CA2153336C

CA2153336C - Recombinant virus immunotherapy

Info

Publication number: CA2153336C
Application number: CA002153336A
Authority: CA
Inventors: Enzo Paoletti; James Tartaglia; William I. Cox
Original assignee: Virogenetics Corp
Current assignee: Connaught Technology Corp
Priority date: 1993-01-21
Filing date: 1994-01-21
Publication date: 2006-12-12
Anticipated expiration: 2014-01-21
Also published as: CA2153336A1

Abstract

Attenuated recombinant viruses containing DNA coding for a cytokine and/or a tumor associated antigen, as well as methods and compositions employing the viruses, are disclosed and claimed. The recombinant viruses can be NYVAC or ALVAC recombinant viruses.
The DNA can code for at least one of: human tumor necrosis factor, nuclear phosphoprotein p53, wildtype or mutant; human melanoma-associated antigen; IL-2; IFN-.gamma.; IL.-4; GMCSF; IL-12; B7; erb-B-2 and carcinoembryonic antigen. The recombinant viruses and gene products therefrom are useful for cancer therapy.

Description

RECOMBINANT VIRUS IMMUNOTHERAPY
F~EhD OF THE I_NV~F ~O_N, The present invention xelatas to a modified poxvirus and to methods of making and using the same_ More in s particular, the invention relates to improved vectors for the insertion and expression of foreign genes for use as safe immunization ~rehicles to protect against a variety of pathogens, as well as for use in iaesunotherapy.
Several publications are referenced in this 1o application. Full citation to these references is found at the end of the specification immediately preceding the claims or where t::e w:.z.iblicat.icn is ~nenticred.. ':hese publicaticns re~.ate to Che art to whic:: t he invent=on pertains.
15 _ ELKHOUND OF THE INV'E~IT~.ON
Vaccinia virus and more recently other poxviruses have been used far the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves 20 recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologcus sequences present in the rescuing poxvirus (Piccini et al . , 1980 .
Specifically, the recombinant poxviruses are 25 constructed in two steps known in the art and analogous to the methods for. c~aating syntheic recombinants of poxviruses such as the vaccinia v=rus and avipox virus described in U.S. patent Nos. 4,769,330, 4,772,848, 4,603,11:2, S,IOO,:aB'', and 5,179,993, the disclosures of 30 which are incorporated herein by reference.
Firs;., the DNa gene sequence tc be inserted int:, the virus, pa=ticular~~r an open readirc frame :rom a ner.-pcx source, is placed into an ... ccii plasmid const_uct intc which DN~~ hemc? ogous to a section of DltA cf the poxwirus 3 S has been inserted . ~epar a to 1',r, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-Gene .215 3.3 ~~~ _ ;,. -. . 2 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 1o 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 2o homologous sections of DNA between two strands of DNA.
In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell 3o 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 s antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies have to recently been reported for generating recombinant vaccinia virus (Scheiflinger et al., 1992; Merchlinsky and Moss, 1992).
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two i5 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 2o 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 25 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 30 (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 3s have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., WO 94116716 '. . . PCT/US94100888 21 ~'~~~~~.

1990a,b).
The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of s Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).
1o 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 is lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.
A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue 2o 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).
2s 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.
3o 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 3s 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 s both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK- recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK+ vaccinia virus, indicating to 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 1s 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 20 (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 25 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 3o 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 3s 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
s gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA- vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990;
Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).
Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of 2o 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 2s 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 a gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR
3o vaccinia virus recombinant in which the a 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 a gene is intact (Zhou et al., 1990). The a 3s 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., _ ~~ ~.~~~~~~~s 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 s 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 io 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
15 region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).
A variety of spontaneous (Altenburger et al., 1989;
2o 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 2s of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes 3o 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 N1L
reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa 3s protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly WO 94116716 , .~ PCT/US94/00888 the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.
The left end of the vaccinia genome includes two genes which have been identified as host range genes, 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).
Two additional vaccine vector systems involve the is use of naturally host-restricted poxviruses, avipo~viruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus 2o 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 2s literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and 3o human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 3s 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a WO 94/16716 , ~ ~~~ CTIUS94100888 3 6~

heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus hare also been developed (Taylor et al., 1990; Edbauer et al., 1990).
s Despite the host-restriction for replication of FPV
and CPV to avian systems, recombinants deri~red from these viruses were found to express extrinsic pros=Bins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed io towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993 a,b;
Taylor et al., 1992; 1991b; 1988b).
In the past, viruses have been shown to have utility 15 in cancer immunotherapy, in that, they provide a means of enhancing tumor immunoresponsiveness. Examples exist showing that viruses such as Newcastle disease virus (Cassel et al., 1983), influenza virus (Lindenmann, 1974;
Lindenmann, 1967), and vaccinia virus (Wallack et al., 20 1986; Shimizu et al., 1988; Shimizu et al. 1984; Fujiwara et al., 1984) may act as tumor-modifying antigens or adjuvants resulting in inducing tumor-specific and tumor-nonspecific immune effector mechanisms. Due to advances in the fields of immunology, tumor biology, and molecular 25 biology, however, such approaches have yielded to more directed immunotherapeutic approaches for cancer.
Genetic modification of tumor cells and immune effector cells (i.e. tumor-infiltrating lymphocytes; TILs) to express, for instance cytokines, have provided 3o encouraging results in animal models and humans with respect to augmenting tumor-directed immune responses (Pardoll, 1992; Rosenberg, 1992). Further, the definition of tumor-associated antigens (TAAs) has provided the opportunity to investigate their role in the 3s immunobiology of certain cancers which may eventually be applied to their use in cancer prevention or therapy (van der Bruggen, 1992).

r WO 94!16716 PCT/US94/00888 2 ~i53~3~ -io Advances in the use of eukaryotic vaccine vectors have provided a renewed interest in viruses in cancer prevention and therapy. Among the viruses engineered to express foreign gene products are adenoviruses, adeno-associated virus, baculovirus, herpesviruses, poxviruses, and retroviruses. Most notably, retrovirus-, adenovirus and poxvirus-based recombinant viruses have been developed with the intent of in vivo utilization in the areas of vector-based vaccines, gene therapy, and cancer to therapy (Tartaglia, in press; Tartaglia, 1990).
Immunotherapeutic approaches to combat cancers or neoplasia can take the form of classical vaccination schemes or cell-based therapies. Immunotherapeutic vaccination is the concept of inducing or enhancing i5 immune responses of the cancer patient to antigenic determinants that are uniquely expressed or expressed at increased levels on tumor cells. Tumor-associated antigens (TAAs) are usually of such weak immunogenicity as to allow progression of the tumor unhindered by the 2o patient's immune system. Under normal circumstances, the severity of the disease-state associated with the tumor progresses more rapidly than the elaboration of immune responses, if any, to the tumor cells. Consequently, the patient may succumb to the neoplasia before a sufficient 2s immune response is mounted to control and prevent growth and spread of the tumor.
Poxvirus vector technology has been utilized to elicit immunological responses to TAAs. Examples exist demonstrating the effectiveness of poxvirus-based 3o recombinant viruses expressing TAAs in animal models in the immunoprophylaxis and immunotherapy of.
experimentally-induced tumors. The gene encoding carcinoembryonic antigen (CEA) was isolated from human colon tumor cells and inserted into the vaccinia virus 3s genome (Kaufman et al., 1991). Inoculation of the vaccinia-based CEA recombinant elicited CEA-specific, antibodies and an antitumor effect in a murine mouse 1l ~ .
model. This recombinant virus has been shown to elicit humoral and cell-mediated responses in rhesus macaques (Kantor et al., 1993). The human melanoma TAA, p97, has also been inserted into vaccinia virus and shown to s protect mice from tumor transplants (Hu et al., 1988;
Estin et al., 1988). A further example was described by Bernards et al. (1987). These investigators constructed a vaccinia recombinant that expressed the extracellular domain of the rat neu-encoded transmembrane glycoprotein, to p185. Mice immunized with this recombinant virus developed a strong humoral response against the neu gene product and were protected against subsequent tumor challenge. vaccinia virus recombinants expressing either a secreted or membrane-anchored form of a breast cancer-15 associated epithelial tumor antigen (ETA) have been generated for evaluation in the active immunotherapy of breast cancer (Hareuveni et al., 1991; 1990). These recombinant viruses have been shown to elicit anti-ETA
antibodies in mice and to protect mice against a 2o tumorigenic challenge with a ras-transformed Fischer rat fibroblast line expressing either form of ETA (Hareuveni et al., 1990). Further, vaccinia virus recombinants expressing the polyoma virus-derived T-Ag were shown efficacious for prevention and therapy in a mouse tumor 2s model system (Lathe et al., 1987).
Recombinant vaccinia viruses have also been used to express cytokine genes (Reviewed by Ruby et al., 1992).
Expression of certain cytokines (IL-2, IFN-a, TNF-a) lead to self-limiting vaccinia virus infection in mice and, in 3o essence, act to attenuate the virus. Expression of other cytokines (i.e. IL-5, IL-6) were found to modulate the immune response to co-expressed extrinsic immunogens (Reviewed by Ruby et al., 1992).
Frequently, immune responses against tumor cells are 35 mediated by T cells, particularly cytotoxic T lymphocytes (CTLs); white blood cells capable of killing tumor cells and virus-infected cells (Greenberg, 1991). The behavior WO 94116716 ~ i PCTIUS94/00888 of CTLs is regulated by soluble factors termed cytokines.
Cytokines direct the growth, differentiation, and functional properties of CTLs, as well as, other immune effector cells.
Cell-based immunotherapy has been shown to provide -effective therapy for viruses and tumors in animal models (Greenberg, 1991; Pardoll, 1992; Riddel et al., 1992). ' Cytomegalovirus (CMV)-specific CTL clones from bone marrow donors have recently been isolated. These clones to were propagated and expanded in vitro and ultimately returned to immunodeficient bone marrow patients. These transferred CMV-specific CTL clones provided no toxic-effects and provided persistent reconstitution of CD8+
CMV-specific CTL responses preventing CMV infection in the transplant patient (Riddel et al., 1992).
There exists two forms of cell-based immunotherapy.
These are adoptive immunotherapy, which involves the expansion of tumor reactive lymphocytes in vitro and reinfusion into the host, and active immunotherapy, which 2o involves immunization of tumor cells to potentially enhance existing or to elicit novel tumor-specific immune responses and provide systemic anti-tumor immunity.
Immunotherapeutic vaccination is the concept of inducing or enhancing immune responses of the cancer patient to 2s antigenic determinants that are uniquely expressed or expressed at increased levels on tumor cells.
It can be appreciated that provision of novel strains, such as NYVAC, ALVAC, and TROVAC having enhanced safety would be a highly desirable advance over the 3o 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 35 provide modified recombinant viruses, which viruses have enhanced safety, and to provide a method of making such recombinant viruses.

,. 3~

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.
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 to 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 is 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 2o has attenuated virulence and enhanced safety. The functions can be non-essential, or associated with virulence. The virus is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified 2s recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.
3o In another aspect, the present invention relates to a vaccine for inducing an antigenic 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 35 functions so that the recombinant virus has attenuated virulence and enhanced safety. The virus used in the vaccine according to the present invention is WO 94116716 , # .. -, PCT/US94/00888 ~, ,. :.
21~~ '~ 3'~ ~6~ ~ __ advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus and canarypox virus. The modified recombinant virus can include, within a non-essential region of the virus genome, a s heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.
In yet another aspect, the present invention relates to an immunogenic composition containing a modified 1o 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
is sequence which encodes an antigenic protein (e. g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof) wherein the composition, when administered to a host, is capable of inducing an immunological response specific to the 2o 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 in vitro by introducing into the cell a modified recombinant virus having attenuated virulence and 25 enhanced safety. The modified recombinant virus can include, within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from a pathogen, a tumor associated antigen, a cytokine, or combination thereof.
3o In a still further aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the virus has attenuated virulence, and wherein the modified recombinant virus further contains 3s DNA from a heterologous source in a nonessential region of the virus genome. The DNA can code for a tumor associated antigen, a cytokine, or a combination thereof.

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 5 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 I4L (by the terminology reported in Goebel et al., 10 1990a,b); and, the combination thereof. 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; or, the combination 15 thereof. The modified Copenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia et al., 1992).
According to another aspect of the present invention, there is provided a recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus is a recombinant vaccinia virus in which the regions C7L-K1L, J2R, B13R+B14R, A56R and I4L have been deleted therefrom.
According to still another aspect of the present invention, there is provided a recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus is a recombinant vaccinia virus wherein the open reading frames for the thymidine kinase gene, the hemorrhagic region, the A type inclusion body region, the hemagglutinin gene, the host range gene region, and the 15a large subunit ribonucleotide reductase gene have been deleted therefrom.
According to yet another aspect of the present invention, there is provided a recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus vector is a recombinant NYVAC, wherein the open reading frames for the thymidine kinase gene, the hemorrhagic region, the A type inclusion body region, the hemagglutinin gene, the host range gene region, and the large subunit ribonucleotide reductase gene have been deleted therefrom.
According to a further aspect of the present invention, there is provided a recombinant poxvirus containing therein exogenous DNA coding for a cytokine, exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen in a non-essential region of the poxvirus genome, wherein the poxvirus expresses the exogenous DNA, wherein the poxvirus vector is the recombinant vaccinia virus NYVAC.
According to still a further aspect of the present invention, there is provided a recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus vector is ALVAC.
According to another aspect of the present invention, there is provided a recombinant poxvirus selected from the group consisting of vP1200, vP1101, vP1098, vP1239, vP1241, vP1237, vP1244, vP1243, vP1248, NYVAC+IFNy+IL-2, 15b vP1250, vP1246, NYVAC+IL-12, vP1230, vP1245, NYVAC+IFNY+IR7, vP1234, vP1233, vP1100 and vP1096.
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 pMP4940 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 WO 94/16716 : ~ ~ ' PCTIUS94I00888 2 ~'~ ~

hemagglutinin gene and generation of recombinant vaccinia virus vP723;
FIG. 5 schematically shows a method for the construction of plasmid pMPCKl~ 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 1o 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 shows the DNA sequence (SEQ ID N0:68) of a canarypox PvuII fragment containing the C5 ORF.
FIGS. 9A and 9B schematically show a method for the construction of recombinant canarypox virus vCP65 (ALVAC-RG);
2o FIG. 10 shows schematically the ORFs deleted to generate NYVAC;
FIG. 11 shows the nucleotide sequence (SEQ ID N0:77) of a fragment of TROVAC DNA containing an F8 ORF;
FIG. 12 shows the DNA sequence (SEQ ID N0:78) of a 2356 base pair fragment of TROVAC DNA containing the F7 ORF;
FIGS. 13A to 13D show graphs of rabies neutralizing antibody titers (REFIT, IU/ml), booster effect of HDC and vCP65 (105-5 TCID50) in volunteers previously immunized 3o 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. 14A to 14C show the nucleotide sequence of a 7351 by fragment containing the ALVAC C3 insertion site (SEQ ID N0:127);
FIG. 15 shows the nucleotide sequences of H6/TNF-a expression cassette and flanking regions from vCP245 (SEQ

WO 94116716 ~. ~ ~ ~ ~ ~ PCT/US94I00888 - 17 ,, ID N0:79);
FIG. 16 shows the nucleotide sequence of the H6/TNF-a expression cassette and flanking regions from vP1200 (SEQ ID N0:89);
FIG. 17 shows the nucleotide sequence of the H6/p53 (wildtype) expression cassette and flanking regions from vP1101 (SEQ ID N0:99);
FIG. 18 shows the nucleotide sequence of the H6/p53 (wildtype) expression cassette and flanking regions from vCP207 (SEQ ID N0:99);
FIG. 19 shows the nucleotide sequence of the H6/MAGE-1 expression cassette and flanking region from vCP235 (SEQ ID N0:109);
FIG. 20 shows the nucleotide sequence of the H6/MAGE-1 expression cassette and flanking regions from pMAW037 (SEQ ID NO:110);
FIG. 21A and B show the nucleotide sequence of the p126.15 SERA cDNA insert along with the predicted amino acid sequence (SEQ ID NOS:119; 120);
2o FIG. 22 shows the nucleotide sequence of the H6/CEA
expression cassette and flanking regions from pH6.CEA.C3.2 (SEQ ID N0:144);
FIG. 23 shows the nucleotide sequence of the H6/CEA
expression cassette and flanking regions from pH6.CEA.HA
(SEQ ID N0:145);
FIG. 24 shows the nucleotide sequence of murine IL-2 from the translation initiation codon through the stop codon (SEQ ID N0:150);
FIG. 25 shows the corrected nucleotide sequence of 3o human IL-2 from the translation initiation codon through the stop codon (SEQ ID N0:159);
FIG. 26 shows the nucleotide sequence of the I3L/murine IFN~y expression cassette (SEQ ID N0:163);
FIG. 27 shows the nucleotide sequence of the I3L/human IFNy expression cassette (SEQ ID N0:168);
FIG. 28 shows the nucleotide sequence of the canarypox insert in pC6HIII3kb (SEQ ID N0:169);

.., 2~~_~ .,:
FIG. 29 shows the nucleotide sequence pC6L (SEQ ID
N0:174);
FIG. 30 shows the nucleotide sequence of the E3L/murine IL-4 expression cassette (SEQ ID N0:178);
s FIG. 31 shows the nucleotide sequence of the expression cassette comprising the E3L promoted human IL-4 gene (SEQ ID N0:186);
FIG. 32 shows the nucleotide sequence of the vaccinia E3L/hGMCSF expression cassette (SEQ ID N0:191);
1o FIG. 33 shows the sequence of the EPV 42kDa/human IL-12 P40 expression cassette (SEQ ID N0:194);
FIG. 34 shows the nucleotide sequence of the vaccinia E3L/human IL-12 P35 expression cassette (SEQ ID
N0:199);
15 FIG. 35 shows the nucleotide sequence of the murine B7 gene (SEQ ID N0:202);
FIG. 36 shows flow cytometric analysis of murine B7 expression in NYVAC and ALVAC infected murine tumor cell lines;
2o FIG. 37 shows the nucleotide sequence for the human B7 gene (SEQ ID N0:207);
FIG. 38 shows the murine p53 gene (SEQ ID N0:214);
and FIG. 39 shows the coding sequence for the human p53 25 gene (SEQ ID N0:215).
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 30 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.
35 The deletion loci were also engineered as recipient loci for the insertion of foreign genes.
The regions deleted in NYVAC are listed below. Also WO 94116716 ~ ~ , PCTIUS94100888 ,= _ listed are the abbreviations and open reading frame designations for the deleted regions (Goebe7_ et al., 1990a,b) and the designation of the vaccini<< recombinant (vP) containing all deletions through the deletion s specified:
(1) thymidine kinase gene (TK; J2R) vF~410;
(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 - KiL) vP804; and (6) large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC).
NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of is eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC is highly attenuated by a number of criteria including i) decreased virulence after intracerebral inoculation in newborn mice, ii) inocuity in genetically (nu+/nu+) or 2o chemically (cyclophosphamide) immunocompromised mice, iii) failure to cause disseminated infection in immunocompromised mice, iv) lack of significant induration and ulceration on rabbit skin, v) rapid clearance from the site of inoculation, and vi) greatly 2s reduced replication competency on a number of tissue culture cell lines including those of human origin.
Nevertheless, NYVAC based vectors induce excellent responses to extrinsic immunogens and provided protective immunity.
3o TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chicks. ALVAC is an attenuated canarypox virus-based vector that was a plaque-cloned derivative of 3s the licensed canarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has some general properties which are the same as some general properties of Kanapox. ALVAC-WO 94/16716 ~ v PCTIUS94/00888 t~
~j ~,> '-based recombinant viruses expressing extrinsic immunogens have also been demonstrated efficacious as vaccine vectors (Tartaglia et al., 1993 a,b). This avipox vector is restricted to avian species for productive s replication. On human cell cultures, canarypox virus replication is aborted early in the viral replication cycle prior to viral DNA synthesis. Nevertheless, when engineered to express extrinsic immunogens, authentic expression and processing is observed in vitro in mammalian cells and inoculation into numerous mammalian species induces antibody and cellular immune responses to the extrinsic immunogen and provides protection against challenge with the cognate pathogen (Taylor et al., 1992;
Taylor et al., 1991). Recent Phase I clinical trials in 1s both Europe and the United States of a canarypox/rabies glycoprotein recombinant (ALVAC-RG) demonstrated that the experimental vaccine was well tolerated and induced protective levels of rabiesvirus neutralizing antibody titers (Cadoz et al., 1992; Fries et al., 1992).
2o Additionally, peripheral blood mononuclear cells (PBMCs) derived from the ALVAC-RG vaccine demonstrated significant levels of lymphocyte proliferation when stimulated with purified rabies virus (Fries et al., 1992).
NYVAC, ALVAC and TROVAC have also been recognized as unique among all poxviruses in that the National Institutes of Health ("NIH")(U.S. Public Health Service), Recombinant DNA Advisory Committee, which issues guidelines for the physical containment of genetic 3o material such as viruses and vectors, i.e., guidelines for safety procedures for the use of such viruses and vectors which are based upon the pathogenicity of the particular virus or vector, granted a reduction in physical containment level: from BL2 to BL1. No other 3s poxvirus has a BL1 physical containment level. Even the Copenhagen strain of vaccinia virus - the common smallpox vaccine - has a higher physical containment level;

WO 94/16716 'e .~~: . PCTIUS94I00888 namely, BL2. Accordingly, the art has recognized that NYVAC, ALVAC and TROVAC have a lower pathogenicity than any other poxvirus. .
Both NYVAC- and ALVAC-based recombinant viruses have been shown to stimulate in vitro specific CD8+ CTLs from human PBMCs (Tartaglia et al., 1993a). Mice immunized with NYVAC or ALVAC recombinants expressing various forms of the HIV-1 envelope glycoprotein generated both primary and memory HIV specific CTL responses which could be to recalled by a second inoculation (Tartaglia et al., 1993a). ALVAC-env and NYVAC-env recombinants (expressing the HIV-1 envelope glycoprotein) stimulated strong HIV-specific CTL responses from peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals (Tartaglia et al., 1993a). Acutely infected autologous PBMC were used as stimulator cells for the remaining PBMC. After 10 days incubation in the absence of exogenous IL-2, the cells were evaluated for CTL activities. NYVAC-env and ALVAC-env stimulated high levels of anti-HIV activities.
2o Thus, these vectors lend themselves well to ex vivo stimulation of antigen reactive lymphocytes; for example, adoptive immunotherapy such as the ex vivo expression of tumor reactive lymphocytes and reinfusion into the host (patient).
Immunization of the patient with NYVAC-, ALVAC-, or TROVAC-based recombinant viruses expressing TAAs produced by the patient's tumor cells can elicit anti-tumor immune responses more rapidly and to sufficient levels to impede or halt tumor spread and potentially eliminate the tumor 3o burden .
Clearly based on the attenuation profiles of the NYVAC, ALVAC, and TROVAC vectors and their demonstrated ability to elicit both humoral and cellular immunological responses to extrinsic immunogens (Tartaglia et al., 1993a,b; Taylor et al., 1992; Konishi et al., 1992) such recombinant viruses offer a distinct advantage over previously described vaccinia-based recombinant viruses.

2 ~ ~~~~~~:

The immunization procedure for such recombinant viruses as immunotherapeutic vaccines or compositions may be via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response against the specific TAA(s).
Alternatively, the vaccine or composition may be administered directly into the tumor mass (intratumor).
Such a route of administration can enhance the anti-tumor activities of lymphocytes specifically associated with 1o tumors (Rosenberg, 1992). Immunization of the patient with NYVAC-, ALVAC- or TROVAC-based recombinant viruses expressing TAAs produced by the patient's tumor cells can elicit anti-tumor immune. responses more rapidly and to sufficient levels to impede or halt tumor spread and potentially eliminate the tumor burden. The heightened tumor-specific immune response resulting from vaccinations with these poxvirus-based recombinant vaccines can result in remission of the tumor, including permanent remission of the tumor. Examples of known TAAs 2o for which recombinant poxviruses can be generated and employed with immunotherapeutic value in accordance with this invention include, but are not limited to p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), and the melanoma-associated antigens IMAGE-1; MZE-2) (van der Bruggen et al., 1991), and p97 (Hu et al., 1988) and the carcinoembryonic antigen (CEA) associated with colorecteal cancer (Kantor et al., 1993; Fishbein et al., 1992; Kaufman et al., 1991).
3o More generally, the inventive vaccines or compositions (vaccines or compositions containing the poxvirus recombinants of the invention) can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such vaccines or compositions can be administered to a patient in need . of such administration in dosages and by techniques well known to those skilled in the medical arts taking into fVO 94116716 PCTlUS94100888 ..

consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration. The vaccines or compositions can be co-administered or sequentially administered with other s antineoplastic, anti-tumor or anti-cancer agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents; again taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and, the 1o route of administration.
Examples of vaccines or compositions of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, etc., administration such as suspensions, syrups or elixirs; and, preparations for 1s parental, subcutaneous, intradermal, intramuscular or intravenous administration (e. g., injectable administration) such as sterile suspensions or emulsions.
In such compositions the recombinant poxvirus may be in admixture with a suitable carrier, diluent, or excipient 2o such as sterile water, physiological saline, glucose or the like. The recombinant poxvirus of the invention can be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. Further, the invention also comprehends a kit wherein the 25 recombinant poxvirus is provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit can also include an additional anti-cancer, anti-tumor or antineoplastic agent and/or an agent which reduces or alleviates ill effects of 3o antineoplastic, anti-tumor or anti-cancer agents for co-or sequential-administration. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.
The poxvirus vector technology provides an appealing s5 approach towards manipulating lymphocytes and tumor cells for use in cell-based immunotherapeutic modalities for cancer. Characteristics of the NYVAC, ALVAC and TROVAC

WO 94116716 ° . _ _ PCTIUS94I00888 21~3$~36~~ -vectors providing the impetus for such applications include 1) their apparent independence for specific receptors for entry into cells, 2) their ability to express foreign genes in cell substrates despite their s species- or tissue-specific origin, 3) their ability to express foreign genes independent of host cell regulation, 4) the demonstrated ability of using poxvirus recombinant viruses to amplify specific CTL reactivities from peripheral blood mononuclear cells (PBMCs), and 5) 1o their highly attenuated properties compared to existing vaccinia virus vaccine strains (Reviewed by Tartaglia et al., 1993a; Tartaglia et al., 1990).
The expression of specific cytokines or the co-expression of specific cytokines with TAAs by NYVAC-, 15 ALVAC-, and TROVAC-based recombinant viruses can enhance the numbers and anti-tumor activities of CTLs associated with tumor cell depletion or elimination. Examples of cytokines which have a beneficial effect in this regard include tumor necrosis factor-a (TNF-a). interferon-gamma 20 (INF-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-7 (IL-7) (reviewed by Pardoll, 1992).
Cytokine interleukin 2 (IL-2) plays a major role in promoting cell mediated immunity. Secreted by the TH1 subset of lymphocytes, IL-2 is a T cell growth factor 25 which stimulates division of both CD4+ and CD8+ T cells.
In addition, IL-2 also has been shown to activate B
cells, monocytes and natural killer cells. To a large degree the biological effects of IL-2 are due to its role in inducing production of IFNy. Recombinant vaccinia 3o virus expressing IL-2 is attenuated in mice compared to wild-type vaccinia virus. This is due to the ability of the vaccinia-expressed IL-2 to stimulate mouse NK cells to produce IFNy, which limits the growth of the recombinant vaccinia virus (Karupiah et al., 1990).
35 Similarly, it has been shown that inoculation of immunodeficient athymic nude mice with recombinant vaccinia virus expressing both IL-2 and the HA gene of WO 94116716 ~ ~ ~ PCTIUS94/00888 ~' r 1R
influenza can protect these mice from subsequent challenge with influenza virus (Karupiah et al., 1992).
Cytokine interferon y (IFNy) is secreted by the TH1 subset of lymphocytes. IFNy promotes the TH1 cell 5 mediated immune response, while inhibiting the TH2 (antibody) response. IFNY induces the expression of major histocompatibility complex (MHC) molecules on antigen presenting cells, and induces the expression of the B7 costimulatory molecule on macrophages. In io addition to enhancing the phagocytic activity of macrophages, IFNy enhances the cytotoxic activity of NK
cells. When expressed in replicating recombinant vaccinia virus, IFN~y limits the growth of the recombinant virus. This allows T cell immunodeficient mice to 15 resolve the infection (Kohonen-Corish et al., 1990).
Cytokine interleukin 4 (IL-4) is secreted by the TH2 subset of lymphocytes. IL-4 promotes the TH2 (antibody) response, while inhibiting the TH1 cell mediated immune response. Recombinant vaccinia virus expressing IL-4 2o shows increased pathogenicity in mice compared to wild-type vaccinia virus (Ramshaw et al., 1992).
Cytokine granulocyte macrophage colony stimulating factor (GMCSF) is pleiotropic. In addition to stimulating the proliferation of cells of both the 25 granulocyte and macrophage cell lineages, GMCSF, in cross-competition with interleukins 3 and 5 (IL-3 and IL-5), influences many other aspects of hematopoiesis and may play a role in facilitation of tumor cell growth (Lopez et al., 1992). GMCSF is used clinically for 3o hematopoietic reconstitution following bone marrow transplantation.
Cytokine interleukin 12 (IL-12), formerly known as natural killer (NK) cell stimulatory factor, is a heterodimer composed of 35kDa and 40kDa subunits. IL-12 is produced by monocytes, macrophages, B cells and other accessory cells. IL-12 has pleiotropic effects on both NK cells and T cells. Partly through its role in '~i ~ .
;, inducing IFNy production, IL-12 plays a major role in promoting the TH1 cell mediated immune response, while inhibiting the TH2 response (reviewed in Trinchieri, 1993). Recently, recombinant murine IL-12 has been demonstrated to have potent antitumor and antimetastatic effects in mice (Brunda et al., 1993).
B7(BB-1), a member of the immunoglobin superfamily, is present on the surface of antigen presenting cells.
Interaction of the B7 molecule on antigen presenting 1o cells with its receptors on T cells provides costimulatory signals, including IL-2, which are necessary for T cell activation (Schwartz, 1992).
Recently it was shown that experimental co-expression of B7 along with a tumor antigen on murine melanoma cells can lead to regression of tumors in mice. This was accomplished by the B7-assisted activation of tumor-specific cytotoxic T cells (Chen et al, 1992).
The c-erb-B-2 gene, which is conserved among vertebrates, encodes a possible receptor protein. The 185 kDa translation product contains a kinase domain which is highly homologous to the kinase domain of the epidermal growth factor (EGF) receptor. The c-erb-B-2 gene is conserved among vertebrates, and is the same as the rat neu gene, which has been detected in a number of 2s rat neuro/glioblastomas. The human c-erb-B-2 gene, also known as HER2, is amplified in certain neoplasias, most notably breast cancer. In the gastric cancer cell line, MKN-7, both the normal 4.6 kb transcript encoding c-erb-B-2 and a 2.3 kb transcript which specifies only the 3o extracellular domain of the putative receptor are synthesized at elevated levels (Yamamoto et al., 1986).
The extracellular domain has been suggested as a potential immunogen for active specific immunotherapy of breast cancer (Fendly et al., 1990).
35 Utility of NYVAC-, ALVAC-, and TROVAC-based recombinant viruses expressing TAAs plus or minus specific cytokines for adoptive immunotherapy can take several forms. For one, genetic modification of PBMCs can be accomplished by vector-mediated introduction of TAAs, cytokine genes, or other genes and then directly reintroduced into the patient. Such administration relies on the drainage or movement of modified PBMCs to lymphoid tissue (i.e. spleen; lymph nodes) via the reticuloendothelial system (RES) for elicitation of the tumor-specific immune response. PBMCs modified by infection with the pertinent NYVAC-, ALVAC-, and TROVAC-1o based recombinant can be employed, for instance, in vitro, to expand TAA-specific CTLs for reinfusion into the patient. Tumor-infiltrating lymphocytes (TILs) derived from the tumor mass can be isolated, expanded, and modified to express pertinent genes using NYVAC-, ALVAC-, or TROVAC-based recombinants viruses prior to reinfusion into the patient. TILs retain the capability of returning to tumors (homing) when re-introduced into the subject (Rosenberg, 1992). Thus, they provide a convenient vehicle for delivery of cytotoxic or 2o cytostatic cytokines to tumor masses.
Cell-based active immunotherapy can also take on several potential modalities using the NYVAC-, ALVAC-, and TROVAC vectors. Tumor cells can be modified to express TAAs, cytokines, or other novel antigens (i.e.
class I or class II major histocompatibility genes).
Such modified tumor cells can subsequently be utilized for active immunization. The therapeutic potential for such an administration is based on the ability of these modified tumor cells to secrete cytokines and to alter 3o the presentation of TAAs to achieve systemic anti-tumor activity. The modified tumor cells can also be utilized to expand tumor-specific CTLs in vitro for reinfusion into the patient.
A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

