US20060275796A1

US20060275796A1 - Canine herpesvirus thymidine kinase nucleic acid molecules

Info

Publication number: US20060275796A1
Application number: US11/374,406
Authority: US
Inventors: Elizabeth Haanes; Rexann Frank
Original assignee: Individual
Current assignee: Merial Ltd
Priority date: 1996-02-15
Filing date: 2006-03-13
Publication date: 2006-12-07
Also published as: PT910406E; NZ331079A; US6159478A; DE69737058T2; AU2327897A; JP2000505297A; US20030049844A1; EP0910406A1; US7041813B2; US5804197A; AU728676B2; EP0910406B1; EP0910406A4; DK0910406T3; DE69737058D1; WO1997029772A1; ATE347375T1; US5753235A; ES2281098T3; CA2243662A1

Abstract

The present invention includes novel recombinant canine herpes virus (CHV) and novel recombinant CHV genomes, and particularly to those CHV and CHV genomes that contain heterologous nucleic acid molecules. The present invention also relates to the use of such genomes and viruses in a variety of applications, including as therapeutic compositions to protect animals from disease. The present invention also relates to novel isolated CHV nucleic acid molecules, to CHV proteins encoded by such nucleic acid molecules, and to antibodies raised against such CHV proteins as well as to the use of such CHV nucleic acid molecules, proteins and antibodies as therapeutic compositions to protect an animal from CHV. The present invention also includes constructs comprising CHV nucleic acid molecules that include heterologous nucleic acid molecules, to recombinant vectors including such constructs, and to the use of such constructs and vectors in the production of recombinant CHV and recombinant CHV genomes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of pending U.S. patent application Ser. No. 08/602,010, entitled “Recombinant Canine Herpesviruses”, filed Feb. 15, 1996, and which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to canine herpesvirus (CHV), and particularly to novel recombinant CHV and recombinant CHV genomes, including those that contain heterologous nucleic acid molecules. The present invention also relates to the use of such genomes and viruses in a variety of applications, including as therapeutic compositions to protect animals from disease. The present invention also relates to novel isolated CHV nucleic acid molecules, to proteins encoded by such nucleic acid molecules, and to use of such CHV nucleic acid molecules to insert heterologous nucleic acid molecules into CHV genomes.

BACKGROUND OF THE INVENTION

Dogs and other canids are affected by a number of diseases against which it would be desirable to develop protective vaccines. Live vaccines, and particularly live viral vector vaccines, are attractive vaccine vector candidates as they appear to be associated with longer-lasting immunity than inactivated virus vaccines or subunit vaccines. One disadvantage of live vaccines, however, has been that attenuated virus strains often revert to virulence. Another disadvantage has been the host range of a number of viral vaccines. In an attempt to deliver genes to an animal, several viral and bacterial systems, such as poxviruses, adenoviruses, Salmonella,. and BCG (Bacillus Calmette-Guerin), have been genetically manipulated to generate vectors containing heterologous antigen genes in order to immunize a host with a vaccine in which the antigens are presented in a “live” configuration. See, for example, the following two review articles: Esposito et al., pp. 195-247, 1989, Advances in Veterinary Science and Comparative Medicine, Vol. 33; Dougan et al., pp. 271-300, 1989, Advances in Veterinary Science and Comparative Medicine, Vol. 33.
Several herpes virus vaccines, such as those based on bovine herpes virus (BHV), cytomegalovirus (CMV), Epstein Barr virus (EBV), equine herpes virus (EHV), feline herpes virus (FHV), herpes simplex virus (HSV), Marek's disease virus (MDV), pseudorabies virus (PRV), turkey herpes virus (HVT), and varicella zoster virus (VZV) have been developed and several have shown at least some efficacy as vaccines against the virus per se or as vectors carrying other genes in certain indications. The listed herpes viruses, however, also have the drawback that even if attenuated, they are subject to reversion.
Canine herpes virus (CHV) infection is a relatively benign infection except in newborn puppies. A few vaccines to protect against CHV infection have been reported including a small-plague variant CHV vaccine disclosed in U.S. Pat. No. 4,213,965, by Carmichael, issued Jul. 22, 1980. The nucleotide sequences of CHV genes encoding gB, gC, gD and UL45 homologs have been reported by Limbach et al., 1994, J. Gen. Virol. 75, 2029-2039, but these proteins, while proposed as vaccine candidates against CHV, were not tested as such by Limbach et al., ibid.
The inventors are not aware of any reports which describe the use of CHV as a vaccine vector, either with respect to inactivating genes in the CHV genome using recombinant DNA techniques, and/or to delivering protective compounds to a canid, in spite of the need to develop safe and efficacious delivery systems to protect canids, and especially dogs, from disease. Two U.S. patents (i.e., U.S. Pat. No. 5,266,489, by Rey-Senelonge et al., issued Nov. 30, 1993; and U.S. Pat. No. 5,223,424, by Cochran et al., issued Jun. 29, 1993) at best speculate on the insertion of genes into certain CHV loci, but neither claims CHV vectors or vaccines, nor provides data supporting such speculations. U.S. Pat. No. 5,266,489, ibid., claimed HVT having a foreign gene inserted into the ribonucleotide reductase (RR) small subunit gene of the HVT genome, but also disclosed without support the insertion of foreign genes into the RR small subunit genes of BHV, CHV, CMV, duck herpes virus, EBV, EHV, FHV, HSV, PRV and VZV. The inventors, however, have demonstrated the inaccuracy of this disclosure in that the inventors have found, and disclosed in the present application, that the CHV genome lacks the RR small subunit gene. That is, the CHV RR small subunit gene does not exist to provide a target for the insertion of foreign genes.
U.S. Pat. No. 5;223,424, ibid., claimed specific hybrid PRV constructs having deletions in the TK, repeat, or and/or gX regions and heterologous sequences inserted into the repeat and/or gX regions, but also proposed without data the ability to insert foreign genes into the repeat region of the CHV genome, even though that genome had not yet been mapped. Also disclosed were certain BHV and HVT constructs and proposals, without data, to delete and insert genes in other herpesviruses, such as EHV and FHV, HSV and MDV. It is also of note that a patent application (now U.S. Pat. No. 5,273,876, by Hock et al, issued Dec. 28, 1993) that was filed significantly later than U.S. Pat. No. 5,223,424, ibid., and shares two co-inventors with U.S. Pat. No. 5,223,424, ibid., states in column 2, lines 57-61, “Among the herpesviruses, only four herpesviruses (herpes simplex of humans, herpes saimiri of monkeys, pseudorabies virus and varicella-zoster virus) have been engineered to contain foreign DNA sequences previous to this disclosure,” thereby indicating the lack of CHV, or a number of other, herpes virus vectors.
Thus, there remains a need for new and improved methods to vaccinate canids to protect them from diseases, such as those caused by genetic or metabolic disorders as well as those caused by infectious agents such as protozoan parasites, helminth parasites, ectoparasites, fungi, bacteria, and viruses.

