| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 10.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 12.10.99 |
| PATENT TITLE |
Chicken infectious anemia virus vaccine |
| PATENT ABSTRACT |
Novel sequences in the genome of a wild type isolate of chicken infectious anemia virus are described, The amino acid sequence of a polypeptide, VP1, encoded by a novel sequence is also disclosed. Additionally, disclosed are the unexpected properties of the isolate which are related to novel amino acids positioned in the amino acid sequence of this isolate's VP1, as compared to the sequence of VP1 found in cell culture-adapted strains; and use of the novel sequences and their respective polypeptides in strategies to control chicken infectious anemia such as by vaccination. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 16.04.98 |
| PATENT REFERENCES CITED |
Claessens et al., "Molecular Cloning and sequence analysis of the genome of chicken anaemia agent", Journal of General Virology, 72, 1991, pp. 2003-2006. Noteborn et al., "Characterization of Cloned Chicken Anemia Virus DNA That Contains All Elements for the Infectious Replication Cycle", Journal of General Virology, 72, 1991, pp. 2003-2006. Meehan et al., "Characterization of viral DNAs from cells infected with chicken anaemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments", Archives of Virology, 124 (1992) pp. 301-319. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A vaccine formulation comprising an immunologically effective amount of at least one chicken infectious anemia virus polypeptide selected from the group consisting of SEQ ID NO:1, or SEQ ID NO:1 in combination with SEQ ID NO:2. 2. The vaccine formulation according to claim 1, in which the at least one polypeptide was produced recombinantly from a host cell system genetically engineered to include an expression vector containing a nucleotide sequence selected from the group consisting: (a) ORF-1 as shown in SEQ ID NO:1; (b) ORF-2 as shown in SEQ ID NO:2; (c) a combination of (a), and (b); wherein said host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, insect cell lines, avian cells, and mammalian cell lines. 3. The vaccine formulation according to claim 1, further comprising a pharmaceutical carrier. 4. The vaccine according to claim 1, further comprising a recombinant vector containing a nucleotide sequence, wherein the nucleotide sequence encodes said at least one polypeptide, and is operatively linked to an expression control element; and wherein the vector is selected from the group consisting of a viral vector and a plasmid. 5. The vaccine according to claim 4, wherein the viral vector comprises a live attenuated virus selected from the group consisting of Marek's disease virus, infectious laryngotracheitis virus, chicken fowlpox virus, and herpesvirus of turkey. 6. The vaccine formulation according to claim 4, wherein the vaccine is a combined vaccine and further comprises one or more nucleotide sequences operatively linked to an expression control element and encoding one or more antigens useful against a viral disease of poultry other than chicken infectious anemia, wherein said viral disease is selected from the group consisting of infectious laryngotracheitis, chicken fowlpox, Newcastle disease, and Marek's disease. 7. The vaccine formulation according to claim 3, wherein the vaccine is a combined vaccine and further comprises one or more nucleotide sequences operatively linked to an expression control element and encoding one or more antigens useful against a viral disease of poultry other than chicken infectious anemia, wherein said viral disease is selected from the group consisting of infectious laryngotracheitis, chicken fowlpox, Newcastle disease, and Marek's disease. 8. A method of immunizing poultry against chicken infectious anemia virus comprising administering a prophylactically effective amount of the vaccine formulation according to claim 1. 9. A method of immunizing poultry against chicken infectious anemia virus comprising administering a prophylactically effective amount of the vaccine formulation according to claim 2. 10. A method of immunizing poultry against chicken infectious anemia virus comprising administering a prophylactically effective amount of the vaccine formulation according to claim 3. 11. A method of immunizing poultry against chicken infectious anemia virus comprising administering a prophylactically effective amount of the vaccine formulation according to claim 4. 12. A method of immunizing poultry against chicken infectious anemia virus comprising administering a prophylactically effective amount of the vaccine formulation according to claim 7. -------------------------------------------------------------------------------- Description |
