WO1995021255A1

WO1995021255A1 - Compositions useful in controlling marek's disease

Info

Publication number: WO1995021255A1
Application number: PCT/US1995/001615
Authority: WO
Inventors: Karel A. Schat; Kazuhiko Ohashi; Priscilla H. O'connell
Original assignee: Cornell Research Foundation, Inc.
Priority date: 1994-02-07
Filing date: 1995-02-06
Publication date: 1995-08-10
Also published as: EP0750670A4; EP0750670A1

Abstract

A novel sequence in the genome of Marek's disease virus (MDV) containing an immediate early gene associated with lytic infection and/or tumor cell development of MDV-infected cells, is described. The location of this sequence in the MDV genome is shown in the figure. The deduced amino acid sequence of the 94 amino acid protein, encoded by this novel sequence, is also disclosed. Disclosed are the use of the novel sequence and the 94 amino acid protein in strategies to control Marek's disease such as by vaccination.

Description

COMPOSITIONS USEFUL IN CONTROLLING MAREK'S DISEASE

1. Background of the Invention 1.1 Field of the Invention

The present invention relates to a composition comprising a novel protein expressed by cells infected with Marek's disease virus (MDV) . More particularly, mechanisms of protection of chickens against Marek's disease are provided by using this novel protein as a vaccine antigen.

1.2 Description of the Background and Related Art

Marek's disease is a lymphoproliferative disease of chickens caused by MDV. MDV, a naturally occurring herpesvirus, infects bursa-derived and thymus-derived lymphocytes in chickens, and can subsequently cause malignant T-cell lymphomas in chickens. Although the MDV genome is present in tumors induced in the MDV-infected chickens, these tumors are generally free of virus particles indicating that latency has been established. MDV-infected chickens may also exhibit neural involvement characterized by nerve paralysis. Since

Marek's disease is contagious, the virus has become an important pathogen of chickens, particularly in an environment of large scale breeding such as in the poultry industry. Because of a lack of effective therapeutic drugs for treatment of Marek's disease, approaches to prevent the disease have focused on vaccine development. One such vaccine, described in U.S. Patent No. 4,160,024, involves a strain of MDV which is naturally nononcogenic and unattenuated. Another vaccine, disclosed in U.S.

Patent No. 4,980,162, is a combined vaccine consisting of cultured cells infected with an attenuated infectious laryngotracheitis virus, and cultured cells infected with attenuated MDV or herpesvirus of turkey (HVT) . A process for preparing a plasmid vector which contains the MDV Type I BarriHI-H fragment, into which is incorporated a structural gene encoding an exogenous protein, is disclosed in U.S. Patent No. 5,171,677. Currently, Marek's disease is controlled by vaccination of embryos at 17-19 days of incubation, or one day old chicks with either HVT; serotype 2 strains of MDV; attenuated (of low pathogenicity) and/or nononcogenic strains of MDV serotype 1, or combinations thereof. However, recently vaccination breaks have been reported in chickens vaccinated with the bivalent vaccine consisting of serotype 2 MDV or HVT (R.L. Witter in Proceedings XIX World's Poultry Congress. Amsterdam, The Netherlands, 1993, 1:298-304), suggesting that new approaches are needed to control Marek's disease. Increased virulence of MDV isolates, decreased genetic resistance of chicken stocks, immunosuppression by other microbial pathogens, and poor management of vaccination procedures may be factors which have contributed to recent vaccination breaks.

2. summary of the Invention

A novel sequence, comprising a small open reading frame (ORF) , has been identified in the MDV genome. RNA transcripts from this sequence have been detected in Marek's disease tumor cell lines, which have a limited number of MDV-specific transcripts. Also, transcription from this sequence occurs in lytically-infected cells. The novel sequence of the present invention may be non- essential for virus replication, and therefore may be used as a region for insertion and expression in MDV of other endogenous (MDV) genes or exogenous genes.

Accordingly, one object of the present invention is to provide an approach to control MDV infection by preventing the development of Marek's disease through the induction of a protective antibody and/or a cell- mediated immune response to a protein which may be associated with lytic infection and/or tumor cell development of MDV-infected cells. Another object of the present invention is to provide a region in the MDV genome, wherein the region is non-essential for virus replication, into which an extra copy(s) of an endogenous (MDV) gene or exogenous gene can be inserted for expression from this recombinant viral vector.

Other objects and advantages of the invention will become readily apparent from the ensuing description.

3• Brief Description of the Figures FIG. 1 is a schematic representation of the genomic structure of MDV with restriction map position of the BairiHI-A region, and the location of the BairiHI-A-specific cDNA clone A41. The A41 cDNA clone is shown as an open box, and the clone's open reading frame (ORF) encoding 94 amino acids is shown below as a line bar.

The location of the polyadenylation signal (AATAAA) in the A41 clone is also shown.

FIG. 2 represents Northern blot analyses to confirm the origins of the A41 cDNA clone. Sizes of transcripts are shown in kilobases.

