Methods for producing hepatitis c virus envelope proteins

Abstract

The present disclosure provides methods of producing Hepatitis C Virus E1 and E2 polypeptides. The methods comprise culturing genetically modified mammalian cells in vitro in a liquid culture medium, wherein 100% oxygen is perfused into the liquid culture medium.

Description

METHODS FOR PRODUCING HEPATITIS C VIRUS ENVELOPE PROTEINSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 538,347, filed September 14, 2023, which application is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “UALB- 060WO_SEQ_LIST” created on August 28, 2024, and having a size of 129,614 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

[0003] Hepatitis C virus (HCV) is a blood-borne pathogen that is estimated to infect 150-200 million people worldwide. Infection by HCV may be non-symptomatic, and can be cleared by patients, sometimes without medical intervention. However, the majority of patients develop a chronic HCV infection, which may lead to liver inflammation, scarring, and even to liver failure or liver cancer. In the United States alone, over 3 million people have a chronic infection.

[0004] The HCV virion contains a positive-sense single stranded RNA genome of about 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030 amino acids. The structural proteins comprise a core protein forming the viral nucleocapsid and two envelope glycoproteins, El and E2.

[0005] There is a need in the art for methods of producing HCV envelope proteins El and E2.SUMMARY

[0006] The present disclosure provides methods of producing HCV El and E2 polypeptides.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGs. 1A-1C provide an amino acid sequence alignment of examples of the core-El-E2 coding regions of an HCV genotype 1 virus, specifically representative HCV 1A, IB and 1C genotypes. Genbank database sequences for the coding region core-El-E2 were aligned using Geneious software v5.6.4. Numbering of amino acids is according to strain NP_671941 (H77). Consensus: SEQ ID NO:1; AVIlal29: SEQ ID NO:2; NP_671491 (H77): SEQ ID NO:3; EU155269: SEQ ID NO:4; EU781810:SEQ ID NO:5; EU781771: SEQ ID NO:6; AB250610: SEQ ID NO:7; EU781752: SEQ ID NO:8;EU781759: SEQ ID NO:9; EF407439: SEQ ID NO:10; EF407427: SEQ ID NO:11; EU362905: SEQ IDNO: 11; EF407413: SEQ ID NO:11; EU781808: SEQ ID NO:14; EU78170: SEQ ID NO:15; AJ238799 (Coni): SEQ ID NO:16; AAK97744: SEQ ID NO:17; AF139594: SEQ ID NO:18; AF176573: SEQ ID NO:19; BAA19625: SEQ ID NO:20; BAA25076: SEQ ID NO:21; BAC54896: SEQ ID NO:22;BAD91386: SEQ ID NO:23; BAF46764: SEQ ID NO:24; BAG30950: SEQ ID NO:24; CAB41951: SEQ ID NO:26; AAK95832: SEQ ID NO:27; AAT69968: SEQ ID NO:28; and BAA03581: SEQ ID NO:29.

[0008] FIGs. 2A-2C provide an alignment of amino acid sequences of the core-El-E2 coding region of representative HCV 2A and HCV2B subtypes. Genbank database sequences for the coding region core- E1-E2 were aligned using Geneious software v5.6.4. The amino acid numbering depicted is in accordance with the common HCV strains: D00994 (HC-J6) for HCV 2A; ABO47639 (JFH1) and HPCJ8G-J8 (J8) for HCV2A and HCV2B, respectively. Consensus: SEQ ID NO: 30; AB047639 (JFH1): SEQ ID NO:31; AB047645: SEQ ID NO:32; AF169005: SEQ ID NO:33; AF238482: SEQ ID NO:34;AY746460: SEQ ID NO:35; HPCPOLP: SEQ ID NO:36; NC_009823: SEQ ID NO:37; HPCJ8G HC-J8: SEQ ID NO:38; AB030907: SEQ ID NO:39; AY232730: SEQ ID NO:40; AY232747: SEQ ID NO:41; and DQ430817: SEQ ID NO:42.

[0009] FIGs. 3A-3C provide an amino acid sequence alignment of the core-El-E2 coding region for representative HCV 3A, 3B, 3C, and 3K genotypes. Genbank database sequences for the coding region core-El-E2 were aligned using Geneious software v5.6.4. Consensus: SEQ ID NO:43; AVI3al77: SEQ ID NO:44; ADF97232(S52): SEQ ID NO:45; YP_0014696: SEQ ID NO:46; CAA54244: SEQ ID NO:47; AAC03058: SEQ ID NO:48; AAY29642: SEQ ID NO:49; ABD85062: SEQ ID NO:50; ABD85063: SEQ ID NO:50; ABD97104: SEQ ID NO:52; BAA06044: SEQ ID NO:53; BAA08372: SEQ ID NO:54; and BAA09890: SEQ ID NO:55.

[0010] FIGs. 4A-4B provide an amino acid sequence of the core-El-E2 coding region for HCV genotype 7a. Amino acid sequence for the coding region core-El-E2 of genotype 7a (isolate QC69; Genbank: ABN05226.1; SEQ ID NO:56) is shown according to the numbering scheme of the reference strain, NP_671941 (H77).

[0011] FIGs. 5A-5C provide amino acid sequences of immunoglobulin (Ig) Fc polypeptides (SEQ ID NOs:57-64, respectively).DEFINITIONS

[0012] The term “hepalilis C virus” (“HCV”), as used herein, refers to any one of a number of different genotypes and isolates of hepalilis C virus. Thus, “HCV” encompasses any of a number of genotypes, subtypes, or quasispecies, of HCV, including, e.g., genotype 1, 2, 3, 4, 5, 6, 7, etc. and subtypes (e.g., la, lb, 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies. Representative HCV genotypes and isolates include: HCV-1, H77, J6, Coni, isolate 1, BK, ECI, EC10, HC-J2, HC-J5; HC-J6, HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, “Hunan”, “Japanese”, “Taiwan”, TH, type 1, type la, H77 type lb, type 1c, type Id, type le, type If, type 10, type 2, type 2a, type 2b, type 2c, type 2d, type 2f, type 3, type 3a, type 3b, type 3g, type 4, type 4a, type 4c, type 4d, type 4f, type 4h, type 4k, type 5, type 5a, type 6, type 6a, and type 7a.

