Mammalian cell lines expressing bovine adenovirus functions

Details Mammalian cell lines expressing bovine adenovirus functions Mammalian cell lines expressing bovine adenovirus functions

1999-11-05

2002-04-30

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S325000, C435S455000

Reexamination Certificate

active

06379944

ABSTRACT:

TECHNICAL FIELD
The present invention relates 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.
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 and contains a genome of approximately 35 kb (Kurokawa et al (1978)
J. Virol.
28:212-218). The locations of hexon (Hu et al (1984)
J. Viol.
49:604-608) and proteinase (Cai et al., (1990)
Nuc. Acids Res.,
18:5568), genes in the BAV3 genome have been identified and sequenced. However, the location and sequences of other genes such as early region 1 (E1) and 3 (E3) in the BAV genome have not been reported.
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.
It is assumed that an indigenous adenovirus vector would be better suited for use as a live. recombinant virus vaccine in different animal species compared to an adenovirus of human origin. Non-human adenovirus-based expression vectors have not been reported so far. If like HAds E3, the E3 regions in other adenoviruses are not essential for virus replication in cultured cells, adenovirus recombinants containing foreign gene inserts in the E3 region could be generated.
BAV3 is a common pathogen of cattle usually resulting in subclinical infection 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). 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.
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 regions 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 exon 1 encoded regions of E1A proteins (Yee and Branton, 1985
Virology
147:142-153; Harlow et al., 1986
Mol. Cell Biol.
6:1579-1589; Barbeau et al., 1992
Biochem. Cell Biol.
70:1123-1134). Interaction of E1A with these cellular proteins has been implicated as the mechanism through which E1A participate

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