MXPA98004669A - Preparation of marek's disease viruses using mamif continuous cell lines - Google Patents
Preparation of marek's disease viruses using mamif continuous cell linesInfo
- Publication number
- MXPA98004669A MXPA98004669A MXPA/A/1998/004669A MX9804669A MXPA98004669A MX PA98004669 A MXPA98004669 A MX PA98004669A MX 9804669 A MX9804669 A MX 9804669A MX PA98004669 A MXPA98004669 A MX PA98004669A
- Authority
- MX
- Mexico
- Prior art keywords
- mdv
- marek
- disease
- nucleic acid
- cells
- Prior art date
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Abstract
The present invention relates to the use of continuous mammalian cell lines for the production of recombinant and non-recombinant viruses of Marek's disease, for the production of genetically altered or produced Marek's disease viruses and virus vectors of the Marek's disease, continuous mammalian cell lines infected with Marek's disease virus, Marek's disease virus vaccines produced using said processes and procedures to protect animals, such as birds, against diseases caused by the disease virus of Marek's disease. Marek and / or r pathogens, particularly, mammalian cell lines capable of infection or transfection with MDV are feline kidney cell lines.
Description
PREPARATION PE VIRUS OF MAREK DISEASE USING CONTINUOUS MAMMALIAN CELL LINES
FIELD OF THE INVENTION
The present invention is directed to continuous mammalian cell lines infected or transfected with Marek's disease virus or with Marek's disease virus vectors, to vaccines produced using Marek's disease virus or disease virus vectors of Marek prepared by culturing said lines of infected or transfected cells. and to methods for vaccinating birds against diseases resulting from infection with Marek's disease virus and / or other pathogens "by administering said vaccines produced using said methods. Particularly »the lines of mammalian cells capable of infection or transfection with Marek's disease viruses are feline kidney cell lines.
BACKGROUND OF THE INVENTION
Marek's disease (MD) is an acute oncogenic disease of chickens »that causes lymphoas» visceral tumors »nerve injuries and immunosuppression. The disease is global and ubiquitous. The etiological agent is a herpesvirus »the Marek's disease virus. Marek's disease has been an important cause of deaths and sentences in colonies of broiler chickens CB.W. Calnek and R.L. Witter »Diseases of Poultry» 9th edition »Io a State University Press» Ames »or a» p. 3-42-385 (1992) 3. There are three serotypes of the disease virus of
Marek. Serotype 1 includes all pathogenic strains and their attenuated derivatives. Serotype 2 consists of viruses of chickens of avirulent nature while serotype 3 »also known as turkey herpesvirus (HVT). it includes avirulent viruses of turkeys that are capable of replicating in chickens. The three serotypes are partially cross-protective but can be distinguished using monoclonal antibodies CR.F. Silva and L.F. Read »Viro !.» vol. 36 »p. 307-320 (1984) 3 and other known methods. The first vaccines against Marek's disease consisted of live serotype 3 viruses. Although initially "the emergence of highly virulent strains of Marek's disease virus" defined by their ability to break vaccination with HVT, they initiated the development of vaccines of serotype 2 and serotype 1 attenuated. as well as bivalent and trivalent combinations of serotypes 1 »2 and 3 CB.W. Calnek and R.L. Witter »D sease of Poultry» 9th Edition, Iowa State University Press »Ames, Io» p. 342-385 (1991) 1. It seems that the Marek's disease virus is less recogenic than other herpesviruses and the nature associated with virus cells leads to the purification of recombinant plaques from the problematic of parental viruses. However, recombinant viruses of 1 to Marek's disease have been generated from serotype 1 CK. Sonoda et al., Vaccine »vol. 14, p. 277-284 (1996); M.S. Parcel Is et al. "J. Virol., Vol. 69, p. 7,888-7,898 (1995); M.S. Parcel Is et al., Virus Genes »vol. 9, p. 5-13 (1994); M.S. Parcel Is et al., J. Viro!., Vol. 68, p. 8.239-8.253 (1994); M. Sakaguchi, Vaccine, vol. 12, p. 953-957 (1994); S.K. Reddy et al., Vaccine, vol. 14, p. 469-477 (1996) 3; from serotype 2 CD.R. Marshall et al., Viro!., Vol. 195, p. 638-643 (1993); R.F. Silva »14s International Herpesvirus Symposium (Summary) (1989) 3; and from serotype 3 CS.K. Reddy et al., Vaccine, vol. 14, p. 469-477 (1996); PCT patent application WO 95/29248 (1995); R.F. Sil a, 1 2 International Herpesvirus Symposium (Summary) (1989); R. Darteil et al., Viro!., Vol. 211, p. 481-90 (1995); United States Patent No. 5,187,087 granted in 1995; V. Zelnik et al., J. Gen. Virol. Vol. 76, p. 2,903-2,907 (1995); PCT patent application WO 93/25665, published in 1993; L.J.IM. Ross et al., J. Gen. Virol. "Vol. 74, p. 371-377 (1993); P.J.A. Sondermeijer et al., Vaccine, vol. 11, p. 349-358 (1993); European Patent No. 431,668B1 »published in 1995» R.W. Morgan et al. »Avian Dis.» Vol. 36, p. 85B-S70 (1992); P.K. Bandyopadhyay et al. »13th Herpesvirus International Symposium 323 (Summary) (1988) 3. In all the aforementioned references "all of which are incorporated herein by reference" the viruses were capable of replication and occurred in primary poultry cells. Commercially available vaccines of Marek's disease virus "with the exception of some monovalent formulations of HVT" consist mainly of primary cells of chicken embryo fibroblasts (CEF) infected with live Marek's disease virus. A significant problem associated with the use of primary chicken cells infected with whole live Marek's disease virus to develop Marek's disease virus for use in vaccines, is that CEF cells must be conserved at liquid nitrogen temperatures. and administered by injection to be effective. Previously, whole live cell vaccines had been necessary since all three serotypes of Marek's disease are strongly associated with cells in cell cultures and in most tissues of an infected bird. The spread of infection in birds can be carried out by direct cell-to-cell contact, with few or no cell-free viruses being released. Infectious virions are produced only in the epithelium of the follicles of the feathers (FFE) and are responsible for bird-to-bird transmission CB.W. Calnek et al., Avian Dis., Vol. 14, p. 219-233 (1970); R.L. Witter et al., J. Nati. Cancer Inst. »Vol. 49 »p. 1.121-1.130 (1972); C.S. Eidson et al. »J. Nati. Cancer Inst. »Vol. 47 »p. 113-120 (1971) 3. Commercial vaccines of the disease virus
Marek-free cells are made by cell culture. However, the production of cell-free Marek's disease virus vaccines has been very limited to vaccines produced using only serotype 3 Marek's disease virus. This is because only serotype 3 of the Marek's disease virus produces free virions in sufficient quantities for the production of Marek's disease virus vaccines. It has been suggested that the lack of expression of the viral glycoprotein gene D (gD) may be involved in limiting the release of cell-free virions Ct PCT patent application WO 95/2924B published in 1995; X. Tan and L.F. Velicer »182 Herpesvirus International Symposium A» 145 (Summary) (1993) 3. In addition to the CEF cells, other primary cells of birds have been used to develop Marek's disease virus, including chicken kidney (CK) and duck embryo (DEF) fibroblasts. only two lines of continuous bird cells have been described as capable of developing certain serotypes of Marek's disease virus. It has been described that serotype 3 of the Marek's disease virus (Marek's disease virus 3) develops in a chemically transformed quail cell line, called QT35 CM. Niikura, T. IMarita et al., J. Vet. Med. Sci. »Vol. 53, p. 439-446 (1991) 3. It has been described that a line of chemically transformed CEF cells, called CHCC-0U2 CH. Ogura and T. Fuji ara, Acta Med. Okayama, vol. 41, p. 141-143 (1987) 3 allows the development of virus 1 of Marek CA disease.
