WO2016141201A2 - Rational vaccine design for hepatitis c virus - Google Patents

Abstract

Compositions and methods are provided relating to HCV E2 protein and modifications thereto which enhance the immunogenicity of the protein for vaccine development with respect to the generation of a neutralizing immune response.

Description

RATIONAL VACCINE DESIGN FOR HEPATITIS C VIRUS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/127,911 , filed March 4, 2015, and U.S. Provisional Patent Application No. 62/216,485, filed September 10, 2015, which applications are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2412-423) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, Asn417Ser, is associated with a glycosylation shift from Asn417 to Asn415 that enables HCV to escape neutralization by monoclonal antibodies (mAbs) such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the Asn417Ser mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn415, we determined the structure of this broadly neutralizing mAb in complex with its E2412-423 epitope to 2.0 A resolution. The conformation of E2412-423 bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, due to disruption of the β-hairpin through interactions with the unusually long complementarity- determining region 3 (CDR3) of the HC33.1 heavy chain. Whereas Asn415 is buried by HCV1 and AP33, it is solvent exposed in the HC33.1-E2412-423 complex, such that glycosylation of Asn415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2412- 423 epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

[0003] Hepatitis C virus (HCV), a member of the Flaviviridae family of positive-stranded RNA viruses, infects at least 2% of the world population and is a major cause of liver cirrhosis, liver failure, and hepatocellular carcinoma. The global burden is estimated at 170 million infected individuals with an annual increase of 3-4 million new infections. The recent introduction of successful HCV-specific direct-acting antiviral agents (DAAs) represents a major advance in HCV therapy. However, the high cost of DAAs precludes their availability to the large majority of HCV-infected individuals living in developing countries. In addition, health care workers with occupational risk for blood-borne pathogens and injection drug users will remain at risk for repeated exposure to HCV, even after successful DAA treatment. Thus, there is a compelling need for a preventive HCV vaccine. The key challenge in HCV vaccine development is to overcome the high diversity of this virus and its potential to escape from host immune responses.

[0004] HCV is composed of a nucleocapsid core enveloped by a lipid bilayer in which two surface glycoproteins, E1 and E2, are anchored. Entry of HCV into hepatocytes is mediated by interactions between the E1E2 heterodimer and at least four cellular receptors: the tetraspanin CD81 , scavenger receptor class B type 1 (SR-B1), and the tight junction proteins occludin and claudin 1. An effective vaccine must include conserved epitopes of E1E2 that are able to elicit broadly neutralizing antibodies. Accordingly, much effort has been focused on the identification of conserved regions of E1E2 mediating virus neutralization through the isolation and characterization of monoclonal antibodies (mAbs) from HCV-infected individuals and experimentally immunized mice. The large majority of these mAbs recognize E2, which is more immunogenic than E1. In addition, E2 contains binding sites for CD81 and SR-B1.

[0005] A significant challenge for vaccine development is defining conserved epitopes that i) are capable of eliciting protective antibodies in this highly diverse virus, and ii) are resistant to development of escape mutants. Treatment of HCV and the development of vaccines that broadly protect against highly diverse HCV genotypes and subtypes are of interest in the field. The present invention addresses this issue.

SUMMARY OF THE INVENTION

[0006] Compositions and methods are provided relating to human anti-HCV vaccines for prophylaxis against infection with different genotypes and subtypes of HCV. HCV E2 glycoprotein, which is the major target of neutralizing antibody response to virus, is modified to enhance the protective immune response. Such modified polypeptides, which may be referred to as an HCV E2 antigen, or antigenic polypeptide, are typically at least about 50 amino acids of contiguous E2 sequence, at least about 100 amino acids, at least about 200 amino acids, up to substantially all of the E2 protein. These polypeptides find use in screening assays, generation of monoclonal antibodies, and in vaccines. For the purposes of discussion, reference is made to the sequence of HCV set forth in SEQ ID NO:1 , which provides an exemplary polyprotein from HCV, where the E2 protein extends from amino acids 394-746 (NCBI refseq ID NP_751921.1). The modifications to the E2 polypeptide described herein can be utilized in various formats and within the context of different HCV E2 genotypes.

[0007] In some embodiments of the invention, a modified HCV E2 polypeptide is provided. In other embodiments, a polynucleotide encoding such a modified HCV E2 polypeptide is provided. The polypeptide and/or the nucleic acid can be used in the formulation of a vaccine, e.g. a virus-like particle, a recombinant protein vaccine which can be formulated with an adjuvant, a vector vaccine, and the like. In some embodiments, a vaccine formulation comprising a polypeptide or a polynucleotide of the invention is provided.

[0008] In some embodiments, an HCV provides for a partial or complete deletion of the HVR1 region. The deletion may comprise some or all of amino acids 384-410 (for convenience, the numbering is shown in Figure 17). In some embodiments, the HCV E2“backbone” comprises E^{2 sequence, amino acid residues 408-746, herein referenced as ^HVR} _{408. In some} embodiments the HCV E2 backbone sequence comprises amino acid residues 411-746, herein referenced as ^HVR1₄₁₁. In some embodiments the HVR1 sequence is not deleted, and the backbone comprises amino acid residues 384-746, herein referenced as sE2. Optionally any of these backbone sequences may further comprise a deletion of the C-terminal region, amino acid residues 662-746.

[0009] In some embodiments, a backbone HCV E2 polypeptide as defined above comprises one or both of the following modifications: (a) amino acid substitution N415P; (b) amino acid substitution N417P; (c) amino acid substitution A439P; (d) amino acid substitution H445P. In some such embodiments the amino acid substitution is one or both of N415P and H445P. These amino acid substitutions affect the conformation of the antigenic polypeptide. Data provided herein suggests that HCV structural flexibility provides an immune evasion strategy that can be overcome by amino acid substitutions that decrease flexibility.

[0010] In some embodiments of the invention, a backbone HCV E2 polypeptide, as defined above, which is optionally combined with one or more amino acid substitutions that affect conformation described above, is modified to provide for insertion of a glycan. Highly immunodominant epitopes that are associated with viral escape or non-neutralizing antibodies can be masked so as to focus an immune response to epitopes that are less immunodominant, but which are essential for virus entry and therefore are less likely to be altered in virus escape mutation and selection. In this modification, amino acid substitutions are made to generate a motif for N-glycosylation. Such modification may comprise replacing residue Y632 with asparagine, i.e. Y632N and introduction of a serine or threonine at G634, i.e. G634S or G634T. Alternatively, R630 can be replaced with asparagine, R630N and Y632 can be replaced with serine or threonine, i.e. Y632S or Y632T. Polypeptides incorporating these changes are expressed in a cell that provides for correct N-glycosylation, including without limitation mammalian cells.

[0011] In some embodiments, modifications comprising: deletion of HVR1 region, introduction of one or more proline residues to stabilize confirmation, and introduction of residues to generate a motif for glycosylation, are all introduced in the HCV E2 polypeptide to generate an HCV E2 antigen. In other embodiments, one or more modifications from each group are introduced. In other embodiments, one or more modifications from a single group, from two groups, or from 3 groups are introduced.

[0012] The modified E2 polypeptide may further comprise one or more of the following amino acid changes: N417Q, N423Q, N448Q and N532Q, as described in published US Patent application 2015/0086580, herein specifically incorporated by reference. These mutations are to remove specific glycans near regions that elicit broadly neutralizing antibodies.

[0013] Other aspects and features will be readily apparent to the ordinarily skilled artisan upon reading the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The invention is best understood from the following detailed description of exemplary embodiments when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to- scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

[0015] Figure 1 , Panels A-C. Location of antigenic domains on the HCV E2 envelope glycoprotein. The E2 structure (PDB code 4MWF) is shown as a molecular surface shaded according to antigenic domains A-E (A: 581-584, 627-633; B: 431-439, 529-535; C: 544-549; D: 420-428, 441-443; E: 408-423).

[0016] Figure 2. Energetic footprint mapping of epitope domain E. A panel of antibodies was used to map the energetic footprint on E2 epitope domain E via alanine scanning mutagenesis. Alanine substitution mutants were constructed in plasmids carrying the 1a H77C E1E2 coding sequence (GenBank accession nos. AF009606) as previously described. All the mutations were confirmed by DNA sequence analysis (Sequetech, Mountain View, CA) for the desired mutation and for exclusion of unexpected residue changes in the full-length E1E2 encoding sequence. E2 mutant proteins were expressed in 293T cells and cell lysates were analyzed by ELISA. Individual protein expression was normalized by binding of CBH-17, an HCV E2 Human mAb to a linear epitope. The mutated constructs were designated X#Y, where # is the residue location in H77C, X denotes the single-letter code for the H77C amino acid, and Y denotes the altered amino acid. Data for H77.16, which binds HVR1 proximal to epitope domain E, are shown for comparison.

[0017] Figure 3. Energetic footprint mapping of epitope domain D. A panel of antibodies (HC84.20, HC84.24, HC84.26) was used to map the energetic footprint on E2 epitope domain D via alanine scanning mutagenesis. Alanine substitution mutants were constructed in plasmids carrying the 1a H77C E1E2 coding sequence (GenBank accession nos. AF009606) as previously described. All the mutations were confirmed by DNA sequence analysis (Sequetech, Mountain View, CA) for the desired mutation and for exclusion of unexpected residue changes in the full-length E1E2 encoding sequence. E2 mutant proteins were expressed in 293T cells and cell lysates were analyzed by ELISA. Individual protein expression was normalized by binding of CBH-17, an HCV E2 human monoclonal antibody to a linear epitope. The mutated constructs were designated X#Y, where # is the residue location in H77C, X denotes the single- letter code for the H77C amino acid, and Y denotes the altered amino acid. Data for non- epitope domain D mAbs HC-1, CBH-4D, and CBH-7 are shown for comparison.

