNA encoding 51 bases of the DEN 5' untranslated region, C, prM and amino-terminal 36% E
was derived by cloning a i#r' dIII-SacI fragment of DEN cDNA

WO 92/1_ 2 PCT/US92/01906 (nucleotides 20-1447, Mason et al., 1987b) into HindIII-SacI
digested IBI25. Plasmid DEN23 containing DEN cDNA encoding the carboxy-terminal 55% NS2A and amino-terminal 28% NS2B
(nucleotides 3745-4213) was derived by cloning a XbaI-SphI
fragment of DEN cDNA into XbaI-SphI digested IBI25. Plasmid DEN20 containing DEN cDNA encoding the carboxy-terminal 55%
NS2A, NS2B and amino-terminal 24 amino acids NS3 (nucleotides 3745-4563) was derived by cloning a XbaI to EcoRI fragment of DEN cDNA into XbaI-EcoRI digested IBI25.
Oligonucleotide-directed mutagenesis (Kunkel, 1985) was used to change the following potential vaccinia virus early transcription termination signals (Yuen et al., 1987). The two T5NT seqeunces in the prM gene in DEN4 were mutagenized (1) 29 aa from the carboxy-terminus (nucleotides 822-828 TTTTTCT to TATTTCT) and (2) 13 aa from the carboxy-terminus (nucleotides 870-875 TTTTTAT to TATTTAT) creating plasmid DEN47. The single T5NT sequence in the NS1 gene in DEN6 17 aa from the amino-terminus was mutagenized (nucleotides 2448-2454 TTTTTGT to TATTTGT) creating plasmid DEN7.
Oligonucleotide-directed mutagenesis as described above was used (1) to insert an EagI and EcoRI site at the carboxy-terminus of NS2A (nucleotide 4102) in plasmid DEN23 creating DEN24, (2) to insert a SmaI site and ATG 15 aa from the carboxy-terminus of E in DEN7 (nucleotide 2348) creating DEN10,( 3) to insert an EagI and HindI I I site at the carboxy-terminus of NS2B (nucleotide 4492) in plasmid DEN20 creating plasmid DEN21, and (4) to replace nucleotides 63-67 in plasmid DEN15 with part of the vaccinia virus early/late H6 promoter (positions -1 to -21, Perkus et al., 1989) creating DEN16 (containing DEN nucleotides 20-59, EcoRV site to -1 of the H6 promoter and DEN nucleotides 68-1447).
A SacI-XhoI fragment from DEN7 (nucleotides 1447-2579) was substituted for the corresponding region in DEN3 generating DEN19 containing DEN cDNA encoding the carboxy-terminal 64% E and amino-terminal 45% NS2A (nucleotides 1447-3745) with nucleotide 2467 present and the modified transcription termination signal (nucleotides 2448-2454). A
XhoI-XbaI fragment from DEN19 (nucleotides 2579-3745) and a Xbal-HindIII fragment from DEN24 (XbaI nucleotide 3745 DEN

92/15672 PCT/l.'S92i,. 4U6 through indII I in IBI25 ) were ligated to ~ioI-~,i indI II
digested IBI25 creating DEN25 containing DEN cDNA encoding the carboxy-terminal 82% NS1, NS2A and amino-terminal 28%
NS2B (nucleotides 2579-4213) with a EagI site at 4102, nucleotide 2467 present and mutagenized transcription termination signal (nucleotides 2448-2454). The XhoI-XbaI
fragment from DEN19 (nucleotides 2579-3745) was ligated to XhoI (within IBI25) and XbaI (DEN nucleotide 3745) digested DEN21 creating DEN22 encoding the carboxy-terminal 82% NS1, NS2A, NS2B and amino-terminal 24 aa NS3 (nucleotides 2579-4564) with nucleotide 2467 present, modified transcription termination signal (nucleotides 2448-2454) and EactI site at 4492.
A HindIII-PstI fragment of DEN16 (nucleotides 20-494) was ligated to a HindIII-PstI fragment from DEN47 (encoding the carboxy-terminal 83% prM and amino-terminal 36% of E
nucleotides 494-1447 and plasmid origin of replication) generating DEN17 encoding C, prM and amino-terminal 36% E
with part of the H6 promoter and EcoRV site preceding the amino-terminus of C. A HindIII-BalII fragment from DEN17 encoding the carboxy-terminal 13 aa C, prM and amino-terminal 3.6% E (nucleotides 370-1447) was ligated to annealed oligonucleotides SPill and SP112 (containing a disabled HindIII sticky end, coRV site to -1 of the H6 promoter, and DEN nucleotides 350-369 with a Bc1II sticky end) creating DEN33 encoding the EcoRV site to -1 of the H6 promoter, carboxy-terminal 20 aa C, prM and amino-terminal 36% E.
SmaI-EacxI digested pTP15 (Mason et al., 1991) was ligated to a SmaI-SacI fragment from DEN4 encoding the carboxy-terminal 11 aa C, prM and amino-terminal 36% E
(nucleotides 377-1447) and SacI-EaaI fragment from DEN3 encoding the carboxy-terminal 64% E, NS1 and amino-terminal 45% NS2A generating DENL. The SacI-XhoI fragment from DEN7 encoding the carboxy-terminal 64% E and amino-terminal 18%
NS1 (nucleotides 1447-2579) was ligated to a BstEII-SacI
fragment from DEN47 (encoding the carboxy-terminal 55% prM
and amino-terminal 36% E nucleotides 631-1447) and aBstEII-XhoI fragment from DENL (containing the carboxy-terminal 11 WO 92/15. 1 PCT/US92/01906 aa C, amino-terminal 45% prM, carboxy-terminal 82% NS1, carboxy-terminal 45% NS2A, origin of replication and vaccinia sequences) generating DEN8. A unique SmaI site (located between the H6 promoter and ATG) was removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating DEN8VC in which the H6 promoter immediately preceded the ATG start codon.
An EcoRV-SacI fragment from DEN17 (positions -21 to -1 H6 promoter DEN nucleotides 68-1447) was ligated to an EcoRV
-SacI fragment of DEN8VC (containing vaccinia sequences, H6 promoter from -21 to -124, origin of replication and amino-terminal 64% E, NS1, amino-terminal 45% NS2A nucleotides 1447-3745) generating DEN18. A XhoI-EagI fragment from DEN25 (nucleotides 2579-4102) was ligated to an XhoI-EacI
fragment of DEN18 (containing the origin of replication, vaccinia sequences and DEN C, prM, E and amino-terminal 18%
NS1 nucleotides 68-2579) generating DEN26. An EcoRV-SacI
fragment from DEN8VC (positions -21 to -1 H6 promoter DEN
nucleotides 377-1447) was ligated to an EcoRV-SacI fragment of DEN26 (containing the origin of replication, vaccinia sequences and DEN region encoding the carboxy-terminal 64%
E, NS1 and NS2A with a base missing in NS1 at nucleotide 2894) generating DEN32. DEN32 was transfected into vP410 infected cells to generate the recombinant vP867 (FIG. 20).

A SacI-XhoI fragment from DEN10 (nucleotides 1447-2579) was substituted for the corresponding region in DEN3 generating DEN11 containing DEN cDNA encoding the carboxy-terminal 64% E, NS1 and amino-terminal 45% NS2A with a SmaI
site and ATG 15 aa from the carboxy-terminus of E. A SmaI-EagI fragment from DEN11 (encoding the carboxy-terminal 15 aa E, NS1 and amino-terminal 45% NS2A nucleotides 2348-3745) was ligated to SmaI-EagI digested pTP15 generating DEN12.
A XhoI-Ea~I fragment from DEN22 (nucleotides 2579-4492) was ligated to the XhoI-EagI fragment from DEN18 described above generating DEN27. An EcoRV-PstI fragment from DEN12 (positions -21 to -1 H6 promoter DEN nucleotides 2348-3447) was ligated to an EcoRV-PstI fragment from DEN27 (containing the origin of replication, vaccinia sequences, H6 promoter -92/1561/2 PCT/US92/, i()6 21 to -124 and DEN cDNA encoding NS2A and NS2B) generating DEN31.
An coRV-XhoI fragment from DEN8VC (positions -21 to -1 H6 promoter DEN nucleotides 377-2579 encoding the carboxy-terminal 11 aa C, prM E, amino-terminal 18% NS1) was ligated to an EcoRV-XhoI fragment from DEN31 (containing the origin of replication, vaccinia sequences and DEN cDNA encoding the carboxy-terminal 82% NS1, NS2A, NS2B with the base present in NS1 at position 2894) generating DEN35. DEN35 was transfected into vP410 infected cells generating the recombinant vP955 (FIG. 20). An EcoRV-SacI fragment from DEN33 (positions -21 to -1 H6 promoter DEN nucleotides 350-1447 encoding the carboxy-terminal 20 aa C, prM and amino-terminal 36% E) and a SacI-XhoI fragment from DEN32 (encoding the carboxy-terminal 64% E and amino-terminal 18%
NS1 nucleotides 1447-2579) were ligated to the EcoRV-SacI
fragment from DEN31 described above generating DEN34. DEN34 was transfected into vP410 infected cells generating the recombinant vP962 (FIG. 20). Oligonucleotides DEN 1 (SEQ ID
NO:125), DEN 2 (SEQ ID N0:126), DEN9 (SEQ ID NO:127), DEN10 (SEQ ID NO:128), SP111 (SEQ ID NO:129), and SP112 (SEQ ID
NO:130) are as follows:
DEN1 5'- CTAGA tga TTTTTAT CGGCCG A -3' DEN2 3'- T ACT AAAAATA GCCGGC TTCGA -5' XbaI EaaI HindIII

DEN9 5' AGCTT CCCGGG atg CTCCTCATGCTGCTGCCC
DEN10 3' A GGGCCC TAC GAGGAGTACGACGACGGG
indIII SmaI

ACAGCCCTGGCGTTCCATCTGACCACCCGAG T -3' TGTCGGGACCGCAAGGTAGACTGGTGGGCTC AGATC -5' vaI XbaI

SPill 5' AGCT GATATCCGTTAAGTTTGTATCGTA atg AACAGGA
SP112 3' A CTATAGGCAATTCAAACATAGCAT TAC TTGTCCT
HindIiI EcoRV
GGAAA A -3' CCTTT TCTAG-5' Dglll Example 17 - CONSTRUCTION OF MODIFIED NYVAC VIRUSES
NYVAC was modified by increasing to different extents the size of the [C7L - K1L] deletion near the left terminus of vaccinia and by introducing a deletion near the right WO 92/15. .. PCT/L'S92/01906 terminus. All deletions were accomplished using the E. coli guanine phosphoribosyl transferase gene and mycophenolic acid in a transient selection system.
Transient Dominant Selection. Using circular donor plasmid, recombination with vaccinia virus was performed by the standard method of transfection of calcium phosphate precipitated plasmid DNA into vaccinia-infected Vero cells.
After 24 h, the infected cells were harvested and the lysate plated in the presence of 1 microgram/ml mycophenolic acid (MPA). Individual plaques were picked and amplified on Vero cells in the presence of MPA. Virus was harvested and plaque purified by two rounds of plaque picking in the absence of MPA. Plaques picked from each round without MPA
were plated on Vero cells and filters hybridized for the presence of pertinent genes.
NYVAC.1. The [C7L - K1LJ deletion present in vP866 (NYVAC) was expanded to include the next two ORFs to the right, K2L and K3L. The putative translation product for the K2L ORF shows homology to the family of serine protease inhibitors (Boursnell et al., 1988). However, transcriptional mapping of this region of the vaccinia genome suggests that the K2L ORF is not expressed (Morgan et al., 1984).
The translation product for K3L shows 28 % homology to eukaryotic initiation factor 2 alpha (eIF-2 alpha) over an 87 amino acid overlap spanning the serine (amino acid 51) phosphorylation site. Phosphorylation of eIF-2 alpha is a step in the antiviral state induced by interferon, suggesting that the vaccinia K3L gene product may be involved in the mechanism by which vaccinia evades the effects of interferon. The K3L gene from Copenhagen strain of vaccinia has been deleted (Beattie et al., 1991). The resulting virus exhibited heightened sensitivity to interferon in vitro as measured both by inhibition of viral induction of protein synthesis and inhibition of viral replication. This suggests that deletion of K3L from vaccinia virus could result in a safer vaccine strain which could be controlled by interferon treatment in the event of vaccination complications.

92/15672 PCT/US92. )06 Construction of Plasmid vMPC7K3GPT for Deletion of C7L
Through K3L. The left and right vaccinia arms flanking the [C7L - K3L] deletion were assembled separately. The left arm was derived from pSD420 (Perkus et al., 1990) and assembled in intermediate deletion plasmid pMP256/257 (Perkus et al., 1991). Synthetic oligonucleotides MPSYN379 (SEQ ID NO:131), MPSYN380 (SEQ ID NO:132) indiII SalI BamHI
MPSYN379 5' TTCCCAAGCTTGTCGACGATAATATGGATCCTCATGAC 3' BctlII
MPSYN380 5' TTCCCAGATCTATGAGTATAGTGTTAAATGAC 3' were used as primers in a PCR reaction using plasmid pSD420 as template. The resulting 0.14 kb fragment was cut with HindIII/BalII and inserted into pMP256/257, replacing the left arm of the plasmid. The resulting plasmid was designated pMP379/380. A 0.7 kb SalI/BamHI fragment was isolated from pSD420 and ligated into pMP379/380 cut with SalI/BamHI, forming plasmid pMPC7F4.

To construct a right deletion junction containing sequences to the right of K3L, synthetic oligonucleotides MPSYN381/MPSYN382 (SEQ ID NO:133/SEQ ID NO:134) BamHI HDaI coRV Smal coRI
MPSYN381 5' GATCCTTGTTAACCCGATATCCCGGG 3' MPSYN382 3' GAACAATTGGGCTATAGGGCCCTTAA 5' were annealed and ligated into pUCB cut with BamHI/EcoRI, forming plasmid pMP381/382. A 1.0 kb HDaI (partial) /EcoRV
fragment was isolated from cloned vaccinia HindIII K and ligated into pMP381/382, forming plasmid pMPK3R, which contains the entire right vaccinia flanking arm. The left vaccinia flanking arm was isolated from pMPC7F4 as a 0.8 kb BalII(partial)/HindIII fragment, and inserted into pMPK3R
cut with BamHI/HindIII. The resulting plasmid, pMPC7K3, is deleted for 14 genes [C7L - K3L].
For use as a selectable marker, the E. coli gene encoding guanine phosphoribosyl transferase (Ecogpt) (Pratt et al., 1983) was placed under the control of a poxvirus WO 92/1. 2 PCT/US92/01906 promoter. A 31 bp promoter element immediately upstream from a gene encoding an entomopox 42 kDa protein can function as a strong promoter in recombinant vaccinia virus at early time post infection. Annealed synthetic oligonucleotides MPSYN369/370 (SEQ ID NO:135/SEQ ID NO:136) Xhol coRI SmaI
MPSYN369 5' TCGAGAATTCCCGGGTCAAAATTGAAAATATATAATTACAA
Bcr1II
TATAAAATA 3' MPSYN370 3' CTTAAGGGCCCAGTTTTAACTTTTATATATTAATGTTA
TATTTTATCTAG 5' containing the 31 bp EPV 42 kDa promoter were ligated upstream from the Ecogpt gene in a pBS-SK background, resulting in plasmid pMP42GPT. A SmaI fragment containing the 42 kDa promoter/Ecogpt gene expression cassette was isolated from pMP42GPT and inserted into vaccinia deletion plasmid pMPC7K3 in the SmaI site at the pUC/vaccinia junction. The resulting plasmid, pMPC7K3GPT was transfected into vP866 (NYVAC). Mycophenolic acid was used in the culture medium for selection of intermediate products of recombination in a transient dominant selection system (Falkner et al., 1990). After removal of selective pressure, progeny virus were screened by plaque hybridization for loss of K2L DNA sequences and retention of K4L. The fidelity of the deletion junction was verified by PCR and DNA restriction and sequence analysis. Recombinant vaccinia virus vP954 (NYVAC.1) contains the [C7L - K3L]
deletion, as well the other deletions present in NYVAC (TK, HA, ATI, 14L, [B13 - B14]).
NYVAC.2. The [C7L - K1L] deletion present in NYVAC was expanded in both directions to include a total of 38 ORFs, [C23L - F4L]. This is the same deletion previously reported in vaccinia deletion mutant vP796 (Perkus et al., 1991).
Noteworthy ORFs removed in the expanded deletion region include the vaccinia growth factor (VGF; C11R) located to the left of the NYVAC deletion. In contrast to WR strain of vaccinia which contains two copies of the VGF, C11R is the only ORF encoding the VGF in Copenhagen strain of vaccinia.
Deletion of both copies of the vaccinia growth factor from 92/15672 PC"T/US92, I06 WR has been shown to reduce the severity of skin lesions upon intradermal inoculation of rabbits and to reduce neurovirulence of the virus in mice (Buller et al., 1988).
The rightmost ORF in the [C23L - F4L] deletion, F4L, encodes the gene for the small subunit of ribonucleotide reductase (Slabaugh et al., 1988). Also included in this deletion is ORF F2L, which shows homology to E. coli dUTPase, another enzyme involved in nucleotide metabolism (Goebel et al., 1990a,b). F2L also shows homology to retroviral protease (Slabaugh et al., 1989).
Construction of Plasmid pMPTRF4GPT for Deletion of C23L
Through F4L. Plasmid pMPLENDA, which was used as an intermediate in the generation of vaccinia deletion mutant vP796 (Perkus et al., 1991) was modified by the addition of the SmaI expression cassette containing the EPV 42 kDa promoter/Ecogpt gene at the pUC/vaccinia junction. The resulting plasmid, pMPTRF4GPT, was transfected into NYVAC.
Following selection using MPA, progeny virus were screened by plaque hybridization for loss of F4L DNA sequences and retention of F5L. Fidelity of the deletion junction was verified by PCR and DNA restriction analysis. Recombinant virus vP938 (NYVAC.2) contains the [C23L - F4L] deletion as well as the other deletions present in NYVAC.
Deletion of ORFs B13R - B29R. The u deletion [B13R -B14R] present in NYVAC was expanded to include all ORFs to the right, a total of 17 ORFs [B13R - B29R]. This is the same deletion previously reported in vaccinia deletion mutant vP759 (Perkus et al., 1991). The expanded deletion region includes two genes whose products show 20 % amino acid identity with each other (Smith et al., 1991). The ORFs encoding these gene products are designated B16R and B19R in Copenhagen (Goebel et al., 1990a,b), which correspond to ORFs B15R and B18R, respectively, in the WR
strain (Smith et al., 1991). Unlike the WR strain of vaccinia, in which both gene products contain typical signal sequences, the predicted translation product of Copenhagen ORF B16 is truncated at the amino terminus and does not contain a signal sequence. B19R encodes a vaccinia surface protein (S antigen) expressed at early times post infection WO 92/1. 2 PCT/US92/01906 (Ueda et al., 1990). Both B16R and B19R show homology to the immunoglobin superfamily, especially the IL-1 receptor.
It has been suggested that one or both of the vaccinia gene products may help vaccinia evade the immune system by binding cytokines and thus diminishing the host inflammatory response (Smith et al., 1991).
Construction of Plasmid PMPTRB13GPT for Deletion of B13R Through B29R. Plasmid pMPRENDA, which was used as an intermediate in the generation of vaccinia deletion mutants vP759 and vP811 (Perkus et al., 1991) was modified by the addition of the SmaI expression cassette containing the EPV
42 kDa promoter/Ecogot gene at the pUC/vaccinia junction.
The resulting plasmid, pMPTRB13GPT, was transfected into NYVAC. Following selection using MPA, progeny virus were screened by plaque hybridization for loss of B15 DNA
sequences and retention of B12. Fidelity of the deletion junction was verified by PCR and DNA restriction analysis.
Recombinant virus vP953 contains the [B13R - B29R) deletion as well as the other deletions present in NYVAC.
Combining the Left [C23L - F4L1 and Right [B13R - B29R1 Deletions. The generation of vaccinia deletion mutant vP811, which contains deletions at both the left [C23L -F4L] and right [B13R - B29R) termini of vaccinia virus has been described (Perkus et al., 1991). vP811 contains both the vaccinia host range gene, C7L, and the selectable marker Ecogpt. To generate a virus containing the large terminal deletions without C7L or cogpt in a NYVAC background, pMPTRF4GPT was used as donor plasmid for recombination with vP953. Progeny virus is being selected by MPA in the transient dominant selection system described above and screened by plaque hybridization for loss of F4L DNA
sequences and retention of F5L. Recombinant virus vP977 contains deletions for [B13R-B29R] and [C23L-F4L] as well as the other deletions present in NYVAC. Like vP811, vP977 is deleted for all genes from both copies of the vaccinia terminal repeats.

EBample 18 - E7CPRE88ION OF HIV GENE PRODUCTS BY
HOST-RESTRICTED POXVIRUSES

This Example describes the generation of host-restricted pbxviruses that express HIV-1 gene products. The vectors employed are NYVAC and ALVAC.
Cells and Virus. NYVAC and ALVAC viral vectors and their derivatives were propagated as described previously (Piccini et al., 1989; Taylor et al., 1988a, b). Vero cells and primary chick embryo fibroblasts (CEF) were propagated as described previously (Taylor et al., 1988a, b). P815 murine mastocytoma cells (H-2d) were obtained from ATCC
(#TIB64) and maintained in Eagles MEM supplemented with 10%
fetal bovine serum CFBS and 100 Iu/ml penicillin and 100 g streptomycin per ml.
Mice. Female BALB/cJ (H-2d) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained on mouse chow and water ad libitum. All mice were used between the ages of 6 and 15 weeks of age.
Media. Assay Medium for immunological assays was comprised of RPMI 1640 medium supplemented with 10% FBS, 4 mM L-glutamine, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate), 5x10-5 M 2-mercaptoethanol, 100 IU
penicillin per ml, and 100 g/mi streptomycin. Stim Medium was comprised of Eagle's Minimum Essential Medium supplemented with 10% FBS, 4 mM L-glutamine, 10-4 M
2-mercaptoethanol, 100 IU penicillin per ml, and 100 g streptomycin per ml.
ALVAC and NYVAC Recombinants Containing the V3 Loop and Epitope 88 of the HIV-1 (IIIB) Envelope. A 150 bp fragment encompassing the V3 loop (amino acids 299-344; Javeherian et al., 1989) of HIV-1 (IIIB) was derived by PCR using oligonucleotides HIV3BL5 (SEQ ID NO:137) (5'-ATGGTAGAAA
TTAATTGTAC-3') and HIV3BL3 (SEQ ID NO:138) (5'-ATCATCGAATTCA
AGCTTATTATTTTGCTCTACTAATGTTAC-3') with pHXB.2D (III) as template (provided by Dr. R. C. Gallo, NCI-NIH, Bethesda, MD). Oligonucleotides HIV88A (SEQ ID NO:139) (5'-ATGAATGTGACAGAAAATTTTAACATGTGGAAAAATGTAGAAATTAATTGTACAAGACCC
-3') and HIV88B (SEQ ID NO:140) (5'-GGGTCTTGTACAATTAATTTCTACATTTTTCCACATGTTAAAATTTTCTGTCACATTCAT

W092/l. /2 PCT/L'S92/01906 -3') were annealed together to produce a double-stranded fragment containing the HIV-1 epitope 88 (amino acids 95-105, Shaffermann et al., 1989). The 150 bp V3-containing PCR fragment containing the epitope and the 42 bp fragment containing the 88 epitope sequences were fused together by PCR by virtue of the existence of complementary sequences.
The reactions were performed using oligonucleotides HIV88C
(SEQ ID NO:141) (5'-AGTAATGTGACAGAAAATTTTAAC-3') and HIV3BL3 (SEQ ID NO:138). The 192 bp PCR-derived fragment contains the epitope 88 sequences fused upstream to the V3 loop sequences. A termination codon (TAA) was incorporated into oligonucleotide HIV3BL3P to terminate translation of the open reading frame and an initiation codon was incorporated into oligonucleotide HIV88C to serve as the start of translation to express the epitope 88/V3 loop fusion protein. Additionally, oligonucleotide HIV3BL3 was synthesized so that an EcoRI site existed at the 3'-end of the 192 bp PCR fragment.
The entomopoxvirus 42 kDa (early) promoter was generated by PCR using oligonucleotides RG273 (SEQ ID
NO:142) (5'-AGGCAAGCTTTCAAAAAAATATAAATGATTC-3') and RG274 (SEQ ID NO:143) (5'-TTTATATTGTAATTATATATTTTC-3') with plasmid, pAM12, as template. The 108 bp fragment containing the 42 kDa promoter was synthesized to contain a HindIII
site at the 5'-end. The 42 kDa promoter containing segment was kinased and digested with HindIII prior to ligation to the epitope 88/V3 fragment digested with EcoRI and pRW831 digested with HindIII and EcoRI. The resultant plasmid was designated as pC5HIVL88. This plasmid was used in in vitro recombination assays with CPpp as rescue virus to generate vCP95. ALVAC recombinant, vCP95, contains the epitope 88/V3 loop in the de-ORFed C5 locus of CPpp.
The plasmid pC5HIVL88 was digested with HindIII and EcoRI to liberate a 300 bp fragment containing the epitope 88/V3 expression cassette described above. This fragment was excised from a LMP-agarose gel and isolated by phenol extraction (2X) and ether extraction (1X). The isolated fragment was blunt-ended using the Klenow fragment of the E.
coli DNA polymerase in the presence of 2mM dNTPs. The 'N92/15672 PCT/US92. )06 fragment was ligated to pSD550, a derivative of pSD548 (FIG.
6) digested with SmaI to yield plasmid pHIVL88VC. This plasmid was used with vP866 as the rescue virus to generate vP878. vP878 contains the epitope 88/V3 loop cassette in the de-ORFed 14L locus of NYVAC.
ALVAC- and NYVAC-Based Recombinants Expressing the HIV-1(IIIB) Envelope Glycoproteins. An expression cassette composed of the HIV-1 (IIIB) env gene juxtaposed 3' to the vaccinia virus H6 promoter (Guo et al., 1989; Taylor et al., 1988a, b) was engineered for expression of gp160 from HIV-1 by the ALVAC and NYVAC vectors. A 1.4 kb fragment was amplified from pHXB.2D (III) (provided by Dr. R.C. Gallo, NCI-NIH, Bethesda, MD) using oligonucleotides HIV3B1 (SEQ ID
NO:144) (5'-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3') and HIV3B5 (SEQ ID NO:145) (5'-ATCATCTCTAGAATAAAAATTATAGCAAAATCCTTTC-3'). This fragment contains the 3' portion of the env gene. PCR amplification with these primers placed a vaccinia virus early transcription termination T5NT sequence motif following the coding sequence and removed the T5NT motif situated at position 6146 to 6152 (Ratner et al., 1985) without altering the amino acid sequence. This change (T to C) creates an coRl (GAATTC) at this position. This 1.4 kb fragment was digested with coRI (5'- end) and KbaI (3'- end) and inserted into EcoRI and CbaI digested pBS-SK (Stratagene, La Jolla, CA). The resultant plasmid was designated as pBSHIVENVI,5. Nucleotide sequence analysis of this fragment demonstrated that the sequence was entirely correct except for a T to C transition at position 7848. This transition was corrected as follows: A 250 bp fragment was derived by PCR using oligonucleotides HIV3B1 (SEQ ID NO:144) (5'-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3') and HIV3B17 (SEQ
ID NO:146) (5'-TGCTACTCCTAATGGTTC-3') with pHXB.2D (III) as template. This fragment was digested with BRIII and EcoRI.
The fragment was inserted into pBSHIV3B1,5, digested with Bg1II and EcoRI and thus substituted for the region with the incorrect nucleotide to yield plasmid pBSHIV3BBP.
PCR was utilized to derive a 150 bp fragment containing the 5' portion of the env gene with oligonucleotides HIV3B9 WO 92/1_.,1? PCT/1JS92/01906 (SEQ ID NO:147) (5'-CATATGCTTTAGCATCTGATG-3') and HIV3B10 (SEQ ID NO:148) (5'-ATGAAAGAGCAGAAGACAGTG-3') with pHXB.2D
(III) as template. PCR was also used to generate a 128 bp fragment containing the vaccinia virus H6 promoter from pC3FGAG using oligonucleotides VVH65P (SEQ ID NO:149) (5'-ATCATCGGTACCGATTCTTTATTCTATAC-3') and VVH63P (SEQ ID NO:150) (5'-TACGATACAAACTTAACGG-3'). Both fragments were digested with KpnI and the 150 bp fragment was kinased prior to co-insertion of these fragments into pBS-SK digested with KpnI.
The resultant plasmid was designated as pBSH6HIV3B5P.
PCR was used to generate a 600 bp fragment from pHXB.2D
(III) with oligonucleotides HIV3B2 (SEQ ID NO:151) (5'-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3') and HIV3B7 (SEQ ID
NO:152) (5'-CAATAGATAATGATACTAC-3'). This fragment was digested with EcoRI and kinased. PCR was also used to derive a 500 bp fragment with the same template but with oligonucleotides HIV3B6 (SEQ ID NO:153) (5'-GTATTATATCAAGTTTATATAATAATGCATATTC-3') and HIV3B8 (SEQ ID
NO:154) (5'-GTTGATGATCTGTAGTGC-3'). This fragment was digested with KpnI. These fragments together correspond to nucleotide 5878 to 6368 (Ratner et al., 1985). The engineering of these fragments with these primers also removes a T5NT sequence positioned at nucleotide 6322 to 6328 without altering the amino acid sequence. These two fragments were inserted into pBSHIV3B3P digested with KpnI
and EcoRI. This plasmid was designated as pBSHIV3BP2768.
Plasmid pBSH6HIV3B5P was digested with KpnI to liberate a 360 bp fragment containing the H6 promoter and the 5' portion (150 bp) of the HIV-1 env gene. This KpnI fragment was ligated into pBSHIV3B3P2768 digested with KpnI to yield plasmid pBSHIV3BEAII. A 2.8 kb fragment was derived from pBSHIV3BEAII by digestion with XbaI followed by a partial KpnI digestion. This fragment was blunt-ended and inserted into SmaI digested pSD550. The plasmid pI4LH6HIV3B was generated and used in in vitro recombination experiments with vP866 as the rescue virus. This generated vP911 which contains the HIV-1 env gene in the 14L locus of the NYVAC
genome.

