# Patent

## Title
Insertion sites in fowlpox vectors

## Abstract

The present invention provides novel insertion sites for introducing DNA into pox vectors.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 National Phase Entry Application of co-pending International Application PCT/US2004/005077, filed 20 Feb. 2004, which designated the U.S. and which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application 60/448,591, filed 20 Feb. 2003.


INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 375,958 Byte ANSI (Text) file named “701315ReplacementSeqListing_ST25.txt,” created on Jul. 31, 2009.
FIELD OF THE INVENTION
The present invention relates generally to improved pox vectors utilizing novel insertion sites, and their use as an immune stimulating agent, such as a vaccine. The improved poxviruses can be used for example for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system.
BACKGROUND OF THE INVENTION
In recent years, attention has been given to the development of recombinant poxvirus technology. Such poxvirus-based vectors are useful for a range of uses, for example generating immune responses such as vaccines, for the development of new vaccines, for delivery of desired proteins and for gene therapy. The advantages of these vectors include: (i) ease of generation and production; (ii) the large size of the genome permitting insertion of multiple genes, (iii) efficient delivery of genes to multiple cell types, including antigen-presenting cells; (iv) high levels of protein expression; (v) optimal presentation of antigens to the immune system; and (vi) the ability to elicit cell-mediated immune responses as well as antibody responses; and (vii) the long-term experience gained with using this vector in humans as a small pox vaccine.
Poxviruses can be genetically engineered to contain and express foreign DNA with or without impairing the ability of the virus to replicate. Such foreign DNA can encode a wide range of proteins, such as antigens that induce protection against one or more infectious agents, immune modulating proteins such as co-stimulatory molecules, or enzymatic proteins. For example, recombinant vaccinia viruses have been engineered to express immunizing antigens of herpesvirus, hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses (Kieny et al., Nature 312:163-6 (1984); Smith et al., Nature 302: 490-5 (1983); Smith et al., Proc. Natl. Acad. Sci. USA 80:7155-9 (1983); Zagury et al., Nature 326:249-50 (1987); Cooney et al., Lancet 337:567-72 (1991); Graham et al., J. Infect. Dis. 166:244-52 (1992), and have been shown to elicit immune responses against influenza virus, dengue virus, respiratory syncytial virus, and human immunodeficiency virus.
Poxviruses have also been used to generate immune reactions against tumor-associated antigens such as CEA, PSA and MUC.
Poxviruses are also attractive candidates for use in gene therapy for the delivery of genetic material into cells for therapeutic use. See U.S. Pat. No. 5,656,465. Compared to other systems such as retrovirus vectors (including lentiviral vectors), adenoviral vectors, and adeno-associated virus vectors, the large genome of poxviruses enables large genes to be inserted into pox-based vectors. Yet, because of the cytoplasmic nature of the virus integration of foreign DNA into a host cell's chromosomes will not occur.
Historically, poxviruses were used as a vaccine for protection against smallpox. Variola virus is the etiological agent of smallpox. During the smallpox era, overall mortality rates were approximately 30%. Vaccinia virus is a highly effective immunizing agent that enabled the global eradication of smallpox. The last naturally occurring case of smallpox occurred in Somalia in 1977. In May 1980, the World Health Assembly certified that the world was free of naturally occurring smallpox. As a result, smallpox vaccination was halted in the US, except for military personnel and laboratory workers. However, recent world events have resulted in a renewed interest in smallpox vaccination as a response to potential bioterrorism.
Vaccinia virus is the prototype of the genus Orthopoxvirus. It is a double-stranded DNA (deoxyribonucleic acid) virus that has a broad host range under experimental conditions but is rarely isolated from animals outside the laboratory (Fenner et al. Orthopoxviruses. San Diego, Calif.: Academic Press, Inc., 1989; Damaso et al., Virology 277:439-49 (2000)). Multiple strains of vaccinia virus exist that have different levels of virulence for humans and animals. For example, the Temple of Heaven and Copenhagen vaccinia strains are highly pathogenic among animals, whereas the NYCBOH strain, from which the Wyeth vaccine strain was derived, had relatively low pathogenicity (Fenner et al. Smallpox and its eradication. Geneva, Switzerland: World Health Organization, 1988). Dryvax,® the vaccinia (smallpox) vaccine currently licensed in the United States, is a lyophilized, live-virus preparation of infectious vaccinia virus (Wyeth Laboratories, Inc., Marietta, Pa.). However, Dryvax® is associated with adverse effects due to local or generalized vaccinia virus replication in children, the elderly and the immunosuppressed.
Attention has focused on attenuated orthopox such as NYVAC (U.S. Pat. No. 5,364,773) and modified vaccinia Ankara (MVA). MVA was derived from the Ankara vaccinia strain CVA,1 which was used in the 1950s as a smallpox vaccine. In 1958, attenuation experiments were initiated in the laboratory of Dr. Anton Mayr (University of Munich) comprising terminal dilution of CVA in chicken embryo fibroblast (CEF) cells that ultimately resulted in over 500 passages. The resulting MVA is an attenuated, replication-defective virus, which is restricted to replication primarily in avian cells.2 Comparison of the MVA genome to its parent, CVA, revealed 6 major deletions of genomic DNA (deletion I, II, III, IV, V, and VI), totaling 31,000 basepairs. (Meyer et al., J. Gen. Virol. 72:1031-8 (1991)). MVA has been administered to numerous animal species, including monkeys, mice, swine, sheep, cattle, horses and elephants with no local or systemic adverse effects.1,3,4 Over 120,000 humans have been safely vaccinated with MVA by intradermal, subcutaneous or intramuscular injections.3 MVA has also been reported to be avirulent among normal and immunosuppressed animals (Mayr et al., Zentralb. Bakteriol. 167:375-90 (1978). Accordingly, in addition to utility as a smallpox vaccine, these more attenuated strains are particularly attractive poxviruses for use as vectors for immune modulation and gene therapy.
One of the main advantages of poxviruses as vectors is the large size of their genomes, which permits the insertion of a wide range of genetic material including multiple genes (i.e., as a multivalent vector). However, the genetic material must be inserted at an appropriate site within the pox genome for the recombinant virus to remain viable. Thus, the genetic material must be inserted at a site in the viral DNA which is non-essential.
Accordingly, it is desirable to identify specific sites within a poxvirus genome that can accommodate insertion of foreign DNA, while retaining the ability to infect foreign cells and express that DNA, while maintaining the desired immunogenicity and diminished virulence. Certain insertion sites are known in different poxviruses. For example, as described above, MVA contains 6 natural deletion sites, which have been demonstrated to serve as insertion sites. See e.g. U.S. Pat. No. 5,185,146, and U.S. Pat. No. 6,440,422. However, these insertion sites are only found in one specific strain of vaccinia, MVA. It would be desirable to identify insertion sites which are more broadly represented, i.e. are present in other attenuated strains, other vaccinia strains such as CVA, Wyeth and other vaccinia strains in addition to MVA, as well as present in other poxvirus genomes.
Accordingly, there remains a need in the art for improved pox vectors containing novel insertion sites, for use for example for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the PANVAC-F recombinant vector schematic. To generate PANVAC-F, two plasmid vectors were used. The first plasmid, designated pT1154, directs insertion of the wCEA(6D) and wMUC-1(6) coding sequences into the FP14 region of the fowlpox virus genome. The second plasmid, designated pT8150, directs insertion of the LFA-3, ICAM-1, and B7.1 coding sequences (collectively known as TRICOM) into the BamHIJ region of the fowlpox virus genome. The wCEA(6D) gene is under the transcriptional control of the vaccinia 40K promoter. The wMUC-1(6) gene is under the transcriptional control of the synthetic early/late (sE/L) promoter. The LFA-3 gene is under the transcriptional control of the vaccinia 30K promoter, the ICAM-1 gene is under the transcriptional control of the vaccinia I3 promoter, and the B7.1 gene is under the transcriptional control of the synthetic early/late (sE/L) promoter. In addition, pT1154 contains the E. coli lacZ gene, under the control of the vaccinia 40K promoter, which is included as a screen for recombinant progeny, and pT8150 contains the GUS gene, under the control of the 7.5 promoter, for use in screening for recombinant progeny.
SUMMARY OF THE INVENTION
The present invention provides improved pox vectors containing novel insertion sites. The improved poxviruses can be used for example for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system.