[0018] Figure 4. Distinct epitope structures of domain E (aa 412-423). The linear epitope from epitope domain E adopts an extended conformation when engaged by the HC33.1 human mAb (A) [12], versus the β hairpin conformation observed when bound to other antibodies including HCV1 (B), which was obtained via immunization of humanized mice.

[0019] Figure 5. Computational analysis of E2 domain E residues for proline backbone conformation. The HC33.1-E2_412-423 structure (PDB code 4XVJ) was analyzed by a Ramachandran plot analysis tool, which maps residue ϕψ backbone angle conformations onto amino acid backbone probability densities determined from a large set of x-ray crystal structures. This was used to identify positions suitable for proline substitution. The point corresponding to the N415 residue is labeled and shown as a blue square.

[0020] Figure 6. Structural models of N415P and H445P E2 proline mutants. The structures of the HC33.1-E2_412-423 complex (A) and HC84.26AM-E2_434-446 complex (C) were used to generate models of the proline mutant N415P (B) and H445P (D) using the modeling program Rosetta.

[0021] Figure 7. Binding of E2,∆HVR1, and∆HVR1₄₀₈ to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each human mAb Human mAbat 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y-axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0022] Figure 8. Binding of E2∆HVR1,∆HVR1 N417T, and∆HVR1 N415P to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre- coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y- axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0023] Figure 9. Binding of E2, E2∆HVR1,∆HVR1 N417T, and∆HVR1 N415P to the AP33 epitope domain E mAb. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y-axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0024] Figure 10. Binding of E2, N448Q, A439P, A440C/W616C to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y-axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the Sf2 genotype 1b strain.

[0025] Figure 11. Binding of E2 H445P,∆HVR1₄₀₈ H445P, and∆HVR1₄₁₁ H445P to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre- coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y- axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0026] Figure 12. Energetic footprint mapping of epitope domain A. A panel of antibodies (CBH-4D, CBH-4G, CBH-4B, CBH-20, CBH-21, and CBH-22) was used to map the energetic footprint on E2 epitope domain A via alanine scanning mutagenesis. Alanine substitution mutants were constructed in plasmids carrying the 1a H77C E1E2 coding sequence (GenBank accession nos. AF009606) as previously described. All the mutations were confirmed by DNA sequence analysis (Sequetech, Mountain View, CA) for the desired mutation and for exclusion of unexpected residue changes in the full-length E1E2 encoding sequence. E2 mutant proteins were expressed in 293T cells and cell lysates were analyzed by ELISA. Individual protein expression was normalized by binding of CBH-17, an HCV E2 human mAb to a linear epitope. The mutated constructs were designated X#Y, where # is the residue location in H77C, X denotes the single-letter code for the H77C amino acid, and Y denotes the altered amino acid. Data from an antigenic domain B antibody (HC-1) and an epitope domain D antibody (HC84.26) are shown for comparison.

[0027] Figure 13. Structural basis for glycan-mediated antibody binding disruption in epitope domain A. A model of a glycan at E2 position 630, from the double mutant R630N/Y632T was generated using Rosetta and GlyProt. Glycans are shown as sticks and labeled according to their associated Asn residue. The E2 protein is shown using surface representation, with the region corresponding to epitope domain A. The structure of a non-neutralizing antibody that binds this region (2A12) is shown for reference.

[0028] Figure 14. Binding of∆HVR1 F627N/V629T and∆HVR1 R630N/Y632T to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre- coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y- axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0029] Figure 15. Binding of E2 K628N/R630S,∆HVR1₄₀₈ K628N/R630S, and∆HVR1₄₁₁ K628N/R630S to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y-axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0030] Figure 16. Binding of E2 Y632N/G634S,∆HVR1₄₀₈ Y632N/G634S, and∆HVR1₄₁₁ Y632N/G634S to a panel of E2 mAbs. Assays were performed with 2 μg of purified E2/ml that was captured by GNA pre-coated wells, and followed by incubation with each human mAb at 1 and 5 µg/ml (x-axis). Specific binding was detected by alkaline phosphatase-conjugated goat anti-human IgG. The y-axis shows the mean optical density (O.D.) values for triplicate wells, the mean of two experiments ±SD. All E2 constructs were produced starting from the H77c strain.

[0031] Figure 17. Schematic of sequence modifications. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] It is to be understood that the 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.

[0033] 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 limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated 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 or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is 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.

[0034] 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 be used in the practice or testing of the present invention, exemplary 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. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0035] 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 "a cell" includes a plurality of such cells and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth.

[0036] It is further noted that the claims may be drafted to exclude any element which may be optional. 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 the use of a“negative” limitation.

[0037] 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. Definitions

[0038] By“Flaviviridae virus” or“flavivirus” is meant any virus from the Flaviviridae family, including those viruses that infect humans and non-human animals. The polynucleotide and polypeptides sequences encoding these viruses are well known in the art, and may be found at NCBI’s GenBank database, e.g., as Genbank Accession nos. NC_004102, AB031663, D11355, D11168, AJ238800, NC_001809, NC_001437, NC_004355 NC_004119, NC_003996, NC_003690, NC_003687, NC_003675, NC_003676, NC_003218, NC_001563, NC_000943, NC_003679, NC_003678, NC_003677, NC_002657, NC_002032, and NC_001461, the contents of which database entries are incorporated by references herein in their entirety. In general the term“flavivirus” includes any member of the family Flaviviridae, including, but not limited to, Dengue virus, including Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4 (see, e.g., GenBank Accession Nos. M23027, M19197, A34774, and M14931); Yellow Fever Virus; West Nile Virus; Japanese Encephalitis Virus; St. Louis Encephalitis Virus; Bovine Viral Diarrhea Virus (BVDV); and Hepatitis C Virus (HCV); and any serotype, strain, genotype, subtype, quasispecies, or isolate of any of the foregoing. Where the flavivirus is HCV, the HCV is any of a number of genotypes, subtypes, or quasispecies, including, e.g., genotype 1, including 1a and 1b, 2, 3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.

[0039] The terms "hepatitis C virus," "HCV," "non- A non-B hepatitis," or "NANBH" are used interchangeably herein, and include any "genotype" or "subgenotype" (also termed "subtype") of the virion, or portion thereof (e.g., a portion of the E2 protein of genotype Ia of HCV), that is encoded by the RNA of hepatitis C virus or that occurs by natural allelic variation. The HCV genome comprises a 5'-untranslated region that is followed by an open reading frame (ORF) that codes for about 3,010 amino acids. The ORF runs from nucleotide base pair 342 to 8,955 followed by another untranslated region at the 3' end. The amino acids are subdivided into ten proteins in the order from 5' to 3' as follows: C; El; E2; NSl; NS2; NS3; NS4 (a and b); and NS5 (a and b). These proteins are formed from the cleavage of the larger polyprotein by both host and viral proteases. The C, El, and E2 proteins are structural and the NS1-NS5 proteins are nonstructural proteins. The C region codes for the core nucleocapsid protein. El and E2 are glycosylated envelope proteins that coat the virus. NS2 may be a zinc metalloproteinase. NS3 is a helicase. NS4a functions as a serine protease cofactor involved in cleavage between NS4b and NS5a. NS5a is a serine phosphoprotein whose function is unknown. The NS5b region has both RNA-dependent RNA polymerase and terminal transferase activity.

[0040] There are about six major distinct HCV genotypes (e.g., genotypes 1, 2, 3, 4, 5, and 6) that are categorized by variations in the core protein and over 80 subgenotypes which exhibit further variation within each genotype, some of which include: Ia; Ib; Ic; 2a; 2b; 2c; 3a; 3b; 4a; 4b; 4c; 4d; 4e; 5a; and 6a.

[0041] In infected cells, the HCV RNA is directly translated into a continuous 3011-residue polypeptide chain. As a reference, a detailed annotation of the polyprotein, with the boundaries of cleavage products, may be found at Genbank, accession no. P27958, or AF009606, herein specifically incorporated by reference. The polyprotein is subsequently cleaved to yield envelope and core proteins, which assemble into new virus particles, and enzymes essential for viral replication. At least ten proteins are encoded by the HCV genome. Both host cell and virally encoded proteases are required for maturation of the HCV polyprotein. Initial cleavages catalyzed by host proteases liberate individual envelope and capsid proteins, located at the polyprotein N terminus, and the portion of the HCV polyprotein that contains the viral enzymes, or nonstructural (NS) proteins. This polyprotein segment contains the free N terminus of nonstructural protein 2 (NS2)— an integral membrane protein with proteolytic activity. NS2, either alone or in conjunction with NS3, then cleaves the NS2–NS3 scissile bond in what is termed cis processing, because cleavage involves an intramolecular autoproteolysis step. The NS2-mediated autoproteolysis generates the NS3 N terminus, and releases a 1984-residue polyprotein that incorporates a bifunctional 631-residue enzyme (NS3) possessing both serine protease and RNA helicase activities. The NS3 protease is responsible for processing the remaining polyprotein.

[0042] As a reference, the amino acid sequence of the E2 protein, which comprises residues 384-746 of the polyprotein for genotype 1, isolate 1a H77C (Genbank) is provided as SEQ ID NO:1, and modifications to the sequence are made with reference to SEQ ID NO:1. It will be understood by one of skill in the art that corresponding modifications are readily made in other HCV genotypes, by modifying the residue that corresponds to the named position in SEQ ID NO:1, and that for continuity with the literature, SEQ ID NO:1 may be considered to start with amino acid residue 384. In other words, for convenience in comparison to the literature, the proteins of the invention are numbered with respect to the HCV polyprotein, i.e. the starting residue of E2 is 384 and the ending residue is 746. Those of skill in the art will readily determine that numbering of these changes will vary depending on the selection of start site, and can easily be mapped to the reference sequences.