[0013] As used herein, the term “isolated,” in reference to a polypeptide, refers to a polypeptide that is in an environment different from that in which the polypeplide naturally occurs. An isolated polypeptide can be purified. By “purified” is meant a compound of interest (e.g., a polypeptide) has been separated from components that accompany it in nature. “Purified” can also be used to refer to a polypeplide separated from components that can accompany it during production of the polypeptide (e.g., during synthesis in vitro, etc.). In some cases, a polypeptide (or a mixture of polypeptides) is substantially pure when the polypeptide (or mixture of polypeptides) is at least 60% or at least 75% by weight free from organic molecules with which it is naturally associated or with which it is associated during production. In some cases, the polypeptide is from 30% to 60% pure. In some cases, the polypeptide (or mixture of polypeptides) is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. For example, in some cases, an El or an E2 polypeptide (or a mixture of El and E2 polypeptides, e.g., an E1 / E2 heterodimer) is substantially pure when the El or E2 polypeplide (or mixture of El and E2 polypeptides) is at least 60% or at least 75% by weight free from organic molecules with which the polypeptide(s) is naturally associated or with which it is associated during production. In some cases, the El or E2 polypeptide (or mixture of El and E2 polypeptides) is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. In some cases, where a composition comprises an E2 polypeptide, the E2 polypeplide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. In some cases, where a composition comprises an E1 / E2 heterodimeric complex polypeplide, the E1 / E2 heterodimeric complex polypeptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure.

[0014] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term “polypeptide” includes glycosylated polypeptides.

[0015] The term “heterologous” refers to two components that are defined by structures derived from different sources. For example, where “heterologous” is used in the context of a polypeplide, the polypeplide includes operably linked amino acid sequences that can be derived from one or more different polypeptides, e.g., amino acid sequences that are not operably linked to the polypeptide innature. As an example, where a composition comprises an HCV E1 / E2 heterodimer and a “heterologous” polypeptide, the “heterologous polypeptide is a polypeptide other than HCV El or HCV E2.

[0016] A "conservali ve amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid subslilulion will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties, and that thus constitute conservative amino acid subslilulion groups, include: 1) aliphatic side chain-containing amino acids: glycine, alanine, valine, leucine and isoleucine; 2) aliphalic- hydroxyl side chain-containing amino acids: serine and threonine; 3) amino acids with amide-containing side chains: asparagine and glutamine; 4) aromatic side chain-containing amino acids: phenylalanine, tyrosine, and tryptophan; 5) amino acids with basic side chains: lysine, arginine, and histidine; 6) amino acids with acidic side chains: aspartate and glutamate, and 7) amino acids with sulfur-containing side chains: cysteine and methionine. Examples of conservative amino acid substitution groups are: valine- leucine-isoleucine, phenylalanine -tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine -glutamine.

[0017] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practiceor testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0020] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an HCV E1 / E2 heterodimer” includes a plurality of such heterodimer and reference to “the culture medium” includes reference to one or more culture media and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0021] The use of the terms “a,” “an,” and “the,” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

[0022] As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90-110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0023] The term “and / or” as used herein a phrase such as “A and / or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used herein a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0024] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essenlially of’ aspects and embodiments.

[0025] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-coinbinalion. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0026] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.DETAILED DESCRIPTION

[0027] The present disclosure provides methods of producing HCV El and E2 polypeptides. The methods comprise culturing genetically modified mammalian cells in vitro in a liquid culture medium, wherein 100% oxygen is perfused into the liquid culture medium.

[0028] Mammalian cells (e.g., cells of a genetically modified mammalian cell line) are cultured in a liquid culture medium in vitro. The liquid culture medium can have a volume of from about 4 liters to about 100 liters. For example, the genetically modified mammalian cells can be cultured in a volume of liquid culture medium that is from about 4 liters to about 5 liters, from about 5 liters to about 10 liters, from about 10 liters to about 15 liters, from about 15 liters to about 20 liters, from about 20 liters to about 25 liters, from about 25 liters to about 50 liters, or from about 50 liters to about 100 liters. The genetically modified mammalian cells can be cultured in a volume of liquid culture medium that is from about 100 liters to about 20,000 liters. For example, in some cases, the genetically modified mammalian cells can be cultured in a volume of liquid culture medium that is from about 100 liters to about 200 liters, from about 200 liters to about 500 liters, from about 500 liters to about 1000 liters, from about 1000 liters to about 5000 liters, from about 5000 liters to about 10,000 liters, from about 10,000 liters to about 15,000 liters, or from about 15,000 liters to about 20,000 liters. The genetically modifiedmammalian cells can be cultured in spinner flasks. The genetically modified mammalian cells can be cultured in a perfusion bioreactor, a batch bioreactor, or a batch refeed bioreactor.

[0029] Suitable liquid culture media include, e.g., Minimal Essential Media (MEM), Dulbecco’s MEM (DMEM), RMPI 1640, Eagle’s MEM (EMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s F-12, McCoy’s 5A Medium, Lonza ProCHO-4 (e.g., Lonza Bioscience catalog no. BEBP12-O29Q; product code 216932), Lonza ProCHO5, and the like. The culture medium can be supplemented with, e.g., glutamine (e.g., 2 mM to 10 mM glutamine), non-essential amino acids, heparin, and the like. In some cases, the liquid culture medium includes serum, e.g., fetal bovine serum (FBS). In some cases, the liquid culture medium comprises 0.5% FBS. In some cases, the liquid culture medium comprises 0.5% FBS, glucose, and glutamine. In some cases, the liquid culture medium comprises 6 g / L glucose, 4 mM glutamine, and 0.5% FBS. In some cases, the liquid culture medium is Lonza ProCHO4 supplemented to final concentrations of 6 g / L glucose, 4 mM glutamine, and 0.5% FBS.