Abujoub and P.M. Coussens, Virol., Vol. 214, p. 541-549 (1985). Other known methods for producing Marek's disease virus include the use of orogenic or oncogenic cell lines. The lines of nfoblastoid cells 1 transformed by Marek CK disease virus. Na? Erian, Avian Pathol. , vol. 16, p. 527-544 (1987) 3 are derived from lymphoid tumors in chickens infected with Marek's disease virus 1 oncogenes. The viral genome is maintained in a latent or semi-latent state in these cells so that the transmission of the infection to CEF cells co-infected or to DEF cells occurs at a low frequency, if it occurs. Furthermore, these lines of 1 infoblastoid are refractory to super-infection with other Marek's disease viruses. In addition »the lines of infoblastoid cells 1 transformed with Marek's disease virus have not proved useful in the production of non-recombinant (conventional) virus vaccines of Marek's disease or in the preparation of recombinant virus of the disease of Marek's disease. Marek or genetically altered Marek's disease viruses or vectors. Similarly, the cells of CK nfoblastoid cells. Nazerian, Avian Pathol., Vol. 16, p. 527-544 (1987) 3 derived from oncogenic avian retroviruses (avian leukosis virus and reticuloendotheliosis virus) are not useful for the production of commercial vaccines of Marek's disease virus or for the generation of recombinant viruses from Marek's disease »due to the disguise of retroviruses» to the poor development characteristics of the infoblastoid cells and to the low level of productive infection of the Marek's disease virus. No mammalian primary cells and mammalian cell lines have ever been described as suitable substrates for the development of any serotype of Marek's disease virus, nor has it ever been demonstrated that a mammal was productively infected with Marek's disease virus.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 describes the insertion of the gH gene of Marek's disease virus 1 in the pCR 3.1 vector map. Figure 2 is a map of the p6reenLantern2 plot. Figure 3 describes the PCR analysis of reverse transcriptase of NLFK 3G4 continuous cells transfected with the gH gene of Marek's disease virus.
BRIEF DESCRIPTION OF THE INVENTION
In one aspect the present invention relates to a method for preparing MDV which comprises: (a) infecting or transfecting with MDV a continuous mammalian cell line »(b) culturing the line of continuous mammalian cells infected or transfected with MDV and (c) recover MDV from the continuous cell culture. The "MDV" as defined herein may be, for example, a non-recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus or a fragment (s) thereof, a recombinant molecule comprising the sequence of the Marek's disease virus nucleic acid or a fragment (s) thereof, a recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus or a fragment (s) thereof and at least a heterologous gene or a fragment (s) thereof inserted into said nucleic acid sequence, and a recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus in which one or more genes have been deleted, regulatory genetics, or a fragment (s) thereof. For example, the deleted gene may be an essential one for replication. More particularly, the suppressed gene can be the gH gene of Marek's disease virus or a fragment (s) thereof. The MDV can be a molecule comprising the nucleic acid sequence selected from the group consisting of serotype 1, serotype 2 and serotype 3 of the Marek's disease virus or a fragment (s) thereof, taken individually by each serotype or in any combination of the In another aspect the invention relates to the procedure described above in which the MDV is a molecule comprising the nucleic acid sequence of the Marek's disease virus, used for the preparation of a vaccine capable of inducing protection against disease in birds. Preferably, the line of mammalian continuous cells used in the aforementioned procedure is a feline kidney cell line infected with the MDV strain Md5, such as the ATCC cell line CRL12336. Examples of other suitable strains include strains 584A and 652 of MDV-1. Also another aspect of the invention is a line of continuous mammalian cells infected or transfected with MDV. The MDV can be any of the forms of MPV mentioned here. Preferably, the mammalian continuous cell line is a line of feline kidney cells infected with the MDV strain Md5, such as the cell line n ^ ATCC CRL12336. Examples of other suitable strains include strains 584A and 652 of MDV-1. An example of a line of mammalian infected or transfected continuous cells of the present invention is one that is capable of expressing a gene present in natural MDVs that is essential for replication, such as, for example, the gH gene. This cell line is capable of replicating a virus of the IO
Marek's disease that does not have replication capacity. Another aspect of the invention is a method of producing a line of mammalian continuous cells infected or transfected with MDV which comprises: (a) infecting or transfecting continuous mammalian cells with MDV and (b) culturing infected or transfected mammalian cells with MDV to propagate the cell line. Another aspect of the present invention relates to a vaccine for protecting birds against the Marek's disease virus, comprising an immunologically effective amount of MDV produced by the method of the present invention, an immunologically effective amount of the infected cells or transfected of the present invention, or an immunologically effective amount of one or more proteins or polypeptides encoded by said MDV produced by the method of the present invention, and an acceptable veterinary vehicle. Also another aspect of the present invention relates to a vaccine wherein the MDV comprises an immunologically effective amount of a nucleic acid sequence or a protein or polypeptide encoded by said nucleic acid sequence., wherein said nucleic acid sequence or protein or polypeptide are capable of inducing a protective response against a disease or pathological condition distinct from Marek's disease, which affects birds. Examples of disease or pathological conditions other than Marek's disease affecting birds are those caused by Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), »infectious bronchitis virus (IBV), Chicken anemia virus (CAV) »Infectious laryngotracheitis virus (ILTV)» avian leukosis virus (ALV), reticuloendothelium iosis virus (REV) and avian influenza virus (AIV). The MDV of vaccines of the present invention can be any of the forms of MDV mentioned herein. Preferably the vaccine is prepared using a mammalian continuous cell line which is a line of feline kidney cells infected with the MDV Md5 strain, such as the cell line n2 ATCC CRL12336. Examples of other suitable strains include strains 5S4A and 652 of MDV-1. Also another aspect of the invention is u? R \ recombinant MDV produced using the methods of the present invention. The recombinant MDV may comprise, for example, the nucleic acid sequence of the non-recombinant Marek's disease virus, in which one or more genes have been deleted, as a gene essential for replication. An example of such an essential gene is the glycoprotein H (gH) gene »or a fragment thereof» of the MDV genome. The resulting incapacitated virus »that has no ability to replicate» is infectious for a single simple cycle provided a line of complementary cells »genetically produced to contain the gene essential for viral and viral replication. therefore »the product of expression of the deleted gene» is available to propagate the incapacitated virus. The production of incapacitated viruses and their use as vaccines are described in PCT patent application GB-91/01632 (publication nos. WO 92/05263). which is incorporated here in its entirety. Also another aspect of the invention is a MDV vector produced using the methods of the present invention. Optionally the MDV vector has a 3 r flanking region comprising the nucleic acid sequence SEQ ID Ns 2. Also optionally the MDV vector can have a 5"flanking region comprising the nucleic acid sequence SEQ ID N2 1. In addition, the MDV vector can have both the 3T flanking region comprising the nucleic acid sequence described in SEQ ID Na 2 »and the flanking region 5» comprising the nucleic acid sequence described in SEQ ID NO 1. Also another aspect of the invention refers to a method for vaccinating a bird against the Marek's disease virus which comprises administering to the bird an immunologically effective amount of MDV produced by the method of the present invention or an immunologically effective amount of the infected or transfectants of the present invention and an acceptable veterinary vehicle, and another aspect of the invention relates to a to produce a vaccine to protect birds against the Marek's disease virus, which comprises combining an immunologically effective amount of MDV produced by the methods of the present invention, an immunologically effective amount of the infected or transfected cells of the present invention, or an immunologically effective amount of one or more proteins or polypeptides encoded by said MDV produced by the methods of the present invention, and a veterinarily acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to lines of continuous mammalian cells infected or transfected with MDV or MDV vectors, to MDV and vaccines produced using MDV or MDV vectors prepared by culturing said infected or transfected cell lines, and to methods for vaccinating birds against diseases resulting from infection with the Marek's disease virus and / or other pathogenic agents, by administering said vaccines produced using said methods. As used herein, the term "MDV" refers to a nucleic acid sequence comprising the genome of the Marek's disease virus or any fragment or fragments of said fragment (s) thereof. MDV includes, for example, nucleic acid sequences obtained from: natural Marek's disease virus (non-recombinant Marek's disease virus) or any fragment or fragments thereof; Marek's disease virus produced recombinantly (recombinant Marek's disease virus) or any fragment or fragments thereof; and Marek's disease virus produced or genetically altered "or any fragment or fragments thereof. The fragment or fragments of the nucleic acid sequence of non-recombinant viruses of Marek's disease »of recombinant virus before Marek's disease, or of Marek's disease virus produced or genetically deleted may include one or more genes of the Marek's disease virus or parts thereof. Preferably said fragment (s) encode (s) antigens, such as proteins and polypeptides of the Marek's disease virus, which are useful as vaccines against the Marek's disease virus. In addition, MDV comprises any of the aforementioned forms of the Marek's disease virus and »in addition. one or more heterologous genes or parts thereof, incorporated in an insertion region located in the MDV nucleic acid sequence. Preferably, the heterologous gene or genes encode antigens "such as proteins and polypeptides" of other disease-causing agents "such as" for example "from other causative agents of bird diseases" which are useful as vaccines against said other disease-causing agents. The MDV may be a molecule comprising the nucleic acid sequence of serotypes 1, 2 or 3 of the Marek's disease virus, or a fragment (s) thereof taken individually or in any combination thereof. In addition, the present invention encompasses MDV comprising, for example, substitutions, insertions, inversions, additions and deletions in the Marek's disease virus nucleic acid sequence and any combinations thereof, as mutants of the eukaryotic virus Marek's disease and Marek's disease virus in which a non-essential region is lacking »as a non-essential gene, the genome of Marek's disease virus or, alternatively, an essential region of the disease virus genome of Marek. Said variations can be natural or can be produced using conventional recombinant techniques to produce said variations. The production and manipulation of recombinant viruses of Marek's disease or a fragment (s) thereof of Marek's disease virus produced or genetically altered or a fragment (s) thereof, and of recombinantly produced variations of viruses of natural Marek's disease are within the knowledge of the art and can be performed according to recombinant techniques described, among other references, in Maniatis et al. »Molecular Cloning, A Laboratory Manual» Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY, 1989; Sambrook et al. »Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Innis et al. (ed.), PCR Strategies, Academic Press Inc. »San Diego» 1995; and Erlich (ed.) »PCR Technology, Oxford University Press» New York »1992; all of which are incorporated here as a reference. The methods for preparing Marek's disease recombinant viruses or fragments thereof »produced or genetically altered Marek's disease viruses or fragment (s) thereof» and recombinantly produced variations of natural Marek's disease viruses containing Heterologous genes are within the knowledge of the technique and can be performed according to recombinant techniques described in Maniatis et al. and in the other references indicated above. The preparation of various forms of Marek's disease recombinant viruses and Marek's disease virus vectors using recombinant techniques is described, for example, in U.S. Patent No. 5,231,023 issued July 27, 1993, which describes a viral vector of Marek's disease virus prepared by inserting a heterologous gene in a non-essential region of the DNA genome of the Marek's disease virus »patent which is incorporated herein by reference.