FIG. 2A represents a Northern blot using pol (A) ⁺ RNA isolated from either CU41 (lane 1) , or the negative control line CU91 (lane 2) , hybridized with a probe prepared from the A41 cDNA clone. FIG. 2B represents a Northern blot using total cellular RNA extracted from either uninfected chicken kidney cells (CKC) (lane 1) , or MDV strain RB1B-infected KC (lane 2) , hybridized with the same probe as used in FIG. 2A. FIG. 3 is a representation of the nucleotide sequence of the BairiHI-A-specific cDNA clone and its upstream genomic sequence obtained from analysis of the A41 clone. The predicted amino acid sequence of the ORF is shown by the single-letter code above the nucleotide sequence, with amino acid numbers in parentheses. The location of a potential polyadenylation sequence is underlined and indicated (AATAAA) . The locations of primers (#1 and #2) used to obtain the A41 clone, and restriction enzyme sites within the sequence are also underlined and indicated. FIG. 4 is a schematic representation showing the locations of the two probes used in the RNase protection assay.

FIG. 5 represents RNase protection assays of total RNAs from RB1B-infected CKC and CU41. Lane 1 represents size markers (in bp) ; lane 2 represents each undigested full-length probe; lane 3 represents RNA from uninfected CKC; lane 4 represents RNA from uninfected CKC; lane 5 represents RNA from CU197; lane 6 represents RNA from RB1B-infected CKC; and lane 7 represents RNA from CU41. FIG. 5A represents the RNase protection assay using probe 1 in the hybridization to the RNAs. FIG. 5B represents the RNase protection assay using probe 2 in the hybridization to the RNAs. FIG. 6 represents a photograph of immunofluorescence staining of RB1B-infected CKC with chicken anti-A41 antibody.

4. Detailed Description of the Invention

The MDV genome is a linear 180 kilobase pair double stranded molecule consisting of two unique regions: a unique short region (US) , and a unique long region (UL) . Each of the unique regions is flanked by inverted repeats: a long terminal repeat (TRL) and internal long inverted repeat (IRL) for UL, and a short internal inverted repeat (IRS) and short terminal repeat (TRS) for US (Figure 1) . Presently, the transformation process of T-cells by MDV is poorly understood. In ly phoblastoid cell lines established from Marek's disease tumors, viral gene expression is very limited whereas many viral transcripts are found in lytically infected cells

(Silver et al. , 1979, Virology 93:127-133). For example, at least 66 discrete transcripts were detected by Northern blot analysis of lytically-infected chicken embryo fibroblasts (CEF) , whereas less than 8 transcripts were detected in lymphoblastoid cell lines (Schat et al., 1989, Int. J. Cancer 44: 101-109). This observation suggests that MDV oncogenicity and latency are closely related, and that the few transcripts found in Marek's disease tumors, and their corresponding gene products, may be important for the initiation and/or maintenance of latency and/or tumor cell development by MDV. Recent studies by Schat et al. (1989, supra) and Sugaya et al. (1990, J. Virol . 64:5773-5782) demonstrated that the regions actively transcribed in tumor cell lines are the US and IRS regions, which correspond to the BairiHI-H, -I₂, -L, and -A DNA fragments according to the restriction enzyme map of MDV (Fukuchi et al., 1984, J. Virol . 51:102-109).

Although it is known that the BairiHI-A region of the MDV genome is actively transcribed in MDV-induced lymphoblastoid tumors and tumor cell lines, information is lacking about the identification and characterization within that region of sequence(s), and their corresponding gene products, which may be associated with the initiation and/or maintenance of tumor cell development. Additionally, there is a lack of information as to the identification and characterization of MDV transcripts in lytically- infected cells of chickens. The present invention relates to the identification and characterization of a MDV-specific cDNA clone, "A41", the sequence of which originates in the IRS region near the IRS-US junction as represented in Figure 1. The cDNA clone was isolated from a cDNA library prepared from a Marek's disease lymphoblastoid cell line, MDCC-CU41 (CU41) . CU41 is a non-expression cell line which contains none or only a few cells expressing viral antigens. However, A41 transcripts appear to be expressed in relatively higher amounts in lytically- infected cells (Fig. 2B, lane 2) than in the Marek's disease lymphoblastoid cell line CU41 (Fig. 2A, lane 1) . From the DNA sequence of A41, one open reading frame (A41 ORF) was identified which can encode 94 amino acids (SEQ ID No. 1) having a calculated molecular weight of 10,600 daltons. The A41 ORF, and its corresponding gene product of 94 amino acids, may be associated with initiation and maintenance of tumor cell development and/or establishment of latency in MDV-infected cells.