SUMMARY OF THE INVENTION

The present invention relates to a new method to protect animals from disease using a recombinant virus or virus genome. When a recombinant CHV of the present invention is administered to an animal, the virus is able to infect cells within the animal. Infected cells are able to express nucleic acid sequences present on the recombinant CHV genome to produce protective compounds, such as proteins and RNAs, capable of protecting the animal from a variety of diseases. Using methods taught in the present invention, vaccines can be generated that are capable of protecting an animal from any disease for which a protective compound can be produced. As such, the present invention is of extremely broad scope and includes a wide variety of vaccines that have a variety of applications.
The present invention includes a recombinant CHV that comprises a recombinant CHV genome. The invention also includes recombinant CHV genomes. In one embodiment, a recombinant CHV has an inactive gene within its genome, with a preferred recombinant CHV in this embodiment being a CdUTPase negative CHV, a CgC negative CHV, a CgE negative CHV, a CgG negative CHV, a CgI negative CHV, a CPK negative CHV, a CTK negative CHV, a CIR6 negative CHV, a CUS2 negative CHV, a CUS9 negative CHV, a CUL49 negative CHV, a CUL51 negative CHV, a CUL45 negative CHV, a CgD negative CHV, a CgB negative CHV, a CUL48 negative CHV, a CUL52 negative CHV, a CgL negative CHV, a CUL49.5 negative CHV, a CICP0 negative CHV, a CICP4 negative CHV, and/or a CUS8.5 negative CHV. In the case of the CUS8.5 negative CHV, a CUS8.5 negative CHV refers to a CHV in which the CUS8.5 open reading frame is disrupted.
The present invention also includes a recombinant CHV genome that comprises a heterologous nucleic acid molecule, which preferably encodes a protective compound that protects a canid from disease. Such a heterologous nucleic acid molecule can be located in an essential gene, a nonessential gene, and/or in an intergenic region. Insertion of a heterologous nucleic acid into a CHV genome can, but need not, inactivate a gene within that genome. Also included in the present invention are methods to produce a recombinant CHV and a recombinant CHV genome, as well as a canine cell line that complements a defect in a gene essential to CHV reproduction in vitro.
One embodiment of the present invention is a method to increase recombinant CHV plaque forming efficiency. Such a method can include the steps of introducing a recombinant CHV genome into a canine cell expressing CHV alpha transinducing factor and culturing the cell to produce recombinant CHV. In another embodiment, the method includes the steps of co-introducing a recombinant CHV genome and a CHV alpha transinducing factor gene into a canine cell and culturing the cell to produce recombinant CHV. The present invention also includes a canine cell line comprising a CHV alpha transinducing factor gene.
One embodiment of the present invention is a therapeutic composition that includes a recombinant CHV, a recombinant CHV genome, or a mixture thereof. The present invention also includes a method to protect an animal from disease by administering such a therapeutic composition to the animal.
The present invention also includes an isolated CHV nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid regions: a CdUTPase gene, a CgE gene, a CgG gene, a CgI gene, a CPK gene, a CTK gene, a CIR6 gene, a CUS2 gene, a CUS9 gene, a CUL49 gene, a CUL51 gene, a CUL48 gene, a CUL52 gene, a CgL gene, a CUL49.5 gene, a CICP4 gene, a CUS8.5 open reading frame, and/or a region of the CHV genome spanning from about the 3′ end of the coding region of the CUL41 gene through about the 3′ end of the coding region of the CUL38 gene. Also included are recombinant molecules, recombinant viruses, recombinant vectors, and recombinant cells that include such CHV nucleic acid molecules. The present invention also includes CHV proteins encoded by such CHV nucleic acid molecules and antibodies that selectively bind to such CHV proteins, as well as methods to produce such CHV nucleic acid molecules, recombinant molecules, recombinant viruses, recombinant vectors, recombinant cells, CHV proteins and antibodies. Also included in the present invention are therapeutic compositions including such CHV nucleic acid molecules, CHV proteins and antibodies, as well as methods to use such compositions to protect an animal from disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic map of two contiguous HindIII fragments that include at least-portions of CHV UL48 UL49, dUTPase, UL51 and UL52 genes.
FIG. 2 depicts a schematic map of the AscI fragment that includes the unique short region of CHV.
FIG. 3 depicts a schematic map of the AscI/blunt fragment that includes the terminal inverted repeat region of CHV.
FIG. 4 depicts a schematic map of a region of the CHV genome spanning at least from the CUS9 gene through the CIR6 gene.
FIG. 5 depicts a schematic map of a region of the CHV genome spanning at least from a portion of the CUL52 gene through at least a portion of the CUL48 gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to canine herpesviruses (CHV), and particularly to recombinant CHV and recombinant CHV genomes as well as to methods to produce and use such viruses and genomes. The present invention also includes identification of regions of the CHV genome that are preferred targets for deletion and/or for insertion of heterologous nucleic acid molecules. As used herein, the term “canine” refers to “of the family Canidae” and, as such, includes reference to dogs (including wild and domesticated dogs), foxes, wolves, jackals, coyotes, and other members of the family Canidae. Similarly, a “canid” is any member of the family Canidae. As used herein, the term CHV refers to any herpesvirus endogenous to the family Canidae. A particularly preferred CHV of the present invention is a recombinant CHV that infects dogs.
One embodiment of the present invention is the use of a recombinant CHV, and particularly of a CHV having a recombinant CHV genome that includes a (i.e., one or more than one) heterologous nucleic acid molecule, as a live CHV-based therapeutic composition, or vaccine, to protect an animal from a disease against which the heterologous nucleic acid molecule is targeted. Live vaccines are advantageous because they are believed to confer more vigorous and longer-lasting immunity than subunit or killed vaccines. While not being bound by theory, it is believed that such advantages are due to the ability of the genetic information carried by the virus to enter cells of the treated animal, replicate itself, and direct the expression of a protective compound, such as a protective protein or a protective RNA, for extended periods of time. Thus, the therapeutic composition need not be administered frequently and, at least in some embodiments, the virus vaccine can function as a self-booster.
CHV is particularly useful as a delivery vehicle for a heterologous nucleic acid molecule to protect canids from a disease for a variety of reasons, including, but not limited to, CHV's limited host range and pathogenicity. CHV, to the inventors' knowledge, has the most limited host range of any herpesvirus, including FHV, in that CHV's host range appears to be substantially limited to canine cells. As far as the inventors are aware, the only report of CHV infection of a cell type other than canine cells is limited infection of mink lung cells; see, for example, Peterson et al, 1988, Comp. Immunol. Microbiol. Infect. Diseases 11, 93-98. This limited host range illustrates the safety of CHV in that the virus apparently cannot be transmitted to a variety of species unrelated to canids.
Furthermore, CHV exhibits only limited pathogenicity. Although the virus has been shown to cause a fatal hemorrhagic disease in hypothermic neonatal pups (i.e., essentially all pups experimentally infected with CHV and maintained at room temperature (i.e., from about 25° C. to 27° C. ) within a week of birth die from the infection), CHV causes insignificant respiratory infection in adult dogs; see, for example, Carmichael, 1970, J. Am. Vet. Med. Assn. 156, 1714-1721. Moreover, prolonged survival or recovery of experimentally infected neonatal pups maintained at 38.4° C. to 39.5° C. was observed. Adult dogs exposed to CHV do, however, become infected since virus shedding has been shown to occur for at least two weeks post-inoculation; and latency is postulated to occur, since CHV has been isolated from primary cultured cells of normal healthy dogs; see, for example, Carmichael, ibid. Furthermore, maternal antibody, or passive transfer of antibody from seropositive dogs has been shown to protect puppies from an otherwise fatal CHV challenge; see, for example, Carmichael, ibid. Due to its limited pathogenicity, CHV apparently need not be attenuated to the extent required for other viruses used as live vaccine vectors. In addition, vaccination of a dam with CHV can lead to passive protection in her pups.
Another advantage of CHV is its limited temperature range. CHV grows well at temperatures ranging from about 34° C. to about 36° C., with optimal growth occurring at about 35° C. CHV, however, does not grow well at temperatures less than or equal to about 33° C. or at temperatures greater than or equal to about 37° C. As such CHV is significantly more temperature sensitive than any other known wild type herpesvirus, including FHV.
Yet another advantage of CHV is its potential for use as a single, multivalent therapeutic composition against a variety of canine pathogens. That is, the CHV genome can be manipulated to incorporate multiple heterologous nucleic acid molecules without disrupting the ability of the genome to be packaged (i.e., assembled) into a live virus. Examples of multivalent therapeutic compositions are described below.
As far as the inventors are aware, this application is the first report of the genetic engineering of a CHV genome, particularly for the development of efficacious canid vaccines, in spite of a long felt need for efficacious vaccines against canine pathogens. The inventors have developed methods to identify CHV genes and intergenic regions, particularly those having utility as targets for the insertion of heterologous nucleic acid molecules, despite the difficulty of using known herpesvirus sequences to identify such regions due to the AT-rich nature of the CHV genome. The CHV genome contains about 70% adenosine and thymidine residues, compared to other known herpesvirus genomes which, on the average, contain from about 30% to about 58% adenosine and thymidine residues (e.g., HSV, BHV, and PRV contain about 30%, EHV about 54%, and FHV about 58%, adenosine and thymidine residues). As such, it is very difficult to design primers or probes using known herpesvirus sequences to identify CHV analogs.
One embodiment of the present invention is a recombinant CHV. As used herein, a recombinant CHV is a CHV that comprises (i.e., has or includes) a genome that has been genetically engineered (i.e., subjected to recombinant nucleic acid (i.e., DNA or RNA) techniques, such as those disclosed in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press; Sambrook et al., ibid., is incorporated by reference herein in its entirety) to differ from the genome of a natural CHV isolate (i.e., a herpesvirus endogenous to the family Canidae). Such a genetically engineered genome is referred to herein as a recombinant CHV genome and is described in more detail below.
A recombinant CHV of the present invention includes not only a recombinant CHV genome but also an envelope and capsid in which the genome is packaged. The viral envelope and capsid are preferably a CHV envelope and a CHV capsid, encoded at least in part by CHV genes, thereby imparting to the recombinant CHV the host range of a natural CHV isolate. It is to be noted, however, that the present invention also includes recombinant CHV having envelopes and/or capsids that have been modified to, for example, alter (e.g., broaden, narrow, or completely change) the host range of the recombinant CHV genome. Such modifications can be accomplished by one skilled in the art by, for example, modifying CHV envelope and/or capsid genes and/or replacing such genes with those of another virus. Altered genes can be located on the CHV genome itself and/or in the genome of the cell in which the recombinant virus is produced.
A recombinant CHV genome of the present invention is a CHV genome in which nucleotides have been deleted, inserted, substituted or inverted using recombinant techniques known to those skilled in the art such that the recombinant CHV genome is no longer the same as a natural CHV genome. A recombinant CHV genome-of the present invention is capable of effecting expression (e.g., transcription, translation) of coding regions that are operatively linked to regulatory sequences within the genome. As used herein, a coding region is a stretch of nucleotides that encodes an RNA molecule and/or a protein. Coding regions can be endogenous to CHV or can be heterologous nucleic acid molecules of the present invention, which are described in more detail below. The phrase operatively linked refers to the positioning of a coding region in the CHV genome such that the coding region is able to be expressed when the genome is inside a cell. Regulatory sequences include transcription control sequences, translation control sequences, and other regulatory sequences that control the expression of coding regions. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable regulatory sequences include any regulatory sequence that can function in the present invention. Preferred regulatory sequences are disclosed herein.
A recombinant CHV genome of the present invention can include a gene that has been inactivated. As used herein, a gene includes a coding region as well as the regulatory sequences involved in expression of that coding region. An inactive gene refers to a gene that no longer exhibits the function of its natural counterpart. Methods to inactivate a gene include, but are not limited to, deletion of one or more nucleotides within the gene, insertion of one or more nucleotides into the gene, replacement of one or more nucleotides within the gene by other nucleotides (i.e., nucleotide substitution), and/or inversion of nucleotides within the gene such that the resulting gene no longer has the function of the corresponding-natural-gene. Such alterations can be effected anywhere within the gene, such as within the coding region, within the regulatory sequences and/or in regions surrounding the coding region or regulatory sequences such that the alteration(s) cause gene inactivation. In one embodiment, an entire gene or the coding region and/or regulatory sequences thereof can be deleted or replaced.
One embodiment of the present invention is an attenuated recombinant CHV. As used herein, an attenuated CHV is a CHV that does not cause 100% mortality if used to infect canid neonates less than 1 week old that are maintained in room temperature. A preferred attenuated CHV of the present invention causes less than about 90% and preferably less than about 70% mortality when used to infect canid neonates less than 1 week of age maintained at room temperature.
An attenuated recombinant CHV can be produced by inactivating a CHV gene that, due to that gene's inactivation, results in an attenuated virus. Methods to inactivate a gene are disclosed above. An attenuated CHV can be identified by exposing pups less than 1 week old to the recombinant virus to be tested and determining the percentage of exposed pups that die; such an exposure method is disclosed, for example, in Carmichael, ibid. If less than 100% percent of the pups die, the virus being tested is attenuated in accordance with the present invention. Suitable CHV genes to inactivate in order to produce an attenuated CHV include any gene that when inactivated leads to an attenuated virus, as determined using an assay as disclosed above. A preferred attenuated recombinant CHV of the present invention is a CHV having a recombinant genome in which a heterologous nucleic acid molecule is inserted into a gene, the insertion resulting in an attenuated virus.
An attenuated recombinant CHV has utility, for example, as a therapeutic composition to protect an animal from CHV infection and/or as a live CHV-based vaccine carrying a heterologous nucleic acid molecule. It is to be noted, however, that, as disclosed above, it is believed that CHV need not be attenuated for use as a live vaccine vector due to the low pathogenicity of natural CHV, particularly as compared to that of other herpesviruses.
One embodiment of the present invention is a recombinant CHV that can reproduce (i.e., grow) in tissue culture; that is, the virus is a reproduction competent CHV. A reproduction competent CHV is a CHV that upon in vitro infection of an appropriate host cell is able to use host cell machinery, as well as its own regulatory control regions and/or encoded enzymes, to effect self-reproduction, i.e., to form infectious virus.
Reproduction competent recombinant CHV genomes can have gene alterations in one or more genes non-essential for growth in vitro. Suitable gene targets (i.e., genes to alter) include any non-essential CHV gene. A non-essential CHV gene can be identified by altering a CHV gene within a CHV genome (e.g., by genetic engineering or classical mutagenesis) and demonstrating that the altered genome is capable of effecting self-reproduction in tissue culture. Preferred non-essential CHV genes to target include, but are not limited to, a CHV deoxyuridine triphosphate pyrophosphatase (CdUTPase) gene, a CHV glycoprotein C (CgC) gene, a CHV glycoprotein E (CgE) gene, a CHV glycoprotein G (CgG) gene, a CHV glycoprotein I (CgI) gene, a CHV serine-threonine protein kinase US3 (CPK) gene, a CHV thymidine kinase (CTK) gene, a CHV IR6 (CIR6) gene, a CHV US2 (CUS2) gene, a CHV tegument phosphoprotein US9 (CUS9) gene, a CHV membrane protein UL49 (CUL49) gene, a CHV membrane protein CHV UL49.5 (CUL49.5) gene, a CHV regulatory protein ICP0 (cICP0) gene, a CHV membrane protein UL51 (CUL51) gene, and a CHV membrane protein UL45 (CUL45) gene. Particularly preferred non-essential genes to target include a CdUTPase gene, a CgC gene, a CgE gene, a CgG gene, a CgI gene, a CUS2 gene and a CUS9 gene. It is to be noted that CHV regions and genes disclosed herein are named in accordance with herpesvirus nomenclature in that the names include “C” for canine and the rest of the name indicating the corresponding herpes simplex virus (HSV) homolog (e.g., “dUTPase”). For example, the CHV unique short region (CUS) is the shorter region of the CHV genome that has unique sequences, analogous to the US region of HSV; the CHV unique long region (CUL) is the longer region of CHV that has unique sequences, analogous to the UL region of HSV; and the CHV inverted repeat regions (CIRs) are analogous to the HSV IR regions. CUS genes are CHV genes that are homologs of (i.e., genes that share some degree of similarity with) HSV genes located in the US region of the HSV genome. CUL genes are CHV genes that are homologs of HSV genes located in the UL region of the HSV genome. CIR genes are CHV genes that are homologs of HSV genes located in the IR regions of the HSV or EHV genome. It is also to be noted that although the UL, US, and IR designations refer to the respective genes' locations in the HSV genome, they do not necessarily refer to the respective genes' locations in the CHV genome. For example, while CUS2 is partially in the IR region of CHV, HSV US2 is entirely in the US region of HSV.
Another embodiment of the present invention is a recombinant CHV that is defective for reproduction in tissue culture. A reproduction defective CHV is a CHV that when inserted into an appropriate host cell is unable to form infectious virus. Such a defective CHV has at least one inactive gene that encodes a protein essential for reproduction, including, but not limited to proteins essential for viral entry, immediate early or early gene expression, DNA replication, capsid assembly, and viral egress. Suitable gene targets include any essential CHV gene, with preferred targets being genes encoding proteins involved in viral entry and/or egress. CHV defective in viral entry and/or egress are easy to complement and are advantageous over most other reproduction defective mutants in that such virus are able to undergo one round of viral replication. An essential CHV gene can be identified by altering a CHV gene within a CHV genome and demonstrating (a) that the altered genome is not capable of effecting self-reproduction in tissue culture under wild type conditions or, if a temperature sensitive mutant, at a non-permissive temperature; and (b) that the altered genome can reproduce in a complementing cell line that expresses an active protein corresponding to the essential gene defect on the CHV genome (assuming the defect can be complemented in trans), or at a permissive temperature. Preferred essential CHV genes to target include, but are not limited to, a CHV glycoprotein D (CgD) gene, a CHV glycoprotein B (CgB) gene, a CHV alpha trans-inducing factor UL48 (CUL48) gene, a CHV helicase/primase UL52 (CUL52) gene, a CHV glycoprotein L (CgL) gene and a CHV ICP4 (cICP4) gene. Particularly preferred essential genes to target include a CgD gene and a CgB gene.
The present invention also includes cell lines that complement replication defective CHVs and use of such cell lines to produce replication defective viruses. As such, the present invention includes canine cell lines that complement, or supplement, a CHV defect in a gene encoding CgD, CgB, CUL48, or CUL52, and/or CgL. Such cell lines can be produced by a variety of means known to those skilled in the art. For example, a cell capable of complementing a CgD negative, or CgD-, CHV (i.e., a virus with a CHV genome having an inactive CgD gene), can be produced by stable integration of an active CgD gene into the cellular genome or by co- transfection of the CgD- CHV with a nucleic acid molecule capable of complementing the defective CgD gene. Such a nucleic acid molecule can be a nucleic acid containing an active CgD gene operatively linked to regulatory sequences to enable expression of the CgD gene in the transfected cell. In another embodiment, such a nucleic acid molecule can be incorporated into a virus that is co-infected with the CgD- CHV. Such methods can also be used to produce cell lines complementing other replication defective CHVs of the present invention. Any canine cell line that CHV can infect and that expresses the complementary active protein can be used in the production of reproduction-defective CHV. Examples include, but are not limited to, the following cell lines available from American Type Culture Collection (ATCC), Rockville, Md.: ATCC CRL-1542 A-72 (Tumor, canine), ATCC CRL-1430 Cf2Th (Thymus, canine, Canis familiaris), ATCC CRL-10389 DH82 (Monocyte-macrophage, canine), ATCC CRL-8468 D17 (Osteogenic sarcoma, canine), ATCC CCL-183 D-17 (Primary osteogenic sarcoma, canine, Canis familiaris), ATCC CCL-34.1 DoCl1 (S+L-) (Kidney, canine, Canis familiaris), ATCC CCL-34.MDCK (NBL-2) (Kidney, canine, Canis familiaris), and ATCC CCL-34.2 MDCK/SF (Kidney, canine, Canis familiaris), such cell lines expressing, preferably in a stable manner, the desired essential gene(s) for complementation. Particularly preferred complementing cell lines include MDCK cells that stably express CgD, CgB, CUL48, CUL52 and/or CgL.
An additional preferred CHV open reading frame to inactivate, or disrupt, includes a CUS8.5 open reading frame.
While not being bound by theory, it is believed that a reproductive defective virus-based vaccine may be safer than a reproduction competent virus-based vaccine. On the other hand, a reproduction competent virus-based vaccine may be more efficacious than a reproduction defective virus-based vaccine. Since CHV, as disclosed above, exhibits low pathogenicity, a reproduction competent recombinant CHV is a preferred embodiment of the present invention.