An example of MDV lacking an essential gene is a MareK disease virus that has no replication capacity. A particular example of an MDV that does not have replication capability is the genome of the Marek's disease virus in which the gH gene has been deleted using recombinant techniques before. As used herein, the term "infection" or "transfection" refers to the transfer of nucleic acids from viruses »genes or a fragment (s) or part (s) thereof to mammalian cells. Such transfer of nucleic acids includes the transfer of any of the aforementioned forms of MDV, such as recombinant and non-recombinant viruses of Marek's disease, to mammalian cell lines, such as lines of feline kidney cells. Methods for gene transference to mammalian cells are within the skill in the art and are described, for example, in Watson et al. "Recombinant DNA, 2 = edition, W.H. Freeman and Company, N.Y. (1992). which is incorporated here as a reference. As used herein, the term "MDV vector" refers to a recombinant expression vector comprising a nucleotide sequence that encodes any proteins or polypeptides encoded by MDV, wherein the nucleotide sequence is operatively associated with one or more regulatory elements »under conditions that lead to the expression of the protein or polypeptide. The MDV vector can be any expression vector that contains appropriate regulatory sequences necessary for the expression of one or more of the proteins or polypeptides encoded by MDV (such as the genome of non-recombinant viruses of Marek's disease or any fragment thereof). »Of Marek's disease recombinant viruses or any fragment thereof» or of Marek's disease virus produced or genetically altered or any fragment or fragments thereof »and may also contain one or more heterologous genes (genes other than naturally associated with the Marek's disease virus) that are capable of being expressed on the lines of infected or transfected mammalian continuous cells of the present invention. The MDV vector may contain, for example, the nucleic acid sequence of Marek's disease recombinant viruses and one or more heterologous genes encoding proteins and polypeptides as antigens, of agents other than the Marek's disease virus causing the disease. diseases, such as other agents that cause bird diseases. The heterologous gene or genes can be inserted into the MDV vector using recombinant techniques that are within the skill of the art »as described» among other references »in Maniatis et al. and / or in the other references mentioned above. Said gene or heterologous genes can be expressed in the mammalian infected or transfected continuous cell lines of the present invention and, once administered to animals as birds, they can elicit an immune response both against Marek's disease recombinant viruses and against the heterologous gene or genes or the expression products or against proteins or polypeptides, such as antigens. A suitable and functional promoter must be linked to the heterologous gene or genes so that the MDV vector is capable of expressing the antigens, such as polypeptides and proteins encoded by the heterologous gene or genes. The promoter can be any eukaryotic, prokaryotic or viral promoter capable of directing transcription in cells infected with the recombinant MDV vector. The production and manipulation of recombinant vectors, including the preparation and use of promoters, are within the knowledge of the art and can be carried out in accordance with the recombinant technique described in
Maniatis et al. and / or in the other references mentioned above. For example, a gene of Marek's disease virus necessary for replication may be missing in the MDV vector., that is, it may not have the capacity to replicate and contain one or more heterologous genes that encode antigens of other disease-causing agents, such as other agents that cause bird diseases. Optionally, the MDV vector has a 3"flanking region comprising the nucleic acid sequence SEQ ID N2 2. Also optionally, the MDV vector can have a 5T flanking region comprising the nucleic acid sequence SEQ ID N2 1. Furthermore, the MDV vector can have both a flanking region 3 comprising the nucleic acid sequence described in SEQ ID N2 2, and a flanking region 5r comprising the nucleic acid sequence described in SEQ ID 1: 1. invention encompasses methods for producing MDV and MDV vectors by infecting or transfecting mammalian continuous cell lines and cell lines that are subcloned or derived in any other way from said mammalian continuous cell lines, and culturing said continuous or infected cell lines. transfected from mammal, such as, for example, feline kidney cell lines. infected or transfected mammalian ones to produce MDV, or being useful in the production of MDV vectors. Then, the MDV produced in said cell lines can be isolated or recovered using isolation and recovery techniques that are within the skill in the art, such as those described in Maniatis et al. and / or in the other references mentioned above. The present invention encompasses all MDVs and MDV vectors produced using the methods of the present invention. The present invention encompasses continuous mammalian cell lines infected or transfected with MDV. As used herein, the term "mammalian continuous cell line" refers to mortalized i cells of a mammal that can be maintained in vitro, preferably for at least 15 to about 20 trans erences. Most preferably, said mammalian continuous cell lines are capable of sustained development for about 50 or more transfers. As used herein, the term "1 line of mammalian continuous cells infected or transfected with MDV" refers to cells of a mammal that can be maintained in vitro and are capable of producing MDV or proteins or polypeptides encoded by said MDV , in a sustained manner, preferably for at least 15 to approximately 20 transfers. Most preferably said lines of infected or transfected cells are capable of producing said MDV or proteins or polypeptides encoded by said MDV for about 50 or more transfers. Preferably, the line of mammalian continuous cells used for infection or transfection with MDV is further characterized as substantially free of mammalian or avian viruses. Preferably, the line of mammalian continuous cells is a line of kidney cells. The preferred line of mammalian kidney continuous cells for infection or transfection with MDV is a feline kidney cell line. To practice the methods of the present invention, any feline kidney cell line can be used, such as, for example, feline kidney cell lines designated CRFK, deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. , and called ATCC CCL-94. A preferred cell line is a feline kidney cell line and any clones thereof, called the NLFK cell line (Norden Laboratories Feline Kidney) (from Pfizer Inc.), deposited on April 8, 1997 in the American Type Culture Collection and named ATCC CRL-12336. The ATCC cell line CRL-12336 is a line of feline kidney cells infected with the Md5 strain of Marek's disease virus. The Md5 strain was obtained from the American Type Culture Collection, having the designation ATCC VR 987. Lines of mammalian cells can be grown and maintained as "lines of mammalian kidney cells or lines of feline kidney cells" using known techniques of mammalian cell culture, such as described in Julio Celis (ed.) »Cell Biology, Laboratory Handbook, Academic Press» NY (1994). More variable conditions of mammalian cell culture can be selected and optimized "including the formulations of the media for specific nutrients" oxygen tension »C02 and reduced serum» using methods known in the art. Mammalian cells can be cultured and expanded from a single cell using various commercially available culture media, under known conditions suitable for propagating mammalian cells. For example, feline kidney cells, such as NLFK cells, kept frozen until use. can be heated to a temperature of about 37 (?) C to about 3B »5 (?) C and added to a suitable growth medium» such as OptiMEM (Life Technologies Inc.) containing 2% fetal bovine serum (FBS) . The cells can be incubated at a temperature of about 37 (?) C to about 38 »5 (?) C in a hybrid incubator with approximately 5% C02 until they become confluent. For the transfer of the cells, the growth medium can be separated and trypsin can be added to the cells at 0 to 05% and 0 to 53 mM EDTA. The cells are separated and the cell suspension can be collected in centrifuge tubes and centrifuged to obtain cell deposits. The trypsin solution can be separated and the cell deposits resuspended in fresh growth medium. Then the cells can be further propagated in additional growth vessels at a desired density. Infection or transfection can occur by cocultivation with MDV-infected cells "in which the MDV is non-recombinant" recombinant, genetically produced or is a vector of the MVD. The cells used for the cocultivation can be MDV infected cells, such as CEF cells or DEF cells, nfoblastoid cells 1, peripheral blood mononucleated cells, CHCC-0U2 cells, QT35 cells and feline kidney cell clones, such as clone 3G4. described in the examples. Alternatively, mammalian cell lines can be infected with MVD by introduction of purified MDV DNA or DNA from MDV-infected cells into mammalian cell lines using procedures that are known to those skilled in the art. as coprecipitation of calcium phosphate »dieti laminoeti Idextrano» polybrene »lipofection and electroporation. Preferably, DEF or CEF cells infected with MDV are used to infect the mammalian continuous cell lines of the present invention. The culture of CEF cells »DEF. 1 infoblastoids. Mononuclear cells of peripheral blood »CHCC-0U2 and QT35 infected with MDV is known to those skilled in the art. For example, feline kidney cells can be infected with MDV by developing the cells to a confluence of about 50 to about 8? and adding DEF cells infected with MDV to the cells were iconfluent. The cells can be incubated at a temperature of about 37 to about 38 »5 ()) C for about 48 hours. The means of development can be separated and replaced by new means. The cells can then be incubated for