[0043] The antigenic protein of the invention is optionally truncated at the C-terminus, such that the protein comprises residues 384 to 746 (or 661). The antigenic protein of the invention is optionally truncated at the amino terminus, such that the protein comprises residues 408 to 746 (or 408 to 661); or residues 411 to 746 (or residues 411 to 661).

[0044] As used herein, the terms "neutralizes HCV," "inhibits HCV," and "blocks HCV" are used interchangeably to refer to the ability of an antibody of the invention to prevent HCV from infecting a given cell.

[0045] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system.

[0046] "Polypeptide" and "protein" as used interchangeably herein, can encompass peptides and oligopeptides. Where "polypeptide" is recited herein to refer to an amino acid sequence of a naturally-occurring protein molecule, "polypeptide" and like terms are not necessarily limited to the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule, but instead can encompass biologically active variants or fragments, including polypeptides having substantial sequence similarity or sequence identify relative to the amino acid sequences provided herein. In general, fragments or variants retain a biological activity of the parent polypeptide from which their sequence is derived.

[0047] As used herein, " polypeptide" refers to an amino acid sequence of a recombinant or non-recombinant polypeptide having an amino acid sequence of i) a native polypeptide, ii) a biologically active fragment of an polypeptide, or iii) a biologically active variant of an polypeptide. Polypeptides suitable for use can be obtained from any species, e.g. , mammalian or non-mammalian (e.g. , reptiles, amphibians, avian (e.g. , chicken)), particularly mammalian, including human, rodent (e.g., murine or rat), bovine, ovine, porcine, murine, or equine, particularly rat or human, from any source whether natural, synthetic, semi-synthetic or recombinant. In general, polypeptides comprising a sequence of a human polypeptide are of particular interest.

[0048] The term "derived from" indicates molecule that is obtained directly from the indicated source (e.g., when a protein directly purified from a cell, the protein is "derived from" the cell) or information is obtained from the source, e.g. nucleotide or amino acid sequence, from which the molecule can be synthesized from materials other than the source of information.

[0049] The term "isolated" indicates that the recited material (e.g, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature (e.g., in a cell). A material (e.g., polypeptide, nucleic acid, etc.) that is isolated constitutes at least about 0.1 %, at least about 0.5%, at least about 1 % or at least about 5% by weight of the total material of the same type (e.g., total protein, total nucleic acid) in a given sample.

[0050] The terms "subject" and "patient" are used interchangeably herein to mean a member or members of any mammalian or non-mammalian species that may have a need for the pharmaceutical methods, compositions and treatments described herein. Subjects and patients thus include, without limitation, primate (including humans), canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)), avian, and other subjects. Humans and non-human animals having commercial importance (e.g. , livestock and domesticated animals) are of particular interest. As will be evidence from the context in which the term is used, subject and patient refer to a subject or patient susceptible to infection by a Flaviviridae virus, particularly HCV.

[0051 ] "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, particularly humans. Non-human animal models, particularly mammals, e.g. primate, murine, lagomorpha, etc. may be used for experimental investigations.

[0052] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. [0053] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0054] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and is usually free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal and the like.

[0055] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. "Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[0056] As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0057] Unless specifically indicated to the contrary, the term "conjugate" as described and claimed herein is defined as a heterogeneous molecule formed by the covalent attachment of one or more polypeptide fragment(s) to one or more polymer molecule(s), wherein the heterogeneous molecule is water soluble, i.e. soluble in physiological fluids such as blood, and wherein the heterogeneous molecule is free of any structured aggregate. A conjugate of interest is PEG. In the context of the foregoing definition, the term "structured aggregate" refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure, such that the heterogeneous molecule is not in a micelle or other emulsion structure, and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insolubilized form, such as a chromatography bead matrix, that does not release the heterogeneous molecule into solution upon contact with an aqueous phase. Accordingly, the term "conjugate" as defined herein encompasses the aforementioned heterogeneous molecule in a precipitate, sediment, bioerodible matrix or other solid capable of releasing the heterogeneous molecule into aqueous solution upon hydration of the solid.

[0058] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by cell culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made in an immortalized B cell or hybridoma thereof, may be made by recombinant DNA methods, including without limitation yeast display.

[0059] The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody. The label may itself be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0060] By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

[0061] By the term "vaccine" as used herein, is meant a composition; a formulation comprising a modified polypeptide of the invention; a virus or virus-like particle comprising a modified polypeptide of the invention complex; or a DNA encoding a modified polypeptide of the invention complex, which, when administered to a subject, induces cellular or humoral immune responses as described herein.

[0062] Some embodiments of the invention provide a method of stimulating an immune response in a mammal, which can be a human or a preclinical model for human disease, e.g. mouse, ape, monkey etc. "Stimulating an immune response" includes, but is not limited to, inducing a therapeutic or prophylactic effect that is mediated by the immune system of the mammal. More specifically, stimulating an immune response in the context of the invention refers to eliciting cellular or humoral immune responses, thereby inducing downstream effects such as production of antibodies, antibody heavy chain class switching, maturation of APCs, and stimulation of cytolytic T cells, T helper cells and both T and B memory cells.

[0063] As appreciated by skilled artisans, vaccine compositions are suitably formulated to be compatible with the intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH of the composition can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Systemic administration of the composition is also suitably accomplished by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.

[0064] Vaccine compositions may include an aqueous medium, pharmaceutically acceptable inert excipient such as lactose, starch, calcium carbonate, and sodium citrate. Vaccine compositions may also include an adjuvant, for example Freud's adjuvant. Vaccines may be administered alone or in combination with a physiologically acceptable vehicle that is suitable for administration to humans. Vaccines may be delivered orally, parenterally, intramuscularly, intranasally or intravenously. Oral delivery may encompass, for example, adding the compositions to the feed or drink of the mammals. Factors bearing on the vaccine dosage include, for example, the weight and age of the mammal. Compositions for parenteral or intravenous delivery may also include emulsifying or suspending agents or diluents to control the delivery and dose amount of the vaccine. [0065] The modified polypeptides of the invention and polynucleotides that encode such modified polypeptides can be used in various HCV vaccine formulations known in the art, as a substitution for the wild-type HCV E2 sequence.

[0066] HCV vaccines include, without limitation, formulation of isolate polypeptides, e.g. E2 alone or in combination with E1 as separate molecules or as heterodimeric E1E2, and an adjuvant. A protein complex of HCV proteins, including E2 of the present invention, can be formulated with T-cell adjuvant immunostimulating complex matrix (IMX). The polypeptides of the invention can be fragmented to generate a peptide vaccine, e.g. administered with poly-L- arginine, can be formulated as a vaccine. Polynucleotides encoding the modified polypeptides of the invention can be administered in plasmid form, in a virus genome, including adenovirus, alphaviruses, canary pox, ovine atadenovirus and semliki-like viral particles. Advances in molecular virology have enabled the manipulation of viruses for delivery of foreign genetic material to mammalian cells. Their highly evolved mechanisms for cell entry and gene expression within the host cell remain intact and viral vectors can be rendered non-pathogenic and non-replicative by deletions at specific locus.

[0067] In some embodiments, the polypeptides of the invention are formulated for vaccine delivery as virus-like particles (VLPs).

[0068] Adenoviral (Ad) vectors are the best characterized viral vectors and have emerged as the most potent at T-cell priming in non-human primates (NHPs) and humans. Ad-based vaccines are particularly attractive gene vehicles as they can stably express large foreign inserts (∼10 kbp), they remain epichromosomal and can be easily rendered replication defective by deletion of the E1 locus.

[0069] Other definitions of terms appear throughout the specification. POLYPEPTIDE AND POLYNUCLEOTIDE COMPOSITIONS

[0070] The application discloses herein a modified HCV E2 polypeptide, which is altered from the wild-type in various ways to increase desired immune responses, which generate neutralizing antibodies across multiple HCV genotypes; and the reduce undesirable immune responses that are readily avoided by escape mechanisms. These modified polypeptides find use in screening assays, generation of monoclonal antibodies, and in vaccines.

[0071] All or a portion of the HCV E2 protein is provided, e.g. as the full-length E2 protein, or a modified peptide derived therefrom, including peptides derived from residues 408-746, residues 411-746, residues 384-746 of HCV E2 protein, where the epitope is of sufficient length to provide for binding specificity substantially similar to the specificity of binding to the native protein, e.g. a peptide of at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids up to the full length of the E2 protein, where the peptide may be a contiguous or non-contiguous sequence of an HCV E2 protein.

[0072] Peptides can be produced using techniques well known in the art. Such techniques include chemical and biochemical synthesis. Examples of techniques for chemical synthesis of peptides are provided in Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990. Examples of techniques for biochemical synthesis involving the introduction of a nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, and Sambrook, et al in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989.

[0073] HCV E2 polypeptides of the invention comprise one or more of the following modifications: deletion of all or part of the HVR1 region; one or more of (a) amino acid substitution N415P; (b) amino acid substitution N417P; (c) amino acid substitution A439P; (d) amino acid substitution H445P; and amino acid substitutions are made to generate a motif for N-glycosylation. Such modification may comprise replacing residue Y632 with asparagine, i.e. Y632N and introduction of a serine or threonine at G634, i.e. G634S or G634T. Alternatively, R630 can be replaced with asparagine, R630N and Y632 can be replaced with serine or threonine, i.e. Y632S or Y632T. Polypeptides incorporating these changes are expressed in a cell that provides for correct N-glycosylation, including without limitation mammalian cells.