Because of the lack of effective drugs to therapeutically treat chickens with Marek's disease, and with recent reports of vaccination breaks in chickens vaccinated with virus, alternative approaches to controlling Marek's disease should be considered. One embodiment of the present invention relates to vaccination with the 94 amino acid gene product of the A4l ORF. In one variation of this embodiment, chicks or chickens are immunized with a vaccine comprising the 94 amino acid protein which may elicit protection by inducing an immune response that would recognize cells lytically-infected with MDV to prevent the development of latency or tumor cells subsequently induced by MDV infection. In another variation of this embodiment, the vaccine comprises a vector, such as a recombinant viral vector, containing the A41 ORF under the control of a strong promoter so that the 94 amino acid protein is expressed. In a second variation of this embodiment, the vaccine comprises a vector such as a recombinant viral vector, containing those nucleotide sequences of this A41 ORF coding for epitopes inducing humoral (approximately 15 amino acids) or cell-mediated (approximately 9 amino acids) immune responses. A second embodiment is the development of a Marek's disease virus which lacks oncogenicity. As noted in U.S. Patent No. 4,160,024 to Schat et al.. which patent is assigned to the assignee of the present invention and is incorporated herein by reference, unattenuated pathogenic strains of MDV have been unacceptable as vaccines for poultry because of their oncogenic potential. In one variation of this embodiment, the A41 ORF of an unattenuated pathogenic strain of MDV can be genetically engineered not to express the 94 amino acid protein. Since the A41 ORF appears non-essential for virus replication, expression of the 94 amino acid protein can be interrupted by the insertion of an exogenous sequence into the A41 ORF, resulting in a recombinant MDV that may be unattenuated, apathogenic, and non-oncogenic. In another variation of this embodiment, the recombinant MDV can be used as a vector in a multivalent vaccine by inserting one or more exogenous genes into the A41 ORF. An exogenous gene would encode protein which acts as an effective antigen in inducing a protective immune response against its corresponding organism of origin. Thus, the recombinant MDV could serve as a combined vaccine against Marek's disease and against other diseases of poultry. In another variation of this embodiment, one or more copies of an endogenous gene, whose gene product is an antigen capable of eliciting a protective immune response, can be inserted under the control of a strong constitutive promoter into the A41 ORF to increase antigen expression for vaccine purposes. For example, glycoprotein B (gB) is a major glycoprotein produced by MDV, which when inoculated into chickens results in the production of neutralizing antibody. Multiple copies of the gene encoding gB can be inserted in the A41 ORF to make a more effective recombinant vaccine against Marek's disease.

EXAMPLE 1 Isolation and Characterization of the A41 ORF The A41 ORF is a sequence derived from the sequence of an MDV-specific cDNA clone, A41, obtained through screening of the CU41 cDNA library with the gel-purified BairiHI-A fragment as a probe. CU41, a Marek's disease lymphoblastoid cell line established from a RBIB-induced tumor (MDV strain RBIB is a very highly oncogenic strain described previously by Schat et al., 1982, Avian Pathol . 11:593-605), is a non-expression cell line which contains none or only a few cells expressing viral antigens (Calnek et al., 1981, Infect . Im un . 34:483- 491) . CU41 was selected for making the cDNA library because only six MDV-specific transcripts, including 3.7, 1.7, 1.1 and 0.9-kb BairiHI-A transcripts, were detected in this cell line (Schat et al., 1989, supra) . For the isolation of cDNA clone A41, a cDNA library was prepared from the poly(A) ⁺ RNA obtained from CU41 cells. The library was screened by plaque hybridization with a ³²P-labeled MDV (strain GA) BairiHI-A fragment, and the hybridizing phages were plaque-purified. Subsequent in vivo excision and rescue of pBluescript (SK)™ carrying cDNA inserts were performed.

Southern blot analysis was performed to determine the approximate map position of the cDNA clone A41. The BairiHI-A fragment was digested by either Bgrll, Smal, EcαRI, HindiII, or Xhol fractionated on 0.8% agarose gels, and transferred to nylon hybridization membranes. Southern blot analyses were performed using ³²P-labeled probes, prepared by using gel-purified cDNA inserts as templates, for hybridization with the membranes. As shown in Figure 1, from Southern blot analysis it was determined that the A41 cDNA clone hybridized with both the 6.1 kb and the 1.7 kb EcoRI subfragments, and both the 1.6 kb and the 0.5 kb Xhol subf agments, of the BairiHI-A fragment. By aligning the subfragments with the restriction map of the BairiHI-A fragment, the exact location of the subfragments could be determined, as shown in Figure 1. It was thus determined by mapping that the A41 clone was localized within the inverted repeat region (IRS) of the BamHl-A fragment.

In order to characterize the transcripts coded for by A41, a radiolabeled probe was prepared from the A41 cDNA clone, and used for Northern blot analysis. In this experiment, total cellular RNA was isolated from uninfected chicken kidney cells (CKC) , RBIB-infected CKCs, CU41, and CU91 (a reticuloendotheliosis virus transformed T-lymphoblastoid cell line representing a negative control for CU41) using the method of Chomczynski et al. (1987, Anal. Biochem. 162:156-159). Poly(A)⁺ RNA, purified from the total cellular RNA preparations, were used in the Northern blot analysis of CU41 and CU91. For Northern blotting, 2 μg of each of the poly(A) ⁺ RNA fractions were fractionated by electrophoresis in 1.2 % agarose gel containing 2.2 M formaldehyde (15), and blotted onto nylon membranes.