[0030] The in vitro culture (liquid culture medium comprising genetically modified mammalian cells) is perfused with 100% oxygen. The amount of HCV El and / or E2 polypeptide that can be produced by perfusing the culture with 100% oxygen is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 75%, at least 100% (or two-fold), at least 2.5-fold, at least 5 -fold, at least 10-fold, at least 25 -fold, at least 50-fold, or at least 100-fold, greater than the amount of the HCV El and / or E2 polypeptide that is produced over the same period of time when the culture is perfused with air (where air comprises about 15-20% oxygen).

[0031] The in vitro culture (liquid culture medium comprising genetically modified mammalian cells) is perfused with 100% oxygen. In some cases, the dissolved oxygen in the culture is from about 10% to about 65%. For example, in some cases, the dissolved oxygen in the culture is from about 10% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, or from about 50% to about 65%. In some cases, the dissolved oxygen in the culture (where the culture medium is perfused with 100% oxygen) is higher than the dissolved oxygen in a culture that is perfused with air (where air comprises about 15-20% oxygen). For example, in some cases, the dissolved oxygen in a culture of the present disclosure (where the culture medium is perfused with 100% oxygen) is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50% higher than the dissolved oxygen in a culture that is perfused with air.

[0032] A method of the present disclosure provides for production of an HCV El and / or E2 polypeptide in an amount of from about 50 mg / kg cells to about 1000 mg / kg cells. For example, a method of the present disclosure provides for production of an HCV El and / or E2 polypeptide in an amount of from about 50 mg / kg cells to about 75 mg / kg, from about 75 mg / kg to about 100 mg / kg, from about 100 mg / kg to about 125 mg / kg, from about 125 mg / kg to about 150 mg / kg, from about 150 mg / kg to about 175 mg / kg, from about 175 mg / kg to about 200 mg / kg, from about 200 mg / kg to about 225 mg / kg, fromabout 225 mg / kg to about 250 mg / kg, from about 250 mg / kg to about 275 mg / kg, from about 275 mg / kg to about 300 mg / kg, from about 300 mg / kg to about 325 mg / kg, from about 325 mg / kg to about 350 mg / kg, from about 350 mg / kg to about 375 mg / kg, from about 375 mg / kg to about 400 mg / kg, from about 400 mg / kg to about 425 mg / kg, from about 425 mg / kg to about 450 mg / kg, from about 450 mg / kg to about 475 mg / kg, from about 475 mg / kg to about 500 mg / kg, or from about 500 mg / kg to about 1000 mg / kg.

[0033] The starting cell density can be from about 0.2 x 106cells / mL culture medium to about 2 x 106cells / mL. The cells can be grown (e.g., maintained) at a density of from about 1 x 106cells / mL to about 20 x 106cells / mL, e.g., from about 1 x 106cells / mL to about 2 x 106cells / mL, from about 2 x 106cells / mL to about 5 x 106cells / mL, from about 5 x 106cells / mL to about 10 x 106cells / mL, from about 10 x 106cells / mL to about 15 x 106cells / mL, or from about 15 x 106cells / mL to about 20 x 106cells / mL.

[0034] The genetically modified mammalian cells can be cultured for a period of time of from about 2 weeks to about 24 months. For example, the genetically modified mammalian cells can be cultured for a period of time of from about 2 weeks to about 4 weeks, from about 4 weeks to about 8 weeks, from about 2 months to about 4 months, from about 4 months to about 8 months, from about 8 months to about 10 months, from about 10 months to about 12 months, from about 12 months to about 18 months, or from about 18 months to about 24 months. In some cases, cells are harvested every 3 to 4 days. In some cases, cells are harvested every 3 to 4 days, and from about 20% to about 80% (e.g., from about 20% to about 30%, from about 30% to about 50%, or from about 50% to about 80%) of the culture volume (including from about 20% to about 30%, from about 30% to about 50%, or from about 50% to about 80%, of the total number of cells in the culture) is removed, and the culture volume is replenished with fresh culture medium. In some cases, cells are harvested every 3 to 4 days, and from about 70% to about 80% of the culture volume (including from about 70% to about 80% of the total number of cells in the culture) is removed, and the culture volume is replenished with fresh culture medium. In some cases, cells are harvested every 2 to 4 days, and from about 20% to about 90% of the culture volume (including from about 20% to about 90% of the total number of cells in the culture) is removed, and the culture volume is replenished with fresh culture medium.

[0035] The viability of the genetically modified mammalian cells, when cultured using a method of the present disclosure (where the culture medium is perfused with 100% oxygen) is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 50% higher, than the viability of the cells when cultured over the same period of time in a culture medium perfused with air.

[0036] As noted above, a mammalian cell line genetically modified to produce HCV El and / or E2 polypeptides is cultured in a liquid culture medium. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, MRC4 fibroblast cells, PER.C6 cells (see, e.g., Fallaux et al. (1998) Hum. Gene Ther. 9:1909; and Pau et al. (2001) Vaccine 19:2716), and the like. In some cases, the cells are genetically modified CHO cells. In some cases, the cells are genetically modified PER.C6 cells.HCV El polypeptides, HCV E2 polypeptides, and HCV E1 / E2 heterodimers

[0037] As noted above, in some cases, an immunogenic composition of the present disclosure comprises an HCV El polypeptide, an HCV E2 polypeptide, or an HCV E1 / E2 heterodimer.El polypeptides

[0038] An HCV El polypeptide suitable for inclusion in an immunogenic composition of the present disclosure, or for inclusion in an HCV E1 / E2 heterodimer present in an immunogenic composition of the present disclosure, can have a length of from about 100 amino acids (aa) to about 150 aa, from about 150 aa to about 175 aa, from about 175 aa to about 195 aa, from about 131 aa to about 175 aa, or from about 175 aa to about 193 aa. In some cases, an HCV El polypeptide is an HCV El ectodomain polypeptide. In some cases, an HCV El polypeptide is a full-length HCV El polypeptide.