As heretofore disclosed, one embodiment of the present invention is a recombinant CHV having one or more inactive genes. Preferred recombinant CHV of the present invention include genomes in which one or more of the following CHV genes have been inactivated, preferably using recombinant techniques: a CdUTPase gene, a CgC gene, a CgE gene, a CgG gene, a CgI gene, a CPK gene, a CTK gene, a CIR6 gene, a CUS2 gene, a CUS9 gene, a CUL49 gene, a CUL51 gene, a CUL45 gene, a CgD gene, a CgB gene, a CUL48 gene, and a CUL52 gene, a CgL gene, a CUL49.5 gene, a CICP0 gene, a CICP4 gene, and a CUS8.5 open reading frame. Other than the genes encoding CgD, CgB, CUL48, CUL52, CgL and CICP4, the preferred genes to inactivate are nonessential genes. Each of the preferred genes to inactivate is a preferred target for the insertion of heterologous nucleic acid molecules; such insertion is a preferred method to inactivate these genes. More preferred recombinant CHV include genomes in which one or more of the following CHV genes have been inactivated, preferably using recombinant techniques: a CdUTPase gene, a CgC gene, a CgE gene, a CgG gene, a CgI gene, a CUS2 gene, a CUS9 gene, a CgD gene, and/or a CgB gene being more preferred. Also preferred are the corresponding recombinant CHV genomes.
Particularly preferred recombinant CHV of this embodiment include a CdUTPase negative CHV, a CgC negative CHV, a CgE negative CHV, a CgG negative CHV, a CgI negative CHV, a CPK negative CHV, a CTK negative CHV, a CIR6 negative CHV, a CUS2 negative CHV, a CUS9 negative CHV, a CUL49 negative CHV, a CUL51 negative CHV, a CUL45 negative CHV, a CgD negative CHV, a CgB negative CHV, a CUL48 negative CHV, a CUL52 negative CHV, a CgL negative CHV, a CUL49.5 negative CHV, a CICP0 negative CHV, a CICP4 negative CHV, and a CUS8.5 negative CHV. Particularly preferred recombinant CHV include a CdUTPase negative CHV, a CgC negative CHV, a CgE negative CHV, a CgG negative CHV, a CgI negative CHV, a CUS2 negative CHV, a CUS9 negative CHV, a CgD negative CHV, and a CgB negative CHV. Also preferred are the corresponding recombinant CHV genomes, as well as recombinant CHV and CHV genomes having more than one of these preferred genes inactivated. Examples of such CHV include, but are not limited to: a CUS2 negative, CdUTPase negative CHV; a CUS2 negative, CdUTPase negative, CgG negative CHV; and a CUS2 negative, CdUTPase negative, CgG negative, CgC negative CHV, a CUL49.5, CdUTPase negative CHV.
One embodiment of the present invention includes a recombinant CHV comprising a recombinant CHV genome comprising a heterologous nucleic acid molecule; i.e., the recombinant CHV genome includes one or more heterologous nucleic acid molecule(s) located, or positioned, in the CHV genome. The present invention also includes a recombinant CHV genome having a heterologous nucleic acid molecule in the genome. Also-included is the use of such a CHV and/or CHV genome as a therapeutic composition as well as in the production of a compound encoded by the heterologous nucleic acid molecule(s).
As used herein, a heterologous nucleic acid molecule is a nucleic acid molecule that is not derived from CHV; that is, a heterologous nucleic acid molecule is isolated from a source other than CHV. An isolated nucleic acid molecule of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology (e.g., by amplification, such as by polymerase chain reaction (PCR) amplification and/or cloning) or can be produced by chemical synthesis.
In accordance with the present invention, a heterologous nucleic acid molecule can be inserted into a CHV genome simply to inactivate a CHV gene. A heterologous nucleic acid molecule can also be inserted into a CHV genome to serve as a target for the insertion of a second heterologous nucleic acid molecule, such as to introduce a restriction enzyme site or a recombination site otherwise not present in the CHV genome. For example, CHV strain D 004 (available from ATCC), which does not contain a Sse83871, I-Sce-I, or NotI restriction enzyme site, can be genetically engineered to include one or more sites by inserting into the genome a heterologous nucleic acid molecule containing a Sse83871, I-Sce-I, and/or NotI site(s). Such a heterologous nucleic acid molecule is a good target for the insertion of another heterologous nucleic acid molecule. Other restriction enzyme sites lacking in CHV can be identified using techniques known to those skilled in the art. Without being bound by theory, it is believed that CHV is more likely to lack GC-rich restriction enzyme sites than AT-rich sites, since the CHV genome is AT-rich, as disclosed above.
Another example of a heterologous nucleic acid molecule to insert into a CHV genome is a gene encoding a selectable marker, such as, but not limited to, an E. coli lacZ gene, a green fluorescent protein gene, a chloramphenicol transacetylase gene, a xanthine-guanine phosphoribosyl transferase gene, a β-glucuronidase gene, a neomycin resistance gene, an E. coli hygromycin resistance gene, and a heterologous thymidine kinase gene (e.g., HSV, FHV; assuming the CHV genome is a CTK negative genome). The presence of such a gene in a CHV genome allows for the selection of recombinant CHV having such a marker gene, and includes the ability to distinguish recombinant CHV from natural CHV isolates. In addition, a second heterologous nucleic acid molecule can be inserted into such a selectable marker gene, thereby inactivating the protein encoded by the marker gene, allowing for yet another method to select for CHV having a desirable heterologous nucleic acid molecule. Methods to select CHV having selectable marker genes, as well as for the inactivation of such markers, is known to those skilled in the art.
A preferred embodiment of the present invention is a recombinant CHV genome, and corresponding virus, in which the genome contains a heterologous nucleic acid molecule operatively linked to a transcription control sequence. As such, the heterologous nucleic acid molecule can be transcribed when transfected into a cell. A heterologous nucleic acid molecule can be joined to CHV transcription control sequences, can be joined to its own or other homologous transcription control sequences, and/or can be joined to transcription control sequences heterologous to both the heterologous nucleic acid molecule and CHV. The heterologous nucleic acid molecule can also be operatively linked to other regulatory sequences. Suitable regulatory sequences include any regulatory sequence that can function in the present invention. Preferred transcription control sequences include those sequences that can function in canine cells, including, but not limited to: mammalian, preferably canine; viral; or natural (i.e., endogenous to the heterologous nucleic acid molecule) transcription control sequences. Examples of transcription control sequences include antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus, actin, Rous sarcoma virus, heat shock, and mammalian hormone transcription control sequences. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). In one embodiment, expression of a heterologous nucleic acid molecule inserted into a CHV genome is mediated, at least in part, by a human cytomegalovirus (CMV) immediate early promoter and a bovine growth hormone polyadenylation site.
A heterologous nucleic acid molecule of the present invention can be located in any region of the CHV genome (i.e., in the UL, US, and/or IR regions), including, but not limited to, in an essential gene, in a non-essential gene, or in an intergenic region. As such, a heterologous nucleic acid molecule can be located in a coding region, a regulatory region, an intron, an untranslated region, or a non-transcribed region of a gene. A heterologous nucleic acid molecule can also be located in a direct or inverted repeat, including direct and/or inverted repeats within the IR, US or UL regions of CHV. For example, a heterologous nucleic acid molecule can be located in one or more CHV origins of replication (Cori), such as in CoriS.
In one embodiment, a heterologous nucleic acid molecule is located in a CHV genome such that a gene is inactivated. Suitable and preferred gene targets are as disclosed above, with non-essential gene targets being preferred.
In another embodiment, a heterologous nucleic acid molecule is located in a region of the CHV genome spanning from about the 3′ end of the coding region of the CUL41 gene through about the 3′ end of the coding region of the CUL38 gene. CUL41 gene refers to the CHV homolog of the HSV virion host shutoff protein UL41 gene. CUL38 gene refers to the CHV homolog of the HSV capsid protein VP19C UL38 gene. In most herpesviruses analyzed by cloning and sequencing techniques to date, the region between UL41 and UL38 contains genes encoding the large and small subunits of ribonucleotide reductase. The inventors have found, however, that the ribonucleotide reductase genes appear to be at least partially deleted in CHV in such a manner that there is an intergenic target in that region for heterologous nucleic acid molecule insertion.
A preferred recombinant CHV genome of the present invention comprises a heterologous nucleic acid molecule located in a region of the genome such that the heterologous nucleic acid molecule-containing CHV genome can be easily distinguished from a CHV genome not containing the heterologous nucleic acid molecule; that is, the heterologous nucleic acid molecule is inserted into a selectable region of the genome. Suitable selectable regions include any region of the CHV genome that, upon introduction of a heterologous nucleic acid molecule, leads to a detectable (e.g., growth-related, biochemical, or molecular) change in the CHV genome or CHV containing the genome. Examples of such selectable regions include, but are not limited to, a CTK gene and a CdUTPase gene. CHV genomes in which a heterologous nucleic acid molecule is inserted into a CTK gene or a CdUTPase gene can be selected using methods known to those skilled in the art; see for example, Kit et al, 1983, Virology 130, 381-389; and Holliday et al, 1991, Antiviral Research 16, 197-203.
Additional examples of selectable regions include restriction endonuclease sites, such as the HindIII site or XbaI site in the CdUTPase gene and the AscI site in the CUS2 gene. The AscI site is particularly preferred as there are no other AscI sites in the CHV genome. As such, a particularly preferred CHV of the present invention is a recombinant CHV having a CHV genome including a heterologous nucleic acid molecule in an AscI site in the CHV genome. Even more preferred is recombinant CHV strain D 004 having a heterologous nucleic acid molecule in an AscI site in the CHV genome. Also preferred are the corresponding genomes. Examples of methods to insert a heterologous nucleic acid molecule into a genome, including into restriction endonuclease site(s) in the genome, are disclosed herein. Such methods are known to those skilled in the art.
One embodiment of the present invention is a recombinant CHV genome having a heterologous nucleic acid molecule in one of the following regions of the CHV genome: a region spanning the 9,300 nucleotide AscI restriction endonuclease fragment, denoted herein as nCAsc₉₃₀₀₁that apparently includes the entire US region; a region spanning the 10,000 nucleotide fragment from AscI to the end of the genome, denoted herein as nCAsc₁₀₀₀₀, that apparently essentially comprises an IR region, including the CIR6 gene; a region spanning the 3,000 nucleotide HindIII fragment, denoted herein as nCHin₃₀₀₀, that spans from a portion of CUL48 through a portion of the CdUTPase gene; a region spanning the 1,900 nucleotide HindIII fragment, denoted herein as nCHin₁₉₀₀, that includes the remainder of the CdUTPase gene through a portion of CUL52; a region spanning the 5,500 nucleotide HindIII fragment, denoted herein as nCHin₅₅₀₀, that includes at least a portion of CgL, CICP0, and CICP4; and/or a region spanning the 8,500 nucleotide HindIII fragment, denoted herein as nCHin₈₅₀₀, that includes a portion of CUL48, CUL45 and CgC. Details regarding the production of these and certain other nucleic acid molecules of the present invention are provided in the Examples section.
Also included in the present invention is a recombinant CHV genome having a heterologous nucleic acid molecule in a region of the genome spanning at least one of the following: a CHV US region comprising nCUS₅₄₉₅, a CHV UL region comprising nCgC/CUL45₂₁₀₀, a CgE gene comprising nCgE₇₅₀, a CgI gene comprising nCgI₁₆₁, a CUS9 gene comprising nCUS9₅₇₉, a CHV UL region comprising nCdUTP/CUL51₇₄₃, a CTK gene comprising nCTK₂₈₀, a CUL48 gene comprising nCUL48₂₉₄, a CUL49 gene included in nCHin₃₀₀₀₁a CUL52 gene comprising nCUL52₁₄₆, a CHV UL region comprising nCUL₁₈₂₃, a CHV UL region comprising nCUL49/CUL48₂₀₄₄, a CHV IR region comprising nCICP4₆₂₆, a CHV UL region comprising nCgL₆₅₅, a CHV UL region comprising nCUL52₇₄₉, a CHV UL region comprising nCdUTP₃₂₀₀, as well as allelic variants of such (i.e., said, any of these) regions. As such, the present invention also includes a recombinant CHV genome having a heterologous nucleic acid molecule in a region of the genome spanning at least one of the following: a CIR6 gene including nCIR6₅₅₂, a CUS2 gene including nCUS2₁₁₇₆, a CPK gene including nCPK₁₂₀₃, a CgG gene including nCgG₁₂₄₈, a CgD gene including nCgD₃₅₇, a CdUTPase gene including nCdUTP₄₅₉, a CUL51 gene including nCUL51₂₆₁, a CgD gene including nCgD₁₀₃₈, a CgI gene including nCgI₁₀₉₅, a CgE gene including nCgE₁₅₆₉, a CUS8.5 open reading frame including nCUS8.5₂₃₇, a CUS9 gene including nCUS9₃₆₀, a CUL49 gene including nCUL49₄₂₀, a CUL48 gene including nCUL48₁₂₆₉, a CICP4 gene including nCICP4₆₂₆, a CgL gene including nCgL₆₅₅, a CdUTPase gene including nCdUTP₉₁₈, a CUL49 gene including nCUL49₂₅₅, a CUL49.5 gene including nCUL49.5₂₆₁, and a CUL52 gene including nCUL52₇₄₉, as well as allelic variants of such regions.
As used herein, an allelic variant of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs at essentially the same locus in another CHV genome as the nucleic acid molecule in CHV strain D 004, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions. Allelic variants are well known to those skilled in the art and would be expected to be found among the family of canine herpesviruses.
The present invention also includes a recombinant CHV genome having a heterologous nucleic acid molecule in a region of the genome spanning at least one of the; following nucleic acid molecules: nCUS₅₄₉₅(and, as such, nCIR6₅₅₂, nCUS2₁₁₇₆, nCPK₁₂₀₃, nCgG₁₂₄₈, and/or nCgD₃₅₇), nCgC/CUL45₂₁₀₀, nCgE₇₅₀, nCgI₁₆₁, nCUS9₅₇₉, nCdUTP/CUL51₇₄₃(and, as such, nCdUTP₄₅₉and nCUL51₂₆₁), nCTK₂₈₀, nCUL48₂₉₄, nCUL52₁₄₆, nCUS₁₀₅₉₂, nCgD₁₀₃₈, nCgI₁₀₉₅, nCgE₁₅₆₉, nCUS8.5₂₃₇, nCUS9₃₆₀, nCUL49/CUL48₂₀₄₄, nCUL49₄₂₀, nCUL48₁₂₆₉, nCICP4₆₂₆, nCgL₆₅₅, nCUL₁₈₂₃, nCdUTP₉₁₈, nCUL49.5₂₆₁, nCUL49₂₅₅, nCUL52₇₄₉, and/or nCdUTP₃₂₀₀, as well as allelic variants of such regions.
A particularly preferred CHV genome of the present invention includes a heterologous nucleic acid molecule located in a region of the CHV genome comprising (e.g., including, represented by, or identified by) at least one of the following nucleic acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO:87, as well as complements of such regions and allelic variants of such regions. The relationships between certain nucleic acid molecules and nucleic acid sequences of the present invention are detailed below. It should be noted that since nucleic acid sequencing technology is not entirely error-free, the sequences represented by the SEQ ID NOs in the present invention at best represent apparent nucleic acid or amino acid sequences of CHV nucleic acid molecules or proteins of the present invention. It is also to be noted that a double-stranded nucleic acid molecule of the present invention for which a nucleic acid sequence has been determined for one strand that is represented by a SEQ ID NO also comprises a complementary strand having a sequence that is a complement of that SEQ ID NO. As such, nucleic acid molecules of the present invention, which can be either double-stranded or single-stranded, include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with either a given SEQ ID NO denoted herein and/or with the complement of that SEQ ID NO, which may or may not be denoted herein. Methods to deduce a complementary sequence are known to those skilled in the art.
The present invention includes a recombinant CHV genome that includes a heterologous nucleic acid molecule that encodes a protective compound; the present invention also includes a recombinant CHV containing such a genome. As used herein, a protective compound is a compound that when administered to an animal protects that animal from a disease corresponding to that compound. For example, a compound derived from Dirofilaria immitis protects an animal from heartworm and other related infections, whereas a compound derived from a virus protects an animal from disease caused by that and related viruses. As used herein, the ability of a compound to protect an animal from a disease refers to the ability of that protective compound to treat, ameliorate and/or prevent the disease.
A protective compound of the present invention includes, but is not limited to, a protective protein and a protective RNA species. Essentially any heterologous nucleic acid molecule that encodes a protective protein or RNA can be used in the present invention. A protective protein of the present invention can be, for example, an immunogen that elicits an immune response which will protect an animal from the corresponding disease or some other compound (e.g., an immunomodulator, a toxin, an enzyme, an antibody, or other binding protein) that neutralizes and/or reduces the disease. A protective RNA of the present invention can be, for example, an RNA-based drug, a ribozyme, a molecule capable of triple helix formation, or an antisense RNA that effectively prevents the expression of a detrimental protein, thereby protecting an animal from disease.
It is within the scope of the present invention to produce therapeutic compositions against a variety of diseases, including infectious diseases, genetic diseases, and other metabolic diseases, including diseases that lead to abnormal cell growth, degenerative processes, and/or immunological defects. Therapeutic compositions of the present invention can protect animals from a variety of diseases including, but not limited to, allergies, autoimmune diseases, cancers, cardiovascular diseases, graft rejection, hematopoietic disorders, immunodeficiency diseases, immunoproliferative diseases, immunosuppressive disorders, infectious diseases, inflammatory diseases, jaundice, septic shock, other immunological defects, as well as other genetic or metabolic defects.
One preferred embodiment of the present invention is a recombinant CHV having a heterologous nucleic acid molecule within its genome that encodes a compound-that protects a canid, or other animal susceptible to CHV infection, from infectious disease. Such disease can be caused by a variety of infectious agents, including, but not limited to, helminth parasites, protozoan parasites, ectoparasites, fungi (including yeast), bacteria and/or viruses. It should also be noted that although some infectious agents have not been definitively classified into one of these groups, such infectious agents are also included in the present invention. A preferred protective compound is derived from (e.g., obtained from natural source or produced using recombinant or synthetic chemistry techniques) an infectious agent.
Preferred helminth infectious agents to target include nematodes, cestodes and trematodes, with filariid, ascarid, capillarid, strongylid, strongyloides, trichostrongyle, and trichurid, parasitic helminths being more preferred, and filariid nematodes being even more preferred. More preferred parasitic helminths to target include the following: Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria (e.g., D. immitis), Dracunculus, Echinococcus, Enterobius, Filaroides, Haemonchus, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum, Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirometra, Spirocera, Stephanofilaria, Strongyloides, Strongylus, Taenia, Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris. Uncinaria, and Wuchereria. More preferred helminths to target, particularly with respect to being canine pathogens include Ancylostoma, Angiostrongylus, Brugia, Capillaria, Crenosoma, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria, Echinococcus, Filaroides, Nanophyetus, Opisthorchis, Paragonimus, Physaloptera, Spirometra, Spirocera, Strongyloides, Taenia, Toxascaris., Toxocara,. Trichinella, Trichuris, and Uncinaria.
Preferred protozoal infectious agents to target include Acetospora, Apicoplexa, Ciliophora, Labyrinthomorphorpha, Microspora, Myxozoa and Sarcomastigophora, with Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma, being more preferred. More preferred protozoans to target, particularly with respect to being canine pathogens, include Babesia, Balantidium, Besnoitia, Cryptosporidium, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Pentatrichomonas, Pneumocystis, Sarcocystis, Schistosoma, Toxoplasma, and Trypanosoma.
Preferred ectoparasite infectious agents to target include fleas, flies, mosquitoes, ticks, mites, lice, spiders, ants and true bugs, with fleas being more preferred. More preferred are fleas of the genera Ctenocephalides, Ctopsyllus, Diamanus, Echidnophaga Nosopsyllus, Pulex, Tunga, and Xenopsylla, with Ctenocephalides canis and Ctenocephalides felis fleas being particularly preferred.
Preferred fungal agents include Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemonium, Phialophora, Prottotheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha.
Preferred bacterial infectious agents to target include Actinomyces, Bacillus, Bacteroides, Bordetella, Bartonella (e.g., B. henselae), Borrelia (e.g., B. burgdorferi), Brucella, Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella, Dermatophilus, Enterococcus, Ehrlichia (e.g., E. canis), Escherichia, Francisella, Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia (e.g., Y. pestis).
Preferred virus infectious agents to target include adenoviruses, caliciviruses, coronaviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, oncogenic viruses, panleukopenia viruses, papilloma viruses, parainfluenza viruses, parvoviruses, rabies viruses, and reoviruses, as well as other cancer-causing or cancer-related viruses.