RNA blots were prehybridized for 3 hours at 42° C in 50% ^■ formamide, 5X SSPE, 5X Denhardt's solution, 0.1% SDS and 100 μg/ml denatured salmon sperm DNA. Hybridization was performed for 18-20 hours at 42°C in the same solution with addition of the radiolabeled A41 probe. After hybridization, the membranes were washed twice with 2X SSPE containing 0.5% SDS at 65°C for 60 minutes. The hybridized filters were autoradiographed at -80°C with Kodak X-Omat AR film and intensifying screens. The results of the Northern blot analysis of the poly(A) ⁺

RNA fractions from CU41 and CU91 are shown in Figure 2A. Two A41-specific transcripts of 2.5 kb, and 1.2 kb were detected readily, and two weakly hybridizing transcripts of 8.0 kb and 4.8 kb were also detected (Fig. 2A, lane 1) . Total RNA extracted from lytically-infected cells were also used to confirm the presence of the transcripts found in CU41 using the A41 probe. The results of the Northern blot analysis of the total RNA fractions from uninfected chicken kidney cells (CKC) , and RBIB-infected CKCs, are shown in Figure 2B. Hybridization with the A41 probe resulted in the detection of major transcripts of 2.5 kb and 1.2 kb, and minor transcripts of 4.8 kb and 0.9 kb, in RBIB-infected CKC (Fig. 2B, lane 2) but no transcripts were detected in uninfected CKC (Fig. 2B, lane 1) . The amounts of these transcripts detected by Northern blot analyses using the A41 probe appear to be relatively more abundant in the RNA sample of RBIB-infected CKC than in that of CU41. A comparison of A41-specific RNA transcripts detected in CU41 and RBIB-infected CKC is shown in Table 1.

Table 1

Source of RNA

CU41 RBIB-infected CKC

8.0 kb -

4.8 kb 4.8 kb

2.5 kb 2.5 kb

1.2 kb 1.2 kb

- 0.9 kb

Previous studies of MDV-transcripts coded for by the BairiHI-A region in CU41 indicated the existence of transcripts of 3.7 kb, 1.7 kb, 1.1 kb, and 0.9 kb (Schat et al., 1989, supra) . The A41 cDNA clone (618 bp) was subcloned into pBluescript KS(+)™ and sequenced using the dideoxy- sequencing method. The sequence of the A41 cDNA clone is shown in Figure 3. The A41 clone was derived from a nonspliced mRNA. Sequence analysis from the subclone resulted in identification of one ORF (A41 ORF) which can encode a predicted polypeptide of 94 amino acids (Figure 3, SEQ ID No. 1). The calculated molecular size of the polypeptide is 10,600 daltons. A potential translation initiation site (ATG) of the ORF was located at positions 799-801. A 323-base 3' untranslated sequence was present in the cDNA, and a putative poly(A) signal, AATAAA, was present 19 nucleotides upstream to the start of the poly(A) tail. To map the 5' end(s) of the A41 transcripts, RNase protection assays were performed using two different probes which covered the 5' portion of the A41 ORF and its upstream region, as shown in Figure 4. Two subclones, a 301 bp Bgrlll-Xhol fragment, and a 296 bp hol-Λfscl fragment, were used as templates for synthesizing ³²P-labeled antisense RNA probes. Each of these probes (3 X 10⁵ cpm) was annealed to a total RNA sample (50 μg) prepared from either CU41, CU197 (a reticuloendotheliosis-transformed T lymphoblastoid cell line), RBlB-infected CKC, or uninfected CKC for 18 hours at 42° C, and then digested with RNase. Protected fragments were resolved on 6% polyacrylamide-8M urea sequencing gels and detected by autoradiography. In this determination of 5' end of the A41 transcript, some undigested probes were present due to incomplete digestion by RNase after hybridization. However, it was clear from this determination that these two probes specif-ically hybridized with RNAs from CU41 and RBIB-infected CKC, but not with those from uninfected CKC and CU197. The Xhol -Mscl region (probe 2) was fully protected when hybridized with the RNA sample from either CU41, or RBIB-infected CKC (Fig. 5B, lanes 7 and 6, respectively) . On the other hand, in addition to a fully protected 300-base fragment, a smaller fragment of approximately 225 bases was detected when the probe 1, corresponding to the Bgrlll- hol fragment, was hybridized with the RNA sample from either CU41, or RBIB-infected CKC. Together with the result that the A41 probe hybridized with a 1.2 kb transcript, it was concluded that the protected fragment of approximately 225 bases should correspond to the region immediately upstream of the .Xnol site.