[0074] The polypeptide may optionally comprise one or more of the following amino acid substitutions: N417Q, N423Q, N448Q and N532Q. These mutations are to remove specific glycans near regions that elicit broadly neutralizing antibodies. [0075] The invention also provides isolated nucleic acids encoding the modified HCV E2 polypeptide, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the modified polypeptide. As is known in the art, various polynucleotides can be devised with respect to codon usage to produce a desired polypeptide, and one of skill in the art can readily generate a polynucleotide sequence that encodes a modified E2 protein. As an optional starting point, the sequence of isolates, 1a H77C (Genbank AF009606) and 1bSF (Genbank JN118490) can be used, without limitation. In some embodiments a contiguous nucleotide sequence is at least about 20 nt., at least about 25 nt, at least about 50 nt., at least about 75 nt, at least about 100 nt, and up to the complete coding sequence may be used.

[0076] For recombinant production of the modified polypeptide, the nucleic acid encoding it is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the modified polypeptide is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0077] The modified polypeptides of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous or homologous polypeptide, which include a signal sequence or other polypeptide having a specific cleavage site at the N- terminus of the mature protein or polypeptide, and the like. A heterologous signal sequence selected preferably may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected.

[0078] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0079] The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0080] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. [0081] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0082] Suitable host cells for cloning or expressing the DNA are the prokaryote, yeast, or higher eukaryote cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1.982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0083] Host cells are transformed with the above-described expression or cloning vectors for modified polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0084] The modified polypeptide composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and the like as known in the art. For example, antibodies against E2 protein can be used as affinity reagents for purification. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE^TM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. [0085] In some embodiments of the invention, the modified polypeptide(s) are used in a screening method to select for antibodies optimized for affinity, specificity, and the like. In such screening methods, random or directed mutagenesis is utilized to generate changes in the amino acid structure of the variable region or regions, where such variable regions will initially comprise one or more of the provided CDR sequences, e.g. a framework variable region comprising CDR1, CDR2, CDR3 from the heavy and light chain sequences. Methods for selection of antibodies with optimized specificity, affinity, etc., are known and practiced in the art, e.g. including methods described by Presta (2006) Adv Drug Deliv Rev. 58(5-6):640-56; Levin and Weiss (2006) Mol Biosyst. 2(1):49-57; Rothe et al. (2006) Expert Opin Biol Ther. 6(2):177-87; Ladner et al. (2001) Curr Opin Biotechnol.12(4):406-10; Amstutz et al. (2001) Curr Opin Biotechnol. 12(4):400-5; Nakamura and Takeo (1998) J Chromatogr B Biomed Sci Appl. 715(1):125-36 each herein specifically incorporated by reference for teaching methods of mutagenesis selection. Such methods are exemplified by Wu et al. (2005) J. Mol. Biol. (2005) 350, 126–144.

[0086] In other embodiments, the modified polypeptide of the invention is used as an immunogen, including without limitation vaccine preparation. Methods of Use

[0087] In the methods disclosed herein, an immunologically effective amount of one or more modified polypeptides of the invention, which may be conjugated to a suitable carrier molecule, polynucleotides encoding such modified polypeptides, including viral vectors, are administered to a patient by administrations of a vaccine, in a manner effective to result in an improvement in the patient's condition. successive, spaced

[0088] In an exemplary embodiment, of carrier proteins represented by virus capsid proteins that have the capability to self-assemble into virus-like particles (VLPs) are utilized in combination with the modified polypeptide of the invention. Examples of VLPs used as peptide carriers are hepatitis B virus surface antigen and core antigen, hepatitis E virus particles, polyoma virus, bovine papilloma virus, and the like.

[0089] In another embodiment, modified polypeptides of the invention are coupled to one of a number of carrier molecules, known to those of skill in the art. A carrier protein must be of sufficient size for the immune system of the subject to which it is administered to recognize its foreign nature and develop antibodies to it.

[0090] In some cases the carrier molecule is directly coupled to the immunogenic peptide. In other cases, there is a linker molecule inserted between the carrier molecule and the immunogenic peptide. For example, the coupling reaction may require a free sulfhydryl group on the peptide. In such cases, an N-terminal cysteine residue is added to the peptide when the peptide is synthesized. In an exemplary embodiment, traditional succinimide chemistry is used to link the peptide to a carrier protein. Methods for preparing such peptidexarrier protein conjugates are generally known to those of skill in the art and reagents for such methods are commercially available (e.g., from Sigma Chemical Co.). Generally about 5-30 peptide molecules are conjugated per molecule of carrier protein.

[0091] Exemplary carrier molecules include proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), flagellin, influenza subunit proteins, tetanus toxoid (TT), diphtheria toxoid (DT), cholera toxoid (CT), a variety of bacterial heat shock proteins, glutathione reductase (GST), or natural proteins such as thyroglobulin, and the like. One of skill in the art can readily select an appropriate carrier molecule. In some cases, the carrier molecule is a non-protein, such as Ficoll 70 or Ficoll 400 (a synthetic copolymer of sucrose and epichlorohydrin), a polyglucose such as Dextran T 70.

[0092] A peptide vaccine composition may comprise single or multiple copies of the same or different modified polypeptide of the invention. In one aspect of this embodiment, the peptide vaccine composition may contain different immunogenic peptides with or without flanking sequences, combined sequentially into a polypeptide and coupled to the same carrier. Alternatively, immunogenic peptides, may be coupled individually as peptides to the same or a different carrier, and the resulting immunogenic peptide-carrier conjugates blended together to form a single composition, or administered individually at the same or different times.

[0093] In general, peptide vaccine compositions are administered with a vehicle. The purpose of the vehicle is to emulsify the vaccine preparation. Numerous vehicles are known to those of skill in the art, and any vehicle which functions as an effective emulsifying agent finds utility in the present invention. To further increase the magnitude of the immune response resulting from administration of the vaccine, an immunological adjuvant may be included in the vaccine formulation. Exemplary adjuvants known to those of skill in the art include water/oil emulsions, non-ionic copolymer adjuvants, e.g., CRL 1005 (Optivax; Vaxcel Inc., Norcross, Ga.), aluminum phosphate, aluminum hydroxide, aqueous suspensions of aluminum and magnesium hydroxides, bacterial endotoxins, polynucleotides, polyelectrolytes, lipophilic adjuvants and synthetic muramyl dipeptide (norMDP) analogs.

[0094] Suitable pharmaceutically acceptable carriers for use in an immunogenic proteinaceous composition of the invention are well known to those of skill in the art. Such carriers include, for example, phosphate buffered saline, or any physiologically compatible medium, suitable for introducing the vaccine into a subject.

[0095] Numerous drug delivery mechanisms known to those of skill in the art may be employed to administer the immunogenic peptides of the invention. Controlled release preparations may be achieved by the use of polymers to complex or absorb the peptides or antibodies. Controlled delivery may accomplished using macromolecules such as, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate, the concentration of which can alter the rate of release of the peptide vaccine.

[0096] In some cases, the peptides may be incorporated into polymeric particles composed of e.g., polyesters, polyamino acids, hydrogels, polylactic acid, or ethylene vinylacetate copolymers. Alternatively, the peptide vaccine is entrapped in microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or macroemulsions, using methods generally known to those of skill in the art.

[0097] In some embodiments, the vaccine is a vector. As used herein, a“vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. In one example, the techniques and products described in U.S. Pat. No. 5,990,091, International Publication Nos. WO 99/60164 and WO98/00166, van Ginkel et al.,“Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene,” J Immunol 159(2):685-93, 1997; and Osterhaus et al,“Vaccination against acute respiratory virus infections and measles in man,” Immunobiology 184(2-3):180-92, 1992, which contain information concerning expressed gene products, antibodies and uses thereof, vectors for in vivo and in vitro expression of exogenous nucleic acid molecules, promoters for driving expression or for operatively linking to nucleic acid molecules to be expressed, method and documents for producing such vectors, compositions comprising such vectors or nucleic acid molecules or antibodies, dosages, and modes and/or routes of administration (including compositions for nasal administration), inter alia, can be employed in the practice of this invention and are incorporated by herein reference in their entireties.

[0098] The vector can be a viral vector, a bacterial vector, a protozoan vector, a retrotransposon, a transposon, a virus shell, or a DNA vector. In another aspect, the viral vector, the bacterial vector, the protozoan vector and the DNA vector can be recombinant vectors. The vector can be an adenovirus. In one example, the adenovirus recombinant can include E1-defective, E3-defective, and/or E4-defective adenovirus vectors, or the“gutless” adenovirus vector, where all viral genes are deleted. Specific sequence motifs such as the RGD motif can be inserted into the H-I loop of an adenovirus vector to enhance its infectivity. An adenovirus recombinant is constructed by cloning specific transgenes or fragments of transgenes into any of the adenovirus vectors such as those described above. In one example, the adenovirus recombinant can be used to transduce epidermal or mucosal cells of a subject in a noninvasive mode for use as an immunizing agent. In one example, the adenovirus vector can be defective in its E1 region. In another example, the adenovirus vector can be defective in its E3 region. In a further example, the adenovirus vector can be defective in its E1 and E3 regions. In another example, the DNA is in plasmid form.

[0099] A vaccine of the present invention can be administered to patient by different routes such as intravenous, intraperitoneal, subcutaneous, intramuscular, or orally. A preferred route is intramuscular or oral. Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the subject; the route of administration; the desired effect; and the particular conjugate employed (e.g., the peptide, the peptide loading on the carrier, etc.). The vaccine can be used in multi-dose vaccination formats.