A preferred recombinant CHV of the present invention includes a recombinant CHV genome having a heterologous nucleic acid molecule encoding a protective compound that elicits an immune response. As used herein, a protective compound that elicits an immune response refers to a compound that when administered to an animal in an appropriate manner, known to those skilled in the art, leads to the production of an immune response in that animal against the protective compound. The immune response, which can include humoral and/or cell-mediated components, preferably protects the immunized animal against the disease(s) targeted by the protective compound. As such, preferred protective compounds include, but are not limited to, antigens associated with diseases, as disclosed herein. Particularly preferred protective compounds of the present invention include Dirofilaria immitis antigens, Toxoplasma gondii antigens, Sarcocystis antigens, Bartonella henselae antigens, Yersinia pestis antigens, flea antigens, flea allergens, viral antigens, and cancer antigens. Even more preferred protective compounds are those disclosed in the following patent applications and patent publications, which are incorporated by reference herein in their entireties: U.S. Pat. No. 5,356,622, issued Oct. 18, 1994; U.S. Pat. No. 5,399,485, issued Mar. 21, 1995; U.S. Pat. No. 5,418,137, issued May 23, 1995; PCT Publication No. WO 92/13560, published Aug. 20, 1992; PCT Publication No. WO 93/10225, published May 27, 1993; PCT Publication No. WO 93/23077, published Nov. 25, 1993; PCT Publication No. WO 94/15593, published Jul. 21, 1994; PCT Publication No. WO 94/17813, published Aug. 18, 1994; PCT Publication No. WO 94/17824, published Aug. 18, 1994; PCT Publication No. WO 95/24198, published Sep. 14, 1995; PCT Publication No. WO 95/32988, published Dec. 7, 1995; U.S. Ser. No. 08/401,509, filed Mar. 9, 1995; U.S. Ser. No. 08/415,365, filed Mar. 30, 1995; U.S. Ser. No. 08/450,944, filed May 23, 1995; U.S. Ser. No. 08/473,034, filed Jun. 6, 1995; U.S. Ser. No. 08/482,304, filed Jun. 7, 1995; U.S. Ser. No. 08/485,434, filed Jun. 7, 1995; U.S. Ser. No. 08/486,036, filed Jun. 7, 1995; U.S. Ser. No. 08/558,735 filed Nov. 16, 1995; PCT Ser. No. PCT/US95/13200, filed Oct. 6, 1995; PCT Ser. No. PCT/US95/14442, filed Oct. 18, 1995; U.S. Ser. No. 08/630,822, filed Apr. 10, 1996; U.S. Ser. No. 08/602,262, filed Feb. 15, 1996; PCT Ser. No.: PCT/US96/03133, filed Mar. 8, 1996;. U.S. Ser. No. 08/639,075, filed Apr. 24, 1996; PCT Ser. No. PCT/US96/07709, filed May 23, 1996; and PCT Ser. No. PCT/US96/09848, filed Jun. 7, 1996, and related filings.
Another preferred protective compound of the present invention is an immunomodulator. Suitable immunomodulators include compounds that enhance the immune response as well as compounds that suppress the immune response. Compounds that enhance the immune response include compounds that preferentially enhance humoral immunity as well as compounds that preferentially enhance cell-mediated immunity. Suitable compounds can be selected depending on the disease being targeted. Suitable immunomodulators include, but are not limited to, cytokines, chemokines, superantigens, and other immunomodulators as well as compounds that induce the production of cytokines, chemokines and other immunomodulators. Examples of such protective compounds 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), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta (TGF-β), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF).
One preferred embodiment of the present invention is a recombinant CHV having more than one heterologous nucleic acid molecule included in the CHV genome. Such a CHV can include two or more heterologous nucleic acid molecules encoding two or more protective compounds to protect an animal from a given disease (e.g., two or more heartworm antigens), or can include two or more heterologous nucleic acid molecules encoding protective compounds each targeted against a different disease (e.g., a compound to protect an animal against heartworm and a compound to protect an animals against a viral infection). A preferred multivalent CHV can also include an heterologous nucleic acid molecule encoding a protective compound that elicits an immune response as well as an heterologous nucleic acid molecule encoding an immunomodulator to enhance the desired immune response. Also included in the present invention are protective compounds that are fusion, or multivalent, proteins comprising more than one functional domain.
The present invention also includes recombinant CHV genomes. As such, the invention includes any CHV genome disclosed herein, including those included in recombinant CHV of the present invention. Also included in the present invention are cells comprising recombinant CHV genomes of the present invention. As used herein, a cell comprising a recombinant CHV genome is a cell into which a recombinant CHV genome has been introduced. Such introduction can be accomplished by any method by which a nucleic acid molecule can be inserted into a cell. Such methods, known to those skilled in the art, include, but are not limited to, infection (i.e., with a virus comprising the genome), transfection, transformation, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A preferred cell comprises a recombinant CHV having a heterologous nucleic acid molecule, which preferably is operatively linked to a transcription control sequence. Cells containing CHV genomes are useful in the production of recombinant CHV. Methods to produce recombinant CHV are disclosed herein.
The present invention also includes isolated CHV nucleic acid molecules. As used herein, a CHV nucleic acid molecule is a nucleic acid molecule that is derived from CHV. As such, the nucleic acid molecule can be produced, for example, by recovery of such a nucleic acid molecule directly from a CHV genome, by recombinant DNA techniques, or by chemical synthesis. That the CHV nucleic acid molecule is isolated indicates that the molecule is removed from its natural milieu. An isolated CHV nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.
A preferred CHV nucleic acid molecule of the present invention is a CHV nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following: with a CdUTPase gene, a CgC gene, a CgE gene, a CgG gene, a CgI gene, a CPK gene, a CTK gene, a CIR6 gene, a CUS2 gene, a CUS9 gene, a CUL49 gene, a CUL51 gene, a CUL45 gene, a CgD gene, a CUL48 gene, a CgL gene, a CUL49.5 gene, a CICP4 gene, a CUS8.5 open reading frame, and/or a CUL52 gene; with other regions of a CUS; and/or with a region of the CHV genome spanning from about the 3′ end of the coding region of the CUL41 gene through about the 3′ end of the coding region of the CUL38 gene. The identifying characteristics of such regions, including the CHV genes listed, are heretofore described.
As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify molecules having similar nucleic acid sequences. Stringent hybridization conditions typically permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity.with the nucleic acid molecule being used as a probe in the hybridization reaction. Formulae to calculate the appropriate hybridizations and wash conditions to achieve hybridization permitting 30% or less mis-match between two nucleic acid molecules are disclosed, for example, in Meinkoth et al, 1984, Anal. Biochem 138, 267-284; Meinkoth et al, ibid, is incorporated by reference herein in its entirety. An example of such conditions includes, but is not limited to, the following: Oligonucleotide probes of about 18-25 nucleotides in length with T_m's ranging from about 50° C. to about 65° C., for example, can be hybridized to nucleic acid molecules typically immobilized on a filter (e.g., nitrocellulose filter) in a solution containing 2×SSPE, 1% Sarkosyl, 5× Denhardts and 0.1 mg/ml denatured salmon sperm DNA at a temperature as calculated using the formulae of Meinkoth et al., ibid. for about 2 to about 12 hours. The filters are then washed 3 times in a wash solution containing 2×SSPE, 1% Sarkosyl at about 55° C. for about 15 minutes each. The filters can be further washed in a wash solution containing 2×SSPE, 1% Sarkosyl at about 55° C. for about 15 minutes per wash.
A CHV nucleic acid molecule of the present invention can include an isolated natural CHV gene or a homolog thereof, the latter of which is described in more detail below. A CHV nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a CHV nucleic acid molecule of the present invention is the minimal size that can form a stable hybrid with one of the aforementioned CHV genes' and other regions under stringent hybridization conditions.
Isolated CHV nucleic acid molecules include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a CHV protein of the present invention or to form stable hybrids under stringent conditions with natural gene isolates.
A CHV nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). CHV nucleic acid molecule homologs can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid molecule and/or by hybridization with a CHV region as defined above.
An isolated CHV nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one CHV protein of the present invention; such proteins are discussed in further detail below. Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a CHV protein.
One embodiment of the present invention is a CHV nucleic acid molecule that, when administered to an animal, is capable of protecting that animal from CHV infection. Such a CHV nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In an additional embodiment, a CHV nucleic acid molecule of the present invention can encode a protective protein, the nucleic acid molecule being delivered to the animal by direct injection (i.e, as a naked nucleic acid) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.
One embodiment of the present invention is a CHV nucleic acid molecule that hybridizes under stringent hybridization conditions with nCUS₅₄₉₅, nCgC/CUL45₂₁₀₀, nCgE₇₅₀, nCgI₁₆₁, nCUS9₅₇₉, nCdUTP/CUL51₇₄₃, nCTK₂₈₀, nCUL48₂₉₄, a nCUL49 included in nCHin₃₀₀₀, nCUL52₁₄₆, nCgI₁₀₉₅, nCgE₁₅₆₉, nCUS8.5₂₃₇, nCUS9₃₆₀, nCUL49/CUL48₂₀₄₄, nCUL49₄₂₀, nCUL48₁₂₆₉, nCICP4₆₂₆, nCgL₆₅₅, nCUL₁₈₂₃, nCdUTP₉₁₈, nCUL49.5₂₅₅, nCUL49₂₅₅, and/or nCUL52₇₄₉. Such a CHV nucleic acid molecule can also hybridize under stringent hybridization conditions with nCAsc₉₃₀₀, nCAsc₁₀₀₀₀, nCHin₃₀₀₀, nCHin₁₉₀₀, nCHin₅₅₀₀, nCHin₈₅₀₀, nCIR6₅₅₂, nCUS2₁₁₇₆, nCPK₁₂₀₃, nCgG₁₂₄₈, nCgD₃₅₇, nCdUTP₄₅₉, nCTK₂₇₉, nCUS9₄₅₀, nCUL48₂₉₁, nCUL51₂₆₁, nCUL52₁₄₄, nCgI₁₅₉, nCUS₁₀₅₉₂, nCICP4₆₂₄, nCgL₅₁₆, nCUL52₇₄₇, nCdUTP₈₅₈, and/or nCdUTP₃₂₀₀. At least some of such CHV nucleic acid molecules can hybridize under stringent hybridization conditions with a nucleic acid molecule having nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO:87, as well as complements of such sequences.
SEQ ID NO:1 and SEQ ID NO:2 represent the deduced nucleic acid sequences of the two complementary strands of nCUS₅₄₉₅. Translation of SEQ ID NO:1 and SEQ ID NO:2 indicates that nucleic acid molecule nCUS₅₄₉₅encodes CIR6, CUS2, CPK and CgG proteins as well as a portion of a CgD protein. Specifically, SEQ ID NO:2 includes: a coding region for a CIR6 protein of about 183 amino acids, denoted herein as nCIR6₅₅₂and represented by SEQ ID NO:3, assuming a start codon spanning about nucleotides 4566-4568 and a stop codon spanning about nucleotides 5115-5117 of SEQ ID NO:2; and a coding region for a CUS2 protein of about 391 amino acids, denoted herein as nCUS2₁₁₇₆and represented by SEQ ID NO:5, assuming a start codon spanning about nucleotides 3232-3234 and a stop codon spanning about nucleotides 4405-4407 of SEQ ID NO:2. The amino acid sequences of the respective encoded proteins PCIR6₁₈₃and PCUS2₃₉₁are represented by SEQ ID NO:4 and SEQ ID NO:6. SEQ ID NO:1 includes: a coding region for a CPK protein of about 400 amino acids, denoted herein as nCPK₁₂₀₃and represented by SEQ ID NO:7, assuming a start codon spanning about nucleotides 2384-2386 and a stop codon spanning about nucleotides 3584-3586 of SEQ ID NO:1; a coding region for a CgG protein of about 415 amino acids, denoted herein as nCgG₁₂₄₈and represented by SEQ ID NO: 9, assuming a start codon spanning about nucleotides 3698-3700 and a stop codon spanning about nucleotides 4943-4945 of SEQ ID NO:1; and a partial coding region for a CgD protein of about 119 amino acids, denoted herein as nCgD₃₅₇and represented by SEQ ID NO:11, assuming a start codon spanning about nucleotides 5137-5139. The amino acid sequences of the respective encoded proteins PCPK₄₀₀, PCgG₄₁₅, and PCgD₁₁₉are represented by SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.
SEQ ID NO:13 and SEQ ID NO:14 represent the deduced nucleic acid sequences of the two complementary strands of nCdUTP/CUL51₇₄₃. SEQ ID NO:13 includes a partial coding region for a CdUTPase protein of about 152 amino acids, denoted herein as nCdUTP₄₅₉and represented by SEQ ID NO:15, assuming a first in-frame codon spanning about nucleotides 3-5, and a stop codon spanning about nucleotides 459-461 of SEQ ID NO:13. The amino acid sequence of the encoded protein PCdUTP₁₅₂is represented by SEQ ID NO:16. SEQ ID NO:14 includes a partial coding region for a CUL51 protein of about 86 amino acids, denoted herein as nCUL51₂₆₁and represented by SEQ ID NO:33, assuming a first in-frame codon spanning about nucleotides 1-3, and a stop codon spanning about nucleotides 259-261 of SEQ ID NO:14. The amino acid sequence of the encoded protein PCUL51₈₆is represented by SEQ ID NO:34.
SEQ ID NO:17 and SEQ ID NO:19 represent the deduced nucleic acid sequences of the two complementary strands of nCUS9₅₇₉. SEQ ID NO:17 includes a coding region for a CUS9 protein of about 149 amino acids, denoted herein as nCUS9₄₅₀and represented by SEQ ID NO:20, assuming a start codon spanning about nucleotides 54-56 and a stop codon spanning about nucleotides 501-503 of SEQ ID NO:17. The amino acid sequence of the encoded protein PCUS9₁₄₉is represented by SEQ ID NO:18.
SEQ ID NO:21 and SEQ ID NO:23 represent the deduced nucleic acid sequences of the two complementary strands of nCUL48₂₉₄. SEQ ID NO:21 includes a partial coding region for a CUL48 protein of about 97 amino acids, denoted herein as nCUL48₂₉₁and represented by SEQ ID NO:24, assuming a first in-frame codon spanning about nucleotides 3-5 of SEQ ID NO:21. The amino acid sequence of the encoded protein PCUL48₉₇is represented by SEQ ID NO:22.
SEQ ID NO:25 and SEQ ID NO:27 represent the deduced nucleic acid sequences of the two complementary strands of nCUL52₁₄₆. SEQ ID NO:25 includes a partial coding region for a CUL52 protein of about 48 amino acids, denoted herein as nCUL52₁₄₄and represented by SEQ ID NO:28, assuming a first in-frame codon spanning about nucleotides 1-3 of SEQ ID NO:25. The amino acid sequence of the encoded protein PCUL52₄₈is represented by SEQ ID NO:26.
SEQ ID NO:29 and SEQ ID NO:31 represent the deduced nucleic acid sequences of the two complementary strands of nCgI₁₆₁. SEQ ID NO:29 includes a partial coding region for a CgI protein of about 53 amino acids, denoted herein as nCgI₁₅₉and represented by SEQ ID NO:32, assuming a first in-frame codon spanning about nucleotides 3-5 of SEQ ID NO:29. The amino acid sequence of the encoded protein PCgI₅₃is represented by SEQ ID NO:30.
SEQ ID NO:35 and SEQ ID NO:37 represent the deduced nucleic acid sequences of the two complementary strands of nCTK₂₈₀. SEQ ID NO:35 includes a partial coding region for a CTK protein of about 93 amino acids, denoted herein as nCTK₂₇₉and represented by SEQ ID NO:38, assuming a first in-frame codon spanning about nucleotides 2-4 of SEQ ID NO:35. The amino acid sequence of the encoded protein PCTK₉₃is represented by SEQ ID NO:36.
The identities of additional nucleic acid molecules, nucleic acid sequences, proteins, and amino acid sequences are presented in the Examples.
Comparison of the CHV nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87 with known sequences indicates that none of these CHV nucleic acid sequences share more than about 70% identity (many, if not all, sharing significantly less identity) with a known nucleic acid sequence. As such, a preferred CHV nucleic acid molecule has a nucleic acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, and even more preferably at least about 99% identical to nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO:87, as well as complements of such sequences.
A more preferred CHV nucleic acid molecule of the present invention includes at least a portion of CHV nucleic acid molecule nCAsc₉₃₀₀, nCAsc₁₀₀₀₀, nCHin₃₀₀₀, nCHin₁₉₀₀, nCHin₅₅₀₀, nCHin₈₅₀₀, nCUS₅₄₉₅, nCIR6₅₅₂, nCUS2₁₁₇₆, nCPK₁₂₀₃, nCgG₁₂₄₈, nCdUTP/CUL51₇₄₃, nCdUTP₄₅₉, nCUS9₅₇₉, nCUS9₄₅₀, nCUL48₂₉₄, nCUL48₂₉₁, nCUL52₁₄₆, nCUL52₁₄₄, nCgI161, nCgI₁₅₉, nCgE₇₅₀, nCTK₂₈₀, nCTK₂₇₉, nCUL51₂₆₁, nCUS₁₀₅₉₂, nCgI₁₀₉₅, nCgE₁₅₆₉, nCUS8.5₂₃₇, nCUS9₃₆₀, nCUL49/CUL48₂₀₄₄, nCUL49₄₂₀, nCUL48₁₂₆₉, nCICP4₆₂₆, nCICP4₆₂₄, nCgL₆₅₅, nCgL₅₁₆, nCUL₁₈₂₃, nCdUTP₉₁₈, nCUL49.5₂₆₁, nCUL49₂₅₅, nCUL52₇₄₉, nCUL52₇₄₇, nCdUTP₈₅₈, and/or nCdUTP₃₂₀₀, as well as allelic variants of those CHV nucleic acid molecules. Such CHV nucleic acid molecules can include nucleotides in addition to those included in the defined fragments; examples of such CHV nucleic acid molecules include full-length genes, full-length coding regions, or nucleic acid molecules encoding multivalent proteins. Particularly preferred CHV nucleic acid molecules are nCAsc₉₃₀₀, nCAsc₁₀₀₀₀, nCHin₃₀₀₀, nCHin₁₉₀₀, nCHin₅₅₀₀, nCHin₈₅₀₀, nCUS₅₄₉₅, nCIR6,₅₅₂, nCUS2₁₁₇₆, nCPK₁₂₀₃, nCgG₁₂₄₈, nCdUTP/CUL51₇₄₃, nCdUTP₄₅₉, nCUS9₅₇₉, nCUS9₄₅₀, nCUL48₂₉₄, nCUL48₂₉₁, nCUL52₁₄₆, nCUL52₁₄₄, nCgI₁₆₁, nCgI₁₅₉, nCgE₇₅₀, nCTK₂₈₀, nCTK₂₇₉, nCUL51₂₆₁, nCUS₁₀₅₉₂, nCgI₁₀₉₅, nCgE₁₅₆₉, nCUS8.5₂₃₇, nCUS9₃₆₀, nCUL49/CUL48₂₀₄₄, nCUL49₄₂₀, nCUL48₁₂₆₉, nCICP4₆₂₆, nCICP₆₂₄, nCgL₆₅₅, nCgL₅₁₆, nCUL₁₈₂₃, nCdUTP₉₁₈, nCUL49.5₂₆₁, nCUL49₂₅₅, nCUL52₇₄₉, nCUL52₇₄₇, nCdUTP₈₅₈, and/or nCdUTP₃₂₀₀,
Similarly, a preferred CHV nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ It NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO:87; or a complement of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and/or SEQ ID NO:87; as well as allelic variants of such nucleic acid molecules. More preferred is a nucleic acid molecule that includes at least one of the following nucleic acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, and/or SEQ ID NO:87; or a complement of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and/or SEQ ID NO:87; also included are nucleic acid molecules that are allelic variants of nucleic acid molecules having those nucleic acid sequences. Such nucleic acid molecules can include nucleotides in addition to those included in the SEQ ID NOs.
The present invention also includes CHV nucleic acid molecules encoding a protein, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed. CHV proteins of the present invention are described in more detail below. Particularly preferred nucleic acid molecules are those that encode a protein having at least one of the following amino acid sequences: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and/or SEQ ID NO:88.
The present invention also includes CHV nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, CHV nucleic acid molecules of the present invention. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein. The size of the oligonucleotide must also be sufficient for the use of the oligonucleotide in accordance with the present invention. Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit CHV infection as disclosed herein.
The present invention also includes an isolated CHV protein encoded by a CHV nucleic acid molecule of the present invention. As such, the present invention includes a CHV protein encoded by a CHV nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following: a CdUTPase gene, a CgC gene, a CgE gene, a CgG gene, a CgI gene, a CPK gene, a CTK gene, a CIR6 gene, a CUS2 gene, a CUS9 gene, a CUL49 gene, a CUL51 gene, a CUL45 gene, a CgD gene, a CUL48 gene, and/or a CUL52 gene; and/or with other portions of a CUS region; and/or with a CgL gene, a CUL49.5 gene, a CICP4 gene, and/or a CUS8.5 open reading frame.
According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology or can be produced by chemical synthesis. As used herein, a CHV protein can be a full-length protein or any homolog of such a protein. Examples of CHV homologs include CHV proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog retains a desired activity of the natural protein, such as, but not limited to, enzymatic activity, activity important for viral growth, and/or ability to elicit an immune response. These activities can be measured using techniques known to those skilled in the art.
CHV protein homologs can be the result of natural allelic variation or natural mutation. CHV protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
The minimal size of a CHV protein homolog of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridize under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. As such, the size of the nucleic acid molecule encoding such a protein homolog is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence. It should also be noted that the extent of homology required to form a stable hybrid can vary depending on whether the homologous sequences are interspersed throughout the nucleic acid molecules or are clustered (i.e., localized) in distinct regions on the nucleic acid molecules. The minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode a CHV protein homolog of the present invention is from about 12 to about 18 nucleotides in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. Similarly, the minimal size of a CHV protein homolog of the present invention is from about 4 to about 6 amino acids in length, with preferred sizes depending on whether full-length, fusion, or other functional portions of such proteins are desired.
One embodiment of the present invention is a CHV protein that can protect an animal from disease, preferably by eliciting an immune response against CHV, and/or can detect CHV infection in an animal. The minimum size of such a protein is a minimum size sufficient to form an epitope, a size that typically is at least from about 5 to about 9 amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope.
The present invention also includes mimetopes of CHV proteins that can be used in accordance with methods as disclosed for CHV proteins of the present invention. As used herein, a mimetope of a CHV protein of the present invention refers to any compound that is able to mimic the activity of such a CHV protein, often because the mimetope has a structure that mimics the CHV protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
One embodiment of the present invention is a fusion, or multivalent, protein that includes a CHV protein-containing domain attached to another functional domain. Such a domain can be an entire protein, or function portion thereof. Examples of such domains include not only protective compounds as disclosed above, but also domains that enhance a protein's stability (e.g., during production, storage and/or use) or that aid in protein purification.
A preferred isolated protein of the present invention is a protein encoded by a nucleic acid molecule that. hybridizes under stringent hybridization conditions with nCIR6₅₅₂, nCUS2₁₁₇₆, nCPK₁₂₀₃, nCgG₁₂₄₈, nCgD₃₅₇, nCgC/CUL45₂₁₀₀, nCdUTP₄₅₉, nCUS9₄₅₀, nCUL48₂₉₁, nCUL52₁₄₄, nCgI₁₅₉, nCgE₇₅₀, nCTK₂₇₉, nCUL51₂₆₁, nCgI₁₀₉₅, nCgE₁₅₆₉, nCUS8.5₂₃₇, nCUS9₃₆₀, nCUL49/CUL48₂₀₄₄, nCUL49₄₂₀, nCUL48₁₂₆₉, nCICP4₆₂₆, nCgL₆₅₅, nCUL₁₈₂₃, nCdUTP₉₁₈, nCUL49.5₂₆₁, nCUL49₂₅₅, and/or nCUL52₇₄₉. Also included is a protein that hybridizes under stringent hybridization conditions with nCAsc₉₃₀₀, nCAsc₁₀₀₀₀, nCHin₃₀₀₀, nCHin₁₉₀₀, nCHin₅₅₀₀, and/or nCHin8500. A further preferred isolated protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having the complement of nucleic acid sequence SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and/or SEQ ID NO:87, or by an allelic variant of any of such nucleic acid molecules. Proteins encoded by nucleic acid sequences SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and SEQ ID NO:87 have amino acid sequences SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:88, respectively.
Comparison of the CHV amino acid sequences SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and SEQ ID NO:88 with known sequences indicates that none of these CHV amino acid sequences share more than about 75% identity (many, if not all, sharing significantly less identity) with a known amino acid sequence.
A preferred CHV protein has an amino acid sequence that is at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95%, and even more preferably at least about 99% identical to amino acid sequence SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and/or SEQ ID NO:88.