| PATENT DESCRIPTION |
1. BACKGROUND OF THE INVENTION 1.1 Field of the Invention The present invention relates to a particular strain (CIA-1) of chicken infectious anemia virus (CIAV) having a polypeptide comprising a novel amino acid sequence which can be used to vaccinate poultry against CIAV. More particularly, the invention relates to a novel, naturally occurring CIAV isolate which has not been adapted to cell culture; and which, due to the novel amino acid sequence of a major viral polypeptide, exhibits a difference in pathogenic potential and cell tropism as compared to cell culture-adapted strains. 1.2 Description of the Background and Related Art Chicken infectious anemia virus (CIAV) was first isolated in Japan in 1979 during an investigation of a Marek's disease vaccination break (Yuasa et al., 1979, Avian Dis. 23: 366-385). Since that time, CIAV has been detected in commercial poultry in all major poultry producing countries (van Bulow et al., 1991, pp.690-699) in Diseases of Poultry, 9th edition, Iowa State University Press). CIAV can cause clinical diseases, characterized by anemia, hemorrhages and immunosuppressive, in young susceptible chickens. Atrophy of the thymus and of the bone marrow are characteristic and consistent lesions of CIAV-infected chickens. Lymphocyte depletion in the thymus, and occasionally in the bursa of Fabricius, results in immunosuppressive and increased susceptibility to secondary viral, bacterial, or fungal infections which then complicate the course of the disease. The immunosuppressive may cause aggravated disease after infection with one or more of Marek's disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus. It has been reported that pathogenesis of MDV is enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the 38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it has been reported that CIAV aggravates the signs of infectious bursal disease (Rosenberger et al., 1989, Avian Dis. 33: 707-713). Additionally, subclinical CIAV infection in older chickens is correlated with decreased performance in broiler flocks (McNulty et al., 1991, Avian Dis. 35: 26314 268). CIAV is highly resistant to environmental inactivation and some common disinfectants, characteristics that may potentiate disease transmission. The economic impact of CIAV infection on the poultry industry is reflected by mortality of 10% to 30% in disease outbreaks, a possible role in vaccine failures, and lower performance of infected flocks due to subclinical infection. CIAV typically can be propagated in embryonated eggs and lymphoblastoid cell lines, such as the Marek's disease virus-transformed chicken lymphoblastoid cell line MDCC-MSB-1 (MSB-1; Akiyama et al., 1974, Biken J. 17: 105-116), but not in chicken embryo fibroblasts, chicken kidney cells, or other primary chicken cells. However, not all strains can be adapted to cell culture as evidenced by the CIA-1 strain (Lucio et al., 1990, Avian Dis. 34: 14614 153). Thus, biological differences may exist between strains. CIAV is a small, non-enveloped icosahedral virus of 25 nm diameter, and contains a genome consisting of 2.3 kb circular, single-stranded DNA. Two polypeptides have been detected in purified virus preparations; a major polypeptide of about 50 kilodaltons (kDa) termed VP1, and a 24 kDa polypeptide termed VP2. These two polypeptides together form a major epitope for the production of virus-neutralizing antibodies. Genomic DNA sequences of several different isolates of CIAV have been reported. Isolate Cux-1 was sequenced by Noteborn et al. (1991, J. Virol. 65: 3131-3139; herein incorporated by reference) revealing 3 open reading frames (ORFS) that potentially encode polypeptides of 51.6 kDa, 24.0 kDa, and 13.6 kDa. There was only one promoter-enhancer region upstream of the ORFs, and a single polyadenylation signal downstream of the ORFs. A single unspliced mRNA of 2100 bases is transcribed from the Cux-1 genome (Noteborn et al., 1992, Gene 118: 267-271; herein incorporated by reference). Another group also sequenced the Cux-1 strain; however, differences were noted between their sequence data and those from Noteborn et al. (Meehan et al., 1992, Arch. Virol. 124: 301-319; herein incorporated by reference). The nucleotide sequence of strain 26P4, isolated in the U.S.A., also showed a number of nucleotide differences when compared with sequences of Cux-1 (Claessens et al., 1991, J. Gen. Virol. 