77396wy za EXAMPLES
nNb r~onina anc~ Synthesis. Plasmids were constructed, screened and grown by standard procedures (Maniat:is et al., 5.982; Perkus et al., 1985; Piccini et s 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 palymerase was obtained from Boehringer Mannheim 1o Hiochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as saec~.~:ied by the various suppliers.
Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 car Applied Biosystems 3808 DNA
15 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., 198"') as previously described (Guo et al., 1989). DNA amplification by polymerase chain reaction 20 (PCR) for sequence verification (Engelke et al., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwal:~, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycles. Excess DNA sequences were.
25 deleted :from plasmids by restriction endanuclease digestion followed by limited digestion by BAL-31 exonuclease and mr.~tagenesis (Mandecki, 1986) using synthetics oligonucleotides.
Cells, Virus, and Transfection. The origins anal 3o conditions of cultvivation of the Copenhagen strain cf vaccini.a virus has been previously described (Guo et al., 1989) . Generati:,~n ci recc~;.binant virus b~r recombination, fi i_~. sitr~ hybridization o_ nitrccell°slose f filters and SCr°_e:21.?1C~ fOr B-yalaCtOSldaS2 aC~lVlty as? aS preVlOll~~ V
35 described (P:iccini et al., 1987).
The origins a.nd conditions o~ cultivation of th.e Copenhagen strain o' vaccinia v2=us and N'~VAC has been * Trade-mar:c WO 94116716 ~r~'~ t PCTIUS94l00888 f ~ ~.. n d!: ~r 7 previously described (Guo et al., 1989; Tartaglia et al., 1992). 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).
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 io 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.
The strain of fowlpox virus (FPV) designated FP-1 has been described previously (Taylor et al., 1988a). It is an attenuated vaccine strain useful in vaccination of day old chickens. The parental virus strain Duvette was obtained in France as a fowlpox scale from a chicken.
The virus was attenuated by approximately 50 serial passages in chicken embryonated eggs followed by 25 passages on chicken embryo fibroblast cells. The virus was subjected to four successive plaque purifications.
One plaque isolate was further amplified in primary CEF
cells and a stock virus, designated as TROVAC, established.
NYVAC, ALVAC and TROVAC viral vectors and their derivatives were propagated as described previously (Piccini et al., 1987; Taylor et al., 1988a,b). Vero cells and chick embryo fibroblasts (CEF) were propagated as described previously (Taylor et al., 1988a,b).
Example 1 - CONSTRUCTION OF PLASMID pSD460 FOR
DELETION OF THYMIDINE RINASE GENE (J2R) Referring now to FIG. 1, plasmid pSD406 contains vaccinia HindIII J (pos. 83359 - 88377) cloned into pUC8.
pSD406 was cut with HindIII and PvuII, and the 1.7 kb .~.a .;:
~'; g. , ;, ~ ;.
VVO 94116716 -' '' PCT/US94I00888 fragment from the left side of HindIII J cloned into pUC8 cut with HindIII/SmaI, forming pSD447. pSD447 contains the entire gene for J2R (pos. 83855 - 84385). The initiation codon is contained within an NIaIII site and s the termination codon is contained within an SSpI 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 1o digested with NIaIII and a 0.5 kb HindIII/NIaIII fragment isolated. Annealed synthetic oligonucleotides MPSYN43/MPSYN44 (SEQ ID NO:1/SEQ ID N0:2) SmaI
MPSYN43 5' TAATTAACTAGCTACCCGGG 3' 15 MPSYN44 3' GTACATTAATTGATCGATGGGCCCTTAA 5' NIaIII EcoRI
were ligated with the 0.5 kb HindIII/NIaIII fragment into pUCl8 vector plasmid cut with HindIII/EcoRI, generating 2o plasmid pSD449.
To obtain a restriction fragment containing a vaccinia right flanking arm and pUC vector sequences, pSD447 was cut with SSpI (partial) within vaccinia sequences and HindIII at the pUC/vaccinia junction, and a 25 2.9 kb vector fragment isolated. This vector fragment was ligated with annealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID N0:3/SEQ ID N0:4) HindIII SmaI
MPSYN45 5' AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAA
30 MPSYN46 3' AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT
NotI SSpI
ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT 3' MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA 5' MPSYN46 generating pSD459.
To combine the left and right flanking arms into one plasmid, a 0.5 kb HindIII/SmaI fragment was isolated from pSD449 and ligated with pSD459 vector plasmid cut with 4o HindIII/SmaI, generating plasmid pSD460. pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2. 32P

WO 94116716 . ; ., ~~ s.: s; Y'; a.

labelled probe was synthesized by primer extension using MPSYN45 (SEQ ID N0:3) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID N0:5) (5' TTAGTTAATTAGGCGGCCGC 3') as primer. Recombinant s virus vP410 was identified by plaque hybridization.
Example 2 - CONSTROCTION OF PLASMID p8D486 FOR
- DELETION OF HEMORRHAGIC REGION
(B13R + B14R) Referring now to FIG. 2, plasmid pSD419 contains to 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 is 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 a region is indicated by an arrow in FIG. 2.
To remove unwanted sequences from pSD419, sequences 2o 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 2s HpaI at the 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 a region 3o in pSD477 is indicated by a triangle. The remaining B13R
coding sequences in pSD477 were removed by digestion with ClaI/HpaI, and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer (SEQ ID N0:6/SEQ ID N0:7) 3s ClaI BamHI HpaI
SD22mer 5' CGATTACTATGAAGGATCCGTT 3' SD20mer 3' TAATGATACTTCCTAGGCAA 5' generating pSD479. pSD479 contains an initiation codon ~.t ~. ,~~1 y'~ r ? ~ a..
a .~

(underlined) followed by a BamHI site. To place E. coli Beta-galactosidase in the B13-B14 (u) deletion locus under the control of the a 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 1o 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 a deletion junction, was utilized. First the ClaI/H~aI
vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID N0:8/SEQ ID N0:9) Clal Sacl Xhol Hpal 2o SD42mer 5' CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT 3' SD40mer 3' TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA 5' B~c III Smal BamHl generating plasmid pSD478. Next the EcoRI site at the pUC/vaccinia junction was destroyed by digestion of pSD478 with EcoRI followed by blunt ending with Klenow fragment of E. coli polymerase and ligation, generating plasmid pSD478E-. pSD478E- was digested with BamHI and HpaI and ligated with annealed synthetic 3o oligonucleotides HEMS/HEM6 (SEQ ID NO:10/SEQ ID N0:11) BamHI EcoRI HbaI
HEMS 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.

WO 94116716 21 ~3~3 PCT/US94100888 _. ,, Example 3 - CONSTROCTION OF PLABMID pMP494~
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 sequenOes (pos. 137,079) and with HindIII
at the pUC/vaccinia junction, then blunt ended with Klenow fragment of E. coli polymerase and ligated, 1o 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 N0:12/SEQ ID N0:13) 2o Ndei AT13 5'TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAAAAAATAAGT
AT14 3' ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTTTTTTATTCA
B~c III EcoRl Hpal TATATAAATAGATCTGAATTCGTT 3' ATI3 ATATATTTATCTAGACTTAAGCAA 5' ATI4 reconstructing the region upstream from A26L and replacing the A26L ORF with a short polylinker region 3o containing the restriction sites BalII, EcoRI and HpaI, as indicated above. The resulting plasmid was designated pSD485. Since the BQ1II and EcoRI sites in the polylinker region of pSD485 are not unique, unwanted BalII and EcoRI sites were removed from plasmid pSD483 (described above) by digestion with BctlII (pos. 140,136) and with EcoRI at the pUC/vaccinia junction, followed by blunt ending with Klenow fragment of E. coli polymerase and ligation. The resulting plasmid was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcoRV (pos.
139,048) fragment from pSD489 containing the A26L ORF was replaced with the corresponding 0.7 kb polylinker-containing ClaI/EcoRV fragment from pSD485, generating pSD492. The BQ1II and EcoRI sites in the pc>lylinker region of pSD492 are unique.
A 3.3 kb BQ1II 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 BcrlII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus to 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, pMP4940, vaccinia DNA encompassing 2o 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 HEMAGGLUTININ GENE (A56R) Referring now to FIG. 4, vaccinia SalI G restriction fragment (pos. 160,744-173,351) crosses the HindIII A/B
3o 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 HindIII B were removed by digestion of pSD419 with HindIII within vaccinia sequences and at the pUC/vaccinia junction followed by ligation. The resulting plasmid, pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to WO 94/16716 ~ ~ ~ ~ PCT/US94100888 the right. A56R coding sequences were removed by cutting pSD456 with RsaI (partial; pos. 161,090) upstream from A56R coding sequences, and with Ea~I (pos. 162,054) near the end of the gene. The 3.6 kb RsaI/Ea4I vector 5 fragment from pSD456 was isolated and ligated with annealed synthetic oligonucleotides MPSYN59 (SEQ ID
N0:15), MPSYN62 (SEQ ID N0:16), MPSYN60 (SEQ ID N0:17), and MPSYN61 (SEQ ID N0:18) RsaI
l0 MPSYN59 5' ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT-MPSYN62 3' TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA-MPSYN59 AGTTGATAGAACAAAATACATAATTT 3' MPSYN62 TCAACTATCT 5' MPSYN60 5' TGTAAAAATAAATCACTTTTTATA-MPSYN61 3' TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT-BqlII SmaI PstI EagI
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 BctlII/BamHI (partial) cassette containing 3o 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 4o pSD466, except that EcoRI, SmaI and BamHI sites were removed from the pUC/vaccinia junction by digestion. of pSD466 with EcoRI/BamHI followed by blunt ending with WO 94/16716 . °' PCTIUS94100888 2 ~~=~ ~~ ~ ~~~ _ 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.
s Example 5 - CONSTRUCTION OF PLA8MID pMPC8R1A
FOR DELETION OF OPEN READING FRAMES
fC7L-R1L1 Referring now to FIG. 5, the following vaccinia io clones were utilized in the construction of pMPCSKl~.
pSD420 is SalI H cloned into pUC8. pSD435 is KpnI F
cloned into pUCl8. pSD435 was cut with SphI and religated, forming pSD451. In pSD451, DNA sequences to the left of the SphI site (pos. 27,416) in HindIII M are 1s removed (Perkus et al., 1990). pSD409 is HindIII M
cloned into pUC8.
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
2o deletion locus (Guo et al., 1990) as follows. To eliminate the BqlII site in pSD409, the plasmid was cut with BqlII in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphI site 25 (pos. 27,416). M2L coding sequences were removed by mutagenesis (Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide BalII
MPSYN82 (SEQ ID N0:19) 5' TTTCTGTATATTTGCACCAATTTAGATCTT-3o ACTCAAAATATGTAACAATA 3' The resulting plasmid, pMP409D, contains a unique BQ1II
site inserted into the M2L deletion locus as indicated above. A 3.2 kb BamHI (partial)/BQ1II cassette 3s containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the 1l kDa promoter.
(Bertholet et al., 1985) was inserted into pMP409D cut with BqlII. The resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination 4o with rescuing vaccinia virus vP723. Recombinant vaccinia WO 94116716 ~~4. PCTIUS94100888 virus, vP784, containing Beta-galactosidase inserted into the M2L deletion locus, was isolated as a blue plaque in the presence of X-gal.
A plasmid deleted for vaccinia genes [C7L-KiL] was assembled in pUC8 cut with SmaI, HindIII and blunt ended with Klenow fragment of E. coli polymerase. The left flanking arm consisting of vaccinia HindII:C C sequences was obtained by digestion of pSD420 with XbaI (pos.
18,628) followed by blunt ending with Klenow fragment of 1o 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 BqlII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid, pMP581CK is deleted for vaccinia sequences between the BglII site (pos. 19,706) in HindIII
C and the BQ1II 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 2o 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 N0:20) 5'-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3'.
The resulting plasmid, pMPCSKl~, is deleted for vaccinia sequences positions 18,805-29,108, encompassing 12 vaccinia open reading frames [C7L - K1L]. Recombination between pMPCSKl~ and the Beta-galactosidase containing 3o 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 PLASMID pSD548 FOR DELETION
OF LARGE SUBUNIT, RIBONUCLEOTIDE REDUCTASE

Referring now to FIG. 6, plasmid pSD405 contains vaccinia HindIII I (pos. 63,875-70,367) cloned in pUC8.
pSD405 was digested with EcoRV within vaccinia sequences WO 94116716 215 3 3 ~ ~ PCTlUS94/00888 (pos. 67,933) and with SmaI at the pUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 was used as the source of all the vaccinia restriction fragments used in the construction of pSD548.
s The vaccinia I4L gene extends from position 67,371-65,059. Direction of transcription for I4L is indicated by an arrow in FIG. 6. To obtain a vector plasmid fragment deleted for a portion of the I4L coding sequences, pSD518 was digested with BamHI (pos. 65,381) 1o and HpaI (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 15 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 I4L gene, 2o was isolated as a blue plaque in the presence of X-gal.
To delete Beta-galactosidase and the remainder of the I4L ORF from vP855, deletion plasmid pSD548 was constructed. The left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and 25 presented schematically in FIG. 6.
To construct a vector plasmid to accept the left vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518A1/518A2 (SEQ ID N0:21/SEQ ID N0:22) 3o BamHl Rsai 518A1 5' GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAT
518A2 3' GACTCATGAAACATTATATTACTATATATAAAAGTGAAATAGAGTA
B~c III EcoRl 35 TTGAGAATAAAAAGATCTTAGG 3' S18A1 AACTCTTATTTTTCTAGAATCCTTAA 5' 518A2 forming plasmid pSD531. pSD531 was cut with RsaI
(partial) and BamHI and a 2.7 kb vector fragment 4o isolated. pSD518 was cut with BctlII (pos. 64,459)/ RsaI

WO 94/16716 5 ~ '~ ~ ~ PCTIUS94/00888 jf-,r= ;, ~\; ,, 39 ~ ' , (pos. 64,994) and a 0.5 kb fragment isolated. The two fragments were ligated together, forming pSD537, which contains the complete vaccinia flanking arm left of the I4L coding sequences.
s To construct a vector plasmid to accept the right vaccinia flanking arm, pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID N0:23/SEQ ID N0:24) BamHl Bgll Smal io 51881 5' GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCATTAATAG
518B2 3' GTCTAGAGGGCCCTTTTTTTAATAAATTGAAAAGTAATTATC
Rsal _EcoRt GGATTTGACGTATGTAGCGTACTAGG 3' S18B1 1 s CCTAAACTGCATACTACGCATGATCCTTAA 5' S18B2 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.
20 67,436) and EcoRI at the vaccinia/pUC junction, and a 0.6 kb fragment isolated. The two fragments were ligated together, forming pSD538, which contains the complete vaccinia flanking arm to the right of I4L coding sequences.
2s 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/BglII. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a polylinker region, which is 3o 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 3s portion of pSD539 with Beta-galactosidase sequences in recombinant vaccinia virus vP855, the vaccinia I4L
deletion cassette was moved from pSD539 into pRCll, a pUC
derivative from which all Beta-galactosidase sequences have been removed and replaced with a polylinker region 40 (Colinas et al., 1990). pSD539 was cut with EcoRI/PstI

,v ' ~2r15~3 3 3 ~ 40 and the 1.2 kb fragment isolated. This fragment was ligated into pRCll cut with EcoRI/PstI (2.35 kb), forming pSD548. Recombination between pSD548 and the Beta-galactosidase containing vaccinia recombinant, vP855, s 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 1o patterns were as expected. Polymerase chain reactions (PCR) (Engelke et al., 1988) using vP866 as template and primers flanking the six deletion loci detailed above produced DNA fragments of the expected sizes. Sequence analysis of the PCR generated fragments around the areas 15 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

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.
25 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 3o VQ1A/VQ1B (SEQ ID N0:25/SEQ ID N0:26) SmaI BglII XhoI PstI NarI BamHI
VQ1A 5' GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3' VQ1B 3' CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5' 35 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 pRV~i842.
4o 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 io the vaccinated individual and also diminishes transmission from vaccinated to unvaccinated individuals or contamination of the environment.
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 EXPRESSING THE
FUSION AND HEMAGGLUTININ-NEURAMINIDASE
GLYCOPROTEINS OF NEWCASTLE DISEASE VIRUS
This example describes the development of TROVAC, a fowlpox virus vector and, of a fowlpox Newcastle Disease Virus recombinant designated TROVAC-NDV and its safety and efficacy. 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 3o 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 a vaccine strain useful in vaccination of day old chickens. The parental virus 2~.~3~~~~

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 s 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 to 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 1s pNDV81 (Taylor et al., 1990) and inserted at the BamHI
site of pUClB to form pCEl3. The vaccinia virus H6 promoter previously described (Taylor et al., 1988a,b;
Guo et al., 1989; Perkus et al., 1989) was inserted into pCEl3 by digesting pCEl3 with SalI, filling in the sticky 2o ends with Klenow fragment of E. coli DNA polymerase and digesting with HindIII. A HindIII - EcoRV fragment containing the H6 promoter sequence was then inserted into pCEl3 to form pCE38. A perfect 5' end was generated by digesting pCE38 with KpnI and NruI and inserting the 2s annealed and kinased oligonucleotides CE75 (SEQ ID N0:27) and CE76 (SEQ ID N0:28) to generate pCE47.
CE75:
CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGATCCCGG
TAC
3o CE76:
CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGATATCG.
In order to remove non-coding sequence from the 3' end of the NDV-F a SmaI to PstI fragment from pCEl3 was inserted into the SmaI and PstI sites of pUCl8 to form pCE23. The 3s non-coding sequences were removed by sequential digestion of pCE23 with SacI, BamHI, Exonuclease III, SI nuclease and EcoRI. The annealed and kinased oligonucleotides VVO 94116716 ~ ~ PCTIUS94/00888 CE42 (SEQ ID N0:29) and CE43 (SEQ ID N0:30) 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 PstI - 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 HindIII - 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 2o into pCE54 by digesting pCE54 with SacI, partially digesting with BamHI and inserting the annealed and kinased oligonucleotides CE166 (SEQ ID N0:31) and CE167 (SEQ ID N0:32) to generate pCE58.
CE166: CTTTTTATAAAAAGTTAACTACGTAG
CE167: GATCCTACGTAGTTAACTTTTTATAAAAAGAGCT
A perfect 3' end for NDV-F was obtained by using the polymerase chain reaction (PCR) with pCE54 as template and oligonucleotides CE182 (SEQ ID N0:33) and CE183 (SEQ
ID N0:34) as primers.
3o 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 pCE7l, the final cassette for NDV-F. Plasmi.d pRW846 is essentially equivalent to plasmid pJCA002 (described below) but containing the H6 promoter and transcription s and translation stop signals. Digestion of pRW846 with HindIII and HpaI 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 1o 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 15 pRW830. A perfect 3' end for NDV-HN was obtained by inserting the annealed and kinased oligonucleotides CE162 (SEQ ID N0:35) and CE163 (SEQ ID N0:36) into the EcoRI
site of pRW830 to form pCE59, the final cassette for NDV-HN.

AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATTTAATTTT
TATAAGCTTG
CE163:
AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTAGTAAAGG

Construction of FPV Insertion Vector. Plasmid pRW731-15 contains a lOkb PvuII - PvuII fragment cloned from genomic DNA. The nucleotide sequence was determined on both strands for a 3660 by PvuII - EcoRV fragment.
3o 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 by EcoRV - EcoRV fragment.
The F8 ORF was entirely contained between an XbaI site and an SspI site in pRW761. In order to create an 3s 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 SSpI. A
3700 by 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
5 N0:38).
JCA017:5' CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGATCCTTATA
CGGGGAATAAT
JCA018:5' l0 ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATATTAAAAAACA
TAAAGTGT
The plasmid resulting from this ligation was designated pJCA002.
Construction of Double Insertion Vector for NDV F
15 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 2o digested with NdeI and partially digested with BalII to generate an NdeI - BctlII 4760 by 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 by PvuII - HindII fragment from 25 pRW731-15 into the SmaI and HindII sites of pBSSK+.
Plasmid pJCA021 was then digested with NdeI and BalII and ligated to the 4760 by NdeI - BglII fragment of pCE80 to form pJCA024. Plasmid pJCA024 therefore contains the NDV-F and HN genes inserted in opposite orientation with 30 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 by of FPV sequence. The left flanking arm adjacent to the NDV-HN sequence consists of 1700 by of FPV
35 sequence.
Development of TROVAC-NDV. Plasmid pJCA024 was transfected into TROVAC infected primary CEF cells by :..,:~ i.~.~~3:~ 5 _ 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 s 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).
to 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.
15 Immunoprecipitation. Immunoprecipitation reactions were performed as described (Taylor et al., 1990) using a polyclonal anti-NDV serum obtained from SPAFAS Inc., Storrs, CT.
The stock virus was screened by in situ plaque 2o 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 25 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 3o 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 been determined that amino acids at the cleavage site in the NDV-F sequence inserted into 35 FPV to form recombinant vFP29 had the sequence Arg - Arg -Gln - Arg - Arg (SEQ ID N0:39) (Taylor et al., 1990) which conforms to the sequence found to be a requirement WO 94116716 ; . ~~ ~ ~ ~ PCTIUS94/00888 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 Queens:land 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 io 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.
2o 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 3o 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 they are recognized by NDV immune serum.

WO 94116716 ~ PCTIUS94100888 21533~G' Immunoprecipitation analysis indicated 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 to 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 2o be responsible for eliciting virus neutralizing antibody (Avery et al., 1979) and both glycoproteins, when expressed individually in a fowlpox vector are able to induce a protective immune response, it can be appreciated that the most efficacious NDV vaccine should 2s express both glycoproteins.
Example 9 - CONSTRUCTION OF ALVAC RECOMBINANTS
EXPRESSING RABIES VIRUS GLYCOPROTEIN G
This example describes the development of ALVAC, a 3o canarypox virus vector and, 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.
35 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 21 ~ 3 3 ~ ~ , , ~T~S94/00888 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 by canarypox PvuII fragment was cloned between the PvuII sites of pUC9 to form pRW764.5. The sequence of this fragment is shown in FIG. 8 between positions 1372 1o and 2251. The limits of an open reading frame designated as C5 were defined. It was determined that the open reading frame was initiated at position 166 within the fragment and terminated at position 487. The C5 deletion was made without interruption of open reading frames.
Bases from position 167 through position 455 were replaced with the sequence (SEQ ID N0:39) GCTTCCCGGGAATTCTAGCTAGCTAGTTT. This replacement sequence contains HindIII, SmaI and EcoRI insertion sites followed by translation stops and a transcription termination 2o 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 N0:40):
ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA
RW146 (SEQ ID N0:41):
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 WO 94116716 ,. . t PCTIUS94/00888 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, s starting at NruI, through the HindIII site of rabies G
followed by BalII. Sequences of oligonucleotides A
through E ((SEQ ID N0:42)-(SEQ ID N0:46)) are:
A (SEO ID N0:42): CTGAAATTATTTCATTATCGCGATATCCGTTAA
GTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT
to B (SEA ID N0:43): CATTACGATACAAACTTAACGGATATCGCGATAA
TGAAATAATTTCAG
C (SEO ID N0:44): ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAA
TTCCCTATTTACACGATCCCAGACAAGCTTAGATCTCAG
D (SEO ID N0:45): CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATA

E (SEQ ID N0:46): CAACGGAAAAACCAGAAGGGGTACAAACAGGAGA
GCCTGAGGAAC
Th.e diagram of annealed oligonucleotides A through E is as follows:
2o A C
B E D
Oligonucleotides A through E were kinased, annealed 25 (95°C for 5 minutes, then cooled to room temperature), and inserted between the PvuII sites of pUC9. The resulting plasmid, pRW737, was cut with HindIII and BctlII
and used as a vector for the 1.6 kbp HindIII-BalII
fragment of ptg155PR0 (Kieny et al., 1984) generating 3o pRW739. The ptg155PR0 HindIII site is 86 by downstream of the rabies G translation initiation codon. BctlII is downstream of the rabies G translation stop codon in ptg155PR0. pRW739 was partially cut with NruI, completely cut with BctlII, and a 1.7 kbp NruI-BalII
3s~ fragment, containing the 3' end of the H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., WO 94116716 ~ ~ ~ ~ PCTIUS94100888 :-, ,.
51 . . s 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 SmaI is (SEQ ID
N0:47): GGATCCCCGGG. pRW824 is a plasmid that contains a nonpertinent gene linked precisely to the vaccinia virus H6 promoter. Digestion with NruI and BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832 SmaI
1o fragment, containing H6 promoted rabies G, was inserted into the SmaI of pRW831, to form plasmid pRW838.
Development of ALVAC-RG. Plasmid pRW838 was transfected into ALVAC infected primary CEF cells by using the calcium phosphate precipitation method 1s 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 2o plaque was then amplified and the resulting ALVAC
recombinant was designated ALVAC-RG (vCP65) (see also FIG. 9). The correct insertion of the rabies G gene into the ALVAC genome without subsequent mutation was confirmed by sequence analysis.
25 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 3o 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.
3s Immunofluorescence was performed as previously described (Taylor et al., 1990) using a rabies G monoclonal antibody. Strong surface fluorescence was detected on 215~~3G -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 1o as previously described (Taylor et al., 1990).
Immunoprecipitation reactions were performed using a rabies G specific monoclonal antibody. Efficient expression of a rabies specific glycoprotein with a molecular weight of approximately 67 kDa was detected with the recombinant ALVAC-RG. No rabies specific products were detected in uninfected cells or cells infected with the parental ALVAC virus.
Secruential PassaginQ Experiment. In studies with ALVAC virus in a range of non-avian species no 2o 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 10 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 3o 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.

WO 94/16716 ~ ~ ~ ~ ~ ~ PCTIUS94/00888 ,.
j. ga ' , , t w 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 t0 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 io 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 t0, t7 and t7A) and onto one dish of primary CEF cells. Samples t0, 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 1 and 2.
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 3o 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 1 and 2 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.

WO 94116716 PCTlUS94100888 ., 215 3 ~'3'~~ ". . _ 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 t0 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 (CPE) or at 7 days if no CPE was evident. The results of this experiment are shown in Table 3. 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.
2o Inoculation of Macagues. Four HIV seropositive macaques were initially inoculated with ALVAC-RG as described in Table 4. 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 3o chimpanzees (50 to 65 kg weight range) were inoculated intramuscularly or subcutaneously with 1 X 10~ 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.

2~ ~~3,,3~f~ .
_ i ; ~.., Inoculation of Mice. Groups of mice were inoculated with 50 to 100 ~,1 of a range of dilutions of different batches of vCP65. Mice were inoculated in the footpad.
On day 14, mice were challenged by intracranial s inoculation of from 15 to 43 mouse LDSO of the virulent CVS strain of rabies virus. Survival of mice was monitored and a protective dose 50% (PDSO) calculated at 28 days post-inoculation.
Inoculation of Dogs and Cats. Ten beagle dogs, 5 1o months old, and 10 cats, 4 months old, were inoculated subcutaneously with either 6.7 or 7.7 loglo TCIDSO of ALVAC-RG. Four dogs and four cats were not inoculated.
Animals were bled at 14 and 28 days post-inoculation and anti-rabies antibody assessed in an RFFI test. The 15 animals receiving 6.7 loglo TCIDSO of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 loglo mouse LDSO (dogs) or 4.3 loglo mouse LDSO (cats) of the NYGS rabies virus challenge strain.
Inoculation of Squirrel Monkeys. Three groups of 2o 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 25 feline leukemia virus. Inoculations were performed under ketamine anaesthesia. Each animal received at the same time: (1) 20 ~1 instilled on the surface of the right eye without scarification; (2) 100 ~,1 as several droplets in the mouth; (3) 100 ~,1 in each of two intradermal 3o injection sites in the shaven skin of the external face of the right arm; and (4) 100 ~1 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 35 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 WO 94/16716 '" ~ ~' ~'' ~- PCTIUS94/00888 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 io 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, 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 3o 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 WO 94/16716 PCTlUS94100888 21~~33 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 s 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 io 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
15 7.5, 200 mM EDTA) 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
2o 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 2s 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.
3o 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 35 thawing. Virus yield was assessed by plaque titration on CEF monolayers.

WO 94/16716 ,~. . .-~ PCT/US94/00888 215 3;~'~ 6~v it r v _ C. Analysis of expression of Rabies G gene.
Dishes were inoculated with recombinant or parental virus at a multiplicity of l0 pfu/cell, allowing an additional dish as an uninfected virus control.
s 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 io 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.
1s 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 5. The results indicate that while a productive infection can be attained in the avian cells, no increase in virus yield can be detected 2o 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 2s 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 3o 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 3s hours post-infection exhibited a strong band in the region of approximately 350 kbp representing ALVAC-specific viral DNA accumulation. No such band is 'WO 94/16716 ~ ri ,_ ,~.. F v 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.
1o 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 is 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 limits of detection of this method have not been fully ascertained and viral DNA accumulation may 2o 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 Expression. To determine if 2s 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 3o 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 MRC-5, WISH and Detroit cells infected with ALVAC-RG. No such specific rabies 35 gene products were detected in any of the uninfected and parentally infected cell lysates.

WO 94/16716 ~~ ~ ~ PCTIUS94/00888 215333'° ~' 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 s 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 io 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.
15 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 model system. A similar test was 2o 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 TCIDSO per ml were serially diluted and 50 to 2s 100 ~.1 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 ~1 of the CVS strain of rabies virus containing from 15 to 43 mouse LD5a as determined by lethality titration in a control group of 3o 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 6. The results indicated that ALVAC-RG was consistently able to protect mice against rabies virus challenge with a PD5o 35 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 ~C1 of virus WO 94/16716 , PCT/US94/00888 containing 6.0 loglo TCIDSO 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 to animals in each species were not vaccinated. Five animals received 6.7 loglo TCIDSO subcutaneously and five animals received 7.7 loglo TCIDSO by the same route.
Animals were bled for analysis for anti-rabies antibody.
Animals receiving no inoculation or 6.7 loglo TCIDSO of ALVAC-RG were challenged at 29 days post-vaccination with 3.7 loglo mouse LDSO (dogs, in the temporal muscle) or 4.3 loglo mouse LDSO (cats, in the neck) of the NYGS rabies virus challenge strain. The results of the experiment are shown in Table 7.
2o 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 TCIDSO 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 TCIDSO 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 3o which killed all controls. All cats immunized with 7.7 loglo TCIDSO 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 .~; ,, WO 94116716 ~ . '-.~ PCT/US94100888 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 1o 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 TCIDSO of ALVAC-RG. Sera were analyzed for the presence of anti-rabies antibody. The results are shown in Table 8.
Four of the five monkeys naive to rabies developed a serological response by seven days post-inoculation with 2o ALVAC-RG. All five monkeys had detectable antibody by 11 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 2s 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 3o 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.
35 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 9, WO 94!16716 ~ PCT/US94/00888 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 s 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 io 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 15 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 10.
No adverse reactivity to inoculation was noted by either intramuscular or subcutaneous routes. Both 2o chimpanzees responded to primary inoculation by 14 days and a strongly rising response was detected following re-vaccination.

WO 94/16716 ~ PCT/US94I00888 2Z~333~~ _ Table 1. Sequential Passage of ALVAC in Avian and non-Avian Cells.
CEF Vero M RC-5 Pass 1 Sample toe 2.4 3.0 2.6 t7 7.0 1.4 0.4 t7A' 1.2 1.2 0.4 io 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 .

i5 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.

2o 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 2s 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 30 ~.g/ml of Cytosine arabinoside and harvested at 7 days post infection.
d: Not detectable 215 3 ,~ 3 ~ PCTIUS94/00888 '. ~ ' Table 2. Sequential Passage of ALVAC-RGAvian and non-Avian in Cells CEF Vero MRC-5 Pass 1 Sample 3.0 2.9 2.9 t0a t7b 7.1 1.0 1.4 t7A' 1.8 1.4 1.2 Pass 2 to Sample 5.1 0.4 0.4 t0 t7 7.1 N.D.d N.D.

t7A 3.8 N.D. N.D.

Pass 3 Sample 5.1 0.4 N.D.
t0 t7 7.2 N.D. N.D.

t7A 3.6 N.D. N.D.

Pass 4 Sample 5.1 N.D. N.D.
t0 t7 7.0 N.D. N.D.

2o t7A 4.0 N.D. N.D

a: This sample was harvested at ero time and z represents virus. The titer is the residual input expressed as loglopfu per ml.

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

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

d: Not detectable.

WO 94116716 21 ~ 3 3 ,~ ~ ,y '.~' ~1 . ~T~S94I00888 Table 3. Amplification of residual virus by passage in CEF cells CEF Vero MRC-5 s a) ALVAC

Pass 2$ 7.0b 6.0 5.2 3 7.5 4.1 4.9 4 7.5 N.D.' N.D.