[00100] The timing of doses depends upon factors well known in the art. After the initial administration one or more booster doses may subsequently be administered to maintain antibody titers. An example of a dosing regimen would be a dose on day 1, a second dose at or 2 months, a third dose at either 4, 6 or 12 months, and additional booster doses at distant times as needed.

[00101] In one aspect, the invention provides a means for classifying the immune response to peptide vaccine, e.g., 9 to 15 weeks after administration of the vaccine; by measuring the level of antibodies against the immunogenic peptide of the vaccine. Formulations

[00102] The vaccine formulations of the present invention may be used in immunization for the various HCV associated diseases. In some embodiments, the recipient is at a high risk of infection.

[00103] The vaccine formulation is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the vaccine formulation is suitably administered by pulse infusion, particularly with declining doses of the vaccine.

[00104] For the prevention or treatment of disease, the appropriate dosage of vaccine will depend on the type of disease to be treated, the severity and course of the disease, whether the vaccine is administered for preventive purposes, previous therapy, the patient's clinical history and response to the vaccine, and the discretion of the attending physician. The vaccine is suitably administered to the patient at one time or over a series of treatments.

[00105] In another embodiment of the invention, an article of manufacture containing materials useful for the vaccination described above is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition is one or more antibodies in a formulation of the invention as described above. The label on, or associated with, the container indicates that the composition is used for treating the condition of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[00106] Therapeutic formulations are prepared for storage by mixing the vaccine having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The vaccine composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. The "therapeutically effective amount" of the vaccine to be administered will be governed by clinical considerations, and is the minimum amount necessary to reduce virus titer in an infected individual.

[00107] One may adjust dosage based on the amount of peptide delivered. An immunologically effective dose is one that stimulates the immune system of the patient to establish a level immunological memory sufficient to provide long term protection against disease caused by infection with HCV. More precise dosages should be determined by assessing the immunogenicity of the vaccine produced so that an immunologically effective dose is delivered.

[00108] The therapeutic dose may be at least about 0.01 μg/kg body weight, at least about 0.05 μg/kg body weight; at least about 0.1 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 1 μg/kg body weight, at least about 2.5 μg/kg body weight, at least about 5 μg/kg body weight, and not more than about 100 μg/kg body weight. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, e.g. in the use of vaccine fragments, or in the use of vaccine conjugates. The dosage may also be varied for localized administration, or for systemic administration, e.g. i.m., i.p., i.v., and the like.

[00109] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[00110] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[00111] Where the formulation is an aqueous suspension, such can contain the active agent in a mixture with a suitable excipient(s). Such excipients can be, as appropriate, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia); dispersing or wetting agents; preservatives; coloring agents; and/or flavoring agents.

[00112] Suppositories, e.g., for rectal administration of agents, can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. [00113] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. EXAMPLES

[00114] 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 Centigrade, and pressure is at or near atmospheric.

[00115] 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. Example 1

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a

Broadly Neutralizing Human Antibody

[00116] The extracellular domains of both E1 and E2 are heavily glycosylated, with up to 5 sites on E1 and up to 11 sites on E2 modified by N-linked glycosylation. Some of these glycans are critical for protein folding and/or HCV infectivity. In addition, a number of studies have highlighted the role of N-linked glycosylation in shielding E2 epitopes from recognition by broadly neutralizing antibodies, as observed for human immunodeficiency virus (HIV) and influenza virus. In a particularly revealing recent example, deep sequencing analysis of HCV resistance to neutralization by mAbs HCV1 and AP33 identified mutations at asparagine 417 of E2 (Asn417Ser and Asn417Thr), an N-glycosylation site within the E2412–423 epitope recognized by these mAbs, as responsible for virus escape. Comparison of the glycosylation status of E2 containing these resistance mutations revealed a glycosylation shift from Asn417 to Asn415 in the Asn417Ser and Asn417Thr E2 proteins. Crystal structures of the wild-type E2412–423 epitope bound to HCV1 and AP33 showed that, whereas the side chain of Asn417 points away from the antibody, Asn415 is buried within the peptide–antibody interface. Consequently, attachment of a glycan at Asn415, but not at Asn417, would create major steric clashes with HCV1 and AP33, resulting in abrogation of antibody binding and HCV escape, as observed in resistance selection studies. Notably, liver transplantation patients with HCV who were treated with HCV1 showed rebounds in viral load at various times following transplantation. These rebounds were consistently associated with mutations in the E2412–423 epitope known to confer resistance to HCV1, in particular Asn417Ser. In sharp contrast to mAbs HCV1 and AP33, HC33.1, which was isolated from an HCV- infected blood donor, can neutralize virus bearing the E2 Asn417Ser and Asn417Thr adaptive mutations with the glycosylation shift to Asn415. Moreover, infectious HCV virions containing the Asn417Ser mutation displayed increased sensitivity to neutralization by HC33.1 and related antibodies, suggesting that the glycosylation shift actually enhanced binding. To understand how HC33.1 is able to penetrate the glycan shield of HCV created by this shift, we determined the crystal structure of this broadly neutralizing human mAb in complex with its E2412–423 epitope.

[00117] Immunogenic determinants on the E2 glycoprotein are roughly segregated into the hypervariable region 1 (HVR1), and at least five clusters of overlapping linear and nonlinear epitopes, designated antigenic domains A–E. HVR1 is a major decoy that elicits isolate-specific neutralizing antibodies from which the virus can readily escape. Antigenic domain A is associated with non-neutralizing antibodies and constitutes another major decoy. By contrast, some antigenic domain B epitopes, and most D and E epitopes, elicit antibodies that are broadly neutralizing among the major HCV genotypes and subtypes. Antibodies to domain C epitopes neutralize HCV with more restricted genotype and subtype profiles. Antigenic domain E is composed of overlapping linear epitopes located within amino acids 412–423 (QLINTNGSWHIN) of E2 (E2412– 423). This region is involved in HCV binding to the CD81 entry receptor, which likely explains why it is so highly conserved among >5,500 E2 sequences in the GenBank database. Accordingly, antibodies to E2412–423 hold considerable promise for vaccine development. Broadly neutralizing mAbs targeting this region have been isolated from immunized mice and HCV-infected individuals. These antibodies, which block the interaction of E2 with CD81, include the rodent mAbs AP33 and 3/11, and human mAbs HCV1 and HC33.1. [00118] EXPERIMENTAL PROCEDURES Protein Expression and Purification–The HC33.1 antibody was expressed as a single-chain Fv fragment (scFv) by in vitro folding from inclusion bodies produced in Escherichia coli. The scFv construct consisted of the heavy chain variable (VH) region (residues Glu1–Ser127) connected to the light chain variable (VL) region (residues Gln1–Leu110) by an 18-residue linker (GSTGGGGSGGGGSGGGGS). The HC33.1 scFv was cloned into the expression vector pET- 26b (Novagen) and expressed as inclusion bodies in BL21(DE3) E. coli cells (Novagen). Bacteria were grown at 37ºC in LB medium to an absorbance of 0.6–0.8 at 600 nm, and induced with 1 mM isopropyl-β-D-thiogalactoside. After incubation for 3 h, the bacteria were harvested by centrifugation and resuspended in 50 mM Tris- HCl (pH 8.0) containing 0.1 M NaCl and 2 mM EDTA; cells were disrupted by sonication. Inclusion bodies were washed extensively with 50 mM Tris-HCl (pH 8.0) and 2% (v/v) Triton X- 100, then dissolved in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT.

[00119] For in vitro folding, inclusion bodies were diluted into ice-cold folding buffer containing 1 M L-arginine-HCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3 mM reduced glutathione, and 0.9 mM oxidized glutathione, to a final protein concentration of 60 mg/l. After 72 h at 4°C, the folding mixture was concentrated 50-fold, dialyzed against 50 mM MES (pH 6.0), and centrifuged to remove aggregates. Correctly folded HC33.1 scFv was then purified using sequential Superdex 75 HR and MonoQ columns (GE Healthcare).

[00120] Crystallization and Data Collection–For crystallization of the HC33.1–E2412–423 complex, HC33.1 scFv (10 mg/ml) was mixed with E2412–423 peptide (GenScript) in a 1:5 molar ratio. The complex crystallized in sitting drops at room temperature in 20% (w/v) polyethylene glycol (PEG) 8000, 3% (v/v) 2-methyl-2,4-pentanediol, and 0.1 M imidazole (pH 6.5). For data collection, crystals were transferred to a cryoprotectant solution of mother liquor containing 20% (v/v) glycerol, prior to flash cooling in a nitrogen stream. X-ray diffraction data were collected in- house at 100 K using a Rigaku R-AXIS IV++ area detector. The HC33.1–E2412–423 crystal belongs to space group P212121 with one complex molecule per asymmetric unit. Diffraction data were indexed, integrated, and scaled with the program CrystalClear (Rigaku). The data set is 94.9% complete to 2.0 Å resolution with Rmerge = 7.9%. Data collection statistics are shown in Table 1.

[00121] Structure Determination and Refinement– The structure of the HC33.1–E2412–423 complex was solved by molecular replacement with Phaser using an ensemble search model constructed by combining aligned structures of the anti-severe acute respiratory syndrome spike protein scFv 80R (PDB accession code 2GHW), the anti-gankyrin scFv F5 (4NIK), and the anti-influenza hemagglutinin scFv F10 (3FKU). After model building for HC33.1 scFv was almost complete, extra density corresponding to the E2412–423 peptide was very clear, and peptide residues were built manually without any ambiguity. All refinements were carried out using CNS1.1, including iterative cycles of simulated annealing, positional refinement, and B factor refinement, interspersed with model rebuilding into σAweighted Fo– Fc and 2Fo– Fc electron density maps using XtalView. Refinement statistics are summarized in Table 1. Stereochemical parameters were evaluated with PROCHECK.

