Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-06-23
2001-11-20
Mosher, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S472000, C435S475000, C435S235100, C435S320100, C435S477000, C424S199100, C424S093200
Reexamination Certificate
active
06319716
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel bovine adenovirus (BAV) expression vector systems in which one or both of the early region 1 (E1) and the early region 3 (E3) gene deletions are replaced by a foreign gene and novel recombinant mammalian cell lines stably transformed with BAV E1 sequences, and therefore, expresses E1 gene products, to allow a bovine adenovirus with an E1 gene deletion replaced by a foreign gene to replicate therein. These materials are used in production of recombinant BAV expressing heterologous (antigenic) polypeptides or fragments for the purpose of live recombinant virus or subunit vaccines or for other therapies.
The present invention also relates to novel bovine adenovirus (BAV) expression vector systems comprising BAV genome sequences disclosed herein. The BAV genome sequences can be replaced by one or more foreign genes to generate recombinant BAV expressing heterologous (antigenic) polypeptides or fragments for the purpose of producing live recombinant virus or subunit vaccines or for other therapies. Further, various BAV transcriptional and translational regulatory signals can be used to modulate the expression of foreign genes that have been inserted into the vector systems of the invention. Additionally, the novel sequences of the present invention can be used for diagnostic purposes, to determine the presence of BAV in a subject or biological sample.
BACKGROUND OF THE INVENTION
The adenoviruses cause enteric or respiratory infection in humans as well as in domestic and laboratory animals.
The bovine adenoviruses (BAVs) comprise at least nine serotypes divided into two subgroups. These subgroups have been characterized based on enzyme-linked immunoassays (ELISA), serologic studies with immunofluorescence assays, virus-neutralization tests, immunoelectron microscopy, by their host specificity and clinical syndromes. Subgroup 1 viruses include BAV 1, 2, 3 and 9 and grow relatively well in established bovine cells compared to subgroup 2 which includes BAV 4,5,6,7 and 8.
BAV3 was first isolated in 1965 and is the best characterized of the BAV genotypes, containing a genome of approximately 35 kb (Kurokawa et al (1978)
J. Virol.
28:212-218). BAV3, a representative of subgroup 1 of BAVs (Bartha (1969)
Acta Vet. Acad. Sci. Hung.
19:319-321), is a common pathogen of cattle usually resulting in subclinical infection (Darbyshire et al. (1965).
J. Comp. Pathol.
75:327-330), though occasionally associated with a more serious respiratory tract infection (Darbyshire et al., 1966
Res. Vet Sci
7:81-93; Mattson et al., 1988
J. Vet Res
49:67-69). Like other adenoviruses, BAV3 is a non-enveloped icosahedral particle of 75 nm in diameter (Niiyama et al. (1975)
J. Virol.
16:621-633) containing a linear double-stranded DNA molecule. BAV3 can produce tumors when injected into hamsters (Darbyshire, 1966
Nature
211:102) and viral DNA can efficiently effect morphological transformation of mouse, hamster or rat cells in culture (Tsukamoto and Sugino, 1972
J. Virol.
9:465-473; Motoi et al., 1972
Gann
63:415-418; M. Hitt, personal communication). Cross hybridization was observed between BAV3 and human adenovirus type 2 (HAd2) (Hu et al., 1984
J. Virol.
49:604-608) in most regions of the genome including some regions near but not at the left end of the genome.
In the human adenovirus (HAd) genome there are two important regions: E1 and E3 in which foreign genes can be inserted to generate recombinant adenoviruses (Berkner and Sharp (1984)
Nuc. Acid Res.,
12:1925-1941 and Haj-Ahmad and Graham (1986)
J. Virol.,
57:267-274). E1 proteins are essential for virus replication in tissue culture, however, conditional-helper adenovirus recombinants containing foreign DNA in the E1 region, can be generated in a cell line which constitutively expresses E1 (Graham et al., (1977)
J. Gen. Virol.,
36:59-72). In contrast, E3 gene products of HAd 2 and HAd 5 are not required for in vitro or in vivo infectious virion production, but have an important role in host immune responses to virus infection (Andersson et al (1985)
Cell
43:215-222; Burgert et al (1987)
EMBO J.
6:2019-2026; Carlin et al (1989)
Cell
57:135-144; Ginsberg et al (1989)
PNAS, USA
86:3823-3827; Gooding et al (1988)
Cell
53:341-346; Tollefson et al (1991)
J. Virol.
65:3095-3105; Wold and Gooding (1989)
Mol. Biol. Med.