[0039] In FIG. 1A-1C, the amino acid sequence of El is amino acid 192 to amino acid 383. In FIG. 2A- 2C, the amino acid sequence of El is amino acid 192 to amino acid 383. In FIG. 3A-3C, the amino acid sequence of El is amino acid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of El is amino acid 192 to amino acid 383. Amino acids at around 170 through approximately 191 serve as a signal sequence for El. As used herein, "El polypeptide" includes a precursor El protein, including the signal sequence; includes a mature El polypeptide which lacks this sequence; and includes an El polypeptide with a heterologous signal sequence. An El polypeptide can include a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 360-383 (see, e.g., WO 96 / 04301). In some cases, a suitable El polypeptide lacks a C-terminal portion that includes a transmembrane region. For example, in some cases, a suitable El polypeptide lacks the C-terminal portion from amino acid 330 to amino acid 384, or from amino acid 360 to amino acid 384. El polypeptides can be an El polypeptide of any genotype, subtype or isolate of HCV. El polypeptides of genotype 1 and El polypeptides of genotype 3 are included in an E1 / E2 heterodimer of the present disclosure.

[0040] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

[0041] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 1A-1C. For example, an El polypeptide of genotype 1A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 1 A and depicted in FIG. 1A-1C. For example, an El polypeptide of genotype IB can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as IB and depicted in FIG. 1A-1C. For example, an El polypeptide of genotype 1C can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192- 383 of an amino acid sequence identified as 1C and depicted in FIG. 1A-1C.

[0042] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 2A-2C. For example, an El polypeptide of genotype 2 A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 2 A and depicted in FIG. 2A-2C. For example, an El polypeptide of genotype 2B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 2B and depicted in FIG. 2A-2C.

[0043] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the consensus El polypeptide amino acid sequence depicted in FIG. 3A-3C.

[0044] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 4A-4B. For example, an El polypeptide of genotype 7A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of the amino acid sequence depicted in FIG. 4A-4B.E2 polypeptides

[0045] An E2 polypeptide suitable for inclusion in an immunogenic composition of the present disclosure, or for inclusion in an HCV E1 / E2 heterodimer present in an immunogenic composition of the present disclosure, can have a length of from about 200 amino acids (aa) to about 250 aa, from about 250 aa to about 275 aa, from about 275 aa to about 300 aa, from about 300 aa to about 325 aa, from about 325 aa to about 350 aa, or from about 350 aa to about 365 aa. In some cases, an HCV E2 polypeptide is an HCV E2 ectodomain polypeptide. In some cases, an HCV E2 polypeptide is a full-length HCV E2 polypeptide.

[0046] In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to amino acid 746. In FIG. 2A- 2B, the amino acid sequence of E2 is amino acid 384 to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is amino acid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2 is amino acid 384 to amino acid 750. As used herein, an "E2 polypeptide" includes a precursor E2 protein, including the signal sequence; includes a mature E2 polypeptide which lacks this sequence; and includes an E2 polypeptide with a heterologous signal sequence. An E2 polypeptide can include a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 715-730 and may extend as far as approximately amino acid residue 746 (see, Lin et al., J. Virol. (1994) 68:5063-5073).

[0047] In some cases, a E2 polypeptide lacks a portion of its C-terminal region, e.g., from about amino acid 715 to the C-terminus; from about amino acid 625 to the C-terminus; from about amino acid 661 to the C-terminus; from about amino acid 655 to the C-terminus; from about amino acid 500 to the C- terminus, where the amino acid numbering is with reference to the numbering in FIG. 1A-1C. See, e.g., U.S. Patent No. 6,521,423.

[0048] An E2 polypeptide suitable for inclusion in an immunogenic composition of the present disclosure can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B. An E2 polypeptide suitable for inclusion in an immunogenic composition of the present disclosure can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, or at least about 75%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

[0049] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C. For example, an E2 polypeptide of genotype 1 can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-746 of an amino acid sequence depicted in FIG. 1A-1C. For example, an E2 polypeptide of genotype 1 A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-746 of an amino acid sequence identified as 1A and depicted in FIG. 1A-1C. For example, an E2 polypeptide of genotype IB can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids 384-746 of an amino acid sequence identified as IB and depicted in FIG. 1A-1C. For example, an E2 polypeptide of genotype 1C can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-746 of an amino acid sequence identified as 1C and depicted in FIG. 1A-1C.

[0050] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 2A-2C. For example, an E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-751 of an amino acid sequence depicted in FIG. 2A-2C. For example, an E2 polypeptide of genotype 2A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-751 of the “consensus” amino acid sequence depicted in FIG. 2A- 2C. For example, an E2 polypeptide of genotype 2B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-751 of the “consensus” amino acid sequence depicted in FIG. 2A-2C.

[0051] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 3A-3C. For example, an E2 polypeptide of genotype 3 can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of an amino acid sequence depicted in FIG. 3A-3C. For example, an E2 polypeptide of genotype 3A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of an amino acid sequence identified as 3A and depicted in FIG. 3A-3C. For example, an E2 polypeptide of genotype 3B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of the amino acid sequence identified as 3B and depicted in FIG. 3A-3C. For example, an E2 polypeptide of genotype 3K can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of the amino acid sequence identified as 3K and depicted in FIG. 3A-3C.