7.1 N.D. N.D.

to 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.
2o b: Titer expressed as loglo pfu per ml c: Not Detectable WO 94/16716 215 3 3.,~ ?~ , ~ ..4 r . PCT/US94100888 Table 4. Schedule of inoculation of rhesus macaques with ALVAC-RG
(vCP65) Animal Inoculation 176L Primary: 1 X pfu of vCP65 orally in TANG

Secondary: 1 X pfu of vCP65 plus 1 X 10' pfu 10' of vCP82a by SC route l0 185 L Primary: 1 X pfu of vCP65 orally in Tang Secondary: 1 X pfu of vCP65 plus 1 X 10' pfu 10' of vCP82 by SC

route 177 L Primary: 5 X pfu SC of vCP65 by SC route 10' Secondary: 1 X pfu of vCP65 plus 1 X 10' pfu 10' of vCP82 by SC

i 5 route 186L Primary: 5 X pfu of vCP65 by SC route 10' Secondary: 1 X pfu of vCP65 plus 1 X 10' pfu 10' of vCP82 by SC

route 178L Primary: 1 X pfu of vCP65 by SC route 10' 20 182L Primary: 1 X pfu of vCP65 by IM route 10' 179L Primary: 1 X pfu of vCP65 by SC route 183L Primary: 1 X pfu of vCP65 by IM route 180L Primary: 1 X pfu of vCP65 by SC route 184L Primary: 1 X pfu of vCP65 by IM route 25 187L Primary 1 X pfu of vCP65 orally 10' a: vCP82 is a canarypox virus recombinant expressing the measles virus fusion and hemagglutinin genes.

215 3..3x3;=sw - , y PCTIUS94100888 Table 5. Analysis of yield in avian and non-avian cells inoculated with ALVAC-RG
Sample Time Cell Type t0 t72 t72A°
Expt 1 CEF 3.3a 7.4 1.7 Vero 3.0 1.4 1.7 to 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 loglo pfu per ml b: Culture incubated in the presence of 40 ~,g/ml of Cytosine arabinoside WO 94/16716 ~ , PCT/US94100888 Table 6. Potency of ALVAC-RG as tested in mice Test Challenge Dosea PDSO°
s 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 io Vaccine Batch L 15 4.03 Vaccine Batch M 15 3.32 Vaccine Batch N 15 3.39 Vaccine Batch J 23 3.42 15 a: Expressed as mouse LD5o b: Expressed as loglo TCIDSo Table 7. Efficacy of ALVAC-RG in dogs and cats Doas Cats Dose Antibodya Survival° 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 1o geometric mean titer in International Units.
b: Expressed as a ratio of survivors over animals challenged 21~,~~' Table 8. Anti-rabies serological response of Squirrel monkeys inoculated with canarypox recombinants Monkey Previous Rabies serum-neutralizing antibodya # Exposure -196° 0 3 7 11 21 28 22 ALVAC' NT9 < < < 2.1 2.3 2.2 1.2 1.2 1.2 51 ALVAC' NT < < 1.7 2.2 2.2 2.2 1.2 1.2 39 vCP37 NT < < 1.7 2.1 2.2 N.T.9 1.2 1.2 55 vCP37 NT < < 1.7 2.2 2.1 N.T.
1.2 1.2 37 ALVAC-RGe 2.2 < < 3.2 3.5 3.5 3.2 1.2 1.2 53 ALVAC-RGe 2.2 < < 3.6 3.6 3.6 3.4 1.2 1.2 38 ALVAC-RG' 2.7 < < 3.2 3.8 3.6 N.T.
1.7 1.7 54 ALVAC-RG' 3.2 < < 3.6 4.2 4.0 3.6 1.7 1.5 57 None NT < < 1.7 2.7 2.7 2.3 1.2 1.2 a: As determined by RFFI test on days indicated and expressed in Internat ionalUnits b: Day-196 represents se rum after primary from day 2o vaccination c: Animals received 5.0 logloTCIDSO ALVAC
of d: Animals received 5.0 logloTCIDSO vCP37 of e: Animals received 5.0 logloTCIDSO ALVAC-RG
of f: Animals received 7.0 logloTCID50 ALVAC-RG
of g: Not tested.

Table 9. Inoculation of rhesus macaques with ALVAC-RGa C~ Days Route of Primary Inoculation post inocu G~t lation or Tan SC SC SC IM SC IM SC IM OR

~ 1-87L~

_g4 . --g - . . . . _ s _ _ 11 - - 1sd 129 75 - 64 128 - .

99~ - - 64 256 - . . _ - -2 - - 32 256 - - - - . . -6 - - 512 512 - - - - . . -a: See Table 9 for schedule of inoculations.
b: Animals 176L and 185L received 8.0 loglo pfu by the 35 oral route in 5 ml Tang. Animal 187L received 7.0 loglo pfu by oral route not in Tang.
c: Day of re-vaccination for animals 176L, 185L, 177L
and 186L by S.C. route, and primary vaccination for 4o animals 178L, 182L, 179L, 183L, 180L, 184L and 187L.
d: Titers expressed as reciprocal of last dilution showing inhibition of fluorescence in an RFFI test.

WO 94/16716 ~~ PCTIUS94100888 Table 10. Inoculation of chimpanzees with ALVAC-RG
Weeks post- Animal 431 Animal 457 Inoculation I.M. S.C.
0 <88 <8 1 <8 <8 l0 8 16 32 a: Titer expressed as reciprocal of last dilution showing inhibition of fluorescence in an RFFI test b: Day of re-inoculation Example 10 - IMMUNIZATION OF HUMANS USING CANARYPOX
EXPRESSING RABIES GLYCOPROTEIN
(ALVAC-RG; yCP65) G~ 5 ALVAC-RG (vCP65) was generated as described in Example 9 and FIGS. 9A and 9B. For scaling-up and vaccine manufacturing ALVAC-RG (vCP65) was grown in primary CEF derived from specified pathogen free eggs.
Cells were infected at a multiplicity of 0.01 and io incubated at 37°C for three days.
The vaccine virus suspension was obtained by ultrasonic disruption in serum free medium of the infected cells; cell debris were then removed by centrifugation and filtration. The resulting clarified 15 suspension was supplemented with lyophilization stabilizer (mixture of amino-acids), dispensed in single dose vials and freeze dried. Three batches of decreasing titer were prepared by ten-fold serial dilutions of the virus suspension in a mixture of serum free medium and 20 lyophilization stabilizer, prior to lyophilization.
Quality control tests were applied to the cell substrates, media and virus seeds and final product with emphasis on the search for adventitious agents and innocuity in laboratory rodents. No undesirable trait 25 was found.
Preclinical data. Studies in vitro indicated that VERO or MRC-5 cells do not support the growth of ALVAC-RG
(vCP65); a series of eight (VERO) and 10 (MRC) blind serial passages caused no detectable adaptation of the 3o virus to grow in these non avian lines. Analyses of human cell lines (MRC-5, WISH, Detroit 532, HEL, HNK or EBV-transformed lymphoblastoid cells) infected or inoculated with ALVAC-RG (vCP65) showed no accumulation of virus specific DNA suggesting that in these cells the 3s block in replication occurs prior to DNA synthesis.
Significantly, however, the expression of the rabies virus glycoprotein gene in all cell lines tested indicating that the abortive step in the canarypox replication cycle occurs prior to viral DNA replication.

WO 94/16716 ~ l 5 3 3 3 ~ PCT/US94100888 The safety and efficacy of ALVAC-RG (vC:P65) were documented in a series of experiments in animals. A
number of species including canaries, chickens, ducks, geese, laboratory rodents (suckling and adult mice), 5 hamsters, guinea-pigs, rabbits, cats and dogs, squirrel monkeys, rhesus macaques and chimpanzees, wE:re inoculated with doses ranging from 105 to 108 pfu. A variety of routes were used, most commonly subcutaneous, intramuscular and intradermal but also oral (monkeys and to mice) and intracerebral (mice).
In canaries, ALVAC-RG (vCP65) caused a "take" lesion at the site of scarification with no indication of disease or death. Intradermal inoculation of rabbits resulted in a typical poxvirus inoculation reaction which 15 did not spread and healed in seven to ten days. There was no adverse side effects due to canarypox in any of the animal tests. Immunogenicity was documented by the development of anti-rabies antibodies following inoculation of ALVAC-RG (vCP65) in rodents, dogs, cats, 2o and primates, as measured by Rapid Fluorescent Focus Inhibition Test (RFFIT). Protection was also demonstrated by rabies virus challenge experiments in mice, dogs, and cats immunized with ALVAC-RG (vCP65).
Volunteers. Twenty-five healthy adults aged 20-45 25 with no previous history of rabies immunization were enrolled. Their health status was assessed by complete medical histories, physical examinations, hematological and blood chemistry analyses. Exclusion criteria included pregnancy, allergies, immune depression of any 3o kind, chronic debilitating disease, cancer, injection of immune globins in the past three months, and seropositivity to human immunodeficiency virus (HIV) or to hepatitis B virus surface antigen.
Study design. Participants were randomly allocated 35 to receive either standard Human Diploid Cell Rabies Vaccine (HDC) batch no E0751 (Pasteur Merieux Serums &
Vaccine, Lyon, France) or the study vaccine ALVAC-RG

(vCP65).
The trial was designated as a dose escalation study.
Three batches of experimental ALVAC-RG (vCP65) vaccine were used sequentially in three groups of volunteers s (Groups A, B and C) with two week intervals between each step. The concentration of the three batches was 1035, 104'5, 105'5 Tissue Culture Infectious Dose (TCID50) per dose, respectively.
Each volunteer received two doses of the same 1o vaccine subcutaneously in the deltoid region at an interval of four weeks. The nature of the injected vaccine was not known by the participants at the time of the first injection but was known by the investigator.
In order to minimize the risk of immediate 15 hypersensitivity at the time of the second injection, the volunteers of Group B allocated to the medium dose of experimental vaccine were injected 1 h previously with the lower dose and those allocated to the higher dose (Group C) received successively the lower and the medium 2o dose at hourly intervals.
Six months later, the recipients of the highest dosage of ALVAC-RG (vCP65) (Group C) and HDC vaccine were offered a third dose of vaccine; they were then randomized to receive either the same vaccine as 2s previously or the alternate vaccine. As a result, four groups were formed corresponding to the following immunization scheme: 1. HDC, HDC - HDC; 2. HDC, HDC -ALVAC-RG (vCP65); 3. ALVAC-RG (vCP65), ALVAC-RG (vCP65) - HDC; 4. ALVAC-RG (vCP65), ALVAC-RG (vCP65), ALVAC-RG
30 (vCP65).
Monitoring of Side Effects. All subjects were monitored for 1 h after injection and re-examined every day for the next five days. They were asked to record local and systemic reactions for the next three weeks and 35 were questioned by telephone two times a week.
Laboratory Investigators. Blood specimens were obtained before enrollment and two, four and six days WO 94/16716 ~ ~ ~ PCTlUS94100888 after each injection. Analysis included complete blood'' cell count, liver enzymes and creative kinase assays.
Antibody assays. Antibody assays were performed seven days prior to the first injection and at days 7 28, 35, 56, 173, 187 and 208 of the study.
The levels of neutralizing antibodies to rabies were determined using the Rapid Fluorescent Focus Inhibition test (RFFIT) (Smith & Yaeger, In Laboratory Techniques on Rabies). Canarypox antibodies were measured by direct to ELISA. The antigen, a suspension of purified canarypox virus disrupted with 0.1% Triton X100, was coated in microplates. Fixed dilutions of the sera were reacted for two hours at room temperature and reacting antibodies were revealed with a peroxidase labelled anti-human IgG
goat serum. The results are expressed as the optical density read at 490nm.
Analysis. Twenty-five subjects were enrolled and completed the study. There were 10 males and 15 females and the mean age was 31.9 (2l to 48). All but three 2o subjects had evidence of previous smallpox vaccination;
the three remaining subjects had no typical scar and vaccination history. Three subjects received each of the lower doses of experimental vaccine (103'5 and 104-s TCIDSO), nine subjects received 105'5 TCIDSO and ten received the HDC vaccine.
Safety (Table 11). During the primary series of immunization, fever greater than 37.7°C was noted within 24 hours after injection in one HDC recipient (37.8°C) and in one vCP65 105'5 TCIDSO recipient (38°C). No other 3o systemic reaction attributable to vaccination was observed in any participant.
Local reactions were noted in 9/10 recipients of HDC
vaccine injected subcutaneously and in 0/3, 1/3 and 9/9 recipients of vCP65 103'5, 104'5, 105'5 TCIDSO, respectively.
Tenderness was the most common symptoms and was always mild. Other local symptoms included redness and WO 94/16716 w PCT/US94/00888 induration which were also mild and transient. All symptoms usually subsided within 24 hours and never lasted more than 72 hours.
There was no significant change in blood cell s counts, liver enzymes or creatine kinase values.
Immune Responses; Neutralizing Antibodies to Rabies (Table 121. Twenty eight days after the first injection all the HDC recipients had protective titers (>_0.5 IU/ml). By contrast none in groups A and B (103-5 and 104'5 TCIDS~) and only 2/9 in group C (105'5 TCIDSO) ALVAC-RG (vCP65) recipients reached this protective titer.
At day 56 (i.e. 28 days after the second injection) protective titers were achieved in 0/3 of Group A, 2/3 of Group B and 9/9 of Group C recipients of ALVAC-RG (vCP65) vaccine and persisted in all 10 HDC recipients.
At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and 11.5 IU/ml in groups A, B, C and HDC
respectively.
At day 180, the rabies antibody titers had 2o substantially decreased in all subjects but remained above the minimum protective titer of 0.5 IU/ml in 5/10 HCD recipients and in 5/9 ALVAC-RG (vCP65) recipients;
the geometric mean titers were 0.51 and 0.45 IU/ml in groups HCD and C, respectively.
Antibodies to the Canarypox virus yTable 13~. The pre-immune titers observed varied widely with titers varying from 0.22 to 1.23 O.D. units despite the absence of any previous contact with canary birds in those subjects with the highest titers. When defined as a 3o greater than two-fold increase between preimmunization and post second injection titers, a seroconversion was obtained in 1/3 subjects in group B and in 9/9 subjects in group C whereas no subject seroconverted in groups A
or HDC.
Booster Infection. The vaccine was similarly well tolerated six months later, at the time of the booster injection: fever was noted in 2/9 HDC booster recipients WO 94/16716 ~ ~ PCT/US94100888 and in 1/10 ALVAC-RG (vCP65) booster recipients. Local reactions were present in 5/9 recipients of HDC booster and in 6/10 recipients of the ALVAC-RG (vCP65) booster.
Observations. FIG. 13 shows graphs of rabies s neutralizing antibody titers (Rapid Fluorescent Focus Inhibition Test or RFFIT, IU/ml): Booster effect of HDC
and vCP65 (105'5 TCIDSO) in volunteers previously immunized with either the same or the alternate vaccine.
Vaccines were given at days 0, 28 and 180. Antibody 1o titers were measured at days 0, 7, 28, 35, 56, 173, and 187 and 208.
As shown in FIGS. 13A to 13D, the booster dose given resulted in a further increase in rabies antibody titers in every subject whatever the immunization scheme.
15 However, the ALVAC-RG (vCP65) booster globally elicited lower immune responses than the HDC booster and the ALVAC-RG (vCP65), ALVAC-RG (vCP65) - ALVAC-RG (vCP65) group had significantly lower titers than the three other groups. Similarly, the ALVAC-RG (vCP65) booster 2o injection resulted in an increase in canarypox antibody titers in 3/5 subjects who had previously received the HDC vaccine and in all five subjects previously immunized with ALVAC-RG (vCP65).
In general, none of the local side effects from 25 administration of vCP65 was indicative of a local replication of the virus. In particular, lesions of the skin such as those observed after injection of vaccine were absent. In spite of the apparent absence of replication of the virus, the injection resulted in the 3o volunteers generating significant amounts of antibodies to both the canarypox vector and to the expressed rabies glycoprotein.
Rabies neutralizing antibodies were assayed with the Rapid Fluorescent Focus Inhibition Test (RFFIT) which is 3s known to correlate well with the sero neutralization test in mice. Of 9 recipients of 105'5 TCIDSO, five had low level responses after the first dose. Protective titers WO 94116716 ' PCT/US94100888 cl.~~ of rabies antibodies were obtained after the second injection in all recipients of the highest dose tested and even in 2 of the 3 recipients of the medium dose. In ~1~ this study, both vaccines were given subcutaneously as s usually recommended for live vaccines, but not for the inactivated HDC vaccine. This route of injection was selected as it best allowed a careful examination of the injection site, but this could explain the late appearance of antibodies in HDC recipients: indeed, none to of the HDC recipients had an antibody increase at day 7, whereas, in most studies where HDC vaccine is give intramuscularly a significant proportion of subjects do (Klietmann et al., Int'1 Green Cross - Geneva, 1981;
Kuwert et al., Int'1 Green Cross - Geneva, 1981).
15 However, this invention is not necessarily limited to the subcutaneous route of administration.
The GMT (geometric mean titers) of rabies neutralizing antibodies was lower with the investigational vaccine than with the HDC control 2o vaccine, but still well above the minimum titer required for protection. The clear dose effect response obtained with the three dosages used in this study suggest that a higher dosage might induce a stronger response.
Certainly from this disclosure the skilled artisan can 2s select an appropriate dosage for a given patient.
The ability to boost the antibody response is another important result of this Example; indeed, an increase in rabies antibody titers was obtained in every subject after the 6 month dose whatever the immunization 3o scheme, showing that preexisting immunity elicited by either the canarypox vector or the rabies glycoprotein had no blocking effect on the booster with the recombinant vaccine candidate or the conventional HDC
rabies vaccine. This contrasts findings of others with 3s vaccinia recombinants in humans that immune response may be blocked by pre-existing immunity (Gooney et al., Lancet 1991, 337:567-72; Etlinger et al., Vaccine 9:470-WO 94116716 215 3 3 3 6 pCTNS94/00888 72, 1991).
Thus, this Example clearly demonstrates that a non-'.
replicating poxvirus can serve as an immunizing vector inJ:
humans, with all of the advantages that replicating s agents confer on the immune response, but without the safety problem created by a fully permissive virus.
TABLE 11: Reactions in the 5 days following vaccination to vCP65 dosage1Q3'5 104.5 l0s.s H
(TCID50) D
C
control Injection 1 st 2nd 1 2nd 1 2nd 1 2nd st st st No. vaccinees3 3 3 3 9 9 10 10 temp > 37.7C0 0 0 0 0 1 1 0 15 soreness 0 0 1 1 6 8 8 6 redness 0 0 0 0 0 4 5 4 I

induration 0 0 0 0 0 4 5 4 WO 94/16716 PCTNS94l00888 c~~ TABLEl2: Rabieseutralizingantibodies (REFIT;U/ml) dual nd geom n rs (GMT) I Indivi titers etric a mean tite Days No. TCID50/dose0 7 28 35 56 1 103'5 < 0.1 < 0.1 < 0.1 < 0.1 0.2 3 103.5 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 4 103.5 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 G.M.T. < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 6 104.5 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 7 104.s < 0.1 < 0.1 < 0.1 2.4 1.9 10 10'x.' < 0.1 < 0.1 < 0.1 1.6 1.1 G.M.T. < 0.1 < 0.1 0.1 0.58 0.47 11 105' < 0.1 < 0.1 1.0 3.2 4.3 13 10~' < 0.1 < 0.1 0.3 6.0 8.8 14 1055 < 0.1 < 0.1 0.2 2.1 9.4 17 a 105.5 < 0.1 < 0.1 < 0.1 1.2 2.5 18 105.5 < 0.1 < 0.1 0.7 8.3 12.5 105-'' < 0.1 < 0.1 < 0.1 0.3 3.7 21 105.5 < 0.1 < 0.1 0.2 2.6 3.9 2 0 23 105 5 < 0.1 < 0.1 < 0.1 1.7 4.2 105 5 < 0.1 < 0.1 < 0.1 0.6 0.9 G.M.T. < 0.1 < 0.1 0.16 1.9 4.4*

2 HDC < 0.1 < 0.1 0.8 7.1 7.2 5 HDC < 0.1 < 0.1 9.9 12.8 18.7 25 8 HDC < 0.1 < 0.1 12.7 21.1 16.5 9 HDC < 0.1 < 0.1 6.0 9.9 14.3 12 HDC < 0.1 < 0.1 5.0 9.2 25.3 15 HDC < 0.1 < 0.1 2.2 5.2 8.6 i6 HDC <: 0.1 < 0.1 2.7 7.7 20.7 19 HDC < 0.1 < 0.1 2.6 9.9 9.1 22 HDC < 0.1 < 0.1 1.4 8.6 6.6 24 HDC < 0.1 < 0.1 0.8 5.8 4.7 G.M.T. < 0.1 < 0.1 2.96 9.0 11.5*

N = u.um smaern ~ iest 215 3 3 3 G ~T~S94100888 TABLE 13: Canarypox antibodies: ELISA Geometric Mean Titers*
Days vCP65 dosage 0 7 28 35 56 TCID50/dose 1035 0.69 ND 0.76 ND 0.68 1045 0.49 0.45 0.56 0.63 0.87 105'5 0.38 0.38 0.77 1.42 1.63 HDC control 0.45 0.39 0.40 0.35 0.39 to * optical density at 1 /25 dilution WO 94/16716 , PCT/US94100888 Example 11 - COMPARISON OF THE LDSO 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.
1o 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 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 3o 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 is similarly to the normal mice except that an WO 94!16716 215 3 3 3 ~ pCT~S94100888 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 5 inoculation were excluded due to potential death by trauma.
The lethal dose required to produce mortality for 50% of the experimental population (LDSO) was determined by the proportional method of Reed and Muench.
1o Comparison of the LDSO of ALVAC and NYVAC with Various Vaccinia Virus Strains for Normal Youna Outbred Mice by the is Route. In young, normal mice, the virulence of NYVAC and ALVAC were several orders of magnitude lower than the other vaccinia virus strains 15 tested (Table 14). 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 2o 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 (3.85x10 PFUs, ALVAC and 3x108 PFUs, NYVAC) by an 25 undetermined mechanism by this route of inoculation.
Comparison of the LDSO of ALVAC and NYVAC with Various Vaccinia Virus Strains for Newborn Outbred Mice by the is Route. The relative virulence of 5 poxvirus strains for normal, newborn mice was tested by titration 3o in an intracranial (ic) challenge model system (Table 15). With mortality as the endpoint, LDSO values indicated that ALVAC is over 100,000 times less virulent than the Wyeth vaccine strain of vaccinia virus; over 200,000 times less virulent than the Copenhagen VC-2 35 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, 6.3x10 PFUs, 1000 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 LDSO) was 6.7 ~ 1.5 days. When compared to WR(L) at a challenge dose of 5 LDSO, wherein MST is 4.8 ~ 0.6 days, the MST of ALVAC challenged mice was significantly longer (P=0.001).
1o 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 6x10 PFUs, caused 1000 mortality. However, the MST
i5 of mice challenged with the highest dose, corresponding to 380 LD5o, 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 LDSO, survived to day 4.

WO 94/16716 21 ~ 3 3 ~ ~ ~~594/00888 Table 14. Calculated 50% Lethal Dose for mice by various vaccinia virus strains and for canarypox virus (ALVAC) by the is route.

POXVIRUS CALCULATED

STRAIN LDP (PFUs) WR(L) 2.5 VC-2 1.26x10 io WYETH 5.00x10' NYVAC 1.58x108 ALVAC 1.58x108 Table 15. Calculated 50% Lethal Dose for newborn mice by various vaccinia virus strains and for canarypox virus (ALVAC) by the is route.

POXVIRUS CALCULATED

STRAIN LDSO (PFUs) WR(L) 0.4 3 o VC-2 0.1 WYETH 1.6 NYVAC 1.58x10 ALVAC 1.00x10' FxamDle Z2 - EVALDATION O$ PYVAC (vP866y AND NYQAC-RG
(vP879) ~mmuno8recinitatzons. Preformed monalayers of avian or non-avian cells were inoculated with l0 pfu per cell of parental NYVAC (vp866) or NYVAC-RG (vP879) virus. The inoculation was performed in EME'~i free of methionine awd supplemented wit.' 2 j dialyzed fetal bovine serum. After a one hour incubat:.on , the inoculum was removed and the 1o medium replaced with E~ (methionine free) containing 20 ~Ci/ml of 35S-methionine. After an overnight incubation of approximately ~6 hours, cells were lysed by the addition of Buffer A (1a NGnidet P-40, 10 mM Tris pH7.4, 150 mM NaCl ,, ? ~nM ~DTA, 0. t; I ~ scd:.um azide, 500 uni is per is ml of aprcti.-~n, and 0.0~~ phenyl met:'~y1 sulfonyl fluoride). Immunoprecipitation was performed using a rabies glycoproteiz speci.i.c monoclonal antibody designated 24-3F10 supplied by Dr. C. Trinarchi, Griff ith Laboratories, New York State Department of Health, zo Albany, New York, and a rat anti-mouse conjugate obtained from Boehri.nger Mannheim Corporation (Cat. X605-500).
Protein A Sepharose CL-43 obtained from Pharmacia LKB
Biotechnology Inc., Piscataway, New Jersey, was used as a suppor t mat:-ix . L;~:.iunoprQcipi t3t2S Wer a fr ac tionated on 25 10% palyacrylami~~e gels according to the method of Dreyfuss et. al. (.984). ~e!s were fixed, treated far fluoragraphy wi~~h 1M Na-salicylate for one hour, and exposed to Kodak XAR-~ film to visualize the immunoprecipitated protein species.
3o Seu~ces of Animals. New Zealand White rabbits were obtained from Hare-Marland (Hewitt, New Jersey). Three week old male Swiss Webst~r autbred mice, timed pregnant female Swiss We~~ste= outbr=_d mice, and four week oLd Swiss wecstsr nude (nu nu mice were obtained from 3~ Taconic Far,:~s, inc. (Ge;~nant~wn, Ne~H Yor:~c) . A' 1 arn:rals were maintained according to NIH guvdelroes. All aE~imal prctocpls were approved by the ir.stit::tiora'_ IAC~C. Whet deemed necessary%, trice which were obv~'_ousl~~~ terminally * . y ode - max:c WO 94/16716 PC'T/US94/00888 ill were euthanized.
Evaluation of Lesions in Rabbits. Each of two rabbits was inoculated intradermally at multiple sites with 0.1 ml of PBS containing 104, 105, 106, 10~, or 108 pfu of each test virus or with PBS alone. The rabbits were observed daily from day 4 until lesion resolution.
Indurations and ulcerations were measured and recorded.
Virus Recovery from Inoculation Sites. A single rabbit was inoculated intradermally at multiple sites of 1o 0/1 ml of PBS containing 106, 10~, or 108 pfu of each test virus or with PBS alone. After 11 days, the rabbit was euthanized and skin biopsy specimens taken from each of the inoculation sites were aseptically prepared by mechanical disruption and indirect sonication for virus recovery. Infectious virus was assayed by plaque titration on CEF monolayers.
Virulence in Mice. Groups of ten mice, or five in the nude mice experiment, were inoculated ip with one of several dilutions of virus in 0.5 ml of sterile PBS.
2o Reference is also made to Example 11.
Cyclophosphamide ICY) Treatment. Mice were injected by the ip route with 4 mg (0.02 ml) of CY (SIGMA) on day -2, followed by virus injection on day 0. On the following days post infection, mice were injected ip with CY: 4 mg on day 1; 2 mg on days 4, 7 and 11; 3 mg on days 14, 18, 21, 25 and 28. Immunosuppression was indirectly monitored by enumerating white blood cells with a Coulter Counter on day 11. The average white blood cell count was 13,500 cells per ~1 for untreated mice (n=4) and 4,220 cells per ~,l for CY-treated control mice (n=5).
Calculation of LDSO. The lethal dose required to produce 50% mortality (LDSO) was determined by the proportional method of Reed and Muench (Reed and Mu~ench 1938 ) .
Potency Testing of NYVAC-RG in Mice. Four to six week old mice were inoculated in the footpad with 50 to 100 ~,1 of a range of dilutions (2.0 - 8.0 loglo tissue culture infective dose 50% (TCIDSO)) of either VV-RG
(Kieny et al., 1984), ALVAC-RG (Taylor et al., 1991b), or the NYVAC-RG. Each group consisted of eight mice. At 14 days post-vaccination, the mice were challenged by s intracranial inoculation with 15 LDSO of the rabies virus -CVS strain (0.03 ml). On day 28, surviving mice were counted and protective does 50% (PDSO) calculated.
Derivation of NYVAC (vP866). The NYVAC strain of vaccinia virus was generated from VC-2, a plaque cloned 1o isolate of the COPENHAGEN vaccine strain. To generate NYVAC from VC-2, eighteen vaccinia ORFs, including a number of viral functions associated with virulence, were precisely deleted in a series of sequential manipulations as described earlier in this disclosure. These deletions 1s were constructed in a manner designed to prevent the appearance of novel unwanted open reading frames. FIG.
10 schematically depicts the ORFs deleted to generate NYVAC. At the top of FIG. 10 is depicted the HindIII
restriction map of the vaccinia virus genome (VC-2 plaque 20 isolate, COPENHAGEN strain). Expanded are the six regions of VC-2 that were sequentially deleted in the generation of NYVAC. The deletions were described earlier in this disclosure (Examples 1 through 6). Below such deletion locus is listed the ORFs which were deleted 2s from that locus, along with the functions or homologies and molecular weight of their gene products.
Replication Studies of NYVAC and ALVAC on Human Tissue Cell Lines. In order to determine the level of replication of NYVAC strain of vaccinia virus (vP866) in 3o cells of human origin, six cell lines were inoculated at an input multiplicity of 0.1 pfu per cell under liquid culture and incubated for 72 hours. The COPENHAGEN
parental clone (VC-2) was inoculated in parallel.
Primary chick embryo fibroblast (CEF) cells (obtained 3s from 10-11 day old embryonated eggs of SPF origin, Spafas, Inc., Storrs, CT) were included to represent a permissive cell substrate for all viruses. Cultures were WO 94/16716 215 3 3 3 ~ ~T~S94100888 analyzed on the basis of two criteria: the occurrence of productive viral replication and expression of an extrinsic antigen. r-The replication potential of NYVAC in a number of human derived cells are shown in Table 16. Both VC-2 and NYVAC are capable of productive replication in CEF cells, although NYVAC with slightly reduced yields. VC-2 is also capable of productive replication in the six human derived cell lines tested with comparable yields except to in the EBV transformed lymphoblastoid cell line JT-1 (human lymphoblastoid cell line transformed with Epstein-Barr virus, see Rickinson et al., 1984). In contract, NYVAC is highly attenuated in its ability to productively replicate in any of the human derived cell lines tested.
Small increases of infectious virus above residual virus levels were obtained from NYVAC-infected MRC-5 (ATCC
#CCL171, human embryonic lung origin), DETROIT 532 (ATCC
#CCL54, human foreskin, Downs Syndrome), HEL 299 (ATCC
#CCL137, human embryonic lung cells) and HNK (human 2o neonatal kidney cells, Whittiker Bioproducts, Inc.
Walkersville, MD, Cat #70-151) cells. Replication on these cell lines was significantly reduced when compared to virus yields obtained from NYVAC-infected CEF cells or with parental VC-2 (Table 16). It should be noted that the yields at 24 hours in CEF cells for both NYVAC and VC-2 is equivalent to the 72-hour yield. Allowing the human cell line cultures to incubate an additional 48 hours (another two viral growth cycles) may, therefore, have amplified the relative virus yield obtained.
3o Consistent with the low levels of virus yields obtained in the human-derived cell lines, MRC-5 and DETROIT 532, detectable but reduced levels of NYVAC-specific DNA accumulation were noted. The level of DNA
accumulation in the MRC-5 and DETROIT 532 NYVAC-infected cell lines relative to that observed in NYVAC-infected CEF cells paralleled the relative virus yields. NYVAC-specific viral DNA accumulation was not observed in~any WO 94116716 " , PCTIUS94/00888 of the other human-derived cells.
An equivalent experiment was also performed using ~t the avipox virus, ALVAC. The results of virus replication are also shown in Table 16. No progeny virus s was detectable in any of the human cell lines consistent with the host range restriction of canarypox virus to avian species. Also consistent with a lack of productive replication of ALVAC in these human-derived cells is the observation that no ALVAC-specific DNA accumulation was 1o detectable in any of the human-derived cell lines.
Expression of Rabies Glycoprotein by NYVAC-RG
1vP879) in Human Cells. In order to determine whether efficient expression of a foreign gene could be obtained in the absence of significant levels of productive viral is replication, the same cell lines were inoculated with the NYVAC recombinant expressing the rabies virus glycoprotein (vP879, Example 7) in the presence of 35S-methionine. Immunoprecipitation of the rabies glycoprotein was performed from the radiolabelled culture 20 lysate using a monoclonal antibody specific for the rabies glycoprotein. Immunoprecipitation of a 67kDa protein was detected consistent with a fully glycosylated form of the rabies glycoprotein. No serologically crossreactive product was detected in uninfected or 2s parental NYVAC infected cell lysates. Equivalent results were obtained with all other human cells analyzed.
Inoculations on the Rabbit Skin. The induction and nature of skin lesions on rabbits following intradermal (id) inoculations has been previously used as a measure 30 of pathogenicity of vaccinia virus strains (Buller et al., 1988; Child et al., 1990; Fenner, 1958, Flexner et al., 1987; Ghendon and Chernos 1964). Therefore, the nature of lesions associated with id inoculations with the vaccinia strains WR (ATCC #VR119 plaque purified on 3s CV-1 cells, ATCC #CCL70, and a plaque isolate designated L variant, ATCC #VR2035 selected, as described in Panicali et al., 1981)), WYETH (ATCC #VR325 marketed as WO 94/16716 215 3 3 3 6 ~TNS94100888 DRYVAC by Wyeth Laboratories, Marietta, PA), COPENHAGEN
(VC-2), and NYVAC was evaluated by inoculation of two rabbits (A069 and A128). The two rabbits displayed different overall sensitivities to the viruses, with rabbit A128 displaying less severe reactions than rabbit A069. In rabbit A128, lesions were relatively small and resolved by 27 days post-inoculation. On rabbit A069, lesions were intense, especially for the WR inoculation sites, and resolved only after 49 days. Intensity of the to lesions was also dependent on the location of the inoculation sites relative to the lymph drainage network.
In particular, all sites located above the backspine displayed more intense lesions and required longer times to resolve the lesions located on the flanks. All lesions were measured daily from day 4 to the disappearance of the last lesion, and the means of maximum lesion size and days to resolution were calculated (Table 17). No local reactions were observed from sites injected with the control PBS. Ulcerative lesions were observed at sites injected with WR, VC-2 and WYETH vaccinia virus strains. Significantly, no induration or ulcerative lesions were observed at sites of inoculation with NYVAC.
Persistence of Infectious Virus at the Site of Inoculation. To assess the relative persistence of these viruses at the site of inoculation, a rabbit was inoculated intradermally at multiple sites with 0.1 ml PBS containing 106, 10~ or 108 pfu of VC-2, WR, WYETH or NYVAC. For each virus, the 10~ pfu dose was located above 3o the backspine, flanked by the 106 and 108 doses. Sites of inoculation were observed daily for 11 days. WR elicited the most intense response, followed by VC-2 and WYETH
(Table 18). Ulceration was first observed at day 9 for WR and WYETH and day 10 for VC-2. Sites inoculated with NYVAC or control PBS displayed no induration or ulceration. At day 11 after inoculation, skin samples from the sites of inoculation were excised, mechanically disrupted, and virus was titrated on CEF cells. The results are shown in Table 18. In no case was more virus recovered at this timepoint than was administered.
Recovery of vaccinia strain, WR, was approximately 106 s pfu of virus at each site irrespective of amount of virus administered. Recovery of vaccinia strains WYETH and VC-2 was 103 to 104 pfu regardless of amount administered.
No infectious virus was recovered from sites inoculated with NYVAC.
1o Inoculation of Genetically or Chemically Immune Deficient Mice. Intraperitoneal inoculation of high doses of NYVAC (5 X 108 pfu) or ALVAC (109 pfu) into nude mice caused no deaths, no lesions, and no apparent disease through the 100 day observation period. In 15 contrast, mice inoculated with WR (103 to 104 pfu), WYETH
(5 x log or 5 x 108 pfu) or VC-2 (104 to 109 pfu) displayed disseminated lesions typical of poxviruses first on the toes, then on the tail, followed by severe orchitis in some animals. In mice infected with WR or 2o WYETH, the appearance of disseminated lesions generally led to eventual death, whereas most mice infected with VC-2 eventually recovered. Calculated LDso values are given in Table 19.
In particular, mice inoculated with VC-2 began to 2s display lesions on their toes (red papules) and 1 to 2 days later on the tail. These lesions occurred between 11 and 13 days post-inoculation (pi) in mice given the highest doses (109, 108, 10~ and 106 pfu), on day 16 pi in mice given los pfu and on day 21 pi in mice given 104 pfu.
3o No lesions were observed in mice inoculated with 103 and 102 pfu during the 100 day observation period. Orchitis was noticed on day 23 pi in mice given 109 and 108 pfu, and approximately 7 days later in the other groups (10~ to io4 pfu). Orchitis was especially intense in the 109 and 3s 108 pfu groups and, although receding, was observed until the end of the 100 day observation period. Some pox-like lesions were noticed on the skin of a few mice, occurring WO 94/16716 215 3 3 3 5 P~~S94100888 around 30-35 days pi. Most pox lesions healed normally, between GO-90 days pi. Only one mouse died in the group inoculated with 109 pfu (Day 34 pi) and one mouse died in.
the group inoculated with 108 pfu (Day 94 pi). No other 5 deaths were observed in the VC-2 inoculated mice.
Mice inoculated with 104 pfu of the WR strain of vaccinia started to display pox lesions on Day 17 pi.
These lesions appeared identical to the lesions displayed by the VC-2 injected mice (swollen toes, tail). Mice to inoculated with 103 pfu of the WR strain did not develop lesions until 34 days pi. Orchitis was noticed only in the mice inoculated with the highest dose of WR (104 pfu). During the latter stages of the observation period, lesions appeared around the mouth and the mice 1s stopped eating. All mice inoculated with 104 pfu of WR
died or were euthanized when deemed necessary between 21 days and 31 days pi. Four out of the 5 mice injected with 103 pfu of WR died or were euthanized when deemed necessary between 35 days and 57 days pi. No deaths were 20 observed in mice inoculated with lower doses of WR (1 to 100 pfu).
Mice inoculated with the WYETH strain of vaccinia virus at higher doses 5 x 10~ and 5 x 108 pfu) showed lesions on toes and tails, developed orchitis, and died.
25 Mice injected with 5 x 106 pfu or less of WYETH showed no signs of disease or lesions.
As shown in Table 19, CY-treated mice provided a more sensitive model for assaying poxvirus virulence than did nude mice. LDSO values for the WR, WYETH, and VC-2 3o vaccinia virus strains were significantly lower in this model system than in the nude mouse model. Additionally, lesions developed in mice injected with WYETH, WR and VC-2 vaccinia viruses, as noted below, with higher doses of each virus resulting in more rapid formation of lesions.
35 As was seen with nude mice, CY-treated mice injected with NYVAC or ALVAC did not develop lesions. However, unlike nude mice, some deaths were observed in CY-treated mice challenged with NYVAC or ALVAC, regardless of the dose.
These random incidences are suspect as to the cause of death.
Mice injected with all doses of WYETH (9.5 x 104 to 9.5 x 108 pfu) displayed pox lesions on their tail and/or on their toes between 7 and 15 days pi. In addition, the tails and toes were swollen. Evolution of lesions on the tail was typical of pox lesions with formation of a papule, ulceration and finally formation of a scab. Mice 1o inoculated with all doses of VC-2 (1.65 x 105 to 1.65 x 109) also developed pox lesions on their tails and/or their toes analogous to those of WYETH injected mice.
These lesions were observed between 7-12 days post inoculation. No lesions were observed on mice injected is with lower doses of WR virus, although deaths occurred in these groups.
Potency Testinq of NYVAC-RG. In order to determine that attenuation of the COPENHAGEN strain of vaccinia virus had been effected without significantly altering 2o the ability of the resulting NYVAC strain to be a useful vector, comparative potency tests were performed. In order to monitor the immunogenic potential of the vector during the sequential genetic manipulations performed to attenuate the virus, a rabiesvirus glycoprotein was used 25 as a reporter extrinsic antigen. The protective efficacy of the vectors expressing the rabies glycoprotein gene was evaluated in the standard NIH mouse potency test for rabies (Seligmann, 1973). Table 20 demonstrates that the PDSO values obtained with the highly attenuated NYVAC
3o vector are identical to those obtained using a COPENHAGEN-based recombinant containing the rabies glycoprotein gene in the tk locus (Kieny et al., 1984) and similar to PDSO values obtained with ALVAC-RG, a canarypox based vector restricted to replication to avian 35 species.
Observations. NYVAC, deleted of known virulence genes and having restricted in vitro growth WO 94/16716 21 ~ 3 3 3 6 ~T~S94100888 characteristics, was analyzed in animal model systems to-~
assess its attenuation characteristics. These studies were performed in comparison with the neurovirulent vaccinia virus laboratory strain, WR, two vaccinia virus vaccine strains, WYETH (New York City Board of Health) and COPENHAGEN (VC-2), as well as with a canarypox virus strain, ALVAC (See also Example 11). Together, these viruses provided a spectrum of relative pathogenic potentials in the mouse challenge model and the rabbit 1o skin model, with WR being the most virulent strain, WYETH
and COPENHAGEN (VC-2) providing previously utilized attenuated vaccine strains with documented characteristics, and ALVAC providing an example of a poxvirus whose replication is restricted to avian species. Results from these in vivo analyses clearly demonstrate the highly attenuated properties of NYVAC
relative to the vaccinia virus strains, WR, WYETH and COPENHAGEN (VC-2) (Tables 14-20). Significantly, the LD5o values for NYVAC were comparable to those observed with 2o the avian host restricted avipoxvirus, ALVAC. Deaths due to NYVAC, as well as ALVAC, were observed only when extremely high doses of virus were administered via the intracranial route (Example 11, Tables 14, 15, 19). It has not yet been established whether these deaths were due to nonspecific consequences of inoculation of a high protein mass. Results from analyses in immunocompromised mouse models (nude and CY-treated) also demonstrate the relatively high attenuation characteristics of NYVAC, as compared to WR, WYETH and COPENHAGEN strains (Tables 17 3o and 18). Significantly, no evidence of disseminated - vaccinia infection or vaccinial disease was observed in NYVAC-inoculated animals or ALVAC-inoculated animals over the observation period. The deletion of multiple virulence-associated genes in NYVAC shows a synergistic 3s effect with respect to pathogenicity. Another measure of the innocuity of NYVAC was provided by the intradermal administration on rabbit skin (Tables 17 and 18).