about 48 to 72 hours, preferably for 48 hours, at a temperature of about 37 to 3S »5 ()) C. The medium can then be separated and replaced by medium containing 25% FBS and incubated for at least 24 hours. The cells can then be divided by trypsinization as described above. Infected cells can be monitored by indirect fluorescent antibodies (IFA) using an MVD-specific antibody. The specific MVD antibody can be produced using known monoclonal antibodies. Techniques for the production of monoclonal antibodies and antibody fragments are well known in the art and are described in depth "among other references" in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and in J.W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, London, 1986, which are incorporated herein by reference. Afterwards, a frozen material of cells infected with MDV can be stored until used. The present invention also relates to a method for producing a line of mammalian continuous cells infected or transfected with MDV, comprising: (a) infecting or transfecting continuous mammalian cells with MDV and (b) culturing infected mammalian cells or transfected with MDV »to propagate the cell line. The present invention further relates to a vaccine for protecting birds against Marek's disease virus which comprises a munologically effective amount of MDV produced by the method of the present invention, an immunologically effective amount of the infected or transfected cells of the present invention "or an immunologically effective amount of one or more proteins or polypeptides encoded by said MDV produced by the method of the present invention, and an acceptable veterinary drug vehicle.The present invention also relates to a vaccine in which the MDV comprises an immunologically effective amount of a sequence of a nucleic acid or a protein or polypeptide encoded by said nucleic acid sequence, wherein said nucleic acid or protein or Polypeptide is able to induce a protective response against a disease or pathological disease distinct from Marek's disease "that affects birds. Examples of diseases or pathological conditions other than Marek's disease that affect birds are those caused by Newcastle disease virus (NDV) »infectious bursal disease virus (IBDV)» infectious bronchitis virus (IBV) » chicken anemia virus (CAV) »infectious laryngotracheitis virus (ILTV). avian leukosis virus (ALV). Reticuloendotel osis virus (REV) and avian influenza virus (AIV). The MDV used in the vaccines of the present invention may be any of the forms of MDV mentioned above. For example, "MDV can be a recombinant molecule comprising the MDV nucleic acid sequence in which one or more genes" regulatory genetic elements, or a fragment (s) thereof, have been deleted. The MDV may also include at least one heterologous gene or a fragment (s) thereof inserted into the MDV nucleic acid sequence. For example, the MDV may be one in which the deleted gene is the gH gene or a fragment (s) thereof.
As used herein, the term "immunologically effective amount" refers to the amount of antigen, eg, protein, polypeptide, polynucleotide molecule or modified cells, capable of inducing a protective response against Marek's disease when administered to a member of an avian species, such as chickens »after a single administration or after several administrations. The phrase "capable of inducing a protective response" is used herein in general "to include the induction or intensification of any immune response in the animal in response to vaccination, including an antibody or a cell-mediated immune response or both, which it serves to protect the vaccinated animal against Marek's disease. The terms "protective response" and "protect", as used herein, refer not only to the absolute prevention of Marek's disease or to the absolute prevention of infection with a Marek's disease virus, but also to any reduction detectable of the degree or rate of infection by said virus, or any detectable reduction in the severity of the disease or of any symptom or disease resulting from infection by the virus in the vaccinated animal compared to an unvaccinated infected animal of the same species. The compositions of the vaccines of the present invention can be formulated following accepted conventions and include veterinary vehicles which are acceptable, such as stabilizing buffers, preservatives and / or conventional solvents, and can also be formulated to facilitate a release. sustained The diluents include water, saline, dextrose, ethanol, glycerol and similar diluents. Isotonicity additives include, among others, sodium chloride, dextrose, mannitol, sorbitol and lactose. Other suitable vehicles and additives of the vaccines, including those that are particularly useful for formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th edition, Mack Publishing, 1990, which is incorporated herein by reference. The vaccine of the present invention may further comprise one or more additional munomodulatory components "such as" for example, an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton »MT)» alumina »mineral gels such as aluminum hydroxide gel» oil-in-water emulsions »water-in-oil emulsions such as» for example »complete and incomplete Freund's adjuvants» block-forming copolymers (CytRx Atlanta »GA)» QS-21 (Cambridge Biotech Inc. »Cambridge» MA) »SAF-M (Chiron» Emeryville »CA)» adjuvant AMPHIGEN1- »saponin. Quil A or another fraction of saponin. monophosphorus 1-1 ipido A and adjuvant Avridine lipidamine. Specific non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1/2 ormulations. The modified SEAM62 formulation is an oil-in-water emulsion containing 5% (v / v) of squalene (Sigma) »l% (v / v) detergent SPAN1-85 (ICI Surfactans). 0.7? 4 (v / v) of detergent TWEEN1-80 (ICI Surfactans), 2.5"(v / v) of ethanol» 200 (?) G / ml of Quil A, 100 (?) G / ml of cholesterol and 0.5% (v / v) of lecithin. The modified SEAM 1/2 formulation is an oil-in-water emulsion comprising 5% (v / v) of squalene »15i (v / v) detergent SPA ^ B5» 0 »7W (v / v) TWEENr detergent" SO, 2.5? (V / v) of ethanol, 100 (?) G / ml of Quil A and 50 (?) G / ml of cholesterol Other immunomodulatory agents that can be included in the vaccine include »for example» one or more terleucins »interferons or other known cytokines The invention is further directed to a method for producing a vaccine to protect birds against the Marek's disease virus which comprises combining an immunologically effective amount of MDV produced by the methods of the present invention, an immunologically effective amount of the infected or transfected cells of the present invention, or an immunologically effective amount of one or more proteins or polypeptides encoded by said MDV produced by the method according to claim 1 , and a vehicle veterinary entity acceptable.
The method for producing vaccines of the present invention comprises culturing the infected or transfected mammalian continuous cell lines of the present invention that are capable of producing proteins or polypeptides useful as vaccines against MDV and / or other disease-causing agents, such as other causative agents of bird diseases, and recovering said proteins and polypeptides to incorporate them into vaccines. MDV-infected mammalian continuous cells can be used as vaccines against MDV or, alternatively, MDVs free of cells »isolated from cells» can be used as vaccines. The method for producing the vaccines of the present invention may include infecting or transfecting lines of continuous mammalian cells, such as lines of feline kidney cells, with an MDV vector comprising the nucleic acid sequence of the disease virus. de Marek or a fragment (s) thereof, and further comprises one or more heterologous genes from other disease-causing pathogens, so that the infected or transfected MDV vector is capable of expressing the proteins or polypeptides encoded by said genes heterologous in addition to expressing one or more of the proteins or polypeptides encoded by the Marek's disease virus or a fragment (s) thereof. The expressed proteins and polypeptides can then be used as vaccines against the Marek's disease virus and other disease-causing agents. For example, vaccines can be produced by infecting or transfecting mammalian continuous cell lines with an MDV vector comprising MDV that has no replication capability, and genes or parts thereof of one or more heterologous proteins or polypeptides, which are agents against agents other than MDV that cause diseases, as agents that cause bird diseases. The present invention further relates to a method for vaccinating a bird against the Marek's disease virus, which comprises administering to the bird a munologically effective amount of MDV produced by the method of the present invention, or an immunologically effective amount of the infected or transfected cells of the present invention, and a veten * nari amenté acceptable vehicle. MDV expressing MDV and / or one or more heterologous proteins or heterologous agents of disease-causing agents of birds, such as pathogens, can be used to vaccinate birds, such as chickens and turkeys, susceptible to said disease-causing agents. Vaccination with MDV or with other antigens produced using the methods of the present invention results in a protective immune response whereby the inoculated animals are protected against further infection by these disease-causing agents. The following examples illustrate more but do not limit the present invention.
EXAMPLE 1 PREPARATION OF A LINE OF CONTINUOUS CELLS NOT INFECTED OF FELINE RIFLE
Fourteen lines of cloned cells were derived from NLFK stem cells by endpoint dilution in 96-well tissue culture plates. The cells expanded into wells derived from a single cell. After several transfers »the cells from each clone were frozen and stored at 70 (?) C. All feline kidney cells were propagated in OptiMEM supplemented with 3% fetal bovine serum (FBS) and 22.5 (?) G / ml gentamicin. Monolayers of NLFK cells were maintained in OptiMEM with the varying concentrations of FBS described in the following examples.