92/15672 PCT/US92/ )06 To insert the HIV-1 env gene into an ALVAC vector, pBSHIV3BEAII was digested with NruI and XbaI. The derived 2.7 kb fragment was blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs.
This fragment contains the entire HIV-1 env gene juxtaposed 3' to the 3'-most 21 bp (to NruI site) of the vaccinia H6 promoter. This fragment was ligated to a 3.1 kb fragment derived by digestion of pRW838 with NruI and EcoRI with subsequent blunt-ending with Klenow. The pRW838 derived fragment contains the homologous arms derived from canarypox to direct the foreign gene to the C5 locus. It also contains the 5'-most 100 bp of the H6 promoter. Therefore, ligation of these fragments resulted in an insertion plasmid containing an expression cassette for the HIV-1 env gene and was designated pC5HIV3BE. This plasmid was used in in vitro recombination experiments with ALVAC as the rescue virus to generate vCP112.
NYVAC-Based Recombinants Expressing the HIV-1 (IIIB) =120. The plasmid pBSHIV3BEAII was digested with EcoRI and XbaI to liberate a 4.3 kb fragment. This fragment contains the vaccinia virus H6 promoter linked to the HIV-1 env gene to nucleotide 6946 (Ratner et al., 1985). The 4.3 kb fragment was ligated to 300 bp coRI/CbaI digested PCR-derived fragment corresponding to the 3' portion of the gp120 coding sequence. The 300 bp PCR fragment was derived using oligonucleotides HIV1-120A (SEQ ID NO:155) (5' -ATCATCTCTAGAATAAAAATTATGGTTCAATTTTTACTACTTTTATATTATATATTTC-3') and HIV1-120B (SEQ ID NO:156) (5'-CAATAATCTTTAAGCAAATCCTC-3') with pHXB.2D as template. The ligation of the 4.3 kb CbaI/ZgoRI fragment and the 300 bp XbaI/EcoRI fragment yielded plasmid pBSHIVB120.
A 1.6 kb KDnI/XbaI fragment was derived from pBSHIVB120 by initially linearizing the plasmid with XbaI followed by a partial KpnI digestion. The 1.6 kb fragment was blunt-ended by treatment with the Klenow fragment of the E. coli DNA
polymerase in the presence of 2mM dNTPs. This fragment was inserted into pSD54IVC digested with SmaI to yield pATIHIVB120. This plasmid was used in in vitro recombination experiments to generate vP921. This WO 92/1. 2 PCT/US92/01906 recombinant contains the portion of the HIV-1 env gene encoding gp120 in the ATI locus of NYVAC.
Immunoprecitiitation. To determine the authenticity of the HIV-1 gerie products expressed by vP911, vP921 and vCP112, immunoprecipitation analyses were performed. Vero cells monolayers were either mock infected, infected with the parental virus vP866, or infected with recombinant virus at an m.o.i. of 10 PFU/cell. Following a 1 hr adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of MEM (minus methionine containing 2%
FBS and [35S)-methionine (20 uCi/mi). Cells were harvested at 18 hr post infection by the addition of 1 ml of 3X Buffer -A (3% NP-40, 30mM Tris pH 7.4, 150 mM NaCl, 3mM EDTA, 0.03%
Na Azide, and 0.6 mg/ml PMSF) with subsequent scraping of the cell monolayers.
Lysates derived from the infected cells were analyzed for HIV-i env gene expression using pooled serum from HIV-1 seropositive individuals (obtained from Dr. Genoveffa Franchini NCI-NIH, Bethesda MD). The sera was preadsorbed with vP866-infected Vero cells. The preadsorbed human sera was bound to protein A-sepharose in an overnight incubation at 4 C. In some cases a monoclonal antiserum specific to gp120 (Dupont) was used as the primary serum and a rat anti-mouse as the second antibody. Following this incubation period, the material was washed 4 times with iX Buffer A.
Lysates precleared with normal human sera and protein A-Sepharose were then incubated overnight at 4 C with the human sera from seropositive individuals bound to protein A-Sepharose. Following the overnight incubation period, the samples were washed four times with 1X Buffer A and 2X with LiC12/urea buffer. Precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20% glycerol; 10% 2-mercaptoethanol) and boiling for 5 minutes. Proteins were fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M Na - salicylate for fluorography.
The results of immunoprecipitation using sera pooled from HIV-1 seropositive individuals showed specific precipitation of the gp120 and gp4l mature forms of the gp160 envelope glycoprotein from vP911 infected cell lysates. No such specific gene products were detected in the parentally (NYVAC; vP866) infected cell lysates.
Specific precipitation of gp120 was also found in vP921 infected cell lysates.
Immunofluorescence analysis with the same sera illustrated that the gp160 and gp120 species expressed by vP911 and vP921, respectively, were present on the surface of infected cells.
Immunoprecipitation was also performed with vCP112 infected CEF cells. No HIV-specific polypeptides were precipitated with a monoclonal antibody directed against the gp120 extracellular moiety from cells infected with the ALVAC parental virus and uninfected CEF cells. Two HIV-specific polypeptides species were, however, precipitated from vCP112 infected cells. These species migrated with apparent mobilities of 160 kDa and 120 kDa, corresponding to the precursor env gene product and the mature extracellular form, respectively.
inoculations. Mice were intravenously inoculated with 5x107 plaque forming units (PFU) in 0.1 ml of phosphate-buffered saline via the lateral tail vein.
Spleen Cell PreRarations. Following euthanasia by cervical dislocation, the spleens of mice were aseptically transferred to a sterile plastic bag containing Hank's Balanced Salt Solution. Individual spleens or pooled spleens from a single experimental group were processed to single cell suspensions by a 1 minute cycle in a Stomacher blender. The spleen cell suspensions were washed several times in either Assay Medium or Stim Medium, as appropriate.
The spleen cells were enumerated by the use of a Coulter Counter or by trypan blue dye exclusion using a hemacytometer and microscope.
Sera. Mice were lightly anesthetized with ether and blood was collected from the retroorbital plexus. Blood from mice comprising an experimental group was pooled and allowed to clot. The serum was collected and stored at -70 C until use.

WO 92/1 '2 PCT/US92/01906 In Vitro Stimulation for the Generation of Secondary Cytotoxic T Lymphocytes (CTL). The pooled spleen cells from the various experimental groups (responders) were diluted to 5x106 cells/ml in Stim Medium. The spleen cells from syngeneic, naive mice (stimulators) were diluted to 1x107 cells per ml and infected for 1 hour in tissue culture medium containing 2% FBS at 37 C with the appropriate vaccinia virus at a m.o.i. of 25 pfu"per cell. Following infection, the stimulator cells were washed several times in Stim Medium and diluted to 1x106 cells per ml with Stim Medium. Five mis of stimulator cells and 5 mis of responder cells were added to a 25 cm3 tissue culture flask and incubated upright at 37 C, in 5% CO2 for 5 days. On the day of the assay, the spleen cells were washed several times in Assay Medium and counted on a hemacytometer in trypan blue with the use of a microscope.
Target Cell Preparation. For vaccinia specific CTL
activity, tissue culture cells were infected overnight by incubation at 1x107 cells per ml in tissue culture medium containing 2% FBS at a m.o.i. of 25 pfu per cell for 1 hour at 37 C. Following incubation, the cells were diluted to between 1 - 2x106 cells per ml with tissue culture medium containing 10% FBS and further incubated at 37 C, in 5% CO2 until use. For HIV specific CTL activity, tissue culture cells were incubated overnight with 20 g/ml of peptide HBX2 (American Biotechnologies, Cambridge, MA), SF2 (American Biotechnologies, Cambridge, MA) or MN (American Biotechnologies, Cambridge, MA) corresponding to the V3 loop region of gp120 of HIV-1 isolates IIIB, SF2, and MN, respectively. On the day of the assay, the targets were washed several times by centrifugation in Assay Medium.
After the final wash, the cells were resuspended in approximately 100 Ci of Na251CrO4 (51Cr). Following incubation at 37 C for 1 hr, the cells were washed at least 3 times in Assay Medium by centrifugation, counted on a hemacytometer, and diluted to 1x105/ml in Assay Medium.
Cytotoxicity Assays. For primary CTL assays, freshly prepared spleen cells were diluted with Assay Medium to 1x107 cells per ml. For secondary CTL assays (following 92/15672 PCT/US92i i06 either in vivo inoculation or in vi_tro stimulation), the spleen cells were diluted to 2x106/ml in Assay Medium. One tenth ml of spleen cell suspension was added to 51Cr labelled target cells in the wells of a 96 well, round-bottom microtiter plate (EXP). In most cases, the spleen cells being assayed for primary CTL activity were further 2-fold diluted in the wells of the microtiter plate prior to the addition of the target cells. As a measure of spontaneous release of 51Cr (SR), target cells were incubated in only Assay Medium. To determine the maximum release of 51Cr (MAX), target cells were deliberately lysed at the beginning of the assay by adding 0.1 ml of 10% sodium dodecyl sulfate to the appropriate wells. To initiate the assay, the microtiter plates were centrifuged at 200 x g for 2 min and incubated for 4 or 5 hrs at 37 C, in 5% CO2.
Following incubation, the culture supernatants of each well were collected using the Skatron Supernatant Collection System. Released 51Cr was determined by a Beckman 5500B
gamma counter. The percent specific cytotoxicity was determined from the counts by the following formula:
% CYTOTOXICITY = (EXP-SR)/(MAX-SR) X 100 Depletion of T Helper Cells and Cytotoxic T Lymphocytes Using Monoclonal Anti-CD4 and Monoclonal Anti-CD8. Spleen cell suspensions were diluted to a density of 107/ml in cytotoxicity medium (RPMI 1640 containing 0.2% BSA and 5 mM
HEPES) containing a 1:5 dilution of anti-CD4 (monoclonal antibody 172.4) or a 1:200 dilution of anti-CD8 (monoclonal antibody anti-Lyt 2.2) and a 1:16 dilution of Cedar Lane Low-Tox rabbit complement. Appropriate controls for the single components (complement, anti-CD4, anti-CD8) were included.
Anti-HIV-1 gp160 ELISA. The wells of ELISA plates (Immulon II) were coated overnight at 4 C with 100 ng of purified HIV-1 gp160 (provided by Dr. D. Bolognesi, Duke University, Durham NC) in carbonate buffer, pH 9.6. The plates were then washed with phosphate-buffered saline containing 0.05% Tween 20 (PBST). The plates were then blocked for 2 hrs at 37 C with PBST containing 1% bovine serum albumin (BSA). After washing with PBST, sera were WO 92/1_ 2 PCT/US92/01906 initially diluted 1:20 with PBST containing 0.1% BSA
(dilution buffer). The sera were further.2-fold serially diluted in the wells of the ELISA plate. The plates were incubated at 37 C for 2 hrs and washed with PBST.
Horseradish peroxidase conjugated rabbit anti-mouse immunoglobulins (DAKO) was diluted 1:2000 in dilution buffer and added to the wells of the ELISA plate and incubated at 37 C for 1 hour. After washing with PBST, OPD (o-phenylenediamine dihydrochloride) in substrate buffer was added and the color was allowed to develop at ambient temperature for about 20 minutes. The reaction was extinguished by the addition of 2.5 M H2S04. The absorbance at 490 nm was determined on a Bio-Tek EL-309 ELISA reader.
The serum endpoint was defined as the reciprocal of the dilution giving an absorbance value of 1Ø
Lvmphocyte Proliferation Assays. Single cell suspensions of the spleen cells of individual mice were diluted to 2x106/ml in Assay Medium and 0.1 ml was added to the wells of 96 well, flat-bottom microtiter plates containing Assay Medium alone, 1, 5, or 10 g of HIV-1 peptide T1, 1, 5, or 10 g of HIV-1 peptide T2, and 1 or 10 g of purified HIV-1 gp160 (Immuno). The cells were incubated for 5 days at 37 C, in 5% CO2. To each well was added 1.0 Ci of [3H]-thymidine for the final 6 hrs of incubation and then harvested onto Beckman Ready Filters using a Cambridge PHD cell harvester. The filter disks were dry-counted in a liquid scintillation counter.
STIMULATION INDEX = CPMsEXp/CPMsMEDIUM
Results: A Recombinant Vaccinia Virus Expressincr HIV qp120 Elicits Primary HIV-specific Cytotoxic T Lvmphocvte Activity. Following iv administration with 5x107 PFUs of vaccinia virus recombinants vP878, vP911, or vP921, or, as a control, with NYVAC, the vector, splenic CTL activity of BALB/c mice was assessed against syngeneic P815 cells which had been incubated overnight with peptide HBX2 (Table 20).
Modest, but significant (P<0.05) primary CTL activity was generated in the spleens of mice administered vP921, expressing HIV gp120. No other recombinant vaccinia virus nor the NYVAC parent vector was able to elicit primary HIV-~ 92/15672 PCT/US92/, J6 specific CTL activity. This was not due to inadequate infection as each group of mice administered a vaccinia virus responded with primary vaccinia-specific CTL activity.
Control, unimmunized mice responded to neither target.
Recombinant Poxviruses Expressing HIV env Peptides Generate HIV-Specific Memory Cytotoxic T LymDhocvtes. At least one month following a single inoculation with one of the recombinant vaccinia viruses, mouse spleen cells were stimulated in vitro with syngeneic, naive spleen cells previously infected with NYVAC or with each of the HIV
recombinant vaccinia viruses (Table 21). Strong HIV-specific CTL activity was detected only in the spleen cell cultures of mice immunized with vP878, vP911, and vP921 which were restimulated in vitro by cells infected with one of the same vaccinia virus HIV recombinants (vP878, vP911, or vP921).- The vaccinia virus recombinants expressing HIV
gp120 or gp160 were better able to generate memory CTLs than the vaccinia virus recombinant expressing only the V3 loop fused to the 88 epitope. HIV-specific memory CTL activity could not be elicited from unimmunized control or NYVAC
immunized spleen cells. The absence of HIV-specific CTL
activity from vector immunized mice could not be attributed to poor immunization since vaccinia-specific memory CTL
activity was apparent after in vitro stimulation with spleen cells infected with any of the vaccinia viruses used.
In a similar study, the ability of a canarypox recombinant expressing the V3 loop region fused to the 88 epitope (vCP95) to generate HIV-specific memory CTLs was examined (Table 22). Three weeks following a single inoculation of 108 PFLTs of vCP95 or the ALVAC vector, CPpp, HIV-specific memory CTL responses were compared to that elicited by the recombinant vaccinia virus analog, vP878.
Vaccinia and canarypox CTL responses were included as controls for proper immunization. Only spleen cells from vP878 and vCP95 immunized mice produced HIV-specific memory CTL activity which could be stimulated by vP878. The inability of vCP95 to stimulate existing memory CTLs to functional cytolytic CTLs may have been related to the in vitro conditions employed which were maximized based upon WO 92/1 _ .12 PCT/US92/01906 the use of vaccinia virus recombinants. Nonetheless, vCP95 was fully capable of generating significant HIV-specific memory CTLs in the spleens of immunized mice.
Characterization of the In Vitro Stimulated Cytotoxic Cells. It is conceivable that the cells mediating cytotoxicity against the HIV peptide-pulsed target cells represent a population of nonspecific effector cells unrelated to CTLs, such as natural killer cells. To test this, the spleen cells of mice immunized with vP921 and restimulated in vitro with vP921 infected spleen cells were depleted of T-lymphocytes bearing surface antigens characteristic of T helper lymphocytes (CD4) or of cytotoxic T lymphocytes (CD8) and assayed against V3 loop peptide pulsed target cells (Table 23). As before, only vP921 immunized mice generated memory HIV-specific CTL activity which could be stimulated in vitro with vP921 infected syngeneic spleen cells. Although the complement preparation (C') and the monoclonal anti-CD4 and anti-CD8 produced some toxic effects, only the cultures depleted of CD8-bearing cells (anti-CD8 + C') were also depleted of HIV-specific cytotoxic effector cells. Thus, the cells mediating cytolytic activity against the HIV peptide-pulsed target cells possessed CD8 antigens on their cell membranes, a characteristic of MHC class I restricted CTLs.
Specificity of CTL Antigen Receptor Recocrnition of the V3 Loop Region of HIV qp120. T lymphocyte antigen receptors are exquisitely sensitive to small alterations in the primary amino acid sequence of the epitope fragment. The V3 loop region of HIV gp120 is hypervariable and differs immunologically among HIV isolates. The hypervariability resides in substitutions and additions of only a few amino acids. To examine the specificity of cytotoxic cells generated by HIV vaccinia virus recombinants, susceptibility to CTL activity was compared among P815 target cells pulsed with peptides corresponding the V3 loop region of HIV
isolates IIIB, SF2, and MN. Only immunization with vP911 and vP921 induced HIV specific primary CTL activity (Table 24). Furthermore, HIV specific CTL activity was confined only to P815 target cells pulsed with peptide corresponding 92/15672 PCT/US92/. 06 to the V3 loop of HIV isolate IIIB. Similar results were obtained with in vitro stimulated, HIV specific secondary CTL activity induced by immunization with the vaccinia virus recombinants vP878, vP911, and vP921 (Table 25). Thus, HIV
specific CTLs elicited by recombinant vaccinia viruses expressing various portions of the env gene of HIV isolate IIIB recognize only target epitopes derived from the same antigenic isolate.
Lymohocyte Proliferation Responses to HIV Epitoties Following Immunization with Vaccinia Virus HIV Recombinants.
Lymphocyte proliferation to antigens is an in vitro correlate of cell-mediated immunity. Presentation of the appropriate antigen induces cellular proliferation in the immune population of cells expressing receptors for the antigen. The initiation and continuation of proliferation requires the involvement of T helper lymphocytes via soluble mediators. To evaluate cell-mediated immunity to HIV
antigens in mice immunized with recombinant vaccinia viruses expressing HIV antigens, spleen cells from mice immunized 27 days earlier were incubated for 5 days with peptides correlating to T helper lymphocyte epitopes designated T1 and TZ, as well as with purified HIV gp160 (Table 26). No proliferative responses to the T helper cell epitopes T1 and T2 were observed in any of the spleen cell cultures.
However, the spleen cells of mice previously immunized with vP921 vigorously responded to HIV gp160 as determined by the incorporation of [3H]-thymidine. A stimulation index (SI) of greater than 2.0 is considered indicative of immunity.
Thus, inoculation of mice with vP921 elicited cell-mediated immunity to HIV gp160.
Antibody Resgonses of Mice Inoculated with Vaccinia Virus HIV Recombinants. To evaluate humoral responses to HIV, mice were immunized at day 0 with one of the vaccinia virus HIV recombinants and received a secondary immunization at week S. The mice were bled at various intervals through 9 weeks after the initial immunization. Pooled sera from each treatment group were assayed for antibodies to HIV by ELISA employing purified gp160 as antigen (Table 27).
Primary antibody responses were generally modest, but WO 92/1_ 2 PCT/US92/01906 detectable with the highest levels induced by vP911.
Following the secondary immunization, the antibody titers of mice immunized with vP911 and vP921 increased and peaked at week 7 with titers of over 4,600 and 3,200, respectively, before declining slightly by week 9. Thus, two vaccinia virus HIV recombinants, vP911 and vP921, were capable of inducing a significant antibody response.

~ 92/15672 PCT/US92/- _ Y06 Table 20. Primary CTL activity of spleen cells from mice immunized with vaccinia virus recombinants against vaccinia virus infected targets and targets pulsed with peptide corresponding to the V3 loop region of HIV-1 gp120.

PERCENT CYTOTOXICITY
TARGET

NONE -3.5 -0.6 -4.8 2.0 1.5 1.6 NYVAC -4.4 9.5 * -5.9 1.9 3.2 1.7 vP878 -4.9 7.1 * -4.0 1.8 2.2 1.2 vP911 -4.0 4.6 * 1.4 2.5 2.0 5.1 vP921 -3.4 10.7 * 15.5 0.9 1.5 2.8 E:T = 100:1 ~ P<0.05 vs appropriate controls, Student's t-test WO 92/15.., 2 PCT/US92/01906 Table 21. Secondary CTL activity of spleen cells following in vitro stimulation with vaccinia virus recombinants.

PERCENT CYTOTOXICITY
IMMIINIZATION TARGET
in vivo in vitro P815 VAC HIV V3 NONE NONE -0.1 1.9 0.5 NYVAC 3.7 8.9 3.8 vP878 4.6 9.0 5.5 vP911 -1.7 2.9 4.8 vP921 2.9 2.9 1.5 NYVAC NONE 0.0 4.4 1.1 NYVAC 3.5 47.8 * 9.2 vP878 6.3 44.1 * 14.4 vP911 7.9 48.6 * 10.6 vP921 6.8 50.8 * 7.9 vP878 NONE 0.1 1.7 1.3 NYVAC 10.2 58.5 * 13.0 vP878 11.6 57.9 * 59.9 0 vP911 7.8 56.2 * 40.8 *
vP921 4.9 42.0 * 14.8 vP911 NONE 0.3 2.9 4.0 NYVAC 6.2 50.7 * 8.5 vP878 5.9 50.9 * 77.4 *
vP911 5.0 54.2 * 82.6 *
vP921 10.9 55.0 * 87.8 *
vP921 NONE 2.9 5.0 9.4 NYVAC 8.3 54.4 * 22.7 vP878 10.4 56.2 * 85.6 *
vP911 8.7 58.2 * 86.5 *
vP921 7.8 55.2 * 81.0 *
BALB/cJ spleen cells from mice immunized approximately 1 month earlier with the indicated vaccinia virus recombinants were incubated with infected syngeneic spleen cells for 5 days and assayed for cytotoxicity at an effector to target cell ratio of 20:1.
~ P<0.05 compared to controls, Student's t-test.

92/15672 PCf/US92A,.906 Table 22. Anamnestic CTL responses of the spleen cells of mice administered a single inoculation of recombinant vaccinia or canarypox virus expressing the V3 loop of HIV gp120.

IXII[UNIZATION
TARGET
PRIMARY BOOSTER
in vivo in vitro P815 Vac CP HIV V3 NONE NONE 0.4 -2.5 -2.3 -1.5 vP804 0.5 8.8 0.7 0.8 vP878 1.8 6.1 0.4 1.6 CP 5.8 4.2 4.9 0.4 vCP95 4.4 2.6 6.1 0.1 SB135 -0.2 -0.7 -0.4 0.5 vP804 NONE 0.7 1.7 0.1 1.3 vP804 5.5 43.5 * 5.8 3.5 vP878 3.6 42.5 * 1.6 -0.3 CP 8.5 7.0 5.6 3.9 vCP95 5.8 5.3 4.4 4.0 SB135 1.2 -0.9 -0.5 -0.2 vP878 NONE 0.2 -2.9 -0.8 -0.2 vP804 5.3 56.4 * 7.5 4.1 vP878 6.7 60.2 * 7.7 41.7 ~
CP 8.7 13.4 9.4 4.7 vCP95 7.1 10.5 8.7 19.0 SB135 1.9 -0.7 -0.2 -1.4 CP NONE 4.6 -0.6 2.3 -0.0 vP804 11.0 17.7 * 5.7 6.1 vP878 7.1 14.6 0 12.3 5.5 CP 7.4 5.9 19.3 * 3.1 vCP95 6.8 5.4 20.4 o 2.8 SB135 1.4 -0.4 0.8 -1.4 vCP95 NONE -0.8 -2.2 -1.3 0.3 vP804 9.4 26.4 * 9.3 6.6 vP878 10.4 22.5 * 16.9 32.1 ~
CP 8.8 7.2 20.0 * 3.2 vCP95 5.1 4.2 19.6 * 7.8 SB135 1.9 -1.5 -0.3 -1.2 Twenty-three days after immunization, the spleen cells were stimulated in vitro for 5 days with virus infected or peptide-pulsed syngeneic spleen cells and then assayed for specific cytotoxicity against virus infected or peptide-pulsed P815 target cells at an effector to target cell ratio of 20:1.
n P<0.05 compared to appropriate controls, Student's t-test.

WO 92/1: 2 PCT/US92/01906 Table 23. Depletion of cytotoxic activity with monoclonal antibodies to CD8 plus complement.

PERCENT CYTOTOZICITY
TARGETS
IXKUNIZATION
in vivo in vitro TREATMENT P815 VAC HIV V3 NONE NONE NONE 1.1 1.5 -0.3 NONE NYVAC NONE -7.4 0.4 -0.4 NONE vP921 NONE -0.2 1.1 -0.7 NYVAC NONE NONE -3.1 -0.3 -1.4 NYVAC NYVAC NONE -2.6 40.5 -0.3 NYVAC vP921 NONE 3.3 31.4 -2.9 vP921 NONE NONE 3.0 -1.3 -0.1 vP921 NYVAC NONE -4.9 25.9 12.2 vP921 vP921 NONE -0.2 21.3 30.5 vP921 vP921 C' 4.6 20.1 22.9 vP921 vP921 anti-CD4 4.2 22.6 23.2 vP921 vP921 anti-CD8 -5.0 22.5 26.9 vP921 vP921 anti-CD4+C' 10.0 26.6 30.1 vP921 vP921 anti-CD8+C' 9.2 7.1 2.3 92/15672 PCT/US92i,. - )06 Table 24. Specificity of primary CTL activity for the V3 loop of HIV-1 isolate IIIB following a single inoculation with HIV recombinant vaccinia viruses.

PERCENT CYTOTOXICITY
TARGET

IIrIIKIINIZATION P815 IIIB SF2 MN
NONE -2.7 -1.9 -0.9 -1.2 0.5 0.5 0.5 0.5 NYVAC -1.6 -0.3 -0.6 -0.3 0.5 0.8 0.7 0.2 vP878 -2.8 0.5 -0.5 -1.2 0.8 1.0 0.6 0.5 vP911 -2.6 7.5 * -0.5 -1.1 0.2 3.2 0.5 0.4 vP921 -2.5 12.5 * -0.1 -1.2 0.7 3.6 0.5 0.5 Mice were administered a single iv inoculation with the indicated vaccinia virus recombinant and assayed f or CTL activity 7 days later against P815 targets and P815 targets pulsed with one of three peptides corresponding to the V3 loop region of HIV-1 isolates IIIB, SF2, and MN. Although assayed at effector to target cell ratios of 100:1, 50:1, and 25:1, only the 100:1 data are shown.
P<0.05 vs appropriate controls, Student's t-test Table 25. Specificity of secondary CTL activity for the V3 loop of HIV-1 isolate IIIB
following a single inoculation with HIV
recombinant vaccinia viruses.

PERC$NT CYTOTOXICITY
TARGET
Il+IIXIINIZATION V3 PEPTIDE
in vivo in vitro P815 IIIB SF2 MN
NONE NONE 1.0 1.1 0.5 -0.0 NYVAC 0.4 0.5 -0.6 -0.3 vP878 0.2 0.2 -0.5 -1.0 vP911 -1.5 0.3 -0.5 0.2 vP921 -0.6 1.4 0.1 -0.5 NYVAC NONE -2.2 0.2 0.5 -1.0 NYVAC 3.2 2.2 3.9 2.5 vP878 4.4 5.9 5.0 6.1 vP911 5.8 11.1 5.0 5.3 vP921 5.0 6.5 2.9 2.9 vP878 NONE 0.1 -0.2 -0.9 -1.0 NYVAC 3.0 4.8 4.4 4.5 vP878 7.9 20.2 7.8 8.6 vP911 4.8 7.8 4.5 4.7 vP921 2.7 6.9 2.8 3.0 vP911 NONE 0.9 1.8 1.4 0.5 NYVAC 8.8 8.3 8.1 6.6 vP878 6.6 57.2 6.8 8.2 vP911 4.6 63.7 2.9 4.2 vP921 7.2 63.6 4.1 4.9 vP921 NONE 0.5 0.8 1.2 0.6 NYVAC 4.4 7.9 7.5 6.0 vP878 8.1 59.0 7.1 7.5 vP911 6.4 71.4 7.9 6.6 vP921 9.3 63.4 9.0 8.1 92/15672 PCf/US92/L i06 31 goa 'no ~ o E: w w ~S v, w ~ N O P w F r O N
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92/15672 PCT/US92 ')06 Example 19 - EXPRESSION OF THE HIV-1 (ARV-2 OR SF-2 STRAIN) env GENE IN ALVAC, TROVAC AND NYVAC
VECTORS

Plasmid Constructions. The lambda clone containing the entire HIV-1 (ARV-2 or SF-2 strain) genome was provided by J. Levy and was described previously (Sanchez-Pescador et al., 1985). The env sequences were subcloned into pUC13, creating plasmid pMP7MX373, which contains the sequences from -1 relative to the initiation codon (ATG) of the env gene product to 715 bp downstream of the termination codon (TAA) of the env gene. These env sequences were excised from pMP7MX373 by digestion with EcoRI and HindiII and inserted into the plasmid vector, pIBI25 (International Biotechnologies, Inc., New Haven, CT), generating plasmid pIBI25env.
Recombinant plasmid pIBI25env was used to transform competent E. coli CJ236 (dut- ung-) cells. Single-stranded DNA was isolated from phage derived by infection of the transformed E. coli CJ236 cells with the helper phage, MG408. This single-stranded template was used in vitro mutagenesis reactions (Kunkel et al., 1985) with oligonucleotide MUENVT12 (SEQ ID NO:157) (5'-AGAGGGG
AATTCTTCTACTGCAATACA-31). Mutagenesis with this oligonucleotide generates a T to C transition and disrupts the T5CT motif at nucleotide positions 6929-6935 of the ARV-2 genome (Sanchez-Pescador et al, 1985). This mutation does not alter the amino acid sequence of the env gene and creates an EcoRI site, which was used to screen for mutagenized plasmid clones. Sequence of confirmation was done by the dideoxynucleotide chain termination method (Sanger et al., 1977). The resultant mutagenized plasmid was designated as pIBI25mutenvll.
A 1.45 kb BalII fragment was derived from pIBI25mutenvll. This fragment contained the mutated env sequences. It was used to substitute for the corresponding unmutated fragment in pIBIenv. The resultant plasmid was designated as pIBI25mutenv8. Further modifications were made to pIBImutenv8. In vitro mutageneses were performed to remove the sequence coding for the rex protein and the LTR
sequence (LTR region) from the 3'-end of the gene and to delete the putative immuno-suppressive (IS) region amino acids 583 through 599) (SEQ ID NO:158) Leu-G ln-Ala-Arg-Val-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Arg-Asp-Gln-Gln-L,eu) (Klasse et al., 1988). These reactions were done with the single-stranded template derived from pIBImutenv8 with oligonucleotides LTR2 (SEQ ID N0:159) (5'-TTGGAAAGGCTTTTG-GCATGCCACGCGTC-3') and MUENSVISR (SEQ ID NO:160) (5'-ACAG
TCTGGGGCATCAAGCAGCTAGGGATTTGGGGTTGCTCT-3'). Mutagenized clones were identified by hybridization and restriction analysis. A clone mutagenized such that it was deleted both of the IS and the LTR region and another deleted of the LTR
was confirmed by nucleotide sequence analysis and designated pIBI25mut3env40 and pIBI25mut2env22, respectively.
A 3.4 kb SmaI/HindIII fragment containing the entire env gene was derived from pIBI25mut3env40 and from pIBImut2env22 and inserted into pCPCVl, digested with Smal/HindIII. The plasmid pCPCVl is an insertion plasmid which enables the generation of CP recombinants. The foreign genes were directed to the C3 insertion locus.
Plasmids pCPCVl and pFPCV2 have been described previously in PCT International Publication No. WO 89/034.29 published April 20, 1989.
Oligonucleotide PROVECNS (SEQ ID NO:161) (5'-CCGTTA
AGTTTGTATCGTAATGAAAGTGAAGGGGACCAGG-3') was used for in vitro mutagenesis reactions via the method of Mandecki (1986) to made a precise ATG:ATG construction with the VVH6 promoter and the env sequences. Potential mutants were screened for the loss of the Smal site. Plasmid clones devoid of a SmaI
site were identified and confirmed by nucleotide sequence analysis. Properly mutagenized plasmid clones were identified and designated as pCPenvIS+ or pCVenvIS- and pFPenvIS+ or pFPenvIS-.
The HIV-1 env genes were excised from pCPenvIS- by digestion with Nrul and HindIII. The two env fragmentsof 2.5 kb (envIS+) and 2.4 kb (envIS-), respectively, were isolated and blunt-ended by reaction with the Klenow fragment of the E. coli DNA polymerase in the presence Pf 2 mMdNTPs. These fragments were ligated with the 3.5 kb fragment derived by digestion of pSIVenvVV with NruI ail 92/15t, PCT/US920906 PstI with a subsequent blunting step with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. The plasmid pSIVenvVV contains the SIV env gene expression cassette regulated by the vaccinia virus H6 promoter in the ATI insertion locus. Digestion of pSIV env VV with NruI and PstI excises the entire SIV env coding sequences and the 3'-most 20 bp of the promoter element.
Ligation to the env IS- and env IS+ fragments restores the 20 bp of the H6 promoter and inserts the HIV-1 env gene into the ATI insertion plasmid. The resultant plasmids were designated as pAR5VV+ and pAR6VV- for env IS+ and env IS-, respectively.
In Vitro Recombination and Purification of Recombinants. Recombination was performed introducing plasmid DNA into infected cells by calcium phosphate precipitation both for CP (ALVAC) and for FP (TROVAC) recombinants, as previously described (Piccini et al., 1987). Plasmids pCPenvIS+ and pCPenvIS- (C5 locus, FIG. 16) were used to make recombinants vCP61 and vCP60 respectively.
Plasmids pFPenvIS+ and pFPenvIS- (F7 locus, FIG. 22) were used to make recombinants vFP63.and vFP62, respectively.
The plasmids pAR5VV+ and pAR6VV were used in in vitro recombination experiments with vP866 (NYVAC) as rescue to yield vP939 and vP940, respectively. Recombinant plaques were selected by autoradiography after hybridization with a 32P-labeled env specific probe and passaged serially three times to assure purity, as previously described (Piccini et al., 1987).
Radioimmunoprecipitation Analysis. Cell monolayers were infected at 10 pfu/cell in modified Eagle's methionine-free medium (MEM met-). At 2 hours post-infection, 20 uCi/ml of [35S] -methionine were added in MEM (-met) containing 2% dialysed fetal bovine serum (Flow). Cells were harvested at 15 hrs post-infection by resuspending them in lysis buffer (150 mM NaCl, 1mM EDTA pH 8, 10 mM Tris-HC1 pH 7.4, 0.2 mg/ml PMSF, 1% NP40, 0.01% Na Azide) and 50 l aprotinin, scraped into eppendorf tubes and the lysate was clarified by spinning 20 minutes at 4 C. One third of the supernatant of a 60 mm diameter Petri dish was incubated WO 92/i" '' PCT/US92/01906 with 1 l normal human serum and 100 l of protein A-Sepharose CL-4B (SPA) (Pharmacia) for 2 hours at room temperature. After spinning for 1 minute, the supernatant was incubated for 1 h 30 minutes at 4 C with 2 l preadsorbed human serum from HIV seropositive individuals (heat-inactivated) and 100 l SPA.
The pellet was washed four times with lysis buffer and two times with lithium chloride/urea buffer (0.2 M LiCl, 2 M
urea, 10 mM Tris-HC1 pH 8) and the precipitated proteins were dissolved in 60 l Laemmli buffer (Laemmli, 1970).
After heating for 5 minutes at 100 C and spinning for 1 minute to remove Sepharose, proteins in the supernatant were resolved on an SDS 10% polyacrylamide gel and fluorographed.
Expression of the HIV-1 env Gene. Six different recombinant viruses were prepared where the HIV env gene of the ARV-2 or SF-2 strain was inserted downstream from a vaccinia early-late promoter, H6. For simplicity, the two ALVAC-based recombinant viruses, vCP61 and vCP60, will be referred to as CPIS+ and CPIS-, the two TROVAC-based recombinants, vFP63 and vFP62, as FPIS+ and FPIS-, and the two NYVAC-based recombinants vP939 and vp940 as VV- and VV+, respectively.
All the constructs were precise, in that, the ATG
initiation codon of the HIV-1 env gene was superimposed on the ATG of the vaccinia H6 promoter. Moreover, all extraneous genetic information 3' to the termination codon was eliminated. CPIS-, FPIS-, and VV- were all obtained by deletion of a 51 bp region, corresponding to amino acids 583-599, located near the 5' portion of the gp4l gene product. This region shares homology with putative immunosuppressive regions (Kiasse et al., 1988, Ruegg et al., 1989b) occurring in the transmembrane polypeptide of other retrovirus glycoproteins (Cianciolo et al., 1985;
Ruegg et al., 1989a,b).
Expression analyses with all six recombinant viruses were performed in CEF, Vero, and MRC-5 cell monolayers.
Immuno-precipitation experiments using sera pooled from HIV
seropositive individuals were performed as described in Materials and Methods. All six recombinants directed the 92/15672 PC'T/US92' 106 synthesis of the HIV-1 gp161 envelope precursor. The efficiency of processing of gp160 to gp120 and gp4l, however, varied between cell types and was also affected by deletion of the immunosuppressive region. Recognition of gp41 by the pooled sera from HIV seropositive individuals =
also varied with the virus background and the cell type.
Example 20 - EYPREBBION OF THE HIV-2 (IBBY BTRAIN) env GENE IN NYVAC