,~~ Considering the results with ALVAC, a virus unable to replicate in nonavian species, the ability to replicate at the site of inoculation is not the sole correlate with reactivity, since intradermal inoculation of ALVAC caused s areas of induration in a dose dependent manner.
Therefore, it is likely that factors other than the replicative capacity of the virus contribute to the formation of the lesions. Deletion of genes in NYVAC
prevents lesion occurrence.
1o Together, the results in this Example and in foregoing Examples, including Example 11, demonstrate the highly attenuated nature of NYVAC relative to WR, and the previously utilized vaccinia virus vaccine strains, WYETH
and COPENHAGEN. In fact, the pathogenic profile of 15 NYVAC, in the animal model systems tested, was similar to that of ALVAC, a poxvirus known to productively replicate only in avian species. The apparently restricted capacity of NYVAC to productively replicate on cells derived from humans (Table 16) and other species, 2o including the mouse, swine, dog and horse, provides a considerable barrier that limits or prevents potential transmission to unvaccinated contacts or to the general environment in addition to providing a vector with reduced probability of dissemination within the 25 vaccinated individual.
Significantly, NYVAC-based vaccine candidates have been shown to be efficacious. NYVAC recombinants expressing foreign gene products from a number of pathogens have elicited immunological responses towards 3o the foreign gene products in several animal species, including primates. In particular, a NYVAC-based recombinant expressing the rabies glycoprotein was able to protect mice against a lethal rabies challenge. The potency of the NYVAC-based rabies glycoprotein 35 recombinant was comparable to the PDSO value for a COPENHAGEN-based recombinant containing the rabies glycoprotein in the tk locus (Table 20). NYVAC-based WO 94!16716 21 ~ 3 3 3 fi PCT~S94/00888 recombinants have also been shown to elicit measles virus r neutralizing antibodies in rabbits and protection against pseudorabies virus and Japanese encephalitis virus challenge in swine. The highly attenuated NYVAC strain s confers safety advantages with human and veterinary applications (Tartaglia et al., 1990). Furthermore, the use of NYVAC as a general laboratory expression vector system may greatly reduce the biological hazards associated with using vaccinia virus.
1o By the following criteria, the results of this Example and the Examples herein, including Example 11, show NYVAC to be highly attenuated: a) no detectable induration or ulceration at site of inoculation (rabbit skin); b) rapid clearance of infectious virus from 15 intradermal site of inoculation (rabbit skin); c) absence of testicular inflammation (nude mice); d) greatly reduced virulence (intracranial challenge, both three-week old and newborn mice); e) greatly reduced pathogenicity and failure to disseminate in 2o immunodeficient subjects (nude and cyclophosphamide treated mice); and f) dramatically reduced ability to replicate on a variety of human tissue culture cells.
Yet, in spite of being highly attenuated, NYVAC, as a vector, retains the ability to induce strong immune 25 responses to extrinsic antigens.

WO 94/16716 : . PCTIUS94100888 loo TABLE Replication 16 of COPENHAGEN
(VC-2), NYVAC and ALVAC in avian or human ines derived cell l Cells Hours post- Yields % Yield infection s CEF 0 3.8 3.7 4.5 24 8.3 7.8 6.6 48 8.6 7.9 7.7 72 8.3 7.7 7.5 25 72A' < 1.8 3.1 1.4 io MRC-5 0 3.8 3.8 4.7 72 7.2 4.6 3.8 0.25 72A 2.2 2.2 3.7 WISH 0 3.4 3.4 4.3 72 7.6 2.2 3.1 0.0004 1 72A -d 1.9 2.9 s DETROIT 0 3.8 3.7 4.4 72 7.2 5.4 3.4 1.6 72A 1.7 1.7 2.9 HEL 0 3.8 3.5 4.3 2 72 7.5 4.6 3.3 0.125 72A 2.5 2.1 3.6 JT-1 0 3.1 3.1 4.1 72 6.5 3.1. 4.2 0.039 72A 2.4 2.1 4.4 2s HNK 0 3.8 3.7 4.7 72 7.6 4.5 3.6 0.079 72A 3.1 2.7 3.7 a: Yield of NYVAC at 72 hours post-infection expressed as a percentage of 3o yield of VAC-2 after 72 hours on the same cell line.
b: Titer expressed as LOGso pfu per ml.
c: Sample was incubated in the presence of 40~g/ml of cytosine arabinoside.
d: Not determined.
*: ATCC #CCL25 Human amnionic cells.

WO 94/16716 215 3 3 3 ~ PCT/US94/00888 Table 17. Induration and ulceration at the site of intradermal inoculation of the rabbit skin INDURA TION ULCER ATION

VIRUS DOSEa STRAIN Sizeb Days' Size Days 10' 877 35 204 35 WYETH 10 32 5 --d --105 116 15 __ __ 10~' 267 17 3 15 10~ 202 17 3 24 10s 240 29 12 31 105 86 8 __ __ 10~ 136 17 -- --10~ 167 21 6 10 __ __ __ __ 10s __ __ __ __ 2 5 10~ __ __ __ __ 1OA __ __ __ __ a pfu of indicated vaccinia virus in 0.1 ml PBS inoculated intradermally into one site.
h mean maximum size of lesions (mm2~
' mean time after inoculation for complete healing of lesion.
" no lesions discernable.

~"a io2 Table 18. Persistence of poxviruses at the site of intradermal inoculation Virus Inoculum Dose Total Virus Recovered s W R 8.08 6.14 7.0 6.26 6.0 6.21 WYETH 8.0 3.66 7.0 4.10 i 6.0 3.59 o VC-2 8.0 4.47 7.0 4.74 6.0 3.97 NYVAC 8.0 0 15 7.0 0 6.0 0 a: expressed as logo pfu.

215~33f WO 94/16716 PCTlUS94/00888 Table 19. Virulence studies in immunocompromised mice POxviruS LDsoa Strain Nude mice Cyclophosphamide treated mice s W R 422 42 VC-2 > 109 < 1.65 x 10'' WYETH 1.58 x 10' 1.83 x 106 NYVAC > 5.50 x 108 7.23 x 108 ALVAC > 109 25.00 x 108 to a: Calculated 50% lethal dose (pfu) for nude or cyclophosphamide treated mice by the indicated vaccinia viruses and for ALVAC by intraperitoneal route.
is b: 5 out of 10 mice died at the highest dose of 5 x 108 pfu.
2o Table 20. Comparative efficacy of NYVAC-RG and ALVAC-RG in mice Recombinant PDsoa W-RG 3.74 ALVAC-RG 3.86 2 NYVAC-RG 3.70 a: Four to six week old mice were inoculated in the footpad with 50-1001 of a range of dilutions (2.0 - 8.0 logo tissue culture infection dose 50% (TCIDso) of either the W-RG (Kieny et al., 1984), ALVAC-RG (vCP65) or NYVAC-RG
30 (vP879). At day 14, mice of each group were challenged by intracranial inoculation of 301 of a live CVS strain rabies virus corresponding to 15 lethal dose 50% (LDSO) per mouse. At day 28, surviving mice were counted and a protective dose 50% (PDso) was calculated.

io4 Example 13 - CONSTRUCTION OF TROVAC RECOMBINANTS
C1' EXPRESSING THE HEMAGGLUTININ GLYCOPROTEINS
OF AVIAN INFLUENZA VIRUSES
s 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 1o from Dr. Robert Webster, St. Jude Children's Research Hospital, Memphis, Tennessee. The strain of FPV
designated FP-1 has been described previously (Taylor et al., 1988a, b). It is a vaccine strain useful in vaccination of day old chickens. The parental virus 15 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 2o 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, 2s 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 3o 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 3s sequence was determined on both strands for a 3659 by PvuII-EcoRV fragment. This sequence is shown in FIG. 11 (SEQ ID N0:77). The limits of an open reading frame designated in this laboratory as F8 were determined within this sequence. The open reading frame is _ WO 94/16716 ~ ~ ~ ,~ PCT/US94100888 initiated at position 495 and terminates at position w 1887. A deletion was made from position 779 to position 1926, as described below. '~..r Plasmid pRW761 is a sub-clone of pRW731.15 containing a 2430 by EcoRV-EcoRV fragment. Plasmid pRW761 was completely digested with XbaI and partially digested with SSnI. A 3700 by XbaI-SSpI band was isolated and ligated with the annealed double-stranded oligonucleotides JCA017 (SEQ ID N0:37) and JCA018 (SEQ ID
l0 N0:38) .
JCA017 (SEO ID N0:37) 5' CTAGACACTTTATGTTTTTTAATATCCGGTCTT
AAAAGCTTCCCGGGGATCCTTATACGGGGAATAAT 3' JCA018 (SEO ID N0:38) 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
N0:48) and RW179 (SEQ ID N0:49) were cut with EcoRV and BamHI and inserted between the EcoRV and BamHI sites of JCA004 to form pRW846.
RW178 (SEO ID N0:48): 5' TCATTATCGCGATATCCGTGTTAACTAGCTA
GCTAATTTTTATTCCCGGGATCCTTATCA 3' RW179 (SEC? ID N0:49): 5' GTATAAGGATCCCGGGAATAAAAATTAGCT
3o AGCTAGTTAACACGGATATCGCGATAATGA 3' Plasmid pRW846 therefore contains the H6 promoter 5' of EcoRV in the de-ORFed F8 locus. The HincII 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.

WO 94116716 ~ PCT/US94I00888 1o6 Construction of Fowlt~ox 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. 12 (SEQ ID N0:78) was determined for a 2356 by region encompassing the unique HincII site. Analysis of this sequence revealed that the unique HincII site (FIG. 12, underlined) was situated 1o 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 . 12 ) .
The arms for the de-ORFed insertion plasmid were derived by PCR using pRW731.13 as template. A 596 by arm (designated as HB) corresponding to the region upstream from the ORF was amplified with oligonucleotides F73PH2 (SEQ ID N0:50) (5'-GACAATCTAAGTCCTATATTAGAC-3') and F73PB (SEQ ID N0:51) (5'-GGATTTTTAGGTAGACAC-3'). A
270 by arm (designated as EH) corresponding to the region downstream from the ORF was amplified using oligonucleotides F75PE (SEQ ID N0:52) (5'-TCATCGTCTTCATCATCG-3') and F73PH1 (SEQ ID N0:53) (5'-GTCTTAAACTTATTGTAAGGGTATACCTG-3').
Fragment EH was digested with EcoRV to generate a 126 by 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 3o plasmid pF7Dl. 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 by 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 BglII

WO 94/16716 21 ~ 3 ~ 3 ~ ~T~S94/00888 to generate a 600 by fragment. This fragment was inserted into pBS-SK that was digested with ApaI, 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 by 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 to plasmid was ligated with annealed oligonucleotides F7MCSB
(SEQ ID N0:54) (5'-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTTTTTATTAGTTTAGTT
AGTC-3') and F7MCSA (SEQ ID N0:55) (5'-GACTAACTAACTAATAAAAAA
CCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAATCGTT-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 pF7D0.
2o 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 3o plasmid pRW828 was partially cut with BanII, the linear product isolated and recut with HindIII. Plasmid pRW828 now with a 100 by HindIII-BanII deletion was used as a vector for the synthetic oligonucleotides RW152 (SEQ ID
N0:56) and RW153 (SEQ ID N0:57). 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.

WO 94/16716 ~ . . PCTIUS94/00888 RW152 (SEO ID N0:56): 5' GCACGGAACAAAGCTTATCGCGATATCCGTTA
AGTTTGTATCGTAATGCTATCAATCACGATTCTGTTCC
TGCTCATAGCAGAGGGCTCATCTCAGAAT3' RW153 (SEQ ID N0:57): 5' ATTCTGAGATGAGCCCTCTGCTATGAGCAGGA
ACAGAATCGTGATTGATAGCATTACGATACAAACTTA
ACGGATATCGCGATAAGCTTTGTTCCGTGC 3' The oligonucleotides were annealed, cut with BanII and HindIII and inserted into the HindIII-BanII deleted pRW828 vector described above. The resulting plasmid to 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 Hemactglutinin at the F8 Locus. A cDNA clone of avian influenza H5 derived from A/Turkey/Ireland/1378/83 was 2o received in plasmid pTH29 from Dr. R. Webster. Synthetic oligonucleotides RW10 (SEQ ID N0:58) through RW13 (SEQ ID
NO: G1) were designed to overlap the translation initiation codon of the previously described vaccinia virus H6 promoter with the ATG of the H5 gene. The 2s sequence continues through the 5' SalI site of the H5 gene and begins again at the 3' H5 DraI site containing the H5 stop codon.
RW10 (SEQ ID N0:58): 5' GAAAAATTTAAAGTCGACCTGTTTTGTTGAGT
TGTTTGCGTGGTAACCAATGCAAATCTGGTC
3o ACT 3' RW11 (SEO ID N0:59): 5' TCTAGCAAGACTGACTATTGCAAAAAGAAGCA
CTATTTCCTCCATTACGATACAAACTTAACG
GAT 3' RW12 (SEQ ID N0:60): 5' ATCCGTTAAGTTTGTATCGTAATGGAGGAAA

GACCAGATTTGCATTGGT 3' to RW13 (SEO ID N0:61): 5' TACCACGCAAACAACTCAACAAAACAGGTCG ~.
ACTTTAAATTTTTCTGCA 3' The oligonucleotides were annealed at 95°C for three~v minutes followed by slow cooling at room temperature.
This results in the following double strand structure with the indicated ends.
EcoRV _Pstl J RW12 ~ RW13 I
RW11 ( RW10 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 2o 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
2s 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
3o fragment from pTH29. Plasmid pRW744 was partially digested with DraI, the linear fragment isolated, recut with SalI and the plasmid now with eight bases deleted between SalI and DraI was used as a vector for the 1.6 kpb pTH29 SalI and DraI fragment. The resulting plasmid 3s pRW759 was cut with EcoRV and DraI. The 1.7 kbp PRW759 EcoRV-DraI fragment containing the 3' H6 promoter and the H5 gene was inserted between the EcoRV and HincII sites of pRW846 (previously described). The resulting plasmid pRW849 contains the HG promoted avian influenza virus H5 WO 94116716 ; ~ PCTIUS94I00888 ilo C~ gene in the de-ORFed F8 locus.
Construction of Insertion Vector for H7 Hema9glutinin at the F7 Locus. Plasmid pCVH71 containing the H7 hemagglutinin from A/CK/VIC/1/85 was received from s Dr. R. Webster. An EcoRI-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 N0:62) and RW166 (SEQ ID N0:63) were annealed, cut with HincII
io and stvI and inserted between the EcoRV and StvI sites of pRW827 to generate pRW845.
RW165 (SEQ ID N0:62): 5' GTACAGGTCGACAAGCTTCCCGGGTATCGCG
ATATCCGTTAAGTTTGTATCGTAATGAATACTCAAATT
CTAATACTCACTCTTGTGGCAGCCATTCACACAAATG
i 5 CAGACAAAATCTGCCTTGGACATCAT 3' RW166 (SEO ID N0:63): 5' ATGATGTCCAAGGCAGATTTTGTCTGCATTTG
TGTGAATGGCTGCCACAAGAGTGAGTATTAGAATTTG
AGTATTCATTACGATACAAACTTAACGGATATCGCGA
TACCCGGGAAGCTTGTCGACCTGTAC3' 2o Oligonucleotides RW165 (SEQ ID N0:62) and RW166 (SEQ
ID N0:63) 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 A~~aLI
digestion of pRW845, recutting it with EcoRI, isolating 25 the largest fragment and annealing with synthetic oligonucleotides RW227 (SEQ ID N0:64) and RW228 (SEQ ID
N0:65). The resulting plasmid was pRW854.
RW227 (SEO ID N0:64): 5' ATAACATGCGGTGCACCATTTGTATAT
AAGTTAACGAATTCCAAGTCAAGC 3' 3o RW228 (SEO ID N0:65): 5' GCTTGACTTGGAATTCGTTAACTTATA
TACAAATGGTGCACCGCATGTTAT 3' The stop codon of H7 in PRW854 is followed by an HpaI
site. The intermediate H6 promoted H7 construct in the de-ORFed F7 locus (described below) was generated by 35 moving the pRW854 EcoRV-H~aI fragment into pRW858 which had been cut with EcoRV and blunt-ended at its PstI site.

WO 94/16716 215 3 3 3 6 ~~594100888 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 by SmaI/HpaI fragment, containing the H6 promoter linked to a non-pertinent gene, into the SmaI site of pF7D0 described previously. The non-pertinent sequences were excised by digestion of pRW858 with EcoRV (site 24 by upstream of the 3'-end of the H6 promoter) and PstI.
The 3.5 kb resultant fragment was isolated and blunt-to 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 by EcoRV/HpaI
fragment derived from pRW854 (described previously).
This EcoRV/H~aI fragment contains the entire AIV HA (H7) gene juxtaposed 3' to the 3'-most 24 by of the W H6 promoter. The resultant plasmid was designated pRW861.
The 12G by EH arm (defined previously) was lengthened in pRW861 to increase the recombination frequency with genomic TROVAC DNA. To accomplish this, a 575 by 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 by and a HB arm of 404 by flanking the AIV H7 gene, was designated 2s 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 by of TROVAC sequence and the right flanking arm of 725 by of TROVAC sequence which directs insertion to the de-3o 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 35 using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis ' , PCTIUS94100888 ~

~'3 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.

s Plasmid pRW849 was used in an in vitro recombination test to produce recombinant TROVAC-AIHS (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 1o 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.

i5 During passage to the plasma membrane it undergoes extensive post-translational modification culminating in proteolytic cleavage into the disulphide linked HA1 and HA2 subunits and insertion into the host cell membrane where it is subsequently incorporated into mature viral 2o 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 2s al., 1990). Surface expression of the H5 hemagglutinin in TROVAC-AIHS, 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 3o specific for the HSHA. 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 35 the sequence at and around the cleavage site of the hemagglutinin molecule plays an important role in determining viral virulence since cleavage of the WO 94/16716 215 3 3 3 ~ PCT~S94/00888 11~
hemagglutinin polypeptide is necessary for virus particles to be infectious. The hemagglutinin proteins of the virulent H5 and H7 viruses possess more than one basic amino acid at the carboxy terminus of HA1. It is s 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 1o 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 1s the specific reagents described above.
Immunoprecipitation analysis of the H5 hemagglutinin expressed by TROVAC-AIHS (vFP89) showed that the glycoprotein is evident as the two cleavage products HA1 and HA2 with approximate molecular weights of 44 and 23 2o 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 2s 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 3o 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. Generation of recombinant virus by 35 recombination, in situ hybridization of nitrocellulose filters and screening for B-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et ii4 al., 1989).
Example 14 - GENERATION OF NYVAC- AND ALVAC-BASED
RECOMBINANT CONTAINING THE GENE ENCODING
IiUMAN TUMOR NECROSIS FACTOR - a (TNF-a ) TNF-a is a cytokine produced by CTLs. It is one of the products of these cells that is responsible for killing tumor cells during an immune response. It has previously been shown that the injection of recombinant 1o TNF could mediate the necrosis and regression of a variety of established murine cancers (Asher et al., 1987). The exact mechanisms for this anti-tumor activity remain unclear, although TNF apparently affects the vascular supply of tumors (Asher et al., 1987). Both the secreted and membrane-bound forms of TNF-a may be critical for its anti-tumor activities (Kriegler et al., 1987 ) .
Plasmid pE4, containing the human necrosis factor -a gene was extracted from E. coli transformed with this 2o plasmid. The pE4 transformed E. coli were obtained from ATCC (ATCC #CLN-39894). PCR fragment PCR-TNF4 (755 bp) was generated using pE4 as template and oligonucleotides TNF3 (SEQ ID N0:66); 5'-ATCATCTCGCGATATCCGTTAAGTTTGTATCGTAATGAGCACTGAAAGCATGATC-3') containing the 3'-most region of the vaccinia H6 promoter (from the NruI site to the end; Perkus et al., 1989) and the first 21 by of the TNF-a coding sequence, and oligonucleotide TNF2 (SEQ ID N0:67) (5'-ATCATCTCTAGAATAAAAATCACAGGGCAATGATCCC-3'), containing the last 15 by of the TNF-a coding sequence, a vaccinia early transcription termination signal (TSNT; Yuen and Moss, 198G), and an XbaI restriction site. A complete NruI/XbaI digestion was performed and the resultant 735 by fragment was isolated and inserted into the 4.8 kb NruI/XbaI fragment obtained by the digestion of the generic insertion plasmid pVQH6C5LSP. The resultant plasmid was designated pMAW048. The nucleotide sequence of the entire H6-TNF-a expression cassette was confirmed as described by Goebel et al. (1990).

WO 94/16716 ~ ~ PCT/US94100888 lls Plasmid pVQH6C5LSP was derived in the following manner:
A C5 insertion vector containing 1535 by upstream of C5, polylinker containing KpnI, SmaI, XbaI, and NotI
s sites, and 404 by of canarypox DNA (31 by of C5 coding sequence and 373 by of downstream sequence) was derived in the following manner. A genomic library of canarypox DNA was constructed in the cosmid vector pVK102 (Knauf et al., 1982) probed with pRW764.5 and a clone containing a io 29 kb insert identified (pHCOSl). A 3.3 kb ClaI fragment from pHCOSl containing the C5 region was identified.
Sequence analysis of the ClaI fragment was used to extend the sequence in FIG. 8 (SEQ ID N0:68) from nucleotides 1-1372.
15 The C5 insertion vector was constructed as follows.
The 1535 by upstream sequence was generated by PCR
amplification using oligonucleotides C5A (SEQ ID N0:69) (5'-ATCATCGAATTCTGAATGTTAAATGTTATACTTG-3') and C5B (SEQ
ID N0:75) (5'-GGGGGTACCTTTGAGAGTACCACTTCAG-3') and 2o purified genomic canarypox DNA as template. This fragment was digested with EcoRI (within oligo C5A) and cloned into EcoRI/SmaI digested pUCB generating pCSLAB.
The 404 by arm was generated by PCR amplification using oligonucleotides C5C (SEQ ID N0:71) 25 (5'-GGGTCTAGAGCGGCCGCTTATAAAGATCTAAAATGCATAATTTC-3') and C5DA (SEQ ID N0:72) (5'-ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3'). This fragment was digested with PstI (within oligo CSDA) and cloned into SmaI/PstI digested pCSLAB generating pCSL.
so pCSL was digested within the polylinker with Asp718 and NotI, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP26 (SEQ ID
N0:73) (5'-GTACGTGACTAATTAGCTATAAAAAGGATCCGGTACCCTCGAGTCTAGAATCG
3s ATCCCGGGTTTTTATGACTAGTTAATCAC-3') and CP27 (SEQ ID N0:74) (5'-GGCCGTGATTAACTAGTCATAAAAACCCGGGATCGATTCTAGACTCGAGGGTA
CCGGATCCTTTTTATAGCTAATTAGTCAC-3') (containing a disabled Asp718 site, translation stop codons in six reading frames, vaccinia early transcription termination signal (Yuen and Moss, 1987), BamHI, KpnI, XhoI, XbaI, ClaI, and SmaI restriction sites, vaccinia early transcription s termination signal, translation stop codons in six reading frames, and a disabled NotI site) generating plasmid pCSLSP.

The early/late H6 vaccinia virus promoter (Perkus et al., 1989) was derived by PCR from a plasmid containing to the promoter using oligonucleotides CP30 (SEQ ID N0:75) (5'-TCGGGATCCGGGTTAATTAATTAGTCATCAGGCAGGGCG-3') and CP31 (SEQ ID N0:76) (5'-TAGCTCGAGGGTACCTACGATACAAACTTAACGGATATCG-3'). The PCR product was digested with BamHI and XhoI (sites is created at the 5' and 3' termini by the PCR) and ligated to similarly digested pCSLSP generating pVQH6C5LSP.