A more preferred CHV protein includes at least a portion of PCIR6₁₈₃, PCUS2₃₉₁, PCPK₄₀₀, PCgG₄₁₅, PCdUTP₁₅₂, PCUS9₁₄₉, PCUL48₉₇, PCUL52₄₈; PCUL51₈₆; PCgI₁₃, PCTK₉₃, PCgI₃₆₄, PCgE₅₂₂, PCUS8.5_78,PCUS9₁₁₉, PCUL49₁₃₉, PCUL48₄₂₂, PCICP4₂₀₈, PCgL₁₇₁, PCdUTP₃₀₅, PCUL49.5₈₆, PCUL49₈₅, and/or PCUL52₂₄₉, as well as proteins encoded by allelic variants of the nucleic acid molecules encoding such proteins. Also preferred are proteins including at least a portion of CgE and/or CUL49 proteins. A particularly preferred CHV protein includes PCIR6₁₈₃, PCUS2₃₉₁, PCPK₄₀₀, PCgG₄₁₅, PCdUTP₁₅₂, PCUS9₁₄₉, PCUL48₉₇, PCUL52₄₈, PCUL51₈₆, PCgI₅₃, PCTK₉₃, PCgI₃₆₄, PCgE₅₂₂, PCUS8.5₇₈, PCUS9₁₁₉, PCUL49₁₃₉, PCUL48₄₂₂, PCICP4₂₀₈, PCgL₁₇₁, PCdUTP₃₀₅, PCUL49.5₈₆, PCUL49₈₅, and/or PCUL52₂₄₉(including, but not limited to the encoded proteins, full-length proteins, processed proteins, multivalent proteins).
Similarly, a preferred CHV protein of the present invention includes at least a portion of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:l6, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:581 SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and/or SEQ ID NO:88. A particularly preferred CHV protein includes SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, and/or SEQ ID NO:88. Also included are proteins encoded by allelic variants of the nucleic acid molecules encoding such proteins.
The present invention also includes a recombinant vector, which includes at least one isolated CHV nucleic acid molecule inserted into any vector capable of delivering the CHV nucleic acid molecule into a host cell. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of CHV nucleic acid molecules of the present invention. Suitable and preferred CHV nucleic acid molecules to include in a recombinant vector are disclosed herein.
One embodiment of the present invention is a recombinant vector comprising an inactive CHV gene. Such a recombinant vector, also referred to as a transfer vector, can be used to produce a CHV comprising a CHV genome having an inactive gene by, for example, co-transfecting such a transfer vector with a CHV genome into a host cell and selecting for a CHV comprising a recombinant CHV genome having an inactive gene. Such a recombinant CHV genome is produced in the host cell by homologous recombination between the inactive gene on the transfer vector and the corresponding active gene on the transfected CHV genome. Transfection, culturing and purification methods to obtain recombinant CHV and CHV genomes are known to the art; see, for example, Graham et al., 1973, Virology 52:456-467; Graham et al, ibid. is incorporated by reference herein in its entirety.
Another embodiment of the present invention is a recombinant vector comprising a CHV nucleic acid molecule that includes a heterologous nucleic acid molecule (i.e., a heterologous nucleic acid molecule is located within a CHV nucleic acid molecule). Suitable and preferred heterologous nucleic acid molecules are disclosed herein. Such a heterologous nucleic acid molecule can be operatively linked to a transcription control sequence, as disclosed above. A recombinant vector comprising a CHV nucleic acid molecule into which a heterologous nucleic acid molecule is inserted is also a transfer vector. Such a transfer vector can be co-transfected with a CHV genome into a host cell to produce a recombinant CHV having a CHV genome including a heterologous nucleic acid molecule, using methods as described above. Recombinant CHV can be selected by identifying those CHV that have the heterologous nucleic acid molecule. If the recombinant vector comprises a selectable marker into which the heterologous nucleic acid molecule is inserted, selection methods as disclosed herein can also be used to identify recombinant CHV. A preferred embodiment is a recombinant vector comprising a CHV nucleic acid molecule having a heterologous nucleic acid molecule in which a majority of the CHV nucleic acid molecule is deleted; a sufficient size of the CHV nucleic acid molecule is retained to allow homologous recombination to occur with the corresponding target gene on the CHV genome. Examples of insertion of a heterologous nucleic acid molecule into a CHV genomic restriction site and into a CHV gene, as well as use of a selectable marker are provided in the Examples section.
Transfer vectors of the present invention are preferably able to replicate in bacterial, and particularly E. coli, hosts, thereby enabling easy manipulation of the CHV nucleic acid molecules, and, if included, heterologous nucleic acid molecules, prior to insertion of such CHV nucleic acid molecules into a CHV genome. Such manipulations, including culturing of E. coli comprising such vectors, is described, for example, in Sambrook et al, ibid.
In one embodiment, recombinant CHV are produced by co-transfection of a set of overlapping cosmid clones comprising the entire viral CHV genome, at least one of the cosmid clones having been genetically engineered to, for example, contain an inactive CHV gene and/or a heterologous nucleic acid molecule. Details of such a method are presented in the Examples.
Any canid host cell is suitable for recombinant CHV production. Examples of suitable and preferred host cells are provided herein. After transfection, transfected cells are cultured in an effective medium, using techniques such as those described in Graham et al, ibid. As used herein, an effective medium refers to any medium in which the transfected cells can produce recombinant CHV. An effective medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins, growth factors and hormones. Culturing is carried out at a temperature, pH and oxygen content appropriate for the transfected cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.
Recombinant CHV can be recovered from the cultured transfected cells using a combination of standard techniques such as, but not limited to, freeze/thaw cycles, sonication, sucrose gradient centrifugation, and/or high speed centrifugation. Recombinant CHV genomes can be recovered from the cultured transfected cells using a combination of standard techniques such as, but not limited to, those described by Walboomers et al, 1976, Virology 74, 256-258.
Preferably, a recombinant CHV or recombinant CHV genome of the present invention is recovered in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the recombinant CHV or CHV genome as a vaccine without substantial negative side effects.
The present invention also includes a method to increase recombinant CHV plaque forming efficiency, thereby facilitating the production of recombinant CHV and CHV genomes. The phrase to increase recombinant CHV plaque forming efficiency refers to the ability to increase, or enhance, the production of CHV above the frequency of plaque forming units produced when a CHV genome is transfected into a cell in the absence of additional factors. The method includes the step of expressing the CHV alpha transinducing factor gene (i.e., CUL48 gene, also called the CHV alpha-tif gene) when a CHV genome is introduced into a canine cell.
Herpesviruses studied to date carry into infected cells a transcriptional activator called alpha transinducing factor (also called VP16 or Vmw65); see, for example, Batterson et al., 1983, J. Virol., 46:371-377. This transactivator protein interacts, along with cellular proteins, with a cis-acting sequence element present in the upstream regulatory regions of herpesvirus immediate early genes. Since purified herpesvirus DNA is infectious in the absence of alpha-tif, it is not absolutely required for expression of immediate early genes, but its presence in stably transfected cell lines has been shown to enhance infectivity of herpes simplex virus type-1 viral DNA; see, for example, Werstuck et al., 1990, J. Virol., 64:984-991.
Expression of the CHV alpha-tif gene can be accomplished in a variety of ways, including, but not limited to, the following. In one embodiment, a recombinant CHV genome is introduced into a canine cell expressing CHV alpha transinducing factor and the cells are cultured to produce recombinant CHV. A canine cell expressing CHV alpha-tif is a canine cell that has been genetically engineered to produce CHV alpha-tif in either a stable or transient manner. Such a canine cell can be produced by introducing a CUL48 nucleic acid molecule of the present invention into a canine cell line in such a manner that the CUL48 nucleic acid molecule can direct the expression of active alpha-tif. An example of a-useful CUL48 nucleic acid molecule is such nCUL48₁₂₆₉. Details regarding the production of an alpha-tif-expressing canine cell and the use such a cell in the production of recombinant CHV and CHV genomes is present in the Examples.
In another-embodiment, recombinant CHV plaque forming efficiency is increased by co-introducing a recombinant CHV genome and a CHV alpha transinducing factor gene into a canine cell and culturing the cell to produce recombinant CHV. The CHV alpha transinducing factor gene can be introduced as part of a recombinant molecule of the present invention. Details regarding this method are also presented in the Examples.
The present invention all includes canine cell lines that include a CHV alpha-tif gene; such cell lines are able to express alpha-tif, thereby facilitating CHV production. One example of such a cell line is a canine cell line transfected with CHV nucleic acid molecule nCUL48₁₂₆₉.
One embodiment of the present invention is a recombinant molecule that includes a CHV nucleic acid molecule operatively linked to a transcription control sequence. Such a recombinant molecule, when introduced into a host cell, can direct the expression of the CHV, nucleic acid molecule(s), thereby leading to the production of one or more CHV protein of the present invention. Such a recombinant molecule preferably is replication competent. Suitable and preferred CHV nucleic acid molecules to include in such a recombinant molecule are as disclosed herein for suitable and preferred CHV nucleic acid molecules per se.
Isolated CHV proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell that is capable of expressing one or more CHV proteins, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a, multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Suitable and preferred CHV nucleic acid molecules with which to transform a cell are as disclosed herein for suitable and preferred CHV nucleic acid molecules per se.
Suitable host cells to transform include any cell that can be transformed with a CHV-nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing CHV proteins of the present invention or can be capable of producing such proteins after being transformed with at least one CHV nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), insect, other animal and plant cells. Preferred host cells include bacterial, yeast, insect and mammalian cells. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p_Land lambda p_Rand fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus, phosphate-regulated and nitrate-regulated transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells, including those disclosed herein-for expression of heterologous nucleic acid molecules, including endogenous CHV transcription control regions.
Recombinant cells of the present invention can be used to produce one or more proteins by culturing such cells under conditions effective to produce such a protein, and recovering the protein. Effective conditions to produce a protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production. Such culturing conditions are well within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant CHV proteins may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
The phrase “recovering the protein” refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating the production of antibodies in a treated animal.
The present invention also includes isolated antibodies capable of selectively binding to a CHV protein of the present invention or to a mimetope thereof. Such antibodies are also referred to herein as anti-CHV antibodies. Isolated antibodies are antibodies that have been removed from their natural milieu. The term “isolated” does not refer to the state of purity of such antibodies. As such, isolated antibodies can include anti-sera containing such antibodies, or antibodies that have been purified to varying degrees.
As used herein, the term “selectively binds to” refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods known to those skilled in the art.
Antibodies of the present invention can be either polyclonal or monoclonal antibodies. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein or mimetope used to obtain the antibodies. Antibodies of the present invention also include chimeric antibodies that can bind to more than one epitope. Preferred antibodies are raised in response to proteins, or mimetopes thereof, that are encoded, at least in part, by a nucleic acid molecule of the present invention.
A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce CHV proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or an assay to monitor recombinant CHV administration, or side effects if used in a therapeutic composition.
Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as compounds to monitor recombinant CHV or recombinant CHV genome administration, (b) as therapeutic compounds to passively immunize an animal in order to protect the animal from CHV infection, and/or (c) as reagents in assays to detect CHV infection.
One embodiment of the present invention is a therapeutic composition that includes a recombinant CHV, a recombinant CHV genome, or a mixture (i.e., combination) of one or more recombinant CHVs and/or recombinant CHV genomes.
As used herein, a therapeutic composition, or vaccine, is a formulation that, when administered to an animal in an effective manner, is capable of protecting that animal from a disease against which the therapeutic composition is targeted. Some therapeutic compositions of the present invention can modulate (i.e., either elicit or suppress) an immune response to protect an animal from disease, whereas other therapeutic compositions can protect an animal in other ways, as disclosed herein. A therapeutic composition of the present invention can be used to prevent and/or treat a disease depending on whether the composition is administered as a prophylactic or after the animal has the disease.
A therapeutic composition of the present invention, when administered to an animal in an effective manner, can infect the cells of the animal (in a manner essentially harmless to the animal) and direct the production of a protective compound able to protect the animal from a disease targeted by the therapeutic composition. If a recombinant CHV includes a protective compound in its envelope and/or capsid, the CHV can function as an immunogen per se.
As disclosed herein, therapeutic compositions can be designed to target a variety of diseases, depending on the nature of the heterologous nucleic acid molecule(s) included in such recombinant CHV and recombinant CHV genomes. Examples of such diseases, and the nature of the corresponding heterologous nucleic acid molecules, are disclosed herein. Furthermore, therapeutic compositions of the present invention can comprise multivalent vaccines. For example, a CHV genome can encode a variety of protective compounds, and/or more than one recombinant CHV genome and/or recombinant CHV can be administered. The present invention also includes the use of a recombinant CHV or recombinant CHV genome to protect an animal against CHV infection. Such a therapeutic composition can, but need not, include a heterologous nucleic acid molecule.
A therapeutic composition of the present invention is preferably administered to a canid, due to the host range specificity of CHV. Suitable canids include dogs (including domesticated and wild dogs), foxes, wolves, jackals, coyotes, and other members of the family Canidae. Particularly preferred canids to treat include domesticated dogs.
It is, however, also within the scope of the present invention to administer therapeutic compositions to other animals. Recombinant CHV can be administered to any animal susceptible to such therapy. Without being bound by theory, it is believed that sufficiently high doses of a recombinant CHV composition of the present invention may be infectious in other animals, particularly other mammals. Mink and other mink-like mammals (e.g., those of the family Mustelidae, such as ermines, ferrets, fishers, martens, otters, and weasels), in particular, may be susceptible to CHV infection, as suggested by the ability of CHV to infect mink lung cells. The host range of recombinant CHV of the present invention can also be altered, as disclosed herein, to infect other animal cells. It is also to be noted that CHV genomes of the present invention can be administered as naked DNA vaccines or in association with other carriers (e.g., liposomes). In accordance with these embodiments, any animal, including, but not limited to, mammals, birds, amphibians, and arthropods (including arachnids and insects) can be administered a therapeutic composition of the present invention. Preferred animals to treat include dogs, cats, humans, ferrets, prairie dogs, other rodents, horses, cattle, sheep, pigs, and poultry, as well as other pets, work animals, economic food animals and zoo animals.
Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, the vaccine can also include an adjuvant and/or a carrier. One advantage of live virus-based vaccines, such as the recombinant CHVs of the present invention, is that adjuvants and carriers are not required to produce an efficacious vaccine, and in some cases, the advantages of recombinant CHV vaccines of the present invention would be precluded by the use of some adjuvants. However, it should be noted that use of adjuvants or carriers is not precluded by the present invention. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, other bacterial cell wall components; aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; other viral coat proteins; other bacterial-derived preparations; block copolymer adjuvants, such as Hunter's Titermax™ adjuvant (Vaxcel™, Inc. Norcross, Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.
In one embodiment, a therapeutic composition of the present invention is administered to an animal in an effective manner to enable the animal to produce sufficient protective compound(s) and/or to directly mount a sufficient immune response to protect the animal from disease. Acceptable protocols to administer therapeutic compositions in an effective manner include enumeration of individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period.
A preferred single dose of a recombinant CHV of the present invention is from about 1×10⁴to about 1×10⁷virus plaque forming units (pfu) per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original vaccination. Booster vaccinations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 1×10⁴to about 1×10⁷virus plaque forming units per kilogram (kg) body weight of the animal are administered from about 1 to about 2 times over a time period of from about 12 to about 18 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal and intramuscular routes.
A recombinant CHV genome can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intranasal and oral routes of administration being preferred. A preferred single dose of a recombinant CHV genome ranges from about 1 nanogram (ng) to about 100 micrograms (μg), depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art; see, for example, Wolff et al., 1990, Science 247, 1465-1468. Suitable delivery methods include, for example, injection, as drops, aerosolized and/or topical administration. Suitable excipients include, for example, physiologically acceptable aqueous solutions (e.g., phosphate buffered saline as well as others disclosed above), liposomes (including neutral or cationic liposomes), and other lipid membrane-based vehicles (e.g., micelles or cellular membranes).
In one embodiment, a therapeutic composition of the present invention is administered to a dam to protect her offspring from disease. In this method, the dam is administered the therapeutic composition at such a time as to be able to develop an immune response such that she can passively transfer antibodies produced against a protective compound of the present invention to her offspring. Such a method can also be used to protect offspring from CHV infection and is particularly useful since neonates are most affected by CHV infection.
The efficacy of a therapeutic composition of the present invention to protect an animal from disease can be tested in a variety of ways including, but not limited to, detection of protective protein or RNA within the treated animal, detection of protective antibodies within the treated animal, detection of cellular immunity within the treated animal, or challenge of the treated animal with an appropriate infectious agent, or other disease component, to determine whether the animal is now protected from the disease caused by such an agent or other component. Such techniques are known to those skilled in the art. In one embodiment, anti-CHV antibodies of the present invention are used to monitor recombinant CHV infection and can be used to distinguish wild type infections from infections using recombinant CHV of the present invention (i.e., by using antibodies that specifically recognize either recombinant CHV of the present invention or wild type virus).
In one embodiment, the efficacy of a therapeutic composition of the present invention may be improved by co-administering (a) a recombinant CHV or recombinant CHV genome and (b) a protective compound (e.g., subunit vaccine) encoded by a CHV nucleic acid molecule or heterologous nucleic acid molecule present in the CHV genome. While not being bound by theory, it is believed that administration of a protective compound in conjunction with the recombinant CHV or CHV genome can boost the immune response, particularly the antibody titer. The protective compound can be administered prior to, concomitant with, and/or following administration of the recombinant CHV or CHV genome. The protective compound can be either produced naturally, recombinantly, or synthetically. The protective compound should be sufficiently pure to allow for effective use of the compound as a vaccine; i.e., it should not cause substantial side effects. The protective compound can be joined (i.e., conjugated) to a carrier or other material that enhances the immunogenicity of the compound.
The present invention also includes the use of CHV nucleic acid molecules, CHV proteins, and anti-CHV antibodies as therapeutic compositions to protect animals from CHV infection. Methods to administer such compositions to canids are known to those skilled in the art. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition is from about 1 microgram (μg) to about 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster vaccinations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 μg to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal and intramuscular routes. A CHV nucleic acid molecule, including recombinant molecules, can be administered as described herein for administration of CHV genomes or CHV of the present invention. Recombinant molecules including heterologous nucleic acid molecules can also be used as therapeutic compositions to protect an animal from disease, using methods as disclosed herein.
It is also within the scope of the present invention to use isolated CHV proteins, mimetopes, CHV nucleic acid molecules and anti-CHV antibodies of the present invention as diagnostic reagents to detect CHV infection. Methods to use such diagnostic reagents to diagnose infection are well known to those skilled in the art.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecular biology, virology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid.; Ausubel et al, 1993, Current Protocols in Molecular Biology, Greene/Wiley Interscience, New York, N.Y.; Graham et al, ibid.; and related references. Ausubel et al, ibid. is incorporated by reference herein in its entirety. Nucleic acid and amino acid sequences of the present invention were compared to known sequences using BLAST (NCBI) and DNAsis (Hitachi Software, San Bruno, Calif.).