[0052] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence of the E2 polypeptide depicted in FIG. 4A-4B. For example, an E2 polypeptide of genotype 7A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-750 of the amino acid sequence depicted in FIG. 4A- 4B.HCV E1 / E2 heterodimers

[0053] HCV E1 / E2 heterodimers suitable for use in an immunogenic composition of the present disclosure include HCV E1 / E2 heterodimers comprising wild-type HCV El polypeptides; HCV E1 / E2 heterodimers comprising wild-type HCV E2 polypeptides; HCV E1 / E2 heterodimers comprising variant HCV El polypeptides; and HCV E1 / E2 heterodimers comprising variant HCV E2 polypeptides. HCV E2polypeptides suitable for use in an immunogenic composition of the present disclosure include wild-type E2 polypeptides and variant E2 polypeptides. HCV El polypeptides suitable for use in an immunogenic composition of the present disclosure include wild-type El polypeptides and variant El polypeprides. An HCV E1 / E2 heterodimer can include any of the HCV El polypeptides discussed above and any of the HCV E2 polypeptides discussed above. In some cases, the HCV E1 / E2 polypeptides are not fusion polypeptides, i.e., neither the El polypeptide nor the E2 polypeptide includes a heterologous fusion partner.Fusion polypeptides

[0054] In some cases, an HCV El polypeptide is a fusion polypeptide comprising: a) an HCV El polypeptide; and ii) a heterologous fusion partner (“heterologous polypeptide”). In some cases, an HCV E2 polypeptide is a fusion polypeptide comprising: a) an HCV E2 polypeptide; and ii) a heterologous fusion partner (“heterologous polypeptide”).

[0055] In some cases, the heterologous fusion partner is an affinity tag. For example, in some cases, the HCV El and / or E2 polypeptide is produced as a fusion polypeptide comprising: a) the HCV El and / or E2 polypeptide; and b) a fusion partner, where the fusion partner is an affinity tag. Suitable affinity tags include, e.g., immunoglobulin (Ig) Fc polypeptides, a poly(histidine) tag (e.g., Hise; SEQ ID NO:68), a maltose binding protein (MBP), a glutathione-S-transferase (GST) polypeptide, calmodulin-binding peptide (CBP), Streptavidin-binding peptide (SBP), Strep-tag II, FLAG (e.g., DYKDDDDK (SEQ ID NO:65), hemagglutinin (HA) (e.g., YPYDVPDYA (SEQ ID NO:66), c-myc T7 ((e.g., EQKLISEEDL; SEQ ID NO:67), Glu-Glu, starch-binding domain (SBD), Flag-Acidic-Target Tag (FATT), Protein A, Protein G, a hybrid Protein A / Protein G polypeptide, and the like. The affinity tag can be at the N- terminus of the HCV El and / or E2 polypeptide or at the C-terminus of the HCV El and / or E2 polypeptide.

[0056] In some cases, the HCV El and / or E2 polypeptide is produced as a fusion polypeptide comprising: a) the HCV El and / or E2 polypeptide; b) a proteolytically cleavable linker; and c) a fusion partner (e.g., where the fusion partner is an Ig Fc polypeptide).

[0057] The proteolytically cleavable linker can include a protease recognition sequence recognized by a protease selected from the group consisting of alanine carboxypeptidase, Armillaria mellea astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, IgA-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E, picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogenactivator, thrombin, tissue kallikrein, tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB, and Xaa-pro aminopeptidase.

[0058] For example, the proleolylically cleavable linker can comprise a matrix metalloproteinase cleavage site, e.g., a cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-1, -8, and - 13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue), e.g., Pro-X-X-Hy-(Ser / Thr), e.g., Pro-Leu / Gln-Gly-Met-Thr-Ser (SEQ ID NO:69) or Pro- Leu / Gln-Gly-Met-Thr (SEQ ID NO:70). Another example of a protease cleavage site is a plasminogen activator cleavage site, e.g., a uPA or a (issue plasminogen activator (tPA) cleavage site. In some cases, the cleavage site is a furin cleavage site. Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO:71), where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in a proleolylically cleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ ID NO:72), where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., LVPR (SEQ ID NO:73). Additional suitable linkers comprising protease cleavage sites include linkers comprising one or more of the following amino acid sequences: LEVLFQGP (SEQ ID NO:74), cleaved by PreScission protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase; Walker et al. (1994) Biotechnol. 12:601); a thrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:75); SLLKSRMVPNFN (SEQ ID NO:76) or SLLIARRMPNFN (SEQ ID NO:77), cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO:78) or SSYLKASDAPDN (SEQ ID NO:79), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO: 80) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO: 81) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO: 82) cleaved by MMP-9; DVDERDVRGFASFL SEQ ID NO:83) cleaved by a thermolysin-like MMP; SLPLGLWAPNFN (SEQ ID NO:84) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO:85) cleaved by cathespin L; SGVVIATVIVIT (SEQ ID NO:86) cleaved by cathepsin D; SLGPQGIWGQFN (SEQ ID NO:87) cleaved by matrix metalloproteinase l(MMP-l); KKSPGRVVGGSV (SEQ ID NO: 88) cleaved by urokinase -type plasminogen activator; PQGLLGAPGILG (SEQ ID NO:89) cleaved by membrane type 1 matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO:90) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin- 1; GPQGLAGQRGIV (SEQ ID NO:91) cleaved by matrix metalloproteinase 13 (collagenase-3);GGSGQRGRKALE (SEQ ID NO:92) cleaved by lissue-lype plasminogen activator(tPA);SLSALLSSDIFN (SEQ ID NO:93) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO:94) cleaved by kallikrein (hK3); SEEGIAVPGNFN (SEQ ID NO:95) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:96) cleaved by calpain (calcium activated neutral protease).