72: 2003-2006; herein incorporated by reference). Despite the differences in nucleotide sequences found in various isolates from around the world, only minor differences in amino acid sequences have been noted. For these reasons, it has been assumed that CIAV is a highly conserved virus. Exposing hens to CIAV may induce maternal antibody in chickens which may help protect against CIAV infections in their progeny. However, such vaccination with any of the CIAV strains has inherent problems including the potential of vertical (through the egg) transmission, and contamination of the environment. It is therefore desirable to develop a vaccine having as the immunogen a purified polypeptide(s) associated with CIAV. 2. SUMMARY OF THE INVENTION According to the present invention, three open reading frames (ORF-1, ORF-2, ORF-3) , have been identified in the genome of CIAV strain CIA-1 , a non-cell culture-adapted strain. Comparing the 3 ORFs from CIA-1 with other CIAV isolates, particularly the prototypic cell culture-adapted strain Cux-1 (C), reveals that the polypeptide (VP1) encoded by ORF-1 contains at least 4 unique amino acid changes that could potentially affect conformational changes in VP1. Because CIA-1 fails to replicate in the Cornell subline of MSB-1 cells in culture, it is likely that one or more unique amino acid changes in VP1 is responsible for the differences in viral biology affecting cell pathology ("pathogenicity"). Additionally, the inability of CIA-1 to infect certain cultured cell lines ("cell tropism") indicates that one or more unique amino acid changes in VP1 is responsible for such tropism. Accordingly, one object of the present invention is to identify the polypeptide(s) involved in the observed differences in phenotype of this novel isolate. Another object of the present invention is to provide an approach to control CIAV infection by interfering with the establishment of infection, or disease progression, in poultry susceptible to CIAV. Another object of the present invention is to prevent the development of disease caused by CIAV by inducing protective antibody and/or a cell-mediated immune response to one or more polypeptides which are associated with the virus. Another object of the present invention is to induce protective antibody and/or a cell-mediated immune response to a viral polypeptide wherein the polypeptide reflects wild type strains of CIAV more so than that in cell culture-adapted strains. Another object of the present invention is to provide one or more nucleic acid sequences, encoding respective CIAV polypeptide(s), which can be inserted for expression into a recombinant viral vector, or introduced directly into susceptible chickens. 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 organization of ORF-1, ORF-2, ORF-3, the promoter-enhancer region (P-E) and the polyadenylation signal (PAS) in the CIA-1 genome. FIG. 2 is a hydrophilicity plot for the polypeptide encoded by CIA-1 ORF-1. Note three changes: At position A, there are 2 turns in the secondary structure versus 3 for Cux-1. At position B, a hydrophilicity shift in the CIA-1 polypeptide is indicated. At position C, Cux-1 has an additional turn compared to the CIA-1 polypeptide. FIG. 3 is a schematic representation showing construction of various chimeric viral clones using CIA-1 DNA and Cux-1 DNA. FIG. 4 is a graph indicating the percentage of cells, transfected with the respective chimeric viral clone, which are positive for CIAV protein production by immunofluorescence as plotted against the number of days post-transfection. FIG. 5 is a schematic representation showing construction of various chimeric viral clones using CIA-1 DNA and Cux-1; and an adjoining table showing the percentage of MSB-1 (S) cells versus MSB-1 (L) cells infected with the viral clones which were positive for VP3 by immunofluorescence assay. Dashes indicate a negative result in immunofluorescence; n.d. indicates that immunofluorescence assays were not performed. 4. DETAILED DESCRIPTION OF THE INVENTION The CIAV genome is a circular single stranded molecule of about 2,300 nucleotides in length. It consists of three overlapping reading frames (ORFs) which are identified herein as ORF-1, ORF-2, and ORF-3, and potentially encode polypeptides of 51.6 kDa (VP1), 24.0 kDa (VP1), and 13.6 kDa (VP3), respectively. As positioned in the genome, the three ORFs either partially or completely overlap one another and appear to use the same upstream region as a promoter and enhancer, and the same polyadenylation signal located downstream of the ORFs (FIG. 1). Presently, the process by which CIAV causes chicken infectious anemia is poorly understood. When CIA-1 is introduced into susceptible 1-day-old chicks, CIA-1 produced signs and lesions characteristic of chicken infectious anemia including low hematocrit values, depletion of erythrocytes and lymphoid cells in the bone marrow, depletion of lymphoid cells of the medulla