[00122] Modeling of Epitope Mutants and Glycans–Rosetta version 2.3 was used to perform a computational alanine scan and escape mutant simulations of the E2412–423 epitope using the interface mutagenesis module, removing the N-terminal arginine residue from the HC33.1– E2412–423 complex structure prior to modeling to avoid any influence of non-epitope residues on results. Extra side chain rotamers were included to ensure sufficient sampling (“-ex1–ex2– ex3”), and a separate simulation for non-alanine mutants was performed incorporating backbone and off- rotamer side chain minimization of the complex before and after mutation (“- min_interface–int_bb–int_chi”). Glycans were modeled using the GlyProt web server, using oligomannose glycans as high mannose glycans were found to be predominant in native E2. As with the Rosetta simulations, the engineered N terminal arginine residue was removed from the peptide in the complex structure prior to input into the server.

[00123] Protein Data Bank Accession Codes– Coordinates and structure factors for the HC33.1– E2412–423 complex have been deposited under accession code 4XVJ. RESULTS

[00124] Overview of the HC33.1–E2412–423 Complex–We expressed the HC33.1 antibody as an scFv by in vitro folding from bacterial inclusion bodies. The scFv construct consisted of the VH region connected to the VL region by an 18-residue linker. The HC33.1 scFv was crystallized with a 13-mer peptide (RQLINTNGSWHIN) representing the E2412–423 epitope to which an Nterminal arginine was added to increase solubility. We determined the structure of the HC33.1–E2412– 423 complex to 2.0 Å resolution by molecular replacement using an ensemble search model built from three scFv structures (Table 1). Clear electron density corresponding to the E2412–423 peptide was found in the binding site, as evident from the 2Fo– Fc electron density map. All scFv residues were visible, except for six residues in the linker connecting VH and VL. The antibody–peptide interface was unambiguous. The conformation of the E2412–423 peptide bound to H33.1 is very different from the β-hairpin conformation of this same peptide bound to HCV1 or AP33, or to the humanized and affinity-matured mAbs MRC10.v362 (derived from AP33) and hu5B3.v3. It is also distinct from the extended conformation of E2412–423 observed in the complex with mAb 3/11. This rat antibody, like HCV1 or AP33 but unlike HC33.1, cannot recognize E2 with a glycosylation shift from Asn417 to Asn415.

[00125] In the HC33.1–E2412–423 complex, residues 414–415 of the peptide form an anti-parallel β-sheet with β-strand F of the VH domain, while the rest of the peptide adopts a coil conformation. In the HCV1–E2412–423 and AP33–E2412–423 complexes, by contrast, these residues form part of the first strand of the β-hairpin. In the HC33.1–E2412–423 structure, the N- terminal coil preceding the β-strand of the bound peptide (residues 412–413) extends behind the FG β-sheet of the HC33.1 VH domain. The peptide makes a turn at residues 416–419, surrounding the F strand of VH, while its C-terminus sits loosely in front of the FG β-sheet. The peptide interacts predominantly with the HC33.1 H chain, which mediates 90% of the 186 total contacts. Interactions with the L chain are restricted to the side chains of Leu413, Trp420 and His421, at the N- and C-termini of E2412–423. HC33.1 has an unusually long VH complementarity- determining region 3 (CDR3) (22 residues compared to 14 and 18 residues for AP33 and HCV1, respectively). In particular, the C-terminus of strand F and N terminus of strand G, which constitute part of this VHCDR3, are each 3–4 residues longer in HC33.1 than in HCV1 or AP33. The extended F strand permits E2412–423 to“clip” onto the antibody through formation of the anti- parallel β-sheet described above. VHCDR1 and VHCDR2 mainly contact the β-turn of the bound peptide.

[00126] Conformation of the E2412–423 Epitope– Since the conformation of E2412–423 bound to HC33.1 is distinct from the β-hairpin or extended conformations of this peptide bound to other mAbs, it would be of interest to know the conformation of unbound E2412–423 in the context of the native E2 protein. However, residues 412–420 were disordered in one of the two recently reported E2 core structures, and were not included in the crystallization construct used for the other E2 structure. Accordingly, we used several prediction methods to assess the preferred unbound conformation(s) of the E2412–423 epitope. Peptide structure prediction using the PEP- FOLD server, an ab initio peptide folding method, resulted in models with a β-hairpin conformation very similar to that in the HCV1 and AP33 complexes with a type I’ β-turn at residues 416–419, and backbone root mean- square-distance (RMSD) of 2.3 Å between the HCV1-bound epitope and the top model. The I-TASSER server, with homologous templates excluded to prevent usage of E2412–423-containing structures as templates, produced a model that was nearly identical to the HCV1-bound β-hairpin (1.0 Å RMSD). Given these modeling results, in addition to the existence of the β-hairpin in the majority of mAb-bound structures, E2412–423 likely adopts a β- hairpin conformation in the E2 protein, and maintains a similar structure after binding to AP33 or HCV1. However, this β-hairpin structure can be disrupted through interactions with certain antibodies, such as 3/11 or HC33.1.

[00127] We next asked whether any epitope substructures are shared between the β-hairpin structure bound by HCV1 or AP33 and the HC33.1-bound structure. Indeed, secondary structural analysis identified a conserved β-turn conformation at residues 416–419 (TNGS) of E2412–423 in complex with each of these mAbs. Superposing the HC33.1-bound peptide onto the HCV1-bound peptide through residues 416–419 gave an RMSD of 0.35 Å in the position of their Cα atoms (RMSD = 0.34 for a comparison of the HC33.1- and AP33-bound peptides), indicating close similarity. An analogous comparison of the HC33.1-bound and 3/11-bound E2412–423 epitopes failed to identify regions of comparable sub-structural similarity. This result, together with the β-strand preceding the 416–419 turn in the HC33.1, HCV1 and AP33, but not 3/11, complexes, implies that the HC33.1- bound conformation of E2412–423 is structurally intermediate between the HCV1/AP33- and 3/11- bound conformations.

[00128] In the β-hairpin structure of E2412–423 bound to HCV1 or AP33, the antiparallel β-strands are stabilized by main-chain– main-chain hydrogen bonds between Ile414 and His421, and between Thr416 and Ser419. By contrast, in the HC33.1–E2412–423 complex, Ile414 forms main- chain–main-chain hydrogen bonds with VHSer103 in β-strand F of HC33.1, resulting in an anti- parallel β-sheet that not only stabilizes the antibody–peptide interaction, but also disrupts the β- hairpin conformation of E2412– 423 that likely predominates in the E2 protein.

[00129] The HC33.1–E2412–423 Interface–The HC33.1–E2412–423 complex buries a total solvent accessible surface area of 1744 Å2, with 11 peptide residues contacting 23 antibody residues. The interface is characterized by high shape complementarity, based on a calculated shape correlation statistic (Sc) (53) of 0.85 (Sc = 1.0 for interfaces with geometrically perfect fits). For clarity, the HC33.1–E2412–423 interface may be subdivided into three regions, according to the distribution of contacting residues on the peptide. In region 1, N-terminal residues 412–416 (QLINT) interact exclusively with VHCDR3, except for Leu413, which also contacts VLCDRI Tyr33 and VLCDR2 Asp51 . Region 2, comprising residues 417-419 (NGS) at the peptide β- turn, interacts with VHCDRI and VHCDR2. In region 3, C-terminal residues 420-423 (WHIN) contact HC33.1 over a broader area that includes 1 1 residues from VLCDR3, VH framework region 2 (VHFR2), VHCDR2, VHFR3, and VHCDR3.

1 30] In a previous study (Keck et al. (2013) J. Virol. 87, 37-51), alanine-scanning mutagenesis was used to identify three E2_412-423 residues as critical for binding of the E2 glycoprotein to HC33.1 : Leu413, Gly418 and Trp420. The HC33.1-E2412-423 complex readily explains this result, as these epitope residues constitute key anchor points in the structure. Thus, the side chains of Leu413 and Trp420 point directly into the HC33.1 binding site, with Leu413 92% buried and Trp420 completely buried upon complex formation. Indeed, computational alanine scanning of the HC33.1-E2412-423 complex structure identified Leu413 and Trp420 as critical binding residues (Table 2). The isobutyl group of Leu413 is oriented parallel to the aromatic ring of VLCDRI Tyr33, with which it makes extensive hydrophobic interactions. In addition, the Cδ1 atom of Leu413 contacts the side chain of VLCDR2 Asp51 , while its Cδ2 atom contacts the main chains of VHCDR3 Ser102 and Ser103 in the unusually long β-strand F of HC33.1 . Together, these four antibody residues form a tight, mainly hydrophobic cage encasing Leu413, which probably accounts for the extreme sensitivity of HC33.1 to even conservative substitutions (e.g. lie) at this position (Keck et al. (2014) PLoS Pathog. 10, e 1004297).

