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
2000-10-19
2003-02-18
Salimi, Ali R. (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...
C435S235100, C435S239000, C435S070400, C435S091420, C435S455000, C435S005000, C435S007100, C435S007200, C435S007210
Reexamination Certificate
active
06521423
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention pertains generally to viral proteins. In particular, the invention relates to improved methods for isolating truncated forms of hepatitis C virus E1 and E2 proteins having improved biological properties for use in vaccine compositions and as diagnostic reagents.
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 0.4 to 2.0% of blood donors. Chronic hepatitis develops in about 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. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (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 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).
Full-length E1 and E2 are retained within cells and have been shown to 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, experimenters had previously thought it desirable to produce secreted forms to facilitate purification of the proteins for further use.
For example, an HCV E2 molecule, truncated at amino acid 661 and which is secreted from mammalian cells, has been described. Spaete et al.,
Virology
(1992) 188:819-830. The production of truncated, secreted HCV E1 and E2 molecules has also been disclosed in International Publication No. WO 96/04301, published Feb. 15, 1996. Inudoh et al.,
Vaccine
(1996) 14:1590-1596, describes the production of an HCV E2 molecule lacking the C-terminal hydrophobic domain. This protein was secreted into culture medium and found to be more antigenic than intracellularly produced counterparts.
Depending on the expression system used, such secreted proteins may not retain the native conformation and may include modified glycosylation patterns. Thus, purification of intracellularly produced HCV E1 and E2 proteins has been attempted in order to preserve the native conformation of the proteins. See, e.g., International Publication No. WO 92/08734, published May 29, 1992.
Despite the above attempts at obtaining HCV E1 and E2, a need still exists for alternative methods of efficiently purifying immunogenic HCV E1 and E2 molecules for use in vaccine compositions and as diagnostic reagents.
SUMMARY OF THE INVENTION
The present invention is based on the isolation of HCV E1 and E2 proteins which display improved biological properties. The proteins are truncated and can be produced using recombinant techniques. Such truncated proteins are normally secreted into culture medium. However, the proteins for use herein are isolated from the cells rather than from culture medium. The molecules so isolated display enhanced receptor-binding abilities, perform better in assays designed to measure the ability of proteins to elicit the production of HCV neutralizing antibodies, and are more immunoreactive and therefore provide improved diagnostic reagents, as compared to their secreted counterparts.
Accordingly, in one embodiment, the subject invention is directed to a method for isolating an HCV E1 polypeptide that lacks a portion of its C-terminus beginning at about amino acid 370, numbered with reference to the HCV1 E1 amino acid sequence. The method comprises:
(a) providing a population of host cells transformed with a polynucleotide comprising a coding sequence for the HCV E1 polypeptide, wherein the coding sequence is operably linked to control elements such that the coding sequence can be transcribed and translated in the host cell;
(b) culturing the population of cells under conditions whereby the HCV E1 polypeptide is expressed intracellularly;
(c) disrupting the host cells; and
(d) isolating the HCV E1 polypeptide from the disrupted cells.
In particularly preferred embodiments, the HCV E1 polypeptide produced by the method lacks a portion of its C-terminus beginning at about amino acid 360, numbered with reference to the HCV1 E1 amino acid sequence.
In another embodiment, the invention is directed to a method for isolating an HCV E2 polypeptide that lacks a portion of its C-terminus beginning at about amino acid 730 but not extending beyond about amino acid 625, numbered with reference to the HCV1 E2 amino acid sequence. The method comprises:
(a) providing a population of host cells transformed with a polynucleotide comprising a coding sequence for the HCV E2 polypeptide, wherein the coding sequence is operably linked to control elements such that the coding sequence can be transcribed and translated in the host cell;
(b) culturing the population of cells under conditions whereby the HCV E2 polypeptide is expressed intracellularly;
(c) disrupting the host cells; and
(d) isolating the HCV E2 polypeptide from the disrupted cells.
In particularly preferred embodiments, the HCV E2 polypeptide produced by the method 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, and more particularly, lacks a portion of its C-terminus beginning at about amino acid 715, 661 or 655.
Further embodiments of the subject invention pertain to HCV E1 and HCV E2 polypeptides produced by the above methods, as well as vaccine compositions comprising the HCV polypeptides and methods of preparing the vaccine compositions.
In yet other embodiments, the invention is directed to methods of detecting the presence or absence of HCV infection in a subject suspected of having an HCV infection. The methods comprise:
(a) providing a biological sample from the subject;
(b) providing an HCV E1 polypeptide or an HCV E2 polypeptide, as described above; and
(c) contacting the biological sample with the CV polypeptide, under conditions which allow HCV antibodies, if present in the biological sample, to bind with the HCV polypeptide,
thereby detecting the presence or absence of HCV infection in the subject.
In other embodiments, the invention is directed to immunodiagnostic test kits for detecting HCV infection. The test kits include an HCV E1 or HCV E2 polypeptide, as described above, and instructions for conducting the immunodiagnostic test.
These and other embodiments of the present invention will readily occur to those of ordinary skill in t
Abrignani Sergio
Chien David
Choo Qui-Lim
Glazer Edward
Houghton Michael
Blackburn Robert P.
Chiron Corporation
Herbin Alisa A.
Li Bao Qun
Pasternak Dahna S.
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