and the cortex of the thymus (herein referred as T-cells), and inflammatory changes in the liver, heart and kidney (Lucio et al., 1990, Avian Dis. 34: 146-153). One or more of the polypeptides encoded by the CIAV ORFs may play a role in the pathogenesis of chicken infectious anemia by facilitating invasion into susceptible cells, and/or initiating T-cell apoptosis. The term "unique amino acid change", for purposes of the specification and claims, refers to one or more changes or amino acid substitutions present in the CIA-1 VP1 amino acid sequence, as compared to the amino acid sequence of the prototypic cell culture-adapted strain Cux-1 (C) as well as that of some other cell culture-adapted strains. The term "polypeptide" is used herein, for purposes of the specification and claims, to refer to a chain of amino acids, having a biological function, and does not refer to a specific length of the chain. Thus, polypeptide may be used to refer to peptides, oligopeptides, and proteins containing the amino acid changes unique to CIA-1 as compared to Cux-1, Conn and GA strains of CIAV. Further, if required, the polypeptide can be modified in vivo or in vitro by, for example, glycosylation, amidation, phosphorylation, carboxylation, or substitution without changing the primary biological function. Amino acid substitutions in a polypeptide, particularly resulting in a change in side group polarity and/or charge, can be reflected in the secondary structure of the polypeptide, and thus can alter biological activity of that polypeptide or the virion bearing the polypeptide. This is important, as the deduced amino acid sequence of the CIA-1 polypeptide VP1 of the present invention shows such amino acid substitutions when compared to the VP1 polypeptide of some cell culture-adapted CIAV strains. Also, amino acid substitutions are present in the CIA-1 VP2 and VP3 polypeptides. Functional equivalents of the polypeptides disclosed herein, i.e. polypeptides with the same amino acid sequence, or amino acid replacements between related amino acids (i.e. having similar polarity and/or charge), are within the scope of this invention. Nucleic acid sequences encoding such functional equivalents are included within the scope of this invention: As well known in the art of recombinant DNA technology for the preparation and modification of nucleic acid sequences, degeneracy of the genetic code permits substitution of bases in a codon resulting in another codon which still encodes for the same amino acid. Mutagenesis, insertion, and or synthesis can be used to generate a nucleic acid sequence derivative (functional equivalent) of ORF-1, ORF-2 or ORF-3 which would encode the respective functional equivalent polypeptide. Thus, the information provided in SEQ ID NOs:1-3 allows a person skilled in the art to isolate and identify nucleic acid sequences encoding functional equivalent polypeptides having the biological function and immunologic activity of the polypeptides disclosed in SEQ ID NOs:1-3. Using methods and procedures in accordance with the present invention, and known to those skilled in the art, including virus propagation, nucleic acid sequence amplification, cloning, sequencing, restriction enzyme analysis, and transfection, DNA may be obtained from other CIAV strains to identify ORFs which can encode a polypeptide functionally equivalent to the polypeptides of the present invention disclosed in SEQ ID NOs:1-3. Additionally, fragments of the nucleic acid sequences disclosed in SEQ ID NOs:1-3 (i.e., DNA subsequences) can be used to encode peptides (a subsequence of the disclosed polypeptides and containing one or more of the amino acid changes of CIA-1 ) having one or more immunoreactive/antigenic determinants and/or determinant associated with equivalent biological function of the polypeptides disclosed in SEQ ID NOs:1-3. Thus, peptides that are included within this scope of this invention include those having one or more determinants characteristic of the polypeptides that specifically include one or more of the amino acid changes in the respective polypeptide of CIA-1 as disclosed in the invention (amino acid residue 22, 75, 139, and/or 144 of the polypeptide VP1; hereinafter referred to as "the amino acid changes in CIA-1"), and include the capability of eliciting an immune response in chickens. Any such modifications of the nucleic acid sequences of the present invention, and the polypeptides they encode, will be described in more detail in the embodiments to follow. The present invention particularly relates to the identification and characterization of ORF-1, and its gene product VP1, from CIAV isolate CIA-1. It is noted that CIA-1 is propagated in chicks, and the