Expression of gp160. Oligonucleotides HIV25PA (SEQ ID
NO. 162) (5'-ATGAGTGGTAAAATTCAGCTGCTTGTTGCCTTTCTGCTAACTAGTGCTTGCTTA-3') and HIV25PB (SEQ ID NO:163) (5'-TAAGCAAGCACTAGTTAGCAGAAAGGCAACAAGCAGCTGAATTTTACCACTCAT-3') were annealed to constitute the initial 54 bp of the HIV-2 SBL/ISY isolate (Franchini et al., 1989) env coding sequence. This fragment was fused 3' to a 129 bp fragment derived by PCR with oligonucleotides H65PH (SEQ ID NO:164) (5'-ATCATCAAGCTTGATTCTTTATTCTATAC-3') and H63PHIV2 (SEQ ID
NO:165) (5'-CAGCTGAATTTTACCACTCATTACGATACAAACTTAACG-3') using pTP15 (Guo et al., 1989) as template. The fusion of these two fragments was done by PCR using oligonucleotides HIV25PC (SEQ ID NO:166) (5'-TAAGCAAGCACTAGTTAG-3') and H65PH
(SEQ ID NO:164). The 174 bp PCR derived fragment was digested with HindIII and SacI and inserted into pBS-SK
(Stratagene, La Jolla, CA) digested with I~}' dIII and I.
The resultant fragment was designated pBSH6HIV2. The insert was confirmed by nucleotide sequence analysis.
The 3' portion of the HIV-2 env gene was also derived by PCR. In this reaction a 270 bp fragment was amplified with oligonucleotides HIV2B1 (SEQ ID NO:167) (5'-CCGCCTCTTGACCAGAC-3') and HIV2B2 (SEQ ID NO:168) (5'-ATCATCTCTAGAATAAAAATTACAGGAGGGCAATTTCTG-3') using pISSY-KPN
(provided by Dr. Genoveffa Franchini, NCI-NIH, Bethesda, MD) as template. This fragment was digested with BamHI and XbaI. The 150 bp fragment derived from this digestion contained a 5' DA_mHI and a 3' XbaI cohesive end. The fragment was engineered to contain a TSNT sequence motif known to be recognized as vaccinia virus early transcription WO 92/1' PCT/US92/01906 termination signal (Yuen et-al., 1987), following the termination codon (TAA).
The majority of the HIV-2 env gene was obtained from pISSY-KPN by digestion with SacI and BamHI. The 2.7 kb fragment generated by this digestion was coinserted into pBS-SK digested with SacI and XbaI with the 150 bp BamHI/XbaI fragment corresponding to the 3' end of the gene. The resultant plasmid was designated pBSHIV2ENV.
The 174 bp SpeI/HindIII fragment from pBSH6HIV2 and the 2.5 kb SpeI/XbaI fragment from pBSHIV2ENV were ligated into pBS-SK digested with IiindIII and XbaI to yield pBSH6HIV2ENV.
The 2.7 kb HindIII/XbaI insert from pBSH6HIV2ENV was isolated and blunt-ended with the Klenow fragment of the E.
coli DNA polymerase in the presence of 2mM dNTP. The blunt-ended fragment was inserted into a SmaI digested pSD5HIVC
insertion vector. The resultant plasmid was designated as pATIHIV2ENV. This plasmid was used in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP920.
Immunoprecipitation analysis was performed to determine whether vP920 expresses authentic HIV-2 gp160. Vero cell monolayers were either mock infected, infected with the parental virus vP866, or infected with vP920 at an m.o.i. of PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S] -methionine (20 Ci/ml).
Cells were harvested at 18 hrs post-infection by the addition of 1 ml 3X buffer A(3$ NP-40, 30 mM Tris pH 7.4, 150 mM NaCl, 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) with subsequent scraping of the cell monolayers.
Lysates from the infected cells were analyzed for HIV-2 env gene expression using pooled serum from HIV-2 seropositive individuals (obtained from Dr. Genoveffa Franchini). The sera was preadsorbed with vP866 infected Vero cells. The preadsorbed human sera was bound to Protein A-sepharose in an overnight incubation at 4 C. Following this incubation period, the material was washed 4X with 1X
buffer A. Lysates precleared with normal human sera and P") 92/15672 PCT/US92' 406 protein A-sepharose were then incubated overnight at 4 C
with the human sera from seropositive individuals bound to protein A-sepharose. After the overnight incubation period, the samples were washed 4X with 1X buffer A and 2X with a LiC12/urea buffer. Precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmlis buffer (125mM Tris(pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M Na-salicylate for fluorography.
Human sera from HIV-2 seropositive individuals specifically precipitated the HIV-2 gp160 envelope glycoprotein from vP920 infected cells. Furthermore, the authenticity of the expressed HIV-2 env gene product was confirmed, since the gp160 polyprotein is processed to the mature gp120 and gp41 protein species. No HIV-specific protein species were precipitated from mock-infected cells or cells infected with the NYVAC parental virus. Also, supportive of the proper expression of the HIV-2 env by vP920 was the observation that the gene product is expressed on the surface of vP920 infected cells.
Expression of qp120. The plasmid pBSH6HIV2 containing the vaccinia virus H6 promoter fused to the 5'-end of the HIV-2 env gene was digested with SpeI and HindIII to liberate the 180 bp fragment containing these sequences.
This fragment was ligated into pBS-SK digested with indIII
and XbaI along with the 1.4 kb SpeI/XbaI fragment of pBSHIV2120A to yield pSHIV2120B.
The plasmid pBSHIV2120A was derived by initially deriving the 3' portion of the gp120 coding sequence by PCR.
The PCR was performed using oligonucleotides HIV2120A (SEQ
ID NO:169) (5'-ATCATCTCTAGAATAAAAATTATCTCTTATGTCTCCCTGG-3') and HIV2120B (SEQ ID NO:170) (5'-AATTAACTTTACAGCACC-3') with pISSY-KPN as template. The PCR-derived fragment was digested with EcoRI and XbaI to yield a 300 bp fragment which contained a 5'-EcoRI cohesive end and a 3'-XbaI
cohesive end. The fragment was engineered with a translation termination sequence (TAA) and a T5NT sequence motif just 5' to the XbaI site. The 300 bp XbaI/EcoRI PCR
fragment was ligated into pBS-SK digested with SacI/XbaI
along with a 1.4 kb SacI/EcoRI fragment derived from pISSY-KPN to generate pBSHIV2120A.
The plasmid pBSHIV2120B was digested with ~'ndIII and XbaI to generate a 1.8 kb fragment containing the HIV-2 gp120 coding sequence juxtaposed 3' to the vaccinia virus H6 promoter. This fragment was blunted with the Klenow fragment of the E. coli DNA polymerase in the presence of 2 mM dNTPs. The blunt-ended fragment was ligated to SmaI
digested pSDSHIVC to generate pATI HIV 2120. This plasmid was used in in vitro recombination experiments to yield vP922.
Immunoprecipitation experiments with vP922 infected cells were performed as described above for the expression of the entire HIV-2 env gene. No HIV-specific species were precipitated from mock infected or vP866 infected Vero cells. A protein species of 120 kDa was, however, precipitated from lysates derived from cells infected with vP922. The HIV-2 gp120 expressed by vP920 was found to be present on the cell surface of vP920 infected Vero cells.
EBample 21 - EXPRESSION OF SIV GENES IN NYVAC
Generation of NYVAC/SIV gp140 Recombinant. A plasmid pSSIIE containing the SIV (Macl42) env gene was obtained from Dr. Genoveffa Franchini (NCI-NIH, Bethesda, MD). This plasmid was digested with IiindlII and PstI to liberate a 2.2 kbp fragment containing from nucleotide 220 of the SIV env gene to a region 160 bp downstream from the translation termination codon. It should be noted that an expression cassette containing this fragment will result in the expression of a gp140 protein species rather that a gp160 species. This 40% deletion of the transmembrane region results from a premature termination at nucleotide 7,934 of the genome (Franchini et al., 1987). Such premature terminations of the env gene product are noted after propagation of SIV in culture (Kodama et al., 1989).
The amino portion of the gene was derived by PCR using pSSIIE as template and oligonucleotides SIVENV1 (SEQ ID
N0:171) (5'-CGATATCCGTTAAGTTTGTATCGTAATGGGATGTCTTGGGAATC-3') = 92/15672 PCT/US92, )06 and SIVENV2 (SEQ ID NO:172) (5'-CAAGGCTTTATTGAGGTCTC-3').
The resultant 250 bp fragment contains the 5'-most 230 bp of the SIV env gene juxtaposed downstream from the 3'-most 20 bp of the vaccinia virus H6 promoter (3'-end of ruI site).
A 170 bp fragment was obtained by digestion of the fragment with HindIII, which removes 80 bp of SIV env sequences.
The sequences containing the remainder of the SIV env gene following the premature termination signal were derived by PCR from pSS35E (obtained from Dr. Genoveffa Franchini).
This plasmid contains sequences containing the C-terminal portion of the SIV env gene into the LTR region downstream from the env gene. The oligonucleotides used to derive the 360 bp fragment were SIVENV3 (SEQ ID NO:173) (5'-CCTGGCCTTGGCAGATAG-3') and SIVENV4A (SEQ ID NO:174) (5'-ATCATCGAATTCAAAAATATTACAAAGAGCGTGAGCTCAAGTCCTTGCCTAATCCTCC-3'). This fragment was digested with PstI and EcoRI to generate a 260 bp fragment having a 5' PstI cohesive end and a 3' EcoRI cohesive end.
The 2.2 kb HindIII/PstI fragment from pSSIIE, the 170 bp NruI/HindIII fragment containing the 5' end of the gene, and the 260 bp PstI/EcoRI containing the 3' end of the gene were ligated with a 3.1 kb NruI/EcoRI fragment derived from pRW838. pRW838 contains the vaccinia virus H6 promoter linked to the rabies G gene flanked by canarypox virus sequences which enable the insertion of genes into the C5 locus. Digestion with NruI and coRI liberates the rabies G
gene and removes the 3'-most 20 bp of the H6 promoter. The resultant C5 insertion plasmid containing the SIV env gene linked to the vaccinia H6 promoter was designated as pC5SIVENV.
The plasmid, pC5SIVENV, was digested with HindIII and EcoRI to liberate a 2.2 kb fragment, containing from nucleotide 150 of the SIV env gene to the end of the entire gene. PCR was used to derive the vaccinia H6 promoter/SIV
env linkage from pC5SIVENV with oligonucleotides MPSYN286 (SEQ ID NO:175) (5'-CCCCCCAAGCTTFTTTATTCTATACTT-3') and SIVENV2 (SEQ ID NO:176) (5'-CAAGGCTTTATTGAGGTCTC-3'). The 320 bp fragment was digested with HindIII to derive a 240 bp fragment. The 2.2 kb HindIII/EcoRI and the 240 bp HindIII

WO 92/1? ' PCr/US92/01906 fragment were coligated into pC3I digested with HindIII and coRl. The resultant plasmid containing the indIII
fragment in the proper orientation relative to the SIVenv coding sequence was designated pC3SIVEM. The plasmid pC3I
was derived as follows. The nucleotide sequence analysis of an 2.5 kb BalII canarypoxvirus genomic fragment revealed the entire C3 open reading frame and the 5' and 3' noncoding regions. In order to construct a donor plasmid for insertion of foreign genes into the C3 locus with the complete excision of the C3 open reading frame, PCR primers were used to amplify the 5' and 3' sequences relative to C3.
Primers for the 5' sequences were RG277 (SEQ ID NO:177) (5'-CAGTTGGTACCACTGGTATTTTATTTCAG-3') and RG278 (SEQ ID NO:178) (5'-TATCTGAATTCCTGCAGCCCGGGTTTTTATAGCTAATTAGTCAAATGTGAG
TTAATATTAG-3').
Primers for the 3' sequences were RG279 (SEQ ID NO:179) (5'-TCGCTGAATTCGATATCAAGCTTATCGATTTTTATGACTAGTTAATCAAATA
AAAAGCATACAAGC-3') and RG280 (SEQ ID NO:180) (5'-TTATCGAGCTCTGTAACATCAGTATCTAAC-3'). The primers were designed to include a multiple cloning site flanked by vaccinia transcriptional and translation termination signals. Also included at the 5'-end and 3'-end of the left arm and right arm were appropriate restriction sites (Asp718 and EcoRI for left arm and coRI and SacI for right arm) which enabled the two arms to ligate into ,sp718/SacI
digested pBS-SK plasmid vector. The resultant plasmid was designated as pC3I.
The plasmid pC3SIVEM was linearized by digestion with coRI. Subsequent partial digestion with HindIII liberated a 2.7 kb HindIII/EcoRI fragment. This fragment was blunt-ended by treatment with Klenow fragment of the E. coli DNA
polymerase in the presence of 2mM dNTPs. The fragment was ligated into pSD550VC digested with maI. The resultant plasmid was designated as pSIVEMVC. This plasmid was used in in vitro recombination experiments with vP866 as rescue virus to generate vP873. vP873 contains the-SIV env gene in the I4L locus.
Generation of a NYVAC/gag/Dol and aaa Recombinants. A
plasmid, pSIVAGSSIIG, containing the SIV cDNA sequence ' 92/15672 PCT/US92: 06 encompassing the gag and pol genes was obtained from Dr.
Genoveffa Franchini (NCI-NIH, Bethesda, MD). The gag and pol genes from this plasmid were juxtaposed 3' to the vaccinia 13L promoter between vaccinia tk flanking arms.
This was accomplished by cloning the 4,800 bp CfoI/TaaI
fragment of pSIVGAGSSIIG, containing the gaa and the oligonucleotides SIVL1 (SEQ ID NO:181) (5'-TCGAGTGAGATAAAGTGAAAATATATATCATTATATTACAAGTA
CAATTATTTAGGTTTAATCATGGGCG-3') and SIVL2 (SEQ ID NO:182) (5'-CCCATGATTAAACCTAAATAATTGTACTTTGTAATATAATGCTATATATTTT
CACTTTATCTCAC-3') corresponding to the 13L promoter into the 4,070 bp XhoI/AccI fragment of pSD542, a derivative of pSD460 (FIG. 1). The plasmid generated by this manipulation was designated pSIVG1.
To eliminate the Qol gene, a 215 bp PCR fragment was derived from pSIVGAGSSIIG using oligonucleotides SIVP5 (SEQ
ID NO:183) (5'-AATCAGAGAGCAGGCT-3') and SIVP6 (SEQ ID
NO:184) (5'-TTGGATCCCTATGCCACCTCTCT-3'). The PCR-derived fragment was digested with BamHI and StuI and ligated with the 5,370 bp partial DamHI/S uI fragment of SIVG1. This resulted in the generation of pSIVG2. pSIVG2 was used in in vitro recombination experiments with vP873 as rescue virus to yield vP948.
The plasmid to insert both gact and pol into NYVAC-based vectors was engineered in the following manner. pSIVG1, described above, contains extraneous 3'-noncoding sequences which were eliminated using a 1 kb PCR fragment. This fragment was generated from plasmid pSIVGAGSSIIG with the oligonucleotides SIVP5 and SIVP6. This PCR derived fragment containing the 3' end of the pol gene was digested with BamHI and HpaI. The 1 kb BamHI/HpaI fragment was ligated to the 7,400 bp partial BamHI/HnaI fragment of pSIVG1 to yield pSIVG4.
Sequence analysis of pSIVG4 revealed a single base pair deletion within the pol gene. To correct this error the 2,300 bp Bg1II/StuI fragment from pSIVG1 was inserted into the 6,100 bp partial BalII/StuI fragment of pSIVG4 to yield pSIVG5. The plasmid, pSIVG5, was used in in vitro WO 92/1' '= PCT/US92/01906 recombination experiments with vP873 as rescue to generate vP943.
Generation of NYVAC/SIV D16 and p28 Recombinants. The pol gene and the portion of the Qaa gene encoding p28, p2, p8, pl, and p6 were eliminated from pSIVG1. This was accomplished by cloning the oligonucleotides SIVL10 (SEQ ID
NO:185) (5'-AGACCAACAGCACCATCTAGCGGCAGAGGAGGAAATTACTAATTTTT
ATTCTAGAG-3') and SIVLll (SEQ ID NO:186) (5'-GATCCTCTA
GAATAAAAATTAGTAATTTCCTCCTCTGCCGCTAGATGGTGCTGTTGGT-3') into the 4,430 bp AccI/BamHI fragment of pSIVG1 to generate pSIVG1 to generate pSIVG3. This plasmid contains an expression cassette for the SIV p17 gene product expressed by the vaccinia l3L promoter.
The entomopoxvirus 42 kDa-promoted SIV p28 gene (5' end only) was inserted downstream from the 13L-promoted p17 gene. This was accomplished by cloning the 360 bp BSDMI/BamHI fragaient of pSIVGI, containing the 5' end of the p28 gene, the oligonucleotides pSIVL14 (SEQ ID NO:187) (5'-TAGACAAAATTGAAAATATATAATTACAATATAAAATGCCAGTACAACAAATAGGTGGTA
ACTATGTCCACCTGCCATT-3') and SIVL15 (SEQ ID NO:188) (5'-GCTTAATGGCAGGTGGACATAGTTACCACCTATTTGTTGTACTGGCATTTTATATTGTAA
TTATATATTTTCAATTTTGT-3'), containing the entomopox 42 kDa promoter into the 4,470 bp partial XbaI/BamHI fragment of pSIVG3. The resultant plasmid was designated as pSIVG6.
The 3' portion of the p28 gene was then inserted into pSIVG6. A 290 bp PCR fragment, containing the 3' end of the SIV p28 gene, was derived from pSIVGI using oligonucleotides SIVP12 (SEQ ID NO:189) (5'-TGGATGTACAGACAAC-3') and SIVP13 (SEQ ID NO:190) (5'-AAGGATCCGAATTCTTACATTAATCTAGCCTTC-3').
This fragment was digested with BamHI and ligated to the 4,830 bp BamHI fragment of pSIVG7, was used in in vitro recombination experiments with vP866 and vP873 as rescue experiments to generate vP942 and vP952, respectively.
Expression Analyses. The SIV gp140 env gene product is a typical glycoprotein associated with the plasma membrane of infected cells. It is expressed as a polyprotein of 140 kDa that is proteolytically cleaved to an extracellular species of 112 kDa and a transmembrane region of 28 kDa (Franchini et al., 1987). Immunofluorescence analysis using WO 92/15672 PCT/US92/'"906 sera from rhesus macaques seropositive for SIV followed by fluorescein conjugated rabbit anti-monkey IgG demonstrated expression of the env gene product on the surface of recombinant infected Vero cells. Surface expression was not detectable on the surface of mock infected cells or cells infected with the NYVAC (vP866) parent virus. Furthermore, cells infected with recombinants containing only gaa genes were not shown to express any SIV components on the surface.
Surface expression in cells infected with vP873, vP943, vP948 and vP952 all demonstrated surface expression and significantly, all contain the SIV env gene.
The authenticity of the expressed SIV gene products (env and aacx) in Vero cells infected with the NYVAC/HIV
recombinants was analyzed by immunoprecipitation. Vero cells were infected at an m.o.i. of 10 with the individual recombinant viruses, with the NYVAC parent virus, or were mock infected. After a 1 hour adsorption period, the inoculum was removed and infected cells were overlayed with 2 ml methionine-free media containing [35S]-methionine (20 Ci/ml). All samples were harvested at 17 hours post infection by the addition of 1 ml of 3X Buffer A. Lysates from the infected cells were analyzed with pooled sera from SIV seropositive rhesus macaques or a monoclonal antibody specific for gaa p24 gene product (both obtained from Dr.
Genoveffa Franchini, NCI-NIH, Bethesda MD).
Immunoprecipitation with the SIV seropositive macaque sera was performed in the following manner. The macaque sera were incubated with protein A-sepharose at 4 C for 16 hours. After washing with buffer A, the sera bound to protein A sepharose were added to lysates precleared with normal monkey sera and protein A sepharose. Following an overnight incubation at 4 C the precipitates were washed 4 x with buffer A and 2 x with LiCl/urea buffer. To dissociate the precipitated protein from the antibody, the samples were boiled in 80 l 2 x Laemmli buffer for 5 minutes. The samples were fractionated on a 12.5% gel using the Dreyfuss gel system (Dreyfuss et al., 1984). The gel was fixed and treated with 1 M Na-salycate for fluorography.

WO 92/1; PCT/US92/01906 All the recombinants containing SIV genes were expressing the pertinent gene products. The NYVAC recombinants vP873, vP943, vP948 and vP952 which contain the SIV env gene all expressed the authentic gp140. However, it is difficult to assess the processing of the gp140 protein to the 112 kDa and 28 kDa mature forms. No species with an apparent molecular weight of 140 kDa was precipitated by macaque anti-SIV sera from mock infected Vero cells, vP866 infected Vero cells and Vero cells infected with a NYVAC/SIV
recombinant not containing the SIV env gene. Expression of the SIV gaQ encoded gene products by vP942, vp943, vp948, and vP952 was demonstrated using the pooled sera from macaques infected with SIV and the monoclonal antibody specific to the p28 gaq component. Expression of the entire p55 gag protein without the pol region, which contains the protease function, by NYVAC (vP948) in Vero cells is evident. These results demonstrate that lack of SIV
protease expression prevents the complete proteolysis of p55 into its mature form. This is demonstrated much more clearly when a monoclonal antibody specific to p28 was used to precipitate gaq specific gene products from vP948 infected Vero cells. Contrary to this result, expression of SIV ctact with the pol gene (includes protease) in vP943 infected Vero cells enabled the expressed p55 gag precursor polypeptide to be proteolytically cleaved to its mature f orms .
Expression of both the p16 and p28 SIV gene products in vP942 and vP952 infected Vero cells was demonstrated using the pooled sera from macaques infected with SIV. Using the monoclonal antibody specific to p28 obviously only recognized the p28 expressed component.
Esample 22 - CONSTRUCTION OF TROVAC RECO2+tBINANTB
EXPRESSING THE HEMAGGLIITININ GLYCOPROTEINS OF
AVIAN INFLIIENZA VIRUSES

This Example describes the development of fowlpox virus recombinants expressing the hemagglutinin genes of three serotypes of avian influenza virus.
Cells and Viruses. Plasmids containing cDNA clones of the H4, H5 and H7 hemagglutinin genes were obtained from Dr.
Robert Webster, St. Jude Children's Research Hospital, 92/15672 PCT/L'S92' 106 Memphis, Tennessee. The strain of FPV designated FP-1 has been described previously (Taylor et al., 1988a, b). 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 scab from a chicken. The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on chick embryo fibroblast (CEF) cells. This virus was 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 was subjected to four successive plaque purifications. One plaque isolate was further amplified in primary CEF cells and a stock virus, designated as TROVAC, was established. The stock virus used in the in vitro recombination test to produce TROVAC-AIH5 (vFP89) and TROVAC-AIH4 (vFP92) had been further amplified though 8 passages in primary CEF cells. The stock virus used to produce TROVAC-AIH7 (vFP100) had been further amplified through 12 passages in primary CEF cells.
Construction of Fowlpox Insertion Plasmid at F8 Locus.
Plasmid pRW731.15 contains a 10 kbp PvuII-PvuII fragment cloned from TROVAC genomic DNA. The nucleotide sequence was determined on both strands for a 3661 bp PvuII-EcoRV
fragment. This sequence is shown in FIG. 21. 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 704 and terminates at position 1888. In order not to interfere with neighboring open reading frames, the deletion was made from position 781 to position 1928, as described below.
Plasmid pRW761 is a sub-clone of pRW731.15 containing a 2430 bp EcoRV- coRV fragment. The F8 ORF was entirely contained between an XbaI site and an SSDI site in PRW761.
In order to create an insertion plasmid which, on recombination with TROVAC genomic DNA would eliminate the F8 ORF, the following steps were followed. Plasmid pRW761 was completely digested with XbaI and partially digested with SSDI. A 3700 bp XbaI-SSDI band was isolated and ligated WO 92/1 '2 PCT/US92/01906 with the annealed double-stranded oligonucleotides JCAO17 (SEQ ID NO:191) and JCA018 (SEQ ID NO:192).
JCA017 (SEQ ID NO:191) 5' CTAGACACTTTATGTTTTTTAATATCCGGTCTT
AAAAGCTTCCCGGGGATCCTTATACGGGGAATAAT 3' JCA018 (SEQ ID NO:192) 5' ATTATTCCCCGTATAAGGATCCCCCGGGAA
GCTTTTAAGACCGGATATTAAAAAACATAAAGTGT 3' The plasmid resulting from this ligation was designated pJCA002. Plasmid pJCA004 contains a non-pertinent gene linked to the vaccinia virus H6 promoter in plasmid pJCA002.
The sequence of the vaccinia virus H6 promoter has been previously described (Taylor et al., 1988a, b; Guo et al.
1989; Perkus et al., 1989). Plasmid pJCA004 was digested 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:193) and RW179 (SEQ ID
NO:194) were cut with coRV and BamHI and inserted between the EcoRV and BamHI sites of JCA004 to form pRW846.
RW178 (SEQ ID NO:193): 5' TCATTATCGCGATATCCGTGTTAACTAGCTA
GCTAATTTTTATTCCCGGGATCCTTATCA 3' RW179 (SEQ ID NO:194): 5' GTATAAGGATCCCGGGAATAAAAATTAGCT
AGCTAGTTAACACGGATATCGCGATAATGA 3' Plasmid pRW846 therefore contains the H6 promoter 5' of EcoRV in the de-ORFed F8 locus. The incII site 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 Smai site.
Construction of Fowlvox Insertion Plasmid at F7 Locus.
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 HincII site within these sequences. The nucleotide sequence shown in FIG. 22 was determined for a 2356 bp region encompassing the unique HincII site. Analysis of this sequence revealed that the unique HincII site (FIG. 22, underlined) was situated within an ORF encoding a polypeptide of 90 amino acids. The ORF begins with an ATG at position 1531 and terminates at position 898 (positions marked by arrows in FIG. 22).

= 192/I5672 PC'T/US92 )06 The arms for the de-ORFed insertion plasmid were derived by PCR using pRW731.13 as template. A 596 bp arm (designated as HB) corresponding to the region upstream from the ORF was amplified with oligonucleotides F73PH2 (SEQ ID
NO:195) (5'-GACAATCTAAGTCCTATATTAGAC-3') and F73PB (SEQ ID
NO:196) (5'-GGATTTTTAGGTAGACAC-3'). A 270 bp arm (designated as EH) corresponding to the region downstream from the ORF was amplified using oligonucleotides F75PE (SEQ
ID NO:197) (5'-TCATCGTCTTCATCATCG-3') and F73PH1 (SEQ ID
NO:198) (5'-GTCTTAAACTTATTGTAAGGGTATACCTG-3').
Fragment EH was digested with coRV 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 HincII site.
This fragment was inserted into pBS-SK (Stratagene, La Jolla, CA) digested with HincII to form plasmid pF7D1. The sequence was confirmed by dideoxynucleotide sequence analysis. The plasmid pF7D1 was linearized with ApaI, blunt-ended using T4 DNA polymerase, and ligated to the 596 bp HB fragment. The resultant plasmid was designated as pF7D2. The entire sequence and orientation were confirmed by nucleotide sequence analysis.
The plasmid pF7D2 was digested with EcoRV and Bg1II to generate a*600 bp fragment. This fragment was inserted into pBS-SK that was digested with gpaI, blunt-ended with T4 DNA
polymerase, and subsequently digested with BamHI. The resultant plasmid was designated as pF7D3. This plasmid contains an HB arm of 404 bp and a EH arm of 126 bp.
The plasmid pF7D3 was linearized with XhoI and blunt-ended with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. This linearized plasmid was ligated with annealed oligonucleotides F7MCSB (SEQ ID
NO:199) (5'-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTT
TTTATTAGTTTAGTTAGTC-3') and F7MCSA (SEQ ID NO:200) (5'-GACTAACTAACTAATAAAAAACCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAA
TCGTT-3'). This was performed to insert a multiple cloning region containing the restriction sites for HindIII, PstI
and SmaI between the EH and HB arms. The resultant plasmid was designated as pF7DO.

