Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-10-08
2001-12-04
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S235100, C424S228100, C424S185100, C424S204100, C530S395000, C530S826000
Reexamination Certificate
active
06326171
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention pertains generally to viral proteins. In particular, the invention relates to truncated, secreted forms of hepatitis C virus E1 and E2 proteins and the isolation and recombinant production of the same.
2. Background of the Invention
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis which is transmitted largely through blood transfusion and sexual contact. The virus is present in between 0.4 to 2.0% of blood donors. Chronic hepatitis develops in approximately 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control of the disease is of medical importance.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. In particular, HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. Currently, there are 6 distinct, but related genotypes of HCV based on phylogenetic analyses (Simmonds et al.,
J. Gen. Virol
. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al.,
Science
(1989) 244:359-362; Choo et al.,
Proc. Natl. Acad. Sci. USA
(1991) 88:2451-2455; Han et al.,
Proc. Natl. Acad. Sci. USA
(1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, there are three putative structural proteins, consisting of the N-terminal nucleocapsid protein (termed “core”) and two envelope glycoproteins, “E1” (also known as E) and “E2” (also known as E2/NS1). (See, Houghton et al.,
Hepatology
(1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2.) E1 is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 kDa. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al.,
J. Virol.
(1993) 67:1385-1395; Tomei et al.,
J. Virol
. (1993) 67:4017-4026.). The HCV envelope glycoproteins E1 and E2 form a stable complex that is co-immunoprecipitable (Grakoui et al.,
J. Virol.
(1993) 67:1385-1395; Lanford et al.,
Virology
(1993) 197:225-235; Ralston et al.,
J. Virol.
(1993) 67:6753-6761). The HCV E1 and E2 glycoproteins are of considerable interest because they have been shown to be protective in primate studies. (Choo et al.,
Proc. Natl. Acad. Sci. USA
(1994) 91:1294-1298).
The envelope of the HCV virion remains uncharacterized. Thus, expression studies using recombinant CDNA templates are the only means currently available to study envelope biosynthesis. E1 and E2 are retained within cells and lack complex carbohydrate when expressed stably or in a transient Vaccinia virus system (Spaete et al.,
Virology
(1992) 188:819-830; Ralston et al.,
J. Virol.
(1993) 67:6753-6761). Since the E1 and E2 proteins are normally membrane-bound in these expression systems, it would be desirable to produce secreted forms to facilitate purification of the proteins for further use.
It has been found that removal of the transmembrane domain of the viral cell surface glycoproteins of influenza virus (Sveda, et al.,
Cell
(1982) 30:649-656; Gething and Sambrook,
Nature
(1982) 300:598-603) and vesicular stomatitis virus (Rose and Bergmann,
Cell
(1982) 30:753-762) results in secretion of the truncated glycoprotein from mammalian host cells. See also EPO Publication No. 139,417. Similarly, truncated cytomegalovirus gH is secreted when expressed in baculovirus cells (International Publication No. WO 92/02628, published 20 February 1992). A C-terminally truncated HCV E2 molecule, capable of secretion from mammalian cells, has been described. Spaete et al.,
Virology
(1992) 188:819-830 However, the transmembrane anchor region of E1 has not heretofore been elucidated and hence the production of truncated forms of HCV E1 for secretion has not been previously disclosed. Furthermore, complexes of truncated, secreted E1 and E2 polypeptides have not been previously described.
SUMMARY OF THE INVENTION
The present invention is based on the elucidation of sequences of E1 and E2 important for anchoring the proteins to the endoplasmic reticulum (ER) and for co-precipitation of E2 with E1. Thus, the elimination of these sequences serves to produce truncated forms of the glycoproteins which are secreted rather than retained in the ER membrane. Furthermore, truncation facilitates purification of the E2 protein without associated E1, and vice versa. The truncated E1 and E2 proteins, when expressed together or combined after expression, are capable of forming a complex.
Accordingly, in one embodiment, the subject invention is directed to an HCV E1 polypeptide, lacking all or a portion of its membrane spanning domain such that the polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell. In preferred embodiments, the HCV E1 polypeptide lacks at least a portion of its C-terminus beginning at or near about amino acid 370 or about amino acid 360, numbered with reference to the HCV1 E1 amino acid sequence.
In another embodiment, the invention is directed to an HCV E2 polypeptide lacking at least a portion of its membrane spanning domain such that the polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell, wherein the polypeptide lacks at least a portion of its C-terminus beginning at or near about amino acid 730 but not extending beyond about amino acid 699, numbered with reference to the HCV1 E2 amino acid sequence. In particularly preferred embodiments, the HCV E2 polypeptide lacks at least a portion of its C-terminus beginning at about amino acid 725, numbered with reference to the HCV1 E2 amino acid sequence.
Other embodiments of the subject invention pertain to polynucleotides encoding the above polypeptides, vectors comprising these polynucleotides, host cells transformed with the vectors and methods of recombinantly producing the polypeptides.
In yet another embodiment, the subject invention is directed to a secreted E1/secreted E2 complex comprising:
(a) an HCV E1 polypeptide, lacking all or a portion of its membrane spanning domain such that the E1 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell; and
(b) an HCV E2 polypeptide, lacking all or a portion of its membrane spanning domain such that said E2 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell.
In preferred embodiments, the secreted E1/secreted E2 complex includes an E1 polypeptide which lacks at least a portion of its C-terminus beginning at about amino acid 360 or 370, numbered with reference to the HCV1 E1 amino acid sequence, and said E2 polypeptide lacks at least a portion of its C-terminus beginning at about amino acid 725 or 730, numbered with reference to the HCV1 E2 amino acid sequence.
In still further embodiments, the subject invention is directed to vaccine compositions comprising the truncated HCV E1 and/or E2 polypeptides and/or complexes of the E1 and E2 polypeptides.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
REFERENCES:
patent: 5942234 (1999-08-01), Ralston et al.
patent: WO 89/04669 (1989-06-01), None
patent: WO 90/11089 (1990-10-01), None
patent: WO 90/14436 (1990-11-01), None
patent: WO 92/02628 (1992-02-01), None
patent: WO 92/08734 (1992-05-01), None
patent: WO 96/04385 (1996-02-01), None
Choo et al., “Isolation of a cDNA Clone Derived from a Blood-Borne Non-A, Non-B Viral Hepatitis Genome”,Science244:359-362 (1989).
Choo et al., “Genetic Organization and Diversity of the Hepatitis C Virus”,Proc. Natl. Acad. Sci. USA88:2451-2455 (1991).
Choo et al., “Vaccination of Chimp
Houghton Michael
Selby Mark
Blackburn Robert P.
Brown Stacey S
Chiron Corporation
Harbin Alisa A.
Park Hankyel T.
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