cells of infected chicks are used to isolate the virus; whereas other CIAV strains, such as Cux-1, GA, and Conn, are able to, and were propagated in cell culture in vitro. The relative positioning of the ORFs is represented in FIG. 1. DNA was isolated and then amplified by polymerase chain reaction to facilitate cloning, sequencing, and expression. From the DNA sequence of CIA-1, three open reading frames (ORF-1, SEQ ID NO:1; ORF-2, SEQ ID NO:2; and ORF-3, SEQ ID NO:3) were identified which can encode polypeptides of about 51 kDa (VP1), 24 kDa (VP2) and 13 kDa (VP3) in molecular size, respectively. Because of the lack of effective drugs to therapeutically treat chickens with chicken infectious anemia, and with problems such as vertical transmission if the virus itself is used as a vaccine, alternative approaches to controlling chicken infectious anemia should be considered. One embodiment of the present invention is to control the development of chicken infectious anemia by inhibiting the development of sequelae in CIAV-infected cells. Therefore, in one variation of this embodiment, one or more of the novel polypeptides of the present invention are used to immunize chickens to induce an immune response which may block infection, and/or initiation of T-cell apoptosis upon infection, by CIAV. In another variation of this embodiment, the DNA encoding one or more of VP1, VP2, or VP3 can be injected directly into the tissue of chicks, or embryos between 17 and 21 days of incubation. In another variation of the embodiment, one or more of the three ORFs can be inserted in a direction, and operatively linked to one or more control elements, in any vector useful as a vaccine by itself or as introduced in a live or attenuated microorganism, wherein the respective ORF is expressed to produce its respective polypeptide. Thus, for example, a recombinant viral vaccine vector can be constructed which may either be inoculated into embryos in ovo or into chicks directly after hatching. In yet another variation of this embodiment, one or more of the three ORFs can be inserted in a plasmid vector. The recombinant plasmid is constructed such that in vivo transcription is regulated by either an inducible promoter or a constitutive promoter. The recombinant plasmid is then injected into embryos in ovo or into chicks directly after hatching. Another variation of this embodiment involves the development of transgenic chickens which express one or more of the three ORFs, wherein the transcription is under the control of an inducible promoter or a constitutive promoter. A second embodiment of the present invention relates to vaccination with one or more of the polypeptides disclosed in SEQ ID NOs:1-3. In one variation of this embodiment, chicks or chickens are immunized with a vaccine comprising one or more of the polypeptides which may elicit protection by inducing an immune response that would recognize either CIAV challenge or cells infected with CIAV. In another variation of this embodiment, one or more peptides derived from the polypeptides disclosed herein (and containing one or more of the amino acid changes as found in CIA-1) are used as the immunogen in a vaccine composition. Alternatively, the vaccine may comprise a vector, such as a recombinant viral vector, containing one or more ORFs under the control of a strong promoter so that the respective polypeptide is expressed. In another variation of this embodiment, the vaccine comprises a vector such as a recombinant viral vector, containing those nucleotide sequence fragments of one or more of the ORFs, wherein the nucleotide sequence fragment codes for epitopes inducing humoral (approximately 15 amino acids) or cell-mediated (approximately 9 amino acids) immune responses which contain one or more amino acid changes in CIA-1. The determination of regions containing epitopes inducing humoral immunity can be based on hydrophilicity and secondary structure analyses (See for example, Hopp et al., 1981, Proc. Natl. Acad. Sci. USA 78: 3824-3828; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148). Likewise, determination of epitopes important in cell-mediated immunity can be derived on the basis of analyses of the amino acid structure including amphilicity (See for example, Berzofsky, 1987, Science 235: 1059-1062). Cleavage of the polypeptides with certain enzymes, such as Cathepsin D, is known to generate NH.sub.2 -terminal fragments/peptides having amino acid residues recognized by the major histo-compatibility complex on the surface of antigen-presenting cells (van Noort et al., 1989, J. Biol. Chem. 264: 14159, 1989). |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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