DETAILED DESCRIPTION
We have now discovered improved pox vectors containing novel insertion sites, for inserting foreign nucleic acid. The improved poxviruses can be used for example for parenteral immunization, as a vector system, or in the active or inactivated form as an adjuvant or as a regulator of the unspecific components of the immune system. Preferred pox include orthopoxviruses such as vaccinia virus, avipox such as fowl pox and canary pox, and swine pox. One preferred type of vaccinia is an attenuated vaccinia such as Modified Vaccinia Ankara (MVA) or NYVAC. MVA is preferred. These vectors can be used in virtually any setting.
Poxviruses are well known cytoplasmic viruses. Thus, genetic material expressed by such viral vectors typically remains in the cytoplasm and does not have the potential for inadvertent integration of the genetic material into host cell genes, unless specific steps are taken. As a result of the non-integrative, cytoplasmic nature of the poxvirus, the poxvirus vector system will not result in having term persistance in other cells. Thus, the vector and the transformed cells will not adversely affect cells in the host animal at locations distant from the target cell. Preferably, the pox used is attenuated relative to a vaccinia such as vaccinia Copenhagen in a mammalian cell. This includes both replicative or replication impaired pox relative to the target cell. For example, an avipox in a mammalian cell, a suipox in a human cell, etc.
The recombinant poxviruses having utility in the present invention have a number of attributes, including (i) efficient delivery of genes to multiple cell types, including APC and tumor cells; (ii) high levels of protein expression; (iii) large genomes, allowing delivery of a wide range of genetic material including multiple genes (i.e., as a multivalent vector), (iv) optimal presentation of antigens to the immune system; (v) the ability to elicit cell-mediated immune responses as well as antibody responses; (vi) transient, rather than permanent, genetic modification of cells, and (vii) the ability to use combinations of poxviruses from different genera, as they are not immunologically cross-reactive.
One preferred poxvirus of the present invention is vaccinia virus, including highly attenuated vaccinia viruses such a Modified Vaccinia Ankara (MVA), NYVAC, a derivative of the Wyeth strain, vTBC33, which lacks a functional K1L gene, and the like. A particularly preferred vaccinia virus is an attenuated vaccinia, including NYVAC and MVA. More preferably, MVA.
Another preferred poxvirus of the present invention is an avipox, including but not limited to fowlpox, and canary pox, including ALVAC. Preferably fowlpox.
Insertion Sites
The novel insertion sites of the present invention are located in intragenic regions between known genes, also referred to herein as open reading frames or simply ORFs, and allow the introduction of foreign DNA at the insertion sites.
The present invention provides methods for identifying novel insertion sites in poxviruses. Novel insertion sites can be identified by analyzing the a poxvirus genome to identify sequences with the following characteristics. First, the insertion site should lie in an intergenic space, preferably between non-essential genes. Second, the insertion of foreign DNA at the insertion site should not disrupt any cryptic ORFs in the region, or promoters of adjacent genes or other regulatory elements.
Any putative insertion site identified using the method of the present invention can be tested by construction of a recombinant poxvirus with foreign DNA (e.g. a marker) inserted at the putative site to allow testing of the recombinant poxvirus, to ensure that any essential genes, non-essential genes, and cryptic ORFs have not been affected.
For example, the method for identifying novel insertion sites can be used to identify novel insertion sites in vaccinia. In one embodiment, novel insertion sites can be identified by analyzing the MVA genome (see Antoine et al., 1998) to identify sequences with the characteristics described above—namely, the site should lie in an intergenic space, preferably between non-essential genes, and the insertion of foreign DNA at that site should not disrupt any cryptic ORFs. In addition, assessment of the essential nature of the MVA genes can be determined by comparing the MVA with its vaccinia homologue. Furthermore, there should be essential genes between the native sites of the 40K, 7.5K, and I3 promoters and the insertion site, and T5NT sequences of surrounding genes should be left intact.
Three particularly preferred insertion sites of the present invention in vaccinia are designated insertion site 44/45, insertion site 49/50, and insertion site 124/125, with the numbers designating the ORFs lying on either side of the insertion site. Particularly preferred vaccinia viruses include attenuated vaccinia viruses such as MVA, NYVAC (attenuated), and Wyeth strain, as well as non-attenuated strains such as TROYVAC.
In one preferred embodiment, the novel insertion site in vaccinia is designated insertion site 44/45. In MVA, insertion site 44/45 lies between ORFs 044L and 045L, and the insertion site is between positions 37346-37357 in the MVA genomic sequence (Genbank Accession # U94848). This region is 5′ of the translational start codon of MVA 044L and 3′ of the translational stop codon of MVA 045L. In vaccinia Copenhagen, for insertion site 44/45 the corresponding ORFs are F14L (homologous to MVA 044L) and F15L (MVA 045L), and the insertion site is 5′ of the translational start codon of vaccinia F14L and 3′ of the translations stop codon of vaccinia F15L. Vaccinia Copenhagen, which contains this region and has its sequence available as Genbank Accession number M35027, is a preferred vaccinia. Similarly, insertion site 44/45 can also be used in other vaccinia strains including NYVAC (where the insertion site is not known to be modified) and TROYVAC. In this embodiment, the DNA sequence at the specified insertion site, i.e. between the nucleotides, contains defined inserts representing a sequence of interest; the flanking nucleotides on both sides remain unchanged.
In another preferred embodiment, the novel insertion site in vaccinia is designated insertion site 49/50. In MVA, insertion site 49/50 lies between ORFs 049L and 050L, and the insertion site is between positions 42687-42690 in the MVA genomic sequence (Genbank Accession # U94848). This region is 5′ of the translational start codon of MVA 049L and 3′ of the translational stop codon of MVA 050L. In vaccinia Copenhagen, for insertion site 49/50 the corresponding ORFs are E2L (homologous to MVA 049L) and E3L (MVA 050L), and the insertion site is 5′ of the translational start codon of vaccinia E2L and 3′ of the translations stop codon of vaccinia E3L. Vaccinia Copenhagen is a prototypical vaccinia. Similarly, insertion site 49/50 can also be used in other vaccinia strains including NYVAC (where the insertion site is not known to be modified) and TROYVAC. In this embodiment, the DNA sequence at the specified insertion site, i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
In yet another preferred embodiment, the novel insertion site in vaccinia is designated insertion site 124/125. In MVA, insertion site 124/125 lies between ORFs 124L and 125L, and the insertion site is between positions 118481-118482 in the MVA genomic sequence (Genbank Accession # U94848). This region is 5′ of the translational start codon of MVA 124L and 3′ of the translational stop codon of MVA 125L. In vaccinia Copenhagen, for insertion site 124/125 the corresponding ORFs are A13L (homologous to MVA 124L) and A14L (MVA 125L), and the insertion site is 5′ of the translational start codon of vaccinia A13L and 3′ of the translations stop codon of vaccinia A14L. Similarly, insertion site 124/125 can also be used in other vaccinia strains including NYVAC (where the insertion site is not known to be modified) and TROYVAC. In this embodiment, the DNA sequence at the specified insertion site, i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
Another preferred poxvirus of the present invention is an avipox, including but not limited to fowlpox, and canary pox, including ALVAC.
A particularly preferred avipoxvirus is fowlpox.
Particularly preferred fowlpox insertion sites of the present invention are designated the LUS insertion site, the FP14 insertion site, and the 43K insertion site. These sites are also referred to sometimes as FPV006/FPV007 (LUS insertion site), FPV254/FPV255 (LUS insertion site), FPV060/FPV061 (FP14 insertion site), and FPV107/FPV108 (43K insertion site).
In one preferred embodiment, the novel insertion site in fowlpox is designated the LUS insertion site. In fowlpox, there are two long unique sequences (LUS) at each end of the viral genome (Genbank Accession # AF198100), and thus two LUS insertion sites in each genome. The LUS insertion site at the left end of the genome is between positions 7470-7475 in the fowlpox genomic sequence, and lies 3′ of FPV006 and 5′ of FPV007 125L. The LUS insertion site at the right end of the genome is between positions 281065 and 281070 in the fowlpox genomic sequence, and lies 5′ of FPV254 and 3′ of FPV255. In this embodiment, an insert representing a sequence of interest can be inserted at any position within the specified insertion site.
In another preferred embodiment, the novel insertion site in fowlpox is designated the FP14 insertion site. This site is between positions 67080-67097 in the fowlpox genomic sequence, and lies 5′ of FPV060 and 3′ of FPV061. In this embodiment, the DNA sequence at the specified insertion site, i.e. between the nucleotides, is deleted in the recombinant virus and replaced with defined inserts representing a sequence of interest.
In yet another preferred embodiment, the novel insertion site in fowlpox is designated the 43K insertion site. This site is at position 128178 of the fowlpox genomic sequence, and lies 5′ of FPV107 and 5′ of FPV108. These genes are divergently transcribed, and the insertion site lies between the two promoter elements for the two ORFs. In this embodiment, an insert representing a sequence of interest can be inserted at this position within the fowlpox genome.
Poxviruses
Poxviruses having utility in the present invention include replicating and non-replicating vectors. Such poxviruses include but are not limited to orthopox such as vaccinia, raccoon pox, rabbit pox and the like, avipox (e.g. fowl pox, canary pox), suipox (e.g. swine pox), capripox (e.g. sheep pox), leporipox, and iridoviruses. Other DNA viruses include iridoviruses and the like. Poxviruses may be selected from the group consisting of vaccinia-MVA strain, vaccinia-Copenhagen, vaccinia-Wyeth strain, NYVAC, TROVAC; avipox such as fowl pox or canarypox such as ALVAC; suipox such as swinepox, and the like. In one embodiment, the recombinant vector is a vaccinia virus. Preferably, an attenuated vaccinia such as MVA or NYVAC. Other preferred vectors include an avipox such as fowl pox or canary pox.
Parental poxviruses useful in the method of the present invention include but are not limited to orthopoxvirus such as highly attenuated vaccinia viruses such as modified vaccinia Ankara (MVA) (Sutter and Moss, Proc. Nat'l Acad. Sci. U.S.A., 89:10847-10851; Sutter et al Virology 1994), replicating vaccinia virus (Perkus et al Science 229:981-984, 1985; Kaufman et al Int. J. Cancer 48:900-907, 1991, Moss Science 252:1662, 1991), Wyeth; avipoxviruses such as fowlpoxvirus, canary poxviruses, such as ALVAC and the like (Baxby and Paoletti, Vaccine 10:8-9, 1992; Rinns, M. M. et al (Eds) Recombinant Poxviruses CRC Press, Inc, Boca. Raton 1992-Paoletti, E. Proc. Nat'l Acad. Sci. USA 93:113491-11353, 1996), and suipoxvirus, capripoxvirus and the like.
In one preferred embodiment, the vaccinia virus is a Wyeth strain or derivative thereof. A derivative of the Wyeth strain includes but is not limited to vTBC33 which lacks a functional K1L gene and the like. In yet another embodiment, the virus is Dry-Vax available as a smallpox vaccine from the Centers for Disease Control, Atlanta, Ga. In another embodiment, the parental poxvirus is a strain of fowlpox, for example POXVAC-TC (Schering-Plough Corporation), and the like.