EXAMPLE 2 This embodiment of the present invention relates to vaccination with the 94 amino acid (aa) gene product of the A41 ORF. In one variation of this embodiment, chicks or chickens are immunized with a vaccine comprising the 94 aa protein which may elicit protection by inducing an immune response that would recognize cells lytically- infected with MDV, or tumor cells induced by MDV infection. The 94 aa protein from infected cells, or recombinant protein produced from an expression vector system, can be purified with methods known in the art including detergent extraction and/or immunoaffinity chromatography. Immunopurification of the protein from an expression vector system may be accomplished using methods known in the art for immunoaffinity chromatography including use of 94 aa protein-specific monoclonal antibodies linked to a chromatographic matrix to form an affinity matrix. A protein preparation is then incubated with the affinity matrix allowing the antibodies to bind to the 94 aa protein. The affinity matrix is then washed to remove unbound components and the 94 aa protein is then eluted from the affinity matrix resulting in a purified preparation of 94 aa protein. The purified 94 aa protein may be chemically or enzymatically cleaved into peptides, or alternatively, peptides may be synthesized using the deduced amino acid sequence from the A41 ORF (SEQ ID No. 1) as a reference. Peptides derived from the 94 aa protein may also be useful in a vaccine preparation.

A substantial portion of the 94 aa protein has been purified using an expression system wherein the plasmid expression vector (pGEX2T) directs the synthesis of foreign polypeptides in Escherichia coli as a fusion protein with glutathione-S- ransferase (GST) , a 26 kilodalton protein from Schistosoma japonicum. In this mode of the embodiment, a 569 bp Mscl -Nrul fragment containing 92 of the 94 amino acids of the A41 ORF was recloned in frame into the 3'end of the GST ORF in pGEX- 2T. The resultant recombinant plasmid was designated pGEXA41SB. E. coli strain DH5α were transformed either with pGEXA41SB, or pGEX2T as a negative control. The respective transformants were grown in 3 ml of LB broth with ampicillin at 37°C overnight. Then, the cultures were diluted x 100 in LB broth with ampicillin and incubated for 2 hours at 37°C with shaking. IPTG was added to 2mM and the cultures were incubated for an additional 3 hours. After IPTG induction, the cells were harvested by centrifugation and the fusion protein and GST were purified by affinity chromatography with a glutathione-containing column matrix. Fractions containing the fusion protein were further purified by SDS-PAGE and subsequent electroelution. The purified recombinant protein termed GST A41, and the control protein GST were dialyzed against PBS for at least 24 hours at 4°.

Specific pathogen-free line P-2a chickens were injected subcutaneously with either GST A41 or GST in an adjuvant. Four weeks after this first immunization, the chickens were injected intravenously with the respective protein without adjuvant. The polyclonal antisera, anti-GST A41 and anti-GST, were used in indirect immunofluorescent antibody assays of CU41, RBIB-infected CKC and MSB1 (an expression Marek's disease cell line) . Acetonefixed cells were incubated with antisera against the fusion proteins for 30 minutes at room temperature and then washed twice with phosphate-bufered saline (PBS, pH 7.4) for 10 minutes. The washed cells were then incubated with fluorescein isothiocyanate (FITC) - conjugated rabbit anti-chicken IgG for 30 minutes at room temperature. Cells were examined with a fluorescence microscope after three 5-minute washes with PBS. MSB1 cells were double-stained with anti-GST A41 antibody, and a monoclonal antibody that recognizes MDV- specific phosphoprotems. FITC-conjugated rabbit anti- chicken IgG, and trimethylrhodamine isothiocyanate- conjugated rabbit anti-mouse IgG were used as secondary antibodies. Immunoperoxidase staining of the various cells was also performed. Goat anti-chicken antiserum labeled with peroxidase was used as a secondary reagent in this experiment.

No staining was observed when the anti-GST antiserum was incubated with uninfected CKC, RB1B- infected CKC, CU41, or MSB1 in immunofluorescence or immunoperoxidase assays. No staining was observed when the anti-GST A41 antiserum was incubated with uninfected CKC, CU41, or MSB1 in immunofluorescence or immunoperoxidase assays. In the double staining of MSBl cells, only approximately 1% of the cells were stained with the monoclonal recognizing the MDV-specific phosphoproteins. However, as shown by immunofluorescence in Figure 5, anti-GST A41 antiserum specifically reacted with the cytoplasmic regions of RBIB-infected CKC. Additional studies, using Western blot analysis, have shown that anti-GST A41 antiserum also reacts with CKC infected with herpesvirus of turkeys (HVT) . An expression vector system, useful for vaccine development, comprises a host containing a recombinant vector which expresses the 94 aa protein, or a peptide thereof containing immunogenic epitopes. Such hosts include, but are not limited to, bacterial transformants, yeast transformants, filamentous fungal transformants, and cultured cells that have been either infected or transfected with a vector which encodes the 94 aa protein. The protein or peptide immunogen is included in a vaccine formulation in pharmacologically effective amounts to induce an immune response. The vaccine may further comprise a physiological carrier such as a solution, a polymer or liposomes; and an adjuvant, or a combination thereof. Another mode of this embodiment provides for either a live recombinant viral vaccine, or an inactivated recombinant viral vaccine, comprising a viral vector containing the A41 ORF under the control of a strong promoter so that the 94 amino acid protein is expressed. Examples of such viral vectors known in the art include a live attenuated infectious laryngotracheitis virus, attenuated chick fowlpox virus, or attenuated herpes virus of turkey; i.e. an infectious virus that can be engineered to express vaccine antigens derived from other organisms. The recombinant live virus, which is attenuated or otherwise treated so that it does not cause disease by itself, is used to immunize chicks or chickens. Subsequent replication of the recombinant virus within the host provides a continual stimulation of the immune system with vaccine antigens such as the 94 aa protein, thereby providing long-lasting immunity.