WO 92/1: ~ PCT/US92/01906 Construction of Insertion Plasmid for the H4 Hemaaalutinin at the F8 Locus. A cDNA copy encoding the avian influenza H4 derived from A/Ty/Min/833/80 was obtained from Dr. R. Webster in plasmid pTM4H833. The plasmid was digested with HindIII and NruI and blunt-ended using the Klenow fragment of DNA polymerase in the presence of dNTPs.
The blunt-ended 2.5 kbp HindIII-NruI fragment containing the H4 coding region was inserted into the HincII site of pIBI25 (International Biotechnologies, Inc., New Haven, CT). The resulting plasmid pRW828 was partially cut with BanII, the linear product isolated and recut with HindIII. Plasmid pRW828 now with a 100 bp HindIII-BanII deletion was used as a vector for the synthetic oligonucleotides RW152 (SEQ ID
NO:201) and RW153 (SEQ ID NO:202). These oligonucleotides represent the 3' portion of the H6 promoter from the EcoRV
site and align the ATG of the promoter with the ATG of the H4 cDNA.
RW 152 (SEQ ID NO:201): 5' GCACGGAACAAAGCTTATCGCGATATCCGTTA
AGTTTGTATCGTAATGCTATCAATCACGATTCTGT
TCCTGCTCATAGCAGAGGGCTCATCTCAGAAT 3' RW 153 (SEQ ID NO:202): 5' ATTCTGAGATGAGCCCTCTGCTATGAGCAGGA
ACAGAATCGTGATTGATAGCATTACGATACAAACT
TAACGGATATCGCGATAAGCTTTGTTCCGTGC 3' The oligonucleotides were annealed, cut with BanII and indIlI and inserted into the HindIII-BanII deleted pRW828 vector described above. The resulting plasmid pRW844 was cut with EcoRV and DraI and the 1.7 kbp fragment containing the 3' H6 promoted H4 coding sequence was inserted between the EcoRV and HincII sites of pRW846 (described previously) forming plasmid pRW848. Plasmid pRW848 therefore contains the H4 coding sequence linked to the vaccinia virus H6 promoter in the de-ORFed F8 locus of fowlpox virus.
Construction of Insertion Plasmid for H5 Hemagalutinin at the F8 Locus. A cDNA clone of avian influenza H5 derived from A/Turkey/Ireland/1378/83 was received in plasmid pTH29 from Dr. R. Webster. Synthetic oligonucleotides RW10 (SEQ
ID NO:203) through RW13 (SEQ ID NO:206) were designed to overlap the translation initiation codon of the previously described vaccinia virus H6 promoter with the ATG of the H5 l92/15672 PCT/US92 )06 gene. The sequence continues through the 5' SalI site of the H5 gene and begins again at the 3' H5 Dral site containing the H5 stop codon.
RW10 (SEQ ID NO:203): 5' GAAAAATTTAAAGTCGACCTGTTTTGTTGAGT
TGTTTGCGTGGTAACCAATGCAAATCTGGTC
ACT 3' RW11 (SEQ ID NO:204): 5' TCTAGCAAGACTGACTATTGCAAAAAGAAGCA
CTATTTCCTCCATTACGATACAAACTTAACG
GAT 3' RW12 (SEQ ID NO:205): 5' ATCCGTTAAGTTTGTATCGTAATGGAGGAAA
TAGTGCTTCTTTTTGCAATAGTCAGTCTTGCTAGA
AGTGACCAGATTTGCATTGGT 3' RW13 (SEQ ID NO:206): 5' TACCACGCAAACAACTCAACAAAACAGGTCG
ACTTTAAATTTTTCTGCA 3' The oligonucleotides were annealed at 95 C for three minutes followed by slow cooling at room temperature. This results in the following double strand structure with the indicated ends.
EcoRV PstI

Cloning of oligonucleotides between the EcoRV and PstI
sites of pRW742B resulted in pRW744. Plasmid pRW742B
contains the vaccinia virus H6 promoter linked to a non-pertinent gene inserted at the HincII 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' SalI
site and the 3' sequence from the H5 stop codon (containing a DraI site). Use of the DraI site removes the H5 3' non-coding end. The oligonucleotides add a transcription termination signal recognized by early vaccinia virus RNA
polymerase (Yuen et al., 1987). To complete the H6 promoted H5 construct, the H5 coding region was isolated as a 1.6 kpb SalI-DraI fragment from pTH29. Plasmid pRW744 was partially digested with DraI, the linear fragment isolated, recut with WO 92/1: = PCT/US92/01906 SalI and the plasmid now with eight bases deleted between Sa I and DraI was used as a vector for the 1.6 kpb pTH29 Sa I and DXaI fragment. The resulting plasmid pRW759 was cut with coRV and DraI. The 1.7 kbp PRW759 coRV-2rAI
fragment containing the 3' H6 promoter and the H5 gene was inserted between the EcaRV and HincII sites of pRW846 (previously described). The resulting plasmid pRW849 contains the H6 promoted avian influenza virus H5 gene in the de-ORFed F8 locus.
Construction of Insertion Vector for H7 Hemagclutinin at the F7 Locus. Plasmid pCVH71 containing the H7 hemagglutinin from A/CK/VIC/1/85 was received from Dr. R.
Webster. An LcoRI-BamHI fragment containing the H7 gene was blunt-ended with the Klenow fragment of DNA polymerase and inserted into the HincII site of pIBI25 as PRW827.
Synthetic oligonucleotides RW165 (SEQ ID NO:207) and RW166 (SEQ ID NO:208) were annealed, cut with HincII and StvI and inserted between the coRV and =I sites of pRW827 to generate pRW845.
RW165 (SEQ ID NO:207): 5' GTACAGGTCGACAAGCTTCCCGGGTATCGCG
ATATCCGTTAAGTTTGTATCGTAATGAATACTCAA
ATTCTAATACTCACTCTTGTGGCAGCCATTCACAC
AAATGCAGACAAAATCTGCCTTGGACATCAT 3' RW166 (SEQ ID NO:208): 5' ATGATGTCCAAGGCAGATTTTGTCTGCATTTG
TGTGAATGGCTGCCACAAGAGTGAGTATTAGAATT
TGAGTATTCATTACGATACAAACTTAACGGATATC
GCGATACCCGGGAAGCTTGTCGACCTGTAC 3' Oligonucleotides RW165 (SEQ ID NO:207) and RW166 (SEQ
ID NO:208) link the 3' portion of the H6 promoter to the H7 gene. The 3' non-coding end of the H7 gene was removed by isolating the linear product of an , aLI digestion of pRW845, recutting it with EcoRI, isolating the largest fragment and annealing with synthetic oligonucleotides RW227 (SEQ ID NO:209) and RW228 (SEQ ID NO:210). The resulting plasmid was pRW854.
RW227 (SEQ ID NO:209): 5' ATAACATGCGGTGCACCATTTGTATAT
AAGTTAACGAATTCCAAGTCAAGC 3' RW228 (SEQ ID N0:210): 5' GCTTGACTTGGAATTCGTTAACTTATA
TACAAATGGTGCACCGCATGTTAT 3' -) 92/15672 PCT/US9! 906 The stop codon of H7 in PRW854 is followed by an lipal site.
The intermediate H6 promoted H7 construct in the de-ORFed F7 locus (described below) was generated by moving the pRW854 EcoRV-HpaI fragment into pRW858 which had been cut with coRV and blunt-ended at its PstI site. Plasmid pRW858 (described below) contains the H6 promoter in an F7 de-ORFed insertion plasmid.
The plasmid pRW858 was constructed by insertion of an 850 bp SmaI/HPaI fragment, containing the H6 promoter linked to a non-pertinent gene, into the Smal site of pF7DO
described previously. The non-pertinent sequences were excised by digestion of pRW858 with coRV (site 24 bp upstream of the 3'-end of the H6 promoter) and PstI. The 3.5 kb resultant fragment was isolated and blunt-ended using the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs. This blunt-ended fragment was ligated to a 1700 bp coRV/HpaI fragment derived from pRW854 (described previously). This coRV/HQaI fragment contains the entire AIV HA (H7) gene juxtaposed 3' to the 3'-most 24 bp of the VV H6 promoter. The resultant plasmid was designated pRW861.
The 126 bp EH arm (defined previously) was 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 NaeI 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 designated as 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 consists of 404 bp of TROVAC sequence and the right flanking =arm of 725 bp of TROVAC sequence which directs insertion to the de-ORFed F7 locus.
Development of TROVAC-Avian Influenza Virus Recombinants. Insertion plasmids containing the avian influenza virus HA coding sequences were individually transfected into TROVAC infected primary CEF cells by using the calcium phosphate precipitation method previously WO 92/15 ' PCT/US92/019,06 described (Panicali et al., 1982; Piccini et al., 1987).
Positive plaques were selected on the basis of hybridization to HA specific radiolabelled probes and subjected to sequential rounds of plaque purification until a pure population was achieved. One representative plaque was then amplified to produce a stock virus. Plasmid pRW849 was used in an in vitro recombination test to produce recombinant TROVAC-AIH5 (vFP89) expressing the H5.hemagglutinin.
Plasmid pRW848 was used to produce recombinant TROVAC-AIH4 (vFP92) expressing the H4 hemagglutinin. Plasmid pRW869 was used to produce recombinant TROVAC-AIH7 (vFP100) expressing the H7 hemagglutinin.
Immunofluorescence. In influenza virus infected cells, the HA molecule is synthesized and glycosylated as a precursor molecule at the rough endoplasmic reticulum.
During passage to the plasma membrane it undergoes extensive post-translational modification culminating in proteolytic cleavage into the disuiphide linked HA1 and HA2 subunits and insertion into the host cell membrane where it is subsequently incorporated into mature viral envelopes. To determine whether the HA molecules produced in cells infected with the TROVAC-AIV recombinant viruses were expressed on the cell surface, immunofluorescence studies were performed. Indirect immunofluorescence was performed as described (Taylor et al., 1990). Surface expression of the H5 hemagglutinin in TROVAC-AIH5, H4 hemagglutinin in TROVAC-AIH4 and H7 hemagglutinin in TROVAC-AIH7 was confirmed by indirect immunofluorescence. Expression of the H5 hemagglutinin was detected using a pool of monoclonal antibodies specific for the H5HA. Expression of the H4HA
was analyzed using a goat monospecific anti-H4 serum.
Expression of the H7HA was analyzed using a H7 specific monoclonal antibody preparation.
Immunoprecipitation. It has 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 necessary for virus particles to be infectious. The hemagglutinin proteins of the -~92/ 1 5672 PCT/US92. -06 virulent H5 and H7 viruses possess more than one basic amino acid at the carboxy terminus of HAl. It is thought that this allows cellular proteases which recognize a series of basic amino acids to cleave the hemagglutinin and allow the infectious virus to spread both in vitro and in vivo. The hemagglutinin molecules of H4 avirulent strains are not cleaved in tissue culture unless exogenous trypsin is added.
In order to determine that the hemagglutinin molecules expressed by the TROVAC recombinants were authentically processed, immunoprecipitation experiments were performed as described (Taylor et al., 1990) using the specific reagents described above.
Immunoprecipitation analysis of the H5 hemagglutinin expressed by TROVAC-AIH5 (vFP89) showed that the glycoprotein is evident as the two cleavage products HA1 and HA2 with approximate molecular weights of 44 and 23 kDa, respectively. No such proteins were precipitated from uninfected cells or cells infected with parental TROVAC.
Similarly immunoprecipitation analysis of the hemagglutinin expressed by TROVAC-AIH7 (vFP100) showed specific precipitation of the HA2 cleavage product. The HA1 cleavage product was not recognized. No proteins were specifically precipitated from uninfected CEF cells or TROVAC infected CEF cells. In contrast, immunoprecipitation analysis of the expression product of TROVAC-AIH4 (vFP92) showed expression of only the precursor protein HAo. This is in agreement with the lack of cleavage of the hemagglutinins of avirulent subtypes in tissue culture. No H4 specific proteins were detected in uninfected CEF cells or cells infected with TROVAC.
Egample 23 - DEVELOPMENT OF A TRIPLE RECOMBINANT
E%PRESSING THREE AVIAN INFLUENZA GENES
Plasmid Construction. Plasmid pRW849 has been discussed previously and contains the H6 promoted avian influenza H5 gene. This plasmid was used for the development of vFP89. Plasmid pRW861 was an intermediate plasmid, described previously used in the development of vFP100. The plasmid contains the H6 promoted avian influenza H7 gene. Plasmid pRW849 was digested with SmaI

WO 92/1 '2 PCT/US92/01906 and the resulting 1.9 kbp fragment from the 5' end of the H6 promoter through the H5 gene was inserted at the SmaI site of pRW861 to produce pRW865. In order to insert the H4 coding sequence, plasmid pRW848 was utilized. Plasmid pRW848 was used in the development of vFP92 and contains the H6 promoted H4 gene (previously described). Plasmid pRW848 was digested with SmaI and a 1.9 kbp fragment containing the H6 promoted H4 coding sequence was then inserted into pRW865 at the SmaI site 5' of the H6 promoted H5 sequence. The resulting plasmid pRW872 therefore contains the H4, H5 and H7 coding sequences in the F7 de-ORFed insertion plasmid.
In order to direct insertion of the genes to the de-ORFed F8 locus, pRW872 was partially digested with SmaI, the linear fragment isolated and recut with HindIII. The 5.7 kbp SmaI to HindIII pRW872 fragment containing all three H6 promoted avian influenza genes was blunt-ended and inserted into pCEN100 which had been cut with incII. Plasmid pCEN100 is a de-ORFed F8 insertion vector containing transcription and translation stop signals and multiple insertion sites. Plasmid pCEN100 was generated as described below. Synthetic oligonucleotides CE205 (SEQ ID NO:211) and CE206 (SEQ ID NO:212) were annealed, phosphorylated and inserted into the BamHI and 1'ndIII sites of pJCA002 (previously described) to form pCE72. A BalII to EcoRI
fragment from pCE72 was inserted into the BalII and EcoRI
sites of pJCA021 to form pCEN100.
CE205 (SEQ ID NO:211): 5' GATCAGAAAAACTAGCTAGCTAGTACGTAGTT
AACGTCGACCTGCAGAAGCTTCTAGCTAGCTAGTT
TTTAT 3' CE206 (SEQ ID NO:212): 5' AGCTATAAAAACTAGCTAGCTAGAAGCTTCTG
CAGGTCGACGTTAACTACGTACTAGCTAGCTAGTT
TTTCT 3' Plasmid pJCA021 was obtained by inserting a 4900 bp PvuII-indII fragment from pRW731-15 (previously described) into the SmaI and HindII sites of pBSSKT.
The final insertion plasmid pRW874 had the three avian influenza HA genes transcribed in the same direction as the deleted F8 ORF. The left flanking arm of the plasmid adjacent to the H4 gene consisted of 2350 bp of fowipox 92/15672 PCT/US92. )06 sequence. The right flanking arm adjacent to the H7 gene consisted of 1700 bp of fowipox sequence. A linear representation of the plasmid is shown below.
2350bp FP H6 H4HA H6 H5HA H6 H7HA 1700bp FP
Development of Recombinant vFP122. Plasmid pRW874 was 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 plaques were selected on the basis of hybridization to specific H4, H5 and H7 radiolabelled probes and subjected to 5 sequential rounds of plaque purification until a pure population was achieved. Surface expression of all three glycoproteins was confirmed by plaque immunoscreen using specific reagents previously described. Stability of inserted genes was confirmed after two rounds of amplification and the recombinant was designated as vFP122.
Example 24 - COMPARISON OF THE LD50 OF ALVAC AND NYVAC
WITH VARIOUS VACCINIA VIRUS STRAINS

Mice. Male outbred Swiss Webster mice were purchased from Taconic Farms (Germantown, NY) and maintained on mouse chow and water ad libitum until use at 3 weeks of age ("normal" mice). Newborn outbred Swiss Webster 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.
Viruses. ALVAC was derived by plaque purification of a canarypox virus population and was prepared in primary chick embryo fibroblast cells (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 strain of vaccinia virus was obtained from Pharmaceuticals Calf Lymph Type vaccine Dryvax, control number 302001B. Copenhagen strain vaccinia virus VC-2 was obtained from Institut Merieux, France. Vaccinia virus strain NYVAC was derived from WO 92/1= '. PCT/US92/01906 Copenhagen VC-2. All vaccinia virus strains except the Wyeth strain were cultivated in Vero African green monkey kidney cells, purified by sucrose gradient density centrifugation and enumerated for plaque forming units on Vero cells. The Wyeth strain was grown in CEF cells and enumerated in CEF cells.
Inoculations. Groups of 10 normal mice were inoculated intracranially (ic) with 0.05 ml of one of several dilutions of virus prepared by 10-fold serially diluting the stock preparations in sterile phosphate-buffered saline. In some instances, undiluted stock virus preparation was used for inoculation.
Groups of 10 newborn mice, 1 to 2 days old, were inoculated ic similarly 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 days (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 (LD50) was determined by the proportional method of Reed and Muench.
Comparison of the LD50- of ALVAC and NYVAC withVarious 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 strains tested (Table 28). NYVAC and ALVAC
were found to be over 3,000 times less virulent in normal mice than the Wyeth strain; over 12,500 times less virulent than the parental VC-2 strain; and over 63,000,000 times less virulent than the WR(L) variant. These results would suggest that NYVAC is highly attenuated compared to other vaccinia strains, and that ALVAC is generally nonvirulent for young mice when administered intracranially, although both may cause mortality in mice at extremely high doses (3.85x108 PFUs, ALVAC and 3x108 PFUs, NYVAC) by an undetermined mechanism by this route of inoculation.
Comparison of the LD5n of ALVACand NYVAC with Various Vaccinia Virus Strains for Newborn Outbred Mice by the ic ~ '92/15672 PCT/US92i '06 Route. The relative virulence of 5 poxvirus strains for normal, newborn mice was tested by titration in an intracranial (ic) challenge model system (Table 29). With mortality as the endpoint, LD50 values indicated that ALVAC
is over 100,000 t'imes less virulent than the Wyeth vaccine strain of vaccinia virus; over 200,000 times less virulent than the Copenhagen VC-2 strain of vaccinia virus; and over 25,000,000 times less virulent than the WR-L variant of vaccinia virus. Nonetheless, at the highest dose tested, 6.3x107 PFUs, 100% mortality resulted. Mortality rates of 33.3% were observed at 6.3x106 PFUs. The cause of death, while not actually determined, was not likely of toxicological or traumatic nature since the mean survival time (MST) of mice of the highest dosage group (approximately 6.3 LD50) was 6.7 1.5 days. When compared to WR(L) at a challenge dose of 5 LD50, wherein MST is 4.8 0.6 days, the MST of ALVAC challenged mice was significantly longer (P=0.001).
Relative to NYVAC, Wyeth was found to be over 15,000 times more virulent; VC-2, greater than 35,000 times more virulent; and WR(L), over 3,000,000 times more virulent.
Similar to ALVAC, the two highest doses of NYVAC, 6x108 and 6x107 PFUs; caused 100% mortality. However, the MST of mice challenged with the highest dose, corresponding to 380 LD50, was only 2 days (9 deaths on day 2 and 1 on day 4). In contrast, all mice challenged with the highest dose of WR-L, equivalent to 500 LD50, survived to day 4.

WO 92/1= "2 PC.'T/US92/01906 Table 28. Calculated 50% Lethal Dose for mice by various vaccinia virus strains and for canarypox virus (ALVAC) by the ic route.

POXVIRUS CALCULATED
STRAIN LD50 (PFUs) WR(L) 2.5 VC-2 1.26x104 WYETH 5.00x104 NYVAC 1.58x108 ALVAC 1.58x108 i i Table 29. Calculated 50% Lethal Dose for newborn mice by various vaccinia virus strains and for canarypox virus (ALVAC) by the ic route.

POXVIRUS CALCULATED
STRAIN LD50 (PFUs) WR(L) 0.4 VC-2 0.1 WYETH 1.6 NYVAC 1.58x106 ALVAC 1.00x107 92/15672 PCT/US92/ '06 EsamDle 25 - GENERATION OF NY9AC-BASED RECOMBINANTB
EXPRESSING THE $HV-1 qB, gC AND gD
GLYCOPROTEINS HOMOLOGS

Expression of-the EHV-1 gB glycoprotein was accomplished by putting the EHV-1 gB homolog gene under the control of the vaccinia virus 13L promoter. Expression of the EHV-1 gC glycoprotein was accomplished by putting the EHV-1 gC homolog gene under the control of the vaccinia virus H6 promoter. Expression of the EHV-1 gD glycoprotein was accomplished by putting the EHV-1 gD homolog gene under the control of the entomopox virus 42K gene promoter.
Generation of vP1025 (aB and gC in ATI locus: qD in HA
locus).
Generation of donor plasmid pJCA042. The 430 bp 5'-most region of the EHV-1 gB coding sequence was PCR-derived using the plasmid pJCA011 (cassette H6- EHV-1 gB in ATI
locus) as template and oligonucleotides JCA156 (SEQ ID
NO:223) (5'-ATGTCCTCTGGTTGCCGTTCT-3') and JCA157 (SEQ ID
NO:224) (5'-GACGGTGGATCCGGTAGGCGG-3'), digested with BamHI
and kinased. This 430 bp fragment was fused to a 120 bp PCR-derived fragment containing the 13L promoter element obtained using plasmid pMP691 (I3L101RAB) as template and oligonucleotides JCA158 (SEQ ID NO:225) (5'-TTTTTCTAGACTGCAGCCCGGGACATCATGCAGTGGTTAAAC-3') and MP287 (SEQ ID NO:226) (5'-GATTAAACCTAAATAATTGT-3'). This 120 bp fragment was digested with XbaI and kinased prior to be ligated with the 430 bp 5'-most region of EHV-1 gB fragment.
The resulting plasmid was designated pJCA034. Sequences of the 13L promoter, of the junction 13L-ATG and of the EHV-1 5'-most region were confirmed by direct sequencing of pJCA034. Plasmid pJCA034 was digested with SmaI and BamHI
to excise the 550 bp SmaI-I3L-EHV-1 gB 5'-BamHI fragment (A). Plasmid pMP665 (cassette H6-EHV-1 gB in COPCS system) was digested with BamHI and XhoI to excise the 2530 bp BamHI-EHV-1 gB 3' fragment (B). Fragments A and B were then ligated together into vector pSD541VC (ATI deorfed locus) digested with SmaI and XhoI to produce pJCA037. Plasmid pJCA037 is the donor plasmid containing the cassette 13L-EHV-1 gB in the ATI deorfed locus. Plasmid pJCA037 was digested with SmaI and XhoI to isolate the 3050 bp SmaI-I3L-WO 92/1:" ? PCT/US92/01906 EHV-1 gB- h~oI fragment (C). The 225 bp KpnI-EHV-1 gC 3'end cleaned up- indIII fragment was PCR-derived using plasmid pVHAH6g13 (cassette H6-EHV-1 gC in HA deorfed locus) and oligonucleotides JCA154 (SEQ ID NO:227) (5'-TATAGCTGCATAATAGAG-3') and JCA163 (SEQ ID NO:228) (5'-AATTAAGCTTGATATCACAAAA.ACTAAAAAGTCAGACTTCTTG-3'), digested with KpnI and indIII, and ligated into vector pBS-SK+
digested with KnnI and HindIII to produce pJCA033. Sequence of the cloned PCR fragment was confirmed by direct sequencing of pJCA033. Plasmid pJCA033 was digested with KpnI and EcoRV to isolate the 220 bp KpnI-EHV-1 gC 3'end-EcoRV fragment (D). Plasmid pVHAH6g13 was digested with Bcr1II and KpnI to isolate the 1330 bp Bg1II-H6-EHV-1 gC 5'-KDnI fragment (E).
Fragments C, D and E were finally ligated together into vector pSD541VC digested with BQ1II and XhoI to produce plasmid pJCA042. Plasmid pJCA042 is the donor plasmid to insert the 13L-EHV-1 gB -- H6-EHV-1 gC double construction in the NYVAC ATI deorfed locus. Plasmid pJCA042 was linearized using NotI prior to IVR.
In vitro recombination experiment was performed on Vero cells using pJCA042 as the donor plasmid and vP866 (NYVAC) as the rescue virus. Standard protocols were used to identify and purify the recombinant virus (Piccini et al., 1987). The NYVAC-based recombinant containing the EHV-1 gB
and gC genes in the ATI deorfed locus was designated vP956.
Generation of Donor Plasmid pJCA064. Plasmid pEHV1BamHID (containing the EHV-1 BamHI D fragment) was digested with HindIII to excise the 1240 bp HindIII-HindIII
containing the entire EHV-1 gD coding sequence but the 15 5'-most bp. The 1240 bp HindIII-HindIII fragment was blunt-ended with Klenow polymerase and ligated into vector pCOPCS657 digested with SmaI and phosphatased. The resulting plasmid was designated pJCA006. Plasmid pJCA006 was digested with Bg1II and indIII to excise the 1500 bp HindIII-H6--EHV-1 gD-BalII fragment. This fragment was ligated into vector pIBI24 digested with BamHI and HindIII
to produce plasmid pEHV1gp50a. Plasmid pEHVigp50a was digested with coRV and coI which are both unique sites to )92/15672 PCT/L'S92. )06 excise the 4100 bp fragment. This fragment was ligated with a synthetic double strand oligonucleotide obtained by hybridization between oligonucleotides JCA052 (SEQ ID
NO:229) (5'-ATCCGTTAAGTTTGTATCGTAATGTCTACCTTCAAGCTTATGA
TGGATGGACGTTTGGTTTTTGC-3') and JCA053 (SEQ ID NO:230) (5'-CATGGCAAAAACCAAACGTCCATCCATCATAAGCTTGAAGGTAGACATTACGATACAAAC
TTAAGCGAT-3'). The resulting plasmid was designated pgp50a3-2. The 490 bp EcoRI-EHV-1 gD 3'end cleaned up -HpaI
was PCR-derived using plasmid pJCA006 as template and oligonucleotides JCA041 (SEQ ID NO:231) (5'-TGTGTGA
TGAGAGATCAG-3') and JCA099 (SEQ ID NO:232) (5'-AACTC
GAGTTAACAAAAATTACGGAAGCTGGGTATATTTAACAT-3'). Plasmid pgp50a3-2 was digested with EcoRI and partially digested with HindIII to excise the 850 bp HindIII-H6-EHV-1 gD 5'-EcoRI fragment. This fragment was ligated with the 490 bp EcoRI-HpaI fragment into vector pBS-SK+ digested with HindIII and SmaI to produce plasmid pJCA020. Plasmid pJCA020 contains the cassette H6-EHV-1 gD.
The 720 bp 5'-most region of the EHV-1 gD coding sequence was PCR-derived using plasmid pJCA020 as template and oligonucleotides JCA044 (SEQ ID NO:233) (5'-CTCTAT
GACCTCATCCAC-3') and JCA165 (SEQ ID NO:234) (5'-ATGTCTA
CCTTCAAGCTTATG-3'). This fragment was digested with EcoRI
and kinased. The 107 bp 42K promoter element was PCR-derived using plasmid pAM12 as template and oligonucleotides RG286 (SEQ ID NO:235) (5'-TTTATATTGTAATTATA-3') and JCA164 (SEQ ID NO:236) (5'-TTTGGATCCGTTAACTCAAAAAAATAAATG-3').
This fragment was digested with BamHI, kinased, and ligated with the 720 bp ATG-EHV-1 gD 5'- coRI fragment into vector pBS-SK+ digested with BamHI and EcoRI to produce plasmid pJCA035. Sequences of the 42K promoter, of the junction 42K-EHV-1 gD and of the EHV-1 gD 5' portion were confirmed by direct sequencing of pJCA035.
Plasmid pJCA035 was digested with BamHI and EcoRI to isolate the 830 bp BamHI-42K-EHV-1 gD 5'portion-EcoRI
fragment (F). Plasmid pJCA020 was digested with EcoRI and XbaI to isolate the 500 bp EcoRI-EHV-1 gD 3'end cleaned up-XbaI fragment (G). Fragments F and G were then ligated together into vector pBS-SK+ digested with BamHI and XbaI to C'1'O 92/1. ? PCT/US92/01906 produce plasmid pJCA038. Plasmid pJCA038 is containing the cassette 42K-EHV-1 gD into vector pBS-SK+. Plasmid pJCA038 was digested with BamHI and HpaI to isolate the 1340 bp HDaI-42K-EHV-1 gD-BamHI fragment. This fragment was ligated into plasmid pSD544 (HA deorfed locus) digested with BamHI
and SmaI to produce plasmid pJCA064. Plasmid pJCA064 is the donor plasmid to insert the cassette 42K-EHV-1 gD into the NYVAC HA deorfed locus. Plasmid pJCA064 was linearized using NotI prior to IVR.
In vitro experiment was performed on Vero cells using pJCA064 as the donor plasmid and recombinant vaccinia virus vP956 (NYVAC background) as the rescue virus. This was performed with standard procedures (Piccini et al., 1987).
The NYVAC-based recombinant containing the EHV-1 gB and gC
genes in the ATI deorfed locus and the EHV-1 gD gene in the HA deorfed locus was designated vP1025.
Generation of donor plasmid pJCA043. The 220 bp indIII-EHV-i gB 3'-most region was PCR-derived using plasmid pJCA011 and oligonucleotides JCA159 (SEQ ID NO:237) (5'-AGGCCAAGCTTGAAGAGGCTC-3') and JCA160 (SEQ ID NO:238) (5'-AAAGGATCCGTTAACACAAAAATTAAACCATTTTTTCATT-3'). This fragment was digested with BamHI and HindIII and ligated into vector pBS-SK+ digested with BamHI and HindIII to produce plasmid pJCA036. Sequence of the EHV-1 gB 3'-most region PCR fragment was confirmed by direct sequencing of pJCA036.
Plasmid pJCA033 was digested with EcoRV and KvnI to isolate the KDnI-EHV-1 gC 3'-most region-EcoRV fragment (H).
Plasmid pJCA038 was digested with BamHI and HpaI to isolate the 1360 bp HDaI-42K-EHV-1 gD -BamHI fragment (I). Plasmid pVHAH6g13 was digested with KpnI and XhoI to isolate the 900 bp h~oI-EHV-1 gC central portion-KDnI fragment (J).
Fragments H, I and J were then ligated together into vector pBS-SK+ digested with BamHI and XhoI to produce plasmid pJCA041.
Plasmid pJCA034 was digested with HindIII and XhoI to isolate the 5900 bp linearized vector XhoI-pBS-SK+-I3L-EHV-1 gB -HindIII fragment (K). Plasmid pJCA036 was digested with BApHI and '~idIII to isolate the 220 bp 'ndIII-EHV-1 gB 3--92/15672 PCt/US92/ 06 most region-BamHI fragment (L). Plasmid pVHAH6g13 was digested with BalII and Xhol to isolate the 440 bp BalII-H6-EHV-1 gC 5'portion-XhoI fragment (M). Fragments K, L and M
were then ligated together to produce plasmid pJCA040.
Plasmid pJCA040 was digested with Smal and Xhol to isolate the 3550 bp SmaI-I3L-EHV-1 gB -- H6-EHV-1 gC
5'portion-XhoI fragment (N). Plasmid pJCA041 was digested with B_amHI and Xhol to isolate the 2460 bp X oI-EHV-1 gC
3'portion -- 42K-EHV-1 gD -BamHI fragment (0). Fragments N
and 0 were finally ligated together into plasmid pSD541VC
(NYVAC ATI deorfed locus) digested with BalII and SmaI to produce plasmid pJCA043. Plasmid pJCA043 is the donor plasmid to insert the 13L-EHV-1 gB -- H6-EHV-1 gC -- 42K-EHV-1 gD triple construction in the NYVAC ATI deorfed locus.
Plasmid pJCA043 was linearized using 1otI prior to IVR.
In vitro experiment was performed on primary chick embryo fibroblasts using pJCA043 as the donor plasmid and vP866 (NYVAC) as the rescue virus. Standard procedures were used to identify and purify the generated recombinant (Piccini et al., 1987). The NYVAC-based recombinant containing the EHV-1 gB, gC and gD genes in the ATI deorfed locus was designated vP1043.
Example 26 - GENERATION OF ALVAC-BASED RECOMBINANTS
EXPRESSING THE $HV-1 qBj gC AND gD
GLYCOPROTEINS HOMOLOGS