In another preferred embodiment, the vaccinia virus is a modified vaccinia virus Ankara (MVA) or derivative thereof. MVA has been generated by long-term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., et al., Infection, 3:6-14 (1975). The MVA virus itself may be obtained from a number of public repository sources. For example, MVA was deposited in compliance with the requirements of the Budapest Treaty at CNCM (Institut Pasteur, Collection Nationale de Cultures Microorganisms, 25, rue du Docteur Roux, 75724 Paris Cedex 15) on Dec. 15, 1987 under Depositary No. I-721 (U.S. Pat. No. 5,185,146); MVA virus was deposited in compliance with the Budapest Treaty at the European Collection of Cell Cultures (ECACC) (CAMR, Porton Down, Salisbury, SP4 OJG, UK) on Jan. 27, 1994, under Depository No. V94012707) (U.S. Pat. No. 6,440,422 and United States patent publication number 20030013190). Also, United States patent publication number 0030013190 further discloses particular MVA strains deposited at the ECACC under Depository No. 99101431, and ECACC provisional accession number 01021411. All of the above documents are herein incorporated by reference in their entirety. Therion Biologics brand MVA products, identified by the tradenames Therion-MVA(TM), Therion Prifree(TM) Vectors and Therion M-Series Vectors(TM), are products of Therion Biologics Corporation, Cambridge, Mass., United States.
The poxvirus of the present invention is able to infect, transfect or transduce host cells in a host. The host includes but is not limited to mammals, including humans, birds, fish and the like. The host cells are any cell amenable to infection, transfection or transduction by the poxvirus and capable of expressing the poxvirus; including any foreign genes inserted therein, at functional levels.
The poxviruses of the present invention are sometimes referred to herein as a viral vector or a vector system or simply a vector.
The poxvirus of the present invention preferably has a low replicative efficiency in the target cell. This preferably means that no more than about 1 progeny per cell are produced, still more preferably, no more than 0.1 progeny per cell. Replication efficiency can readily be determined empirically by determining the virus titer after infection of the target cell.
As a result of the low replication efficiency and the non-integrative, cytoplasmic nature of the vector, the vector system will not result in sustained replication and infection of other cells. Thus, the pox vector and transformed cells will not adversely affect cells in the host animal at locations distant from where the target cell is.
The poxvirus gene delivery system described herein can be used for any host. Preferably, the host will be a mammal. Preferred mammals include primates such as humans and chimpanzees, domestic animals such as horses, cows, pigs, etc. and pets such as dogs and cats. More preferably, the host animal is a primate or domestic animal. Still more preferably the host animal is a primate such as a human.
In order to further ensure that the poxvirus vector used for a particular host animal is avirulent in that animal, in addition to the above criteria, one can readily screen for a viral vector by looking at the virus's host range and tissue specificity. For example, one method is looking at a virus' natural host range. Preferably, the virus vector selected would be from a virus whose primary range of infection is for a different host animal than the animal that the gene delivery system is to be used in. For example, swinepox can be used as a viral vector when the host is a primate such as a human. However, for veterinary purposes where the host is a pig it would not be preferable. Certain highly attenuated or modified strains such as modified orthopoxvirus (e.g., the MVA or NYVAC strain of vaccinia or strains genetically modified or selected to be non-virulent in their normal host range or in a desired host cell) that are not virulent in their normal host range can, however, be used. Tissue specificity also can be used to preliminarily screen for infectivity and replication efficiency.
Where the host is human, preferred vectors include pox vectors, for example, suipox, such as swinepox, avipox such as fowlpox, canary pox, or pigeon pox, and capripoxvirus. In addition, iridoviruses such as frog virus, and African swine fever virus are also preferred. Preferred viral vectors for use with human cells are non-lytic, avirulent poxviruses such as avipox [Taylor, et al., Vaccine, 6:497-503 (1985) and Jenkins, et al., AIDS Research And Human Retroviruses 7:991-998 (1991)] and suipox [Feller, et al., Virology 183:578-585 (1991)].
Genes for Insertion into Poxvirus
Any DNA of interest can be inserted into the poxvirus vector of the present invention.
Because poxviruses have a large genome, they can readily be used to deliver a wide range of genetic material including multiple genes (i.e., act as a multivalent vector). The sizes of the poxvirus genomes ranges between about 130-300 kbp with up to 300 genes, depending on the strain of the virus. Therefore, it is possible to insert large fragments of foreign DNA into these viruses and yet maintain stability of the viral genome.
In one embodiment, at least one nucleic acid fragment encoding a gene is inserted into a poxvirus vector. In another embodiment at least two and up to about ten different nucleic acids encoding different genes are inserted into the poxvirus vector.
In one embodiment of the present invention, the recombinant poxvirus has the DNA encoding a disease-related antigen of interest, such as an antigen(s) from a disease causing agent or an antigen associate with a disease state, inserted at its novel insertion site, and expresses that antigen(s).
In another embodiment of the present invention, the recombinant poxvirus has the DNA encoding a co-stimulatory molecule(s) inserted at its novel insertion site, and expresses the co-stimulatory molecule(s).
The, recombinant vectors of the present invention are particularly useful to generate cell-mediated immune reactions. Cell-mediated immunity is crucial to cancer and diseases such as those caused by pathogenic microorganisms, particularly viruses and other intracellular microorganisms. Accordingly, the present invention provides a composition that has at least a first recombinant virus which has incorporated into its genome or portion thereof a gene encoding an antigen from cells of a disease state. The first recombinant poxvirus may also comprise one or more genes encoding one or more immunostimulatory molecules or genes. In one preferred embodiment, the co-stimulatory molecule is a combination of nucleic acids encoding B7 (e.g. B7-1), ICAM-1, and LFA-3, also known as TRICOM, which include activation of both CD4 and cd8 activators. Another embodiment provides a composition that has a second recombinant virus that comprises one or more genes encoding one or more immunostimulatory molecules or genes. A host cell infected with both recombinant viruses expresses both the antigen(s) from a disease causing agent and expresses the immunostimulatory molecule(s). The antigen may be expressed at the cell surface of the infected host cell. The immunostimulatory molecule may be expressed at the cell surface or may be actively secreted by the host cell. The expression of both the antigen and the immunostimulatory molecule provides the necessary MHC restricted peptide to specific T cells and the appropriate signal to the T cell to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells. The overall result is an upregulation of the immune system. In a preferred embodiment the upregulation of the immune response is an increase in antigen specific T-helper lymphocytes and/or cytotoxic lymphocytes, which are able to kill or inhibit the growth of a disease causing agent or a cell infected with a disease causing agent.
The disease-related antigen of interest can be an antigen from a pathogenic microorganism or a tumor associated antigen. The genes can be derived from any organism, including bacteria, parasites, normal or transformed cells, viruses or other microorganisms. Preferred genes are derived from transformed cells. For example, any gene for which a poxvirus-based live vaccine is desired.
Such disease causing agents include but are not limited to cancer and pathogenic microorganisms.
Cancers which may be treated using the recombinant poxvirus of the present invention include but are not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, bladder cancer, colon cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and the like.
The aforementioned cancers can be assessed or treated by methods described in the present application. In the case of cancer, a gene encoding an antigen associated with the cancer is incorporated into the recombinant poxvirus genome or portion thereof along with a gene encoding one or more immunostimulatory molecules. Alternatively, the gene encoding an antigen associated with the cancer and the gene encoding one or more immunostimulatory molecules are incorporated into separate recombinant poxviruses. The antigen associated with the cancer may be expressed on the surface of a cancer cell or may be an internal antigen. In one embodiment the antigen associated with the cancer is a tumor associated antigen (TAA) or portion thereof. Examples of TAA that may be used in the present invention include but are not limited to melanoma TAAs which include but are not limited to MART-1 (Kawakami et al. J. Exp. Med. 180:347-352, 1994), MAGE-1, MAGE-3, GP-100, (Kawakami et al. Proc. Nat'l. Acad. Sci. U.S.A. 91:6458-6462, 1994), CEA and tyrosinase (Brichard et al. J. Exp. Med. 178:489, 1993). In another embodiment the TAAs are MUC-1, MUC-2, the point mutated ras oncogene and the point mutated p53 oncogenes (pancreatic cancer), CA-125 (ovarian cancer), PSA (prostate cancer), c-erb/B2 (breast cancer) and the like (Boon et al., Ann. Rev. Immunol. 12:337, 1994). Other antigens associated with cancer include MN antigen, Jade, and BZLF-1. The present invention is in no way limited to the genes encoding the above listed TAAs. Other TAAs may be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506.
In another preferred embodiment, the target antigen is a tumor associated antigen, a tumor specific antigens, and/or a tissue-specific antigens. In this embodiment, at least one epitope of an antigen is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN-.alpha., IFN-.beta., IFN-.beta 17 mutants, IFN-65, CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD11c, CD16, CD18, CD21, CD28, CD32, CD34, CD35, CD40, CD44, CD45, CD54, CD56, OX40L, 4-1BBL, K2, K1, P.beta., O.alpha., M.alpha., M.beta.2, M.beta.1, Hepsin, Pim-1, LMP1, TAP2, LMP7, TAP1, TRP, O.beta., IA.beta., IA.alpha., IE.beta., IE.beta.2, IE.alpha., CYP21, C4B, CYP21P, C4A, Bf, C2, HSP, G7a/b, TNF-.alpha., TNF-.beta., D, L, Qa, T1a, COL11A2, DP.beta.2, DP.alpha.2, DP.beta.1, DP.alpha.1, DN.alpha., DM.alpha., DM.beta., LMP2, TAPi1, LMP7, DO.beta., DQ.beta.2, DQ.alpha.2, DQ.beta.3, DQ.beta.1, DQ.alpha.1, DR.beta., DR.alpha., G250, HSP-70, HLA-B, HLA-C, HLA-X, HLA-E, HLA-J, HLA-A, HLA-H, HLA-G, HLA-F, nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF-II, TGF-.beta., GM-CSF, M-CSF, G-CSF1, erythropoietin, .beta.-HCG, 4-N-acetylgalactosaminyltransferase, GM2, GD2, GD3, JADE, MART, BAGE, GAGE, MAGE-1, MAGE-2, MAGE-3, XAGE, MUC-1, MUC-2, MUC-3, MUC-4, MUC-18, ICAM-1, C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, LFA-3 (CD58), EpCAM, B7.1, CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K19, HKer 8, pMel 17, TP10, tyrosinase related proteins 1 and 2, p97, p53, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC and MCC, ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abil, C1q, C1r, C1s, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, C1Inh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CR1, CR2, CR3, CR4, C3a/C4a receptor, C5a receptor, Epstein-Barr Virus antigens (EBNA), BZLF-1, BXLF-1, and Nuclear Matrix Proteins, modified TAAs, splice variants of TAAs, functional epitopes, epitope agonists, and degenerate nucleic acid variations thereof.