[00131] Computational simulations of this mutant (Table 2) showed substantial predicted affinity loss, whether or not nearby residues were permitted to minimize to accommodate the Ile side chain; only the Cγ2 methyl group fit into the pocket noted above, yielding a loss of favorable HC33.1 contacts. The side chain of Trp420 binds deep in a β-barrel composed of VH strands C, C’, C”, F and G, and VL strands F and G. It makes numerous hydrophobic interactions with side chains of residues lining the β-barrel, in particular Trp47, Tyr59 and Lys112 from VH strands C’, C” and G, respectively, and Trp92 and Val99 from the VL FG loop. These interactions would be mostly lost upon alanine substitution of Trp420, in agreement with the finding that this mutation abolished binding of E2 to HC33.1. Additionally, Trp420 makes two main-chain–sidechain hydrogen bonds with HC33.1: Trp420 N–Oγ VHCDR2 Ser52 and Trp420 O–Nζ VHCDR3 Lys112. Like Trp420, Gly418 is completely buried in the HC33.1–E2412–423 interface, where it makes 21 close contacts with the backbones of VHCDR1 Asn31 and Phe32, and of VHCDR2 Ser52, Ser53 and Ser54.

[00132] Modeling the structure of the Gly418Ala mutant indicated that the alanine methyl group could in principle be accommodated at this position without substantially clashing with HC33.1, as reflected by its predicted ΔΔG (Table 2). However, Gly418 is located at the tip of the type I’ β-turn of the E2412–423 peptide, and glycine is a highly preferred amino acid at this position for this type of β-turn (54). Accordingly, substitution of Gly418 by other residues could adversely affect folding of the β-turn, which is wedged between the VHCDR1 and VHCDR2 loops.

[00133] Accommodation of the Glycosylation Shift to Asn415 of E2412–423–Escape of HCV from neutralization by the E2412–423-specific mAbs HCV1, AP33 and 3/11 is the result of adaptive mutations at Asn417 (Asn417Ser and Asn417Thr) that cause a glycosylation shift from Asn417 to Asn415 in the E2 protein. In marked contrast to these mAbs, HC33.1 can penetrate the glycan shield at Asn415 originating from the glycosylation shift. Structures of the wildtype E2412– 423 epitope bound to HCV1, AP33 and 3/11 showed that, whereas the side chain of Asn417 points away from the antibody (as does that of Asn423, an additional glycosylation site within this epitope), Asn415 is buried in the antibody–peptide interface. Therefore, attachment of a glycan at Asn415 (but not at Asn417 or Asn423) would create untenable steric clashes with HCV1, AP33 and 3/11, abolishing antibody recognition. The ability of HC33.1 to accommodate the glycosylation shift to Asn415 is explained by the unique conformation of E2412–423 induced (or captured) by this mAb, rather than by HC33.1 binding in a different way to the β-hairpin or extended conformation of E2412–423 recognized by HCV1, AP33 or 3/11. In the HCV1–E2412–423 and AP33–E2412–423 complexes, the buried Asn415 residue is part of the first strand of the E2412– 423 β- hairpin, whereas in the HC33.1–E2412–423 complex, Asn415 is part of a strand that forms an anti-parallel β-sheet with strand F of VH, such that the Asn415 side chain flanks the antibody and is 58% solvent-accessible. A glycan chain can be attached to the side-chain amide of Asn415, based on modeling using the GlyProt server, indicating that glycosylation of E2 at Asn415 would not prevent binding of HC33.1. Indeed, a glycan at this position could mediate additional productive interactions with the mAb, resulting in higher affinity, as suggested by the finding that the neutralization potency of HC33.1 against glycanshifted HCV virions is >10-fold greater than against wild-type virions. While the simulated oligomannose glycan at Asn415 was largely directed away from the antibody due to Asn415 side chain movement by the GlyProt server, we generated an alternate conformation of the Asn415 glycan by using the E2412–423 structure alone as input to GlyProt (removing mAb HC33.1). This resulted in a modeled Asn415 side chain conformation closer to the antibody-flanking conformation observed in the crystal structure, and attached glycan with substantial antibody contacts, including ones with VHCDR1, VHCDR3, and the L chain framework. Importantly, the side chains of Asn417 and Asn423 are also solvent-exposed (60% and 95%, respectively) in the HC33.1–E2412–423 structure, which would permit attachment of glycan chains to these residues as well, without interfering with mAb binding. Modeling of the Asn417 and Asn423 glycans using GlyProt supports this. Thus, HC33.1 binding to the flexible E2412–423 epitope induces a conformation that allows accommodation of multiple glycans in the interface, whether at Asn417 and Asn423 in wild-type E2, or at Asn415 and Asn423 in glycosylation-shifted E2 mutants. [00134] We have identified the structural basis for human antibody recognition of HCV E2412–423 that is capable of neutralizing clinically observed glycan shift mutants in this linear epitope. This provides new insights into the capability of antibodies such as HC33.1 to effectively target HCV, thereby overcoming previously identified escape mechanisms through mutation or glycan attachment at E2 residue 415. Based on initial structures of mAbs bound to E2412–423, it appeared that this epitope was restricted to a β-hairpin conformation. However, our study has revealed a third distinct conformation of E2412–423, intermediate between a β-hairpin and coil, that highlights this epitope’s remarkable structural heterogeneity. Along with sequence variability and N glycosylation, conformational flexibility has been proposed as a mechanism used by viruses such as HIV and influenza to evade humoral immunity. Conformational flexibility is thought to pose a barrier to effective elicitation of broadly neutralizing antibodies to a viral site because antibodies are unable to simultaneously target many possible epitope conformations. For example, murine norovirus was found to escape neutralization by antibodies against the capsid protein by undergoing mutations in two flexible loops of the capsid that switch their conformation to one with poor stereochemical complementarity to neutralizing antibodies.

[00135] Our results suggest that HCV may employ structural flexibility as an immune evasion strategy. The crystal structure of the HCV E2 core revealed that ~60% of all residues are either disordered or in loops, implying considerable overall flexibility. Neutralizing and non-neutralizing mouse mAbs specific for an E2 epitope comprising residues 427–446 were found to bind distinct conformations of this epitope that determined recognition specificity. Here we have shown that the E2412–423 epitope can adopt at least three different conformations, which may contribute to reducing its immunogenicity in HCV-infected individuals. Although structural flexibility may render E2412–423 less antigenic, such flexibility could also be disadvantageous to HCV, insofar as conformational variability provides the immune system with multiple potential targets for antibody neutralization. Thus, if the E2412–423 epitope were restricted to the β-hairpin conformation recognized by HCV1-like antibodies, it could not targeted by HC33.1-like antibodies. As a consequence, the immune system would be unable to counter HCV variants that have undergone the Asn415 glycosylation shift.

[00136] The HC33.1–E2412–423 structure explains the ability of HC33.1 to accommodate glycans at Asn417 and Asn423 in wild-type E2, or at Asn415 and Asn423 in glycosylation-shifted E2 mutants. A possible additional glycan has been proposed for the mutation Ser419Asn, which was observed in the context of other mutations, notably Asn417Thr, in a cell-based study of HC33.1 escape mutants. In combination, the Ser419Asn and Asn417Thr mutations may generate a new glycan at position 419 due to creation of a Thr-X-Asn glycosylation sequon. Although far less common than the Asn-X-Ser/Thr sequon, the Thr-X-Asn sequon is one of several reported alternative motifs for N-glycosylation. In silico mutagenesis of Ser419Asn and Asn417Thr permitted modeling of a glycan at Asn419. As expected from the high solvent exposure (60%) of the wild-type Ser419 side chain in the HCV1-E2412-423 structure, this glycan, if present, is not predicted to abolish antibody binding completely. Indeed, Asn417Ser/Ser419Asn escape variants could still be neutralized by HC33.1 , albeit at higher antibody concentrations, and HC33.1 retained significant binding to recombinant E1 E2 bearing these mutations. Apart from affecting interactions with HC33.1 directly, a glycan at Asn419 may influence the structure or conformational dynamics of the E2412-423 epitope, as noted in studies of peptide dynamics, or interact with other regions of E2. Another possibility is that the side chain change of Ser419 to Asn has a direct effect on HC33.1 recognition; this would be supported by the hydrogen bond of Ser419 with Asn54 on the HC33.1 H chain, as well as some predicted loss of affinity upon in silico mutation (Table 2). From the standpoint of vaccine design, the HC33.1-E2412-423 structure provides critical information regarding a well-conserved site and a new point of viral vulnerability. Having demonstrated how in principle antibodies can effectively target this epitope without including residue 415 in the interface core, vaccine design efforts can attempt to present this epitope structure to the immune system, rather than the likely preferred β-hairpin. Given that additive neutralization was observed between antibodies targeting this site (including HC33.1) and antibodies targeting another well-conserved site that also contains residues critical for binding CD81 , simultaneously inducing robust immune responses to these epitope sites, while minimizing the chances of escape (including glycan shifting to Asn415), is key to an effective HCV vaccine.

Example 2

Modified Hepatitis C E2 Glycoprotein For Vaccine Development

[00137] HCV is an RNA virus in the flavivirus family, with a 9.5 kilobase genome, six distinct genotypes and several subtypes. Even within subtypes, there is remarkable sequence variability, particularly within envelope proteins E1 and E2, which is thought to result from mutation of surface residues to evade immune response in the host. Given its high sequence variability and resultant lack of amenability to targeted approaches, HCV is often compared with HIV, which historically has garnered greater attention from the research community. A recent phase 2 clinical trial of a monoclonal antibody targeting an epitope of HCV E2 demonstrated that HCV actively escapes the immune response via mutation when under immune pressure (Babcock et al. PLoS One 2014, 9(6):e100325).