Generation of donor r)lasmid pJCA049. Plasmid pJCA040 was digested with SmaI and X oI to isolate the 3550 bp SmaI-I3L-EHV-1 gB -- H6-EHV-1 gC 5'portion-XhoI fragment (A).
Plasmid pJCA041 was digested with BamHI and ~hoI to isolate the 2460 bp XhoI-EHV-1 gC 3'portion -- 42K-EHV-1 gD -BamHI
fragment (B). Fragments A and B were ligated together into plasmid pSPVQC3L digested with DamHI and SmaI to produce plasmid pJCA049. Plasmid pJCA049 is the donor plasmid to insert the I3L-EHV-1 gB -- H6-EHV-1 gC -- 42K-EHV-1 gD
triple construction in the ALVAC C3 deorfed locus. Plasmid pJCA049 was linearized using NotI prior to IVR.
in vitro experiment was performed on primary chick embryo fibroblasts using pJCA049 as the donor plasmid and CPpp (ALVAC) as the rescue virus. Standard procedures were followed to identify and purify the generated recombinant '.VO 92/1-- PCT/US92/01906 (Piccini et al., 1987). The ALVAC-based recombinant containing the EHV-1 gB, gC and gD genes in the C3 deorfed locus was designated vCP132.
EYample 27 - EXPRESSION ANALYSIS OF THE NYVAC- AND ALVAC-RECOMBINANTS

Immunofluorescence assays were performed as described previously (Taylor et al., 1990) using monoclonal antibodies specific to EHV-1 gB (16G5 or 3F6), EHV-1 gC (14H7) and EHV-1 gD (20C4). All anti-EHV-1 monoclonals were obtained from George Allen (Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, 40546-0076). Expression of all three EHV-1 specific products was detectable internally in cells infected with either vP1025, vP1043 or vCP132. Only the EHV-1 gC glycoprotein was well expressed on the surface of infected cells. Surface expression for EHV-1 gB glycoprotein was much weaker and surface expression of EHV-1 gD glycoprotein was questionable.
Immunoprecipitations were done using the same monoclonal antibodies to determine the authenticity of the expressed EHV-1 gB, gC and gD gene products. Monoclonal 3F6 specific for EHV-1 gB glycoprotein precipitated proteins with apparent molecular masses on an SDS-PAGE gel system of 138 kDa, 70 kDa and 54 kDa from lysates derived from cells infected with the recombinant viruses vP956, vP1025, vP1043 or vCP132. No protein was precipitated from lysates derived from uninfected cells or from either parental virus (NYVAC
and ALVAC) infected cells. Monoclonal 14H7 specific for EHV-1 gC glycoprotein precipitated a glycoprotein with an apparent molecular mass of 90 kDa from lysates derived from cells infected either with vP956, vP1025 or vP1043. The EHV-1 gC glycoprotein expressed by recombinant vCP132 has an apparent molecular mass slightly smaller (about 2 kDa less) than that expressed by recombinants vP956, vP1025 or vP1043.
Monoclonal antibody 20C4 specific for EHV-1 gD glycoprotein precipitated a glycoprotein with an apparent molecular mass of 55 kDa from lysates derived from cells infected with vP1025, vP1043 or vCP132.
Immunoprecipitations were also done using a rabbit anti-EHV-1 hyperimmune serum obtained from G. Allen. This )92/15672 PCT/US92, i06 serum precipitated all three EHV-1 products from lysates derived from cells infected either with vP1025, vP1043 or vCP132.
Example 28 - PROTECTION DATA OBTAINED USING THE HAMSTER
CHALLENGE MODEL

Challenge experiments (hamster model) have been done at Rhone-Merieux (Lyon, France) to assess the relative level of protection induced by poxvirus EHV-1 recombinants vP956, vP1025 and vCP132. Hamsters were vaccinated on day 0 and boosted on day 14 with various dilutions of the EHV-1 recombinants. All immunized and control animals were challenged on day 28 with a hamster-adapted EHV-1 challenge strain. Final count of dead animals was made on day 35 (7 days post challenge). Results of the challenge experiment are shown below in Table 30:

Table 30 Recombinant EHV-1 genes Dose TCID50 loglo /dead/total vP956 gB + gC 7.6 5.6 3.6 vP1025 gB + gC + gD 7.8 5.8 3.8 vCP132 gB + gC + gD 8.8 6.8 4.8 Control none 4/4 V1'O 92 1: PCT/US92/01906 Example 29 - DURATION OF IMMUNITY STUDIES IN DOGS
The aim of this study was to determine how long a protective immune response would be maintained in dogs after a single inoculation with ALVAC-RG (vCP65). Forty-one beagle dogs of 8 months of age which were free of anti-rabies antibody were inoculated with one dose of 6.7 loglo TCID50 of ALVAC-RG by the subcutaneous route. Dogs were bled on day 0 and at 1, 2, 3, 6 and 12 months after vaccination and sera assayed for the presence of anti-rabies antibody using the RFFI test. All animals were monitored for side-effects of vaccination.
At 6 months post-vaccination, 5 dogs were challenged by intra-muscular inoculation of the virulent NYGS strain of rabies virus. Animals received 103=4 50% mouse lethal doses in the temporal muscle. Three uninoculated control animals received the same inoculation. A second group of 11 vaccinated dogs and 3 non-vaccinated control dogs were challenged in an identical manner at 12 months post-vaccination. The serological results and results of challenge at 6 and 12 months are shown below in Table 31.
None of the dogs vaccinated with ALVAC-RG (vCP65) exhibited adverse reaction to vaccination. All dogs vaccinated'with ALVAC-RG (vCP65) demonstrated the induction of rabies virus neutralizing antibody by 7 days post-vaccination. Maximal titers were achieved between 14 and 28 days post-vaccination after which titers decreased. At the time of challenge at 6 or 12 months post-vaccination titers were low and in some animals, approaching zero. Despite the low titers, all animals survived a lethal rabies challenge in which unvaccinated control dogs succumbed. RFFI titers of animals that survived challenge at 6 months post-vaccination were assessed at 8 months (2 months post-challenge). The serum titers in these animals were 7.4, 7.4, 2.3, 1.8 and 7.4. International Units. These elevated and maintained levels of rabies neutralizing antibody indicate that animals were efficiently primed by the initial single inoculation. The experiment is on-going and the remaining animals will be challenged at 2 or 3 years following vaccination; however, to date, the experiment is 92/ l 5672 PC'T/LJS92, l06 successful and illustrates the utility of the present invention.

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W'0 92/1" '2 PCT/US92/01906 Example 30 - EXPRESSION OF BOVINE HERPESVIRIIS TYPE 1 BHVl GENES IN NYVAC

Generation of NYVAC/BHV1 gIV recombinant. A plasmid, pBHVgIV, was obtained from Rhone Merieux. This plasmid contains the BHV1 gIV gene (Straub strain), encoded on a 3.9 kb PstI fragment, cloned into the PstI site of pBS-SK+. The gIV gene (Tikoo et al., J. Virol. (1990) 64:5132) from this plasmid was cloned between vaccinia virus flanking arms.
This was accomplished by cloning the 2,000 bp PstI-XhoI
fragment of pBHVgIV, containing the gIV gene, into the PstI-XhoI site of pSD542 (defined in Example 32). The plasmid generated by this manipulation is called pBHV1.
The 3'-end of the ir promoter was then cloned upstream of the gIV gene. This was accomplished by cloning the oligonucleotides, BHVL7 (SEQ ID NO:239) (5'-TCGAGCTTAA
GTCTTATTAATATGCAAGGGCCGACATTGGCCGTGCTGGGCGCGCTGCTCGCCGTTGCGG
TGAGCTTGCCTACACCCGCGCCGC-3') and BHVL8 (SEQ ID NO:240) (5'-GGCGCGGGTGTAGGCAAGCTCACCGCAACGGCGAGCAGCGCGCCCAGCAC
GGCCAATGTCGGCCCTTGCATATTAATAAGACTTAAGC-31), encoding the 31-end of the Tr promoter and the 51-end of the gIV gene, into the 5,500 bp partial SstII-XhOI fragment of pBHV1. The plasmid generated by this manipulation is called pBHV3.
Extraneous 3'-noncoding sequence was then eliminated.
This was accomplished by cloning the oligonucleotides, BHVL5 (SEQ ID N0:241) (5'-GGGTGACTGCA-3') and BHVL6 (SEQ ID
NO:242) (5'-GTCACCC-3'), into the 5,200 bp partial SmaI-PstI
fragment of pBHV3. The plasmid generated by this manipulation is called pBHV4.
Extraneous linker sequence was then eliminated. This was accomplished by ligating the 5,200 bp PstI fragment of pBHV4. The plasmid generated by this manipulation is called pBHV5.
The 5'-end of the ff promoter was then cloned into pBHV5. This was accomplished by cloning the 130 bp AflII-XhoI fragment of pPI4, containing the 5'-end of the n promoter, into the 5,200 bp Af1II-XhoI fragment of pBHV5.
The plasmid generated by this manipulation is called pBHV6.
pBHV6 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1051.

Immunoprecipitation analysis was performed to determine whether vP1051 expresses an authentic BHV1 gIV glycoprotein.
Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1051 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2%
fetal bovine serum and [35S]-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A(3$ NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03%
Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 gIV expression using the BHV1 gIV-specific monoclonal antibody, 3402 (obtained from Dr. Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with iX buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the gIV-specific monoclonal antibody, 3402. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C.
After washing this material 5X with 1X buffer A, the BHV1 gIV-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X with 1X
buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M
Na-salicylate for fluorography.
The BHV1 gIV-specific monoclonal antibody, 3402, specifically precipitated the BHV1 gIV glycoprotein from vP1051 infected cells, but did not precipitate BHV1-specific proteins from NYVAC or mock infected cells.
Generation of NYVAC/BHV1 aI and aIV Recombinant. A

WO 92/lk- 1 PCT/US92/01906 plasmid, pBHVgIV, containing the BHV1 gIV gene, was obtained from Rhone Merieux. The gIV gene from this plasmid was cloned between vaccinia virus flanking arms. This was accomplished by cloning the 2,000 bp ~stI-=oI fragment of pBHVgIV, containing the gIV gene, into the pstI-= I site of pSD542. The plasmid generated by this manipulation is called pBHV1.
The 3'-end of the n promoter was then cloned upstream of the gIV gene. This was accomplished by cloning the oligonucleotides, BHVL7 (SEQ ID N0:239) and BHVL8 (SEQ ID
NO:240), encoding the 3'-end of the r promoter and the 5'-end of the gIV gene, into the 5,500 bp partial SstII-Xhol fragment of pBHV1. The plasmid generated by this manipulation is called pBHV3.
Extraneous 3'-noncoding sequence was then eliminated.
This was accomplished by cloning the oligonucleotides, BHVL5 (SEQ ID NO:241) and BHVL6 (SEQ ID N0:242), into the 5,200 bp partial SmaI-,stI fragment of pBHV3. The plasmid generated by this manipulation is called pBHV4.
Extraneous linker sequence was then eliminated. This was accomplished by ligating the 5,200 bp gstl fragment of pBHV4. The plasmid generated by this manipulation is called pBHV5.
The 5'-end of the v promoter was then cloned into pBHV5. This was accomplished by cloning the 130 bp AflII-XhoI fragment of pPI4, containing the 5'-end of the n promoter, into the 5,200 bp AflII- h~oI fragment of pBHV5.
The plasmid generated by this manipulation is called pBHV6.
The BHV1 gI gene was then cloned into pBHV6. This was accomplished by cloning the 2,900 bp BalII fragment of pBHV8, containing the H6-promoted gI gene, into the BalII
site of pBHV6. The plasmid generated by this manipulation is called pBHV9.
pBHVB was generated by the following procedure: a plasmid, pIBRS6, was received from Rhone Merieux. The plasmid contains a 6.6 kb SalI fragment, containing the BHV1 gI gene (Straub strain). The 5'-end of the gI gene (Whitbeck et al., J. Virol. (1988) 62:3319) was cloned downstream of the H6 promoter and between vaccinia virus HA

92/15672 PCT/US92, )06 flanking arms. This was accomplished by cloning the 540 bp SalI-PstI fragment of pIBRS6 into the 4,400 bp SalI-PstI
fragment of pGD5. The plasmid generated by this manipulation is called pIBR2.
The initiation codon of the H6 promoter was then aligned with the initiation codon of the gI gene. This was accomplished by cloning the oligonucleotides, IBRL1 (SEQ ID
NO:243) (5'-ATCCGTTAAGTTTGTATCGTAATGGCCGCTCGCGGCGGTGCTGAA
CGCGCCGC-3') and IBRL2 (SEQ ID NO:244) (5'-GGCGCGTTCAGCA
CCGCCGCGAGCGGCCATTACGATACAAACTTAACGGAT-3'), into the 3,800 bp NruI-SstII fragment of pIBR2. The plasmid generated by this manipulation is called pIBR4.
An NcoI site, necessary for future manipulations, was then generated downstream from the gI sequence. This was accomplished by cloning the oligonucleotides, IBRL3 (SEQ ID
NO:245) (5'-CCATGGTTTAATGCA-3') and IBRL4 (SEQ ID N0:246) (5'-TTAAACCATGGTGCA-3'), into the PstI site of pIBR4. The plasmid generated by this manipulation is called pIBR5.
Additional gI sequence was then cloned into pIBR5.
This was accomplished by cloning the 1,740 bp Tth111I-NcoI
fragment of pIBRS6 into the 3,700 bp Tth111I-NcoI fragment of pIBR5. The plasmid generated by this manipulation is called pIBR7.
A BglII site, necessary for future manipulations, was then generated downstream from the gI sequence. This was accomplished by cloning the oligonucleotides, IBRL5 (SEQ ID
NO:247) (5'-CATGGTTTAAGATCTC-3') and IBRL6 (SEQ ID NO:248) (5'-CATGGAGATCTTAAAC-3'), into the NcoI site of pIBR7. The plasmid generated by this manipulation is called pIBR8.
The 31-end of the gI gene was then cloned into pIBRB.
This was accomplished by cloning the 2,285 bp StuI fragment of pIBRS6 into the E. coli DNA polymerase I(Klenow fragment) filled-in 4,300 bp S uI- c~ ~lII (partial) fragment of pIBR8. The plasmid generated by this manipulation is called pIBR20.
The H6-promoted BHV1 gI gene was then moved to a vaccinia virus donor plasmid. This was accomplished by cloning the E. coli DNA polymerase I(Klenow fragment) filled-in 2,900 bp BalII-IJCoI (partial) fragment of pIBR20 WO 92/1-- '2 PCT/US92/01906 into the SmaI site of pSD542. This places the H6-promoted gI gene between tk flanking arms. The plasmid generated by this manipulation is called pIBR22.
A BalII site was then created upstream from the H6 promoter. This was accomplished by cloning the 2,800 bp I'ndIII- coRV fragment of pIBR22 into the 3,500 bp HindIII-EcoRV fragment of pGD3. (pGD3 is a plasmid that contains a BalII site upstream from an H6-promoted herpes simplex virus type 2 (HSV2) gD gene. This manipulation repalces the HSV2gD sequence with the BHVIgI gene, thereby creating a Bg1II site upstream from the H6-promoted gI gene). The plasmid generated by this manipulation is called pBHV8.
pBHV9 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1074.
Immunoprecipitation analysis was performed to determine whether vP1074 expresses authentic BHV1 gI and gIV
glycoproteins. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1074 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S)-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A (3% NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 gI and gIV
expression using the BHV1 gI-specific monoclonal antibody, 5106, and the gIV-specific monoclonal antibody, 3402 (obtained from Dr. Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with 1X buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the gI-specific monoclonal antibody and the gIV-specific monoclonal antibody. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose 92/15672 PCf/US92. -U6 bound rat anti-mouse antibody overnight at 4 C. After washing this material 5X with 1X buffer A, the gI or gIV-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X with iX
buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M
Na-salicylate for fluorography.
The BHV1 gI and gIV-specific monoclonal antibodies, 5106 and 3402, specifically precipitated the BHV1 gI and gIV
glycoproteins from vP1074 infected cells, but did not precipitate BHV1-specific proteins from mock or NYVAC
infected cells.
Generation of NYVAC/BHV1 gIII recombinant. A plasmid, pBHVgIII, was obtained from Rhone Merieux. This plasmid contains the BHV1 giII gene (Straub strain), encoded on a 3.4 kb BamHI/ indIII fragment cloned into the BamHI/HindIII
site of pBSK+. The gIII gene (Fitzpatrick et al., Virology (1989) 173:146) from this plasmid was cloned between vaccinia virus flanking arms. This was accompslished by cloning the 1,000 bp NcoI-X oI fragment of pBHVgIII, containing the 5'-end of the gIII gene, and the oligonucleotides, BHVL1 (SEQ ID N0:249) (5'-GATCCTGAGAT
AAAGTGAAAATATATATCATTATATTACAAAGTACAATTATTTAGGTTTAAT-31) and BHVL2 (SEQ ID NO:250) (5'-CATGATTAAACCTAAATAATTGT
ACTTTGTAATATAATGATATATATTTTCACTTTATCTCAG-3'), encoding the 13L promoter, into the D_amHI-XhoI site of pSD544. The plasmid generated by this manipulation is called pBHV2.
The 3'-end of the gIII gene was then cloned into pBHV2.
This was accomplished by cloning the oligonucleotides, BHVL15 (SEQ ID NO:251) (5'-TCGAGCCCGGGTAATCCAACCCGGTC
TTACTCGCGCTCGCGCCCTCGGCTCCGCGCCCTAGG-3') and BHVL16 (SEQ ID
N0:252) (5'-GTACCCTAGGGCGCGGAGCCGAGGGCGCGAGCGCGAG
TAAGACCGGGTTGGATTACCCGGGC-3'), encoding the 3'-end of the gIII gene, into the 4,700 bp XhoI-" 718 fragment of pBHV2.

1' '2 PCT/US92/01906 The plasmid generated by this manipulation is called pBHV7.
The rest of the gIII gene was then cloned into pBHV7.
This was accomplished by cloning the 500 bp partial SmaI-XhoI fragment of pBHVgIII, containing an interior portion of the giII gene, into the 4,750 bp partial SmaI-XhoI fragment of pBHV7. The plasmid generated by this manipulation is called pBHV10.
pBHV10 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1073.
Immunoprecipitation analysis was performed to determine whether vP1073 expresses an authentic BHV1 gIII
glycoprotein. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1073 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S]-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A(3$ NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 gIII expression using the BHV1 giII-specific monoclonal antibody, 1507 (obtained from Dr. Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with iX buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the gIII-specific monoclonal antibody, 1507. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C.
After washing this material 5X with IX buffer A, the gIII-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X with 1X
buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the 92/15672 PCT/US92, '06 addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M
Na-salicylate for fluorography.
The BHV1 gIII-specific monoclonal antibody, 1507, specifically precipitated the BHV1 gIII glycoprotein from vP1073 infected cells, but did not precipitate BHV1-specific proteins from mock or NYVAC infected cells.
Generation of NYVAC/BHV1 gIII AND QIV recombinant. A
plasmid, pBHVgIV, containing the BHV1 gIV gene, was obtained from Rhone Merieux. The gIV gene from this plasmid was cloned between vaccinia virus flanking arms. This was accomplished by cloning the 2,000 bp PstI-XhoI fragment of pBHVgIV, containing the gIV gene, into the PstI-XhoI site of pSD542. The plasmid generated by this manipulation is called pBHVi.
The 3'-end of the v promoter was then cloned upstream of the gIV gene. This was accomplished by cloning the oligonucleotides, BHVL7 (SEQ ID NO:239) and BHVL8 (SEQ ID
NO:240), encoding the 3'-end of the n promoter and the 5'-end of the gIV gene, into the 5,500 bp partial SstII-XhoI
fragment of pBHV1. The plasmid generated by this manipulation is called pBHV3.
Extraneous 3'-noncoding sequence was then eliminated.
This was accomplished by cloning the oligonucleotides, BHVL5 (SEQ ID NO:241) and BHVL6 (SEQ ID NO:242), into the 5,200 bp partial SmaI-PstI fragment of pBHV3. The plasmid generated by this manipulation is called pBHV4.
Extraneous linker sequence was then eliminated. This was accomplished by ligating the 5,200 bp PstI fragment of pBHV4. The plasmid generated by this manipulation is called pBHV5.
The 5'-end of the n promoter was then cloned into pBHV5. This was accomplished by cloning the 130 bp Af1II-Xhol fragment of pPI4, containing the 5'-end of the n promoter, into the 5,200 bp Af1II-XhoI fragment of pBHV5.
The plasmid generated by this manipulation is called pBHV6.
The BHV1 giII gene was then cloned into pBHV6. This WO 92/1= '2 PC'T/US92/01906 was accomplished by cloning the 1,600 bp AsD718-BamHI
fragment of pBHV10, containing the 13L-promoted gIII gene, into the 5,300 bp partial BamHI-AsD718 fragment of pBHV6.
The plasmid generated by this manipulation is called pBHVil.
pBHVll was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1083.
Immunoprecipitation analysis was performed to determine whether vP1083 expresses authentic BHV1 gIII and gIV
glycoproteins. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1083 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mls of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S]-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A (3% NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 giII and gIV
expression using the BHV1 gIII-specific monoclonal antibody, 1507, and the gIV-specific monoclonal antibody, 3402 (obtained from Dr. Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with 1X buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the giII-specific monoclonal antibody and the gIV-specific monoclonal antibody. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C. After washing this material 5X with iX buffer A, the BHV1 gIII or gIV-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X with 1X
buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4%

92/15672 PCT/US92/ .06 SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with 1M
Na-salicylate for fluorography.
The BHV1 gIII and gIV-specific monoclonal antibodies, 1507 and 3402, specifically precipitated the BHV1 gIII and gIV glycoproteins from vP1083 infected cells, but did not precipitate BHV1-specific proteins.from mock or NYVAC
infected cel ls .
Generation of NYVAC/BHV1 gI and aIII recombinant. A
plasmid, pBHVgIII, containing the BHV1 gIII gene was obtained from Rhone Merieux. The gIII gene from this plasmid was cloned between vaccinia virus flanking arms.
This was accomplished by cloning the 1,000 bp NcoI-XhoI
fragment of pBHVgIII, containing the 5'-end of the gIII
gene, and the oligonucleotides, BHVL1 (SEQ ID NO:249) and BHVL2 (SEQ ID NO:250), encoding the 13L promoter, into the BgmHI-XhoI site of pSD544VC. The plasmid generated by this manipulation is called pBHV2.
The 3'-end of the gIII gene was then cloned into pBHV2.
This was accomplished by cloning the oligonucleotides, BHVL15 (SEQ ID NO:251) and BHVL16 (SEQ ID NO:252), encoding the 3'-end of the gIII gene, into the 4,700 bp XhoI-Asp718 fragment of pBHV2. The plasmid generated by this manipulation is called pBHV7.
The rest of the gIII gene was then cloned into pBHV7.
This was accomplished by cloning the 500 bp partial maI-X oI fragment of pBHVgIII, containing an interior portion of the gIII gene, into the 4,750 bp partial SmaI- h~oI fragment of pBHV7. The plasmid generated by this manipulation is called pBHV10.
The BHV1 gI gene was then cloned into pBHV10. This was jlII fragment of accomplished by cloning the 2,900 bp Dc pBHVB, containing the H6-promoted gI gene, into the BamHI
site of pBHV10. The plasmid generated by this manipulation is called pBHV12.
pBHV12 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1087.
immunoprecipitation analysis was performed to determine WO 92/1 -- "'2 PCT/US92/01906 whether vP1087 expresses authentic BHV1 gI and gIII
glycoproteins. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1087 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S]-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A (3% NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 gI and gIII
expression using the BHV1 gI-specific monoclonal antibody, 5106, and the gIII-specific monoclonal antibody, 1507 (obtained from Dr. Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with 1X buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the gI-specific monoclonal antibody and the gIII-specific monoclonal antibody. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C. After washing this material 5X with 1X buffer A, the BHV1 gI or gIII-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X with 1X
buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4%
SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with iM
Na-salicylate for fluorography.
The BHV1 gI and gIII-specific monoclonal antibodies, 5106 and 1507, specifically precipitated the BHVi gI and giII glycoproteins from vP1087 infected cells, but did not precipitate BHV1-specific proteins from mock or NYVAC
infected cells.
Generation of NYVAC/BHV1 gI, aIII and ctIV recombinant.
A plasmid,-pBHVgIV, containing the BHV1 gIV gene, was obtained from Rhone Merieux. The gIV gene from this plasmid was cloned between vaccinia virus flanking arms. This was accomplished by cloning the 2,000 bp PstI-XhoI fragment of pBHVgIV, containing the gIV gene, into the PstI-XhoI site of pSD542VCVQ. The plasmid generated by this manipulation is called pBHVl.
The 3'-end of the rt promoter was then cloned upstream of the gIV gene. This was accomplished by cloning the oligonucleotides, BHVL7 and BHVL8, encoding the 3'-end of the n promoter and the 5'-end of the gIV gene, into the 5,500 bp partial SstII-XhoI fragment of pBHVl. The plasmid generated by this manipulation is called pBHV3.
Extraneous 3'-noncoding sequence was then eliminated.
This was accomplished by cloning the oligonucleotides, BHVL5 (SEQ ID NO:241) and BHVL6 (SEQ ID NO:242), into the 5,200 bp partial SmaI-PstI fragment of pBHV3. The plasmid generated by this manipulation is called pBHV4.
Extraneous linker sequences were then eliminated. This was accomplished by ligating the 5,200 bp PstI fragment of pBHV4. The plasmid generated by this manipulation is called pBHV5.
The 5'-end of the n promoter was then cloned into pBHV5. This was accomplished by cloning the 130 bp Af1II-XhoI fragment of pPI4, containing the 5'-end of the n promoter, into the 5,200 bp Af1II-XhoI fragment of pBHV5.
The plasmid generated by this manipulation is called pBHV6.
The BHV1 giII gene was then cloned into pBHV6. This was accomplished by cloning the 1,600 bp AsD718-DamHI
fragment of pBHV10, containing the 13L-promoted gIII gene, into the 5,300 bp partial DamHI-~sp718 fragment of pBHV6.
The plasmid generated by this manipulation is called pBHV11.
The BHV1 gI gene was then cloned into pBHVll. This was accomplished by cloning the 2,900 bp Bct1II fragment of pBHV8, containing the H6-promoted gI gene, into the BalII
site of pBHV11. The plasmid generated by this manipulation WO 92/1" '2 PCT/US92/01906 is called pBHV13.
pBHV13 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1079.
Immunoprecipitation analysis was performed to determine whether vP1079 expresses authentic BHV1 gI, gIII and gIV
glycoproteins. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP1079 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35S]-methionine (20 Ci/ml). Cells were harvested at 7 hrs post-infection by the addition of 1 ml 3X buffer A (3% NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.
Lysates were then analyzed for BHV1 gI, gIII and gIV
expression using the BHV1 gI-specific monoclonal antibody, 5106, the gIII-specific monoclonal antibody, 1507, and the gIV-specific monoclonal antibody, 3402 (obtained from Dr.
Geoffrey Letchworth, U. of Wisconsin, Madison, WI). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A sepharose at room temperature for 4 hours. After washing the material 5X with 1X buffer A, the protein A-sepharose bound rat anti-mouse antibody was bound to the gI, gIII and gIV-specific monoclonal antibodies. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C. After washing this material 5X with 1X buffer A, the BHV1 gI, gIII
and gIV-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was added to the lysate and incubated overnight at 4 C. After washing the samples 4X
with 1X buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM
Tris (pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and 92/15672 PCT/US92 ' )06 treated with 1M Na-salicylate for fluorography.
The BHV1 gI, gIII and gIV-specific monoclonal antibodies, 5106, 1507 and 3402, specifically precipitated the BHV1 gI, gilI and gIV glycoproteins from vP1079 infected cells, but did not precipitate BHV1-specific proteins from mock or NYVAC infected cells.
Esample 31 - EXPRESSION OF BOVINE VIRAL DIARRSEA VIRUS
(BVDV) GENES IN NYVAC

Generation of NYVAC/BVDV crE1/aE2 recombinant. The BVDV
gEl (gp48/gp25) "gene" (Osloss strain) was cloned into pIBI25. This was accomplished by blunt-ending the 1,370 bp EcoRI-BamHI fragment of pSP65-gEl (obtained from Eurogentec, Liege, Belgium; Renard et al., European Patent Application No:86870095) with E. coli DNA polymerase I(Klenow fragment), ligating XhoI linkers onto the ends and cloning the resulting fragment into the XhoI site of pIBI25. The plasmid generated by this manipulation is called pBDVl.
The initiation codon of the H6 promoter was then aligned with the "initiation codon" of the gEl "gene". This was accomplished by cloning the oligonucleotides, BDVM4 (SEQ
ID NO:253) (5'-AGCTTGATATCCGTTAAGTTTGTATCGTAATGGGCAAAC
TAGAGAAAGCCCTGT-3') and BDVM5 (SEQ ID NO:254) (5'-GGGCTTTCTCTAGTTTGCCCATTACGATACAAACTTAACGGATATCA-3'), encoding the 3'-end of the H6 promoter and the 5'-end of the gEl "gene", into the 4,250 bp HindIII-BalI (partial) fragment of pBDV1. The plasmid generated by this manipulation is called pBDV6.
The gEl "gene" was then cloned downstream of the H6+ATI+HA triple promoter (Portetelle et al., Vaccine (1991) 9:194) and between HA flanking arms. This was accomplished by cloning the 1,380 bp EcoRV-PstI (partial) fragment of pBDV6, containing the gEl "gene", into the 3,700 bp EcoRV-PstI fragment of pATI25. The plasmid generated by this manipulation is called pBDV7.
A~amHI site, necessary for future manipulations, was then generated downstream of the BVDV sequence. This was accomplished by cloning the oligonucleotide, BDVM6 (SEQ ID
NO:255) (5'-TCGAGGATCC-3'), into the ~hoI site of pBDV7.
The plasmid generated by this manipulation is called pBDV8.