Genes encoding an antigen of a disease causing agent in which the agent is a pathogenic microorganism include viruses such as HIV (GP-120, p17, GP-160, gag, po1, qp41, gp120, vif, tat, rev, nef, vpr, vpu, vpx antigens), smallpox, influenza (NP, hemagluttinin (HA antigen), neuraminidase, PB1, PB2, PA, NP, M.sub.1, M.sub.2, NS.sub.1, NS.sub.2)), papillomaviruses (E1, E2, E3, E4, E5a, E5b, E6, E7, E8, L1, L2), adenovirus (E1A, E1B, E2, E3, E4, E5, L1, L2, L3, L4, L5), HSV (ribonucleotide reductase, .alpha.-TIF, ICP4, ICP8, 1CP35, LAT-related proteins, gB, gC, gD, gE, gH, gI, gJ, and dD antigens), human papilloma virus, equine encephalitis virus, hepatitis (Hep B Surface Antigen (gp27.sup.S, gp36.sup.S, gp42.sup.S, p22.sup.c, pol, x)) and the like. Pathogenic bacteria include but are not limited to anthrax, Chlamydia, Mycobacteria, Legioniella and the like. Pathogenic protozoans include but are not limited to malaria, Babesia, Schistosomiasis and the like. Pathogenic yeast include Aspergillus, invasive Candida, and the like. In a preferred. embodiment the pathogenic microorganism is an intracellular organism.
For purposes of a vaccine, genes of interest are those which encode immunogenic proteins of a pathogenic organism. In many cases, these are protein components of surface structures such as the bacterial cell wall or viral envelope. In appropriate instances, immunogenic fragments or subunits of the proteins may be used.
One preferred group of nucleic acids for insertion into the poxvirus include co-stimulatory molecules, accessory molecules, and/or genes encoding a cytokine and/or growth factor. Examples of costimulatory molecules include but are not limited to B7-1, B7-2, ICAM-1, CD40, CD40L, LFA-3, CD72, OX40L (with or without OX40), and the like.
Examples of cytokines and growth factors encompassed by the present invention include but are not limited to: granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), tumor necrosis factors (TNF.alpha. and TNF.beta.), transforming growth factors (TGF.alpha. and TGF.beta.), epidermal growth factors (EGF), stem cell factor (SCF), platelet-derived growth factors (PDGF), platelet-derived endothelial cell growth factor, nerve growth factor (NGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF-I and IGF-II), growth hormone, interleukins 1 to 15 (IL-1 to IL-15), interferons .alpha., .beta. and gamma. (IFN-.alpha., IFN-.beta. and IFN-.gamma.), brain-derived neurotrophic factor, neurotrophins 3 and 4, hepatocyte growth factor, erythropoictin, EGF-like mitogens, TGF-like growth factors, PDGF-like growth factors, melanocyte growth factor, mammary-derived growth factor 1, prostate growth factors, cartilage-derived growth factor, chondrocyte growth factor, bone-derived growth factor, osteosarcoma-derived growth factor, glial growth-promoting factor, colostrum basic growth factor, endothelial cell growth factor, tumor angiogenesis factor, hematopoietic stem cell growth factor, B-cell stimulating factor 2, B-cell differentiation factor, leukemia-derived growth factor, myelomonocytic growth factor, macrophage-derived growth factor, macrophage-activating factor, erythroid-potentiating activity, keratinocyte growth factor, ciliary neurotrophic growth factor, Schwann cell-derived growth factor, vaccinia virus growth factor, bombyxin, neu differentiation factor, v-Sis, glial growth factor/acetylcholine receptor-inducing activity, transferrin, bombesin and bombesin-like peptides, angiotensin II, endothelin, atrial natriuretic factor (ANF) and ANF-like peptides, vasoactive intestinal peptide, Bradykinin and related growth factors. Preferred cytokines and growth factors include but are not limited to IL-2, GM-CSF, TNF.alpha., IFN.gamnma., IL-12, RANTES, and the like.
One does not have to use a gene encoding an entire protein, but rather only the domain desired. For example, if an immune reaction is desired, only the fragment necessary to stimulate the immune reaction needs to be encoded. The co-stimulatory molecules, accessory molecules, and cytokines of the present invention are useful as biologic adjuvants, which can be administered systemically to the host via inserting nucleic acids encoding such into the same or different recombinant poxvirus vectors. In one preferred embodiment, one administers a poxvirus vector containing B7, LFA-3 and ICAM-1 in conjunction with the tumor associated antigen. In a further preferred embodiment, the poxvirus also contains OX40L. In another embodiment, the poxvirus contains OX40L alone. In yet another embodiment, the poxvirus encodes both OX40L or OX40 intrabody and OX40.
Poxviruses expressing B7-1, ICAM-1, and LFA-3, also known as TRICOMTM, induce activation of both CD4+ and CD8+T cells. (U.S. Pat. No. 6,045,802; Hodge et al., J. Natl. Cancer Inst. 92: 1228-39 (2000); Hodge et al., Cancer Research 59: 5800-07 (1999)). OX40 is a primary co-stimulator of T cells that have encountered antigen, rather than naïive T cells, and promotes T-cell expansion after T cell tolerance is induced. (Bansal-Pakal et al., Nature Med. 7: 907-12 (2001)). OX40L plays a role during T cell activation by a) sustaining the long-term proliferation of CD4+ and CD8+T cells, b) enhancing the production of Th1 cytokines such as IL-2, IGN-g, and TNF-a from both CD4+ and CD8+T cells without changing IL-4 expression, c) protecting T cells from apoptosis. The combination of B7-1, ICAM-1, LFA-3, and OX40L enhances initial activation and then further potentiates sustained activation of naïve and effector T cells.
Another preferred group of nucleic acids for insertion into the poxvirus encode antibodies. Antibodies have long been used in biomedical science as in vitro tools for the identification, purification and functional manipulation of target antigens. Antibodies have been exploited in vivo for both diagnostic and therapeutic applications. Recent advances in antibody engineering have now allowed the gene encoding antibodies to be manipulated so that the antigen biding domain can also be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with the ability to create large human immunoglobulin libraries and their ability to be stably expressed in precise intracellular location inside mammalian cells, has provided a powerful new family of molecules for gene therapy applications such as the one including a poxvirus vector in the present application. These intracellular antibodies are called “intrabodies”. (Marasco et al. Gene Therapy, 4:11-15, 1997; U.S. Pat. Nos. 5,965,371; 5,851,829; 6,329,173; and 6,072,036). Preferably nucleic acids encoding angiogenesis modulating intrabodies encode a single chain antibody.
In another preferred embodiment, the poxvirus vectors of the present invention can be used to generate a pox-virus vaccine of use to the poultry industry. Examples of genes will be derived from pathogens which are important to the poultry industry and include those pathogens for which vaccines of variable efficacy already exist, namely; infectious bronchitis virus, infectious bursal disease virus, reovirus, Marek's disease virus, Newcastle disease virus, laryngo-tracheitis virus, and avian encephalomyelitis virus. The genes will also be derived from poultry pathogens for which no vaccines currently exist despite the need to control their spread. This list includes: Eimeria species which cause coccidiosis salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium, Staphylococcus auereus, Aspergillus flavus, Escherichia coli, Mycoplasma gallisepticum, Mycoplasma gallinarum, Mycoplasma synoviae, RNA lymphoid leukosis virus, avian influenza, and hemorrhagic enteritis virus.
Methods for Inserting Genes into the Proxrivus Genome
Foreign genes for insertion into the genome of a poxvirus in expressible form can be obtained by any conventional technique for isolating a desired gene.
For organisms which contain a DNA genome, the genes encoding an antigen of interest are isolated from the genomic DNA; for organisms with RNA genomes, the desired gene may be isolated from cDNA copies of the genome. If restriction maps of the genome are available, strategies can be designed for cleaving genomic DNA by restriction endonuclease digestion to yield DNA fragments that contain the gene of interest. In some cases, desired genes may have been previously cloned and thus, the genes can be obtained from the available clones. Alternatively, if the DNA sequence of the gene is known, the gene can be synthesized by any of the conventional techniques for polymerase chain reaction or synthesis of deoxyribonucleic acids (e.g., the phosphate or phosphite triester techniques).
Genes encoding an antigen of interest can be amplified by cloning the gene into a bacterial host. For this purpose, various prokaryotic cloning vectors can be used. Examples are plasmids pBR322 and pEMBL.
The genes encoding the antigen of interest can be prepared for insertion into the poxvirus vectors by standard techniques. In general, the cloned genes can be excised from the prokaryotic cloning vector by restriction enzyme digestion. In most cases, the excised fragment will contain the entire coding region of the gene. The DNA fragment carrying the cloned gene can be modified as needed, for example, to make the ends of the fragment compatible with the insertion sites of the poxvirus vectors, then purified prior to insertion into these vectors at restriction endonuclease cleavage sites (cloning sites) as described below.
The basic techniques of inserting genes into viruses are known to the skilled artisan and involve, for example, recombination between the viral DNA sequences flanking a gene in a donor plasmid and homologous sequences present in the parental virus (Mackett, et al., Proc. Natl. Acad. Sci. USA 79:7415-7419 (1982)). For example, a recombinant virus such as a poxvirus for use in delivering the gene can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the fowlpoxvirus described in U.S. Pat. No. 5,093,258, the disclosure of which is incorporated herein by reference. Other techniques include using a unique restriction endonuclease site that is naturally present or artificially inserted in the parental viral vector.
First, the DNA gene sequence to be inserted into the virus can be placed into a plasmid, e.g., an E. coli plasmid construct, into which DNA homologous to a section of DNA such as that of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA which is the desired insertion region. The resulting plasmid construct is then amplified by growth within E. coli bacteria and isolated. Preferably, the plasmid also contains an origin of replication such as the E. coli origin of replication, and a marker such as an antibiotic resistance gene for selection and propagation in E. coli. 
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g., chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively results in a poxvirus modified by the presence of the promoter-gene construct in its genome, at a site which does not affect virus viability.
Where the embodiment of the present invention provides insertion of more than one nucleic acid (e.g. a tumor antigen and a costimulatory molecule), the first nucleic acid is inserted into the novel insertion sites of the present invention, as described above, and additional nucleic acid(s) can be inserted either into the novel insertion sites described here or other insertion sites.
The gene is preferably inserted into a site or region (insertion region) in the virus which does not affect virus viability of the resultant recombinant virus. The novel insertion sites are intragenic regions between known genes, preferably non-essential genes. The skilled artisan can readily identify such regions in a virus by, for example, randomly testing segments of virus DNA for regions that allow recombinant formation without seriously affecting virus viability of the recombinant. One region that can readily be used and is present in many viruses is the thymidine kinase gene. For example, it has been found in all poxvirus genomes examined [leporipoxvirus: Upton, et al., J. Virology, 60:920 (1986) (shope fibroma virus); capripoxvirus: Gershon, et al., J. Gen. Virol., 70:525 (1989) (Kenya sheep-1); orthopoxvirus: Weir, et al., J. Virol., 46:530 (1983) (vaccinia); Esposito, et al., Virology, 135:561 (1984) (monkeypox and variola virus); Hruby, et al., PNAS, 80:3411 (1983) (vaccinia); Kilpatrick, et al., Virology, 143:399 (1985)(Yaba monkey tumor virus); avipoxvirus: Binns, et al., J. Gen. Virol. 69:1275 (1988) (fowlpox); Boyle, et al., Virology, 156:355 (1987) (fowlpox); Schnitzlein, et al., J. Virological Methods, 20:341 (1988) (fowlpox, quailpox); entomopox (Lytvyn, et al., J. Gen. Virol. 73:3235-3240 (1992)].