Plasmid pMAW048 was used in in vitro recombination assays with ALVAC (CPpp) as rescue virus to yield recombinant virus vCP245. Insertion with this plasmid 2o replaces the two copies of the C5 open reading frame with the human TNF-a expression cassette. Fig. 15 presents the nucleotide sequence of the H6/TNF-a sequence and flanking regions within vCP245 (SEQ ID N0:79). The H6 promoter starts at position 74. The TNF-a start codon is 25 at position 201, and the TNF-a stop codon is at position 902. Positions 1 through 73 and positions 903 through 965 flank the H6/TNF-a expression cassette.

PCR fragment PCR-TNFH6 (156 bp) was amplified from plasmid pBSH6 using oligonucleotides H65PH (SEQ ID N0:80) 30 (5'-ATCATCAAGCTTGATTCTTTATTCTATAC-3') containing a HindIII site in the initial 21 by of the H6 promoter region and TNFH6 (SEQ ID N0:81) (5'-CATGCTTTCAGTGCTCATTACGATACAAACTTAACGG-3') containing the 3'-most 19 nucleotides of the H6 promoter and the 5'-most 35 18 nucleotides of the TNF coding sequence.

Plasmid pBSH6 was generated in the following manner.

The vaccinia H6 promoter through the EcoRV site was WO 94/16716 215, 3 3 s PCTIUS94/00888 derived from a plasmid containing the synthetic H6 promoter (Perkus et al., 1989) using PCR and primers H6PCR2 (SEQ ID N0:82) (5'-TTAACGGATATCGCGATAATG-3') and H6PCR1 (SEQ ID N0:83) (5'-ACTACTAAGCTTCTTTATTCTATACTTAAAAAGTG-3') creating a 5' HindIII site. This 122 by PCR-derived fragment was digested with HindIII and EcoRV followed by ligation to similarly digested pBS-SK+ (Stratagene, La Jolla, CA) generating plasmid pBSH6. The insert was confirmed by io nucleotide sequence analysis.
PCR fragment PCR-TNF (721 bp) was amplified from plasmid pE4 using oligonucleotides TNF1 (SEQ ID N0:84) (5'-ATGAGCACTGAAAGCATG-3') containing the initial 18 nucleotides of the TNF-a coding sequence and TNF2 (SEQ ID
NO:G7). The PCR fragment, PCR-TNF fusion (859 bp), was generated using PCR-TNFH6 and PCR-TNF as templates and oligonucleotides H65PH (SEQ ID N0:80) and TNF2 (SEQ ID
N0:67) as primers. PCR-TNF fusion was digested with HindIII and XbaI and the resultant 841 by fragment was 2o inserted into pBS-SK+ (Stratagene, La Jolla, CA) digested with HindIII and XbaI. The resultant plasmid was designated pMAW047 and the H6-TNF cassette was confirmed by nucleotide sequence analysis as described previously (Goebel et al., 1990).
The 841 by HindIII/XbaI fragment containing the H6-TNF-a expression cassette was isolated from pMAW047, blunt-ended using the Klenow fragment of the E. coli DNA
polymerase in the presence of 2mM dNTPs, and inserted into the vaccinia insertion plasmid pSD541. The 3o resultant plasmid was designated pMAW049.
Plasmid pSD541 was derived in the following manner.
Flanking arms for the ATI region were generated by PCR
using subclones of the Copenhagen HindIII A region 'as WO 94/16716 ~ PCTIUS94100888 ms template. Oligonucleotides MPSYN267 (SEQ ID N0:85) (5'-GGGCTCAAGCTTGCGGCCGCTCATTAGACAAGCGAATGAGGGAC:-3') and MPSYN268 (SEQ ID N0:86) (5'-AGATCTCCCGGGCTCGAGTAATTAATTAATTTTTATTACACCAGAAAAGACGGCTTG
AGATC-3') were used to derive the 420 by vaccinia arm to the right of the ATI deletion. Synthetic oligonucleotides MPSYN269 (SEQ ID N0:87) (5'-TAATTACTCGAGCCCGGGAGATCTAATTTAATTTAATTTATATAACTCATTTTTTGA
ATATACT-3') and MPSYN270 (SEQ ID N0:88) (5'-1o TATCTCGAATTCCCGCGGCTTTAAATGGACGGAACTCTTTTCCCC-3') were used to derive the 420 by 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 HindIII and EcoRI to yield sticky ends, and ligated into pUC8 digested with HindIII and EcoRI to generate pSD541.
The plasmid pMAW047 was used in in vitro recombination assays (Piccini et al., 1987) with NYVAC
(vP866; Tartaglia et al., 1992) as the rescue virus.
Recombination with this plasmid replaces the ATI open reading frame with the H6-TNF-a expression cassette. The NYVAC recombinant virus containing the H6-TNF-a cassette was designated vP1200. Fig. 16 presents the nucleotide sequence of the H6/TNF-a expression cassette incorporated into the NYVAC recombinant, vP1200, and flanking NYVAC
sequences (SEQ ID N0:89). The H6 promoter starts at position 59. The TNF-a start codon is at position 185, and the TNF-a stop codon is at position 884. Positions 1 3o through 58 and positions 885 through 947 flank the H6/TNF-a expression cassette.

Table 21. Expression of Human TNF-a by vP1200 and vCP245 Sample Description TNF-a (ng/ml) vP1196 NYVAC-CMVgB+pp65 0 vP1200 NYVAC-TNF-a >240 CPpp ALVAC 0 vCP245 ALVAC-TNF-a 59 to Expression of TNF-a by vP1200 (NYVAC-TNF-a) and vCP245 (ALVAC-TNF-cz) was measured by ELISA assay, using a commercially available kit (Genzyme Diagnostics, Cambridge, MA, cat.#1915-O1). Samples were prepared by infection of Vero cells (NYVAC recombinants) or primary is chick embryo fibroblasts (ALVAC) with recombinant or parent virus. The cells were harvested when CPE was complete and the infected cell lysates were used for the ELISA assay, after sonication and clarification by centrifugation at 500 xg for l0 min. one control, 2o vP1196, which expresses two cytomegalovirus proteins, gB
and pp65, was prepared in the same manner as the TNF-a recombinants. The other control, ALVAC parent, was a partially purified virus stock. All samples contained approximately 10~ PFU/ml of virus. The results, shown in 2s Table 21, indicate that both vP1200 and vCP245 are expressing human TNF-a. Expression of such levels in vivo can be therapeutic.
Example 15 - NYVAC AND ALVAC-BASED D53 RECOMBINANT
VIRUSES
The nuclear phosphoprotein, p53, is found in normal cells at very low steady state levels. Expression of p53 is tightly regulated throughout the cell cycle and may be involved in controlling cell proliferation. The 3s molecular mechanisms by which p53 exerts its tumor suppressor activity remain unknown, although p53 appears to exist in two conformational states. One form is unique to wildtype p53 and is associated with the ability to block cell cycle progression while the second form is associated with the ability to promote cell proliferation and is common to wildtype and mutant forms (reviewed by Ulrich et al., 1992). p53 is the gene most frequently found to be mutated in a wide variety of human tumors (reviewed by Hollstein et al., 1991).
Probably the most studied cancer associated with p53 mutation is breast cancer. It is known that p53 mutation results in the overexpression of the p53 gene product in to primary breast cancer patients (Davidoff et al., 1991).
The basis for p53 overexpression was found to result from a post-transcriptional mechanism, since p53-specific mRNA
levels were similar in tumors with high and low level protein expression. Further, the p53 mRNA from overexpressing tumors were found to contain missense mutations in highly conserved regions of the gene. These mutations were subsequently found to give rise to more stable p53 protein forms which form complexes with heat shock protein 70 (HSP-70). Since HSP-70 proteins have 2o been implicated in antigen processing, not only may the humoral response to p53 observed in a subset of breast cancer patients have resulted from unique processing/presentation modes for complexes, such an association may also elicit cellular anti-p53 protein responses (Davidoff et al., 1992). Such anti-p53 cellular immune responses may be more germane to the eventual immunotherapy of such cancers.
Generation of Poxvirus-based Recombinant Viruses Expressing Wildtype and Mutant Forms of the Human p53 Gene Product Three plasmids, p53wtXbaISP6/T3, p53-217XbaI, and p53-238XbaI containing wildtype human p53 gene sequences, and two mutant forms of p53, respectively, were obtained from Dr. Jeffrey Marks (Duke University). The p53-217XbaI contains a p53 gene encoding a p53 product lacking codon 217 while p53-238XbaI encodes a p53 gene product with an cysteine to arginine substitution at WO 94/16716 ~ ~ ~ PCT/US94100888 amino acid 238. The sequence of the wildtype p53 cDNA
and the deduced amino acid sequence was described previously (Lamb and Crawford, 1986; FIG. 3;.
All three p53 genes were individually juxtaposed 3' r~
to the modified vaccinia virus H6 promoter described by Perkus et al., 1989. These manipulations wEare performed in the following manner. A 227 by PCR-derived fragment was generated using oligonucleotides MM002 (SEQ ID N0:90) (5'-GATCTGACTGCGGCTCCTCCATTACGATACAAACTTAACGG-3') and l0 RW425 (SEQ ID N0:91) (5'-GTGGGTAAGGGAATTCGGATCCCCGGGTTAATTAATTAGTGATAC-3') and plasmid pRW825 as template. PCR using these oligonucleotides amplifies the vaccinia H6 promoter sequences from pRW825 such that the 3' end of the is promoter is precisely linked to the 5'-most region of the p53 coding sequence. Plasmid pRW825 contains the vaccinia virus H6 promoter (Perkus et al., 1989) linked to a nonpertinent gene.
PCR was also used to generate a 480 by and 250 by 2o fragment from p53wtXbaISP6/T3. The 480 by fragment was derived with oligonucleotides MM003 (SEQ ID N0:92) (5'-GTTTGTATCGTAATGGAGGAGCCGCAGTCAGATC-3') and MM008 (SEQ ID
N0:93) (5'-CATTACGATACAAACTTAACGGATATCGCGACGCGTTCACACAGGGCAGGTCTTGGC
25 -3'). This fragment contains the 3' portion of the vaccinia virus H6 promoter sequences and the 5' portion of the p53 coding sequences through the SctrAI site. The 250 by fragment was derived by amplification with oligonucleotides MM005 (SEQ ID N0:94) (5'-3o TACTACCTCGAGCCCGGGATAAAAAACGCGTTCAGTCTGAGTCAGGCCC-3') and MM007 (SEQ ID N0:95) (5'-GTGTGAACGCGTCGCGATATCCGTTAAGTTTGTATCGTAATGCAGCTGCGTGGGCGT
GAGCGCTTC-3'). This PCR fragment contains the 3' end of the p53 coding sequences beginning at the StuI
35 restriction site. The 480 by and 250 by PCR fragments were generated such that the 5' end of the MM005/MM007-derived (SEQ ID N0:94/95) fragment overlaps the 3'~end of WO 94/16716 , ~~, , ' PCTIUS94I00888 the MM003/MM008-derived (SEQ ID N0:92/93) fragment.
The 227 bp, 480 bp, and 250 by PCR-derived fragments were pooled and fused by PCR using oligonucleotides MM006 (SEQ ID N0:96) (5'-ATCATCGGATCCCCCGGGTTCTTTATTCTATAC-3') and MM005 (SEQ ID N0:94). The 783 by fused PCR product contains the H6 promoter juxtaposed 5' to the 5' portion of the p53 coding sequence (through the SarAI restriction site) followed by the end of the p53 coding sequence beginning at the StuI site. Following the end of the p53 to coding sequence, a TSNT sequence motif providing early vaccinia transcription termination (Yuen and Moss, 1986) and a unique XhoI site were added. It should be noted that the final H6-p53 PCR fusion product (783 bp) does not contain the p53 coding sequences between the SarAI
and StuI restriction sites.
The 783 by fusion was digested with BamHI (5' end) and XhoI (3' end) and inserted into plasmid pSD550 to yield plasmid pMM105. Plasmid pSD550 enables the insertion of foreign genes into the vaccinia I4L locus by 2o replacing the I4L coding sequence. This plasmid was derived from pSD548 (Tartaglia et al., 1992) by first digesting this plasmid with BalII and SmaI. This digested plasmid was then ligated to annealed oligonucleotides 539A (SEQ ID N0:97) (5'-AGAAAAATCAGTTAGCTAAGATCTCCCGGGCTCGAGGGTACCGGATCCTGATTAGTT
AATTTTTGT-3') and 539B (SEQ ID N0:98) (5'-GATCACAAAAATTAACTAATCAGGATCCGGTACCCTCGAGCCCGGGAGATCTTAGCT
AACTGATTTTTCT-3) to generate pSD550.
Plasmids containing intact p53 gene (wildtype or 3o mutant forms) juxtaposed 3' to the H6 promoter were generated by first digesting pMM105 with ScrrAI and StuI.
A 795 by SctrAI/StuI fragment was isolated from p53wtXbaISP6/T3 and p53-238XbaI, while a 792 by fragment was isolated from p53-217XbaI. These fragments were individually ligated to the SarAI/StuI digested pMM105 plasmid to yield pMM106, pMM108, and pMM107, respectively.

WO 94/16716 ~ ~ PCTIUS94/00888 Plasmids pMM106, pMM107, and pMM108 were used in standard in vitro recombination experiments (Piccini et al., 1987) with NYVAC (vP866; Tartaglia et al., 1992) as the rescue virus to generate recombinant viruses vP1101, vP1096, and vP1098, respectively. Fig. 17 presents the nucleotide sequence of the wildtype p53 expression cassette and flanking regions within vP1101 (SEQ ID
N0:99). The H6 promoter starts at position 145. The p53 start codon is at position 269, and the p53 stop codon is 1o at position 1450. Positions 1 through 144 and positions 1451 through 1512 flank the H6/p53 expression cassette.
The sequences within vP1096 and vP1098 are identical except vP1096 contains a 3 base deletion from nucleotide 920 to 922 while vP1101 contains a point mutation at nucleotide 980 (T or C).
Both immunofluorescence and immunoprecipitation assays were performed using a p53-specific monoclonal antibody (pAB1801, Oncogene Science provided by Dr. ,7.
Marks) to demonstrate expression of p53 in vP1101, vP1098 2o and vP1096 infected Vero cells. These assays were performed as described previously (Taylor et al., 1990).
Immunofluorescence assay demonstrated p53-specific fluorescent staining of cells infected with vP1101, vP1096, or vP1098. The p53 antigen was located in both the nucleus and cytoplasm of the infected cells. The nuclear staining, however, was more intense in vP1101 infected cells. These results are similar to those reported by Ronen et al. (1992) using replication-competent vaccinia to express wildtype and mutant forms of p53. No p53-specific fluorescent staining was observed in Vero cells infected with the parental NYVAC
virus, vP866.
ALVAC (CPpp) p53 insertion plasmids were engineered by excising the p53 expression cassettes from pMM106, pMM107, and pMM108 by digestion with BamHI and XhoI and inserting them individually into BamHI/XhoI digested pNVQCSLSP-7. The 1320 by BamHI/XhoI fragment containing WO 94/16?16 ~ PCTIUS94100888 the H6-p53 expression cassette from pMM106 and pMM108 was inserted into pNVQCSLSP-7 to yield pMM110 and pMM112, Ze' respectively, while the 1317 by BamHI/XhoI fragment derived from pMM107 and inserted into pNVQCSLSP-7 yielded s pMMlll.
The plasmid pNVQCSLSP-7 was derived in the following manner. pCSLSP (defined in Example 1) was digested with BamHI and ligated to annealed oligonucleotides CP32 (SEQ
ID NO:100) (5'-l0 CATCTTAATTAATTAGTCATCAGGCAGGGCGAGAACGAAGACTATCTGCTCGTTAAT
TAATTAGGTCGACG-3') and CP33 (SEQ ID NO:101) (5'-CATCCGTCGACCTAATTAATTAACGACGACATAGTCTCGTTCTCGCCTGCCTGATGA
CTAATTAATTAA-3') to generate pVQC5LSP6. pVQC5LSP6 was digested with EcoRI, treated with alkaline phosphatase 15 and ligated to self-annealed kinased oligonucleotide CP29 (SEQ ID N0:102) (5'-AATTGCGGCCGC-3'), digested with NotI
and linear was purified followed by self-ligation. This procedure introduced a NotI site to pVQC5LSP6, generating pNVQCSLSP-7.
2o Insertion plasmids pMM110, pMMlll, and pMM112 were used in standard in vitro recombination experiments (Piccini et al., 198?) with ALVAC (CPpp) as the rescue virus to yield vCP207, vCPl93 and vCP191, respectively.
Confirmation of expression of the p53 gene product was 25 accomplished by immunoprecipitation assays performed as described above. Fig. 18 presents the nucleotide sequence of the H6/p53 (wildtype) expression cassette and flanking regions from vCP207 (SEQ ID N0:103). The H6 promoter starts at position 109. The p53 start codon is 3o at position 233, and the p53 stop codon is at position 1414. Positions 1 through 232 and positions 1415 through 1483 flank the H6/p53 expression cassette. The nucleotide sequence is identical to that within vCPi93 and vCP191 except vCP193 contains a 3 nucleotide deletion 35 from nucleotide 973 to 975 while vCP191 contains a point mutation at nucleotide 94 to (T to C).
A listing of the NYVAC- and ALVAC- based p53 WO 94/16716 ~ PCTIUS94/00888 recombinant viruses is provided in Table 22.
TABLE 22. NYVAC and ALVAC-based p53 recombinant viruses Recombinant Virus Parent Virus Gene Insert vP1101 NYVAC w.t.53 vP1096 NYVAC
p53 (-as 217) vP1098 NYVAC p53 (aa238; C to R) vCP207 ALVAC w.t. 53 vCP193 ALVAC p53 (-as 217) to vCP191 ALVAC
p53 (aa 238; C to R) Example 16 - UTILITY OF NYVAC- AND ALVAC-BASED

GENE
Human melanoma-associated antigen MZ2-E is encoded by the MAGE-1 gene (Reviewed by van der Bruggen and Van der Eynde, 1992). MAGE-1 is expressed in primary melanoma tumor cells, melanoma-derived cell lines, and 2o certain tumors of non-melanoma origins but not in normal cells except in testis (Coulie et al., 1993). Of interest from an immunological perspective, CTLs from melanoma-bearing patients that are of the HLA-A1 MHC
haplotype are known to recognize a nonapeptide from the MZ2-E gene product (Traveseri et al., 1992). Therefore, definition of such an antigen provides a mechanism for targeted immunotherapy for HLA-typed (HLA-A1) melanoma patients.
Generation of NYVAC- and ALVAC-based Recombinant Viruses Containing the MAGE-1 Gene PCR fragment PCR-H6 (162 bp) was synthesized using pBSH6 (described in Example 14) as template and oligonucleotides H65PH (SEQ ID N0:80) and M1-4 (SEQ ID
- N0:104) (5'-CAGACTCCTCTGCTCAAGAGACATTACGATACAAACTTAACG-3') which contains the last 18 by of the H6 promoter and the initial 24 nucleotides of the MAGE-1 gene. A second PCR fragment (PCR-M1) was amplified from plasmid pTZI8RMAGE1 using oligonucleotides M1-1 (SEQ ID N0:105) ,, WO 94/16716 ~ ~ ' PCT/US94/00888 (5'-ATGTCTCTTGAGCAGAGGAGTCTG-3' and M1-2 (SEQ ID N0:106) (5'-CAGGCCATCATAGGAGAGACC-3'). The resultant PCR
fragment represents the initial 546 by of the MAGE-1 coding sequence.
Plasmid pTZI8RMAGE-1 contains a cDNA clone of the MAGE-1 gene. This gene encodes the MZE-2 human melanoma rejection antigen. This plasmid was provided by Dr.
Lloyd Old (Memorial Sloan-Kettering, NY, NY) who obtained the plasmid originally from Dr. Thierry Boon (Ludwig to Inst. for Cancer Research, Brussels, Belgium).
PCR fusion product, PCR-H6M1 was generated using PCR-H6 and PCR-M1 as templates and oligonucleotides H65PH
(SEQ ID N0:80) and M1-2 (SEQ ID N0:106) as primers. A
complete HindIII/BalII digestion of PCR-H6M1 was performed and the resultant 556 by was purified for subsequent cloning steps.
PCR fragment PCR-M3' (535 bp) was amplified from pTZI8RMAGE-1 using oligonucleotides M1-3 (SEQ ID N0:107) (5'-GTGGCTGATTTGGTTGGTTTTCTG-3') which contains 24 2o nucleotides complementary to the MAGE-1 gene at a region approximately 200 by upstream of the M1-2 oligonucleotide sequence and M1-5 (SEQ ID N0:108) (5'-ATCATCTCTAGAAAAAAAATCACATAGCTGGTTTCAG-3') containing the terminal 15 nucleotides of the MAGE-1 coding sequence, a 2s vaccinia early transcription termination signal (T5NT;
Yuen and Moss, 1986) and an XbaI restriction site. PCR-M3' was digested with BQ1II and XbaI. The resultant 414 by fragment was isolated and co-inserted into HindIII/XbaI digested pBS-SK(+) with the 556 by 3o HindIII/BalII digested PCR fragment PCR-H6M1. The resultant plasmid containing the entire H6-MAGE-1 expression cassette was designated pMAW034. The H6-MAGE-1 cassette was confirmed by nucleotide sequence analysis as per Goebel et al., 1990).
3s The 864 by NruI/XbaI fragment from pMAW034 was isolated and inserted into pVQH6C5LSP (described in Example 14) that was digested in a similar fashion. The 21 ~ 3 3 ~ 6 PCT/US94/00888 resultant plasmid was designated pMAW036. This plasmid served as the insertion plasmid for replacing the two C5 ORFs in the ALVAC genome with the H6-MAGE-1 expression cassette.
s Plasmid pMAW036 was used in standard in vitro recombination experiments with ALVAC as the rescuing virus. Recombinant virus were identified by an in situ plaque hybridization assay using MAGE-1-specific radiolabeled DNA probes. Recombinant plaques were plaque 1o purified and amplified. The resultant ALVAC-based recombinant containing the MAGE-1 gene was designated vCP235. Fig. 19 presents the nucleotide sequence of the H6/MAGE-1 expression cassette and flanking region contained within vCP235 (SEQ ID N0:109). The H6 promoter 15 starts at position 74. The MAGE-1 start codon is at position 201, and the MAGE-1 stop codon is at position 1031. Positions 1 through 73 and positions 1032 through 1094 flank the H6/MAGE-1 expression cassette.
The NYVAC (vP866) insertion plasmid pMAW037 was 2o generated by initially digesting pMAW034 with NruI/BamHI.
The resultant 879 by fragment was isolated and inserted into NruI/BamHI digested pSPHAH6. The resultant plasmid was designated pMAW037.
Plasmid pSPHAH6 was generated in the following 25 manner. Plasmid pSD544 (containing vaccinia sequences surrounding the site of the HA gene 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
3o polymerase I and treated with alkaline phosphatase.
SP126 (containing the vaccinia H6 promoter) was digested with HindIII, treated with Klenow and the H6 promoter isolated by digestion with SmaI. Ligation of the H6 promoter fragment to pSD544 generated SPHA-H6 which 35 contained the H6 promoter in the polylinker region (in the direction of HA transcription).
Plasmid pMAW037 was used in standard in vitro WO 94116716 . ' ' PCT/US94100888 tj recombination experiments (Piccini et al., :1987) with NYVAC (vP866) as the rescue virus. Fig. 20 presents the nucleotide sequence of the H6/MAGE-1 expres:~ion cassette and flanking regions within pMAW037 (SEQ ID NO:110). The H6 promoter starts at position 52. The MAGE-1 start codon is at position 179, and the MAGE-1 step codon is at position 1009. Positions 1 through 51 and ~~ositions 1010 through 1084 flank the H6/MAGE-1 expression cassette.
Examule 17 - GENERATION OF AN ALVAC- AND NYVAC-BASED
to CEA RECOMBINANT VIROSES
The CEA gene was provided in plasmid pGEM.CEA, which contains the CEA coding sequence (2,109 nucleotides) as well as 5' and 3' untranslated regions (Dr. J. Schlom, NCI-NIH). The 5' end of the CEA construct was modified to remove the 5' untranslated sequences and place the vaccinia H6 promotor before the ATG initiation codon of CEA. This was accomplished by PCR with the oligonucleotide pair CEA1 (SEQ ID NO:111) (5'-2o TATCGCGATATCCGTTAAGTTTGTATCGTAATGGAGTCTCCCTCG-3') and CEA2 (SEQ ID N0:112) (5'-TGCTAGATCTTTATCTCTCGACCACTGTATG-3') and plasmid pGEM.CEA as template. The resulting fragment links the 3' 30 nucleotides of the H6 promotor to the CEA initiation codon, extends 22 nucleotides past 2s the ApaI site at position 278 of the CEA coding sequence, and terminates with a BalII site introduced by the PCR
primer CEA2 (SEQ ID N0:114). Prior to cloning, this fragment was digested with EcoRV (site located within the 3' end of the H6 promotor) and BalII. The digested 5' 3o PCR fragment was then included in a 3-way ligation with two fragments derived from plasmid pI4L.H6: an NcoI/BqlII
vector fragment and an NcoI/EcoRV fragment which contained the 5' portion of the H6 promotor. The resulting plasmid, designated pI4L.H6.CEA-5', contains ' 3s the full length H6 promotor linked to a 5' CEA fragment extending from the ATG codon through the ApaI site at position 278.
The 3' end of CEA was modified to remove the 3' WO 94116716 215 3 3 3 ~ ~T~S94100888 untranslated region of CEA and place a vaccinia early transcription termination signal (TSNT) followed by a series of restriction sites (XhoI, XbaI, SmaI, HindIII) after the TAG termination codon. This was accomplished by PCR with the oligonucleotide pair CEA3 (SEQ ID N0:113) (5'-CTATGAGTGTGGAATCCAGAACG-3') and CEA4 (SEQ ID N0:114) (5'-TCAGAAGCTTCCCGGGTCTAGACTCGAGATAAAAACTATATCAGAGCAACC-3') and plasmid pGEM.CEA as template. The resulting fragment extends from a position 32 nucleotides 5' of the to CEA HindII site located at position 1203 through the 3' end of the coding sequence. This fragment was cloned as a HindII/HindIII fragment into a HindII/HindIII-digested pGEM.CEA vector fragment. The resulting plasmid, designated pGEM.CEA-3', contains the entire CEA gene as found in pGEM.CEA with a 3' end modified to remove the 3' untranslated region and replace it with a TSNT signal followed by XhoI, XbaI, SmaI, and HindIII restriction sites.
To generate an ALVAC C3 donor plasmid containing 2o CEA, a BamHI/ApaI fragment containing the H6 promotor linked to the 5' end of CEA was obtained from pI4L.H6.CEA-5', an ApaI/XhoI fragment containing the remainder of the CEA coding sequence (plus TSNT) was obtained from pGEM.CEA-3', and a BamHI/XhoI C3 vector fragment was derived from plasmid p126.C3. After subsequent 3-way ligation, the plasmid pH6.CEA.C3 was obtained. This plasmid contains the full length H6/CEA
expression cassette inserted between left and right flanking arms of ALVAC DNA which direct insertion to the 3o C3 sites on the ALVAC genome. Transcription of CEA is oriented from right to left.
Plasmid p126.C3, an ALVAC C3 donor plasmid, was derived as follows. This plasmid contains an insert consisting of cDNA derived from the Plasmodium falciparum SERA gene (Li et al., 1989; Bzik et al., 1989; Knapp et al., 1989; NOTE: SERA is also known as SERP I and p126) under the control of the entomopox virus 42K early . . : . . y.,, i3o promotor.
A. Methodolog3r for generatina t~126. C3 .
1. Construction of P. falciparum FCR3 Strain Blood Stage cDNA Library.
s Total RNA from human erythrocytes infected with P.
falciparum FCR3 strain was provided by Dr. P. Delplace (INSERM-U42). Poly-A+ RNA was isolated from this sample by use of oligo(dT) cellulose (Stratagene, La Jolla, CA.) as described by Aviv and Leder (1972) and modified by 1o Kingston (1987). Briefly, total RNA was mixed with oligo(dT) cellulose in Binding buffer (0.5M NaCl, O.O1M
Tris-Cl, pH 7.5) and incubated for 30 minutes at room temperature. Poly-A+ RNA/oligo(dT) cellulose complexes were pelleted by centrifugation and washed 3 times with i5 Binding buffer. Purified poly-A+ RNA was eluted from the oligo(dT) cellulose in Elution buffer (0.01M Tris-C1, pH
7.5). A second elution with DEPC-treated dH20 was performed, the eluates were pooled, and the poly-A+ RNA
recovered by ethanol precipitation.
2o The purified poly-A+ RNA was used as a template for the synthesis of first strand cDNA by reverse transcriptase in a reaction primed with oligo(dT) (Watson and Jackson, 1985; Klickstein and Neve, 1987). For this reaction, l2ug poly-A+ RNA was incubated with 105 units 25 AMV reverse transcriptase (Life Sciences, Inc., St.
Petersburg, FL.) in 100mM Tris-C1 pH 8.3, 30mM KC1, 6mM
MgCl2, 25mM DTT, 80 units RNasin, 1mM each dNTP, and 24ug/ml oligo(dT)12-is as primer for 2 hours at 42°C.
After organic extractions, double stranded cDNA was 30 obtained by use of DNA polymerase I and RNase H with first strand cDNA as template (Watson and Jackson, 1985:
Klickstein and Neve, 1987). The first strand cDNA was incubated with 25 units DNA polymerase I and 1 unit RNase H in 20mM Tris-C1 pH 6, 5mM MgCl2, lOmM (NH4)2S04, 100mM
35 KC1, 500ug/ml BSA, 25mM DTT, and O.lmM each dNTP at 12°C
for one hour followed by one hour at room temperature to synthesize second strand cDNA. The double stranded cDNA