WO 92/1Y' '2 PC'I'/US92/01906 Approximately 830 bp of gE2 (gp53) sequence (osloss strain) was then cloned downstream of the gEl sequence.
This was accomplished by cloning the 980 bp Bc~II-BamHI
fragment of p7F2-(obtained from Eurogentec, Liege, Belgium;
Renard et al., European Patent Application No:86870095), containing the gE2 sequence, into the 5,100 bp LamHI-BglII
(partial) fragment of pBDVB. The plasmid generated by this manipulation is called pBDV9.
The H6 promoted-gEl/gE2 sequence was then cloned between ATI flanking arms. This was accomplished by cloning the 2,200 bp NruI-BamHI fragment of pBDV9, containing the gEl/gE2 sequence, into the 4,900 bp NruI-BamHI fragment of pPGI7. This places the gEl/gE2 sequence under the transcriptional control of the H6 promoter and into an insertion vector. The plasmid generated by this manipulation is called pBDV23.
Approximately 270 bp of additional gE2 sequence (Osloss strain) was then cloned downstream of the existing BVDV
sequence. This was accomplished by cloning the 1,260 bp = II-DaRHI fragment of pSP65E1+E2-1 (obtained from Eurogentec, Liege, Belgium; Renard et al., European Patent Application No:86870095), containing the gE2 sequence, into the 6,100 bp fragment of pBDV23. The plasmid generated by this manipulation is called pBDV24.
pBDV24 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP972.
Immunoprecipitation analysis was performed to determine whether vP972 expresses authentic BVDV gEl and gE2 glycoproteins. Vero cell monolayers were either mock infected, infected with NYVAC or infected with vP972 at an m.o.i. of 10 PFU/cell. Following an hour adsorption period, the inoculum was aspirated and the cells were overlayed with 2 mis of modified Eagle's medium (minus methionine) containing 2% fetal bovine serum and [35SJ-methionine (20 Ci/ml). Cells were harvested at 18 hrs post-infection by the addition of 1 ml 3X buffer A (3% NP-40, 30mM Tris (pH7.4), 3mM EDTA, 0.03% Na Azide and 0.6 mg/mi PMSF) and 50 mis aprotinin, with subsequent scraping of the cell monolayers.

Lysates were then analyzed for BVDV gEl and gE2 expression using the BVDV gp48-specific monoclonal antibodies, NYC16 and NY12B1, and the BVDV gp53-specific monoclonal antibody, 209D3 (obtained from Rhone Merieux, Lyon, France). This was accomplished by the following procedure: rat anti-mouse sera was bound to protein-A
sepharose at room temperature for 4 hours. After washing the material 5X with 1X buffer A, protein A-sepharose bound rat anti-mouse antibody was bound to the gEl-specific monoclonal antibodies, NYC16 and NY12B1, and the gE2-specific monoclonal antibody, 209D3. The lysates, meanwhile, were precleared by incubating normal mouse sera and the protein A-sepharose bound rat anti-mouse antibody overnight at 4 C. After washing this material 5X with 1X
buffer A, the BVDV gEl or gE2-specific monoclonal antibody, rat anti-mouse, protein A-sepharose conjugate was.added to the lysate and incubated overnight at 4 C. After washing the samples 4X with iX buffer A and 2X with a LiC12/urea buffer, the precipitated proteins were dissociated from the immune complexes by the addition of 2X Laemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins were then fractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixed and treated with iM Na-salicylate for fluorography.
The BVDV gEl or gE2-specific monoclonal antibodies precipitated BVDV-specific glycoproteins from vP972 infected cells, but did not precipitate BVDV-specific proteins from NYVAC or mock infected cells.
Generation of NYVAC/BVDV CAPSID/cxEl/gE2 recombinant.
The BVDV gEl "gene" was cloned into pIBI25. This was accomplished by blunt-ending the 1,370 bp EcoRI-BamHI
fragment of pSP65-gEl, containing the gEl "gene", with E.
coli DNA polymerase I (Klenow fragment), ligating XhoI
linkers onto the ends and cloning the resulting fragment into the XhoI site of pIBI25. The plasmid generated by this manipulation is called pBDV1.
The gEl "gene" was then cloned between u flanking arms.
This was accomplished by cloning the 1,400 bp XhoI fragment WO 92/1" -2 PCT/US92/01906 of pBDV1, containing the gEl sequence, into the XhoI site of pSD486. The plasmid generated by this manipulation is called pBDV11.
The "initiation codon" of the gEl "gene" was then aligned with the initiation codon of u promoter. This was accomplished by cloning the oligonucleotides, BDVM7 (SEQ ID
NO:256) (5'-CGATTACTATGGGCAAACTAGAGAAAGCCCTGT-3') and BDVM8 (SEQ ID NO:257) (5'-GGGCTTTCTCTAGTTTGCCCATAGTAAT-3'), encoding the 3'-end of the u promoter and the 5'-end of the gEl sequence, into the 4,800 bp partial BalI-C1aI fragment of pBDV11. The plasmid generated by this manipulation is called pBDV12.
Part of the BVDV gE2 "gene" was then cloned into pBDV12, downstream from the gEl sequence. This was accomplished by cloning the 1,000 bp BalII-BamHI fragment of p7F2, containing the gE2 sequence, into the 4,650, bp BczlII-BamHI fragment of pBDV12. The plasmid generated by this manipulation is called pBDV14.
The rest of the gE2 "gene" was then cloned into pBDV14.
This was accomplished by cloning the 1,260 bp BalII-BamHI
fragment of pSP65E1+E2-1, containing the gE2 "gene", into the 4,650 bp Bc1II-BamHI fragment of pBDV14. The plasmid generated by this manipulation is called pBDV17.
The capsid "gene" (Osloss strain) was then cloned into pBDV17, upstream from the gEl sequence. This was accomplished in 2 steps. The first step aligned the initiation codon of the u promoter with the "initiation codon" of the capsid "gene". This was accomplished by cloning the oligonucleotides, BDVL12 (SEQ ID NO:258) (5'-CGATTACTATGGAGTTGATTACAAATGAACTTTTATACAAAACATACAAAC
AAAAACCCGCTGGAGTGGAGGAACCAGTATATAACCAAGCAGGTGACCCT-3') and BDVL13 (SEQ ID NO:259) (5'-CTAGAGGGTCACCTGCTTGGTTATATA
CTGGTTCCTCCACTCCAGCGGGTTTTTGTTTGTATGTTTTGTATAAAAGTTCATTTGTAA
TCAACTCCATAGTAAT-3'), encoding the 3'-end of the u promoter and the 5'-end of the capsid sequence, into the 5,200 bp C1aI-XbaI fragment of pBDV17. The plasmid generated by this manipulation is called pBDV25. The second step cloned the rest of the capsid "gene" into pBDV25. This was accomplished by cloning the 1,870 bp BstEII-Bg1II fragment V''q 92/15672 PCT/US92/" 106 of pSP65C-E1-E2 (obtained from Eurogentec, Liege, Belgium;
Renard et al., European Patent Application No:86870095), containing the capsid "gene", into the 4,700 bp BstEII-BcxlII
fragment of pBDV25. The plasmid generated by this manipulation is called pBDV26.
The u-promoted capsid/gEl/gE2 sequence was then cloned between tk flanking arms. This was accomplished by cloning the 3,200 bp SnaBI-BamHI fragment of pBDV26, containing the u-promoted capsid/gEl/gE2 sequence, into the 4,000 bp SmaI-BamHI fragment of pSD542. The plasmid generated by this manipulation is called pBDV27.
pBDV27 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1017.
Immunoprecipitation experiments with vP1017 infected cells were performed as described above for the expression of vP972. No BVDV-specific proteins were precipitated from mock infected or NYVAC infected Vero cells. BVDV-specific proteins were precipitated, however, from lysates of vP1017.
Generation of NYVAC/BVDV gE2 recombinant. The BVDV gEl "gene" was cloned into pIBI25. This was accomplished by blunt-ending the 1,370 bp EcoRI-BamHI fragment of pSP65-gEl, containing the gEl "gene", with E. coli DNA polymerase I
(Klenow fragment), ligating ioI linkers onto the ends and cloning the resulting fragment into the XhoI site of pIBI25.
The plasmid generated by this manipulation is called pBDV1.
The initiation codon of the H6 promoter was then aligned with the putative "initiation codon" of the gEl "gene". This was accomplished by cloning the oligonucleotides, BDVM4 (SEQ ID NO:253) and BDVM5 (SEQ ID
NO:254), encoding the 3'-end of the H6 promoter and the 5'-end of the gEl "gene", into the 4,250 bp HindIII-BalI
(partial) fragment of pBDV1. The plasmid generated by this manipulation is called pBDV6.
The gEl "gene" was then cloned downstream of the H6+ATI+HA triple promoter and between HA flanking arms.
This was accomplished by cloning the 1,380 bp EcoRV-PstI
(partial) fragment of pBDV6, containing the gEl "gene", into the 3,700 bp EcoRV-PstI fragment of pATI25. The plasmid generated by this manipulation is called pBDV7.

WO 92/1 %2 PC'T/US92/01906 A&4_mHI site, necessary for future manipulations, was then generated downstream of the BVDV sequence. This was accomplished by cloning the oligonucleotide, BDVM6 (SEQ ID
NO:255), into the XhoI site of pBDV7. The plasmid generated by this manipulation is called pBDV8.
Approximately 830 bp of BVDV gE2 sequence was then cloned downstream of the gEl "gene". This was accomplished by cloning the 980 bp $gIII-Bam1iI fragment of p7F2, containing the gE2 sequence, into the 5,100 bp DamHI-Bg1II
(partial) fragment of pBDV8. The plasmid generated by this manipulation is called pBDV9.
The gEl/gE2 sequence was then cloned between ATI
flanking arms. This was accomplished by cloning the 2,200 bp NruI-BamHI fragment of pBDV9, containing the H6-promoted gEl/gE2 "genes", into the 4,900 bp NruI-BamHI fragment of pPGI7. The plasmid generated by this manipulation is called pBDV23.
Approximately 270 bp of additional gE2 sequence was then cloned downstream of the existing BVDV sequence. This was accomplished by cloning the 1,260 bp Bg1II-BamHI
fragment of pSP65E1+E2-1, containing the additional gE2 sequence, into the 6,100 bp BamHI-BalII (partial) fragment of pBDV23. The plasmid generated by this manipulation is called pBDV24.
The gEl sequence was then deleted from BDV24. This was accomplished by cloning a 130 bp NruI-Pstl PCR fragment, containing the 3'-end of the H6 promoter and the 5'-end of the gE2 "gene", into the 5,900 bp NruI-PstI fragment of pBDV24. This PCR fragment was generated from the plasmid, pBDV17, with the oligonucleotides, BDVP14 (SEQ ID NO:260) (5'-TTTCGCGATATCCGTTAAGTTTGTATCGTAATGCTCCCAGTTTGCAAACCC-3') and BDVP15 (SEQ ID NO:261) (5'-TCTCCACCTTTACACCACACT-3').
The plasmid generated by this manipulation is called pBDV28.
Sequence analysis revealed that the H6 promoter in pBDV28 contains a 2 bp insertion. To correct this error, the 130 bp NruI-PstI fragment of pBDV28, containing the 3'-end of the H6 promoter and the 5'-end of the gE2 "gene", was cloned into the 5,900 bp NruI-PstI fragment of pBDV24. The plasmid generated by this manipulation is called pBDV29.

192/15672 PCT/US9A' ?06 pBDV29 was used in in vitro recombination experiments with vP866 (NYVAC) as the rescuing virus to yield vP1097.
Immunoprecipitation experiments with vP1097 infected cells are performed as described above to yield BVDV
proteins from cells or lysates. Esample 32 - CLONING AND $YPR$SSION OF HIIKAN
CYTOlYiEGALOVIRIIS (HCIriV) GLYCOPROTEIN ANTIGENS
IN PO%VIRIIS VECTORS

Cloning of the HCMV aB gene into the NYVAC donor plasmid, pSD542. The 4800 bp HindIII-DamHI fragment of the HindIII D fragment of the HCMV DNA was cloned into the 2800 bp HindIII-BamHI fragment of the plasmid pIBI24. By in vitro mutagenesis (Kunkel, 1985; Russel et al., 1986) using the oligonucleotides CMVM5 (SEQ ID NO:262) (5'-GCCTCATCGCTGCT
GGATATCCGTTAAGTTTGTATCGTAATGGAATCCAGGATCTG-3') and CMVM3 (SEQ ID NO:263) (5'-GACAGATTGTGATTTTTATAAGCATCGTAAGC
TGTCA-3'), the gB gene was modified to be expressed under the control of the vaccinia H6 promoter (Taylor et al., 1988a,b; Perkus et al., 1989). The plasmid containing the modified gB was designated 24CMVgB(5+3).
The 2900 bp coRV-DamHI fragment of 24CMVgB(5+3) was cloned into the 3100 bp coRV- c~lII fragment of pSP131. This cloning step put the gB gene under the control of the H6 promoter. The resulting plasmid was designated SP131gB.
To modify the restriction sites flanking the H6 promoted gB in SP131gB the following steps were performed.
Plasmid pMP22BHP contains a subclone of the HindIII F
fragment of vaccinia (WR strain) containing a portion of the HBV sAg in a polylinker region at the DamHI site. pMP22BHP
was digested within the polylinker with HindIII and ligated to a HindIII fragment from SP131CMVgB (containing the H6 promoted gB gene) generating plasmid SAg22CMVgB. SAg22CMVgB
was digested with &amHI and partially digested with HindIII
and ligated to a polylinker derived from pIBI24 by BamHI and HindIII digestion creating plasmid 22CMVgB which contains the H6 promoted gB gene without the HBV sAg.
Plasmid pSD542 (a NYVAC TK locus donor plasmid) was derived from plasmid pSD460 (Tartaglia et al., 1992) by forming vector plasmi-d pSD513 as described above in Example 7. The polylinker region in pSD513 was modified by cutting iVVO 92/1 '2 PCT/US92/01906 with gstI/$amHI and ligating to annealed synthetic oligonucleotides MPSYN288 (SEQ ID NO:264) (5' GGTCGACGGATCCT 3') and MPSYN289 (SEQ ID NO:265) (5' GATCAGGATCCGTCGACCTGCA 3') resulting in plasmid pSD542.
22CMVgB was digested with Dj2HI and siI to generate a fragment containing the H6 promoter and part of the gB gene, and with NsiI and PstI to generate a fragment containing the remainder of the gB gene. These two fragments were ligated to pSD542 that had been digested with $UHI and PstI within its' polylinker creating the NYVAC donor plasmid 542CMVgB.
Cloning of the HCMV aB size into the ALVAC donor plasmid CP3LVOH6. An 8.5kb canarypox $g1II fragment was cloned in the BamHI site of pBS-SK plasmid vector to form pWW5. Nucleotide sequence analysis revealed a reading frame designated C3. In order to construct a donor plasmid for insertion of foreign genes into the C3 locus with.the complete excision of the C3 open reading frame, PCR primers were used to amplify the 5' and 3' sequences relative to C3.
Primers for the 5' sequence were RG277 (SEQ ID NO:177) and RG278 (SEQ ID NO:178).
Primers for the 3' sequences were RG279 (SEQ ID NO:179) and RG280 (SEQ ID N0:180). The primers were designed to include a multiple cloning site flanked by vaccinia transcriptional and translational termination signals. Also included at the 5'-end and 3'-end of the left arm and right arm were appropriate restriction sites (,s 718 and = RI for left arm and EcoRI and SacI for right arm) which enabled the two arms to ligate into ,s 718/SacI digested pBS-SK plasmid vector. The resultant plasmid was designated as pC3I.
A 908 bp fragment of canarypox DNA, immediately upstream of the C3 locus was obtained by digestion of plasmid pWW5 with siI and SspI. A 604 bp fragment of canarypox and DNA was derived by PCR (Engelke et al., 1988) using plasmid pWW5 as template and oligonucleotides CP16 (SEQ ID NO:266) (5'-TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTC
TGC-3') and CP17 (SEQ ID NO:267) (5'-TCGCTCGAGTAG
GATACCTACCTACTACCTACG-3'). The 604 bp fragment was digested with ~sg718 and h~oI (sites present at the 5' ends of oligonucleotides CP16 and CP17, respectively) and cloned V ~ 92/15672 PCT/US92/ )06 into Asp718-XhoI digested and alkaline phosphatase treated IBI25 (International Biotechnologies, Inc., New Haven, CT) generating plasmid SPC3LA. SPC3LA was digested within IBI25 with coRV and within canarypox DNA with siI and ligated to the 908 bp siI-SSDI fragment generating SPCPLAX which =
contains 1444 bp of canarypox DNA upstream of the C3 locus.
A 2178 bp BalII-StvI fragment of canarypox DNA was isolated from plasmids pXX4 (which contains a 6.5 kb NsiI
fragment of canarypox DNA cloned into the PstI site of pBS-SK. A 279 bp fragment of canarypox DNA was isolated by PCR
(Engelke et al., 1988) using plasmid pXX4 as template and oligonucleotides CP19 (SEQ ID NO:268) (5'-TCGCTCGAGCTTTC
TTGACAATAACATAG-3') and CP20 (SEQ ID NO:269) (5'-TAGGAGC
TCTTTATACTACTGGGTTACAAC-3'). The 279 bp fragment was digested with XhoI and SacI (sites present at the 5' ends of oligonucleotides CP19 and CP20, respectively) and,cloned into SacI-XhoI digested and alkaline phosphatase treated IBI25 generating plasmid SPC3RA.
To add additional unique sites to the polylinker, pC3I
was digested within the polylinker region with EcoRI and C1aI, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP12 (SEQ ID NO:272) and CP13 (SEQ ID NO:273) (containing an coRI sticky end, XhoI site, BamHI site and a sticky end compatible with C1aI) generating plasmid SPCP3S.
CP12 (SEQ ID NO:272) 5'-AATTCCTCGAGGGATCC -3' CP13 (SEQ ID NO:273) 3'- GGAGCTCCCTAGGGC-5' EcoRI ChoI BamHI

SPCP3S was digested within the canarypox sequences downstream of the C3 locus with StyI and SacI (pBS-SK) and ligated to a 261 bp BalII-SacI fragment from SPC3RA and the 2178 bp Bcr1II-StvI fragment from pXX4 generating plasmid CPRAL containing 2572 bp of canarypox DNA downstream of the C3 locus. SPCP3S was digested within the canarypox sequences upstream of the C3 locus with Asp718 (in pBS-SK) and AccI
and ligated to a 1436 bp " 718- ccI fragment from SPCPLAX
generating plasmid CPLAL containing 1457 bp of canarypox DNA
upstream of the C3 locus. CPLAL was digested within the canarypox sequences downstream of the C3 locus with StyI and SacI (in pBS-SK) and ligated to a 2438 bp StyI-SacI fragment WO 92/1z '2 PCT/US92/01906 from CPRAL generating plasmid CP3L containing 1457 bp of canarypox DNA upstream of the C3 locus, stop codons in six reading frames, early transcription termination signal, a polylinker region, early transcription termination signal, stop codons in six reading frames, and 2572 bp of canarypox DNA downstream of the C3 locus. The resulting plasmid was designated SPCP3L.
The early/late H6 vaccinia virus promoter (Guo et al., 1989; Perkus et al., 1989) was derived by PCR (Engelke et al., 1988) using pRW838 as template and oligonucleotides CP21 (SEQ ID NO:270) (5'-TCGGGATCCGGGTTAATTAAT
TAGTTATTAGACAAGGTG-3') and CP22 (SEQ ID NO:271) (5'-TAGGAATTCCTCGAGTACGATACAAACTTAAGCGGATATCG-3'). The PCR
product was digested with BamHI and EcoRI (sites present at the 5' ends of oligonucleotides CP21 and CP22, respectively) and ligated to CP3L that was digested with BamHI,and EcoRI
in the polylinker generating plasmid VQH6CP3L.
ALVAC donor plasmid VQH6CP3L was digested within the polylinker with XhoI and within the H6 promoter with NruI
and ligated to aNruI/ '#ndIII fragment from 22CMgB
containing part of the H6 promoter and gB gene and a polylinker derived from pIBI24 by ~ioI and indIII digestion generating the ALVAC donor plasmid CP3LCMVgB.
Example 33 - CONSTRUCTION OF RECOMBINANT VIRUSES:
CYTOMEGALOVIRUS
The CMV (cytomegalovirus) gB gene was inserted into the TK site of NYVAC. The recombinant virus was designated vP1001. The CMV gB gene was inserted into the C3 site of ALVAC. The recombinant was designated vCP139.
Examvle 34 - I1rIIrIIINOFLIIORESCENCE OF CMV GB PROTEIN IN
RECOMBINANT VIRUS INFECTED CELLS
Immunofluorescence studies were performed as described previously (Taylor et al., 1990) using guinea pig polyclonal serum followed by fluorescein isothiocyanate goat anti-guinea pig. Cells infected with vP1001 showed gB expressed on the plasma membrane. Weak internal expression was detected within cells infected with vCP139.

Wo) 92/15672 PCT/US92!'"906 EsamDle 35 - IMIINOPRECIPITATION OF CMV GB IN ABCOMBINANT
INFECTED CELLS

Immunoprecipitation experiments were performed as described previously (Taylor et al., 1990). The CMV gB
glycoprotein produced in CMV infected cells has a molecular weight of 55 kDa with a precursor form of 130 kDa (Gretch et al., 1988). Cells infected with vP1o01 and vCP139 produce two CMV gB coded proteins of approximately 116 kDa and 55 kDa.
Example 36 - NEUTRALIZING ANTIBODIES
Following immunization of CBA mice with vP1001 (NYVAC-HCMV gB), neutralizing antibody titers of the sera of inoculated mice were assessed (Gonczol et al., 1986).
Antibodies capable of neutralizing human cytomegalovirus were detected in the sera of mice 14-21 days later (geometric mean titers of 1:16) and between 28 and 60 days post-immunization (gmt=1:26). Immunization of CBA mice with ALVAC-HCMV gB generated HCMV neutralizing antibody titers of 1:64 gmt (14-21 days pi, 1:91 gmt between 21 and 28 days pi), and 1:111 between 28 and 60 days pi. Thus, immunization of mice with vaccinia virus or canarypox virus recombinants expressing HCMV gB elicited antibodies able to neutralize the infectivity of HCMV.
Esample 37 - CELL MEDIATED I1+II+IIINITY
Besides HCMV neutralizing antibody titers, vCP139 is also capable of eliciting cytotoxic T lymphocytes capable of killing murine L929 cells infected with a recombinant vaccinia virus expressing HCMV gB (vaccinia WR-gB). CBA mice were immunized intraperitoneally with 2.5x108 pfu of vCP139.
Sixteen to 30 days later, spleen cell suspensions of the mice were re-stimulated in vitro by co-incubation with syngeneic spleen cells previously infected with vP1001 at a ratio of 2:1. After 5 days, the spleen cells were counted and, using the 51Cr-release assay (Zinkernagel et al., 1984), assessed for cytotoxicity against uninfected L929 cells or L929 cells infected with adenovirus Ad5dlE3, recombinant adenovirus expressing HCMV gB (Ad-gB), vaccinia virus, and recombinant vaccinia virus expressing HCMV gB.
Only background levels of reactivity were measured against WO 92/1 ' PCT/US92/01906 the uninfected targets as well as the targets infected with Ad5dlE3. In contrast, the in vitro stimulated spleen cells readily killed L929 cells infected with Ad-gB expressing HCMV gB. Although some lytic reactivity was observed against targets infected with the vaccinia virus vector, much higher cytolysis was measured against targets infected with the recombinant vaccinia virus expressing gB. This clearly demonstrated that cytotoxic T lymphocytes specific for epitopes located within HCMV gB were generated by inoculation with the recombinant canarypox virus expressing HCMV gB (vCP139).
Example 38 - NYVAC AND ALVAC DONOR PLASMID CONSTRUCTION:
CANINE PARVOVIRIIS

In order to generate poxvirus recombinants expressing the canine parvovirus VP2 capsid gene, donor plasmids were constructed in which the VP2 gene was amplified from the genome of the CPV-d isolate (CPV-2 antigenic type), coupled to the vaccinia H6 promoter (Perkus et al., 1989) and inserted into NYVAC or ALVAC insertion vectors. The NYVAC
insertion site is the deorfed ATI locus while the ALVAC
insertion site is the deorfed C3 locus.
The VP2 gene sequences were obtained by PCR from the plasmid pBI265(1). This plasmid, obtained from Dr.
Colin R. Parrish, James A. Baker Institute, Cornell University, Ithaca, NY, contains the genome of the CPV-d isolate (Cornell 790320)(antigenic type CPV-2). The DNA
sequence of the VP2 gene from this isolate has been published (Parrish et al., 1988).
Using pBI265(1) as template and synthetic oligonucleotides RG451 (SEQ ID NO:274) (5'-TCGGGT
ACCTCGCGATATCCGTTAAGTTTGTATCGTAATGAGTGATGGAGCAGT-3') and RG452 (SEQ ID NO:275) (5'-TAGGAATTCCTCGAGTTAA
TATAATTTTCTAGGTGC-3') as primers, the complete VP2 open reading frame (ORF) was amplified by PCR. The purified DNA
fragment was cut with Lsp718 and coRI and cloned into the AsD718 and coRI sites in pBluescript SK+, resulting in pDT4. The VP2 gene was confirmed by DNA sequence analysis.
The VP2 gene contains two TTTTTNT sequences within the ORF which could function as early transcriptional stop ~ 92/15672 PCT/US92/ 06 signals (Yuen et al., 1987). To eliminate these signals, PCR site-directed mutagenesis was used to change the nucleotide sequence while retaining the correct amino acid sequence. A 250 bp fragment was amplified from pBI265(1) using synthetic oligonucleotides RG453 (SEQ ID NO:276) (5'-ATCAGATCTGAGACATTGGGTTTCTATCCATG-3') and RG454 (SEQ ID
NO:277) (5'-TTAGTCTACATGGTTTACAATCAAAGAAGAATGTTCCTG-31).
IIII and AccI, and The purified fragment was digested with Dc used to replace the ~g,..lII/= I VP2 fragment in pDT4. The resulting plasmid, pED3, contains the modified VP2 gene in which the TTTTTNT sequences have been changed to TTTCTAT and TTCTTCT.
A NYVAC donor plasmid containing the CPV VP2 capsid gene was constructed as follows. The modified VP2 gene was excised from pED3 with NruI and XhoI and cloned into pMPATIH6HSVTK cut with NruI/ h~oI. pMPATIH6HSVTK ~s a derivative of pSD552 (described elsewhere in this disclosure) in which an expression cassette containing the coding sequences for the HSV-1 thymidine kinase gene under control of the H6 promoter is inserted between the HpaI and XhoI sites in the polylinker region. Cutting this plasmid with ruI and XhoI excises the tk gene, but retains the 5'end of the H6 promoter. Insertion of the modified VP2 gene into this vector as described above generates pATI-VP2.
This NYVAC donor plasmid contains the H6 promoted VP2 gene flanked by the ATI insertion arms.
An ALVAC donor plasmid containing the CPV VP2 capsid gene was constructed as follows. The modified VP2 gene was excised from pED3 with NruI and ~C2oI and the purified fragment was cloned into pVQH6CP3L (plasmid described in Flavivirus section) cut with druI and Xhol. The resulting plasmid, pC3-VP2, contains the H6 promoted VP2 gene flanked by the C3 insertion arms.
ExamRle 39 - GENERATION OP NYVAC AND ALVAC RECOMBINANTB:
CANINE PARVOVIRUS (CPV) The donor plasmid pATI-VP2 was used in in vitro recombination experiments in VERO cells with NYVAC (vP866) and NYVAC-RG (vP879) as rescue viruses to yield vP998 and vP999 respectively (Tartaglia et al., 1992). Recombinant viruses were identified by in situ hybridization procedures (Piccini et al., 1987) using a radiolabelled VP2 specific DNA probe. Recombinant plaques were purified by three rounds of plaque purification and amplified for further analysis.
The donor plasmid pC3-VP2 was used in in vitro recombination experiments in CEF cells with ALVAC (CPpp) and ALVAC-RG (vCP65A) as rescue viruses to yield vCP123 and vCP136 respectively (Taylor et al., 1992). Recombinant viruses were identified by in situ hybridization procedures (Piccini et al., 1987) using a radiolabelled VP2 specific probe (positive signal) and C3 ORF specific probe (negative signal). Recombinant plaques were purified by three rounds of plaque purification and amplified for further analysis.
Ezample 40 - EXPRESSION ANALYSIS OF THE NYVAC- AND ALVAC-All the recombinants containing the CPV VP2 gene were tested for expression by immunofluorescence as previously described (Taylor et al., 1990) using monoclonal antibodies specific for VP2 epitopes or polyclonal CPV dog serum. All sera were obtained from Dr. Colin R. Parrish, James A. Baker Institute, Cornell University, Ithaca, NY. The NYVAC-based recombinants were tested on VERO cells while the ALVAC-based recombinants were tested on CEF cells. Recombinants vP998, vP999, vCP123, and vCP136 all displayed internal fluorescence, with localization in the nucleus. No surface fluorescence was detected. In addition, the two recombinants containing the rabies G gene (vP999 and vCP136) were screened with monoclonal antibodies specific for rabies G epitopes. Both displayed strong fluorescence on the surface of the cell.
To further characterize expression of an authenic VP2 gene product in the above recombinants, immunoprecipitation analysis was done using the same antisera (Taylor et al., 1990). The NYVAC-based recombinants were tested on VERO
cells while the ALVAC-based recombinants were tested on CEF
cells. In all recombinants (vP998, vP999, vCP123, and vCP136) the antisera precipitated a protein of 65 kDa, which is consistent with the size of the native VP2 gene. No ' N92/15672 PCT/US92 106 protein of this size was detected from cell lysates or from either parental virus (NYVAC or ALVAC).
Esample 41 - INSERTION OF EPSTEIN BARR VIRUS (EBV) GENES
INTO ALVAC

Construction of donor plasmid EBV Triple.2. Plasmid EBV Triple.l (Example 11) contains expression cassettes for EBV genes gH, gB and gp340 all inserted into a vaccinia TK
locus insertion plasmid. Plasmid EBV Triple.1 was digested with SmaI/BamHI and a 0.3 kb fragment containing the 42kDa Entomopox virus promoter and the 5' end of the EBV gH gene was isolated. EBV Triple.1 plasmid was also digested with BamHI and a 7.3 kb fragment containing the 3' end of the EBV
gH gene, the EBV gB expression cassette, and the EBV gp340 expression cassette was isolated. These two fragments were then ligated into the ALVAC C5 locus insertion plasmid pNVQC5LSP7 (described herein, see Tetanus example) which had been cut with SmaI/DamHI. The resulting plasmid was designated EBV Triple.2.
Insertion of EBV genes into ALVAC. Plasmid EBV
Triple.2, containing expression cassettes for the three EBV
genes, gH, gB and gp340, in the C5 insertion locus, was used as donor plasmid for recombination with ALVAC, generating ALVAC recombinant vCP167.
Expression of EBV proteins by vP944 and vCP157.
Metabolically labelled lysates from cells infected with ALVAC recombinant vCP167 and vP944, the NYVAC-based recombinant containing the same three genes (Example 11), were subjected to immunoprecipitation using human polyclonal serum to EBV as well as mouse monoclonal antibodies to EBV
gB and gp340. Precipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by radioautography. Proteins of the correct molecular weights and specificities for EBV gB, gH and gp340 were observed for both NYVAC-based recombinant vP944 and ALVAC-based recombinant vCP167.
EBample 42 - CONSTRUCTION OF AN EXPRESSION CASSETTE FOR
INSERTION ON EQUINE INFLUENZA HA
(A1/PRAGIIE/56) INTO NYVAC AND ALVAC

purified EIV (A1/Prague/56) genomic RNA was provided by Rhone-Merieux (Lyon, France). EIV-specif ic cDNA was prepared as described by Gubler and Hoffman (1983).
Oligonucleotide EIVSIP (SEQ ID N0:278) (5'-ATCATCCT
GCAGAGCAAAAGCAGG-3') was used to synthesize first strand cDNA. This oligonucleotide (SEQ ID NO:278) is complementary to the 3'-end of each genomic RNA segment. As per Gubler and Hoffman (1983), the cDNA is dG-tailed and inserted into pMG5 digested with coRV and dC-tailed. Insertion of the cDNA in this manner in pMG5 creates a$amHI site on both plasmid/cDNA sequence borders.
Five hundred colonies from this EIV cDNA library were transferred in duplicate to LB-agar plates containing ampicillin (50 g/ml). The colonies were transferred to nitrocellulose for hybridization with a radiolabeled EIV HA-specific probe. This probe was derived by using radiolabeled first strand cDNA synthesized with oligonucleotide EIVSIP (SEQ ID NO:278) and purified HA
genomic RNA as template. The HA genomic segment was purified from a 1.2% low melting point agarose gel (Bethesda Research Laboratories, Gaithersburg, MD). Total genomic RNA
was fractionated in this gel system run at 2 volts/cm in iX
TBE. HA RNA was recovered by excising the HA band and melting the agarose at 75 C followed by two cycles of phenol extraction, one ether extraction, and ETOH precipitation.
Colony hybridization was performed according to standard procedures (Maniatis et al., 1991) and a cDNA clone containing a 1.4 kb HA cDNA insert was identified. The clone was confirmed to be HA-specific by Northern blot analysis versus genomic RNA and nucleotide sequence analysis. This 1.4 kb fragment was used to generate a radiolabeled HA-specific DNA probe for subsequent cDNA
library screenings.
Using the probe, other HA-specific cDNA clones were identified. The largest were of 1.0 kb, 1.2 kb, and 1.4 kb and they were designated as pEIVAIPHA-1, -10, and -8, respectively. Collectively, these clones contain an entire EIV HA coding sequence as determined by nucleotide sequence analysis. The entire sequence of the EIV HA (A1/Prague/56) determined from these analyses is provided in FIG. 23 (SEQ
ID NO:279).