To illustrate this mode of the embodiment, using molecular biological techniques such as those illustrated in Example 1, the A41 ORF may be inserted into the viral vector genomic DNA at a site which allows for expression of the 94 aa protein epitopes but does not negatively affect the growth or replication of the viral vector. The resultant recombinant virus can be used as the immunogen in a vaccine formulation. The same methods can be used to construct an inactivated recombinant viral vaccine formulation except that the recombinant virus is inactivated, such as by chemical means known in the art, prior to use as an immunogen. Alternatively, the DNA comprising the expression vector containing the A41 ORF, such as for example a recombinant viral vector, can be used in a subunit vaccine. Gene vaccines comprising antigen encoding DNAs have been described in the art. Direct gene transfer into animals resulting in expression of the exogenous gene in vascular endothelial cells, as well as the tissue of major organs, has been demonstrated by techniques such as injecting intravenously an expression plasmid:cationic liposome complex (Zhu et al., 1993, Science 261:209) . Other effective methods for delivering vector DNA into a target cell are known in the art. In one example, purified recombinant plasmid DNA containing viral genes has been used to inoculate (whether parentally, mucosally, or via gene-gun immunization) chickens to induce a protective immune response (Fynan et al., 1993, Proc. Natl . Acad. Sci . USA 90:11478- 11482) . In another example, cells removed from the chick or embryo can be transfected or electroporated by standard procedures known in the art, resulting in the introduction of the vector DNA into the target cell. Cells containing the vector DNA may be selected from those lacking the vector DNA by incorporating a selection marker into the vector such as the neo gene, and growing the cells in the corresponding selection media such as in the presence of G418. Selected cells, containing the recombinant expression vector for expressing the 94 aa protein or peptides derived therefrom, may then be reintroduced into the chick or embryo.

EXAMPLE 3 This embodiment of the present invention provides for the development of a Marek's disease virus which may lack oncogenicity. As noted in U.S. Patent No. 4,160,024 to Schat et al.. unattenuated pathogenic strains of MDV have been unacceptable as vaccines for poultry because of their oncogenic potential. In one mode of this embodiment, the A41 ORF of an unattenuated pathogenic strain of MDV can be genetically engineered not to express the 94 amino acid protein by inserting an exogenous sequence into the A41 ORF to interrupt its expression. For example, the US/IRS junction region from which the A41 clone originates has been deleted in a MDV strain JM clone isolated and characterized recently (Kost et al. 1993, Virology 192:161-169) , thus demonstrating that the A41 ORF is non-essential for virus replication in vitro. It is believed that the immediate early genes, such as the A41 ORF, play an essential role in the initiation and/or maintenance of transformation by MDV. By inserting an exogenous sequence into the A41 ORF, thereby interrupting transcription from the A41 ORF, a recombinant MDV that is unattenuated, pathogenic, and non-oncogenic may be developed which would be useful in a vaccine against MDV. Alternately, for this and other modes of this embodiment, the recombinant viral DNA may be used in the vaccine composition as described according to Example 2.