In fowlpox, in addition to the TK region, other insertion regions include, for example, BamHI J [Jenkins, et al., AIDS Research and Human-Retroviruses 7:991-998 (1991)] the EcoRI-HindIII fragment, BamHI fragment, EcoRV-HindHIII fragment, BamHI fragment and the HindIII fragment set forth in EPO Application No. 0 308 220 A1. [Calvert, et al., J. of Virol. 67:3069-3076 (1993); Taylor, et al., Vaccine 6:497-503 (1988); Spehner, et al., (1990) and Boursnell, et al., J. of Gen. Virol. 71:621-628 (1990)].
In addition to the requirement that the gene be inserted into an insertion site, successful expression of the inserted gene(s) by the modified poxvirus requires the presence of a promoter operably linked to the desired gene, i.e., in the proper relationship to the inserted gene. The promoter must be placed so that it is located upstream from the gene to be expressed. Promoters are well known in the art and can readily be selected depending on the host and the cell type one wishes to target. For example in poxviruses, poxviral promoters should be used, such as the vaccinia 7.5K, 40K, fowlpox. Enhancer elements can also be used in combination to increase the level of expression. Furthermore, the use of inducible promoters, which are also well known in the art, in some embodiments are preferred.
Promoters useful according to the present invention include poxvirus promoters such as, e.g., an entomopox promoter, an avipox promoter, or an orthopox promoter such as a vaccinia promoter, e.g., HH, 11K or Pi. For example, the Pi promoter, from the Ava I H region of vaccinia, is described in Wachsman et al., J. of Inf. Dis. 155, 1188-1197 (1987). More particularly, this promoter is derived from the Ava I H(Xho I G) fragment of the L-variant WR vaccinia strain, in which the promoter directs transcription from right to left. The map location of the promoter is approximately 1.3 Kbp (kilobase pair) from the 5′ end of Ava IH, approximately 12.5 Kbp from the 5′ end of the vaccinia genome, and about 8.5 Kbp 5′ of the Hind III C/N junction. The Hind III H promoter (also “HH” and “H6” herein) sequence is an up-stream of open reading frame H6 by Rosel et al., J. Virol. 60, 436-449 (1986). The 11K promoter is as described by Wittek, J. Virol. 49, 371-378 (1984) and Bertholet, C. et al., Proc. Natl. Acad. Sci. USA 82, 2096-2100 (1985). One can take advantage of whether the promoter is an early or late promoter to time expression of particular genes. Additionally, as discussed below, one can use additional promoters.
Another preferred embodiment provides a poxvirus vector in which the promoter is modulated by an external factor or cue, allowing control of the level of polypeptide being produced by the vectors by activating that external factor or cue. For example, heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature. The promoter of the gene which encodes the metal-containing protein metallothionine is responsive to Cd+ ions. Incorporation of this promoter or another promoter influenced by external cues also make it possible to regulate the production of the proteins.
In another preferred embodiment, the poxvirus genome is modified to carry a nucleic acid encoding at least one gene of interest which is operably linked to an “inducible” promoter. Such inducible systems allow careful regulation of gene expression. See, Miller and Whelan, Human Gene Therapy, 8:803-815 (1997). The phrase “inducible promoter” or “inducible system” as used herein includes systems wherein promoter activity can be regulated using an externally delivered agent. Such systems include, for example, systems using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters (Brown et al. Cell, 49:603-612, 1987); systems using the tetracycline repressor (tetR)(Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551, 1992; Yao et al., Human Gene Ther. 9:1939-1950, 1998; Shokelt et al., Proc. Natl. Acad. Sci. USA 92.6522-6526, 1995). Other such systems include FK506 dimer, VP16 or p65 using castradiol, RU486/mifepristone, diphenol muristerone or rapamycin (see, Miller and Whelan, supra, at FIG. 2). Yet another example is an ecdysone inducible system (see, e.g. Karns et al, MBC Biotechnology 1:11, 2001). Inducible systems are available, e.g., from Invitrogen, Clontech, and Ariad. Systems using a repressor with the operon are preferred. One would adapt these promoters by substituting portions of pox promoters for the mammalian promoter.
One embodiment of the present invention provides the use of a regulatory element such as a transcriptional regulatory element or an enhancer.
In one preferred embodiment of the present invention, a “transcriptional regulatory element” or “TRE” is introduced for regulation of the gene of interest. As used herein, a TRE is a polynucleotide sequence, preferably a DNA sequence, that regulates (i.e., controls) transcription of an operably-linked polynucleotide sequence by an RNA polymerase to form RNA. As used herein, a TRE increases transcription of an operably linked polynucleotide sequence in a host cell that allows the TRE to function. The TRE comprises an enhancer element and/or pox promoter element, which may or may not be derived from the same gene. The promoter and enhancer components of a TRE may be in any orientation and/or distance from the coding sequence of interest, and comprise multimers of the foregoing, as long as the desired, transcriptional activity is obtained. As discussed herein, a TRE may or may not lack a silencer element.
Another preferred embodiment of the present invention provides an. “enhancer” for regulation of the gene of interest. An enhancer is a term well understood in the art and is a polynucleotide sequence derived from a gene which increases transcription of a gene which is operably-linked to a promoter to an extent which is greater than the transcription activation effected by the promoter itself when operably-linked to the gene, i.e. it increases transcription from the promoter. Having “enhancer activity” is a term well understood in the art and means what has been stated, i.e., it increases transcription of a gene which is operably linked to a promoter to an extent which is greater than the increase in transcription effected by the promoter itself when operably linked to the gene, i.e., it increases, transcription from the promoter.
The activity of a regulatory element such as a TRE or an enhancer generally depends upon the presence of transcriptional regulatory factors and/or the absence of transcriptional regulatory inhibitors. Transcriptional activation can be measured in a number of ways known in the art (and described in more detail below), but is generally measured by detection and/or quantization of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) the regulatory element. As discussed herein, the regulatory element can be of varying lengths, and of varying sequence composition. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, more preferably at least about 20-fold. More preferably at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably, at least about 1000-fold. Basal levels are generally the level of activity, if any, in a non-target cells, or the level of activity (if any) of a reporter construct lacking the TRE of interest as tested in a target cell type.
A “functionally-preserved” variant of a TRE is a TRE which differs from another TRE, but still retains ability to increase transcription of an operably linked polynucleotide, especially cell-specific transcription activity. The difference in a TRE can be due to differences in linear sequence, arising from, for example, single or multiple base mutation(s), addition(s), deletion(s), and/or modification(s) of the bases. The difference can also arise from changes in the sugar(s), and/or linkage(s) between the bases of a TRE.
Certain point mutations within sequences of TREs have been shown to decrease transcription factor binding and gene activation. One of skill in the art would recognize that some alterations of bases in and around known the transcription factor binding sites are more likely to negatively affect gene activation and cell-specificity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects. Certain mutations are also capable of increasing TRE activity. Testing of the effects of altering bases may be performed in vitro or in vivo by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in TRE functional and TRE non-functional cells. Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a TRE sequence without altering the ability of the sequence to regulate transcription.
In the present invention, the poxvirus vectors directed at specific target cells may also be generated with the use of TREs that are preferentially functional in the target tumor cells. Non-limiting examples of tumor cell-specific heterologous TREs, and non-limiting examples of respective potential target cells, include TREs from the following genes: .alpha.-fetoprotein (AFP) (liver cancer), mucin-like glycoprotein DF3 (MUC1) (breast carcinoma), carcinoembryonic antigen (CEA) (colorectal, gastric, pancreatic, breast, and lung cancers), plasminogen activator urokinase (uPA) and its receptor gene (breast, colon, and liver cancers), E2F1 (cell cycle S-phase specific promoter) (tumors with disrupted retinoblastoma gene function), HER-2/neu (c-erbB2/neu) (breast, ovarian, stomach, and lung cancers).
In the present invention, tumor-specific TREs may be used in conjunction with tissue-specific TREs from the following exemplary genes (tissue in which the TREs are specifically functional are in parentheses): hypoxia responsive element, vascular endothelial growth factor receptor (endothelium), albumin (liver), factor VII (liver), fatty acid synthase (liver), Von Willebrand factor (brain endothelium), alpha-actin and myosin heavy chain (both in smooth muscle), synthetast I (small intestine), Na—K—Cl transporter (kidney). Additional tissue specific TREs are known in the art.
Accordingly, in one embodiment, the cell specific, heterologous TRE is tumor cell specific. Preferably, both heterologous TREs are tumor cell specific and functional in the same cell. In another embodiment, one of the first heterologous TREs is tumor cell specific and the second heterologous TRE is tissue specific, whereby both TREs are function in the same cell.
Introduction of the viral vector carrying the gene to be delivered to the target host cell may be effected by any method known to those of skill in the art.
Administration of the recombinant poxvirus of the invention can be either “prophylactic” or “therapeutic” depending on the subject. When provided prophylactically, the recombinant poxvirus of the present invention is provided in advance of any symptom, but when one believes the subject is at risk. The prophylactic administration of the recombinant poxvirus serves to prevent or ameliorate any subsequent angiogenic-related condition. When provided therapeutically, the recombinant poxvirus is provided at or after the onset of a symptom of infection or disease. Thus the present invention may be provided to either prior the anticipated exposure to a disease-causing agent or disease state or after the initiation of the infection or disease.
The term “unit dose” as it pertains to the inoculum refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of recombinant poxvirus calculated to produce the desired immunogenic effect in association with the required diluent. The specifications for the novel unit dose of an inoculum of this invention are dictated by and are dependent upon the unique characteristics of the recombinant virus and the particular immunologic effect to be achieved.
Administration
For administration to a subject, the poxvirus of the present invention is prepared as an inoculum. The inoculum is typically prepared as a solution in a tolerable (acceptable) diluent such as saline, phosphate-buffered saline or other physiologically tolerable diluent and the like to form an aqueous pharmaceutical composition.