')92/15672 PC'I'/US92 )06 A full-length cDNA clone of the EIV HA was next generated by splicing segments from different cDNA clones.
The 5'-most 1200 bp of the HA coding sequence was derived from pEIVAIPHA-8 by PCR using this plasmid as template and, oligonucleotides EIVSIP (SEQ ID NO:278) and EIVAIP7A (SEQ ID
NO:280) (5'-GTTGGTTTTTTCTATTAG-3'). This 1200 bp fragment was digested with ~stI creating PstI cohesive ends at both the 5' and 3' termini. The 3'-most 600 bp of the HA coding sequence was derived from pEIVAIPHA-10 by digestion with BamHI and PstI. These two fragments were inserted into pBS-SK (Stratagene, La Jolla, CA) digested with PstI and BamHI.
The plasmid generated containing the entire EIV HA
(A1/Prague/56) coding sequence was designated as pBSEIVAIPHA.
The EIV HA coding sequence (ATG to TAA) was derived by PCR from pBSEIVAIPHA using oligonucleotides EIVAIPHA5P (SEQ
ID NO:281) (5'-CGATATCCGTTAAGTTTGTATCGTAATGAA
GACTCAAATTCTAATATTAGCC-3') and EIVAIPHA3P (SEQ ID NO:282) (5'-ATCATCGGATCCATAAAAATTATATACAAATAGTGCACCG-3'). The oligonucleotide EIVAIPHA5P (SEQ ID NO:281) provides the 3'-most 26 bp (from NruI site) of the vaccinia virus H6 promoter (Goebel et al., 1990a,b). The 1.7 kb PCR-derived fragment was inserted into NruI digested pCPCV1 to yield pC3EIVAIPHA. pCPCV1 is an insertion vector which contains the H6 promoter. Insertion of the 1.7 kb blunt-ended fragment in the proper orientation places the EIV HA 3' to the H6 promoter. The plasmid pCPCV1 was derived as follows.
Plasmid pFeLV1A, which contains a 2.4 kb fragment containing the FeLV env gene (Guilhot et al., 1987) in the PstI site of pTP15 (Guo et al., 1989) was digested with PstI to excise the FeLV sequences and religated to yield plasmid pFeLVF4.
The vaccinia virus H6 promoter element followed by a polylinker region were liberated from pFeLVF4 by digestion with KnnI and HRaI. The 150 bp fragment was blunt-ended using T4 DNA polymerase and inserted into pRW764.2, a plasmid containing a 3.3kb PvuII genomic fragment of canarypox DNA. pRW764.2 was linearized with EcoRI, which recognizes a unique EcoRI site within the canarypox sequences, and blunt-ended using the Kienow fragment of the WO 92/1'. PCT/US92/01906 E. coli DNA polymerase. The resultant plasmid was designated as pCPCV1. This plasmid contains the vaccinia virus H6 promoter followed by a polylinker region and flanked by canarypoxvirus homologous sequences.
EsamDle 43 - CONSTRUCTION OF AN EXPRESSION CASSETTE FOR
INSERTION OF EIV HA (A2/FONTAINEBLBAII/79) INTO NYVAC AND ALVAC

Purified EIV (A2/Fontainebleau/79) genomic RNA was provided by Rhone-Merieux (Lyon, France). EIV-specific cDNA
was prepared as described by Gubler and Hoffman (1983) and as described for the EIV (Al/Prague/56) cDNA preparation.
The oligonucleotide EIVSIP (SEQ ID NO:278) was used for first-strand cDNA synthesis.
To screen bacterial colonies containing full-length cDNA clones of the HA gene, eight pools of transformed colonies were amplified in 500 ml cultures and plasmid DNA
preparations obtained by standard procedures (Sambrook et al., 1989) Total plasmid DNA was used as template in standard PCR reactions with oligonucleotides EIVSIP (SEQ ID
NO:278) and EIVS2H (SEQ ID NO:283) (5'-ATCATCAAGCTTAGTAGAAA
CAAGG-3'). Such a reaction would potentially amplify only full-length cDNA sequences of all eight EIV genomic segments, since these primers were complementary to conserved sequences at the 5' and 3' ends of these eight segments.
Plasmid preparation, pPEIVA2F-5, as template generated a 1.8 kb PCR-derived fragment consistent with the size of a full-length HA-specific fragment. This PCR-derived fragment was re-amplified by PCR for use as a probe against the remainder of the cDNA library. Using this probe, clones pEIVA2FHA-7 and -8 were identified and the cDNA insert analyzed by nucleotide sequence analysis using custom synthesized oligonucleotides (Goebel et al., 1990a).
Nucleotide sequence analysis demonstrated that clones #7 and #8 represented the 3'-most 1200 bp of the EIV
(A2/Fontainebleau/79) HA coding sequence (FIG. 24) (SEQ ID
NO:284).
The 1200 bp EIV sequence was amplified from clone #7 by PCR using oligonucleotides A2F3P (SEQ ID NO:285) ) 92/15672 PCT/US9!. A906 (5'-ATCATCACTAGTATAAAAATCAAATGCAAATGTTGCATCTGATGTTGCC-3') and A2FBAM2 (SEQ ID NO:286) (5'-ATCATCGGATCCATCACCCG
AGCACAAACAATGAGCAG-3'). The 5'-end of this 1200 bp fragment after digestion with BamHI corresponds to nucleotide 617 of the complete EIV (A2/Fontainebleau/79) HA coding sequence in FIG. 24 (SEQ ID NO:284). This fragment was also digested with SpeI which was engineered 3' to the coding sequence using oligonucleotide A2F3P (SEQ ID N0:285). This 1200 bp fragment was to be co-inserted into SmaI/SpeI digested pBS-SK (Stratagene, La Jolla, CA) with a fragment containing the 5'-most 616 bp (defined below). However, screening of potential transformants demonstrated that only the 1200 bp fragment was inserted. Numerous clones were chosen for nucleotide sequence analysis.
After nucleotide sequence analysis of numerous clones, pBSEIVA2FHA-19 was chosen for further manipulation. This clone contained errors near the DamHI site at nucleotide 617 (FIG. 24) (SEQ ID N0:284) and at nucleotide 1570 (FIG. 24) (SEQ ID N0:284). To correct these errors, the following manipulations were made. Plasmid pBSEIVA2FHA-19 was digested with DamHI and SphI and the excised 900 bp fragment was isolated. This fragment was co-inserted into pBS-SK
digested with SneI/BamHI with a 250 bp SDhI/SpeI fragment encompassing the 3'-most region of the HA coding sequence.
This 250 bp PCR fragment was derived using clone #7 (above) as template and oligonucleotides A2F3P (SEQ ID NO:285) and A2F6 (SEQ ID NO:287) (5'-TTGACTTAACAGATGCAG-3'). The resultant plasmid was designated as pEIVH33P.
The 5'-most 616 bp of the HA coding sequence for the EIV HA was generated in the following manner. First, first-stand cDNA was generated as above. This first-strand cDNA
preparation was then used as template to amplify these sequences by PCR using oligonucleotides A2F5P (SEQ ID
NO:288) (5'-ATGAAGACAACCATTATTTTG-3') and A2FBAM1 (SEQ ID
NO:289) (5'-TGTTGAGACTGTTACTCG-3'). This fragment was inserted into HincII digested pBS-SK (Stratagene, La Jolla, CA) and the resultant plasmid called pEIVH35P.
The vaccinia virus H6 promoter sequence (Goebel et al., 1990a,b) and the 5'-most region of the HA coding sequence WO 92/1' PCT/US92/01906 were amplified and fused in the following manner. The H6 sequences were derived from a pBS-based plasmid containing the HIV-1 (IIIB) env gene linked precisely to the H6 promoter called pBSH6IIIBE. These sequences were amplified by PCR using oligonucleotides H65PH (SEQ ID NO:164) (5'-ATCATCAAGCTTGATTCTTTATTCTATAC-3') and H63P (SEQ ID
NO:291) (5'-TACGATACAAACTTAACGG-3'). The 120 bp H6 fragment was used as template with oligonucleotides H65PH (SEQ ID
NO:164) and EIV5PACC (SEQ ID NO:292) (5'-GGTTGGGTTTTGAC
TGTAGACCCAATGGGTCAGTAGTATCAAAATAATGGTTGTCTTCATTACGATACAAACTT
AACGG-3') to yield a 161 bp fragment containing the H6 promoter and the initial 41 bp of the HA coding sequence to the AccI restriction site. This fragment was digested with HindIII and AccI and co-inserted into HindIII/BamHI digested pBS-SK with the 550 bp AccI/BamHI fragment from pEIVH35P.
The resultant plasmid was designated as pH6EIVH35P.
The entire HA expression cassette was derived by co-insertion of the 710 bp HindIII/BamHI fragment from pH6EIVH35P and the 1200 bp BamHI/SpeI fragment from pEIVH33P
into pBS-SK digested with I'n_dIII and EReI. The derived plasmid was designated as pBSA2FHAB.
To correct the base change noted above near the BamHI
site at nucleotide position 617, the Mandecki procedure (Mandecki, 1986) was employed. pBSA2FHAB was linearized with BamHI and the mutagenesis procedure performed using oligonucleotide A2F7 (SEQ ID NO:293) (5'-CAATTTCGATAAAC
TATACATCTGGGGCATCCATCACCCGAGCACAAACAATGAGCAGACAAAATTG-3').
The plasmid containing the corrected version of the HA was designated pBSA2FHA.
Example 44 - CONSTRUCTION OF THE INSERTION PLABMIDB
pEIVCSL AND pEIVEAVQVV IIBED TO GENERATE
vCP128 and vP961. RESPECTIVELY

Plasmid pC3EIVAIPHA was digested with NruI and HindIII
to excise the 1.7kb fragment containing the 3'-most 26 bp of the H6 promoter and the entire EIV (A1/Prague/56) HA coding sequence. Following blunt-ending with Klenow, this fragment was inserted into plasmid pRW838 digested with NruI/EcoRI
and blunt-ended with Klenow to provide plasmid pC5AIPHA.
The plasmid pRW838 contains the rabies G gene (Kieny et al., 1984) fused to the vaccinia H6 promoter in a canarypox V ' 92/15672 PCT/US92/ 06 insertion plasmid (C5 locus). Digestion with NruI and EcoRI
excises the rabies G gene leaving behind the 5'-most 100 bp of the H6 promoter and the C5 flanking arms.
The plasmid pC5AIPHA was digested with SmaI and SacI to excise an 820 bp fragment containing the H6 promoter and the 5'-most 645 bp of the EIV (A1/Prague/56) coding sequence.
This fragment was co-inserted into pBS-SK digested with HindIII and SmaI with a 1.1 kb SacI/HindIII fragment from pC3EIVAIPHA containing the remainder of the HA coding sequence. The resultant plasmid was designated as pBSAIPHAVQ.
Plasmid pBSAIPHAVQ was then linearized with SpeI and SmaI. This 4.7 kb fragment was ligated to a 1.8 kb SpeI/partial HincIl fragment derived from pBSA2FHA. The resultant pBS-based plasmid, containing the EIV
(A1/Prague/56) and (A2/Fontainebleau/79) HA genes in a head to head configuration, was designated as pBSAIPA2FHAVQ.
A totI/ hCoI fragment (3.5 kb) derived from pBSAIP2FHAVQ
containing the two HA genes was isolated and inserted into pSD542 (described below for EIV (A2/Suffolk/89) and pC5L to provide the insertion plasmids pEIVHAVQVV and pEIVC5L, respectively.
The C5L insertion plasmid was derived as follows.
Using the cosmid vector pVK102 (Knauf and Nester, 1982), a genomic library for vCP65 (ALVAC-based rabies G recombinant with rabies in C5 locus) was constructed. This library was probed with the 0.9 kb vuII canarypoxvirus genomic fragment contained within pRW764.5 (C5 locus). These canarypox DNA
sequences contain the original insertion locus. A clone containing a 29 kb insert was grown up and designated pHCOS1. From this cosmid containing C5 sequences, a 3.3 kb Cla fragment was subcloned. Sequence analysis from this C aI fragment was used to extend the map of the C5 locus from 1-1372.
The C5 insertion vector, pC5L, was constructed in two steps. The 1535 bp left arm was generated by PCR
amplification using oligonucleotides C5A (SEQ ID NO:294) (5'-ATCATCGAATTCTGAATGTTAAATGTTATACTTTG) and C5B (SEQ ID
NO:295) (GGGGGTACCTTTGAGAGTACCACTTCAG-3'). The template DNA

WO 92/15672 CA 02558659 1992-03-09 pC7T/US92/01906 was vCP65 genomic DNA. This fragment was cloned into coRI/SmaI digested pUC8. The sequence was confirmed by standard sequencing protocols. The 404 bp right arm was generated by PCR amplification using oligonucleotides C5C
(SEQ ID NO:296) (5'-ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3') and C5DA (SEQ ID NO:297) (5'-ATCATCCTGCAGGTATTC
TAAACTAGGAATAGATG-3'). This fragment was then cloned into the vector previously generated containing the left arm digested with SmaI/PstI. The entire construct was confirmed by standard sequence analysis and designated pC5L. This insertion plasmid enables the insertion of foreign genes into the C5 locus.
E8ample 45 - CONSTRUCTION OF INSERTION PLABMIDS TO
GENERATE ALVAC- AND NYVAC-BASED RECOMBINANTS
EXPRESSING INFLUENZA VIRUS (A2/BIIFFOLIC/89) HEMAGGLIITININ GENE

An M13 clone containing the hemagglutinin (HA) gene from equine influenza virus (A2/Suffolk/89) was provided by Dr. M. Binns (Animal Health Trust, P.O. Box 5, Newmarket, Suffolk, CB8 7DW, United Kingdom). This clone contains a full-length 1.7 kb cDNA fragment containing this HA gene inserted into the M13 vector via the indIII site.
Initially, the equine influenza virus (EIV) HA gene was amplified from the above M13 clone by PCR using oligonucleotides EIVS1 (SEQ ID NO:298) (5'-ATGAAGACAACC
ATTATTTTG-3') and EIVS2 (SEQ ID NO:299) (5'-TCAAATGCAAA
TGTTGCATCT-3'). This 1.7 kb fragment was ligated into pBS-SK (Stratagene, La Jolla, CA) digested with SmaI. Two positive clones were derived and analyzed by nucleotide sequence analysis (Goebel et al., 1990a). Clone A contained one non-conserved base change while clone B contained three such changes compared to the sequence provided in FIG. 25 (SEQ ID NO:300). To generate a full-length correct version of the EIVHA gene, clone B was digested with SacI and MscI
to excise a 390 bp fragment. This fragment was ligated into a 4.3 kb MscI/partial SacI fragment derived from clone A.
This provided a corrected EIVHA and was designated as pBSEIVHS.
The 5'-most 360 bp of the EIVHA coding sequence was derived from pBSEIVHS by PCR using oligonucleotides I3L5EIV

WO 92/15672 CA 02558659 1992-03-09 ~ ~
PCT/L,S9../01906 (SEQ ID NO:301) (5'-GTTTAATCATGAAGACAACCATTATTTTGATAC-3') and EIVPVU (SEQ ID NO:302) (5'-AGCAATTGCTGAAAGCGC-3'). The entire 13L promoter region (Goebel et al., 1990a,b) was derived from pMPI3L101 by PCR using oligonucleotides 13L5B5 (SEQ ID NO:303) (51-ATCATCGGATCCCGGGACATCATGCAGTGGTTAAAC-3') and EIV5I3L (SEQ ID NO:304) (5'-CAAAATAATGGTTGTCTTCATGAT
TAAACCTAAATAATTGTAC-3'). These fragments were fused due to the complementary conferred by the engineering of oligonucleotides I3L5EIV (SEQ ID NO:301) and EIV5I3L (SEQ ID
NO:304) by PCR with oligonucleotides 13L5B5 (SEQ ID NO:303) and EIVPVU (SEQ ID NO:302) to yield a 480 bp fragment.
Plasmid pMPI3L101 contains an expression cassette consisting of the gene encoding the rabies glycoprotein under the control of the I3L promoter, all inserted into a vaccinia insertion plasmid deleted for ORFS C6L-K1L (Goebel et al., 1990a,b). The 13L promoter consists of 101 bases (nt 64,973-65,074 Goebel et al., 1990a,b) immediately upstream from the initiation codon of the ORF 13L.
The above derived fusion fragment linking the 13L
promoter precisely to the 5' region of the EIVHA coding sequence was digested with Ba.mEi:I (5'-end) and AccI (3'-end) and the 400 bp fragment isolated. This fragment was ligated to a 4.6 kb BamHI/partial AccI fragment derived from pBSEIVHS and the resultant plasmid designated as pBSEIVHSI3L.
A 1.8 kb SmaI/XhoI fragment containing the EIVHA
expression cassette was derived from pBSEIVHSI3L. This fragment was inserted into a SmaI/ChoI digested pSD542 (described in Example 32) insertion vector to yield pTKEIVHSI3L.
The 1.8 kb SmaI/XhoI fragment from pBSEIVHEI3L (above) was inserted into the CPpp (ALVAC) insertion plasmid, VQCP3L, digested with SmaI/XhoI. The resultant plasmid was designated as pC3EIVHSI3L.
Insertion plasmid VQCP3L was derived as follows.
VQCPCP3L was derived from pSPCP3L (defined in Example 32) by digestion with XmaI, phosphatase treating the linearized plasmid, and ligation to annealed, kinased oligonucleotides CP23 (SEQ ID NO:305) (5'-CCGGTTAATTAATTAGTTATTAGACAAGG

WO 92/156717-2 CA 02558659 1992-03-09 ~

TGAAAACGAAACTATTTGTAGCTTAATTAATTAGGTCACC-3') and CP24 (SEQ
ID NO:306) (5'-CCGGGGTCGACCTAATTAATTAAGCTACAAATAGTTTCGTTT
TCACCTTGTCTAATAACTAATTAATTAA-3').
ESample 46 - DEVELOPMENT OF ALVAC-EQUINE INFLUENZA VIRUS
RECOMBINANTS

Plasmid pEIVC5L contains the hemagglutinin coding sequences for equine-1, Al/Prague/56 (H7) and equine-2, A2/Fontainebleau/79 (H3). Both genes are linked to the vaccinia virus H6 promoter and inserted at the de-orfed C5 locus. ALVAC virus was used as the rescuing virus in in vitro recombination to rescue the inserted DNA. Positive plaques were selected on the basis of hybridization to H7 and H3 coding sequences. Recombinant plaques were plaque purified until a pure population containing both foreign genes was achieved. At this time the recombinant was declared vCP128 and a stock virus was established..
Immunofluorescence analysis was performed using a monoclonal antibody specific for the H3 hemagglutinin and a polyclonal anti-H7 serum from horse. Surface fluorescence was detected on vCP128 infected VERO cells using both reagents indicating that both antigens were appropriately presented on the infected cell surface.
Immunoprecipitation analysis using the H3 specific monoclonal antibodies demonstrated the presence of a protein of approximately 75 kd in vCP128 infected CEF cells. This potentially represents the HAo precursor glycoprotein. No cleavage products were detected. Immunoprecipitation analysis using the H7 specific polyclonal serum demonstrated the presence of a precursor glycoprotein of approximately 75 kd. The HA1 and HA2 cleavage products with molecular weights of approximately 45 and 30 kd respectively were also visualized.
Plasmid pC3EIVHS13L contains the hemagglutinin coding sequence of the equine-2 A2/Suffolk/89 subtype. The gene is linked to the vaccinia virus 13L promoter and inserted at the de-orfed C3 insertion site. ALVAC virus was used as the rescuing virus in in vitro recombination to rescue the foreign gene. Recombinant plaques were selected on the basis of hybridization to a H3 specific radiolabelled probe.

Positive plaques were plaque purified until a pure recombinant population was achieved. At this time the recombinant was declared vCP159 and a virus stock established. Immunofluorescence analysis on vCP159 infected CEF cells using an H3 specific serum polyclonal from chicken indicated that an immunologically recognized protein was expressed on the infected cell surface.
Example 47 - DEVSLOPI[ENT OF NYVAC RECOMBINANTS CONTAINING
THE HEMAGGLIITININ GLYCOPROTEINS OF EQUINE
INFLUENZA VIRUS SUBTYPES

Plasmid pEIVHAVQVV contains the sequences encoding equine-1, A1/Prague/56 (H7) and equine-2 A2/Fontainebleau/79 (H3). Both genes are linked to the vaccinia virus H6 promoter and inserted at the TK site. NYVAC (vP866) was used as the rescuing virus in in vitro recombination to rescue the foreign genes. Recombinant plaques were selected on the basis of hybridization to radiolabelled H3 and H7 specific proteins. Recombinant progeny virus was plaque purified until a pure population was achieved. At this time the recombinant was declared vP961 and a virus stock established. Immunofluorescence analysis using a H3 specific monoclonal antibody and a polyclonal anti-H7 serum indicated that both glycoproteins were expressed on the infected cell surface. Immunoprecipitation analysis with the same reagents indicated that the H3 glycoprotein was expressed as a precursor glycoprotein with a molecular weight of approximately 75 kd. No cleavage products were evidenced. The H7 glycoprotein was evident as a precursor glycoprotein of approximately 75 kd and HA1 and HA2 cleavage products with molecular weights of approximately 45 and 30 kd, respectively.
Plasmid pTKEIVHSI3L contains the coding sequence of the equine-2 A2/Suffolk/89 hemagglutinin glycoprotein. The coding sequence is linked to the I3L promoter and inserted at the TK site. NYVAC (vP866) was used as the rescuing virus and recombinant plaques selected on the basis of hybridization to a H3 specific radiolabelled probe.
Recombinant plaque progeny were plaque purified until a pure population was achieved. At this time the recombinant was declared vP1063 and a virus stock established.

WO 92/15672 CA 02558659 1992-03-09 pC7T/LS92/01906 Immunofluorescence analysis using a polyclonal anti-H3 serum from chicken indicated that an immunologically recognized protein was expressed on the infected cell surface.
Example 48 - CONSTRUCTION OF VACCINIA VIRIIB/F$LV
INSERTION PLABMIDB

FeLV (Feline Leukemia Virus) env DNA sequences were supplied by Dr. F. Galibert (Laboratories d'Hematologie Experimentale Hospital Saint-Luis, Paris, France) in the form of a 2.4 kbp FeSV-SM DNA (Guilhot et al.., 1987) fragment inserted into an M13mp8 vector (Messing, 1983).
This 2.4 bp PstI/RpnI fragment containing the entire open reading frame (FeLV p70 + p13E) was isolated and inserted into pUC18 (Messing, 1983) for convenience. The KnnI site at the 3' end of the env sequences were converted to a PstI
site and the 2.4 kbp PstI fragment was isolated and ligated into PstI digested pTp15 (Guo et al., 1989). The resultant plasmid was designated pFeLV1A.
In vitro mutagenesis (Mandecki, 1986) was used to convert pFeLV1A to pFeLV1B. This was done using oligonucleotide SPBGLD (SEQ ID N0:307) (5'-AATAAATCAC
TTTTTATACTAATTCTTTATTCTATACTTAAAAAGT-3'). Mutagenesis with this oligonucleotide enabled the removal of the BQ1II site at the border of the H6 promoter and HA sequences. This provides the actual sequences of these DNA segments as found in the virus.
Plasmid pFeLV1B was then mutagenized with oligonucleotide FeLV5P (SEQ ID N0:308) (5'-CGCTATAGG
CAATTCAAACATAGCATGGAAGGTCCAAACGCACCCA-3') to create pFeLV1C.
In vitro mutagenesis was done as described by Mandecki (1986) with the following modification. Following digestion of pFeLV1B at the unique SmaI site, the DNA was digested with Ba131. At times 5 sec., 10 sec., 20 sec., 40 sec., and 80 sec., aliquots were taken and the reaction terminated by adding EGTA to a final concentration of 20mM. The aliquots were pooled and used in the mutagenesis reaction. Resultant plasmid, pFeLV1C, contained the FeLV env gene juxtaposed 3' to the vaccinia virus H6 promoter such that there exists and ATG to ATG substitution. The plasmid, pFeLV1C, was used in in vitro recombination tests with vP425 as the rescuing virus to construct a recombinant vaccinia virus (vP453) which expresses the entire FeLV envelope glycoprotein.
The plasmid pFeLV1C was used as a reagent to generate pFeLV1D. This recombinant plasmid contains the entire FeLV
env gene except it lacks the putative immunosuppressive region (Cianciolo et al., 1985; Mathes et al., 1978). The sequence encoding the immunosuppressive region (nucleotide 2252-2332 of sequence in Guihot et al., 1987) was deleted by in vitro mutagenesis (Mandecki, 1986) in the following manner. The plasmid, pFeLV1C, was linearized with BsmI.
The linearized plasmid was treated with Ba131 and aliquots were taken at 1 min., 2 min., 4 min., and 8 min. and pooled for use in the mutagenesis reaction. In vitro mutagenesis was done using oligonucleotide FeLV1SD (SEQ ID NO:309) (5'-ACCTCCCTCTCTGAGGTAGTCTATGCAGATCACACCGGACTCG
TCCGAGACAATATGGCTAAATTAAGAGAAAGACTAAAACAGCGGCAGCAACTGTTTGACT
CCCAACAG-3'). The resultant plasmid, pFeLV1D, was used in in vitro recombination tests with vP410 as the rescuing virus to generate vP456. This vaccinia virus recombinant was generated to express the entire envelope glycoprotein lacking the putative immunosuppressive region.
Example 49 - CONSTRUCTION OF AVIPOBVIRIIB/FeLV
INSERTION PLABMIDS

For construction of the FP-1 recombinants, the 2.4 kbp H6/FeLV env sequences were excised from pFELV1A (described above) with BalII and by partial digestion with PstI. The BalII site is at the 5' border of the H6 promote sequence.
The PstI site is located 420 bp downstream from the translation termination signal for the envelope glycoprotein open reading frame.
The 2.4 kbp H6/FeLV env sequence was inserted into pCE11 digested with BamHl and PstI. The FP-1 insertion vector pCE11, was derived from pRW731.13 by insertion of a multiple cloning site into the nonessential HindII site.
This insertion vector allows for the generation of FP-1 genome. The recombinant FP-1/FeLV insertion plasmid was then designated pFeLVF1. This FP-1/FeLV insertion plasmid was then designated pFeLVF1. This construction does not provide a precise ATG for ATG substitution.