Example 1

This Example demonstrates the isolation of certain CHV nucleic acid molecules of the present invention.
The disclosed CHV nucleic acid molecules were amplified from a CHV genome by PCR amplification using a variety of primers designed in view of published herpesvirus sequences. The following PCR conditions were used: 0.2 millimolar (mM) dNTPs, 1 μM of each primer, 1× PCR buffer (available from Perkin Elmer Cetus,. Emeryville, Calif.), 50 ng of CHV DNA (isolated from CHV strain D 004 as described in Example 2) and 0.5 μl of a thermostable DNA polymerase, all in an about 100 μl total volume. The PCR reactions included an initial denaturation for 3 minutes at 95° C., five cycles of 1 minute each at 95° C., 35° C. for 1 minute, 72° C. for 1 minute, 35 cycles of 1 minute each at 95° C., 37° C. for 1 minute, 72° C. for 1 minute, and finally 10 minutes at 72° C. The resultant PCR products were directly cloned into the pCRII TA cloning vector (available from Invitrogen Corp., San Diego, Calif.) according to the manufacturer's specifications. Primers which were successful in amplifying fragments from a CHV genome, which was determined to be very AT-rich compared to other herpesvirus genomes, are described below.
A. Isolation of a Nucleic Acid Molecule Including a Partial CHV dUTPase Gene and a Partial CHV UL51 Gene
The following primers were designed using dUTPase protein sequence derived from HSV-1 (McGeoch et al, 1988, J. Gen. Virol. 69, 1531-1574), EHV-4 (Riggio et al, 1993, Arch. Virol. 133, 171-178), BHV-1 (Liang et al, 1993, Virology 195, 42-50), and EBV (Lees et al, 1993, Virology 195, 578-586: Primer 212S.(dUTPase forward) having nucleic acid sequence 5′ GG CGA ATT CCI AAR MGI GAI GAR GAY G 3′, denoted herein as SEQ ID NO:39; and Primer 365A (dUTPase reverse) having nucleic acid sequence 5′ C GCG GAT CCI GTI SWI CCY AAI CC 3′, denoted herein as SEQ ID NO:40. These primers led to the amplification of an about 743 nucleotide fragment, which was significantly larger than expected. Nucleic acid sequence analysis, described in Example 3, indicated that the 743 nucleotide fragment contained part of the CUL51 gene as well as part of the CdUTPase gene; as such, the fragment was denoted nCdUTP/CUL51₇₄₃. Nucleic acid sequence analysis also indicated that the dUTPase reverse primer actually hybridized to a region of the CHV DNA genome within the CUL51 gene rather than within the CdUTPase gene. It is believed that the mispriming was due to nucleotide position 19 of SEQ ID NO:40 being a Y instead of an R; the latter sequence would have matched more closely to the targeted priming region, about 290 nucleotides upstream from the 3′ end of nucleic acid molecule nCdUTP/CUL51₇₄₃. This result demonstrates the sensitivity of PCR amplification to primer design.
B. Isolation of a CHV gE Nucleic Acid Molecule
The following primers were designed using gE protein sequence derived from FHV-1 (Spatz et al, 1994, J Gen. Virol. 75, 1235-1244 ), EHV-1 (Elton et al, 1991, Gene 101, 203-208), and BHV-l (Leung-Tack et al, 1994, Virology 199, 409-421): Primer 197S (gE forward) having nucleic acid sequence 5, GGC GAA TTC TAY CAY WSI CAY GTI TA 3′, denoted herein as SEQ ID NO:41; and Primer 441A (gE reverse) having nucleic acid sequence 5° CGC GGA TCC RTC RTT ISW IGG DAI ISW IGT 3′, denoted herein as SEQ ID NO:42. These primers led to the amplification of an about 750 nucleotide fragment, referred to herein as nCgE₇₅₀.
C. Isolation of a CHV TK Nucleic Acid Molecule
The following primers were designed using TK protein sequence derived from HSV-1 (McGeoch et al., 1988, ibid.), HSV-2 (Kit et al, 1987, Antimicrob. Agents Chemother. 31, 1483-1490, BHV-1 (Kit et al, U.S. Pat. No. 4703011, issued Oct. 27, 1987), FHV-1 (Nunberg et al, 1989, J. Virol. 63, 3240-3249), EHV-1 (Robertson et al, 1988, Nuc. Acids Res. 16, 11303-11317), and PRV (Prieto et al,.1991, J. Gen. Virol. 72, 1435-1439): Primer EJH 002 (TK forward) having nucleic acid sequence 5′ GGC GAA TTC GGI AAR WSI ACI RC 3′, denoted herein as SEQ ID NO:43; and Primer EJH004 (TK reverse) having nucleic acid sequence 5′ GGC GGA TCC GGT TGI CKR TC 3′, denoted herein as SEQ ID NO:44. These primers led to the amplification of an about 280 nucleotide fragment, referred to herein as nCTK₂₈₀.
D. Isolation of a Nucleic Acid Molecule Including CHV gC and CHV UL45 Genes
The following primers were designed using gC and UL45 sequences derived from Limbach et al., ibid.: Primer gC sense, having nucleic acid sequence 5′ CGCGGATCCAAGGTAATAAGTCAAAATGAG 3′, denoted herein as SEQ ID NO:45; and Primer gC ant, having nucleic acid sequence 5′ CGCGGATCCGACAAAAACAAAAAGTAATG 3′, denoted herein as SEQ ID NO:46. These primers led to the amplification of an about 2100 nucleotide fragment, referred to herein as nCgC/CUL45₂₁₀₀.
E. Attempt to Isolate a CHV Ribonucleotide Reductase Gene
The following primers were designed using ribonucleotide subunit small subunit protein sequence derived from HSV-1 (McGeoch et al, 1988, ibid.), PRV (Dewind et al, 1993, J. Gen. Virol. 74, 351-359), EHV-1 (Telford et al, 1992, Virology 189, 04-316) and BHV-1 (Simard et al, 1992, Virology 190, 689-701): Primer EJH 021 (RR forward) having nucleic acid sequence 5′ CCG AAT TCY TIA TGA THY TIA THG ARG G 3′, denoted herein as SEQ ID NO:47; and Primer EJH022 (RR reverse) having nucleic acid sequence 5′ CCG GAT CCY TCR AAR AAR TTI GTR TGY TT 3′, denoted herein as SEQ ID NO:48. These primers did not lead to the amplification of a fragment under a variety of magnesium and amplification conditions, suggesting the lack of a coding region for a ribonucleotide reductase small subunit in CHV. As a control, these primers were shown to be able to easily amplify a ribonucleotide reductase small subunit fragment from an FHV genome.

Example 2

This Example describes the production of CHV genomic libraries.
Canine herpesvirus strain D 004 (Binn, et al., 1967, Proc. Soc. Exp. Biol. Med. 126, 140) was obtained from ATCC. Virus were propagated on Madin-Darby Canine Kidney (MDCK) cells according to standard virological procedures. Viral DNA was prepare from CHV-infected MDCK cells by previously described methods; see, for example, Walboomers et al, ibid. The viral DNA was digested with restriction endonucleases HindIII, PstI, EcoRI or XbaI, and the resultant digests were cloned into either vector pSP72 (available from Promega Corp., Madison, Wis.), or pLitmus 28 or 38 (available from New England Biolabs, Beverly, Mass.). DNA was prepared from the resultant recombinant plasmids and the inserts were sorted according to size.

Example 3

This Example describes the isolation of genomic HindIII restriction fragment nucleic acid molecules containing CdUTPase nucleic acid sequences and the nucleic acid sequencing of at least regions of these nucleic acid molecules.
Nucleic acid molecule nCdUTP/CUL51₇₄₃, produced as described in Example 1, was labelled with either ³²p or digoxigenin (available from Boehringer Mannheim Biochemicals (BMB), Indianapolis, Ind.) and used to screen the CHV HindIII fragment-containing library, either by colony hybridization or Southern blotting. Two clones hybridized with nCdUTP/CUL51₇₄₃, one containing a HindIII fragment of about 3000 nucleotides, referred to herein as nCHin₃₀₀₀, and another containing a HindIII fragment of about 1,900 nucleotides, referred to herein as nCHin₁₉₀₀. The HindIII fragments were further characterized by restriction mapping, as shown in FIG. 1. Also shown is the relative location of nCdUTP/CUL51₇₄₃(referred to therein as nCdUTP₇₄₃) with respect to the HindIII fragments.
Nucleic acid molecules nCdUTP/CUL51₇₄₃, nCHin₃₀₀₀, and nCHin₁₉₀₀were submitted to DNA sequence analysis, using a sequencing strategy as depicted in FIG. 1. Among sequences obtained were SEQ ID NO:13 and SEQ ID NO:14, which include SEQ ID NO:15 and SEQ ID NO:33, as well as SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27. The relative locations of genes included in these sequences (i.e., the CUL52, CUL51, CdUTPase and CUL48 genes) are shown in FIG. 1. Also shown is the apparent location of the CUL49 gene.
SEQ ID NO:13 and SEQ ID NO:14 represent the deduced nucleic acid sequences of the two complementary strands of nCdUTP/CUL51₇₄₃. SEQ ID NO:13 includes a partial coding region for a CdUTPase protein of about 152 amino acids, denoted herein as nCdUTP₄₅₉and represented by SEQ ID NO:15, assuming a first in-frame codon spanning about nucleotides 3-5, and a stop codon spanning about nucleotides 459-461 of SEQ ID NO:13. The amino acid sequence of the encoded protein PCdUTP₁₅₂is represented by SEQ ID NO:16. SEQ ID NO:14 includes a partial coding region for a CUL51 protein of about 86 amino acids, denoted herein as nCUL51₂₆₁and represented by SEQ ID NO:33, assuming a first in-frame codon spanning about nucleotides 1-3, and a stop codon spanning about nucleotides 259-261 of SEQ ID NO:14. The amino acid sequence of the encoded protein PCUL51₈₆is represented by SEQ ID NO:34.
SEQ ID NO:21 and SEQ ID NO:23 represent the deduced nucleic acid sequences of the two complementary strands of nCUL48₂₉₄. SEQ ID NO:21 includes a partial coding region for a CUL48 protein of about 97 amino acids, denoted herein as nCUL48₂₉₁and represented by SEQ ID NO:24, assuming a first in-frame codon spanning about nucleotides 3-5 of SEQ ID NO:21. The amino acid sequence of the encoded protein PCUL48₉₇is represented by SEQ ID NO:22.
SEQ ID NO:25 and SEQ ID NO:27 represent the deduced nucleic acid sequences of the two complementary strands of nCUL52₁₄₆. SEQ ID NO:25 includes a partial coding region for a CUL52 protein of about 48 amino acids, denoted herein as nCUL52₁₄₄and represented by SEQ ID NO:28, assuming a first in-frame codon spanning about nucleotides 1-3 of SEQ ID NO:25. The amino acid sequence of the encoded protein PCUL52₄₈is represented by SEQ ID NO:26.

Example 4

This Example describes a method to identify restriction enzymes that effect limited cleavage of the CHV genome. Also described is the isolation and sequencing of an AscI fragment containing the entire US region of CHV.
CHV DNA was evaluated for recognition sites for several restriction enzymes with 8-base, or larger, recognition sites. No sites were found for enzymes Sse8387I, I-Sce-I, or NotI. Two AscI sites were found, which were mapped to sites within the inverted repeats surrounding the US region, specifically within CHV gene US2 (see FIG. 2). The AscI fragments, of 9.3, 10.0 and approximately 100-120 kilobases (kb), were cloned into pLitmus 38 and submitted to restriction enzyme mapping analysis. The restriction map of the 9.3-kb AscI fragment, denoted herein as nCAsc9300 is shown in FIG. 2; this fragment apparently contains the entire CUS region, as well as small portions of the internal and terminal IR regions. The 10.0-kb AscI-blunt fragment, denoted herein as nCAsc10000, contains most of the terminal IR region and is shown in FIG. 3. The about 100-120-kb AscI-blunt fragment contains the remainder of the CHV genome. Both AscI sites are located just 5′ of the diploid copies of the CHV homolog of the EHV-1 IR6 gene. (Breeden et al, 1992, Virology 191,.649-660), and the orf1 of BHV-1 (Leung-Tack et al, ibid.). The AscI sites appear to be located within an open reading frame that is the homolog of the EHV US1 gene (Breeden et al ibid.) and the HSV-1 US2 gene (McGeoch et al, 1985, J. Mol. Biol 181, 1-13), although the size is somewhat different from these other genes. No function has been ascribed to the HSV-1 US2 gene product, but it has been shown to be dispensable for growth in tissue culture and to attenuate the virus (Meignier et al, 1988, Virology 162, 251-254).
Nucleic acid molecules nCAsc₉₃₀₀and nCAsc₁₀₀₀₀were submitted to DNA sequence analysis using a strategy as depicted in FIG. 2 and FIG. 3. Among sequences obtained were SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:29, and SEQ ID NO:31 The relative locations of genes included in these sequences (i.e., the CUS9, CgI, CgD, CgG, CPK, CUS2, and C!R6 genes) are shown in FIG. 2 and FIG. 3. Also shown is the putative location of the CgE gene.
SEQ ID NO:1 and SEQ ID NO:2 represent the deduced nucleic acid sequences of the two complementary strands of nCUS₅₄₉₅. Translation of SEQ ID NO:1 and SEQ ID NO:2 indicates that nucleic acid molecule nCUS₁₄₉₅, encodes CIR6, CUS2, CPK and CgG proteins as well as a portion of a CgD protein. Specifically, SEQ ID NO:2 includes: a coding region for a CIR6 protein of about 183 amino acids, denoted herein as nCIR6₅₅₂and represented by SEQ ID NO:3, assuming a start codon spanning about nucleotides 4566-4568 and a stop codon spanning about nucleotides 5115-5117 of SEQ ID NO:2; and a coding region for a CUS2 protein of about 391 amino acids, denoted herein as nCUS2,₁₇₆and represented by SEQ ID NO:5, assuming a start codon spanning about nucleotides 3232-3234 and a stop codon spanning about nucleotides 4405-4407 of SEQ ID NO:2. The amino acid sequences of the respective encoded proteins PCIR6₁₈₃and PCUS2₃₉₁are represented by SEQ ID NO:4 and SEQ ID NO:6. SEQ ID NO:1 includes: a coding region for a CPK protein of about 400 amino acids, denoted herein as nCPK₁₂₀₃and represented by SEQ ID NO:7, assuming a start codon spanning about nucleotides 2384-2386 and a-stop codon spanning about nucleotides 3584-3586 of SEQ ID NO:1; a coding region for a CgG protein of about 415 amino acids, denoted herein as nCgG₁₂₄₈and represented by SEQ ID NO:9, assuming a start codon spanning about nucleotides 3698-3700 and a stop codon spanning about nucleotides 4943-4945 of SEQ ID NO:1; and a partial coding region for a CgD protein of about 119 amino acids, denoted herein as nCgD₃₅₇and represented by SEQ ID NO:11, assuming a start codon spanning about nucleotides 5137-5139. The amino acid sequences of the respective encoded proteins PCPK₄₀₀, PCgG₄₁₅, and PCgD₁₁₉are represented by SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.
SEQ ID NO:17 and SEQ ID NO:19 represent the deduced nucleic acid sequences of the two complementary strands of nCUS9₅₇₉. SEQ ID NO:17 includes a coding region for a CUS9 protein of about 149 amino acids, denoted herein as nCUS9₄₅₀and represented by SEQ ID NO:20, assuming a start codon spanning about nucleotides 54-56 and a stop codon spanning about nucleotides 501-503 of SEQ ID NO:17. The amino acid sequence of the encoded protein PCUS9₁₄₉is represented by SEQ ID NO:18.
SEQ ID NO:29 and SEQ ID NO:31 represent the deduced nucleic acid sequences of the two complementary strands of nCgI₁₈₁. SEQ ID NO:29 includes a partial coding region for a CgI protein of about 53 amino acids, denoted herein as nCgI₁₅₉and represented by SEQ ID NO:32,assuming a first in-frame codon spanning about nucleotides 3-5 of SEQ ID NO:29. The amino acid sequence of the encoded protein PCgI₅₃is represented by SEQ ID NO:30.