EXAMPLE 2 INFECTION OF MONOCAPS AND CULTIVATION NEWLY CELLED SEEDS
NLFK
Cultures of DEF cells or non-cloned NLFK cells were inoculated with the Md5 strain of MDV serotype 1. The preparation and propagation of DEF cells was performed as described by J.J. Solomon, Tissue Culture Association, vol. 1, p. 7-11 (1975), which is incorporated herein by reference. The embryonated duck eggs were obtained from Cornell University.
The virus was detected with an indirect fluorescent antibody (IFA) assay using MDV-specific monoclonal antibodies CR.F. Silva and L.F. Lee, Virol., Vol. 36 »p. 307-320 (1984) 3 to gl coprotein B (1AN86) or to phosphoprotein 38 (H19). These antibodies were provided by Dr. Lucy Lee of the Laboratory of Oncology and Avian Diseases of the United States Department of Agriculture (East Lansing, MI). Briefly, the cells to be examined were fixed with 80 JÍ acetone, incubated with one or more monoclonal antibodies, washed and incubated with mouse anti-FITC conjugate (Kirkegaard &Perry Laboratories). The stained cell preparations were examined with an ultraviolet light microscope and the positive cells were indicated by the fluorescence characteristic of FITC. For a positive control, one MDV seeding consisted of infected DEF cells in which approximately half of the cells expressed MDV antigen when stained with IFA. Seeding was diluted to approximately 100 IFA positive cells estimated per well. Duplicate wells were stained with IFA periodically to detect the presence of MDV infection. Not all wells containing NLFK cells demonstrated the presence of replicating MDV. Only 3 of a total of 48 wells demonstrated by IFA the development of MDV. Dilution of inoculum from MDV-infected DEF cells used in this experiment resulted in single cells uniformly distributed at the bottom of a 2-4-well plate containing no pre-existing cell monolayer. Therefore »the presence of fluorescent patches of several cells indicated replication of the virus in the feline cells. A study was conducted to determine if MDV replicated better in newly seeded cells or in cells in a confluent layer. The cultures of the monolayers had been planted 4 days before the infection »while the newly plated cells had been planted 4 hours before the infection. The inoculum of DEF cells infected with MDV contained approximately 3,000 positive IFA cells per well in a 6-well plate. As a control, only inoculum was added to several wells. The presence of MDV antigen in cells was periodically monitored by staining with IFA. Five days after the inoculation »12 wells were trypsinized and used to seed a 75 cm2 flask and more 24-well plates. Five days later, the 75 cm2 flasks and half of the wells of the 24-well plates were realigned with fresh medium. The presence of MDV antigen in the 24-well plates was monitored by staining with IFA 4 days and 12 days after the change of medium (7 days after transfer). These cells were monitored by staining with IFA five days later. There was little difference in the ability of MDV to replicate in monolayers of NLFK cells or in newly seeded cells. In addition, this experiment demonstrated that, in the absence of NLFK cells, infection of DEF cells by MDV decreased and was lost in the third transfer. In the presence of NLFK cells, MDV infection is maintained and may be enhanced during the transfer of infected cells. This study supports the suggestion that the replication of MDV in cells NLFK is intensified by a change of the medium during infection. Both the number and the size of the fluorescent bulbs increased when the medium was changed 5 days after the transfer.
EXAMPLE 3 INFECTION OF PE CELLS FELINE KIDNEY CLONDS OF LINE OF
NLFK CELLS
Fifteen clones of feline cells derived from the line of NLFK cells were evaluated for their ability to allow the development of MDV Md5 isolate. The clones were grown in 75 cm2 flasks. To each flask was added one milliliter of a suspension of DEF cells infected with MDV containing approximately 5,000 IFA positive cells. As controls, flasks were also inoculated containing the NLFK stem cell line or no cells were inoculated. After 7 days, the flasks were transferred to other 75 cm2 flasks and 24-well plates. The presence of MDV antigens in the NLFK cells was monitored periodically by IFA staining in the 24-well plates. After 6 days, the flasks were made with new medium. From the results of 3ß
staining with IFA, the four clones that showed the most aggressive MDV replication were chosen to transfer to the next level. Eight clones showed little or no propagation of MDV while the three remaining cell clones gave intermediate results. Two days after the change of medium, these flasks were trypsinized and used to seed two 75 cm2 flasks and other 24-well plates, in which the presence of MDV was periodically monitored by staining with IFA. These results suggest that the reason why non-cloned NLFK cells initially showed only a few positive wells is that a subpopulation of NLFK cells is capable of allowing replication of MDV. Although it appears that several clones are capable of allowing up to a certain extent the development of MDV, four clones demonstrated good replication of MVD under the conditions used in these experiments. These were clones 3G4, 9, 7 and KKCL6. Clones 3G4 and 9 were the best of the four and were selected for more transfers. It should also be noted that the DEF cell inoculum lost the MDV infection in the first trans erenc a. As indicated above, changing the medium during incubation intensified the spread of MDV infection.
EXAMPLE 4 PREPARATION OF FELINE KIDNEY CELL MATERIALS
INFECTED WITH MDV
After several additional transfers, cultures of clones 3G4 and 9 of NLFK cells were obtained in which almost all cells express the gB and pp38 antigens, as determined by IFA. These cultures do not show the typical CPE induced by MDV seen in cultures of infected DEF cells. Frozen cell materials were prepared from transfers 5 and 6 and used as seed material for the in vivo experimentation described below.
EXAMPLE 5 INOCULATION OF CHICKENS WITH MDV VIRUSES AND WITH NLFK CELLS
INFECTED WITH MDV
In vivo experiments were performed using specific chickens free of pathogens (strain RF1-5) obtained from Hy-Vac. The feline cell line used in this work was obtained from Pfizer Animal Heath, Lincoln, as NLFK cells (Norden Laboratories Feline Kidney). Fifteen cell lines cloned from the original NLFK cell line were derived by end-point dilution in 96-well tissue culture plates. Two of the NLFK cell clones, termed clone 3G4 and clone 9, were used in the studies. CHCC-0U2 CH cells. Ogura and T. Fujiwara, Acta Med. Okayama, vol. 41, p. 141-143 (1987) 3 were provided by Dr. Douglas N. Foster of the University of Minnesota. The highly virulent MDV strain CR.L. Witter et al., Avian Res., Vol. 24, p. 210-232 (1980) 3 was purchased from the American Type Culture Collection (ATCC VR-987) and used in this study at the cell culture transfer level 11. Six groups of 10 chicks 3 days old were inoculated intraperitoneally with: (1) a mixture of clone 3G4 and clone 9 of uninfected NLFK cells (control cells), (2) DEF cells infected with Md5 (positive control); (3) 0U2 cells infected with Md5, (4) clone 3G-4 of NLFK cells infected with Md5 and (5) clone 9 of NLFK cells infected with Md5. The remaining group (6) was a negative control not inoculated. The birds were inoculated with a dose between 500 and 2,000 TCID50 of MDV per chick. Virus titration was determined using the TCID50 procedure in monolayers of secondary DEF cells fixed with 80% acetone 6-8 days after infection and examined by IFA as described above. The infected and uninfected cells were developed at 38.5 (?) C and 554 C02 in humidified incubators, in OptiMEM (Life Technologies) supplemented with gentamicin and appropriate amounts of fetal bovine serum (O-5%). S died during the study, the chicks were necropsied and the signs of MDV infection were examined. In the surviving animals, the kidneys, spleen, liver and brain were extracted for histological evaluation. Blood was drawn on day 14 after inoculation to isolate peripheral blood lymphocyte virus (ante-colored coatings) and plated on monolayers of DEF cells. All the chickens injected with cells infected with Md5 (groups 2-5) developed classic signs of very virulent MDV and most of these birds died of Marek's disease. The negative control and cell control groups did not develop signs of Marek's disease during the duration of the experiment (8 weeks of age). The results are shown in table 1.
C07ADR01 Number of surviving chicks (percentages) after the test with MDV-1 (strain Md5), per week after the test
The histopathological evaluation of the tissues (kidney, spleen, liver, heart and brain) revealed lesions characteristic of Marek's disease in surviving birds of the infected groups. No such lesions were seen in birds of the control group or the negative control group. To reisolate virus, between 0.5 and 2 ml of blood was collected in heparinized tubes by puncture in the chick-wing vein on the 14th day after inoculation. The blood from each experimental group was collected and centrifuged. The ante-colored coating cells were removed from each tube and used to inoculate monolayers of DEF cells. On day 4 after inoculation the cells were fixed with 80% acetone and prepared for the IFA assay. The cell control and negative control groups were MDV negative in the IFA assay. The positive control groups, 0U2, clone 3G4 of NLFK cells and clone 9 of NLFK cells were MDV positive in the IFA assay. EXAMPLE ß
Two additional MDV-1 isolates, designated 652 and 5S4A, were adapted to be developed in clone 3G4 of NLFK cells. These isolates are more recent and more virulent than the isolate of Md5 CR. L. Witter, Avian Dis., Vol. 41, p. 149-163 (1997) 3. The procedures used to adapt these isolates to their development in 3G4 cells are essentially identical to those described in example 3 for adapting the Md5 isolate to its development in various clones of NLFK cells.