215333fi 131 " -was recovered by organic extractions and ethanol precipitation.
The double-stranded blood stage cDNA was then sequentially treated with T4 DNA polymerase to create s blunt ends and EcoRI methylase to protect internal EcoRI
sites. EcoRI linkers were then added followed by digestion with EcoRI and size selection on a 5-25%
sucrose gradient. Fractions containing long cDNAs (1-10 Kb) were pooled and ligated into coRI cleaved Lambda to ZAPII vector (Stratagene, La Jolla, CA.). The resulting phage were packaged and used to infect the XL-1 Blue E.
coli strain (Stratagene). The phage were then harvested from these cells and amplified by one additional cycle of infection of XL-1 Blue to produce a high titer FCR3 is strain blood stage cDNA library.
2. Screen of cDNA Library for SERA cDNA Clones.
The FCR3 strain cDNA library was screened by plaque hybridization with 32P end-labelled oligonucleotides derived from published sequences of SERA to detect cDNA.
2o The cDNA library was plagued on lawns of XL-1 Blue (Stratagene) in 150mm dishes at a density of 100,000 plaques per dish. Plaques were transferred to nitrocellulose filters which were then soaked in 1.5M
NaCl/0.5M NaOH for 2 minutes, 1.5M NaCl/0.5M Tris-C1 pH 8 25 for 5 minutes, 0.2M Tris-C1 pH 7.5/2X SSC for one minute, and baked for 2 hours in an 80°C vacuum oven. Filters were prehybridized in 6X SSC, SX.Denhardts, 20mM NaH2P04, 500ug/ml salmon sperm DNA for two hours at 42°C.
Hybridizations were performed in 0.4% SDS, 6X SSC, 20mM
3o NaH2P04, 500ug/ml salmon sperm DNA for 18 hours at 42°C
after the addition 32P-labelled oligonucleotide. After hybridization, filters were rinsed 3 times with 6X SSC, 0.1% SDS, washed for 10 minutes at room temperature, and washed for 5 minutes at 58°C. Filters were then exposed 35 to X-ray film at -70°C.
Plaques hybridizing with the oligonucleotide probe were cored from plates and resuspended in SM buffer (100mM NaCl, 8mM MgS04, SOm~M Tris-C1 pH 7.5, 0.01%
gelatin) captaining 4% chloroform. Dilutions of such phage stocks were used to infect XL-1 Blue, plaques were transferred to nitrocellulose, and the filters were hybridized with 3ZP-labelled oligonucleotides. Well isolated positive plaques were selected and subjected to two additional rounds of purif icatian as just described.
3. '0 0 positive Phaqe Clones.
I0 SERA cDNAs i,n t."~e pBluescript plasznid vector (Stratagene) were obtained by an in vivo excision protocol developed for use with the lambda ZAPIT vector (Stratagene) . Br=efly, purified recombinant lambda phage stocks were incubated with XL-1 glue cells and 8408 filamentous helper phage fo:r 1~ minutes a- 37°C. After the addition of 2X Y'" media (1% NaCI, 1% yeast extract, 7..6% Bacto-tryptane), incubation was continued for 3 hours at 37°c~ fol:?owed by 20 minutes at 70°C. After' centrifugation, f ilamentous phage particles containing 2o pBluescript phagemid (with cDNA insert) were recovered in the supernatant. D.ilutions of the recovered f ilamentous phage stock were mixed with XL-1 Blue and plated to obtain colonies containing pBluescript plasmids with SERA
cDNA inserts.
z5 4. Generation of Malaria cDNA by PCR.
By use cf the polymerase chain reaction (PCR), the 5' portion of the ceding sequence of SERA was amplified with specif is olic~cnucleotide primers and first strand cDNA as template ~'Saiki et al. 1988, Frohman et al.
3o 1988) . SERA-specific first strand cflNA was synthesized by reverse transcriptase using the reaction conditions ~. - o described. above a-:d s~ec;_.c a~~.ccnuc_eo~les as pri.n rs.
. RNA was subsequen~.ly e'_~minated by treatment with RNase A
prior to PCR. The GeneAmp+LNA a~sp'_iFication kit (Per kin i5 Elmer Cetus, Norwal:t, C"~.) was used for PC~. Brielv, f it s t s trend cDNA i z SOnM KCB , lOmM Tr is-C 1 pH 3 . 3 , 1. 5:n1~
MgCl2, 0.01 % gel atin was mixed wit: 200uM each dNT?, luNl *Trace-mark '17386-:.~

of each primer, and 2.5 units Taq polymerase. Reactions were processed in a Thermal Cycler (Perkin Elmer Cetus) with 1 cycle of denaturation, annealing, and extension at 94°C for 2 minutes, 43°C for 3 minutes, and 72°C for 40 minutes; 40 cycles at 94°C for 1 minute, 43°C for 2 minutes, and 72°C for 4 minutes followed by a final extension at 72°C for 20 minutes.
The inclusion of restriction sites in primers used for PCR allowed the cloning of amplified SERA cDNA into 1o plasmid vectors. Clones cantaining cDNAs derived from t~~ro independent PCRs were obtained for eac."1 SERA cDNA
that was amplified in order to contzol fcr Taq polymerase errors.
B. Results.
1. Isolation clonincr and characterization of SERA
cDNA.
We have isolated overlapping cDNA clones spanning the SERA coding sequence from the PCR3 strain of P.
2o falciparum. The p125.6 cDNA, Which extends from the coRI site at position 1892 (numbering based on SERF I
gene of FCBR strain; Knapp eat al. , 1989) through the 3' end of the coding sequence, was isolated from the blood stage cDNA Lambda ~APLI cDNA library by hybridization to a SERA-specific o.Ligonucleotide JAT2 (SEQ ID N0:115) (5'-GTCTCAGAACGTGTTCATGT-3'), which a derived from the 3' end of th a SERA coding sequence (Bzik et al., 1989; Knapp et al., 1989). Clones derived from the 5' end of the SERA coding sequence were obtained by PCR wish primers JAT15 (SEQ ID N0:116) (5'-CACGGATCCATGAAGTCATATATTTCCTT-3' ) and JAT16 (SEQ ID NO:11'') (5'-- GTGAAGCTTAATCCATAATCTTC.~AT.'~:,TT-3 ' ) and SERA f it s t sgrand cDNA templa to (obtained with oligonucleotide primer JAT1%
( SEQ ID fC : 1:.8 ) ( 7' _~.~T J~.=~GC~."~'='TATACAT?~1CAGA.~TA.~CA-3' ; ~~
and were cloned i:~t;~ pUCl9 (New England Biolabs, BPv~art~~.=~, N.L~..). These 1923 by cDNAs e:xten~ from the initiation colon to a point 3'... by 3' o~ the internal EcoR~ site ( pos i tic:: l c 3 2 ) . C::e such c~i3? , F=2 5 . 8 , was found by DI~n *Trade-mar'.~c WO 94/16716 '~, ; ~ ~ ' PCTIUS94100888 sequence analysis to contain a Taq polymerase error at nucleotide 1357. This error, an A to G substitution, resides within the 315 by KpnI/NdeI restriction fragment.
A second SERA 5' cDNA, p126.9, has no mutations within s this KpnI/NdeI fragment. An unmutated 5' SERA cDNA was generated by replacing the 315 by Kpn_I/NdeI fragment in p126.8 with the analogous fragment from p126.9 to generate p126.14. Full length SERA cDNA was generated by ligating the p126.14 5' cDNA as an XmaI/EcoRI fragment to into a partial EcoRI/XmaI digested p126.6 vector fragment to generate p126.15.
The complete nucleotide sequence of the p126.15 SERA
cDNA insert was determined and is shown in Figures 21A
and 21B (SEQ ID N0:119) along with the predicted amino 15 acid sequence (SEQ ID N0:120). This cDNA contains a 2955 by open reading frame encoding 984 amino acids that is identical to the SERA allele II gene in the FCR3 strain and the FCBR SERP I gene (Li et al., 1989, Knapp et al., 1989 ) .
2o The SERA cDNA was isolated from p126.15 as a 3 Kb XmaI/EcoRV fragment and the XmaI end ligated into an XmaI/BglII digested pCOPCS-5H vector fragment. DNA
polymerase I Klenow fragment was used to fill in the pCOPCS-5H BalII site which was subsequently ligated to 2s the EcoRV end to generate p126.16. In this plasmid, SERA
is under the control of the early/late vaccinia H6 promotor.
2. Modification of SERA cDNA.
The 3' end of the SERA cDNA was modified to place a 3o vaccinia early transcription termination signal (T5NT;
Yuen and Moss, 1987) and a series of restriction sites (XhoI, SmaI, SacI) immediately after the TAA termination codon. This was accomplished by PCR with oligonucleotides JAT51 (SEQ ID N0:121) (5'-35 TAGAATCTGCAGGAACTTCAA-3'), JAT52 (SEQ ID N0:122) (5'-CTACACGAGCTCCCGGGCTCGAGATAAAAATTATACATAACAGAAATAACATTC-3'), and plasmid p126.16 as template. The resulting 300 by amplified fragment was cloned as a PstI/SacI fragment into p126.16 digested with PstI and SacI to generate p126.17.
The 5' end of the SERA cDNA in p126.17 was modified to place several restriction sites (HindIII, SmaI, BamHI) and the 42K entomopox promotor before the ATG initiation codon. This was accomplished by PCR with oligonucleotides JAT53 (SEQ ID N0:123) (5'-CTAGAGAAGCTTCCCGGGATCCTCAAAATTGAAAATATATAATTACAATATAAAATG
l0 AAGTCATATATTTCCTTGT-3'), JAT54 (SEQ ID N0:124) (5'-ACTTCCGGGTTGACTTGCT-3'), and plasmid p126.16 as template.
The resulting 250 by amplified fragment was cloned as a HindIII/HindII fragment into p126.17 digested with HindIII and HindII to generate p126.18. This plasmid contains a cassette consisting of the SERA cDNA
controlled by the 42K entomopox promotor, with a vaccinia early transcription termination signal, and flanked by restriction sites at the 5' (HindIII, SmaI, BamHI) and 3' (XhoI, SmaI, SacI) ends.
2o The 42K promotor/SERA cassette was isolated from p126.18 as a BamHI/XhoI fragment and cloned into a BamHI/XhoI digested pSD553 vector fragment. The resulting plasmid is designated p126.ATI.
3. Generation of p126.C3.
2s The 42K/SERA expression cassette was isolated from p126.ATI as a BamHI/XhoI fragment and cloned into a BamHI/XhoI-digested VQCP3L vector fragment. The resulting plasmid, designated p126.C3, is an ALVAC C3 donor plasmid.
30 4. Derivation of pSD553.
- The pSD553 vaccinia donor plasmid was used for the generation of p126.ATI. It contains the vaccinia K1L
host range gene (Gillard et al., 1986) within flanking Copenhagen vaccinia arms, replacing the ATI region (orfs 35 A25L, A26L; Goebel et al., 1990a,b). pSD553 was constructed as follows.
Left and right vaccinia flanking arms were constructed by PCR using pSD414, a pUCB-based clone of vaccinia SalI B (Goebel et al., 1990a,b) as template.
~t~ The left arm was synthesized using synthetic deoxyoligonucleotides MPSYN267 (SEQ ID N0:85) (5'-GGGCTGAAGCTTGCTGGCCGCTCATTAGACAAGCGAATGAGGGAC-3') and MPSYN268 (SEQ ID N0:86) (5'-AGA TCT CCC GGG CTC GAG TAA
TTA ATT AAT TTT TAT TAC ACC AGA AAA GAC GGC TTG AGA TC-3') as primers. The right arm was synthesized using synthetic deoxyoligonucleotides MPSYN269 (SEQ ID N0:87) (5'-TAA TTA CTC GAG CCC GGG AGA TCT AAT TTA ATT TAA TTT
ATA TAA CTC ATT TTT TGA ATA TAC T-3') and MPSYN270 (SEQ
ID N0:88) (5'-TAT CTC GAA TTC CCG CGG CTT TAA ATG GAC GGA
ACT CTT TTC CCC-3') as primers. The two PCR-derived DNA
fragments containing the left and right arms were combined in a further PCR reaction. The resulting product was cut with EcoRI/HindIII and a 0.9kb fragment isolated. The 0.9kb fragment was ligated with pUC8 cut with EcoRI/HindIII, resulting in plasmid pSD541. The polylinker region located at the vaccinia deletion locus 2o was expanded as follows. pSD541 was cut with BalII/XhoI
and ligated with annealed complementary synthetic deoxyoligonucleotides MPSYN333 (SEQ ID N0:125) (5'-GAT
CTT TTG TTA ACA AAA ACT AAT CAG CTA TCG CGA ATC GAT TCC
CGG GGG ATC CGG TAC CC-3')/MPSYN334 (SEQ ID N0:126) (5'-TCG AGG GTA CCG GAT CCC CCG GGA ATC GAT TCG CGA TAG CTG
ATT AGT TTT TGT TAA CAA AA-3') generating plasmid pSD552.
The K1L host range gene was isolated as a !kb BQ1II(partial)/HpaI fragment from plasmid pSD452 (Perkus et al., 1990). pSD552 was cut with BalII/HpaI and ligated with the K1L containing fragment, generating pSD553.
5. Derivation of VQCP3L.
The VQCP3L ALVAC donor plasmid was used for the generation of p126.C3 and was constructed as follows.
Insertion plasmid VQCP3L was derived as follows. An 8.5kb canarypox BalII fragment was cloned in the BamHI
site of pBS-SK plasmid vector to form pWWS. Nucleotide sequence analysis of a 7351 by subgenomic fragment from ALVAC containing the C3 insertion site is presented in Figs. 14A to 14C (SEQ ID N0:127). The C3 ORF is located between nucleotides 1458 to 2897. 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 N0:128) (5'-1o CAGTTGGTACCACTGGTATTTTATTTCAG-3') and RG278 (SEQ ID
N0:129) (5'-TATCTGAATTCCTGCAGCCCGGGTTTTTATAGCTAATTAGTCAAATGTGAGTTAATA
TTAG-3'). Primers for the 3' sequences were RG279 (SEQ
ID N0:130) (5'TCGCTGAATTCGATATCAAGCTTATCGATTTTTATGACTAGTTAATCAAATAAA
AAGCATACAAGC-3') and RG280 (SEQ ID N0:131) (5'-TTATCGAGCTCTGTAACATCAGTATCTAAC-3'). The primers were designed to include a multiple cloning site flanked by vaccinia transcriptional and translational termination 2o 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 EcoRI and SacI for right arm) which enabled the two arms to ligate into Asp718/SacI digested pBS-SK plasmid vector. The 2s resultant plasmid was designated as pC3I. A 908 by fragment of canarypox DNA, immediately upstream of the C3 locus was obtained by digestion of plasmid pWWS with NsiI
and SSpI. A 604 by fragment of canarypox and DNA was derived by PCR using plasmid pWW5 as template and 30 oligonucleotides CP16 (SEQ ID N0:132) (5'-TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTCTGC-3') and CP17 (SEQ
ID N0:133) (5'-TCGCTCGAGTAGGATACCTACCTACTACCTACG-3').
The 604 by fragment was digested with Asp718 and XhoI
(sites present at the 5' ends of oligonucleotides CP16 35 and CP17, respectively) and cloned into A~718-XhoI
digested and alkaline phosphatase treated IBI25 (International Biotechnologies, Inc., New Haven, CT) i3s generating plasmid SPC3LA. SPC3LA was digested within IBI25 with EcoRV and within canarypox DNA with NsiI, and ~t ligated to the 908 by NsiI-SSpI fragment generating SPCPLAX which contains 1444 by of canarypox DNA upstream s of the C3 locus. A 2178 by BqlII-S,tvI 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 by fragment of canarypox DNA was isolated by PCR using plasmid pXX4 as 1o template and oligonucleotides CP19 (SEQ ID N0:134) (5'-TCGCTCGAGCTTTCTTGACAATAACATAG-3') and CP20 (SEQ ID
N0:135) (5'-TAGGAGCTCTTTATACTACTGGGTTACAAC-3'). The 279 by fragment was digested with XhoI and SacI (sites present at the 5' ends of oligonucleotides CP19 and CP20, 15 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 ClaI, treated with alkaline 2o phosphatase and ligated to kinased and annealed oligonucleotides CP12 (SEQ ID N0:136) (5'-AATTCCTCGAGGGATCC-3') and CP13 (SEQ ID N0:137) (5'-CGGGATCCCTCGAGG-3') (containing an EcoRI sticky end, XhoI
site, BamHI site and a sticky end compatible with ClaI) 25 generating plasmid SPCP3S. SPCP3S was digested within the canarypox sequences downstream of the C3 locus with SCI and SacI (pBS-SK) and ligated to a 261 by BalII-SacI
fragment from SPC3RA and the 2178 by BalII-SCI fragment from pXX4 generating plasmid CPRAL containing 2572 by of 3o 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 by AS~718-AccI fragment from SPCPLAX generating plasmid CPLAL containing 1457 by of canarypox DNA
35 upstream of the C3 locus. The derived plasmid was designated as SPCP3L. VQCP3L was derived from pSPCP3L by digestion with XmaI, phosphatase treating the linearized 215333fi plasmid, and ligation to annealed, kinased oligonucleotides CP23 (SEQ ID N0:138) (5'-CCGGTTAATTAATTAGTTATTAGACAAGGTGAAAACGAAACTATTTGTAGCTTAATT y AATTAGGTCACC-3') and CP24 (SEQ ID N0:139) (5'-CCGGGGTCGACCTAATTAATTAAGCTACAAATAGTTTCGTTTTCACCTTGTCTAATA
ACTAATTAATTAA-3').
DNA sequence analysis of pH6.CEA.C3 revealed a one nucleotide deletion (T) at position 1203 of the CEA
coding sequence (eliminating a HindII site) which occurred during a previous cloning step. This deletion was corrected by replacing a 1047 nucleotide MscI
fragment (extending from position 501 to 1548) from pH6.CEA.C3 with the analogous, unmutated MscI fragment from pGEM.CEA. The resulting plasmid was designated pHG.CEA.C3.2.
CEA has been inserted into ALVAC by recombination between NotI-linearized pH6.CEA.C3.2 donor plasmid and ALVAC rescuing virus. Recombinants containing CEA have been identified by plaque hybridization with a DNA probe 2o derived from the CEA coding sequence (an NruI/XhoI
fragment containing the full length CEA coding sequence).
A NruI/XhoI fragment containing the 3' end of the H6 promotor linked to the full length CEA coding sequence was isolated from pH6.CEA.C3.2. This fragment was ligated to an NruI/XhoI-digested pSPHA.H6 vector fragment, which was derived from the pSD544 HA donor plasmid by the insertion of a fragment containing the H6 promotor. The resulting plasmid was designated pH6.CEA.HA and contains the CEA coding sequence linked to 3o the regenerated H6 promotor. The pH6.CEA.HA donor plasmid directs insertion of the H6/CEA expression cassette to the HA site of NYVAC. Transcription of CEA
is oriented from left to right.
Plasmid pSD544 was derived as follows. pSD456 is a subclone of Copenhagen vaccinia DNA containing the HA
gene (ASGR; Goebel et al., 1990a,b) and surrounding regions. pSD456 was used as template in polymerase chain WO 94116716 PCTlUS94100888 reactions for synthesis of left and right vaccinia arms flanking the A56R ORF. The left arm was synthesized using synthetic oligodeoxynucleotides MPSYN279 (SEQ ID
N0:140) (5' CCCCCCGAATTCGTCGACGATTGTTCATGATGGCAAGAT 3') and MPSYN280 (SEQ ID N0:141) (5'-CCCGGGGGATCCCTCGAGGGTACCAAGCTTAATTAATTAAATATTAGTATAAAAAGT
GATTTATTTTT -3') as primers. The right arm was synthesized using MPSYN281 (SEQ ID N0:142) (5'-AAGCTTGGTACCCTCGAGGGATCCCCCGGGTAGCTAGCTAATTTTTCTTTTACGTAT
1o TATATATGTAATAAACGTTC -3') and MPSYN312 (SEQ ID N0:143) (5'- TTTTTTCTGCAGGTAAGTATTTTTAAAACTTCTAACACC -3') as primers. Gel-purified PCR fragments for the left and right arms were combined in a further PCR reaction. The resulting product was cut with EcoRI/HindIII. The is resulting 0.9 kb fragment was gel-purified and ligated into pUC8 cut with EcoRI/HindIII, resulting in plasmid pSD544. Figures 22 and 23 present the nucleotide sequences of the H6/CEA expression cassettes and flanking regions in plasmids pH6.CEA.C3.2 and pH6.CEA.HA, 2o respectively (SEQ ID N0:144/145, respectively). In Fig.
22, the HG promoter begins at position 57. The CEA start codon begins at position 181 and the stop codon ends at position 2289. Positions 1 through 58 and 2290 through 2434 flank the H6/CEA expression cassette. In Fig. 23, 25 the H6 promotor begins at position 60. The CEA start codon begins at position 184 and the stop codon ends at position 2292. Positions 1 through 59 and 2293 through 2349 flank the H6/CEA expression cassette.
Example 18 - MURINE IL-2 INTO ALVAC AND NYVAC
3o Insertion of murine IL-2 into ALVAC. Plasmid pmut-1 (ATCC No. 37553) contains the murine IL-2 gene from American Type Culture Collection, Rockville, MD. The IL-2 gene was placed under the control of the vaccinia H6 promoter (Perkus et al., 1989) and the IL-2 3' noncoding 35 end was removed in the following manner.
Template pRW825, containing the H6 promoter and a nonpertinent gene, was used in a polymerase chain WO 94/16716 2 I 5 3 3 3 G ~T~S94/00888 reaction (PCR) with primers MM104 (SEQ ID N0:146) 5'ATCATCGGATCCCTGCAGCCCGGGTTAATTAATTAGTGATAC 3' and MM105 (SEQ ID N0:147) 5' GAGCTGCATGCTGTACATTACGATACAAACTTAACGGA 3'. The 5' end of MM104 contains BamHI, PstI and SmaI sites followed by a sequence which primes from the H6 promoter 5' end toward the 3' end. The 5' end of MM105 overlaps the IL-2 5' end and MM105 primes from the H6 promoter 3' end toward the 5' end. The resultant 228 base pair PCR derived fragment to contains the H6 promoted 5' most base pairs of IL-2.
Template plasmid pmut-1 was used in a second PCR
with primers MM106 (SEQ ID N0:148) 5' CGTTAAGTTTGTATCGTAATGTACAGCATGCAGCTG 3' and MM107 (SEQ ID
N0:149) 5' GAGGAGGAATTCCCCGGGTTATTGAGGGCTTGTTGAGA 3'.
The 5' end of MM106 overlaps the 3' end of the H6 promoter and primes from the IL-2 5' end toward the 3' end. The 5' end of MM107 contains EcoRI and SmaI sites followed by a sequence which primes from the IL-2 3' end toward the 5' end. The resultant 546 base pair PCR
2o derived fragment was pooled with the above 228 base pair PCR product and primed with MM104 and MM107. The resultant 739 base pair PCR derived fragment, containing the H6 promoted IL-2 gene, was digested with BamHI and EcoRI, generating a 725 base pair fragment, for insertion between the BamHI and EcoRI sites of pBS-SK (Stratagene, LaJolla, California), yielding pMM151.
The 755 base pair pMM151 BamHI-XhoI fragment containing the H6 promoted IL-2 gene was inserted between the BamHI and XhoI sites of the C3 vector pCP3LSA-2. The 3o resultant plasmid pMM153, contains the H6 promoted IL-2 gene in the C3 locus.
The nucleotide sequence of murine IL-2 from the translation initiation codon through the stop codon is given in Figure 24 (SEQ ID N0:150).
C3 vector plasmid pCP3LSA-2 was derived in the following manner. Plasmid SPCP3L (Example 17) was digested with NsiI and NotI and a 6433 by fragment WO 94116716 ' PCT/US94/00888 Cp _ isolated and ligated to annealed oligonucleotides CP34 (SEQ ID N0:151) 5' GGCCGCGTCGACATGCA 3' and CP35 (SEQ ID
N0:152) 5' TGTCGACGC 3', generating plasmid pCP3LSA-2.
Recombination between donor plasmid pMM153 and ALVAC
rescuing virus generated recombinant virus vCP275, which contains the vaccinia H6 promoted murine IL-2 gene in the C3 locus.
Insertion of murine IL-2 into NYVAC. Plasmid pMM151, defined above, was digested with BamHI/XhoI and a 755 base pair fragment containing the H6 promoted IL-2 gene was isolated. This BamHI/XhoI fragment was inserted between the BamHI and XhoI sites of the NYVAC TK vector pSD542. The resultant plasmid pMM154, contains the H6 promoted IL-2 gene in the TK locus.
is Plasmid pSD542 was derived in the following manner.
To modify the polylinker region, TK vector plasmid pSD513 (Example 7) was cut with PstI/BamHI and ligated with annealed synthetic oligonucleotides MPSYN288 (SEQ ID
N0:153) 5' GGTCGACGGATCCT 3' and MPSYN289 (SEQ ID N0:154) 5' GATCAGGATCCGTCGACCTGCA 3', resulting in plasmid pSD542.
Recombination between donor plasmid pMM154 and NYVAC
rescuing virus generated recombinant virus vP1239, which contains the HG promoted murine IL-2 gene in the TK
locus .
Expression of murine IL-2 in ALVAC and NYVAC based recombinants. ELISA assay. The level of expression of murine IL-2 produced by ALVAC and NYVAC based recombinants vCP275 and vP1239 was quantitated using an 3o ELISA kit from Genzyme Corporation, Cambridge, MA.
(InterTest-2XTM Mouse IL-2 ELISA Kit, Genzyme Corporation, Code # 2122-O1). Duplicate dishes containing confluent monolayers of mouse L-929 cells (2 x 106 cells/dish) were infected with recombinant virus vCP275 or vP1239 expressing murine IL-2 or infected with ALVAC or NYVAC
parental virus. Following overnight incubation at 37°C, supernatants were harvested and assayed for expression of WO 94116716 21 ~ 3 3 3 6 ~T~S94100888 143 "
murine IL-2 using the InterTest-2XTM Mouse IL-2 ELISA Kit as specified by the manufacturer (Genzyme Corporation, Cambridge, MA). The InterTest-2XTM Mouse IL-2 ELISA Kit is a solid-phase enzyme-immunoassay employing the multiple antibody sandwich principle. ELISA plates were read at 490 nm. Background from ALVAC or NYVAC samples was subtracted, and values from duplicate dishes were averaged. The quantity of murine IL-2 secreted is expressed as pg/ml, which is equivalent to pg/106 cells (Table 23).
Table 23 Recombinant virus Murine IL-2 secreted vCP275 160 pg/ml vP1239 371 pg/ml Example 19 - HUMAN IL-2 INTO ALVAC AND NYVAC
2o Insertion of Human IL-2 into ALVAC. Plasmid pTCGF-11 (ATCC No. 39673) contains the human IL-2 gene from American Type Culture Collection, Rockville, MD. The IL-2 gene was placed under the control of the vaccinia H6 promoter (Perkus et al., 1989), two codons were 2s corrected, and the IL-2 3' noncoding end was removed in the following manner.
Template plasmid pRW825, containing the H6 promoter and a nonpertinent gene, was used in a polymerase chain reaction (PCR) with primers MM104 (SEQ ID N0:146) 3o 5' ATCATCGGATCCCTGCAGCCCGGGTTAATTAATTAGTGATAC 3' and MM109 (SEQ ID N0:155) 5' GAGTTGCATCCTGTACATTACGATACAAACTTAACGGA 3'. The 5' end of MM104 contains BamHI, Pstl and SmaI sites followed by a sequence which primes from the vaccinia H6 promoter 5' 3s end toward the 3' end. The 5' end of MM109 overlaps the IL-2 5' end, and MM105 primes from the H6 promoter 3' end toward the 5' end. The resultant 230 base pair PCR
derived fragment contains the H6 promoted 5' most base pairs of IL-2.
4o Template plasmid pTCGF-11 was used in a PCR with WO 94/16716 ' PCT/US94100888 primers MM108 (SEQ ID N0:156) 5' C1j CGTTAAGTTTGTATCGTAATGTACAGGATGCAACTC 3' and MM112 (SEQ ID
N0:157) 5' TTGTAGCTGTGTTTTCTTTGTAGAACTTGAAGTAGGTGCACTGTTTGTGACAAGTGC
AAGACTTAGTGCAATGCAAGAC 3'. The 5' end of MM108 overlaps the 3' end of the H6 promoter and primes from the IL-2 5' end toward the 3' end. MM112 primes from position 100, in the human IL-2 sequence (Figure 25), toward the 5' end. The resultant 118 base pair fragment contains the 3' most base pairs of the H6 promoter and 5' 100 by of the IL-2 gene.
Plasmid pTCGF-11 from American Type Culture Collection was sequenced, and the sequence was compared with the published sequence (Clark, et al., 1984). Two mutations resulting in amino acid changes were discovered. Oligonucleotide primers MM111 (SEQ ID
N0:158) 5' TTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTTCTGGATTTACAGATGAT
TTTGAATGGAATTAATAATTAC 3' and MM112 were used to correct 2o these two base changes in pTCGF-11.
The corrected nucleotide sequence of human IL-2 from the translation initiation codon through the stop codon is given in Figure 25 (SEQ ID N0:159).
Except for a silent G to T change in pTCGF-11 at position 114, the sequence in Figure 25 is the same as the IL-2 sequence described in Clark, et al., 1984. The T at position 41 in the sequence in Figure 25 is C in pTCGF-11, and the codon change is from leu to pro. The T
at position 134 in the sequence in Figure 25 is C in 3o pTCGF-11, and the codon change is from leu to ser. The predicted amino acid sequences of other human, bovine, murine, ovine, and porcine IL-2 isolates were compared with the sequence in Clark, et al., 1984; the codons at positions 41 and 134 are both conserved as leu.
Template pTCGF-11 was used in a PCR with primers MM110 (SEQ ID N0:160) 5' GAGGAGGAATTCCCCGGGTCAAGTCAGTGTTGAGATGA 3' and MM111. The WO 94116716 ~ ~ PCTIUS94100888 5' end of MM110 contains EcoRI and SmaI sites followed ~y a sequence which primes from the IL-2 3' end toward the':
5' end. MM111 primes from position 75 toward the IL-2 3'"
end. The resultant 400 base pair PCR derived fragment s was pooled with the above 230 and 118 base pair PCR
products and primed with MM104 and MM110. The resultant 680 base pair PCR derived fragment, containing the vaccinia H6 promoted IL-2 gene, was digested with BamHI
and EcoRI and inserted between the BamHI and EcoRI sites to of pBS-SK (Stratagene, LaJolla, California), yielding pRW956.
Plasmid pRW956 was digested with BamHI/XhoI and a 700 by fragment containing the H6 promoted IL-2 gene was isolated. This fragment was inserted between the BamHI
15 and XhoI sites of the C3 vector plasmid pCP3LSA-2 (Example 18). The resultant plasmid, pRW958, contains the H6 promoted IL-2 gene in the C3 locus.
Recombination between donor plasmid pRW958 and ALVAC
rescuing virus generated recombinant virus vCP277, which 20 contains the H6 promoted human IL-2 gene in the C3 locus.
Insertion of Human IL-2 into NYVAC. Plasmid pRW956, defined above, was digested with BamHI/XhoI and a 700 base pair fragment was isolated. This fragment, containing the vaccinia H6 promoted human IL-2 gene, was 25 inserted between the BamHI and XhoI sites of the NYVAC TK
vector plasmid pSD542 (Example 18). The resultant plasmid, pRW957, contains the H6 promoted human IL-2 gene in the TK locus.
Recombination between donor plasmid pRW957 and NYVAC
3o rescuing virus generated recombinant virus vP1241, which contains the H6 promoted human IL-2 gene in the TK locus.
Expression of human IL-2 in ALVAC and NYVAC based recombinants. ELISA assay. The level of expression of human IL-2 produced by ALVAC and NYVAC based recombinants 35 vCP277 and vP1241 was quantitated using a Human Interleukin-2 ELISA kit from Collaborative Biomedical Products, Inc., Becton Dickinson, Bedford, MA. (IL-ISA 2TM

WO 94/16716 . PCT/US94100888 Cat. No. 30020). Duplicate dishes containing confluent monolayers of human HeLa cells (2 x 106 cells/dish) were infected with recombinant virus vCP277 or vP1241 expressing human IL-2 or infected with ALVAC or NYVAC
s parental virus. Following overnight incubation at 37°C, supernatants were harvested and assayed for expression of human IL-2 using the IL-ISA 2TM Human Interleukin-2 ELISA
kit as specified by the manufacturer (Collaborative Biomedical Products, Inc., Becton Dickinson, Bedford, 1o MA). The IL-ISA 2TM Kit is a solid-phase enzyme-immunoassay employing the multiple antibody sandwich principle. ELISA plates were read at 490 nm. Background from ALVAC or NYVAC samples was subtracted, and values from duplicate dishes were averaged. The IL-ISA 2TM
is KitIL-2 quantitates human IL-2 in Biological Response Modifiers Program (BRMP) units (Gerrard et al., 1993).
The quantity of human IL-2 secreted is expressed as BRMP
u/ml, which is equivalent to BRMP u/106 cells (Table 24).
Table 24 Recombinant virus Human IL-2 secreted vCP277 850 BRMP U/ml vP1241 953 BRMP U/ml Example 20 - MURINE IFNy INTO ALVAC AND NYVAC
Insertion of Murine IFNY into ALVAC. Plasmid pmsl0 was obtained from ATCC (#63170). Plasmid pmsl0 contains cDNA encompassing the entire mouse IFNY coding sequence 3o with flanking region cloned into the PstI site of pBR322.
Plasmid pMPI3H contains the vaccinia I3L promoter (Perkus et al., 1985; Schmitt and Stunnenberg, 1988) in pUC8. Plasmid pMPI3H is designed for cleavage at a H~~aI
site within the promoter and at a site in the downstream 3s polylinker region to allow for downstream addition of the 3' end of the I3L promoter linked to a foreign gene.
Linkaae of murine IFNv cxene with I3L promoter;
Construction of pMPI3mIF. Murine IFNy coding sequences with linkage to I3L promoter were synthesized by PCR