[0059] In some cases, the heterologous polypeptide is an Ig Fc polypeptide. The Fc polypeptide can be a human IgGl Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an Fc polypeptide depicted in FIG. 5A-5C. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgGl Fc polypeptide depicted in FIG. 5A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG2 Fc polypeptide depicted in FIG. 5 A; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide depicted in FIG. 5A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG3 Fc polypeptide depicted in FIG. 5A; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide depicted in FIG. 5A. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 100-327 of the IgG4 Fc polypeptide depicted in FIG. 5C.

[0060] In some cases, an affinity tagged HCV E1 / E2 heterodimer is produced by a genetically modified mammalian cell, where the affinity tagged HCV E1 / E2 heterodimer comprises: a) an HCV El polypeptide; and b) an affinity-tagged HCV E2 polypeptide comprising, in order from N-terminus to C- terminus: i) an Ig Fc polypeptide; ii) a proteolytically cleavable linker; and iii) an HCV E2 polypeptide. In some cases, the proteolytically cleavable linker comprises an amino acid sequence selected from the group consisting of EEVEFQGP (SEQ ID NO:74), ENEYFQS (SEQ ID NO:97), DDDDK (SEQ ID NO:72), I(E / D)GR, and EVPRGS (SEQ ID NO:98).Purification steps

[0061] The HCV El and / or E2 polypeptide can be isolated from cells harvested from a mammalian cell culture of the present disclosure. In some cases, the HCV El and / or E2 polypeptide is isolated and purified from a cell lysate made from the genetically modified host cell. The HCV El polypeptide, HCV E2 polypeptide, or HCV E1 / E2 heterodimer can be isolated from the genetically modified host cell by, e.g., lysis using a non-ionic detergent, or using a freeze-thaw method. See, e.g., Frey et al. (2010) Vaccine 28:6367. Methods of isolating purifying a polypeptide from a cell lysate are known in the art and include, e.g., affinity chromatography, size exclusion chromatography, high-performance liquid chromatograph, and ion-exchange chromatography. One or more of such purification steps can be employed.

[0062] In some cases, where the HCV El and / or E2 polypeptide is a fusion protein comprising the HCV El and / or E2 polypeptide and a fusion partner, the fusion protein is purified on an affinity column comprising an antibody specific for the fusion partner, or other affinity partner that binds the fusion partner, immobilized on an insoluble support. Purification can be achieved using Protein A-immobilized columns or Protein G-immobilized columns ( in the case of an Ig Fc-tagged E1 / E2 heterodimer); Protein L-immobilized columns (in the case of Ig light chain-tagged El and / or E2); Ig-immobilized columns (in the case of Protein A-or Protein G-tagged El and / or E2); nickel columns (Ni-columns) (in the case of poly(His)-tagged El and / or E2); or Glutathione-immobilized columns (in the case of Glutathione-S- transferase-tagged El and / or E2), as examples. In some cases, where the HCV El and / or E2 polypeptide is a fusion protein comprising the HCV El and / or E2 polypeptide and a fusion partner, and where the fusion partner is an Ig Fc polypeptide, the fusion protein can be purified on a Protein A column (i.e., affinity chromatography using Protein A immobilized on an insoluble support). Suitable methods that can be used for purifying an affinity-tagged HCV El polypeptide, an HCV E2 polypeptide, or an HCV E1 / E2 heterodimer, are described in, e.g., U.S. Patent No. 6,121,020; U.S. Patent No. 6,274,148; and Mazzocca et al. (2005) J. Biol. Chem. 280:11329. For example, in some cases, an affinity tagged HCV E1 / E2 heterodimer can be prepared in a method comprising cell disruption and debris removal by microfiltration, followed by purification using three subsequent chromatographic steps: lectin affinity chromatography, hydroxyapatite chromatography, and ion exchange chromatography.

[0063] In some cases, where the HCV El and / or E2 polypeptide is a fusion protein comprising the heterologous polypeptide and a fusion partner, and where the fusion partner is an Ig Fc polypeptide, and where a proteolytically cleavable linker is interposed between the Ig Fc and the HCV El and / or E2 polypeptide, the fusion protein can be purified on a Protein A column (i.e., affinity chromatography using Protein A immobilized on an insoluble support). The fusion protein can be immobilized on the Protein A column; and an enzyme that cleaves a proteolytic cleavage site in the proteolytically cleavablelinker is applied to the column comprising the immobilized fusion protein; the enzyme releases the heterologous polypeptide from the Protein A column.

[0064] In some cases, a method of producing an HCV E1 / E2 heterodimer disclosure comprises: a) contacting a lysate of a genetically modified host cell (where the genetically modified host cell produces an affinity tagged HCV E1 / E2 heterodimer (e.g., an HCV E2 polypeptide and an HCV El-affinity tag fusion polypeptide; an HCV E2-affinity tag fusion polypeptide and an HCV El polypeptide; or an HCV El -affinity tag fusion polypeplide and an HCV E2-affinity tag fusion polypeptide) with an affinity tagbinding polypeptide immobilized on an insoluble support, where the affinity tagged HCV E1 / E2 heterodimer present in the lysate binds to the immobilized affinity tag-binding polypeptide, generating an immobilized affinity tagged HCV E1 / E2 heterodimer; and b) contacting the immobilized affinity tagged HCV E1 / E2 heterodimer with an enzyme that cleaves between the affinity tag and the affinity-tagged HCV El and / or E2 polypeptides, thereby releasing an untagged HCV E1 / E2 heterodimer; and collecting the released untagged HCV E1 / E2 heterodimer. In some cases, the untagged HCV E1 / E2 heterodimer is subjected to one or more further purification steps.

[0065] Where the affinity tag is an Ig Fc polypeptide, suitable Ig Fc binding moieties include, but are not limited to, Protein A (Graille et al. (2000) Proc. Natl. Acad. Sci. USA 97:5399); Protein G (Sjobring et al. (1991) J. Biol. Chem. 266:399); and a Protein A / G fusion polypeptide (Eliasson et al. (1988) J. Biol. Chem. 263:4323).