The route of inoculation may be scarification, intravenous (I.V.), intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.), intratumor and the like, which results in eliciting a protective response against the disease causing agent. The dose is administered at least once. Subsequent doses may be administered as indicated.
In one embodiment, heterologous prime-boost regimens are employed. For example, the host can be immunized at least once with a first vector such as a nucleic acid-based vector. Subsequent immunizations are performed with a poxvirus vector. In another example, the host is first immunized with a first poxvirus vector and then with a second poxvirus vector of a different genus.
In providing a manual with the recombinant poxvirus of the present invention, preferably a human, the dosage of administered recombinant poxvirus will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumor burden and the like.
In general, it is desirable to provide the recipient with a dosage of recombinant virus in the range of about 105 to about 1010 plaque forming units, although a lower or higher dose may be administered.
One would inject a sufficient amount of the viral vectors to obtain a serum concentration in the organ of interest of the protein ranging between about 1 pg/ml to 20 μg/ml. More preferably between about 0.1 μg/ml to 10 μg/ml. Still more preferably, between about 0.5 μg/ml to 10 μg/ml.
Examples of methods for administering the recombinant poxvirus into mammals include, but are not limited to, exposure of tumor cells to the recombinant virus ex vivo, or injection of the recombinant poxvirus into the affected host by intravenous, S.C., I.D. or I.M. administration of the virus. Alternatively the recombinant poxvirus or combination of recombinant vectors may be administered locally by direct injection into the cancerous lesion or tumor or topical application in a pharmaceutically acceptable carrier. The quantity of recombinant poxvirus carrying the nucleic acid sequence of one or more antigens in combination with nucleic acid sequences encoding multiple costimulatory molecules to be administered is based on the titer of virus particles. A preferred range of the immunogen to be administered is 105 to 1010 virus particles per mammal, preferably a human. If the mammal to be immunized is already afflicted with cancer or metastatic cancer, the vaccine can be administered in conjunction with other therapeutic treatments.
The present invention also provides a pharmaceutical composition comprising a poxvirus, including a recombinant poxvirus, and a pharmaceutically acceptable carrier.
The effect of the genetic material delivered can be carefully monitored and regulated using this system. Preferred poxvirus vectors such as swinepox will only express the genetic material for about two weeks. Thus, if the condition being treated is alleviated within that time frame, since the vector system is self limiting, no unnecessary material will be produced after that time period. Where additional dosages will be needed, additional administration of the material can be accomplished by repeating the injection. In certain cases, the addition of a second, third, etc. material can also be added with these vectors.
A preferred embodiment of the present invention relates to a method of modulating immune responses to achieve a desired goal.
One embodiment of the present invention provides treating a subject in need, for example, a subject having cancer, with a poxvirus of the present invention in combination with a chemotherapeutic agent. Preferably, the poxvirus expresses a gene associated with a cancer. The chemotherapeutic agent can be any anti-cancer drug.
Examples of anti-cancer drugs that may be used in the various embodiments of the invention, including pharmaceutical compositions and dosage forms and kits of the invention, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; capsitabine; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleulin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine, mechlorethamine oxide hydrochloride rethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, improsulfan, benzodepa, carboquone, triethylenemelamrine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelainine, chlomaphazine, novembichin, phenesterine, trofosfamide, estermustine, chlorozotocin, gemzar, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, aclacinomycins, actinomycin F(1), azaserine, bleomycin, carubicin, carzinophilin, chromomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, doxorubicin, olivomycin, plicamyciri, porfiromycin, puromycin, tubercidin, zorubicin, denopterin, pteropterin, 6-mercaptopurine, ancitabine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, enocitabine, pulmozyme, aceglatone, aldophosphamide glycoside, bestrabucil, defofamide, demecolcine, elfornithine, elliptinium acetate, etoglucid, flutamide, hydroxyurea, lentinan, phenamet, podophyllinic acid, 2-ethylhydrazide, razoxane, spirogermanium, tamoxifen, taxotere, tenuazonic acid, triaziquone, 2,2′,2′′-trichlorotriethylamine, urethan, vinblastine, vincristine, vindesine and related agents. 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine, edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopraminde; MIF inhibitor; mifepristone; miltefosine; nirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; taxel; taxel analogues; taxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rheniuim Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division ibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil, leucovorin, capsitabine, cyclosphosphamide, and gemcitabine. The magnitude of a prophylactic or therapeutic dose of each active ingredient in the treatment of a patient with a solid tumor will typically vary with the specific active ingredients, the severity and type of tumor, and the route of administration. The dose and the dose frequency may vary according to age, body weight, response, and the past medical history of the patient; the likelihood of mestastic recurrence must also be considered. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference® (54th ed., 2000). Unless otherwise indicated, the magnitude of a prophylactic or therapeutic dose of each pharmaceutical used in an embodiment of the invention will be that which is known to those in the art to be safe and effective, or is regulatory approved.
In one embodiment, a treatment method in accordance with the present invention includes treating a subject in need, for example, a subject having cancer, for example breast cancer, ovarian cancer or prostate cancer, with a first poxvirus vector designed to elicit a cytotoxic T-cell response to a foreign gene. For a cancer patient, the gene can be a tumor associated antigen, such as PSA, PSAM, BRCA1, ras, CEA or MUC. One can also use a foreign gene encoding a viral antigen or antigens to treat an individual having or susceptible to an infectious disease, preferably a viral envelope protein. Cytotoxic T-cells specific for the desired cancer-associated antigen can be generated by administering between about 105-109 pfu of a recombinant poxvirus carrying a sequence encoding a tumor-associated antigen to the individual affected with the tumor. For detailed construction of such vectors, see, e.g., U.S. Pat. No. 5,656,465. Preferably one would also use an immune modulator such as the use of cytokines, e.g., IL-2, or co-stimulatory molecules, e.g., B7.1 or B7.2, as biologic adjuvants, which can be administered systemically to the host via inserting nucleic acids encoding such into the same or different recombinant poxvirus vectors. In one preferred embodiment, one administers a poxvirus vector containing B7, LFA-3, ICAM-1 and Ok40L in conjunction with the foreign gene.
The invention will be further characterized by the following examples which are intended to be exemplary of the invention.
EXAMPLES
Example 1
Vaccinia Insertion Site Studies
The relative paucity of defined-insertion sites in MVA hastened the search for new sites in the MVA genome into which foreign genes can be inserted. Analysis of the published MVA sequence (Antoine et al., 1998) identified several other potential insertion sites hereafter referred to as MVA 44/45; MVA 49/50 and MVA 124/125. The numbers designate the ORF s lying on either side of the insertion site.
Selection of appropriate sites was determined following examination of the sequence to determine whether several criteria were met. First, the insertion site should lie in an intergenic space, preferably between non-essential genes. Assessment of the essential nature of the MVA genes was determined after comparing the MVA gene with its′ vaccinia homologue. Second, there′ should be essential genes between the native sites of the 40K, 7.5K and I3 promoters and the insertion site. Third, the proposed insertion site should not disrupt cryptic ORFs in the region, or promoters of adjacent genes. Similarly T5NT sequences of surrounding genes should be left intact.
Regions corresponding to the 5′ and 3′ flankers of the new vectors were amplified from genomic DNA using Pfu polymerase. These fragments were inserted into plasmid vectors and sequenced. Following confirmation of sequence, the SHIV 89.6P env gene under 40K promoter, and the C1-lacZ cassette were inserted into these vectors to generate test plasmids for IVR: pT5158, pT5164, pT5172. The choice of antigen was dictated by available MVA reagent for comparative immunogenicity studies.
Site 44/45
Site 44/45 in MVA lies between ORFs 044L and 045L—the insertion site is between positions 37346-37357 in the MVA genomic sequence (Genbank Acc # U94848). DNA sequence between these nucleotides is deleted in recombinant virus and replaced with defined inserts. This region is 5′ of the translational start codon of MVA 044L and 3′ of the translational stop codon of MVA 045L. This region is present in the vaccinia Copenhagen sequence (Genbank Accession number M35027). The corresponding vaccina ORFs are F14L (homologous to MVA 044L) and F15L (MVA 045L). Although the DNA sequence of NYVAC is not available in Genbank, there is nothing in the literature to indicate that this region has been modified in the NYVAC genome.
Site 49/50
Site 49/50 in MVA lies between ORFs 049L and 050L—the insertion site is between positions 42687 and 42690 in the MVA genomic sequence. This region is 5′ of the translational'start codon of MVA 049L and 3′ of the translational stop codon of 050L. This region is present in the Copenhagen sequence—the homologues are E2L (MVA 049L) and E3L (MVA 050L). This region has not been modified in the NYVAC genome.
Site 124/125
Site 124/125 in MVA lies between ORFs 124L and 125L—the insertion site is at positions 118481-118482 in the MVA genomic sequence. This lies 5′ of the translational start codon of MVA 124L and 3′ of the translational stop codon of 125L. This region is present in the Copenhagen sequence—the homologues are A13L (MVA 124L) and A14L (MVA 125L). According to the literature, this region has not been modified in the NYVAC genome.
Example 2
FowlPox Insertion Site Studies
Generation of recombinant fowlpox generally involves inserting foreign genes and a lac Z promoter-gene cassette at the unique regions of the fowlpox genome. Recombinant viruses are selected on the basis of colorimetric screening with bluo-gal. Identification of alternative insertion sites in the fowlpox genome is desirable to enable insertion of multiple gene-promoter cassettes into the virus.
Three potential new insertion sites were identified and designated: 43K, FP14 and the Long Unique Sequence (LUS). The 43K insertion site is the subject of another independent study based on plaque selection in a manner similar to that of vaccinia Δ37K.
The overall aim of this project is to identify insertion sites that are at least as stable as those with the foreign gene and lacZ inserted in the BamHI J region. Furthermore, the immunogenicity of recombinant viruses with insertions at these new sites should be equivalent to those with insertions at BamHI J. The organization of the new viruses and controls available for comparative studies are listed in Table 1.