EXAMPLE 7 PREPARATION OF LINE OF NLFK CELLS EXPRESSING THE GENE TH OF THE
CLON 3G4 OF MDV PREPARATION OF CONSTRUCTION PCR3.1GH
The 2.6 kb glycoprotein H (gH) gene was isolated from the Md5 strain of Marek's disease virus by polymerase chain reaction (PCR) and cloned into pCR2 TA cloning vector (available from Invitrogen Inc. ., USA, catalog number K2000-1) by polymerase chain reaction (PCR) using a commercially available PCR reagent system (GIBCO-Life Technologies, catalog number 10198-018), using 5'-GGGGG TACCA AGTTG CATTG GATG6 CTACA TA-3 'as upper PCR primer and 5'-GGGGC TAGCT TAAAG ATCGT CGTAC AGGCT CAA-3' co or lower primer of the PCR. The gH gene can be sequenced using known techniques. The sequence of the gH gene is described by Scott et al., J. Gen. Virol., Vol. 74, p. 1185-1190 (1993). The MDV gH gene was then subcloned into the PCR3.1 vector (available from Invitrogen Inc., USA »catalog number K30000-01) using the Kpnl and Xhol sites of the multiple cloning site (see Figure 1). The pCR3.1 vector is a eukaryotic expression vector containing the immediate early promoter of ci tomegalovi rus (CMV) for efficient expression in mammals, and the neomycin resistance gene to allow positive selection of cells containing the vector . The pCR3.1 vector is characterized by the following: bases of the CMV promoter 1-596; putative transcriptional start bases 520-625; bases of the T7 promoter / priming site 63B-657 »bases of multiple cloning site 670-785; bases of the reverse priming site of pCR3.1 797-815; bases of the polyadenylation site BGH 796-IOIO; bases of origin CoIEl I.IOO- 1.773; promoter bases of SV40 and of origin 3,178-3,515; bases of the neomycin / kanamycin resistance gene (ORF) 2.355-3.143; bases of the polyadenylation site of thymidine kinase 1910-2.18 ?; bases of the ampicillin resistance gene (ORF) 3.595-4.455; and bases of origin fl 4,586-5,042. The construction was confirmed by sequence analysis and restriction analysis using known techniques. Using an in vitro transcription / translation system (available from Pro ega, USA, Sistema TnT T7, catalog number L1170), the construct was able to express a protein of the expected size of approximately 90 kDa. Generation of stable MDV gH transfectants from NLFK 3G4 cells The pCR3.1gH construct was introduced into NLFK 3G4 cells by lipofection using liposome reagent available from Qiagen, USA, Superfect catalog number 301305). Stable transfectant groups were generated using 300 μg / ml G418 selection medium (available from Gibco BRL, USA, catalog number 10131-019) (available from Opti em I Gibco BRL, USA, catalog number 11058-021) and 1% fetal bovine serum (available from Gibco BRL, USA »catalog number 16000-036). Transfected cells express the neomycin resistance gene and will not be affected by G41B which is toxic to NLFK 3G4 cells. Total RNA was isolated from groups of G41B-resistant cells using the Biotex Ultraspec RNA isolation system, catalog number BL-10050. The total RNA (1 μg per sample) of the groups was tested and found positive by the reverse transmerase polymerase chain reaction (RT-PCR) (using the SuperScript amplification system available from Gibco BRL, USA, catalog number 1S089-011, the PCR reagent system available from Gibco BRL »USA, catalog number 10198-018), indicating that mRNA for the MDV gH gene was beginning to be expressed. Individual cell clones were obtained by mitral dilution. After the mean selection expansion G418 (described above), the total RNA was isolated from the cloned cells and assayed as above by RT-PCR (see Figure 3). Frozen cell materials were prepared from the positive RT-PCR cloned cells. According to FIG. 2, a microgram of total RNA was assayed by RT-PCT. Samples were tested in similar pairs with (+ RT) and without (-RT) addition of reverse transcriptase. A negative control containing all the reagents was included but did not include an RNA template to confirm that the reagents were not compromised. In four of the clones shown (1C, 1E, 3A and 3C) the 865 bp gH fragment was amplified in lane + RT. In each sample »the -RT lane is negative» which indicates that the positive signal is from MDV gH mRNA »and not contaminating DNA.
EXAMPLE 8 RECOMBINANT MDV VECTOR PRODUCTION
A construction of a plasmid having a detectable label is prepared. The marker gene is surrounded by the 5T and 3 * flanking regions of the gH gene, which are used for homologous recombination. The 5T flanking region of the gH gene of MDV-1 was cloned by PCR (upper PCR primer: 5T-GGGAT GCATT ACGGT CTCTG AACAA G-3 ', lower PCR primer: 5'-GGGAT GCATA AGGAC GCCA ATTAC TATCT- 3") and placed in the Nsil site treated at the blunt end (described below) of the pGreenLantern-2 plasmid, available from G BCO-Life Technologies, USA (see Figure 2). The 5 'flanking region of the gH gene from MDV-1, available from Gene Bank, USA, contains the TK gene at position 894 to 1925. The plasmid pGreenLantern-2 has the gene encoding the green fluorescence protein of jellyfish (gfp) which is used as a marker protein in The method of generating a recombinant virus having deleted the gH gene The Nsil site of the plasmid pGreenLantern-2, upstream of the CMV promoter of the green luorescence protein gene, is treated with polymerase T4 AG
to create a blunt end for ligation with the 5 'flanking region of the gH gene containing P and 1 ends. A plasmid was obtained from Dr. Meihan Nonoya a, of the Tampa Bay Research Institute »FL, USA, which contained the fragment "F" of Ba Hl of MDV-1"which includes the 3T flanking region of the gH gene CFukuchi et al." J. Virol., 102-109 (1984) 3. The DNA sequence of the 2.4 kb region (referred to herein as the 3 r flanking region) is shown in SEQ ID Ns 2. The 3"flanking region of the MDV-1 gH gene was obtained by PCR cloning (upper primer: 5). '-AAGAT TTTTC CCAAG TCC-3', lower primer: 5 * -TCGTC GAATA ATGTG ATC-3 *) and cloned into the aforementioned pGreenLantern-2 plasmid having the 5 * flanking region of the MDV-1 inserted into the site Nsil The 3 'flanking region was inserted into the Nael site, downstream of the entire fluorescent-green protein, this plasmid having the green fluorescence protein gene flanked by the 5r and 3"flanking regions of the gene gH of MDV-1 is transfected into the line of gH-producing 3G4 cells infected with MDV-1. Green virus plates are selected under the ultraviolet light microscope and purified from parental MDV-l viruses. The resulting new recombinant virus has deleted the gH gene and replaced by the gene encoding the green fluorescence protein. In a second recombination step to separate the green fluorescence protein gene from the virus having the gH gene deleted, heterologous genes from viruses other than MDV, such as, for example, the chicken interferon gene, can optionally be inserted. , Newcastle disease virus or infectious bursal disease virus, etc., which can replace the green fluorescence protein gene. The presence of white plaques of virus in contrast to green parental plaques contain the new recombinant MDV-l virus in which the gH gene has been deleted and the green fluorescence protein gene has been separated.