WO 94116716 215 3 3 3 6 PCT~S94100888 using oligonucleotides MPSYN607 (SEQ ID N0:161) 5' ~ , TAATCATGAACGCTACACACTGC 3' and MPSYN608 (SEQ ID N0:162) 5' CCCGGATCCCTGCAGTTATTGGGACAATCTCTT 3' as primers, and plasmid pmsl0 as template. The PCR product was cut with BamHI and a 510 by fragment was isolated and ligated with vector plasmid pMPI3H cut with HpaI/BamHI. Following sequence verification, the resulting plasmid was designated pMPI3mIF.
Insertion of I3L/murine IFNY cassette into the C3 locus; construction of pMPC3I3mIF. Plasmid pMPI3mIF was cut with HindIII and blunt ended with Klenow fragment of E. coli polymerase. The DNA was then cut with BamHI and a 0.6 kb fragment containing the I3L/murine IFNy cassette was isolated. This fragment was ligated with vector plasmid pVQC3LSA-3 cut with SmaI/BamHI, resulting in insertion plasmid pMPC3I3mIF.
Nucleotide sequence of the I3L/murine IFNy expression cassette is given in Figure 26 (SEQ ID
N0:163). The start codon for the murine IFNy gene is at 2o position 101, and the stop codon is at position 596.
Plasmid pVQC3LSA-3 was derived in the following manner. ALVAC C3 locus insertion plasmid VQCP3L (Example 17) was digested with NsiI and NotI and a 6503 by fragment isolated and ligated to annealed oligonucleotides CP34 (SEQ ID N0:151) 5' GGCCGCGTCGACATGCA 3' and CP35 (SEQ ID N0:152) 5' TGTCGACGC 3', generating plasmid VQCP3LSA-3. (Note:
Plasmid VQCP3LSA-3 is identical to plasmids VQCP3LSA-5 and VQCP3LSA, used in subsequent examples; see, e.a., 3o Examples 24, 25, 26.) Recombination between donor plasmid pMPC3I3mIF and ALVAC rescuing virus generated recombinant virus vCP271, which contains the I3L promoted murine IFNy gene in the C3 locus.
Insertion of Murine IFNy into NYVAC. Insertion of I3L/murine IFNy cassette into TK locus; construction of pMPTKI3mIF. Plasmid pMPI3mIF, defined above, was cut WO 94/16716 ~ PCTIUS94/00888 i4s with HindIII and blunt ended with Klenow fragment of E.
coli polymerase. The DNA was then cut with BamHI and a 0.6 kb fragment containing the I3L/murine IFNy cassette was isolated. This fragment was ligated with NYVAC TK
vector plasmid pSD542 (Example 18) cut with SmaI/BamHI, resulting in insertion plasmid pMPTKI3mIF.
Recombination between donor plasmid pMPTKI3mIF and NYVAC rescuing virus generated recombinant virus vP1237, which contains the I3L promoted murine IFNY gene in the TK locus.
Expression of murine IFNv in ALVAC and NYVAC based recombinants. ELISA assay. The level of expression of murine IFNy produced by ALVAC and NYVAC based recombinants vCP271 and vP1237 was quantitated using an ELISA kit from Genzyme Corporation, Cambridge, MA.
(InterTest-y Kit, Genzyme Corporation, cat # 1557-00).
Duplicate dishes containing confluent monolayers of mouse L-929 cells (2 x 106 cells/dish) were infected with recombinant virus vCP271 or vP1237 expressing murine IFN~y or infected with ALVAC or NYVAC parental virus.
Following overnight incubation at 37°C, supernatants were harvested and assayed for expression of murine IFNy using the InterTest-'y Kit as specified by the manufacturer (Genzyme Corporation, Cambridge, MA). The InterTest-y Kit is a solid-phase enzyme-immunoassay employing the multiple antibody sandwich principle. ELISA plates were read at 490 nm. Background from ALVAC or NYVAC samples was subtracted, and values from duplicate dishes were averaged. The quantity of murine IFNY secreted is 3o expressed as nanograms/ml, which is equivalent to ng/106 cells (Table 25).
Table 25 Recombinant virus Murine IFNv secreted vCP271 972 ng/ml vP1237 3359 ng/ml WO 94116716 2 I 5 3 3 3 6 ~T~S94/00888 Biolocrical assay. The biological activity of murine~., IFNY expressed by ALVAC and NYVAC based recombinants was quantitated using a standardized IFNy bio-assay (Vogel et al., 1991). This assay quantitates IFN activity by titrating its ability to protect L-929 cells from VSV
(vesicular stomatitis virus)-induced cytopathic effect (CPE) .
Confluent monolayers of mouse L-929 cells (2 x 106 cells/dish) were mock-infected or infected with NYVAC, 1o vP1237, ALVAC, or vCP271 at an moi of 5. Dishes were inoculated in duplicate. Following the 1 hour adsorption period, 1 ml of fresh medium was added to each dish and they were incubated overnight at 37°C. The supernatants from both dishes were pooled, filtered through a 0.22 ~,m 1s filter and tested for IFN activity as detailed below.
Two-fold serial dilutions of supernatants were tested, beginning at undiluted for mock infected, NYVAC, and ALVAC infected dishes, or 1:100 and 1:1000 for vCP271 and vP1237 infected dishes.
2o In the IFN-y bio-assay, 50 ~.1 medium was added to all wells of a 96-well plate, followed by 50 ~1 of a serial dilution of a stock of commercial murine IFN-y (Genzyme Corporation, MG-IFN, lot # B3649) or culture supernatant as described above. Next, 50 ~.1 of L-929 2s cells (3 x 104 cells) were added to each well, and plates were placed at 37°C overnight. After 24 hours, cells were infected with 100 ~,1 VSV (moi of 0.1). Plates were incubated at 37°C overnight. After 24 hours, CPE was assessed. The well which gave the same CPE as VSV in the 3o absence of interferon was defined as having 1 unit/ml interferon. This method coincided well with the interferon standard.
The interferon concentration in the supernatants is determined as the reciprocal of the dilution which gives 3s similar CPE to the standard at 1 unit/ml interferon. For mock-infected, NYVAC and ALVAC infected cells, less than 20 units/ml interferon is produced. For vP1237, 14,000 i5o units/ml IFN-y is produced, and for vCP271, 3,600 units/ml IFN-y is produced. vP1237 produced four-fold greater levels of IFN-y than vCP271, confirming results seen above by the ELISA assay. The lack of protection s conferred by supernatants from mock-infected or parental virus-infected cells shows that the protective activity in supernatants from vP1237- and vCP271-infected cells is IFN-y. This was confirmed by a neutralization assay which showed that antisera to IFN-y (Genzyme Corporation 1o monoclonal hamster anti-murine IFN-y, 1222-00, lot #B3847) but not antisera to murine IFN-a/Q (Lee Biomolecular Research, INC., San Diego, CA, No. 25301, lot. #89011) or murine IFN-(~ (Lee Biomolecular Research, Inc., No. 25101, lot. #87065) was capable of neutralizing i5 the protective activity produced by these recombinants.
Example 21 - HUMAN IFNy INTO ALVAC AND NYVAC
Insertion of Human IFNv into ALVAC. Plasmid p52 was obtained from ATCC (No. 65949). Plasmid p52 contains cDNA encoding the carboxy terminal 2/3 of the human IFNy 2o coding sequence with untranslated 3' region cloned into the PstI site of pBR322.
Linkage of human IFNy gene with I3L promoter;
Construction of pMPI3hIF.
(A) The missing region of the human IFNy gene was 25 synthesized using long, overlapping PCR primers, MPSYN615 (SEQ ID N0:164) 5' TAATCATGAAATATACAAGTTATATCTTGGCTTTTCAGCTCTGCATCGTTTTGGGTT
CTCTTGGCTGTTACTGCCAGGACCCATATGTAAAAGAAGC 3' and MPSYN616 (SEQ ID N0:165) 5' 3o TTCTTCAAAATGCCTAAGAAAAGAGTTCCATTATCCGCTACATCTGAATGACCTGCA
TTAAAATATTTCTTAAGGTTTTCTGCTTCTTTTACATATGGGTCCTGGC 3' with no extraneous template. The end of MPSYN615 is designed for cloning into the HpaI site of pMPI3H (Example 20).
There are 25 by of overlap between MPSYN615/MPSYN616. A
35 179 by PCR product was isolated.
(B) The remainder of human IFNy gene was synthesized using PCR primers MPSYN617 (SEQ ID N0:166) 5' TCTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAG 3' and MPSYN618 (SEQ ID N0:167) 5' CCCGGATCCCTGCAGTTACTGGGATGCTCTTCGA 3' with plasmid p52 as template. MPSYN617 has 25 by overlap with missing region; MPSYN618 is designed for cloning into the downstream BamHI site of pMPl3H. A ca. 350 by PCR
fragment was isolated.
(A+B) Combination PCR was performed using isolated fragments from (A) and (B), above, and external primers 1o MPSYN615 and MPSYN618. The PCR product was digested with BamHI, and a ca. 510 by fragment was isolated. This fragment was cloned into pMPI3H cut with HpaI/BamHI.
Following sequence confirmation, the resulting plasmid was designated pMPI3hIF. pMPI3hIF contains the human IFNy gene under the control of the I3L promoter.
Insertion of I3L/human IFNy cassette into C3 locus;
construction of pMPC3I3hIF. Plasmid pMPI3hIF was cut with HindIII and blunt ended with Klenow fragment of E.
coli polymerase. The DNA was then cut with BamHI and a 0.6 kb fragment containing the I3L/human IFNy cassette was isolated. This fragment was ligated with ALVAC C3 vector plasmid pVQC3LSA-3 (Example 20) cut with SmaI/BamHI, resulting in ALVAC insertion plasmid pMPC3I3hIF.
Nucleotide sequence of the I3L/human IFNy expression cassette is given in Figure 27 (SEQ ID
N0:168). The start codon for the human IFNy gene is at position 101, and the stop codon is at position 599.
Recombination between donor plasmid pMPC3I3hIF
3o and ALVAC rescuing virus generated recombinant virus vCP278, which contains the I3L promoted human IFNy gene in the C3 locus.
Insertion of Human IFNy into NYVAC. Insertion of I3L/human IFNV cassette into TK locus; construction of pMPTKI3hIF. Plasmid pMPI3hIF, described above, was cut with HindIII and blunt ended with Klenow fragment of E.
coli polymerase. The DNA was then cut with BamHI and a WO 94!16716 PCT/US94100888 0.6 kb fragment containing the I3L/human IFNy cassette was isolated. This fragment was ligated with NYVAC TK

vector plasmid pSD542 (Example 18) cut with SmaI/BamHI, resulting in NYVAC insertion plasmid pMPTKI3hIF.

Recombination between donor plasmid pMPTKI3hIF

and NYVAC rescuing virus generated recombinant virus vP1244, which contains the vaccinia I3L promoted human IFNy gene in the TK locus.

Expression of human IFNy in ALVAC and NYVAC based to recombinants ELISA assay The level of expression of human IFNy produced by ALVAC and NYVAC based recombinants vCP278 and vP1244 was quantitated using a human Interferon y ELISA kit from Genzyme Corporation, Cambridge, MA. (InterTest-yTM Kit, Genzyme Corporation, cat # 1556-00). Duplicate dishes containing confluent monolayers of human HeLa cells (2 x 106 cells/dish) were infected with recombinant virus vCP278 or vP1244 expressing human IFNy or infected with 2o ALVAC or NYVAC parental virus. Following overnight incubation at 37 C, supernatants were harvested and assayed for expression of human IFNy using the InterTest-y Kit as specified by the manufacturer (Genzyme Corporation, Cambridge, MA). The InterTest-y Kit is a solid-phase enzyme-immunoassay employing the multiple antibody sandwich principle. ELISA plates were read at 490 nm. Background from ALVAC or NYVAC samples was subtracted, and values from duplicate dishes were averaged. The quantity of human IFNy secreted is 3o expressed as nanograms/ml, which is equivalent to ng/106 cells (Table 26).

Table 26 Recombinant virus Human IFNy secreted vCP278 9 ng/ml vP1244 l5 ng/ml 21 ~3~36 Example 22 - Murine IL-2 plus IFNv into ALVAC and NYVAC
Insertion of Murine IFNv into C6 locus of ALVAC;
addition to ALVAC-murine IL-2 recombinant virus.
Derivation of C6 insertion vector. ALVAC C6 insertion vector pC6L was derived as follows. A 3.0 kb canarypox HindIII fragment containing the entire C6 ORF was cloned into the HindIII site of pBS-SK (Stratagene) to form plasmid pC6HIII3kb. Nucleotide sequence of the canarypox insert in pC6HIII3kb is presented in Figure 28 (SEQ ID
1o N0:169). In Figure 28, the C6 ORF is located between nucleotides 377 to 2254.
Extension of canarypox sequence to the right of pC6HIII3kb was obtained by sequence analysis of overlapping canarypox clones. In order to construct a i5 donor plasmid for insertion of foreign genes into the C6 locus with the complete excision of the C6 open reading frame, flanking 5' and 3' arms were synthesized by using PCR primers and genomic canarypox DNA as template. The 380 by 5' flanking arm was synthesized using primers C6A1 20 (SEQ ID N0:170) 5' ATCATCGAGCTCGCGGCCGCCTATCAAAAGTCTTAATGAGTT 3' and C6B1 (SEQ ID N0:171) 5' GAATTCCTCGAGCTGCAGCCCGGGTTTTTATAGCTAATTAGTCATTTTTTCGTAAGT
AAGTATTTTTATTTAA 3'. The 1155 by 3' flanking arm was 25 synthesized using primers C6C1 (SEQ ID N0:172) 5' CCCGGGCTGCAGCTCGAGGAATTCTTTTTATTGATTAACTAGTCAAATGAGTATATA
TAATTGAAAAAGTAA 3' and C6D1 (SEQ ID N0:173) 5' GATGATGGTACCTTCATAAATACAAGTTTGATTAAACTTAAGTTG 3'. Left and right flanking arms synthesized above were combined 3o by PCR reaction using primers C6A1 and C6D1, generating a full length product of 1613bp. This PCR product was cut near the ends with SacI/Kt~nI and cloned into pBS-SK cut with SacI/Kt~nI, generating C6 insertion plasmid pC6L.
pC6L contains, in the C6 deletion locus, a multicloning 35 region flanked by translational stop codons and TSNT
transcriptional terminators (Yuen and Moss, 1986). The sequence of pC6L is presented in Figure 29 (SEQ ID

WO 94/16716 . : PCTIUS94/00888 N0:174). In Figure 29, the multicloning region is located between nucleotide 407 and nucleotide 428.
Annealed synthetic oligonucleotides VQC (SEQ ID
N0:175) 5' TTAATCAGGATCCTTAATTAATTAGTTATTAGACAAGGTGAAACGAAACTATTTGTA
GCTTAATTAATTAGCTGCAGCCCGGG 3' and VQN (SEQ ID N0:176) 5' CCCGGGCTGCAGCTAATTAATTAAGCTACAAATAGTTTCGTTTTCACCTTGTCTAAT
AACTAATTAATTAAGGATCCTGATTAA 3' were ligated into pBS-SK
resulting in an intermediate plasmid. Plasmid pMM117 to contains a SmaI/EcoRI polylinker fragment from this intermediate plasmid replacing the SmaI/EcoRI polylinker of pC6L.
Plasmid pMP42GPT contains the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (Ecogpt is gene) (Pratt and Subramani, 1983) under the control of an entomopox promoter (EPV 42kDa). The 31 by EPV 42kDa promoter sequence (SEQ ID N0:177) used in pMP42GPT is 5' CAAAATTGAAAATATATAATTACAATATAAA 3'.
Insertion of 42kDa/Ecogpt cassette into C6 locus;
2o Construction of pMP117Qpt-B. Plasmid pMP42GPT was cut with EcoRI and a 0.7kb fragment containing the 42kDa/Ecogpt expression cassette was isolated. This fragment was inserted into vector plasmid pMM117 cut with EcoRI in both orientations, generating pMP117gpt-A and 2s pMP117gpt-B.
Insertion of I3L/murine IFNy cassette into C6 locus;
construction of pMPC6mIFQpt. Plasmid pMPI3mIF (Example 20) was cut with HindIII and blunt ended with Klenow fragment of E. coli polymerase. The DNA was then cut 3o with PstI (partial digest) and a 0.6 kb fragment containing the I3L/murine IFNy cassette was isolated.
Vector plasmid pMP117gpt-B was cut with SmaI (partial digest) and full length linear DNA was isolated. This was cut with PstI and the largest fragment was isolated.
35 Vector and insert fragments were ligated, resulting in insertion plasmid pMPC6mIFgpt. In addition to the I3L/murine IFNy expression cassette, plasmid pMPC6mIFgpt WO 94116716 21 ~ 3 ~ 3 6 pCT~S94/00888 155 , contains the 42kDa/Ecogpt expression cassette to allow for selection of recombinants through the use of --~°
mycophenolic acid (Boyle and Coupar, 1988; Falkner and Moss, 1988).
Recombination was accomplished between donor plasmid pMPC6mIFgpt and rescuing virus vCP275 (Example 18).
Recombinant virus are plaque purified. The resultant ALVAC based recombinant virus contains the vaccinia I3L
promoted murine IFNY gene, as well as the EPV 42kDa 1o promoted Ecogpt gene, both in the C6 locus and the vaccinia H6 promoted murine IL-2 gene in the C3 locus.
Insertion of Murine IFNy and Murine IL-2 into NYVAC.
Insertion of I3L~ murine IFNy cassette into TK locus;
construction of pMPTKm2IF. Plasmid pMPI3mIF (Example 20) was cut with HindIII and blunt ended with Klenow fragment of E. coli polymerase. The DNA was then cut with BamHI
and a 0.6 kb fragment containing the I3L/murine IFNy cassette was isolated. This fragment was ligated with vector plasmid pMM154 (Example 18) cut with SmaI/BamHI, 2o resulting in insertion plasmid pMPTKm2IF.
Recombination between donor plasmid pMPTKm2IF
and NYVAC rescuing virus generated recombinant virus vP1243, which contains the vaccinia I3L promoted murine IFNy gene and the vaccinia H6 promoted murine IL-2 gene, both in the TK locus.
Expression of murine IL-2 in ALVAC and NYVAC based recombinants: comparison with recombinants coexpressing murine IL-2 and murine IFNY. ELISA assay. The level of 3o expression of murine IL-2 produced by ALVAC based recombinants vCP275 and vCP288, and NYVAC based recombinants vP1239 and vP1243 was quantitated using a Murine Interleukin-2 ELISA kit from Collaborative Biomedical Products, Inc., Becton Dickinson, Bedford, MA.
(Mouse IL-2 ELISA kit Cat. No. 30032). Duplicate dishes containing confluent monolayers of murine L929 cells (2 x 106 cells/dish) were infected with recombinant virus WO 94/16716 ~ . , PCT/US94100888 vCP275 or vP1239 expressing murine IL-2, recombinant virus vCP288 or vP1243 coexpressing murine :CL-2 and murine IFNy, or infected with ALVAC or NYVAC parental virus. Following overnight incubation at 37 C, supernatants were harvested and assayed for expression of murine IL-2 using the mouse Interleukin-2 EhISA kit as specified by the manufacturer (Collaborative Biomedical Products, Inc., Becton Dickinson, Bedford, MA). The mouse Interleukin-2 ELISA kit is a solid-phase enzyme-lo immunoassay employing the multiple antibody sandwich principle. ELISA plates were read at 490 nm. Background from ALVAC or NYVAC samples was subtracted, and values from duplicate dishes were averaged. The mouse Interleukin-2 ELISA kit quantitates murine IL-2 in Biological Response Modifiers Program (BRMP) units (Gerrard et al., 1993). The quantity of murine IL-2 secreted is expressed as BRMP u/ml, which is equivalent to BRMP u/106 cells (Table 27).
2o Table 27 Recombinant virus Cytokines expressed Murine IL-2 secreted vCP275 mIL-2 1838 BRMP u/ml vCP288 mIL-2 + mIFNy 2124 BRMP u/ml vP1239 mIL-2 5030 BRMP u/ml vP1243 mIL-2 + mIFNy 4353 BRMP u/ml From the results reported in Table 27, it is evident that co-expression of murine IFNy does not affect the level of 3o murine IL-2 expression by ALVAC or NYVAC-based recombinants. Also, the level of murine IL-2 expression under the conditions of this assay is approximately twice .
as high for NYVAC based recombinants as it is for ALVAC
based recombinants, in agreement with the results presented in Table 23, which were based on a different murine IL-2 ELISA assay (Intertest-2XTM, Genzyme Corporation, Cambridge, MA).

WO 94116716 ~ PCTIUS94/00888 157 "
Example 23 - HUMAN IL-2 PLU8 IFNy INTO ALVAC AND NYVAC
Insertion of Human IFNY into C6 locus of ALVAC;
addition to ALVAC-Human IL-2 recombinant virus.
Insertion of I3LJhuman IFNY cassette into C6 locus;
construction of pMPC6I3hIF. Plasmid pMPI3hIF (Example 21) was cut with HindIII and blunt ended with Klenow fragment of E. cvli polymerase. The DNA was then cut with BamHI and a 0.6 kb fragment containing the I3L/human IFNy cassette was isolated. ALVAC C6 vector plasmid to pMM117 (Example 22) was cut with BamHI (partial)/SmaI and the largest fragment was isolated. These fragments were ligated, resulting in insertion plasmid pMPC6I3hIF.
Recombination was accomplished between donor plasmid pMPC6I3hIF and rescuing virus vCP277 (Example 19). Recombinants are plaque purified. The resultant ALVAC based recombinant virus contains the vaccinia I3L
promoted human IFNy gene in the C6 locus and the vaccinia H6 promoted human IL-2 gene in the C3 locus (vCP277+IFNy).
2o Insertion of Human IFNv and IL-2 into NYVAC.
Insertion of H6/human IL-2 cassette into the TK locus;
construction of pMPTK2hIF. Plasmid pMPTKI3hIF (Example 21) was cut with PstI and phosphatased with Shrimp alkaline phosphatase. Plasmid pRW858 (Example 19) was cut with PstI and a 700 by fragment isolated.
Phosphatased vector and insertion fragment were ligated, resulting in plasmid pMPTK2hIF. In pMPTK2hIF, the two human cytokine gene cassettes are both contained within the NYVAC TK insertion site, oriented in a tail-to-tail orientation. The human IFNY gene is under the control of the vaccinia I3L promoter, and the human IL-2 gene is under the control of the vaccinia H6 promoter.
Recombination was accomplished between donor plasmid pMPTKh2IF and NYVAC rescuing virus. Recombinants are plaque purified. The resultant NYVAC based recombinant virus contains the vaccinia I3L promoted human IFNy gene and the vaccinia H6 promoted human IL-2 WO 94116716 . . ~' , . .. PCTIUS94100888 gene, both in the TK locus (NYVAC+IFNY+IL-2).

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Murine IL-4 into ALVAC. Plasmid p2A-E3, containing the murine IL-4 gene, (m IL-4) was obtained from the American Type Culture Collection (ATCC No. 37561). The murine IL-4 gene was placed under the control of the vaccinia E3L promoter by PCR as described below.

The vaccinia E3L promoter, a strong early promoter, is located immediately upstream from the 1o vaccinia E3L open reading frame (Goebel et al., 1990).

Nucleotide sequence of the E3L/murine IL-4 expression cassette is presented in Figure 30 (SEQ ID

N0:178). In Figure 30, the start codon of the murine IL-4 gene is at nucleotide position 68, and the stop codon is at nucleotide position 488.

The mIL-4 gene was amplified by PCR with oligonucleotide primers MIL45 (SEQ ID N0:179) 5' CTCACCCGGGTACCGAATTCGAATAAAAAAATGATAAAGTAGGTTCAGTTTTATTGC

TGGTTGTGTTAGTTCTCTCTAAAAATGGGTCTCAACCCCCAG 3' and MIL43 (SEQ ID N0:180) 5' TTAGGGATCCAGATCTCGAGATAAAAACTACGAGTAATCCATTTGCATGATGCTC

3' and plasmid p2A-E3 (ATCC) as template. The resulting fragment contained the mIL-4 gene linked to the vaccinia E3L promotor and flanked by XmaI/KpnI/EcoRI and XhoI/BqlII/BamHI sites at the 5' and 3' ends, respectively. The amplified E3L/mIL-4 fragment was digested with XmaI/BamHI and ligated to an XmaI/BamHI-digested pVQCP3LSA-5 vector fragment. Plasmid pVQCP3LSA-5 (same as VQCP3LSA-3, Example 20) is an ALVAC C3 locus 3o insertion plasmid. The resulting C3 donor plasmid was designated pC3.MIL4.2. An expression cassette consisting of the E. coli gpt gene linked to the entomopox 42kDa promoter was isolated as a SmaI fragment from plasmid pMP42GPT (Example 22), then cloned into a SmaI-digested pC3.MIL4.2 vector fragment. The resulting plasmid was designated pC3MIL4.gpt.

Recombination was accomplished between donor WO 94116716 21 ~ 3 3 3 fi PCTIUS94/00888 159 '.. .
plasmid pC3MIL4.gpt and ALVAC rescuing virus using the mycophenolic acid selection system (Example 22).
Recombinant virus are plaque purified. The resultant recombinant virus contains the murine IL-4 gene under the control of the vaccinia E3L promoter, as well as the Ecogpt gene under the control of the EPV 42kDa promoter, both at the C3 locus of ALVAC.
Murine IL-4 into NYVAC. The mIL-4 gene was amplified by PCR with primers MIL45 (SEQ ID N0:179) and 1o MIL43 (SEQ ID N0:180) and plasmid p2A-E3 (ATCC) as template. The resulting fragment contained the mIL-4 gene linked to the vaccinia E3L promotor and flanked by XmaI/KpnI/EcoRI and XhoI/BqlII/BamHI sites at the 5' and 3' ends, respectively. The amplified E3L/mIL-4 fragment was digested with XmaI/BamHI. NYVAC TK insertion plasmid pSD542 (Example 18) was digested with XmaI/BamHI and ligated to the XmaI/BamHI-digested PCR fragment. The resulting TK donor plasmid was designated pTK-mIL4.
Recombination between donor plasmid pTK-mIL4 2o and NYVAC rescuing virus generated recombinant virus vP1248 which contains the vaccinia E3L promoted murine IL-4 gene in the TK locus.
Example 25 - HUMAN IL-4 INTO ALVAC AND NYVAC
Human IL-4 into ALVAC. Plasmid pcD-hIL-4, containing the human IL-4 gene, was obtained from the American Type Culture Collection (ATCC No. 57593).
PCR fragment PCRhIL4-I was synthesized using plasmid pcD-hIL-4 as template DNA and synthetic oligonucleotides E3LIL4-C (SEQ ID N0:181) 5' 3o GCTGGTTGTGTTAGTTCTCTCTAAAAATGGGTCTCACCTCCCAACTG 3' and E3LIL4-D (SEQ ID N0:182) 5' ATCATCTCTAGAATAAAAATCAGCTCGAACACTTTGAATATTTCTCTCTCATG 3' as primers.
Oligonucleotides E3LIL4-A (SEQ ID N0:183) 5' ATCATCAAGCTTGAATAAAAAAATGATAAAGTAGGTTCAGTTTTATTGCTGGTTGTG
TTAGTTCTCTCTAAAA 3' and E3LIL4-B (SEQ ID N0:184) 5' TTTTAGAGAGAACTAACACAACCAGCAATAAAACTGAACCTACTTTATCATTTTTTT

WO 94!16716 PCTIUS94100888 ATTC 3' were annealed to generate Fragment II containing the vaccinia E3L promoter sequence (Example 24).
A second fusion PCR product (PCRhIL4-II) was ~t obtained using PCR fragment PCRhIL4-I and Fragment II
s (annealed oligos) as DNA template and E3LIL4-D and E3LIL4-E (SEQ ID N0:185) 5' ATCATCAAGCTTGAATAAAAAAATGATAAAGTAGGTTCAG 3' as oligonucleotide primers. A complete HindIII/XbaI digest of PCRhIL4-II yielded an 536 by fragment which was 1o subsequently isolated. A complete HindIII/XbaI digest of pBS-SK+ (Stratagene) was performed and the 2.9 kb fragment isolated. The isolated fragments were ligated, resulting in plasmid pBShIL4.
Figure 31 (SEQ ID N0:186) presents the nucleotide is sequence of the expression cassette consisting of the E3L
promoted human IL-4 gene. The start codon for the human IL-4 gene is at nucleotide position 62, and the stop codon is at nucleotide position 521.
A complete XbaI digest of plasmid pBShIL4 was 2o performed. Ends were filled in using Klenow fragment of E. coli polymerase. This linearized plasmid was then digested with XhoI and the 536 by fragment, containing the E3L promoter and human IL4 gene, was isolated. C3 insertion vector plasmid VQCP3LSA (same as pVQCP3LSA-3, 2s Example 20) was completely digested with XhoI/SmaI and the 6.5 kb fragment isolated. The isolated fragments were ligated, resulting in insertion plasmid pC3hIL4.
Recombination between donor plasmid pC3hIL4 and ALVAC rescuing virus generated recombinant virus vCP290, 3o which contains the vaccinia E3L promoted human IL-4 gene in the C3 locus.
Human IL-4 into NYVAC. Plasmid pBShIL4 (discussed above) contains the E3L/human IL-4 expression cassette in pBS-SK. A complete XbaI digest of plasmid pBShIL4 was 35 performed. Ends were filled in using Klenow fragment of E. coli polymerase. This linearized plasmid was then digested with XhoI and the 536 by fragment, containing the E3L promoter and human IL-4 gene, was isolated.
NYVAC TK insertion vector plasmid pSD542 (Example 18) was completely digested with XhoI/SmaI and the 3.9 kb fragment isolated. The isolated fragments were ligated, resulting in insertion plasmid pTKhIL4.
Recombination between donor plasmid pTKhIL4 and NYVAC rescuing virus generated recombinant virus vP1250, which contains the vaccinia E3L promoted human IL-4 gene in the TK locus.
1o Example 26 - HUMAN GMCSF IN ALVAC AND NYVAC
Human GMCSF into ALVAC. Plasmid GMCSF, containing the gene encoding the human granulocyte-macrophage colony-stimulating factor (hGMCSF), was obtained from the American Type Culture Collection (ATCC No. 39754).
is PCR fragment GMCSF-I was synthesized using plasmid GMCSF as template DNA and E3LGMC-A (SEQ ID N0:187) 5' GCTGGTTGTGTTAGTTCTCTCTAAAAATGTGGCTGCAGAGCCTGCTG 3' and E3LGMC-B (SEQ ID N0:188) 5' ATCATCCTCGAGATAAAAATCACTCCTGGACTGGCTCCCAGCAGTCAAAGGGG
20 3' as oligonucleotide primers.
Synthetic oligonucleotides E3LSMA-B (SEQ ID N0:189) 5' ATCATCCCCGGGGAATAAAAAAATGATAAAGTAGGTTCAGTTTTATTGCTGGTTGTG
TTAGTTCTCTCTAAAA 3' and E3LIL4-B (SEQ ID N0:184; Example 2s 25) were annealed to generate fragment GMCSF-P containing the vaccinia E3L promoter sequence.
A fusion PCR product (GMCSF-II) was obtained using fragments GMCSF-I and GMCSF-P as DNA templates and E3LSMA-A (SEQ ID N0:190) 5' 3o ATCATCCCCGGGGAATAAAAAAATGATAAAGTAGGTTCAG3' and E3LGMC-B
as oligonucleotide primers. A complete XhoI/SmaI digest of GMCSF-II yielded a 0.5 kb fragment which was subsequently isolated. A complete XhoI/SmaI digest of pBS-SK+ was performed and the 2.9 kb fragment isolated.
3s The isolated fragments were ligated, resulting in plasmid pBSGMCSF, which contains the vaccinia E3L/hGMCSF
expression cassette.