[0066] The Ig Fc binding moiety can be immobilized onto a solid support, where the solid support can be of any of a variety of forms, e.g., a bead, a magnetic bead, a plate, and the like. The solid support can be made of any of a variety of materials, including, but not limited to, polystyrene, agarose, polyesters, polyethylene, and the like.

[0067] An untagged HCV E1 / E2 heterodimer can be purified such that the untagged HCV E1 / E2 heterodimer is at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, at least 99% pure, or greater than 99% pure.Examples of Non-Limiting Aspects of the Disclosure

[0068] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0069] Aspect 1. A method of producing a hepatitis C virus (HCV) El polypeptide, an HCV E2 polypeptide, or an E1 / E2 heterodimer, the method comprising culturing genetically modified mammalian cells in vitro in a liquid culture medium, wherein 100% oxygen is perfused into the liquid culture medium, wherein the cells are cultured at a cell density of from about 1 x 106 cells / ml to about 20 x 106 cells / ml, and wherein the cells are genetically modified with one or more recombinant expression vectors comprising nucleotide sequences encoding HCV El and / or E2 polypeptides.

[0070] Aspect 2. The method of aspect 1, wherein the cells are genetically modified Chinese hamster ovary (CHO) cells.

[0071] Aspect 3. The method of aspect 1 or aspect 2, wherein the HCV El polypeptide, the HCV E2 polypeptide, or the HCV E1 / E2 heterodimer is produced in an amount of from about 50 mg / kg cells to about 400 mg / kg cells.

[0072] Aspect 4. The method of any one of aspects 1-3, wherein the cells are cultured for a period of time of up to 2 years.

[0073] Aspect 5. The method of any one of aspects 1-4, comprising replacing from about 20% to about 90% of the culture medium every 2 to 4 days.

[0074] Aspect 6. The method of aspect 5, wherein from about 70% to about 80% of the cells are harvested every 3 to 4 days.

[0075] Aspect 7. The method of aspect 6, comprising lysing the harvested cells.

[0076] Aspect 8. The method of any one of aspects 1-7, comprising isolating the HCV El and / or E2 polypeptides from the cells or from a lysate of the cells.

[0077] Aspect 9. The method of aspect 8, wherein the HCV El and / or E2 polypeptide is purified by a method comprising one or more of: affinity chromatography, size exclusion chromatography, high-performance liquid chromatograph, and ion-exchange chromatography.

[0078] Aspect 10. The method of any one of aspects 1-7, wherein the El and / or the E2 polypeptide is an affinity-tagged fusion polypeptide comprising a heterologous polypeptide that facilitates purification of the E1 / E2 heterodimer.

[0079] Aspect 11. The method of aspect 10, comprising lysing the cells to generate a cell lysate comprising the affinity-tagged E1 / E2 heterodimer.

[0080] Aspect 12. The method of aspect 10 or aspect 11, wherein the affinity-tagged heterodimer comprises: a) an HCV El polypeptide; and b) an HCV E2 polypeptide, wherein one or both of the HCV El and HCV E2 polypeptides is a fusion polypeptide comprising: i) the HCV El polypeptide or the HCV E2 polypeptide; and i) an affinity tag polypeptide, and wherein the fusion polypeptide comprises a proteolytically cleavable peptide linker between the affinity tag polypeptide and the HCV El or HCV E2 polypeptide.

[0081] Aspect 13. The method of aspect 11 or aspect 12, wherein the affinity-tagged heterodimer comprises: a) an HCV El polypeptide; and b) a fusion polypeptide comprising, in order from N-terminus to C-terminus: i) an affinity tag polypeptide; ii) a proteolytically cleavable linker; and iii) an HCV E2 polypeptide.

[0082] Aspect 14. The method of aspect 12 or aspect 13, wherein the proteolytically cleavable linker is selected from the group consisting of DDDDK (SEQ ID NO:72), LEVLFQGP (SEQ ID NO:74), ENLYFQS (SEQ ID NO:97), I(E / D)GR, and LVPRGS (SEQ ID NO:98).

[0083] Aspect 15. The method of any one of aspects 11-14, comprising:

[0084] a) contacting the cell lysate with an affinity tag-binding polypeptide immobilized on an insoluble support, wherein the affinity tagged HCV E1-E2 heterodimer present in the lysate binds to the immobilized affinity tag -binding polypeptide, generating an immobilized affinity tagged HCV E1-E2 heterodimer; and

[0085] b) contacting the immobilized HCV E1-E2 heterodimer with a protease that cleaves the proteolytically cleavable linker present in the immobilized affinity tagged HCV E1-E2 heterodimer, thereby releasing the HCV E1-E2 heterodimer from the affinity tag; and

[0086] c) collecting the E1 / E2 heterodimer.

[0087] Aspect 16. The method of any one of aspects 10-15, wherein the affinity tag polypeptide is an immunoglobulin (Ig) Fc polypeptide.

[0088] Aspect 17. The method of aspect 15 or aspect 16, wherein the affinity tag-binding polypeptide is an Ig Fc-binding polypeptide.

[0089] Aspect 18. The method of aspect 17, wherein the Ig Fc-binding polypeptide is Protein A, Protein G, or a Protein A / G fusion.

[0090] Aspect 19. The method of any one of aspects 15-18, further comprising subjecting the E1 / E2 heterodimer to one or both of size exclusion chromatography and ion-exchange chromatography.EXAMPLES

[0091] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); sor sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal (ly); s.c., subcutaneous(ly); and the like.Example 1:

[0092] Four-liter or 10-liter spinners were batch harvest / batch refed 2 or 3 times weekly. Using the cell density count, the culture harvest volume was calculated to obtain: when fresh medium was added to the remaining culture after harvest, the final volume was again at the starring full 4 or 10 liters volume at an adjusted cell density of 0.2 - 0.3 E6 cells / ml for a 4 day batch growth, and to 0.3 - 0.4E6 cells / ml for a 2 or 3 day batch growth.