LIST OF THE SEQUENCES
(1) GENERAL INFORMATION: (i) APPLICANT: Pfizer Inc. (ii) TITLE OF THE INVENTION: PROCEDURE FOR THE
PREPARATION OF MAREK DISEASE VIRUSES USING CONTINUOUS MAMMALIAN CELL LINES (iii) SEQUENCE NUMBER: 2 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Pfizer Inc. (B) STREET: 235 East 42nd Street (C) ) CITY: New York (D) State: New York (D) COUNTRY: United States of America (F) POSTAL CODE: 10017 (V) LEGIBLE FORM OF THE COMPUTER: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Relay 81.0, til.25 version
(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 60 / 049,055 (B) SUBMISSION DATE: June 10, 1997 (OR CLASSIFICATION: (ii) ATTORNEY / AGENT INFORMATION: (A) NAME : Lorraine B. Ling (B) REGISTRATION NUMBER: 35.251 (C) REFERENCE NUMBER / FILE: PC9888
(2) INFORMATION OF SEQ ID * 2 l: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 2,023 base pairs (B) TYPE: nucleic acid (OR STRING: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: no (iv) ANTICIPATION: no (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID ^ i:
ACGGTCTCTG AACAAGACGG GCGATAATAT TACCCATOTT TCGCATAGCC ßTACCTCCCG 60
TTCTCTCCTG ATTATTTGAA AATGATAAAG TAGCCGTTTT ATTACAAGCT ATAT6ATTCC 120
TACAATCCGT TACGTTAGCA G? CGCC? TJC CACTGCCTCG TTGr? TATGT ATCCTGTTTG 180
T? TTATGACG XTGTAAAATT TTAGGAGTGT CAGTTATCCG TGCTTTATAG TCASACGCGG 2K0
TCCCCAATAT AGAGCATAGT CTATGAAAAT CAOtCACT? T GT? ßCCTTTTC TTTAGGCACA 300
TCACAGGTAG AACAGACAGT TTGCCTCTTG CTACAAAT? C TAACATTCGA CAAATAACGA SCO
TACAATCTGA TCCTTGAGGC GCAATTTGCC CA? TCAGAGA T? TGGAA'rcC AATAACTGCT 420
TTATCCCGQT GAGTCTTTGT TCArGTTTAC TGCGTGTCTT C? GGTTACGA GAAAATTTGC 48Q
AAGTTTTTAß TTCTACAATG ACGCATACTC CATCACAG C TACTT CCAC AAATCACGAG 540
GCAACTTAAA CATGCAAATA C? ATCC8TC tACGTCGT C TAGGTTTACT TCGAAGACC? 600
ATCGAAA? TC CGTCAACTGT TTAAATACAT CTAATACCAT GACCTTOCC * A? AAT? TTGG Í & O
CAAAßCTTCT CCCCGGCCAA TCATACACCT GAGATCCXAS ACACATCGCT TCTGCATAAA 720
GCCCTTTGTA A? AGCGATCG TGACATCG ?? CACC? CCCGC T ?? ACGTCGC TTTCTAAGG? 7S0
CAITCGTATT TACATßCCGT TTaAAATTTC GAGTCCTACT AACCTCTCTG OGArATCTTT 840
TGAGTACGTT CTTCTCTCCC ATTG? ACATG ípCGGAGCCAC AATOGT € CTC GGTAATGGCA 900
TCTCAGATGA CATCTßCACA GCTCATACGT GTATACCTCG ATGGATCAAT GßGTATAGGT 960
AAAAOGTCAA TGTTGAATGA GATACCGAC6 CACTCTTTAA TGGGAST ?? C CßT? TAAAG 1020
CTTTTCGAAC CTATG ?? AT? CTGGCGGTAT TATTTTACTG ATTTGGTCAC GACCßTAAAT 1080
GATACATGTG ATCGTCGTCG CAGGGGAGAG TTTTCT TAT TTCAATCTAG CATGATTGTA 11Í0
ACACCTTTAC AATCAAAGTT TCCAGATCCC TATCTTGTAT TTCATGAGCG CTT? TCGTCG 1200
AAGTGTCATC GCATAACAGG «ACACOTCGC AATCCAT8C TTATATTAAT TCTAGATCGA 1260
CATCCC &TAT CCGCTACCGT ATGTTTTCCC ATTGCTCGAC ATTT? ACtGG AGATTßTTCC 1320
TTGGAGATßC TAATTAGTAT GATAATAAGG TTCCCCCAßG AACCGCCAGG ATGCAACTTG US
GTGATTGTCG ATCTACATSA CGA? AAGGAG CATSWAGCC GTCTATCTrC ACGGAATAGG H-1
ACCGGCGAGA AAACAGATCT ACTAATGCTC AGGGCACrt? ATGCAGTGTA T? CCTGnTA 1500
OTAGACACTA TTATGT? CGC AA? TCATATT TGTCCCTACA GTAAGGATGA ATGGGAATCT 1560
GAATGGTTGC ATCTACCATG sTTTGATACA TCTTTGGCCA CAACGTTTAT AAACG? ACCT 1620
CGTACTGATT ATCGCGGTAG TAGGGtGTC? TTACACCATA CGCTTTTAGC GATATTT? AG 16B0 CGGCGACAAT T? TSTGCCGA AGATCGTAGC TTATCAACAA COCATGCATG GAT? TTGTCß 1.740
GGATtATTAA TGAAACTGCS GAACATTAAC GTCßAACGAT TTAATATTAC TGGCCTGTCC lßOO
ACAAGAAAGT GTGTAflAATC GTTCATGSAT hCSKT & tCGß AßAflATTGG-T AACACATAGT IB 60
? .GCTGGA? TG ATGCCTTCGA GATTGA? GCT GATGTACT? G CCTATAAIAA? GAGATOGCT 1 «0
ATGTAAAACT ACCCATTCAT ATCGCGCTTC TATAATTAGC TTGCCCACAT CACAATGA G 1980
CGG AATATT GACTTAT? T * AAGATAOTAA TTTGCCGTCC TTA 2023
(2) INFORMATION OF THE SEQ ID ^ 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2,236 base pairs (B) TYPE: nucleic acid (OR STRING: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: no (iv) ANTI-SENSE: no (i) DESCRIPTION OF SEQUENCE: SEQ ID N <2 2:
TAAATA ?? GA? CTTTGGGAA TAACAAG TA TGTATAGAAT TTATTTCGCG TGAAGATTTT 60
TCCCAAGTCC GATCACATTT CAGGTATTAC AGCGGTAATA GATCCATGCA TTATCAGGGT 120
TTGACGTATT ATCTCGATTA AGAACATATT GTAATACACC CACTGTTTCT CAAACGAGTO 1 * 0
TCTATCAATG ATATAATACA TTGATGTATC GACT? TAATA CCCCCAATGT TCAAAGGCTG 240
ATAA ?? TG? TATATCTATC CCCCCGCATA GCAATTCIGC GTATCn? CT CCCACCGATT 300
CTCCT? ACGC CACGTCTATG GGATCAATGT CITTATATAG ACCGTCTAGA ATAAGAGCCA 360
GGTTACGTAT CT? SASCTCC TGTATAGATT TTGGTGCAGA TCGTTCGGCC ACATCCAATA -I2Q
AAGTAGTCTÜ GTCTG AAAG CTGATGGAC TAAGAACTCC ATGTTGCTCT TCCAATGAAG 4 SO
AAGTCCAGTT CACAACTAAT TTCAGTAACC ATGCCAAGAA ATAAAATCCT CG AAT ?? AC 5 «U
TGTTTTTTTTC TGCAAGACA? GTCGGCAT? C AGTAGS ATT CCCCCTCA? T GGTAGAGGT? 600
TGAT? ATCGC ACAACTCGCG AATTAAGTCA TAACAATTTG GCAGACGATT TAATATATGT 660
ATATACTGAA GCAAC ?? A ?? CTTCTGACTG GGCGATATAT T? TTGTT? C TGGTCCAAI-T 720
CCAACAAACA TGGATGCGTG TCTTCCAAAT AAAGCATTTG AAATCATCCC CAACTCACTT 760
TGTATAATTT CCAGGTCGGA TTGAGATCCA TTCTCCG? AT AAGATTAG ?? TTTAAATTGA S40
GCATGTTCAT ATTAAAAACC GAGTC? ACTT TCC? GAAG? T TTCCCATAAC TTATTTAGAG 900
A? GTAG? GGG TATACAAGAG CTGGTATCGC AACTCGATAT CT? AAATACG GGTGGTATAT 960
ATTTOATTTG TACCAAACAA CTGAACCC »G CA GTTTTTT CC6TTGTACT GGATTTGTTT 1020
GTTACTGTTC ACGTTCAATT TACCCCGCCT CCAGCCGTCA TATCCCATGC GCGTTGCACA 1080
GTCGTCGTGT TTGCAGCTTT CTTTGCTGTA ACTATAACAT CGACTCGCCT GCCGAATATC 1140
TCTGATCATA ATGCTTCTCT AGGAGTGGGA ATGCCAT AA ATAATCCTTC AACGAGGTC? 1200
CTCAA? GACT TAGGTAATTC AGTCAATC? T GCACAACTTA GCACAAATGC ATCACC? CTG 1260
CACTCA TA CTA ?? T TOA ATATATGTCC GTTATTATAG GGAATTCGCG TATATGAATT 1J20
GTAOGATCAT GTGGAAAATC GTATGCGGCC TGTATCGTTA ACCCAGAAAT TGCATTTGTC 13 «0
GGTACCATAT ACTTTGCTAT ATCCGGATCA TACGTTTCCA GACAGAGAAG CCCACAAAGC 1440
TCACGTTCAC TGCATATACC ATCACGACTT AAC CAGCTA TACTAT GAT GAACAATTCA 1SOD
TTGTCA? CC? AGA ???? GC CCACTTCATA CC CTGCGAA GTAATTCT G GCGAACATGA ISSD
GCGGCCAATG GTTTGGACTG ACCACCACGT AGAACCAACC CAATT? TGC GAGCTCTGGT 1620
AAIACCATCA TCTATACAGC CTGCCTACAG CA? AA ?? C ?? CCGCCGCAA? AAAATACCTT 1680
TATATCCCAT TCCGATACAT AAA? CTG &? C ATTCTATAAC G ??? ACATG CCGTATTTAA 1740
TATOCATTGA CTGTCCTCTC TCGACGTAAC CTATATCACT GTAGCGCAAA TCCAATCCTT 1800
•: 6 • IAACAGCA TTCCGTTAAT C? CTGGGTGC ACGGATTAAC GTGTACGTAT TTACTGTCGC 1BEO
GTCATATGA? CGACAATGAG CTTGGGTATG CAGCTCGTCA TTCA? CGCCA TTTGTGGCAA 1320
AGCAATAAGG GTCTCAGACC ATCACATTAT TCGACGAAT GTACTACAT? GCCCACCCCT 1980
TGTTTAACTA TGTCAAGCAT GGATTTGGAT ACTATGTCAA CAGAAGCTAA TG ?? TATACC 2040
ATCCCCCTC? TGAATTGATG ATGGACGATC GGATACATGC GAAAACT TT GGGTCGTAT 2100
GACCACTATC TGAGGAATTA GATTGGGATG ATATTATGCA CpTCTCTTA TTTAGGCGAT 216D
ATATTTTACA ATCCAACAGC TATGACATAC ATCCTC ??? T CACCCGTATG TTTACTCTTT 2220
GGCTATCTAC TTTGTC 2236
Claims (45)
1. - A method for preparing MDV »comprising: (a) infecting or transfecting with MDV a line of mammalian continuous cells» (b) culturing the continuous mammalian cell line infected or transfected with MDV and (c) recover MDV from continuous cell culture.