In another mode of this embodiment, the recombinant

MDV can be used as a vector in a multivalent vaccine by inserting one or more exogenous genes, under the control of its own promoter or a MDV promoter, into the A41 ORF. As discussed previously, a deletion into the A41 ORF results in a MDV clone still capable of replicating in chicken cells in vi tro, thus it is likely that interruption of the A41 ORF by an insertion would result in a clone of similar capabilities. Successful insertion of the lacZ ORF into the A41 ORF is further evidence that the A41 ORF can serve as a site in the MDV genome for insertion of exogenous genes. This was accomplished by inserting a lacZ cassette containing the SV40 promoter into a A41 ORF-containing plasmid. The final resulting plasmid contained the lacZ in forward orientation inserted in the remaining sequence of the A41 ORF. Total MDV DNA was purified from the CV1988 strain of MDV. MDV fragments containing lacZ were prepared by restriction enzyme digestion of the plasmid containing the lacZ cassette inserted into the A41 ORF. Total MDV DNA and MDV fragments containing lacZ were used to co-transfect chicken embryo fibroblasts (CEF) using the calcium precipitation method. Cultures were stained with 0.2 mg/ml Bluo-Gal™ at 5 days post transfection; and blue staining plaques were removed from the monolayer, trypsinized, and co-cultured with secondary CEF. Recombinant viruses were plaque-purified by repeating selection of lacZ-expressing plaques (usually 4-6 times) until stable recombinants were isolated. Chicken embryo fibroblasts have been lytically-infected with the plaque-purified lacZ- expressing recombinant MDV, thus demonstrating that the A41 ORF is non-essential for virus replication, and can be used as an insertion site for the expression of exogenous genes. Desirably, one or more exogenous gene(s) inserted into the A41 ORF would encode protein which acts as an effective antigen in inducing a protective immune response against its corresponding organism of origin. Thus, the recombinant MDV could serve as a combined vaccine against Marek's disease and against other diseases of poultry. Examples of such antigens useful in a combined vaccine, and their corresponding pathogen and disease caused, are known in the art of veterinary medicine. For example, genes encoding antigens of infectious laryngotracheitis virus causing infectious laryngotracheitis, can be inserted into the A41 ORF for expression. In another example, one or more copies of the gene encoding the viral hemagglutinin glycoprotein of the chicken fowlpox virus causing fowlpox, can be inserted into the A41 ORF for expression. In yet another example, one or more copies of either or both of the gene encoding the HN antigen or the gene encoding the F antigen of Newcastle disease virus causing Newcastle disease, can be inserted in the A41 ORF for expression. Recombinant expression of Newcastle disease viral antigens in HVT has been described previously (Morgan et al. in Proceedings XIX World's Poultry Congress. Amsterdam, The Netherlands, 1993) .

In another mode of this embodiment, one or more copies of an endogenous (MDV) gene, whose gene product is an antigen capable of eliciting a protective immune response, can be inserted within the A41 ORF to increase antigen expression for vaccine purposes. For example, glycoprotein B (gB) is a major glycoprotein produced by MDV, which when inoculated into chickens results in the production of neutralizing antibody (Nazerian et al., 1992, J. Virol . 66:1409-1413). Multiple copies of the gene encoding gB can be inserted in the A41 ORF under the control of a MDV promoter to make a more effective recombinant vaccine against Marek's disease. One or more of the other major viral glycoproteins of MDV, such as gA, may induce a protective immune response consisting of either cellular immunity, or humoral immunity.

It should be understood that while the invention has been described in detail herein, the examples were for illustrative purposes only. Other modifications of the embodiments of the present invention that are obvious to those skilled in the art of molecular biology, veterinary medicine, and related disciplines are intended to be within the scope of the appended claims.

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(A) APPLICATION NUMBER:

(B) FILING DATE:

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: U.S Serial No. 08/192,633 (B) FILING DATE: February 7, 1994 (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Nelson, M. Bud

(B) REGISTRATION NUMBER: 35,300 (C) REFERENCE DOCKET NUMBER: 18617.0000 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (716) 856-4000

(B) TELEFAX: (716) 849-0349 (2) INFORMATION FOR SEQ ID NO. 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1406 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single-stranded (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: yes (iv) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (B) CLONE: clone A41 (v) ORIGINAL SOURCE:

(A) ORGANISM: Marek's Disease Virus

(B) STRAIN: MDCC-CU41

(C) CELL TYPE: virus (vi) FEATURE:

(A) LOCATION: A41 open reading frame, 799-1080

(B) IDENTIFICATION METHOD: by experiment (C) OTHER INFORMATION: (vii) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

AATGCCAGCA TTGCATTTCT AGCACACAAA CTAGACCGGC CCACCGAGTC 50

AAATTGTGTC CTGCTGAACG ATTGCATAGT TTGAGATGGC CATGCCAGTA 100

AACAAATACT TGAATACCAT CGACTAAGCA CAAAAGGCTC TTATCATCTA 150 CTAGCAGAGT TGTGAAGTAG ATGAGCGATT GTGGGGCCCA GCCGACCAAA 200

GCCCTGTAGG CAAACATTCC TGTACTGCCG GTTAAGCAGA TTAAACACTC 250

ATCGCTAGAG CCGCAAATTA TTTCCCGATG ACAACTTTAT GCGGCGGCTG 300

CGGTTTGTTT TAATTAATAC CTAATGGGCA AATATTCACG CTCCCACAAA 350

ATAAACACAA ATCACCCAGT GTTCCCACAT CAAAAATCGC CATGTAGGGA 400 AGATGGATTT GAAGATCGCA GACTCGACAG GGAACGCGCT CAAAAAAAAA 450

AAAACGGAGG GATGGGTCTT TATTCAAAGA AGTGTAAAAG GTAATGCGAC 500

CACTCGAGCA GCAAACATTA ACCGCGCGCT TATGGATACT ACGAGCTAGC 550

ACCGGGAAAG GCAGTGCATG AGTCCCGTTG ATGAATATGG GGGGAGGGGG 600

GAATAGGTCG GTAAAACTTG TGCCCGCAGA CCTCCGACAT GAACAGTAGA 650 CTGTTTAATG TAGAACGAAA TCGAGGAGCC ATACCGGATA CATTTTGCGG 700

GCAAGTTCGC CATCCGGTTT TCAGAAAAAT CAGACTTTTG CTGAACATCG 750

AGAGTATTAT TCATCACAAA TTAAAAACTC GGGCATGTCT GATCCTTC ATG 801 Met

1

AGC TGG CCA AGA GGC GAC AGT AAA AAA AAA ATC GAA GGG GGG 843 Ser Trp Pro Arg Gly Asp Ser Lys Lys Lys lie Glu Gly Gly 5 10 15