6:433-452 and Wold and Gooding (1991)
Virology
184:1-8). The E3-19 kiloDalton (kDa) glycoprotein (gp19) of human adenovirus type 2 (HAd2) binds to the heavy chain of a number of class 1 major histocompatibility complex (MHC) antigens in the endoplasmic reticulum thus inhibiting their transport to the plasma membrane (Andersson et al. (1985)
Cell
43:215-222; Burgert and Kvist, (1985)
Cell
41:987-997; Burgert and Kvist, (1987)
EMBO J.
6:2019-2026). The E3-14.7 kDa protein of HAd2 or HAd5 prevents lysis of virus-infected mouse cells by tumor necrosis factor (TNF) (Gooding et al. (1988)
Cell
53:341-346). In addition, the E3-10.4 kDa and E3-14.5 kDa proteins form a complex to induce endosomal-mediated internalization and degradation of the epidermal growth factor receptor (EGF-R) in virus-infected cells (Carlin et al.
Cell
57:135-144; Tollefson et al. (1991)
J. Virol.
65:3095-3105). The helper-independent recombinant adenoviruses having foreign genes in the E3 region replicate and express very well in every permissive cell line (Chanda et al (1990)
Virology
175:535-547; Dewar et al (1989)
J. Virol.
63:129-136; Johnson et al (1988)
Virology
164:1-14; Lubeck et al (1989)
PNAS, USA
86:6763-6767; McDermott et al (1989)
Virology
169:244-247; Mittal et al (1993)
Virus Res.
28:67-90; Morin et al (1987)
PNAS, USA
84:4626-4630; Prevec et al (1990)
J. Inf. Dis.
161:27-30; Prevec et al (1989)
J. Gen. Virol.
70:429-434; Schneider et al (1989)
J. Gen. Virol.
70:417-427 and Yuasa et al (1991)
J. Gen. Virol.
72:1927-1934). Based on the above studies and the suggestion that adenoviruses can package approximately 105% of the wild-type (wt) adenovirus genome (Bett et al (1993)
J. Virol.
67:5911-5921 and Ghosh-Choudhury et al (1987)
EMBO. J.
6:1733-1739), an insertion of up to 1.8 kb foreign DNA can be packaged into adenovirus particles for use as an expression vector for foreign proteins without any compensating deletion.
The E1A gene products of the group C human adenoviruses have been very extensively studied and shown to mediate transactivation of both viral and cellular genes (Berk et al., 1979
Cell
17:935-944; Jones and Shenk, 1979
Cell
16:683-689; Nevins, 1981
Cell
26:213-220; Nevins, 1982
Cell
29:913-919; reviewed in Berk, 1986
Ann. Res. Genet
20:45-79), to effect transformation of cells in culture (reviewed in Graham, F. L. (1984) “Transformation by and oncogenicity of human adenoviruses. In: The Adenoviruses.” H. S. Ginsberg, Editor. Plenum Press, New York; Branton et al., 1985
Biochim. Biophys. Acta
780:67-94) and induce cell DNA synthesis and mitosis (Zerler et al., 1987
Mol. Cell Biol.
7:821-929; Bellet et al., 1989
J. Virol.
63:303-310; Howe et al., 1990
PNAS, USA
87:5883-5887; Howe and Bayley, 1992
Virology
186:15-24). The E1A transcription unit comprises two coding sequences separated by an intron region which is deleted from all processed E1A transcripts. In the two largest mRNA species produced from the E1A transcription unit, the first coding region is further subdivided into exon 1, a sequence found in both the 12s and 13s mRNA species, and the unique region, which is found only in the 13s mRNA species. By comparisons between E1A proteins of human and simian adenoviruses three regions of somewhat conserved protein sequence (CR) have been defined (Kimelman et al., 1985
J. Virol.
53:399-409). CR1 and CR2 are encoded in exon 1, while CR3 is encoded in the unique sequence and a small portion of exon 2. Binding sites for a number of cellular proteins including the retinoblastoma protein Rb, cyclin A and an associated protein kinase p33
cdk2
, and other, as yet unassigned, proteins have been defined in ex
Babiuk Lorne A.
Baxi Mohit
Reddy Police Seshidhar
Tikoo Suresh Kumar
Zakhartchouk Alexandre
Morrison & Foerster / LLP
Mosher Mary E.
University of Saskatchewan
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