Example 5

This Example describes the nucleic acid sequencing of additional nucleic acid molecules of the present invention.
Nucleic acid molecule nCTK₂₈₀, produced as described in Example 1, was submitted to DNA sequence analysis to obtain the following sequences. SEQ ID NO:35 and SEQ ID NO:37 represent the deduced nucleic acid sequences of the two complementary strands of nCTK₂₈₀. SEQ ID NO:35 includes a partial coding region for a CTK protein of about 93 amino acids, denoted herein as nCTK₂₇₉and represented by SEQ ID NO:38, assuming a first in-frame codon spanning about nucleotides 2-4 of SEQ ID NO:35. The amino acid sequence of the encoded protein PCTK₉₃is represented by SEQ ID NO:36.

Example 6

This Example discloses the production of a recombinant CHV genome and recombinant CHV of the present invention.
A cassette including the human CMV immediate early promoter and the poly-adenylation signal from bovine growth hormone separated by a polylinker was PCR amplified from plasmid pcDNA3 (available from Invitrogen) using forward primer EJH058 having nucleic acid sequence 5′ TCCCCCGGGGGCGCGCCTTGACATTGATTATTGAC 3′, denoted SEQ ID NO:49, and reverse primer EJH059 having nucleic acid sequence 5′ GCCCTTAAGGGGCGCGCCAATGCGATGCAATTTCC 3′, denoted SEQ ID NO:50. EJH058 has SmaI and AscI sites attached to the 5′ end of pcDNA3 homologous sequences, and EJH059 has AflII and AscI sites attached to the 5′ end of pcDNA3 homologous sequences. The resultant PCR amplified fragment of about 930 nucleotides was digested with SmaI and AflII and ligated into the SnaBI and AflII sites of plasmid pLitmus 38. This cloning procedure eliminated the entire polylinker region of pLitmus38. The resulting recombinant plasmid, denoted herein as pAscCMV/BGH, contains the CMV promoter—BGH polyadenylation signal cassette, with its original polylinker, in a plasmid such that the entire cassette can be excised with enzyme AscI. This cassette plasmid can also be prepared with other rare-cutting enzyme sites flanking the cassette.
Plasmid pAscCMV/BGH allows for the insertion of heterologous nucleic acid molecules between the CMV promoter and the BGH polyadenylation signal. The resulting heterologous nucleic acid molecule-containing cassette was excised from the plasmid for insertion into a CHV genome. For example, a heterologous nucleic acid molecule containing a lacZ gene was inserted into the polylinker region of pAscCMV/BGH such that the lacZ gene is expressed by the CMV promoter in a eukaryotic system. The cassette containing the lacZ gene, referred to herein as AscCMV/lacZ/BGH is then excised from the plasmid by AscI digestion and gel purified by standard methods.
About 5-10 μg of CHV DNA is digested with AscI, resulting in three fragments as disclosed herein. The DNA fragments are dephosphorylated with calf intestine alkaline phosphatase (available from BMB) for 10 minutes at 37° C.; the enzyme is then inactivated for by incubation for 10 minutes at 65° C. The phosphatase-treated digested CHV DNA is then subjected to extraction with phenol and phenol/chloroform and precipitated with ethanol.
The phosphatase-treated digested CHV genomic DNA is mixed with the gel-purified AscCMV/lacZ/BGH cassette at a molar ratio of approximately 1:2 under standard ligation conditions. Since the viral DNA is dephosphorylated, it should not be able to self ligate; thus all resultant ligated viral molecules should contain two copies of the inserted cassette. The ligated DNA is then subjected to phenol extraction and ethanol precipitated.
The precipitated ligated DNA is resuspended in hepes-buffered saline and submitted to standard viral transfection conditions, such as that described by Graham et al., ibid., along with appropriate controls (e.g., undigested viral DNA, digested and dephosphorylated viral DNA that was self-ligated, and no DNA). Resultant viral plaques are screened under an X-gal overlay for expression of β-galactosidase.

Example 7

This Example discloses the production of another recombinant CHV genome and recombinant CHV of the present invention.
A recombinant, or transfer, vector to be used in the production of a recombinant CHV having a heterologous nucleic acid molecule in a TK gene of the CHV genome is constructed as follows. A CHV TK nucleic acid molecule of the present invention (e.g., nCTK₂₈₀) is ligated into a pLitmus plasmid to produce a pCTK-Litmus plasmid. An expression cassette including a heterologous nucleic acid molecule (e.g., a lacZ gene or a nucleic acid molecule encoding an antigen isolated from a pathogenic organism) ligated to a CMV immediate early promoter and a BHV polyadenylation site in such a manner that the heterologous nucleic acid molecule is expressed in a eukaryotic cell is inserted into the CTK nucleic acid molecule within pCTK-Litmus such that there are CTK flanking sequences on either side of the expression cassette (e.g., into a restriction site internal to nCTK₂₈₀).
A recombinant CHV is produced by co-transfecting the recombinant vector and CHV DNA into canine MDCK cells using previously described methods; see, for example, Graham et al, ibid. Recombinant TK negative CHV are selected for by passage in bromodeoxyuridine; see, for example, Kit et al., 1983, Virology 130, 381-389. If the heterologous nucleic acid molecule is the lacZ gene, such recombinant CHV can also be selected as described in Example 6.

Example 8

This Example discloses the production of another recombinant CHV genome and recombinant CHV of the present invention.
A recombinant, or transfer, vector to be used in the production of a recombinant CHV having a heterologous nucleic acid molecule in a dUTPase gene of the CHV genome is constructed as follows. A CHV dUTPase nucleic acid molecule of the present invention (e.g., nCdUTP₄₅₉) is ligated into a pLitmus plasmid to produce a pCdUTP-Litmus plasmid. An expression cassette including a heterologous nucleic acid molecule (e.g., a lacZ gene or a nucleic acid molecule encoding an antigen isolated from a pathogenic organism) ligated to a CMV immediate early promoter and a BHV polyadenylation site in such a manner that the heterologous nucleic acid molecule is expressed in a eukaryotic cell is inserted into the CdUTP nucleic acid molecule within pCdUTP-Litmus such that there are CdUTPase flanking sequences on either side of the expression cassette (e.g., into a restriction site internal to nCdUTP₄₅₉).
A recombinant CHV is produced by co-transfecting the recombinant vector and CHV DNA into canine MDCK cells as described in Example 7. Recombinant dUTPase negative CHV are selected for by passage in mercurithio analogs of deoxyuridine; see, for example, Holliday et al, 1991, Antiviral Research 16, 197-203. If the heterologous nucleic acid molecule is the lacZ gene, such recombinant CHV can also be selected as described in Example 6. Recombinant virus carrying the foreign DNA of interest can also be selected by either by plaque hybridizations, or by dot-blot hybridizations of infected cell cultures. Verification of the proper insert within the CHV genome is conducted by Southern hybridization analysis.

Example 9

This Example discloses the production of a recombinant CHV by co-transfection of a set of overlapping cosmid clones comprising the entire viral CHV genome.
A library of CHV cosmid clones is created in the cosmid vector SuperCos (available from Stratagene Cloning Systems, La Jolla, Calif.) according to manufacturer's specifications, except that the vector is modified to contain one,or more restriction sites not present in CHV genomic DNA (e.g., Sse83871, I-Sce-I, or NotI) so that the cosmid inserts can be excised prior to cotransfection. A heterologous nucleic acid molecule in an expression cassette, such as one of those described in Examples 6-8, is inserted into a cosmid clone using standard procedures. A recombinant CHV is produced by co-transfection of a set of overlapping cosmid clones comprising the entire viral CHV genome, including the cosmid comprising a heterologous nucleic acid molecule, using techniques as described in van Zihl et al., 1988, J. Virol. 62, 2191-2195.

Example 10

This Example describes the nucleic acid sequencing of additional nucleic acid molecules of the present invention.
Nucleic acid molecules nCAsc₉₃₀₀and nCAsc₁₀₀₀₀, produced as described in Example 4, were submitted to additional DNA sequence analysis. The resultant nucleic acid sequences were compiled to produce SEQ ID NO:51 and SEQ ID NO:52. SEQ ID NO:51 and SEQ ID NO:52 represent the deduced nucleic acid sequences of the two complementary strands of a nucleic acid molecule referred to herein as nCUS₁₀₅₉₂. Nucleic acid molecule nCUS₁₀₅₉₂includes the entire US region of CHV plus terminal repeat and internal repeat sequences. Analysis of SEQ ID NO:51 indicates that the US region spans from nucleotides about 2047 through about 9724 of SEQ ID NO:51, whereas the terminal repeat sequences and inverted repeat sequences span from nucleotides about 1 through about 2046 and from nucleotides about 9725 through about 10592, respectively, of SEQ ID NO:51.
Translation of SEQ ID NO:51 and SEQ ID NO:52 indicates that nucleic acid molecule nCUS₁₀₅₉₂contains at least the following open reading frames: CIR6, CUS2, CPK, CgG, CgD, CgI, CgE, CUS8.5 and CUS9. The relative locations of these open reading frames is shown in FIG. 4.
Specifically, SEQ ID NO:52 includes: the coding strand of nCIR6₅₅₂(having SEQ ID NO:3) which encodes a CIR6 protein of about 183 amino acids, assuming a start codon spanning about nucleotides 9672-9674 and a stop codon spanning about nucleotides 10221-10223 of SEQ ID NO:52; and the coding strand of nCUS2₁₁₇₆(having SEQ ID NO:5) which encodes a CUS2 protein of about 391 amino acids, assuming a start codon spanning about nucleotides 8338-8340 and a stop codon spanning about nucleotides 9511-9513 of SEQ ID NO:52. Nucleic acid molecules nCIR6₅₅₂and nCUS2₁₁₇₆are also described in Example 4 in relation to nCUS₅₄₉₅, as are the corresponding nucleic acid sequences and amino acid sequences they encode.
SEQ ID NO:51 includes: the coding strand of nCPK₁₂₀₃(having SEQ ID NO:7) which encodes a CPK protein of about 400 amino acids, assuming a start codon spanning about nucleotides 2375-2377 and a stop codon spanning about nucleotides 3575-3577 of SEQ ID NO:51; the coding strand of nCgG₁₂₄₆(having SEQ ID NO:9) which encodes a CgG protein of about 415 amino acids, assuming a start codon spanning about nucleotides 3689-3691 and a stop codon spanning about nucleotides 4934-4936 of SEQ ID NO:51; the coding strand of nCgD₁₀₃₈(having SEQ ID NO:53, and including SEQ ID NO:11) which encodes a CgD protein of about 345 amino acids, assuming a start codon spanning about nucleotides 5128-5130 and a stop codon spanning about nucleotides 6163-6165 of SEQ ID NO:51; the coding strand of nCgI₁₀₉₅(having SEQ ID NO:55, and including SEQ ID NO:29) which encodes a CgI protein-of about 364 amino acids, assuming a start codon spanning about nucleotides 6225-6227 and a stop codon spanning about nucleotides 7317-7319 of SEQ ID NO:51; the coding strand of nCgE₁₅₆₉(having SEQ ID NO:57) which encodes a CgE protein of about 522 amino acids, assuming a start codon spanning about nucleotides 7467-7469 and a stop codon spanning about nucleotides 9033-9035 of SEQ ID NO:51; the coding strand of nCUS8.5₂₃₇(having SEQ ID NO:59) which encodes a CUS8.5 protein of about 78 amino acids, assuming a start codon spanning about nucleotides 9028-9030 and a stop codon spanning about nucleotides 9262-9264 of SEQ ID NO:51; and the coding strand of nCUS9₃₆₀(having SEQ ID NO:61) which encodes a CUS9 protein of about 119 amino acids, assuming a start codon spanning about nucleotides 9376-9378 and a stop codon spanning about nucleotides 9733-9735 of SEQ ID NO:51. SEQ ID NO:61 differs from the coding region reported for CUS9 in Example 4 (e.g., SEQ ID NO:20), in that additional sequence analysis indicated that SEQ ID NO:17 included a sequencing error resulting in a frameshift, leading to a longer deduced open reading frame. Nucleic acid molecules nCPK₁₂₀₃and nCgG₁₂₄₈are also described in Example 4 in relation to nCUS₅₄₉₁, as are the corresponding nucleic acid sequences and amino acid sequences they encode. The amino acid sequences of the respective encoded proteins PCgD₃₄₅, PCgI₃₆₄, PCgE₅₂₂, PCUS8.5₇₈, and PCUS9₁₁₉are represented by SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NO:62, respectively.

Example 11

This Example describes the nucleic acid sequencing of additional nucleic acid molecules of the present invention.
Nucleic acid molecules nCHin₃₀₀₀, and nCHin₁₉₀₀, produced as described in Example 3, were submitted to additional DNA sequence analysis. Also submitted to additional nucleic acid sequence analysis was nucleic acid molecule nCHin₈₅₀₀, a HindIII fragment shown to include the 3′ end of CUL48 as well as CgC and CUL45. The resultant nucleic acid sequences were compiled to produce SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:85, and SEQ ID NO:86. SEQ ID NO:63 and SEQ ID NO:63 represent the deduced nucleic acid sequences of the two complementary strands of a nucleic-acid molecule referred to herein as nCUL49/CUL48₂₀₄₄. Translation of SEQ ID NO:63 and SEQ ID NO:64 indicates that nucleic acid molecule nCUL49/CUL48₂₀₄₄contains at least the following open reading frames: CUL48 and the 3′ end of CUL49. SEQ ID NO:77 and SEQ ID NO:78 represent the deduced nucleic acid sequences of the two complementary strands of a nucleic acid molecule referred to herein as nCUL₁₈₂₃. Translation of SEQ ID NO:77 and SEQ ID NO:78 indicates that nucleic acid molecule nCUL₁₈₂₃contains at least the following open reading frames: CUL51, CdUTPase, CUL49.5, and the 5′ end of CUL49. SEQ ID NO:85 and SEQ ID NO:86 represent the deduced nucleic acid sequences of the two complementary strands of a nucleic acid molecule referred to herein as nCUL52₇₄₉. Translation of SEQ ID NO:85 and SEQ ID NO:86 indicates that nucleic acid molecule nCUL52₇₄₉contains a partial open, reading frame for CUL52, the 3′ end of which is included in SEQ ID NO:28. The relative location of each of these open reading frames is shown in FIG. 5.
Specifically, SEQ ID NO:63 includes: the coding strand of nCUL49₄₂₀(having SEQ ID NO:65) which encodes a non-full length CUL49 protein of about 139 amino acids, assuming a stop codon spanning about nucleotides 419-421 of SEQ ID NO:63; and the coding strand of nCUL48₁₂₆₉(having SEQ ID NO:67, and including SEQ ID NO:24) which encodes a CUL48 protein of about 422 amino acids, assuming a start codon spanning about nucleotides 541-543 and a stop codon spanning about nucleotides 1807-1809 of SEQ ID NO:63. The amino acid sequences of the respective encoded proteins PCUL49₁₃₉and PCUL48₄₂₂are represented by SEQ ID NO:66 and SEQ ID NO:68, respectively.
SEQ ID NO:77 includes the coding strand of nCdUTP₉₁₈(having SEQ ID NO:79 and including SEQ ID NO:15) which encodes a CdUTPase protein of about 305 amino acids, assuming a start codon spanning about nucleotides 624-626 and a stop codon spanning about nucleotides 1539-1541 of SEQ ID NO:77. The amino acid sequence of the respective encoded protein PCdUTP₃₀₅is represented by SEQ ID NO:80.
SEQ ID NO:78 includes: the coding strand of nCUL51₂₆₁(having SEQ ID NO:33) which encodes a non-full length CUL51 protein of about 86 amino acids, assuming translation begins at about nucleotide 1 and a stop codon spanning about nucleotides 259-261 of SEQ ID NO:78; the coding strand of nCUL49.5₂₆₁(having SEQ ID NO:81) which encodes a CUL49.5 protein of about 86 amino acids, assuming a start codon spanning about nucleotides 1175-1177 and a stop codon spanning about nucleotides.1433-1435 of SEQ ID NO:78; and the coding strand of nCUL49₂₅₅(having SEQ ID NO:83) which encodes a non-full length CUL49 protein of about 85 amino acids, assuming a start codon spanning about nucleotides 1586-1588 of SEQ ID NO:78. The amino acid sequence of the respective encoded proteins PCUL51₈₆, PCUL49.5₈₆, and PCUL49₈₅are represented by SEQ ID NO:34, SEQ ID NO:82 and SEQ ID NO:84, respectively.
SEQ ID NO:85 includes the coding strand of nCUL52₇₄₇. (having SEQ ID NO:87) which encodes a non-full length CUL52 protein of about 249 amino acids, assuming translation begins at about nucleotide 1 of SEQ ID NO:85. The amino acid sequence of the respective encoded protein PCUL52₂₄₉is represented by SEQ ID NO:88.