2.- The procedure according to the claim 1 »wherein the MDV is a recombinant molecule comprising the MDV nucleic acid sequence» or a fragment (s) thereof.
3. The method according to claim 1, wherein the MDV is a recombinant molecule comprising the nucleic acid sequence of the MVD "and at least one heterologous gene or a fragment (s) thereof inserted in said MDV nucleic acid sequence.
4. The method according to claim 1, wherein the MDV is a recombinant molecule comprising the nucleic acid sequence of MDV in which one or more genes »regulatory genetic elements» or a fragment (s) have been deleted. ) thereof.
5. The method according to claim 4 »wherein the deleted gene is essential for replication.
6. The method according to claim 5 wherein the deleted gene is the gH gene or a fragment (s) thereof.
7. The method according to claim 1, wherein the MDV is a molecule comprising a sequence of a nucleic acid selected from the group consisting of serotype I, serotype 2 and serotype 3 of the disease virus. Marek "or a fragment (s) thereof" taken individually or in any combination thereof.
8. The method according to claim 1, wherein the MDV is a molecule comprising the nucleic acid sequence of the MareK disease virus used for the preparation of a vaccine capable of inducing protection against disease in birds. .
9. The method according to claim 1, wherein the cell line is a line of feline kidney cells.
10. The procedure according to the rei indication 9 »in which the MDV is the MDV-1 strain Md5.
11. The procedure according to claim 10. wherein the cell line is ATCC CRL-12336.
12. A line of mammalian continuous cells infected or transfected with MDV.
13. The cell line according to claim 12, wherein the MDV is a recombinant molecule comprising the MDV nucleic acid sequence or a fragment (s) thereof.
14. The cell line according to claim 12 »wherein the MDV is a recombinant molecule comprising the MDV nucleic acid sequence or a fragment (s) of the same» and having at least one heterologous gene or a fragment (s) thereof inserted in said MDV nucleic acid sequence.
15. The cell line according to claim 12 »wherein the MDV is a recombinant molecule comprising the nucleic acid sequence of the MDV in which one or more genes »regulatory genetic elements» or a fragment (s) thereof have been deleted.
16. The cell line according to claim 15 »wherein the deleted gene is essential for replication.
17. The cell line according to claim 16 wherein the gene deleted is the gH gene or a fragment (s> thereof
18. The cell line according to claim 12 »in the that MDV is a molecule comprising a nucleic acid sequence selected from the group consisting of serotype 1, serotype 2 and serotype 3 of Marek's disease virus, or a fragment (s) thereof, taken individually or
19. The cell line according to claim 12, wherein the MDV is a molecule comprising the nucleic acid sequence of the Marek's disease virus used for the preparation of a vaccine. able to induce protection against the disease in birds
20. The cell line according to the rei indication 12 »wherein the cell line is a line of feline kidney cells infected or transfected with MDV.
21. The cell line according to the rei indication 20"in which the MDV is the Md5 strain of the MDV-1.
22. The cell line according to claim 21 »wherein the cell line is the ATCC CRL-12336.
23. A method for producing a line of mammalian continuous cells infected or transfected with MDV "comprising: (a) infecting or transfecting mammalian DC cells with continuous MDV and (b) culturing mammalian cells infected or transfected with MDV for propagate the cell line.
24.- A vaccine to protect birds against the Marek's disease virus »comprising an immunologically effective amount of MDV produced by the method according to claim 1, an immunologically effective amount of infected or transfected cells according to the claim 12"or an immunologically effective amount of one or more proteins or polypeptides encoded by said MDV produced by the process according to the indication l" and an acceptable veterinary vehicle.
25. The vaccine according to claim 24 »wherein the MDV comprises a munolically effective amount of a sequence of a nucleic acid or of a protein or polypeptide encoded by said nucleic acid sequence» wherein said sequence of the nucleic acid or protein or polypeptide is capable of inducing a protective response against a disease or pathological condition other than Marek's disease that affects the birds.
26. The vaccine according to claim 26 »wherein the protein or polypeptide capable of inducing a protective response against a disease or pathological condition other than Marek's disease affecting birds is selected from the group consisting of the virus of Newcastle disease (NDV). infectious bursal disease virus (IBDV) »infectious bronchitis virus (IBV)» chicken anemia virus (CAV), infectious laryngotracheitis virus (ILTV). avian leukosis virus (ALV) »reticuloendotel iosis virus (REV) and avian influenza virus (AIV).
27. The vaccine according to claim 25 »26 or 27» wherein the MDV is a recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus or a fragment (s) thereof.
28. The vaccine according to claim 25, 26 or 27, wherein the MDV is a recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus and at least one heterologous gene or fragment (s). of the same inserted in the aforementioned nucleic acid sequence of the Marek's disease virus.
29. The vaccine according to claim 25, 26 or 27 »in which the MDV is a recombinant molecule comprising the nucleic acid sequence of the Marek's disease virus in which one or more genes have been deleted» regulatory genetic elements, or a fragment (s) thereof.
30. The vaccine according to claim 29 »wherein the gene deleted is the gH gene or a fragment (s) thereof.
31.- The vaccine according to claim 25 »26 or 27» wherein the MDV is a molecule comprising a sequence of a nucleic acid selected from the group consisting of serotype 1 »serotype 2 and serotype 3 of the virus of Marek's disease, or a fragment (s) thereof, taken individually or in any combination thereof.
32. The vaccine according to claim 25 »26 or 27, wherein the cell line is a line of feline kidney cells infected or transfected with MDV.
33.- The vaccine according to claim 32 »wherein the MDV is the Md5 strain of MDV-1.
34. The vaccine according to claim 33, wherein the cell line is ATCC CRL-12336. 35.- A recombinant MDV molecule produced by the method according to claim 1. 36.- The recombinant MDV according to claim 35, wherein the recombinant MDV comprises the nucleic acid sequence of the MareK disease virus in which one or more genes have been deleted. 37.- The recombinant MDV according to claim 36 »in which one or more of the deleted genes is an essential gene for replication. 38.- The recombinant MDV according to claim 37, wherein the gene deleted is the gH gene or a fragment (s) thereof. 39.- The recombinant MDV according to the re-indication 35 »in which the cell line is a line of feline kidney cells infected or transfected with MDV. 40.- The recombinant MDV according to the indi cation 39, in which the MDV is the Md5 strain of MDV-1. 41.- The recombinant MDV according to claim 40, wherein the cell line is the ATCC CRL-12336. 42.- A vector of the MDV produced using the method according to claim 1. 43.- The vector of the MDV according to claim 42 »wherein the vector of the MDV has a 3 'flanking region comprising the sequence of the nucleic acid SEQ ID 2 2. 44.- The use of MDV in accordance with requirement 1 »to prepare a vaccine to protect animals against disease. 45.- The use in accordance with the reivinidación 44 »where the animals are birds.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US049055 | 1997-06-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA98004669A true MXPA98004669A (en) | 1999-12-10 |
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