GAA ACA TTG CTC GAT AAT CGC GTA GCC AGA CCG CAC CAT ATT 885 Glu Thr Leu Leu Asp Asn Arg Val Ala Arg Pro His His lie

20 25

CTC CCC CTC CCG CAA ATT CAG AAT TGT ATA AGA GAA CGT AGG 927 Leu Pro Leu Pro Gin lie Gin Asn Cys lie Arg Glu Arg Arg 30 35 40 AAA AAA AAA AGG AAT ATA CAT ACC ACA CAC TTT GAT ATT TTG 969 Lys Lys Lys Arg Asn lie His Thr Thr His Phe Asp lie Leu 45 50 55

GAT GTG TCC CAG GGC AAT GGG AAC AGC AAT CAC TTT CGA ATT 1011 Asp Val Ser Gin Gly Asn Gly Asn Ser Asn His Phe Arg lie 60 65 70

CCT TCA GCC CAA AGC ACA ACC GCG GGT CCA CCG GGA CTC TCC 1053 Pro Ser Ala Gin Ser Thr Thr Ala Gly Pro Pro Gly Leu Ser 75 80 85

GAC ATA GCT CGA GCC AAA AGG GAA TTG TAGCCCAGGC 1090 Asp lie Ala Arg Ala Lys Arg Glu Leu

90

AAGTGCAATG TTTTAATTTG TTCTTCTCCC TCCCCCACAT AAAAAAACCA 1140 CTGGATCGTA CAAGTATACG AGTATATGGG TGGGGTGCCG TTTTATATAA 1190

ACACAGCTTA GTTTGTTTGC CACGTCAAGG AAGGGCGGTG CATATCTGCA 1240

AGTAAACAAA ACTCGGGGTT CTGTACGATT GGCCGGGGTC TTACATGCTC 1290

GCCGAATTGG ATTTGAGAAT CAATCTTCCG ACGGGTTTCC TGACTTGAAC 1340

AGGGGAAAAG AGGAGGGGGA GTGTGTTATC TTGTCGCGAA CCAATAAAAT 1390 AGATTTGTGG CCTAAC 1406

Claims

We claim:

1. A Marek's disease (MDV) virus nucleotide sequence, and the 94 amino acid polypeptide it encodes, which can be useful for vaccine formulations, said sequence and polypeptide consisting essentially of SEQ ID No. l.

2. A vaccine formulation comprising an immunologically effective amount of a 94 amino acid protein encoded by A41 ORF, or a peptide derived therefrom, wherein the protein consists essentially of the amino acid sequence of SEQ ID No. 1.

3. The vaccine formulation according to claim 2, in which the protein was produced recombinantly from a host cell system genetically engineered to include an expression vector containing a nucleotide sequence consisting essentially of SEQ ID No. l, said host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, insect cell lines, and mammalian cell lines.

4. The vaccine formulation according to claim 2, in which the peptide is produced by chemical synthesis.

5. The vaccine formulation according to claim 2, further comprising a pharmaceutical carrier.

6. The vaccine according to claim 2, comprising a recombinant viral vector containing the A41 ORF under the control of a strong promoter so that the 94 amino acid protein is expressed from the vector.

7. The vaccine according to claim 6, wherein the viral vector is a live attenuated virus selected from the group consisting of infectious laryngotracheitis virus, chicken fowlpox virus, and herpesvirus of turkey.

8. A vaccine formulation comprising a recombinant unattenuated pathogenic non-oncogenic strain of MDV, wherein said strain is genetically engineered not to express the 94 amino acid protein of SEQ ID Nol. 1 by interrupting expression from A41 ORF by a mechanism selected from the group consisting of inserting a nucleotide sequence into the A41 ORF of SEQ ID No. 1, or deleting the sequence corresponding to the A41 ORF of SEQ ID No. 1.

9. The vaccine formulation according to claim 8, wherein said recombinant MDV is a multivalent vaccine comprising one or more exogenous sequences inserted into the A41 ORF, and wherein said exogenous sequence encodes an antigen useful against a disease of poultry other than Marek's disease, said sequence is under the control of its own promoter or a MDV promoter.

10. The vaccine formulation according to claim 8, wherein the nucleotide sequence inserted into the A41 ORF comprises one or more genes endogenous to MDV and said gene encodes a major viral glycoprotein of MDV.