Example 12

This Example discloses the production of another recombinant CHV genome and recombinant CHV of the present invention.
A 3.2 kb fragment containing the entire CdUTPase sequence was amplified from CHV genomic DNA using forward primer RSF009 having nucleic acid sequence 5′ GCCGGTACCAGGCTTTGGACGAGATTTAGG 3′, denoted SEQ ID NO:89, and reverse primer RSF008 having nucleic acid sequence 5′ GCCGAATTCAATATAATTAATAAACTCTC3′, denoted SEQ ID NO:90. RSF009 has an Asp718 site attached to the 5′ end of CHV homologous sequences, and RSF008 has an EcoRI site attached to the 5′ end of CHV homologous sequences. The resultant PCR-amplified fragment of about 3.2 kb, referred to herein as nCdUTP₃₂₀₀, was digested with Asp718 and EcoRI and ligated into plitmus28 (available from New England Biolabs). The resultant recombinant plasmid, denoted herein as p28CdUTP₃₂₀₀, was verified by end-sequencing and restriction mapping. Plasmid p28CdUTP₃₂₀₀was then digested with HindIII and XbaI, releasing an 858 base-pair fragment, referred to herein as nCdUTP₈₅₈, containing all of the dUTPase open reading frame except 108 bp at the 3′ end. The XbaI site was found to be 49 nucleotides upstream of the start codon of the dUTPase-ORF (open reading frame), and is also 70 nucleotides into the UL49.5 ORF on the opposite strand, which overlaps the duTPase ORF. Previous studies have shown that the UL49.5 ORF in other herpesviruses encodes a membrane protein, and is nonessential for growth in tissue culture (e.g., Liang et al, ibid.).
The cohesive ends on the remaining 2.3 kb fragment of p28CdUTP₃₂₀₀, minus the 858 bp HindIII/XbaI fragment, were filled in using Klenow fragment and dNTPs according to standard methods. The resulting blunt-ended fragment was gel purified by standard methods. A heterologous nucleic acid sequence operatively linked to transcription control regions, in this case an AscCMV/lacZ/BGH cassette described in Example 6, was isolated from its plasmid by digestion with AscI, and the cohesive ends were filled in by Klenow fragment and dNTPs. This cassette was ligated to the HindIII and XbaI-digested p28CdUTP₃₂₀₀fragment described above by standard methods resulting in a plasmid containing flanking regions to the CHV dUTPase gene and with the lacZ gene, operatively linked to transcription control regions, inserted into a deletion of the dUTPase gene, herein denoted as pdUTP/lacZ.
A recombinant CHV is produced by co-transfecting the plasmid with the deleted dUTPase gene and the inserted heterologous nucleic acid molecule, in this case pdUTP/lacZ, and CHV DNA into canine cells using previously described methods; see, for example, Graham et al, ibid.). Alternatively, a recombinant CHV is produced by transfecting canine cells with the aforementioned plasmid as described, and then infecting the cells with CHV. Recombinant dUTPase-negative CHV are selected for by passage in mercurithio analogs of deoxyuridine as described in Example 8. If the heterologous nucleic acid molecule is the lacZ gene, such a recombinant CHV can be selected as described in Example 6.

Example 13

This example discloses a method for obtaining higher plaque forming efficiencies to facilitate the production of recombinant CHV genomes and recombinant CHV of the present invention. The method involves the expression of the CHV alpha-tif gene in the presence of CHV genomes introduced into canine cells during CHV production.
The alpha-tif gene of CHV (denoted herein as nCUL48₁₂₆₉) was identified to lie on two CHV clones described herein: namely, nCHin₃₀₀₀and nCHin₈₅₀₀. Nucleotide sequencing from the ends of these two fragments using universal primers and internal primers designed based on previous sequences was used to determine the entire sequence. A single fragment including the complete alpha-tif gene was PCR amplified from CHV viral DNA using forward primer RSF014 having nucleic acid sequence 5′ CCGGAATTCGCTTAGTGAGAGTATAAAC.3′, denoted SEQ ID NO 91, and reverse primer RSF016 having nucleic acid sequence 5′ CCGGAATTCCCTCATATTATATACTAAC 3′, denoted SEQ ID NO:92. Both primers have EcoRI sites attached to the 5′ end of the CHV homologous sequences. The resultant PCR amplified fragment of about 1351 base pairs was digested with EcoRI and ligated into EcoRI-digested pCDNA3 (available from Invitrogen, described in Example 6). This plasmid contains a neomycin-resistance gene for selection of stably transformed cell lines by techniques well known to those skilled in the art. Resulting plasmids were checked for the proper orientation by digestion with HindIII. A plasmid consisting of pcDNA3 with the alpha-tif gene inserted in the proper orientation for expression was found, and is designated herein as pATIF₁₃₅₁Expression of CHV alpha-tif from pATIF₁₃₅₁was verified by cell-free transcription/translation using T7 RNA polymerase and a rabbit reticulocyte lysate system. These techniques are readily apparent to those skilled in the art, and are available in kit form, for example, from Amersham. Improved CHV DNA infectivity is then achieved by cotransfection of pATIF₁₃₅₁with from about 1 to about 5 μg of CHV DNA by methods such as described in Examples 6-9 and Example 12.
Alternatively, cell lines that stably express CHV alpha-tif are derived. Three canine cell lines, A72, D17 and CF2Th (all available from ATCC) were transfected with pATIF₁₃₅₁by standard methods, see, for example, Graham et al, ibid. The cells are then subjected to selection using varying amounts of the antibiotic G418 (available from BMB), determined empirically for each cell line. Cloned G418-resistant cell lines are then derived by standard dilution methods. Cell lines stably expressing CHV alpha-tif are then used for standard CHV DNA transfections or co-transfections such as those described in Examples 6-9 and Example 12.

Example 14

This Example describes the identification and sequencing of additional CHV nucleic acid molecules of the present invention.
CHV nucleic acid molecule nCHin₅₅₀₀, a CHV HindIII fragment derived as described in Example 2, was submitted to nucleic acid sequence analysis. Sequences derived from the sequence analysis include SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:73 and SEQ ID NO:74. Translation of these sequences indicated that nCHin₅₅₀₀includes at least a portion of the CgL and CICP4 coding regions. The nucleic acid molecule also apparently contains the CICP0 coding region.
SEQ ID NO:69 and SEQ ID NO:70 represent the deduced nucleic acid sequences of the two complementary strands of a CHV ICP4 nucleic acid molecule referred to herein as nCICP4₆₂₆. Translation of SEQ ID NO:69 indicates that nCICP4₆₂₆encodes a non-full length CICP4 protein of about 208 amino acids, denoted herein as PCICP4₂₀₈, having an amino acid sequence represented by SEQ ID NO:72. The coding region for PCICP4₂₀₈is denoted herein as nCICP4₆₂₄, having nucleic acid sequence SEQ D NO:71.
SEQ ID NO:73 and SEQ ID NO:74 represent the deduced nucleic acid sequences of the two complementary strands of a CHV gL nucleic acid molecule referred to herein as nCgL₆₅₅. Translation of SEQ ID NO:74 indicates that nCgL₆₅₅encodes a non-full length CgL protein of about 171 amino acids, denoted herein as PCgL₁₇₁, having an amino acid sequence represented by SEQ ID NO:76. The coding region for PCgL₁₇₁is denoted herein as nCgL₅₁₆, having nucleic acid sequence SEQ D NO:75, assuming that the open reading frame spans about nucleotides 1 through 516 of SEQ ID NO:74, with a stop codon spanning about nucleotides 514-516.

Example 15

This example describes the mapping of messenger RNA molecules transcribed from nucleic acid molecules of the present invention. These transcripts are useful for verifying expression (or lack of expression) of various genes.
Total RNA was prepared from CHV-infected cells using a phenol/guanidine extraction method familiar to those skilled in the art. See, for example, Chomczynski et al, 1987, Anal. Biochem. 162, 156-159. Multiple samples of RNA were separated on formaldehyde/agarose gels and transferred to nitrocellulose by standard methods. Based on the sequences of nCUS₁₀₅₉₂, designated SEQ ID NO:51 and SEQ ID NO:52, nucleic acid fragments were isolated for use as probes, such that they would hybridize to sequences homologous to the open reading frames nCPK₁₂₀₃(SEQ ID NO:7), nCgG₁₂₄₈(SEQ ID NO:9), nCgD₁₀₃₈(SEQ ID NO:53), nCgI₁₀₉₅(SEQ ID NO:55), nCgE₁₅₆₉(SEQ ID NO:57), nCUS8.5237 (SEQ ID NO:59), nCUS9₃₆₀(SEQ ID NO:61), nCIR6₅₅₂(SEQ ID-NO:3), and nCUS2₁₁₇₆(SEQ ID NO:5). The probes were as follows: to hybridize with an nCIR6₅₅₂(SEQ ID NO:3) transcript, a PstI-HindIII restriction fragment extending from about nucleotide 1 to about nucleotide 685 in nCUS₁₀₅₉₂herein denoted as probe A; to hybridize with an nCUS2₁₁₇₆(SEQ ID NO: 5) transcript, a PCR-amplified fragment extending from about nucleotide 2056 to about nucleotide 2273 in nCUS₁₀₅₉₂herein denoted as probe B; to hybridize with an nCPK₁₂₀₃(SEQ ID NO: 7) transcript, a PstI-DraI restriction fragment extending from about nucleotide 2326 to about nucleotide 27.88 in nCUS₁₀₅₉₂herein denoted as probe C; to hybridize with an nCgG₁₂₄₈(SEQ ID NO:9) transcript, a BarrHI-PstI restriction fragment extending from about nucleotide 3766 to about nucleotide 4494 in nCUS₁₀₅₉₂herein denoted as probe D; to hybridize with an nCgD₁₀₃₈(SEQ ID NO:53) transcript, either a BglII-BglII restriction fragment extending from about nucleotide 5728 to about nucleotide 6561 in nCUS₁₀₅₉₂(this piece slightly overlaps nCgI₁₀₉₅) herein denoted as probe E, or a PCR-amplified fragment that will extend from about nucleotide 5295 to about nucleotide 5885 in nCUS₁₀₅₉₂herein denoted as probe F; to hybridize with an nCgI₁₀₉₅(SEQ ID NO:55) transcript, a BglII-BglII restriction fragment extending from about nucleotide 6561 to about nucleotide 7263 in nCUS₁₀₅₉₂herein denoted as probe G; to hybridize with an nCgE₁₅₆₉(SEQ ID NO:57) transcript, a deletion subclone extending from about nucleotide 7667 to about nucleotide 8425 in nCUS₁₀₅₉₂herein denoted as probe H; to hybridize with a putative ncus85237 (SEQ ID NO:59) transcript, a PCR-amplified fragment extending from about nucleotide 9023 to about nucleotide 9242 in nCUS₁₀₅₉₂herein denoted as probe I; to hybridize to an nCUS9₃₆₀(SEQ ID NO:61) transcript, a PCR-amplified fragment extending from about nucleotide 9447 to about nucleotide 9715 in nCUS₁₀₅₉₂herein denoted as probe J. These probes are shown graphically in FIG. 4. The probes were labelled with ³²p DATP using random priming, a technique well known to those skilled in the art. Hybridization of the probes to the RNA samples on nitrocellulose and washing at stringent temperatures were done according to well known methods.
The main transcript sizes that hybridized to the various probes were as follows: probe A hybridized strongly to a transcript of about 0.7 kb, but also hybridized weakly to transcripts of about 1.7, 2.2 and 2.6 kb; probe B hybridized with a transcript of about 2.2 kb; probe C hybridized to a transcript of about 2.6 kb; probe D hybridized to two transcripts of about 1.9 kb and 2.6 kb; probe E hybridized to two transcripts of about 4.2 kb and 3.2 kb; probe G hybridized to two transcripts of about 4.2 kb and 3.2 kb; probe H hybridized to three transcripts of about 1.9 kb, 3.2 kb, and 4.2 kb; probe I hybridized to the same three transcripts as probe H; probe J hybridized to a transcript of about 2.1 kb. These data suggest that the transcripts of US2, US9, and IR6, although starting at different points, terminated in a common general area in the inverted repeats, since probe A hybridized to transcripts that were generally the same size as the US2 and US9 transcripts. Furthermore, these data suggest that the PK and gG transcripts overlapped, probably terminating at a common 3′ terminus, since probe C hybridized to only a 2.6 kb transcript but probe D hybridized with both the 2.6 kb transcript and the 1.4 kb transcript. This result was even more likely because only one polyadenylation consensus sequence (AATAAA) was found downstream of the gG ORF, but before the gD ORF. This polyadenylation signal was found from nucleotide 4935-4940 of nCUS₁₀₅₉₂. Similarly, these data indicate the gI and gE transcripts overlapped, probably terminating at a common 3′ terminus since probe G hybridized to a transcript of 2.6 kb, while probe H hybridized both to the 2.6 kb transcript and the 1.9 kb transcript. Probes E, G, and H also all hybridized to a 4.2 kb transcript, suggesting that the gD transcript also overlapped the gI and gE transcripts, probably terminating at a common 3′ terminus. Verification of this result is if probe F hybridizes only to the 4.2 kb transcript. Probe I hybridized to only the 4.2, 2.6 and 2.9 kb transcripts suggesting two things; first, that these three transcripts probably terminated at polyadenylation signals at about 9186-9181, 9256-9261 or 9260-9265 of nCUS₁₀₅₉₂, also that the putative US8.5 open reading frame is not transcribed to levels detectable by Northern analysis.

Claims

1-83. (canceled)

84. An isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having the sequence of SEQ ID NO:35 or SEQ ID NO:37.

85. The isolated nucleic acid molecule of claim 84, wherein said CHV nucleic acid molecule comprises a nucleic acid sequence at least about 80% identical to SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

86. The isolated nucleic acid molecule of claim 84, wherein said isolated nucleic acid molecule comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

87. The isolated nucleic acid molecule of claim 84, wherein said CHV nucleic acid molecule comprises a nucleic acid sequence at least about 95% identical to SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

88. The isolated nucleic acid molecule of claim 84, wherein said CHV nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, and a nucleic acid molecule comprising an allelic variant of any of said nucleic acid molecules.

89. The isolated nucleic acid molecule of claim 84, wherein said molecule encodes a protein having thymidine kinase activity.

90. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a protein at least about 95% identical to SEQ ID NO:36.

91. The isolated nucleic acid molecule of claim 90 wherein said protein has thymidine kinase activity.

92. The isolated nucleic acid molecule of claim 90, wherein said nucleic acid sequence encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:36 and an amino acid sequence which is an allelic variant of SEQ ID NO:36.

93. The isolated nucleic acid molecule of claim 90, wherein said nucleic acid sequence encodes a protein having the amino acid sequence of SEQ ID NO:36.

94. The isolated nucleic acid molecule of claim 90, wherein said nucleic acid sequence is at least about 95% identical to SEQ ID NO:35 or SEQ ID NO:38.

95. The isolated nucleic acid molecule of claim 90, wherein said nucleic acid sequence is at least about 99% identical to SEQ ID NO:35 or SEQ ID NO:38.

96. The isolated nucleic acid molecule of claim 90, wherein said nucleic acid sequence is identical to SEQ ID NO:35 or SEQ ID NO:38.

97. An isolated nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence encoding a protein at least about 95% identical to SEQ ID NO:36; and

(b) a nucleic acid sequence fully complimentary to the sequence of (a).

98. The isolated nucleic acid molecule of claim 97, wherein said nucleic acid sequence encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:36 and an amino acid sequence which is an allelic variant of SEQ ID NO:36.

99. The isolated nucleic acid molecule of claim 97, wherein said nucleic acid sequence encodes a protein having the amino acid sequence of SEQ ID NO:36.

100. The isolated nucleic acid molecule of claim 97, wherein said nucleic acid sequence is at least about 95% identical to SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

101. The isolated nucleic acid molecule of claim 97, wherein said nucleic acid sequence is at least about 99% identical to SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:38.

102. A fragment of the isolated nucleic acid molecule of claim 97, wherein said fragment is at least 18 nucleotides in length.