TABLE 1







Structure of the viral recombinants designed to test


alternative insertion sites in the fowlpox genome.











Virus
Insertion site
gene 1
gene 2





TBC-FPV
—
—
—
negative control



43K
40K-BH10 env
C1-lacZ
new site



FP14
40K-BH10 env
C1-lacZ
new site



LUS
40K-BH10 env
C1-lacZ
new site


vT155
Bam J
40K-BH10 env
C1-lacZ
positive control









The 43K Insertion Site

The fowlpox 43K gene has homology to the vaccinia 37K major envelope antigen gene, F13L. Calvert et al (1992) demonstrated that this gene encodes a major membrane protein, which is critical for the generation of extracellular enveloped virions. As an alternative to the selecting recombinant plaques based on the expression of 43K, we have designed a vector which enables the region immediately 3′ of the 43K coding region to be used as an insertion site in the fowlpox genome, where recombinant plaques can be screened for expression of lac Z using a colorimetric screen.
Examination of the available 43K gene sequence, Genbank Accession #M88587, revealed a short intergenic space between the 3′ end of the 43K gene and the 5′ end of the P74 gene. A plasmid was designed where the 5′ flanker corresponded to nt 282-1753 in the 43K gene sequence. This sequence comprises 338 bp of 5′ untranslated sequence and the entire coding region of the 43K gene. The last 12 codons in the 43K gene that are present in the 5′ flanker were wobbled to minimize the possibility of recombination between these and the native sequences that are present in the 3′ flanker as part of the promoter for the 74K gene. The 3′ flanker extends from nt 1718-2233 in the 43K gene sequence and consists of the terminal 12 codons of the 43K coding region, the promoter for p74 and the first 442 bp of the p74 coding region. The terminal part of the 43K coding region includes some motifs that correspond to poxvirus transcriptional sequences, and might presumably be required for function of p74. The test vector—pT5104—was constructed by inserting the 40K-BH10 env and C1-lacZ promoter-gene cassettes between these flanker regions.
Fowlpox has now been completely sequenced, and its genomic sequence is available in Genbank, Acceession No. AF198100. Within this genomic sequence, the 43K insertion site is at position 128178 of the fowlfox genomic sequence, and lies 5′ of FPV107 and 5′ of FPV108. These genes are divergently transcribed, and the insertion site lies between the two promoter elements for the two ORFs.
The FP14 Insertion Site
This insertion site was identified from sequence generated by Tartaglia et al (1990) accession #X17202, corresponding to a 10.5 kb HindIII fragment which has some homologies with the HindIII D region of vaccinia virus. This region was mapped to approximately 70 kb from the left end of the viral genome. Part of this region was also sequenced by Binns et al. (1990), who generated some stable recombinant viruses by inserting lac Z into particular genes in this region. One particular area of the Tartaglia et al (1990) sequence satisfies many of the criteria previously identified as important for a new insertion site: intergenic space with good distance between genes; non-disruptive of promoter regions of adjacent genes and no crypic open reading frames (ORF) in the region.
The area of this new insertion site lies between the end of FP14 and the start of FP18. Each of these genes is transcribed towards the right side of the genome, and the intergenic space is 447 bp. The fowlpox homologues of the vaccinia D genes are located on either side of this region; these genes have been shown in vaccinia to be essential for viral replication. Detailed analysis of this intergenic space identified cryptic minor ORFs on both strands of the DNA, however a potential insertion site was identified 160 bp upstream of the FP18 translational start—this site is not part of any cryptic ORF. Flanker regions of a plasmid transfer vectors were generated by PCR from genomic DNA of TBC-FPV. The 5′ flanker corresponds to sequence between nt 1344 and 1949 and the 3′ flanker to sequence between nt 1964 to 2462 of the Tartaglia sequence. The test vector—pT5105—was constructed by inserting the 40K-BH10 env and C1-lacZ promoter-gene cassettes between these flanker regions.
Within the fowlpox genomic sequence deposited with Genbank, the FP14 insertion site is between positions 67080-67097 in the fowlfox genomic sequence, and lies 5′ of FPV060 and 3′ of FPV061.
The LUS Insertion Site
The Inverted Terminal Repeat (ITR) region of the poxvirus genome is an attractive option for the insertion of foreign genes. Recombination between the ends of the genome offers the possibility of duplicating the genes inserted at one end of the genome, and thus possibly increasing the level of expression of an inserted antigen and consequently the immunogenicity of the recombinant virus. There are two reports in the literature describing sequences in the fowlpox ITR region: Boursnell et al (1990) and Tomley et al (1988). The ITR region of the left end of the fowlpox genome is approximately 10 kb long and comprises two different types of sequence:  ̃4 kb of tandem repeat sequence and  ̃6 kb of Long Unique Sequence (LUS). Boursnell et al (1990) demonstrated that the fusion gene of Newcastle disease virus and the lacZ coding region could be inserted into genes of the LUS and generate a stable recombinant with a double copy of the inserted genes.
Examination of the Tomley et al sequence (Genbank Accession # D00295) reveals a large intergenic space of 410 bp between two identified ORFs. There are cryptic ORFs in this sequence, however a possible insertion site was identified between the promoters of these oppositely transcribed ORFs. Fragments of DNA corresponding to flanker regions were amplified by PCR from TBC-FPV, and a test vector was constructed by inserting the 40K-BH10 env and C1-lacZ cassettes between the flanker regions. The 5′ and 3′ flankers corresponds to nt 1322-1954 and 1961-2660 respectively in the Tomley sequence.
Within the fowlpox genomic sequence in Genbank, there are two long unique sequences (LUS) at each end of the viral genome, and thus two LUS insertion sites in each genome. The LUS insertion site at the left end of the genome is between positions 7470-7475 in the fowlpox genomic sequence, and lies 3′ of FPV006 and 5′ of FPV007 125L. The LUS insertion site at the right end of the genome is between positions 281065 and 281070 in the fowlpox genomic sequence, and lies 5′ of FPV254 and 3′ of FPV255.
Recombinant Selection and Analysis
IVRs will be performed in CEDs and 10 independent plaques will be carried for several rounds of purification. The isolates will be tested by interim back plaque analysis for BH10 env expression and 4 independent isolates will be selected and amplified into small Seed Stocks (2 to 3×15 cm plates for each). After titration they will be passaged twice at moi=0.1 in, CEDs (1×15 cm plate for each isolate and amplification) to observe their stability.
While the 3 independent IVRs may be done independently, the passages will be done in parallel and also include vT155. After the yield of these recombinants and their passage 1 (P1) and passage 2 (P2) is determined, the stability assessed by black plaques assay, the expression of the env assessed by ELISA, the best isolate for each insertion site will be selected for the immunogenicity study.
Immunogenicity Study
A small scale immunogenicity study will determine if the new insertion sites elicit an immune response against HIV-1 BH10 env as good as vT155 in which env is inserted in the BamHI J fragment of FPV. 5 groups of 5 mice each (corresponding to the 5 different viruses) will be injected with 106 or 107 pfu of virus 4 times at 3 weeks interval and bled prior to each injection. All blood sample will be tested for Abs against HIV-1 env by ELISA.
Example 3
Vaccinia and Fowlpox Recombinant Vectors Containing Insertions
MVA recombinant vectors containing a test antigen in the specified intergenic sites were successfully generated and evaluated, i.e. they were used and expressed the appropriate proteins. The DNA inserted into these vectors contained the following elements: the SHIV 89.6P env gene under the control of the 40K pox virus promoter, and the E. coli marker gene LacZ under the control of the C1 pox promoter. The SHIV 89.6 P envelope gene is a chimeric simian/human immunodeficienty virus type 1 isolate (Reimann, K. A. et al., 1996. J. Virol. 70:6922-6928; Yao Q. et al., AIDS Res Hum Retroviruses. 2000 Feb. 10; 16(3):227-36). The following schematic recombinant virus structures represent the insertion of this insert into a) MVA at MVA 44/45; b) MVA at MVA 49/50; and c) MVA at MVA 124/125:

    a)—5′ flanker 44/45-40K-SHIV 89.6P env-C1-LacZ-44/45 3′ flanker—
    b)—5′ flanker 49/50-40K-SHIV 89.6P env-C1-LacZ-49/50 3′ flanker—
    c)—5′ flanker 124/125-40K-SHIV 89.6P env-C1-LacZ-124/125 3′ flanker—


Fowlpox recombinant vectors containing a test antigen were also successfully generated and evaluated. The inserted foreign DNA contained the following elements: the HIV BH-10 envelope gene under the control of the 40K pox virus promoter, and the E. coli marker gene LacZ under the control of the C1 pox promoter. The HIV BH-10 gene has been previously described (Muesing, M. A. et al., 1985 Nature. 313:450-8; Yao Q. et al., AIDS Res Hum Retroviruses. 2000 Feb. 10; 16(3):227-36). The following schematic recombinant virus structures represent the insertion of this insert into a) FPV at FPV 43K; b) FPV at FP14; and c) FPV at FP LUS:

    a)—5′ flanker 43K-40K-BH10 env-C1-7LacZ-43K 3′ flanker—
    b)—5′ flanker FP14-40K-BH10 env-C1-LacZ-FP14 3′ flanker—
    c)—5′ flanker LUS-40K-BH10 env-C1-LacZ-LUS 3′ flanker—


Additional recombinant vectors have been generated to date using these insertion sites for MVA and FPV.
For example a further example of a recombinant vector with an insertion at the novel fowlpox insertion site, FP14, is the recombinant vaccine vector PANVAC-F. PANVAC-F was constructed using two plasmid vectors. A schematic depicting the construction of this vector is provided in FIG. 1. The first plasmid, designated pT1154, directs insertion of the wCEA(6D) and wMUC-1(6) coding sequences into the FP14 region of the fowlpox virus genome. The second plasmid, designated pT8150, directs insertion of the LFA-3, ICAM-1, and B7.1 coding sequences (collectively known as TRICOM) into the BamH I J region of the fowlpox virus genome. The wCEA(6D) gene is under the transcriptional control of the vaccinia 40K promoter. The wMUC-1(6) gene is under the transcriptional control of the synthetic early/late (sE/L) promoter. The LFA-3 gene is under the transcriptional control of the vaccinia 30K promoter, the ICAM-1 gene is under the transcriptional control of the vaccinia I3 promoter, and the B7.1 gene is under the transcriptional control of the synthetic early/late (sE/L) promoter. In addition, pT1154 contains the E. coli lacZ gene, under the control of the vaccinia 40K promoter, which is included as a screen for recombinant progeny, and pT8150 contains the GUS gene, under the control of the 7.5 promoter, for use in screening for recombinant progeny. A plaque-purified isolate from the POXVAC-TC vaccine strain of fowlpox virus was used as the parental virus for this recombinant vector. In vivo recombination between the plasmid vectors and the viral DNA resulted in the formation of a recombinant virus in which pT1154 was integrated at FP14, and pT8150 was integrated at the BamHI J region, as illustrated in FIG. 1.
A further example of a recombinant vector with an insertion at the novel fowlpox insertion site, FP14, is the recombinant fowlpox virus designated rF-OX40L. This vector was generated by insertion of the murine OX40L gene under the control of the 40k promoter into the genome of rF-CEA at the FP14 site in the genome. The OX40L cDNA was amplified from anti-CD40/anti-IgM-activated murine B cells by RT-PCR using primers specific for the 5′-(dGGTACCGGTACCATGGAAGGGGAAGGGGTTC) (SEQ ID NO:1) and the 3′-(dCTCGAGCTCGAGTCACAGTGGTACTTGGTTC) (SEQ ID NO:2) ends of the open reading frame. The cDNA was cloned into a poxvirus transfer vector. Sequence analysis confirmed that no mutations were introduced in the cloning process.
A further example of a recombinant vector with an insertion at the novel fowlpox insertion site, FP14, is the recombinant fowlpox virus designated rF-MUC-1-TRICOM/OX40L. This vector was generated by insertion of the murine OX40L gene under the control of the 40k promoter into the genome of rF-TRICOM at the FP14 site in the genome. Both recombinant fowlpox viruses expressing OX40L were generated by methods previously described (Gritz, L., Destree, A., Cormier, N., Day, E., Stallard, V., Caiazzo, T., Mazzara, G. and Panicali, D., J Virol 64, 5948-57, 1990). These viruses also express the human MUC-1 gene and the bacterial beta-galactosidase gene.
REFERENCES
1. Antoine, G., Scheiflinger, F., Dorner, F., and Faulkner F. G. (1998) The complete genomic sequence of the Modified Vaccinia Ankara Strain: Comparison with other poxviruses. Virology 244: 365-396.
2. Binns et al. (1990) J. Gen. Virol. 71:2873-81.
3. Boursnell et al. (1990) J. Gen. Virol. 71: 621-628.
4. Calvert et al. (1992) Virology 191: 783-792.
5. Tartaglia et al. (1990) J. Gen. Virol 71:1517-1524.
6. Tomley et al. (1988) J. Gen. Virol. 69:1025-1040.
All references cited herein are hereby incorporated by reference.

## Claims

1. A recombinant fowlpox virus containing and capable of expressing at least one foreign gene inserted at an insertion site within the fowlpox virus genome,
wherein the insertion site is located (i) at a position corresponding to position 128178 of the fowlpox genomic sequence, (ii) at a position corresponding to a region between positions 67080 and 67097 in the fowlpox genomic sequence, (iii) at a position corresponding to a region between positions 7470 and 7475 in the fowlpox genomic sequence, or (iv) at a position corresponding to a region between positions 281065 and 281070 in the fowlpox genomic sequence wherein the fowlpox virus genome corresponds to SEQ ID NO:3.

2. The recombinant fowlpox virus of claim 1, wherein the foreign gene encodes a marker, a therapeutic agent, or an antigenic determinant.

3. The recombinant fowlpox virus of claim 2, wherein the foreign gene codes for an antigenic determinant selected from the group consisting of a pathogenic virus, a bacteria, other microorganism, a parasite, and a tumor cell.

4. The recombinant fowlpox virus of claim 2, wherein the foreign gene codes for an antigenic determinant expressed in a pathogen, wherein the pathogen is selected from the group consisting of Plasmodium, Mycobacteriua, Herpes virus, influenza virus, hepatitis, and HIV.

5. The recombinant fowlpox virus of claim 1, wherein the foreign gene is selected from the group of genes encoding hormones, growth factors, immunostimulatory molecules, enzymes, cytokines, receptors, and tumor suppressor genes.

6. A pharmaceutical composition, comprising the recombinant fowlpox virus of claim 1.

7. A method for inducing an immunological response against at least one antigen in a mammal, said method comprising inoculating the mammal with at least a first recombinant fowlpox virus containing and capable of expressing at least the gene encoding said antigen inserted at an insertion site within the fowlpox virus genome,
wherein the insertion site is located (i) at a position corresponding to position 128178 of the fowlpox genomic sequence, (ii) at a position corresponding to a region between positions 67080 and 67097 in the fowlpox genomic sequence, (iii) at a position corresponding to a region between positions 7470 and 7475 in the fowlpox genomic sequence, or (iv) at a position corresponding to a region between positions 281065 and 281070 in the fowlpox genomic sequence wherein the fowlpox virus genome corresponds to SEQ ID NO:3.

8. The method of claim 7, wherein said recombinant fowlpox virus further contains a gene encoding at least one immunostimulatory molecule.

9. The method of claim 7, wherein said method further comprises inoculating the mammal with a second vector containing and capable of expression at least one additional gene.

10. The method of claim 9, wherein said second vector contains a gene encoding at least one immunostimulatory molecule.

11. The method of claim 9, wherein the second vector comprises an adenovirus vector.

12. The method of claim 9, wherein the second vector comprises a recombinant vaccinia virus vector.

13. The method of claim 9, wherein the second recombinant vector comprises a recombinant fowlpox virus vector.