To achieve the precise ATG:ATG construction, a ruI/SstII fragment of approximately 1.4 kbp was derived from the vaccinia virus insertion vector pFeLV1C (described herein). The ruI site occurs within the H6 promoter at a positive 24 bp upstream from the ATG. The SstII site is located 1.4 kbp downstream from the ATG and 1 kbp upstream from the translation termination codon. The NruI/SstII
fragment was ligated to a 9.9 kbp fragment which was generated by digestion of pFeLVF1 with SstII and by partial digestion with NruI. This 9.9 kbp fragment contains the 5.5 kbp FP-1 flanking arms, the pUC vector sequences, 1.4 kbP of FeLV sequence corresponding to the downstream portions of the env gene, and the 5'-most portion (approximately 100 bp) of the H6 promoter. The resultant plasmid was designated pFeLVF2. The precise ATG:ATG construction was confirmed by nucleotide sequence analysis.
A further FP-1 insertion vector, pFeLVF3, was derived from FeLVF2 by removing the FeLV env sequences corresponding to the putative immunosuppressive region (described above).
This was accomplished by isolating as I/SstII fragment of approximately 1 kbp obtained from the vaccinia virus insertion vector, pFeLV1D (described above), and inserting this fragment into a 10.4 kbp ~stI/SstII fragment containing the remaining H6/FeLV env gene derived by digestion of pFeLVF2 with PstI and SstII.
The insertion plasmids, pFeLVF2 and pFeLVF3, were using in in vitro recombination tests with FP-1 as the rescuing virus. Progeny virus was plated on primary chick embryo fibroblast (CEF) monolayers obtained from 10 day old embryonated eggs (SPAFAS, Storrs, CT) and recombinant virus screened for by plaque hybridization on CEF monolayers.
Recombinant progeny identified by hybridization analyses .were selected and subjected to four round of plaque purification to achieve a homogeneous population. An FP-1 recombinant harboring the entire FeLV env gene has been designated vFP25 and an FP-1 recombinant containing designated vFP32.
For construction of the CP recombinants, a 2.2 bp fragment containing the H6/FeLV env sequences were excised from pFeLVF2 and pFeLVF3 by digestion with KpnI and HpaI.
The KDnI site is at the 5' border of the H6 promoter sequence. The HDaI site is located 180 bp downstream from the translation termination signal for the envelope glycoprotein open reading frame. These isolated fragments were blunt-ended. These 2.2 kbp H6/FeLV env sequences were inserted into the nonessential coRI site of the insertion plasmid pRW764.2 following blunt-ending of the EcoRI site.
This insertion vector enables the generation of CP
recombinants harboring foreign genes in the C3 locus of the CP genome. The recombinant CP insertion plasmid was then designated pFeLVCP2 and pFeLVCP3, respectively.
The insertion plasmids, pFeLVCP2 and pFeLVCP3, were used in in vitro recombination tests with CP as the rescuing virus. Progeny of the recombination were plated on primary CEF monolayers obtained from 10 day old embryonated eggs (SPAFAS, Storrs, CT) and recombinant virus selected by hybridization using radiolabeled FeLV DNA as a probe.
Positive hybridizing plaques were selected and subjected to four rounds of plaque purification to achieve a homogeneous population. A recombinant expressing the entire FeLV env gene has been designated vCP35 and a recombinant expressing the entire env gene lacking the immunosuppressive region was designated vCP37.
EBamDle 50 - GENERATION OF AN ALVAC-BABED RECOMBINANT
CONTAINING THE FeLV-B env GENE

Plasmid pFeLV env 24 was obtained from Rhone-Merieux (Lyon, France) and contains the FeLV-B env gene. The plasmid contains a 4.2 kb cDNA derived fragment derived from the NCE161 FeLV strain. Plasmid pFeLV env 34 contains the 4.2 kb FeLV-B-specific insert in the maI site of pBS-SK
(Strategene, La Jolla, CA). The sequence of the FeLV-B env gene is presented in FIG. 26 (SEQ ID NO:310). In this sequence the initiation codon (ATG) and termination codon (TGA) are underlined.
The expression cassette for the FeLV-B env was constructed as follows. The vaccinia virus H6 promoter was derived from plasmid pI4LH6HIV3B (described herein with respect to HIV) by PCR using oligonucleotides H65PH (SEQ ID

NO:164) (5'-ATCATCAAGCTTGATTCTTTATTCTATAC-3') and H63PFB
(SEQ ID NO:311) (5'-GGGTGCGTTGGACCTTCCATTACGATAGAAACTTA
ACGG-3'). Amplification of these sequences with these oligonucleotides generated an H6 promoter with a 5' indIII
site and a 3'-end containing the initial 20 bp of the FeLV-B
env coding sequence.
The 5'-portion of the FeLV-B env gene was derived by PCR from pFeLV-B env 34 using oligonucleotides FB5P (SEQ ID
NO:312) (5'-CCGTTAAGTTTGTATCGTAATGGAAGGTCCAAGCG-3') and FB5PA (SEQ ID NO:313) (5'-GGGTAAATTGCAAGATCAAGG-3'). This PCR-derived fragment contains homology to the 3'-most 23 bp of the H6 promoter (5'-end) and a unique ApaI site at position 546. The H6 promoter was fused to the 5'-end of the FeLV-B env gene by PCR using the two PCR fragments defined above as template and oligonucleotides FB5PA (SEQ ID
NO:313) and H65PH (SEQ ID NO:164). The PCR fusion.fragment was digested with HindIII and ADaI to yield a 680 bp fragment.
Plasmid pFeLV-B env 34 was digested with _UaI and NcoI
to liberate a 740 bp fragment containing the middle portion of the env gene (FIG. 26). The 3'-end of the gene was derived by PCR using pFeLV-b env 34 as template and oligonucleotides FBT5D (SEQ ID NO:314) (5'-CCCCATGCATTT
CCATGGCAGTGCTCAATTGGACCTCTGATTTCTGTGTCTTAATAG-3') and FB3PX
(SEQ ID NO:315) (5'-ATCATCTCTAGAATAAAAATCATGGTCGGTCCG
GATC-3'). PCR amplification of the 3' portion of the env gene with these oligonucleotides eliminated a T5NT element at position 1326-1332. This sequence was altered by making a T to C substitution at position 1329 (FIG. 26). This change does not alter the amino acid sequence of the env gene product. The 3'-end of the gene was engineered with an CbaI site and a T5NT sequence. The 5'-end of this PCR-derived fragment also contains a unique NcoI site (corresponds with that at position 1298; FIG. 26). This fragment was digested with NcoI and XbaI to generate a 707 bp fragment. -The 740 bp NcoI/ApaI and 707 bp NcoI/XbaI fragments were co-inserted into pBS-SK digested with ApaI and XbaI.
The resultant plasmid was designated as pBSFB3P. The 1.5 kb Ar)aI/XbaI fragment from pBSFB3P was then co-inserted into pBS-SK, digested with indIII and XbaI, with the 680 bp PCR
fragment containing the H6 promoted 5'-end of the FeLV-B env gene (above). The resultant plasmid was designated as pB5FEB.
The 2.2 kb HindIII/XbaI fragment from pB5FEB, containing the H6 promoted FeLV-B env gene, was isolated and blunt-ended with the Klenow fragment. This blunt-ended fragment was inserted into pC5L (see discussion regarding HIV herein) digested with SmaI to yield pC5LFEB.
Plasmid pC5LFEB was used in standard in vitro recombination assays with ALVAC(CPpp) as the rescue virus.
Recombinant plaques were identified using FeLV-B env specific DNA probes. Following three round of plaque purification, the virus was propagated and designated as vCP177.
Example 51 - GENERATION OF AN ALVAC-FeLV-A ENV
RECOMBINANT VIRUS

The plasmid pFGA-5 from which the FeLV-A env sequences were derived was provided by Dr. J. Neil (University of Glasgow) and described previously (Stewart et al., 1986).
Initially, the 531 bp PstI/ indIII fragment corresponding to nucleotides 1 to 531 (Stewart et al., 1986) was excised and ligated into pCPCV1 digested with PstI and HindIII and designated as pC3FA-1. The plasmid pCPCV1 was derived as follows. Plasmid pFeLV1A was digested with PstI to excise the FeLV sequences and religated to yield plasmid pFeLVF4.
The vaccinia virus H6 promoter element (Taylor et al., 1988) followed by a polylinker region were liberated from pFeLVF4 by digestion with KpnI and HpaI. The 150 bp fragment was blunt-ended using T4 DNA polymerase and inserted into pRW764.2, a plasmid containing a 3.3kb PvuII genomic fragment of canarypox DNA. pRW764.2 was linearized with EcoRI, which recognizes a unique EcoRI site within the canarypox sequences, and blunt-ended using the Klenow fragment of the E. coli DNA polymerase. The resultant plasmid was designated as pCPCV1. This plasmid contains the vaccinia virus H6 promoter followed by a polylinker region and flanked by canarypoxvirus homologous sequences.

The plasmid pC3FA-1 was linearized with PstI and mutagenized in an in vitro reaction via the Mandecki procedure (1986) using oligonucleotide FENVAH6-1 (SEQ ID
NO:316) (5'-CCGTTAAGTTTGTATCGTAATGGAAAGTCCAACGCAC-3'). The in vitro mutagenesis procedure removed extraneous 5'-noncoding sequences resulting in a precise ATG:ATG
configuration of the vaccinia H6 promoter element and the FeLV-A env sequences. The resultant plasmid was designated as pH6FA-1.
The remainder of the FeLV-A env gene was derived from pFGA-5 by standard PCR using custom synthesized oligonucleotides (Applied Biosystems, San Rafael, CA). An 836 bp PCR fragment was derived using pFGA-5 as template and the oligonucleotides FENVA-2 (SEQ ID NO:317) (5'-CCATAATTCG
ATTAAGACACAGAATTCAGAGGTCCAATTGAGCACC-3') and FENVAH (SEQ ID
NO:318) (5'-CAAGATGGGTTTTGTGCG-3'). This fragment .
corresponds to nucleotides 488 to 1327 of the FeLV-A env gene (Stewart et al., 1986). The use of oligonucleotide FENVA-2 (SEQ ID NO:317) alters the nucleotide sequence at positions 1301 to 1309 from GAT5GT to GAATTCTGT. This alteration eliminates the T5NT sequence motif known to be recognized as a poxvirus early transcription termination signal (Yuen and Moss, 1987) and introduces an coRI
restriction site at this position. These nucleotide manipulations change amino acid 414 from glutamic acid to the conserved amino acid aspartic acid (Stewart et al., 1986). Amino acid 415 is not altered by these nucleotide changes. This 836 bp fragment was digested with HindIII and EcoRI to generate a 770 bp fragment corresponding to nucleotides 532 to 1302 of the FeLV-A env gene.
A 709 bp PCR fragment was derived using pFGA-5 as template and the oligonucleotides FENVA-3 (SEQ ID NO:319) (5'-GGTGCTCAATTGGACCTCTGAATTCTGTGTCTTAATCGAATTATGG-3') and FENVA-4 (SEQ ID NO:320) (5'-ATCATCAAGCTTTCATGGTCGGTCCGG-3').
This fragment corresponds to nucleotides 1281 to 1990 of the FeLV-A env gene (Stewart et al., 1986). Using oligonucleotide FENVA-3 (SEQ ID NO:319) to amplify this fragment also alters the T5NT element and introduces an EcoRI site as above for the 836 bp PCR derived fragment.

WO 92/15672 CA 02558659 1992-03-09 PCI-/US92M"906 The 709 bp fragment was digested with coRI/ indIII and the resultant fragment was co-inserted with the 770 bp FiindIII/ coRI fragment, derived from the 836 bp fragment (above), into pBS-SK (Stratagene, La Jolla, CA) digested with HindIII. The resultant plasmid was designated as pF3BS1-B and the FeLV env sequences were confirmed by nucleotide sequence analysis.
To reconstruct the entire FeLV-A env gene linked precisely to the vaccinia virus H6 promoter, a 1.5 kb HindiII fragment was isolated from pF3BS1-B. This fragment corresponds to nucleotides 532 to 1990 of the FeLV-A env (Stewart et al., 1986). The 1.5 kb H'ndIII fragment was ligated to pH6FA-1 digested with I'ii dIII. Plasmid constructs containing the 1.5 kb HindIII fragment were screened for the proper orientation by restriction analysis and a plasmid clone containing the entire intact FeLV-A env gene linked to the H6 promoter was designated as pH6FA-3.
The 2.2 kb H6/FeLV-A env expression cassette was excised from pH6FA-3 by partial digestion with EcoRI
followed by a partial digestion with indIII. The fragment was inserted into pRW831 digested with HindIII and coRI.
The resultant plasmid was designated as pC5FA.
pRW831 refers to an ALVAC (CPpp) insertion plasmid which enables the insertion of foreign genes into the C5 open reading frame. In the process of insertion into this region, the use of pRW831 causes the deletion of most of the C5 open reading frame. To generate pRW831 the following manipulations were done. An 880 bp vuII genomic fragment from the canarypoxvirus genome was inserted between the ~vuII sites of pUC9. The canarypox sequences contained within the resultant plasmid, pRW764.5, was analyzed by nucleotide sequence analysis and the C5 open reading frame was defined. Previously, insertion between a pair of BalII
sites situated within the C5 ORF was used to engineer recombinants at this locus (Taylor et al., 1992). The sequence of the entire region is provided in FIG. 16 (SEQ ID
NO:220). The nucleotide sequence begins (SEQ ID N0:220) at the PvuII site. The C5 ORF initiates at position 166 and terminates at nucleotide 487. Precise manipulation of these sequences enabled the deletion of nucleotides 167 through 455. Such a deletion was made so as not to interrupt the expression of other viral genes.
The procedure to derive pRW831 is as follows. pRW764.5 was partially digested with saI and the linearized fragment was isolated. The saI linear fragment was redigested with BctlIII. The resultant 2.9 kb RsaI/BalII fragment (deleted of nucleotides 156 through 462) was isolated and ligated to annealed oligonucleotides RW145 (SEQ ID NO:107) and RW146 (SEQ ID NO:108). The resultant plasmid was designated as pRW831 and contains a sequence with unique HindIII. SmaI, and EcoRI sites in place of the C5 sequences.
Plasmid pC5FA was used in recombination experiments with ALVAC(CPpp) as the rescuing virus. Recombinant viruses were identified by in situ plague hybridization using a radiolabeled FeLV-A env-specific probe. Following,three cycles of plaque purification with subsequent hybridization confirmation, the recombinant was designated as vCP83.
Example 52 - GENERATION OF AN ALVAC-FaLV-A ENV RECOMBINANT

REGION OF p15E

The putative immunosuppressive region is situated within the=p15E transmembrane region of the FeLV envelope glycoprotein (Cianciolo et al., 1986; Mathes et al., 1978).
This region was deleted in the following manner. The FeLV-A
env sequences from nucleotide 1282 to 1602 (Stewart et al., 1986) were amplified by PCR from pFGA-5 using oligonucleotides FENVA-3 (SEQ ID NO:320) and IS-A (SEQ ID
NO:468) (5'-TAAGACTACTTCAGAAAG-3'). The env sequences from nucleotide 1684 to 1990 (Stewart et al., 1986) were amplified by PCR from pFGA-5 using oligonucleotides FENVA-4 (SEQ ID NO:320) and IS-B (SEQ ID NO:323) (5'-GCGGATCACA
CCGGACTC-3'). The former PCR-derived fragment was digested with EcoRI and the latter with HindIII and was subsequently kinased with ATP and T4 kinase. These fragments were co-ligated into pBS-SK digested with HindIII and EcoRI. The resultant plasmid was confirmed by nucleotide sequence analysis and designated as pBSFAIS-. Ligation of the above fragments joins the sequences 5' and 3' to the 81 bp DNA
segment encoding the putative immunosuppressive region and, WO 92/15672 PC'T/LJS92/f'Q06 therefore, deletes the sequences encoding the immunosuppressive peptide.
The FeLV-A sequences lacking the region encoding the immunosuppressive region were excised from pC5FA by digestion with SstII/AnaI.
This 381 bp fragment was replaced by the 300 bp SstII/ADaI fragment from pBSFAIS'.
The ligation that was done was with a 4.8kb SstII/ApaI
fragment from pC5FA and the 300 bp fragment described above.
The resultant plasmid was designated pC5FAISD.
The plasmid pC5FAISD was employed in recombination experiments with ALVAC (CPpp) as the rescuing virus.
Recombinant viruses were identified by in situ hybridization using a radiolabeled FeLV-A env specific probe. Following three cycles of plaque purification, the recombinant was designated as vCP87. This recombinant contains the FeLV-A
env gene lacking the region encoding the putative 27 amino acid immunosuppressive region. The gene was inserted into the C5 locus.
ESamDle 53 - GEtBRATION OF ALVAC-FeLV-A gag RECOMBINANT VIRUSES

The FeLV-A gac/pol sequences were derived from plasmid pFGA-2 gag. This plasmid was derived from the FeLV-A
infectious clone pFGA-2 (Stewart et al., 1986) by subcloning the 3.5 kb PstI subfragment containing a portion of the LTR
(651 bp) sequences, the entire gaa gene, and 1272 bp of the pol gene. The 3.5 kb fragment was inserted into gstI
digested pUCB (Bethesda Research Laboratories, Gaithersburg, MD). Initially, this 3.5 kb gstI FeLV-A DNA fragment was isolated and inserted into pBS-SK (Stratagene, La Jolla, CA). The resultant plasmid was designated as pBSGAG. The entire 3.5 kb insert was analyzed by nucleotide sequence (SEQ ID NO:324) (FIG. 27) analysis to confirm position of the initiation codon (nucleotide 652 to 654 underlined in FIG. 27) and pertinent restriction sites defined in the nucleotide sequence of the gaa region previously reported for FeLV-B (Leprevotte et al., 1984).
The plasmid pFGA-2 gaa was digested with BalII and PstI
to liberate a 2.5 kb fragment. BalII recognizes a site at nucleotide position 1076 (SEQ ID NO:324) while PstI

VVO 92/15672 CA 02558659 1992-03-09 pc7T/L,S92/01906 recognizes a site at the end of the FeLV-A insert. The 2.5 kb fragment was isolated and redigested with HindIII and PstI which recognize sites within the co-migrating plasmid sequences. This eliminated the ability of the plasmid sequences to compete in subsequent ligation reactions.
PCR was used to derive the 5' portion of the FeLV-A gaa coding sequences. The plasmid pFGA2 gaq was used as template with oligonucleotides FGAGBGL (SEQ ID NO:325) (5'-GATCTCCATGTAGTAATG-3') and FGAGATG (SEQ ID NO:326) (5'-CGATATCCGTTAAGTTTGTATCGTAATGTCTGGAGCCTCTAGTG).
Oligonucleotide FGAGATG (SEQ ID N0:326) contains the 3'-most 25 nucleotides of the vaccinia virus H6 promoter and includes the 3'-most 3 bp of the ruI site at its' 5'-end.
These H6 sequences are precisely joined at the ATG
(initiation codon) and the nucleotides corresponding to the initial 16 nucleotides of the gaa coding sequence. .
Oligonucleotide FGAGBGL (SEQ ID N0:325) corresponds to the reverse complement of sequences 59 bp downstream from the unique BalII site in the gag sequences (Leprevotte et al., 1984). PCR using these reagents yielded a 500 bp fragment which was subsequently digested with BalII to generate a 450 bp fragment.
The 450 bp Bcr1II digested PCR-derived fragment was coligated with the 2.5 kb BQ1II/PstI fragment, containing the remainder of the gaa gene and a portion of the pol gene, and pCPCV1 (above in env construction) digested with NruI
and Psti. pCPCVl (NruI/PstI) contains the 5' portion of the vaccinia virus H6 promoter including the 5'-most 3 bp of the NruI recognition signal. The resultant plasmid was designated as pC3FGAG.
The plasmid pC3FGAG was linearized with PstI, blunt-ended with T4 DNA polymerase and ligated to a 100 bp SspI/SmaI fragment excised from pSD513 (defined in Example 7). The 100 bp SSDI/SmaI fragment provides termination codons at the 3' end of the FeLV-A gacr/pol sequences. The resultant plasmid was designated as pC3FGAGVQ.
The FeLV-A gag/pol expression cassette was excised from pC3FGAGVQ by digestion with coRI and indIII. The resultant 3.4 kb fragment was isolated and ligated with pC3I

(defined in Example 32) di-gested with coRI and HindIII to yield pC3DOFGAGVQ.
The plasmid pC3DOFGAGVQ was used in in vitro recombination experiments with vCP83 and vCP87 as rescue viruses. The recombinant containing the FeLV-A gaa/pol sequences and the entire FeLV-A env gene was designated as vCP97 while the recombinant containing the same gaQ/pol sequences and the entire FeLV-A env lacking the immunosuppressive region was designated vCP93.
Example 54 - INSERTION OF FoLV-A gag INTO A VACCINIA
VIRUS BACKGROUND

The insertion plasmid pCEN151 was generated by cloning .a 3.3 kb coRI/HindIII fragment from pC3FGAG (above) into the SmaI site of pSD553. This insertion was performed following blunt-ending the fragment with the Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs.
Plasmid pSD553 is a vaccinia deletion/insertion plasmid of the COPAK series. It contains the vaccinia K1L host range gene (Gillard et al., 1986; Perkus et al., 1990) within flanking Copenhagen vaccinia arms, replacing the ATI
region (ORFS A25L, A26L; Goebel et al., 1990a,b). pSD553 was constructed as follows. The polylinker region located at the vaccinia ATI deletion locus of plasmid pSD541 (defined in Examp1e10) was expanded as follows. pSD541 was cut with Bg1II/XhoI and ligated with annealed complementary synthetic deoxyoligonucleotides MPSYN333 (SEQ ID NO:329) (5'-GATCTTTTGTTAACAAAAACTAATCAGCTATCGCGAATCGATT
CCCGGGGGATCCGGTACCC-3') and MPSYN334 (SEQ ID NO:330) (5'-TCGAGGGTACCGGATCCCCCGGGAATCGATTCGCGATAGCTGATTAG
TTTTTGTTAACAAAA-3') generating plasmid pSD552. The K1L host range gene was isolated as a 1 kb $gjII(partial)/HDaI
fragment from plasmid pSD452 (Perkus et al., 1990). pSD552 .was cut with c$~lII/HpaI and ligated with the K1L containing fragment, generating pSD553.
Plasmid pCEN151 was used in in vitro recombination experiments with vP866 as rescue virus to generate vPlOll.
Example 55 - IMMIINOFLOORESCENCE AND INMIINOPRECIPITATION
ANALYSIS OF F LV ENV AND gag GENES IN ALVAC
RECOMBINANT VIRUSES

Immunoprecipitation. Vero cell monolayers were WO 92/1,672 CA 02558659 1992-03-09 PC"T/US92/01906 infected at an m.o.i. equa-l to 10 pfu/cell with parental or recombinant viruses. At 1 hr post-infection, the inoculum was aspirated and methionine-free medium supplemented with (35S) -methionine (DuPont, Boston, MA; 1000 Ci/mmol), 20 Ci/ml was added and further incubated till 18 hr post-infection. Immunoprecipitation and immunofluorescence analyses were performed as described previously (Taylor et al., 1990) using a bovine anti-FeLV serum (Antibodies, Inc., La Jolla, CA) or a monoclonal antibody specific for the p27 core protein (provided by Rhone-Merieux, Inc., Athens, GA).
FeLV Virus Isolation. On day one, 3x104 QN10 cells/well were plated in a 12-well plate in 1 ml of Dulbecco's MEM containing HEPES buffer (DFB), 10% FBS, and 4 g/ml polybrene. The cells were incubated overnight at 37 C. Without removing the medium, 200 l of sample (cat plasma) was added to each well. Following a 2 hr incubation at 37 C, the medium as replaced with 1.5 ml of fresh DFB and allowed to further incubate at 37 C. On day five, plates were examined for transformation. If negative, medium was replaced with 1.5 ml of fresh DFB and again allowed to further incubate at 37 C. On day eight, plates were re-examined for transformation. If negative, cells were subcultured in 5 cm plates by dispersing cells by two washes with trypsin-EDTA and placing in 4 ml DFB for inoculation into a 5 cm plate. Cells were allowed to incubate for four days at 37 C prior to examination for transformation.
Detection of FeLV Antigen By Immunofluorescence. Blood smears were fixed for five min in MeOH at -20 C, washed in dH20, and then air dried. A volume of 24 l of rabbit anti-FeLV antibody was applied to the blood smear within a circle inscribed on the-smear with a diamond pen. The smear was incubated in the presence of the antibody for 1 hr at 37 C
.in a humidified chamber prior to washing three times with PBS and one time with dH20. The smear was then air dried.
A volume of 25 l of goat anti-cat IgG-FITC was applied to the circle and incubated as above with the primary antiserum. The sample was washed and dried as above for the primary antiserum prior to examination for immunofluorescence in a microscope with a ultra violet light source.
FeLV Antibody Neutralization Assay. On day one, 5x104 QN10 cells/well were plated in a 12-well plate in 1 ml DFB
plus 4 g/ml polybrene. The cells were inoculated at 37 C.
Serum dilutions were prepared in round bottom 96 well plates from 1:2 to 1:256 using 50 , l volumes of Leibowitz medium.
Added 50 l FeLV-A at 4x105 focus forming units (ffu) per ml. Two wells were included with medium without serum as a virus control. Plates were incubated for 2 hr at 37 C.
Following the 2 hrs adsorption period, 25 l of each dilution was placed into a well of QN10 cells. Virus control was titrated by diluting 1:2, 1:4, 1:8, and 1:6 in 50 l volumes of Leibowitz medium in the 96-well plate prior to inoculation of QN10 cells with 25 gl onto QN10 cells.
Plates were inoculated at 37 C for three days. On day day, medium was replaced with 1 ml of DFB/well. Two days later, foci were counted under a microscope. Neutralizing antibody titers were estimated as the dilution of serum producing 75%
reduction in focus count compared to virus control.
In order to determine whether the env gene product expressed by vCP83 and vCP87 was transported to the plasma membrane of infected cells, immunofluorescence experiments were performed as described previously (Taylor et al., 1990). Primary CEF monolayers were infected with parental (ALVAC) or recombinant viruses, vCP83 and vCP87 and immunofluorescence was performed at 24 hr post-infection using a bovine anti-FeLV serum. The results demonstrate that cells infected with vCP83 showed strong surface fluorescent staining, whereas cells infected with vCP87 or parental ALVAC virus showed no significant surface staining.
Expression of the FeLV env gene product was also analyzed in immunoprecipitation assays using the bovine anti-FeLV serum. No FeLV-specific protein species were precipitated from lysates derived from uninfected CEFs or CEFs infected with the parental ALVAC virus. Three FeLV-specific proteins were precipitated from vCP83 infected cells with apparent molecular weights of 85 kDa, 70 kDa, and 15 kDa. This result is consistent with expression of the precursor env gene product (85 kDa) and the mature cleavage WO 92/1567 2 CA 02558659 1992-03-09 ~
PCT/L,S92/01906 products p70 and p15E. Immunoprecipitation from lysates derived form vCP88 infected cells demonstrated a single FeLV-specific protein species with an apparent molecular weight of 83 kDa. This is consistent with expression of a non-proteolytically processed env gene product of the size expected following deletion of the putative immunosuppressive region. So, in short, expression of the env lacking the immunosuppressive region was apparently not properly transported to the surface of infected cells nor was it proteolytically cleaved to mature env specific protein forms.
Expression of the FeLV gag-specific gene products was analyzed by immunoprecipitation using monoclonal antibodies specific to an epitope within the p27 core protein (D5) and the bovine anti-FeLV serum. No FeLV-specific proteins were precipitated from lysates derived from uninfected cells or cells infected with parental viruses. Clearly, from vPlOll, vCP93, and vCP97 infected cells, FeLV specific protein species of 55kDa were precipitated with the D5 and bovine anti-FeLV serum. Protein species of these apparent molecular weights are consistent with gag-specific precursor forms. Low levels of a 27 kDa protein species consistent with the size of the mature p27 core protein were also apparent.
Examnle 56 - INVIVO EVALUATION OF vCP93 AND vCP97 The protective efficacy of vCP93 and vCP97 were evaluated by a live FeLV challenge of cats following two inoculations of the recombinant viruses. The ALVAC-based FeLV recombinants were administered via the subcutaneous route with 108 PFU on days 0 and 28. Cats were challenged by an oronasal administration of an homologous FeLV-A strain (Glasgow-1) at seven days following the booster inoculation.
Blood samples were obtained pre- and post-challenge for evaluation of FeLV antigenemia (p27 detection), FeLV
isolation, the presence FeLV antigen in white blood cell (WBC) smears by immunofluorescence, and the induction of FeLV neutralizing activity.
No adverse reactions were observed following vaccination with the ALVAC (canarypoxvirus)-based WO 92/15672 CA 02558659 1992-03-09 pCT/US92/01906 recombinant viruses, vCP93 and vCP97. All six non-vaccinated controls succumbed to the FeLV challenge and developed a persistent viremia by three weeks following the challenge exposure. This was evidenced by detection of p27 antigen in the blood, FeLV isolation and detection of FeLV
antigen by immunofluorescent analysis of WBC smears (Table 32). The non-vaccinated controls remained persistently infected for the remainder of the study (until 12 weeks post-challenge).
A persistent viremia developed in three of six cats vaccinated with vCP93 at three weeks post-challenge. At this timepoint, blood samplings from these three cats were shown to contain p27 antigen and/or live FeLV (Table 32).
One of these cats (No. 2) resolved this infection by six weeks post-infection and remained free of viremia through 12 weeks post-challenge. The other two cats (No. 1 and 5) remained persistently infected by the three criteria, p27 antigenemia, FeLV isolation, and FeLV antigen detection in WBCs. Three of six cats vaccinated with vCP93 (No. 3, 4, and 6) were free of persistent viremia through nine weeks post-challenge (Table 32). Two of these cats (No. 3 and 6) remained free of circulating virus through week 12 post-challenge, while one cat (No. 4) became suddenly infected at 12 weeks. So, partial protection (three of six cats) was afforded protection against persistent viremia by vaccination with vCP93.
Most impressively, all six cats vaccinated with vCP97 were fully protected against the homologous challenge with FeLV-A (Glasgow-1). Only one of these cats (No. 12) evidenced any suggestion of persistent viremia. This occurred at three weeks post-challenge when p27 antigen was detected in the blood sample (Table 32). No live FeLV was ever isolated from the blood of this cat following challenge. All other cats were free of p27 antigenemia, free of live FeLV, and never demonstrated any FeLV antigen in WBC smears for 12 weeks following challenge exposure (Table 32).
Evolution of FeLV Neutralizing Antibodies. Due to the potential role of neutralizing antibodies in protection against FeLV infection (Russell and Jarrett, 1978; Lutz et al., 1980), the generation of such a response was monitored pre- and post-challenge. None of the cats in the study, whether vaccinated with vCP93 or vCP97, demonstrated any neutralizing antibody titers prior to FeLV challenge (Table 33). Following challenge, none of the cats which developed a persistent viremia had any detectable neutralizing antibody titers. Significantly, cats-protected against a persistent infection developed FeLV-specific serum neutralizing titers (Table 33). These titers increased in magnitude in all protected cats following challenge, with the highest level being observed at the terminal time point of the study, at 12 weeks post-challenge (Table 33).

W~ 92/15672 PCT/US92/f=906 Table 32.
Response of cats to challenge with feline leukemia virus Time (weeksl relative to challenge Group Cat -5 -2 0 +3 +6 +9 +12 No. E'V2 EV EV EV F3EV FEV FEV
1 vCP 93: 1 -- -- -- ++ -++ +++ +++

Felv-A 2 -- -- -+ --- --- ---env(IS-) 3 -- -- -- -- --- --- ---+9ao/po 4 -- -- -- -- --- --- ++--- -- -- ++ -++ +++ +++

2. vCP 97: 7 -- -- -- -- --- --- ---Felv-A 8 -- -- -- -- --- --- ---env(IS+) 9 -- -- -- -- --- --- ---+9~/pol 10 -- -- -- --12 -- -- -- +- --- --- ---SUBSTITUTE SHEET

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Claims (22)

CLAIMS:

1. A recombinant virus, comprising exogenous DNA from a non-poxvirus source and encoding an antigenic protein of the source inserted by recombination in a nonessential region of a canarypox virus genome, wherein the non-poxvirus source is rabies virus, Hepatitis B virus, Japanese encephalitis virus, yellow fever virus, Dengue virus, measles virus, pseudorabies virus, Epstein-Barr virus, herpes simplex virus, human immunodeficiency virus, simian immunodeficiency virus, equine herpes virus, bovine herpes virus, bovine viral diarrhea virus, human cytomegalovirus, canine parvovirus, equine influenza virus, feline leukemia virus, feline herpes virus, Hantaan virus, C. tetani, avian influenza virus, mumps virus, or Newcastle Disease virus, and wherein said canarypox virus is attenuated.

2. The recombinant virus as claimed in claim 1, wherein the canarypox virus is attenuated through more than 200 serial passages on chick embryo fibroblasts, a master seed of which after such passages is subjected to four successive plaque purifications under agar, and one plaque clone of which after such purifications is amplified through five additional passages, or is ALVAC.

3. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is rabies virus and the recombinant virus is vCP65 or vCP136.

4. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is human immunodeficiency virus and the recombinant virus is vCP95, vCP112, vCP60, vCP61, vCP125, vCP124, vCP126, vCP144, vCP120, vCP138, vCP117, vCP130, vCP152, vCP155, vCP156, vCP146, vCP148, vCP154, vCP168 or vCP153.

5. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is equine herpes virus and the recombinant virus is vCP132.

6. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is human cytomegalovirus and the recombinant virus is vCP139.

7. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is canine parvovirus and the recombinant virus is vCP123 or vCP136.

8. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is Epstein-Barr virus and the recombinant virus is vCP167.

9. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is equine influenza virus and the recombinant virus is vCP128 or vCP159.

10. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is feline leukemia virus and the recombinant virus is vCP177, vCP83, vCP35, vCP37, vCP87, vCP93, or vCP97.

11. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is feline herpes virus and the recombinant virus is vCP162.

12. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is Hantaan virus and the recombinant virus is vCP114 or vCP119.

13. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is Hepatitis B virus and the recombinant virus is vCP169 or vCP157.

14. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is C. tetani and the recombinant virus is vCP161.

15. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is mumps virus and the recombinant virus is vCP171.

16. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is Japanese encephalitis virus and the recombinant virus is vCP107 or vCP140.

17. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is simian immunodeficiency virus and the recombinant virus is vCP172.

18. The recombinant virus as claimed-in claim 1 or 2, wherein the non-poxvirus source is rabies virus and the recombinant virus is vCP65.

19. The recombinant virus as claimed in claim 1 or 2, wherein the non-poxvirus source is human immunodeficiency virus and the recombinant virus is vCP95, vCP112, vCP60 or vCP61.

20. A vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine comprising a carrier and the recombinant virus as claimed in any one of claims 1 to 18.

21. A vaccine for inducing an immunological response in a human inoculated with the vaccine, said vaccine comprising a carrier and the recombinant virus as claimed in any one of claims 1 to 18.

22. A method for expressing an antigenic protein in a cell cultured in vitro, which method comprises introducing into the cell the recombinant virus as claimed in any one of claims 1 to 18.