[0093] Run conditions of the UA bioreactor process were as follows:1.) Closed system multiple batch harvest / refeed Corning 4 liter spinners or multiple Corning 10 liter spinners.2.) Headspace sweep using 5% CCLin Air.3.) Bubbler sparging using 5% CO2 in Air or 100% oxygen at 12 ml / minute in the 4 liter spinners and 24 to 36 ml / minute for the 10 liter spinners.4.) Closed system batch harvest / refeeds and collections at 2, 3, or 4 day intervals when cells reach a cell density of greater than / equal to 1.0 E6 cells / ml.5.) Refeed cell concentrations of 0.2 to 0.4E6 cells / ml.6.) Allow collected Cell Harvest to settle at 4 degrees C for a maximum 7 days after collection.7.) Centrifugation of settled cells and a IX PBS wash before storage at -80 degrees C for later purification use. No cryoprotectant was used.8.) Lonza ProCHO4 medium was used with added glucose to final 6.0 g / liter, glutamine final 4 mM, and 0.5% FBS.

[0094] A comparison of the cell culture using cell culture media perfused with: i) air (5% CO2 in air); ii) 100% O2 ; iii) initially (days 0 to 13) with air (5% CO2 in air), then switched to 100% oxygen (days 13- 85); or iv) air (5% CO2 in air) days 0 - 13; and continued in air (5% CO2 in air) days 13-85, is provided in the Table below.

[0095] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a hepalilis C virus (HCV) El polypeptide, an HCV E2 polypepride, or an E1 / E2 heterodimer, the method comprising culturing genetically modified mammalian cells in vitro in a liquid culture medium, wherein 100% oxygen is perfused into the liquid culture medium, wherein the cells are cultured at a cell density of from about 1 x 106cells / ml to about 20 x 106cells / ml, and wherein the cells are genetically modified with one or more recombinant expression vectors comprising nucleotide sequences encoding HCV El and / or E2 polypeptides.

2. The method of claim 1, wherein the cells are genetically modified Chinese hamster ovary (CHO) cells.

3. The method of claim 1 or claim 2, wherein the HCV El polypeptide, the HCV E2 polypeptide, or the HCV E1 / E2 heterodimer is produced in an amount of from about 50 mg / kg cells to about 400 mg / kg cells.

4. The method of any one of claims 1-3, wherein the cells are cultured for a period of time of up to 2 years.

5. The method of any one of claims 1-4, comprising replacing from about 20% to about 90% of the culture medium every 2 to 4 days.

6. The method of claim 5, wherein from about 70% to about 80% of the cells are harvested every 3 to 4 days.

7. The method of claim 6, comprising lysing the harvested cells.

8. The method of any one of claims 1-7, comprising isolating the HCV El and / or E2 polypeptides from the cells or from a lysate of the cells.

9. The method of claim 8, wherein the HCV El and / or E2 polypeptide is purified by a method comprising one or more of: affinity chromatography, size exclusion chromatography, high- performance liquid chromatograph, and ion-exchange chromatography.

10. The method of any one of claims 1-7, wherein the El and / or the E2 polypepride is an affinity-tagged fusion polypeptide comprising a heterologous polypeptide that facilitates purification of the E1 / E2 heterodimer.

11. The method of claim 10, comprising lysing the cells to generate a cell lysate comprising the affinity-tagged E1 / E2 heterodimer.

12. The method of claim 10 or claim 11, wherein the affinity-tagged heterodimer comprises: a) an HCV El polypeptide; and b) an HCV E2 polypeptide, wherein one or both of the HCV El and HCV E2 polypeptides is a fusion polypeptide comprising: i) the HCV El polypeptide or the HCV E2 polypeptide; and i) an affinity tag polypeptide, and wherein the fusion polypeptide comprises a proteolytically cleavable peptide linker between the affinity tag polypeptide and the HCV El or HCV E2 polypeptide.

13. The method of claim 11 or claim 12, wherein the affinity-tagged heterodimer comprises: a) an HCV El polypeptide; and b) a fusion polypeptide comprising, in order from N-terminus to C-terminus: i) an affinity tag polypeptide; ii) a proteolytically cleavable linker; and iii) an HCV E2 polypeptide.

14. The method of claim 12 or claim 13, wherein the proteolytically cleavable linker is selected from the group consisting of DDDDK (SEQ ID NO:72), LEVLFQGP (SEQ ID NO:74), ENLYFQS (SEQ ID NO:97), I(E / D)GR, and LVPRGS (SEQ ID NO:98).

15. The method of any one of claims 11-14, comprising: a) contacting the cell lysate with an affinity tag-binding polypeptide immobilized on an insoluble support, wherein the affinity tagged HCV E1-E2 heterodimer present in the lysate binds to the immobilized affinity tag-binding polypeptide, generating an immobilized affinity tagged HCV E1-E2 heterodimer; and b) contacting the immobilized HCV E1-E2 heterodimer with a protease that cleaves the proteolytically cleavable linker present in the immobilized affinity tagged HCV E1-E2 heterodimer, thereby releasing the HCV E1-E2 heterodimer from the affinity tag; and c) collecting the E1 / E2 heterodimer.

16. The method of any one of claims 10-15, wherein the affinity tag polypeptide is an immunoglobulin (Ig) Fc polypeptide.

17. The method of claim 15 or claim 16, wherein the affinity tag-binding polypeptide is an Ig Fc-binding polypeptide.

18. The method of claim 17, wherein the Ig Fc-binding polypeptide is Protein A, Protein G, or a Protein A / G fusion.

19. The method of any one of claims 15-18, further comprising subjecting the E1 / E2 heterodimer to one or both of size exclusion chromatography and ion-exchange chromatography.

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