WO 94!16716 PCT/US94/00888 Nucleotide sequence of the vaccinia E3L/hGMCSF
expression cassette is given in Figure 32 (SEQ ID
''f N0:191). In Figure 32, the start codon for hGMCSF is at nucleotide position 62, the stop codon is at nucleotide position 494.
A complete XhoI/SmaI digest of pBSGMCSF (above) was performed and the 0.5 kb fragment, containing the vaccinia E3L promoter and hGMCSF gene, was isolated.
ALVAC C3 insertion plasmid VQCP3LSA (Example 20) was 1o completely digested with XhoI/SmaI and the 6.5 kb fragment isolated. The isolated fragments were ligated, resulting in plasmid pC3hGMCSF.
Recombination between donor plasmid pC3hGMCSF and ALVAC rescuing virus generated recombinant virus vCP285, which contains the vaccinia E3L promoted human GMCSF gene in the C3 locus.
Human GMCSF into NYVAC. A complete XhoI/SmaI digest of pBSGMCSF was performed and the 0.5 kb fragment, containing the vaccinia E3L promoter and hGMCSF gene, was 2o isolated. pSD542 (Example 18) was completely digested with XhoI/SmaI and the 3.9 kb fragment isolated. The isolated fragments were ligated, resulting in plasmid pTKhGMCSF.
Recombination between donor plasmid pTKhGMCSF and NYVAC rescuing virus generated recombinant virus vP1246, which contains the vaccinia E3L promoted human GMCSF gene in the TK locus.
Expression of human GMCSF in ALVAC and NYVAC based recombinants. ELISA assav. The level of expression of 3o human GMCSF produced by ALVAC and NYVAC based recombinants vCP285 and vP1246 was quantitated using an ELISA kit from Genzyme Corporation, Cambridge, MA.
(Factor-Test Human GM-CSF ELISA Kit, Genzyme Corporation, product code GM-TE.) Duplicate dishes containing confluent monolayers of human HeLa cells (2 x 106 cells/dish) were infected with recombinant virus vCP285 or vP1246 expressing human GMCSF or infected with ALVAC

WO 94/16716 21 ~ 3 3 3 ~G ~T~S94100888 or NYVAC parental virus. Following overnight incubation at 37°C, supernatants were harvested and assayed for expression of human GMCSF using the Factor-Test Human GM-CSF ELISA kit as specified by the manufacturer (Genzyme Corporation, Cambridge, MA). The Factor-Test Human GM-CSF ELISA Kit is a solid-phase enzyme-immunoassay employing the multiple antibody sandwich principle.
ELISA plates were read at 490 nm. Background from ALVAC
or NYVAC samples was subtracted, and values from to duplicate dishes were averaged. The quantity of human GMCSF secreted is expressed as picograms(pg)/ml, which is equivalent to pg/106 cells (Table 28).
Table 28 is Recombinant virus Human GMCSF secreted vCP285 2413 pg/ml vP1246 4216 pg/ml Example 27 - HUMAN IL-12 IN ALVAC AND NYVAC
Derivation of DNA encoding the two subunits of the human IL-12 ctene. First strand cDNA synthesis was performed on total RNA isolated from human EBV
transformed cell line GJBCL stimulated 24 hrs. with 100nM
Phorbol 12,13-Dibutyrate. Oligonucleotide primers used for PCR amplification of the genes encoding the p35 and p40 subunits (below) were based on the published human IL-12 sequence (Gubler et al., 1991).
3o The p40 subunit of the human IL-12 gene (hIL12p40) was obtained as PCR fragment PCR J60 using first strand cDNA from cell line GJBCL as template and oligonucleotides JP202 (SEQ ID N0:192) 5' CATCATATCGATGGTACCTCAAAATTGAAAATATATAATTACAATATAAAATGTGTC
ACCAGCAGTTGG 3' and JP189 (SEQ ID N0:193) 5' TACTACGAGCTCTCAGATAGAAATTATATCTTTTTGGG 3' as primers.
PCR J60 was cut with SacI/ClaI and a 1.0 kb fragment was isolated and ligated with pBSSK+ (Stratagene), cut with SacI/ClaI, generating plasmid PBSHIL12p40II. In plasmid 4o PBSHIL12p40II, hIL12p40 is under the control of the entomopox 42kDa promoter (Example 22).
The sequence of the EPV 42kDa/human IL-12 P40 expression cassette is presented in Figure 33 (SEQ ID
N0:194). In Figure 33, the initiation codon for the s human IL-12 P40 subunit is at nucleotide position 32, the stop codon is at nucleotide position 1017.
The p35 subunit of the human IL-12 gene (hIL12p35) was obtained as PCR fragment PCR J59 using first strand cDNA from cell line GJBCL as template as oligonucleotides l0 JP186 (SEQ ID N0:195) 5' CATCATGGTACCTCAAAATTGAAAATATATAATTACAATATAAAATGTGTCCAGCGC
GCAGCC 3' and JP201 (SEQ ID N0:196) 5' TACTACATCGATTTAGGAAGCATTCAGATAG 3' as primers. PCR J59 was cut with Asp718/Clal and a 0.7 kb fragment was 1s isolated and ligated with pBSSK+ (Stratagene), generating plasmid PG2. The hIL12p35 gene was put under the control of the vaccinia E3L promoter (Example 24) by a PCR
reaction using plasmid PG2 as template and oligonucleotides JP218 (SEQ ID N0:197) 5' TTAGTTCTCTCTAAAAATGTGTCCAGCGCGCAGCC 3' and JP220 (SEQ ID
N0:198) 5' CATCATATCGATTTAGGAAGCATTCAGATAGCTCGTCAC 3' as primers. PCR J62 was cut with Asp718/ClaI and a 0.7 kb fragment was isolated and ligated with pBSSK+
2s (Stratagene), generating plasmid PBSHIL12p35II.
The sequence of the vaccinia E3L/human IL-12 P35 expression cassette is presented in Figure 34 (SEQ ID
N0:199). In Figure 34, the initiation codon for the human IL-12 P35 subunit is at nucleotide position 62, the 3o stop codon is at nucleotide position 719.
A cassette containing poxvirus-promoted genes for both subunits of human IL-12 was assembled in pBSSK+ by ligating a 0.7kb As~718/ClaI fragment from PBSHIL12p35II
and a l.Okb Asp718/SacI fragment from PBSHIL12p40II into 3s pBSSK+ cut with SacI/ClaI. The resulting plasmid was designated PBSHIL12. In PBSHIL12 the EPV 42kDa/hIL12p40 cassette and the vaccinia E3L/hIL12p35 cassette are I
WO 94!16716 2 1 5 3 3 3 fi p~~S94100888 oriented in a head-to-head orientation relative to each other.
Human IL-12 into ALVAC. The combination cassette containing poxvirus-promoted genes for both subunits of human IL-12 was excised as a l.7kb SacI/ClaI fragment from plasmid PBSHIL12. The fragment was blunt-ended by treatment with the Klenow fragment of E. coli polymerase, and cloned into ALVAC C6 vector plasmid pC6L (Example 22) cut with SmaI. The resulting plasmid was designated l0 pC6HIL12.
Recombination was performed between donor plasmid pC6HIL12 and ALVAC rescuing virus. Recombinant virus are plaque purified. The resultant recombinant virus (ALVAC
+ IL-12) contains both of the human IL-12 genes in the C6 locus of ALVAC.
Human IL-12 into NYVAC. The combination cassette containing poxvirus-promoted genes for both subunits of human IL-12 was excised as a l.7kb SacI/ClaI fragment from plasmid PBSHIL12. The fragment was blunt-ended by 2o treatment with the Klenow fragment of E. coli polymerase, and cloned into NYVAC TK vector plasmid pSD542 (Example 18) cut with SmaI. The resulting plasmid was designated pTKHIL12.
Recombination was performed between donor plasmid pTKHIL12 and NYVAC rescuing virus. Recombinant virus are plaque purified. The resultant recombinant virus (NYVAC
+ IL-12) contains both of the human IL-12 genes in the TK
locus of NYVAC.
Example 28 - MURINE B7 IN ALVAC AND NYVAC
3o Murine B7 into ALVAC. Preparation of cDNA for murine B7. Macrophages from a naive Balb/c mouse spleen were stimulated in vitro with Concanavalin A and LPS.
Total RNA from these cells was used as a template for first-strand cDNA synthesis by reverse transcription using oligo dT as a primer. An aliquot of first strand cDNA preparation was used for the specific murine B7 cDNA
amplification by PCR using the primers LF32 (SEQ ID

WO 94116716 ' ' PCTIUS94100888 N0:200) 5' TATCTGGAATTCTATCGCGATATCCGTTAAGTTTGTATCGTAATGGCTTGCAATTGT
CAG 3' and LF33 (SEQ ID N0:201) 5' ATCGTAAGCTTACTAAAGGAAGACGGTCTG 3'. The specific primers LF32 and LF33 were derived from the published sequence of murine B7 (Freeman and al., 1991). Nucleotides 5' to the ATG in LF32 correspond to part of the vaccinia H6 promoter (Perkus et al., 1989). The amplified 951 nucleotide cDNA fragment containing the murine B7 gene 1o was digested by EcoRI and HindIII and subsequently cloned into the corresponding sites of the plasmid pBSSK+
(Statagene). The resulting plasmid, pLFl, was digested with NruI and XhoI, and a 949 by fragment containing part of the vaccinia H6 promoter and the entire murine B7 gene was isolated.
Plasmid pMPC616E6 contains a non relevant gene under the control of the vaccinia H6 promoter in the ALVAC C6 insertion locus. Plasmid pMPC616E6 was digested with NruI and XhoI, and the 4,403 by NruI-XhoI fragment 2o containing the bulk of the H6 promoter in the ALVAC C6 insertion locus was isolated. This vector fragment was ligated with the NruI/XhoI fragment from pLFi. The resulting plasmid was named pLF4.
Nucleotide sequence of the murine B7 gene is given in Figure 35 (SEQ ID N0:202). In Figure 35, the start codon for the murine B7 gene is at nucleotide 1 and the stop codon is at nucleotide 919.
Recombination between donor plasmid pLF4 and ALVAC rescuing virus generated recombinant virus vCP268, 3o which contains the vaccinia H6 promoted murine B7 gene in the C6 locus.
Murine B7 into NYVAC. Plasmid pSIVl2 contains a nonrelevant gene under the control of the vaccinia H6 promoter in the NYVAC I4L insertion locus. Plasmid pSIVl2 was digested with NruI and XhoI, and the 3,557 by NruI-XhoI fragment containing the bulk of the H6 promoter in the NYVAC I4L insertion locus was isolated. This WO 94/16716 PCTlUS94/00888 167 . .~
fragment was ligated to annealed synthetic ~' oligonucleotides LF57 (SEQ ID N0:203) 5' CGACATTTGGATTTCAAGCTTCTACG 3' and LF58 (SEQ ID N0:204) 5' GATCCGTAGAAGCTTGAAATCCAAATGTCG 3' which contain an internal HindIII site. The resulting plasmid, pLF2, was digested with NruI and HindIII, and a 3,659bp vector fragment was isolated. Plasmid pLFl (above) was digested with NruI and HindIII, and a 951 by NruI-HindIII fragment containing part of the vaccinia H6 promoter and the to entire murine B7 gene was isolated. These two fragments were ligated, generating plasmid pLF3.
Plasmid pLF3 corresponds to an I4L NYVAC donor plasmid containing the entire murine B7 coding sequence under the control of the vaccinia H6 promoter.
is Recombination between donor plasmid pLF3 and NYVAC
rescuing virus generated recombinant virus vP1230, which contains the vaccinia H6 promoted murine B7 gene in the I4L locus.
Surface expression of B7 on murine tumor cells 2o infected with ALVAC and NYVAC-based recombinants expressing murine B7. K1735 mouse melanoma cells and CC-36 mouse colon carcinoma cells were infected with 10 pfu per cell of NYVAC-B7 (vP1230), ALVAC-B7 (vCP268), or NYVAC or ALVAC parental virus for 1 hour, washed free of 2s unadsorbed virus by centrifugation, and incubated at 37°C. B16 mouse melanoma cells were treated similarly except that the cells were infected with 5 pfu of virus per cell. After a 1 hr (K1735) or overnight (CC-36;B16) incubation, the cells were washed in PBS by 3o centrifugation and resuspended in 1.0 ml of PBS. To each cell preparation, 0.005 ml of 1:5 diluted Fc Block (Pharmingen, San Diego, CA, cat. 01241A; purified anti-mouse Fcy II receptor) and 0.1 ml of 1:100 diluted FITC-rat anti-mouse B7 monoclonal antibody (Pharmingen, cat.
35 01944D) was added. The cells were incubated for 30 minutes at 4°C, washed twice in cold PBS by centrifugation and analyzed for cell-associated FITC

TWO 94116716 . ' PCTIUS94100888 d' __ i6a fluorescence by flow cytometry (Becton-Dickinson FACScan).
Although K1735 cells infected with NYVAC-B7 (vP1230) or ALVAC-B7 (vCP268) for 1 hour showed only slightly higher fluorescence than control uninfected or NYVAC or ALVAC infected cells, B7 expression in recombinant infected CC-36 and B16 cells was remarkable (Figure 36). As demonstrated by the uninfected control cells, none of the three cell lines endogenously io expresses murine 87. Clearly, infection of established murine tumor cell lines with NYVAC-B7 (vP1230) or ALVAC-B7 (vCP2G8), but not the vectors NYVAC or ALVAC, results in high levels of expression of the murine T-lymphocyte co-activator molecule, BB-1/B7.
Example 29 - HUMAN B7 IN NYVAC
Preparation of cDNA for human B7.
Macrophages from human peripheral blood were stimulated in vitro with Concanavalin A and LPS. Total RNA from these cells was used as a template for first 2o strand cDNA synthesis by reverse transcription using oligo dT as a primer. An aliquot of first strand DNA
preparation was used for specific human B7 cDNA
amplification by PCR using the primers LF62 (SEQ ID
N0:205) 5' ATCGTAAGCTTATTATACAGGGCGTACACTTTC 3' and LF6lbis (SEQ ID N0:206) 5' TATCTGGAATTCTATCGCGATATCCGTTAAGTTTGTATCGTAATGGGCCACACACGG
AGG 3'.
The specific primers LF62 and LF6lbis were derived from the published sequence of human B7 (Freeman and al., 1989). Nucleotides 5' to the ATG in LF6lbis correspond to part of the vaccinia H6 promoter (Perkus et al., 1989). The amplified 997 nucleotide cDNA fragment containing the murine B7 gene was digested by EcoRI and HindIII and subsequently cloned into the corresponding sites of the plasmid pBSSK+ (Stratagene). This plasmid was designated pLF6.
The sequence for the human B7 gene is presented in WO 94/16716 215 3 3 3 ~ ~T~S94I00888 Figure 37 (SEQ ID N0:207). In Figure 37, the start codon for the human B7 gene is at nucleotide position 1 and the stop codon is at nucleotide position 865.
Insertion of Human B7 into NYVAC.
Plasmid pLF3 (Example 28) was digested with NruI and HindIII and a 3652 by vector fragment containing the bulk of the H6 promoter in the NYVAC I4L insertion locus was isolated. Plasmid pLF6 (above) was digested with NruI
and HindIII, and a 897 by fragment containing part of the to vaccinia H6 promoter and the entire human B7 gene was isolated. These two fragments were ligated, generating plasmid pLF7.
Plasmid pLF7 corresponds to an I4L NYVAC donor plasmid containing the entire human B7 coding sequence under the control of the vaccinia H6 promoter.
Recombination between donor plasmid pLF7 and NYVAC
rescuing virus generated recombinant virus vP1245, which contains the vaccinia H6 promoted human B7 gene in the I4L locus.
2o Expression of human B7. FACScan.
Human HeLa cells were infected with recombinant virus vP1245 expressing human B7 or with NYVAC parental virus. A monoclonal antibody specific for human B7 (Anti-BB1(B7), Cat. No. 550024, Becton Dickinson Advanced Cellular Biology, San Jose, CA), was used to detect expression of human B7 on the surface of infected cells by flow cytometry (Becton-Dickinson FACScan) as described in Example 28. B7 was detected on the surface of cells infected with recombinant virus vP1245. B7 was not 3o detected on the surface of uninfected cells or cells infected with NYVAC parental virus.
Immuno~recipitation. NYVAC based recombinant virus vP1245 was assayed for expression of the human B7 gene using immunoprecipitation. Recombinant or parental virus 3s were inoculated onto preformed monolayers of tissue culture cells in the presence of radiolabelled 35S-methionine and treated as previously described (Taylor et 17o al., 1990). Immunoprecipitation reactions were performed using a monoclonal antibody specific for human B7 (Anti-BB1(B7), Cat. No. 550024, Becton Dickinson Advanced Cellular Biolo gy, San Jose, CA). A protein of s between approximately 44 and 54 kDa was precipitated from ~t cells infected with recombinant virus vP1245, in agreement with Freeman et al. (1989). The protein was not immunoprecipitated from uninfected cells or cells infected with NYVAC parental virus.
1o Example 30 - CO-INSERTION OF MURINE IFNy AND MURINE 87 INTO ALVAC AND NYVAC
Co-Insertion of Murine IFNy and Murine B7 into ALVAC. Recombination was accomplished between donor 15 plasmid pLF4 (Example 28) and rescuing virus vCP271 (Example 20). Recombinant virus are plaque purified.
The resultant ALVAC based recombinant virus (ALVAC + IFNy + B7) contains the vaccinia I3L promoted murine IFNy gene in the C3 locus and the vaccinia H6 promoted murine B7 2o gene in the C6 locus.
Co-Insertion of Murine IFNY and Murine B7 into NYVAC. Recombination was accomplished between donor plasmid pMPTKmIF (Example 20) and rescuing virus vP1230 (Example 28). Recombinant virus are plaque purified.
25 The resultant NYVAC based recombinant virus (NYVAC+IFNy+B7) contains the vaccinia I3L promoted murine IFN~y gene in the TK locus and the vaccinia H6 promoted murine B7 gene in the I4L locus.
Example 31 - INSERTION OF WILDTYPE AND MUTANT FORMS OF

The gene for the nuclear phosphoprotein p53 is the gene most frequently found to be mutated in a wide variety of human tumors (reviewed in Hollstein et al., 3s 1991). NYVAC and ALVAC-based p53 recombinant virus. are described in Example 15.
Insertion of wildtype Murine p_53 into ALVAC.
Plasmid p11-4 containing murine wild-type p53 was received from Arnold Levine (Princeton University, WO 94/16716 ~ ~ PCTIUS94I00888 W
Princeton, New Jersey). The p53 sequence is described in Pennica et al., (1984). The murine wild-type p53 gene was placed under the control of the vaccinia H6 promoter and the p53 3' non coding end was removed with PCR-derived fragments.
A fragment containing the H6 promoted 5' end of the p53 gene fused to the 3' end of the p53 gene was generated by several PCRs as described below.
PCR I: Plasmid pRW825, containing the H6 promoter 1o and a nonpertinent gene, was used as template with oligonucleotides MM080 (SEQ ID N0:208) 5' ATTATTATTGGATCCTTAATTAATTAGTGATACGC 3' and MM081 (SEQ ID
N0:209) 5' CTCCTCCATGGCAGTCATTACGATACAAACTTAAC 3' producing a 228bp fragment containing the H6 promoter and the 5'-most base pairs of the murine p53 gene. MM080 anneals to the 5' end of the H6 promoter and primes toward the 3' end.
MM081 anneals to the 3' end of the H6 promoter and primes toward the 5' end.
2o PCR II: Plasmid p11-4 was used as template with oligonucleotides MM082 (SEQ ID N0:210) 5' CGTTAAGTTTGTATCGTAATGACTGCCATGGAGGAGTC 3' and MM083 (SEQ
ID N0:211) 5' TAGTAGTAGTAGTAGCTTCTGGAGGAAGTAGTTTCC 3' to generate a 129bp fragment with the 3'-end of the H6 promoter, the 5' end of the p53 gene followed by l5bp which overlaps PCR
fragment PCRIII (described below). MM082 contains the 3' end of the H6 promoter and primes from the 5' end of the murine p53 gene. MM083 anneals to position 97 (Figure 38) of the murine p53 gene and primes toward the 5' end.
PCRIII: Plasmid p11-4 was used as template with oligonucleotides MM084 (SEQ ID N0:212) 5' CAGAAGCTACTACTACTACTACCCACCTGCACAAGCGCC 3' and MM085 (SEQ
ID N0:213) 5' AACTACTGTCCCGGGATAAAAATCAGTCTGAGTCAGGCCCCAC 3' to generate a 301bp fragment. The 301bp PCR-derived fragment contains the 3' end of the p53 gene, and the 5' end overlaps the 3' end of the PCRII product. MM084 (SEQ
ID N0:212) primes from position 916 of the murine p53 gene toward the 3' end. MM085 (SEQ ID N0:213) primes from position 1173 toward the p53 gene 5' end. The three s PCR products were pooled and primed with MM080 and MM085. ' The resultant 588bp fragment contains a BamHI site followed by the H6 promoted 5' end of the p53 gene fused to the p53 gene 3' end followed by a SmaI site; the 5' end of the p53 gene ends at the XhoI site at position 37, to and the 3' end starts at the SacII site at position 990 (Figure 38). The 588bp PCR-derived fragment was digested with BamHI and SmaI generating a 565bp fragment which was inserted into BamHI/SmaI digested pNC5LSP5 (described below). The resultant plasmid, designated pMM136, was 15 digested with KspI and XhoI to remove a 149bp fragment, and the 953bp KS~I/XhoI fragment from p11-4 was inserted.
The resultant plasmid, pMM148, contains the H6 promoted wild-type murine p53 in the ALVAC C5 insertion locus.
The construction of pNC5LSP5 is as follows. A C5 2o insertion vector plasmid pCSLSP (Example 14) was digested with EcoRI, treated with alkaline phosphatase and ligated to self-annealed oligonucleotide CP29 (SEQ ID N0:102) 5' AATTGCGGCCGC 3', then digested with NotI and linear purified followed by self-ligation. This procedure 25 introduced a NotI site to pCSLSP, generating pNC5LSP5.
The nucleotide sequence of the wildtype murine p53 gene is presented in Figure 38 (SEQ ID N0:214). The start codon is at position 1 and the stop codon is at position 1171.
3o Recombination between donor plasmid pMM148 and ALVAC rescuing virus generated recombinant virus vCP263.
vCP263 contains the wild type murine p53 gene under the control of the vaccinia H6 promoter in the C5 locus.
Insertion of a mutant form of Murine p53 into ALVAC.
35 Plasmid pSVK215 containing a mutant form of the murine p53 gene was received from Arnold Levine (Princeton University, Princeton, New Jersey). The mutation in WO 94116716 3 ~ PCTIUS94100888 pSVKH215 changes the sequence GTAC of the murine p53 coding sequence (Figure 38) nt positions 643 through 646 ...
to CCAAGCTTGG. The insertion between nt positions 643 and 646 changes the predicted amino acid coding sequence s from val-pro to pro-ser-leu-ala; and the insertion replaces a K~nI site with a HindIII site. The construction of pSVKH215 is described in Tan et al., (1986).
Plasmid pMM136 (described above) contains the to vaccinia H6 promoted 5' end of the p53 gene fused to the 3' end of the p53 gene in an ALVAC C5 locus insertion plasmid. pMM136 was digested with KspI and XhoI to remove 149bp, and the 960bp KspI/XhoI fragment containing the mutation described above from pSVKH215 was inserted.
1s The resultant plasmid, pMM149, contains the H6 promoted murine mutant p53 gene in the C5 locus.
Recombination between donor plasmid pMM149 and ALVAC rescuing virus generated recombinant virus vCP267.
vCP267 contains the mutant form of the murine p53 gene 2o under the control of the vaccinia H6 promoter in the C5 locus.
Example 32 - INSERTION OF MUTANT FORMS OF HUMAN P53 INTO AhVAC AND NYVAC
25 Mutant forms of Human o53 into ALVAC.
Figure 18 (Example 15) presented the sequence of the vaccinia H6 promoted human wild type p53 gene cassette in an ALVAC-based recombinant, vCP207. In this example, to facilitate description of the mutant forms of the human 3o p53 gene being described, Figure 39 (SEQ ID N0:215) presents only the coding sequence for the human wild type p53 gene. The start codon is at position 1 and the stop codon is at position 1180.
Plasmid Cx22A, containing a mutant form of the human 3s p53 gene, was received from Arnold Levine (Princeton University, Princeton, New Jersey). Relative to the wild type p53 sequence presented in Figure 39, the G at nucleotide position 524 is substituted with an A, WO 94116716 ~ ' ' ~ PCT/US94100888 changing the arg amino acid at codon 175 of the wild type Zc' protein to a his amino acid in Cx22A.
Plasmid pMM110 (Example 15, Figure 18) contains the vaccinia H6 promoted wildtype human p53 gene in the ALVAC
C5 insertion site. The human p53 gene contains two PflmI
sites. p53 coding sequences upstream from the first PflmI site and downstream from the second PflmI site are the same in pMM110 as in Cx22A. pMM110 was digested with PflmI to remove the 853 central base pairs of the p53 1o gene. The 853bp PflmI fragment from Cx22A containing the base change at position 524 was inserted. The resultant plasmid, pMM143, contains the H6 promoted mutant p53 gene.
Recombination between donor plasmid pMM143 and ALVAC
rescuing virus generated recombinant virus,vCP270.
vCP270 contains the mutant form of the human p53 gene under the control of the vaccinia H6 promoter in the C5 locus.
Plasmid pR4-2 containing a mutant form of the human 2o p53 gene was received from Arnold Levine (Princeton University, Princeton, New Jersey). Relative to the wild type p53 sequence presented in Figure 39, the G at nucleotide position 818 is substituted by an A, changing the arg codon at amino acid position 273 to a his codon in pR4-2.
Plasmid pMM110 (Example 15, Figure 18) contains the vaccinia H6 promoted human wildtype p53 gene in the ALVAC
C5 insertion site. p53 coding sequences upstream from the first PflmI site and p53 coding sequences downstream 3o from the second PflmI site are the same in pMM110 as in pR4-2. pMM110 was digested with PflmI to remove the 853 central base pairs of the p53 gene. The 853bp PflmI
fragment from pR4-2 containing the base change at -nucleotide position 818 was inserted. The resultant plasmid, pMM144, contains the H6 promoted mutant form of the human p53 gene in the C5 insertion locus.
Recombination between donor plasmid pMM144 and ALVAC

WO 94/16716 215 3 3 3 ~ PCT/US94/00888 rescuing virus generated recombinant virus vCP269.
vCP269 contains the mutant form of the human p53 gene under the control of the vaccinia H6 promoter in the C5 locus.
Mutant forms of Human p53 into NYVAC.
Plasmid Cx22A, described above, contains a mutant form of the human p53 gene, in which the G at nucleotide position 524 (Figure 39) is substituted by an A, changing the arg codon at amino acid position 175 to a his codon 1o in Cx22A.
Plasmid pMM106 (Example 15) contains the vaccinia H6 promoted wild-type human p53 gene in the NYVAC I4L
insertion locus. p53 coding sequences upstream from the first PflmI site and p53 coding sequences downstream from the second PflmI site are the same in pMM106 as in Cx22A.
pMM106 was digested with PflmI to remove the 853 central base pairs of the p53 gene. The 853bp PflmI fragment from Cx22A containing the base change at position 524 was inserted. The resultant plasmid, pMM140, contains the H6 2o promoted mutant p53 gene.
Recombination between donor plasmid pMM140 and NYVAC
rescuing virus generated recombinant virus vP1234.
vP1234 contains the mutant form of the human p53 gene under the control of the vaccinia H6 promoter in the I4L
locus .
Plasmid pR4-2, described above, contains a mutant form of the human p53 gene, in which the G at nucleotide position 818 (Figure 39) is substituted by an A, changing the arg codon at amino acid position 273 to a his codon 3o in pR4-2.
pMM106 (Example 15) contains the H6 promoted wild-type human p53 gene in the I4L locus. p53 coding sequences upstream from the first PflmI site and p53 coding sequences downstream from the second PflmI site are the same in pMM106 as in pR4-2. pMM106 was digested with PflmI to remove the 853 central base pairs of the p53 gene. The 853bp PflmI fragment from pR4-2 containing r i76 C~ the base change at position 818 was inserted. The resultant plasmid, pMM141, contains the H6 promoted mutant p53 gene.
Recombination between donor plasmid pMM141 and NYVAC
s rescuing virus generated recombinant virus vP1233.
vP1233 contains the mutant form of the human p53 gene under the control of the vaccinia H6 promoter in the I4L
locus.
A listing of the wildtype and mutant forms of murine 1o p53 and the mutant forms of human p53 present in ALVAC
and NYVAC recombinants described in Examples 31 and 32 is provided in Table 29.
Table 29 15 Recombinant Virus Parent Virus Species Gene Insert vCP263 ALVAC murine w.t. p53 vCP267 ALVAC murine p53 (+3 aa) vCP270 ALVAC human p53 (aa 175; R to H) vCP269 ALVAC human p53 (aa 273; R to H) 2 s vP1234 NYVAC human p53 (aa 175; R to H) vP1233 NYVAC human p53 (aa 273; R to H) 3o Immunoprecipitation.
ALVAC and NYVAC based recombinants vP1101, vP1096, vP1098, vCP207, vCP193, vCP191 (all described in Example 15; Table 22, as well as ALVAC and NYVAC based recombinants vCP270, vCP269, vP1233, vP1234 described in 35 this Example, Table 29), contain wild type or mutant forms of the human p53 gene. All of these recombinant virus were assayed for expression of the human p53 gene using immunoprecipitation.
Recombinant or parental virus were inoculated onto 4o preformed monolayers of tissue culture cells in the presence of radiolabelled 35S-methionine and treated as previously described (Taylor et al., 1990).

Immunoprecipitation reactions were performed using a human p53 specific monoclonal antibody 1801. A protein of between 47 and 53 kDa was precipitated foom cells infected with any of the recombinant viruses, vP1101, vP1096, vP1098, vCP207, vCP193, vCP191, vCP270, vCP269, vP1233, or vP1234, but not from uninfected dwells or cells infected with parental ALVAC or NYVAC virus.
Based upon the properties of the poxvirus vector systems, NYVAC, ALVAC and TROVAC cited above, such 1o vectors expressing either wildtype or mutant forms of p53 provide valuable reagents to determine whether endogenous CTL activities can be detected in patient effector populations (TILs, PBMC, or lymph node cells); and, valuable vehicles for the stimulation or the augmenting of such activities; for instance, augmenting such activities by in vitro or ex vivo stimulation with these recombinant viruses. Further, the highly attenuated properties of both NYVAC and ALVAC allow the recombinants of the invention to be used for interventive 2o immunotherapeutic modalities discussed above, e.g., in vivo interventive immunotherapy Example 33 - ERH-H-2 INTO COPAR
Plasmid ErbB2SphIstop was obtained from Jeffrey Marks (Duke University Center). ErbB2SphIstop contains a 3.8 kb human erb-B-2 cDNA insert cloned in pUCl9. The insert extends from nt 150 through nt 3956 (Yamamoto et al., 1986) and contains the entire erb-B-2 coding sequence. In ErbB2SphIstop, the St~hI site at nt 2038 was mutagenized by the addition of an XbaI linker, creating 3o an in frame stop codon. The remaining, truncated, ORF
thus specifies an extracellular, secretable form of the erb-B-2 gene product, mimicking the translation product of the 2.3 kb mRNA. Plasmid ErbB2SphIstop was digested with XhoI and the 3.8 kb erb-B-2 fragment was isolated.
This isolated fragment was ligated with COPAK vector plasmid pSD555 cut with XhoI, resulting in plasmid pMM113.

17s Plasmid pSD555 was derived as follows. Plasmid pSD553 (Example 17) is a vaccinia deletion/insertion plasmid of the COPAK series. It contains the vaccinia K1L host range gene (Gillard et al., 1986) within flanking Copenhagen vaccinia arms, replacing the ATI
region (orfs A25L, A26L; Goebel et al., 1990).
Plasmid pSD553 was cut with NruI and ligated with a SmaI/NruI fragment containing the synthetic vaccinia H6 promoter element (Perkus et al., 1989) upstream from the 1o NruI site located at -26 relative to the translation initiation codon. The resulting plasmid, pMP553H6, contains the vaccinia H6 promoter element located downstream from the K1L gene within the A26L insertion locus.
To complete the vaccinia H6 promoter and add a multicloning region for the insertion of foreign DNA, plasmid pMP553H6 was cut with NruI/BamHl and ligated with annealed synthetic oligonucleotides MPSYN349 (SEQ ID
N0:216) 5' 2o CGATATCCGTTAAGTTTGTATCGTAATGGAGCTCCTGCAGCCCGGGG 3' and MPSYN350 (SEQ ID N0:217) 5' GATCCCCCGGGCTGCAGGAGCTCCATTACGATACAAACTTAACGGATATCG 3'.
The resulting plasmid, pSD555, contains the entire H6 promoter region followed by a multicloning region.
Recombination between donor plasmid pMM113 and NYVAC
rescuing virus generated recombinant virus vP1100.
vP1100 contains the erb-B-2 gene under the control of the vaccinia H6 promoter in the I4L locus, along with the vaccinia K1L host range gene.
3o Immunoprecit~itation. Preformed monolayers of Vero cells were inoculated at 10 pfu per cell with parental NYVAC virus and recombinant virus vP1100 in the presence of radiolabelled 35S-methionine and treated as previously described (Taylor et al., 1990). Immunoprecipitation reactions were performed using a human erb-B-2 specific monoclonal antibody TA1-1C. A protein of approximately 97 kDa was precipitated from cells infected with vP1100, WO 94116716 ~ ~ PCTIUS94/00888 but not from uninfected cells or cells infected with parental NYVAC virus.
Having thus described in detail preferred embodiments of the present invention, it is to be s understood that the invention defined by the appended claims is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

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Claims

CLAIMS:

1. A recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA
encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus is a recombinant vaccinia virus in which the regions C7L-K1L, J2R, B13R+B14R, A56R and I4L have been deleted therefrom.

2. A recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA
encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus is a recombinant vaccinia virus wherein the open reading frames for the thymidine kinase gene, the hemorrhagic region, the A
type inclusion body region, the hemagglutinin gene, the host range gene region, and the large subunit ribonucleotide reductase gene have been deleted therefrom.

3. A recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA
encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus vector is a recombinant NYVAC, wherein the open reading frames for the thymidine kinase gene, the hemorrhagic region, the A type inclusion body region, the hemagglutinin gene, the host range gene region, and the large subunit ribonucleotide reductase gene have been deleted therefrom.

4. A recombinant poxvirus containing therein exogenous DNA coding for a cytokine, exogenous DNA encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen in a non-essential region of the poxvirus genome, wherein the poxvirus expresses the exogenous DNA, wherein the poxvirus vector is the recombinant vaccinia virus NYVAC.

5. A recombinant poxvirus containing in a non-essential region of the poxvirus genome exogenous DNA
encoding at least one cytokine, immunostimulatory molecule, or tumor-associated antigen, wherein the poxvirus vector is ALVAC.

6. The recombinant poxvirus of any one of claims 1 through 5 wherein the exogenous DNA encodes at least one cytokine and at least one tumor antigen.

7. The recombinant poxvirus of any one of claims 1 through 5 wherein the exogenous DNA encodes at least one cytokine and at least one immunostimulatory molecule.

8. The recombinant poxvirus of any one of claims 1 through 5 wherein the exogenous DNA encodes at least one tumor antigen and at least one immunostimulatory molecule.

9. The recombinant poxvirus of any one of claims 1 through 5, wherein the exogenous DNA encodes at least one of human tumor necrosis factor .alpha., wildtype p53, mutant p53, a human melanoma-associated antigen, IL-2, IFN.gamma., IL-4, GM-CSF, IL-12, B7, erb-B-2 or carcinoembryonic antigen.

10. A recombinant poxvirus selected from the group consisting of vP1200, vP1101, vP1098, vP1239, vP1241, vP1237, vP1244, vP1243, vP1248, NYVAC+IFN.gamma.+IL-2, vP1250, vP1246, NYVAC+IL-12, vP1230, vP1245, NYVAC+IFN.gamma.+IR7, vP1234, vP1233, vP1100 and vP1096.

11. A composition for inducing an immunological response comprising the virus as claimed in any one of claims 1 through 10 in admixture with a suitable carrier.

12. A method for expressing a gene product in a cell cultured in vitro comprising introducing into the cell the virus as claimed in any one of claims 1 to 10.