[00138] A vaccine for HCV must overcome the high sequence variability of this virus, and its capacity to escape, by inducing protective antibodies that target conserved epitopes. The recently described structures of HCV E2 (the primary target of the human antibody response to HCV), and antibodies bound to several E2 epitopes (see, for example, Krey et al. PLoS Pathog 2013, 9(5):e1003364; Meola et al. J Virol 2015, 89(4):2170-2181; Li et al. J Biol Chem 2015, 290(16):10117-10125; Pantua et al. J Mol Biol 2013, 425(11):1899-1914), in conjunction with advanced bioinformatics and structural modeling algorithms, permit the rational structure-based design of optimized variants of the E2 protein for use in an effective vaccine. The present invention describes these designs. Experimental Procedures

[00139] E2 Antigen Expression. H77c E2 (GenBank accession no. AF009606), amino acid (aa) 384 to 661, 411-661 or 408-661 was cloned into the expression vector pSec in-frame with the IgƘ signal peptide sequence and fused with a myc and six-histidine tag at the carboxyl terminus. Amino acids substitution and glycan insertion at specific designed residues were created by using a QuikChange II site-directed mutagenesis kit (Agilent, La Jolla, CA) in accordance with the manufacturer's instructions. Mutations were confirmed by DNA sequence analysis (ElimBiopharm, Hayward, CA). The conservation of native E2 conformation of wild- type (wt) H77c E2 and mutants was confirmed using a panel of neutralizing and non- neutralizing HCV human mAbs to conformational epitopes on E2 by ELISA (Keck et al. J Virol 2008, 82(12):6061-6066).

[00140] ELISA Immunoassays. As described previously microtiter plates were prepared by coating each well with 500 ng of GNA and blocking with 2.5% non-fat dry milk and 2.5% normal goat serum. Purified wt HCV H77c E2 or mutant E2, were captured by GNA onto the plate and later bound by 1 and 5 µg/ml of human mAb. The bound human mAb was detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG followed by incubation with p-nitrophenyl phosphate for color development. Absorbance was measured at 405 nm and 570 nm.

[00141] Isothermal Calorimetry. The thermodynamic parameters for the binding of E2 domain E peptide (aa 412-423) and proline mutants to the HC33.1 antibody were carried out using a MicroCal ITC200 titration microcalorimeter. Purified HC33.1 antibody was exhaustively dialyzed against PBS buffer. In a typical experiment, 2 μl aliquots of 100–500 μM peptide solution were injected from a 40 μl rotating syringe into the sample cell containing 200 μl of 10–30 μM HC33.1 antibody solution at 25 °C. For each titration experiment, an identical buffer dilution correction was conducted; these heats of dilution were subtracted from the corresponding binding experiment. A computerized nonlinear least square fitting method was used to determine the change in enthalpy (∆H), the equilibrium association constant (K_a), and the molar stoichiometry (n). Reversible equilibrium thermodynamics was assumed in calculating values of the change in free energy,∆G, from the relationship∆G =−RTlnK_a. The change in entropy,∆S, was calculated from∆G =∆H− T∆S. Data acquisition and analysis were performed using the software package Origin provided by the manufacturer.

[00142] Surface Plasmon Resonance. The binding affinity of E2∆HVR1₄₁₀ protein (residues 410-661) and mutants to HC33.1 and AP33 mAbs was assessed by SPR using a BIAcore T100 biosensor at 25 °C. HC33.1 or AP33 mAbs (1000–1500 RUs) were immobilized on a BIAcore CM5 chip by amino coupling. An additional flow cell was immobilized with buffer to serve as a blank control. For analysis of E2∆HVR1₄₁₀ protein binding to mAbs, solutions containing different concentrations of E2∆HVR1₄₁₀ protein (1–50 ^M) were flowed sequentially over the chips immobilized with mAbs and the blank flow cell. Sensograms were fitted to a 1:1 binding model using BIAevaluation software (BIAcore) to obtain on- and off-rates. K_D was calculated as k_off/k_on.

[00143] HC84.26AM-E2_434-446 Structure Determination - Protein Expression and Purification–The HC84.26AM antibody was expressed as a single-chain Fv fragment (scFv) by in vitro folding from inclusion bodies produced in Escherichia coli. The scFv construct consisted of the heavy chain variable (V_H) region (residues Glu1–Ser127) connected to the light chain variable (V_L) region (residues Gln1–Leu110) by an 18-residue linker (GSTGGGGSGGGGSGGGGS). The HC84.26AM scFv was cloned into the expression vector pET-26b (Novagen) and expressed as inclusion bodies in BL21(DE3) E. coli cells (Novagen). Bacteria were grown at 37^oC in LB medium to an absorbance of 0.6–0.8 at 600 nm, and induced with 1 mM isopropyl- ^-D- thiogalactoside. After incubation for 3 h, the bacteria were harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl and 2 mM EDTA; cells were disrupted by sonication. Inclusion bodies were washed extensively with 50 mM Tris-HCl (pH 8.0) and 2% (v/v) Triton X-100, then dissolved in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT. For in vitro folding, inclusion bodies were diluted into ice-cold folding buffer containing 1 M L-arginine-HCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3 mM reduced glutathione, and 0.9 mM oxidized glutathione, to a final protein concentration of 60 mg/l. After 72 h at 4 °C, the folding mixture was concentrated 50-fold, dialyzed against 50 mM MES (pH 6.0), and centrifuged to remove aggregates. Correctly folded HC84.26AM scFv was then purified using sequential Superdex 75 HR and MonoQ columns (GE Healthcare).

[00144] Crystallization and Data Collection–For crystallization of the HC84.26AM–E2_434–446 complex, HC84.26AM (10 mg/ml) was mixed with E2_434–446 peptide (NTGWLAGLFYQHK) (GenScript) in a 1:5 molar ratio. Crystals were grown at room temperature in sitting drops containing 1 μl of the complex solution mixed with 1 μl of reservoir solution consisting of 1.6 M sodium phosphate monobasic, 0.4 M potassium phosphate dibasic, and 0.1 M sodium phosphate dibasic/citric acid (pH 4.2). For data collection, crystals were soaked briefly in a cryoprotectant containing mother liquor plus 20% (v/v) 2-methyl-2,4-pentanediol before flash freezing in liquid nitrogen. X-ray diffraction data for the HC84.26AM–E2_434–446 complex were collected at beamline X29A of the Brookhaven National Synchrotron Light Source Laboratory. The HC84.26AM–E2_434–446 crystal belongs to space group P2₁2₁2₁ with one molecule per asymmetric unit. Diffraction data were processed using Mosflm. Scaling and reduction were performed with programs from the CCP4 suite. The data set is 97% complete to 2.0 Å resolution with R_merge = 0.145.

[00145] Structure Determination and Refinement–The structure of the HC84.26AM–E2_434–446 complex was solved by molecular replacement with Phenix.MRage using ensemble search models for the V_H and V_L domains of the HC84.26AM scFv. These ensembles were chosen based on sequence homology to the corresponding V_H (Protein Data Bank accession codes: 4JZO, 4HJO, 3QOT, 4DN3) and V_L (4AIX, 4AJO, 4QHK, 4DAG) chains. A difference electron density map was calculated to reveal the E2_434–446 peptide and model building of the peptide was based exclusively on the visible electron density. The model was refined to an R_work of 0.22 (R_free = 0.26) using iterative cycles of model building in Coot and refinement using Phenix.

[00146] Protein Data Bank Accession Code–Coordinates and structure factors for the HC84.26AM–E2_434–446 complex have been deposited in the Protein Data Bank under accession code 4Z0X. Results

[00147] A panel of human monoclonal antibodies (mAbs) targeting the HCV E2 protein has been isolated and characterized Keck et al. J Virol 2013, 87(1):37-51.) Based on cross-competition studies, epitope mapping, as well as structural studies (Krey et al. PLoS Pathog 2013, 9(5):e1003364; Meola et al. J Virol 2015, 89(4):2170-2181; Li et al. J Biol Chem 2015, 290(16):10117-10125), these antibodies have been found to target several regions of the E2 protein surface, referred to as antigenic domains A-E (Table 1, Figure 1). While antigenic domains B, D, and E are associated with neutralizing antibodies, due at least in part due to overlap with the CD81 binding site critical for human cell entry, antigenic domain A is not associated with neutralizing antibodies.

[00148] ¹Key binding (hotspot) residues shared by some or all of the antibodies that bind to the domain, where mutation to alanine (or glycine in the case of an alanine residue) reduces binding significantly (> 80%) compared to wild-type. Residues in italics are hotspot residues for a subset of antibodies. [00149] With the goal of inducing broadly neutralizing antibodies to conserved epitopes on E2, several complementary rational design strategies were utilized: 1) Conformationally stabilize the E2 structure to favor recognition by antibodies to antigenic domains B, D, and E (“Conformation”); 2) Introduce glycans at specific sites to disrupt recognition by antibodies to domain A (“Glycan Insertion”); 3) Remove the hypervariable region 1 (HVR1) decoy epitope that is associated with viral escape (“∆HVR1”). The basic concepts of these strategies can be referred to as improving immunoprominence of selected epitopes (antigenic domains B, D, and E), while immune silencing (i.e. eliminating the immune response toward) others (antigenic domains A and HVR1). Selected E2 protein mutants with regard to each of these strategies are given in Table 2. These have been characterized experimentally and are described in more detail below.

[00150] The conformation strategy of antigen design entails producing selected mutants near an epitope site, improving its chance of antibody recognition and thus antibody induction by locking the structure into an antibody-bound conformation. Selected mutants will not include key binding (“hotspot”) residues as such mutants would likely reduce antibody binding and/or alter the antigenic specificity of antibodies induced to the site. Hotspot residues were identified via alanine scanning (reducing mAb binding to <20% compared to wild-type affinity), as shown for antigenic domains E and D (Figures 2, 3). Such designs utilize the residue proline to lock the backbone conformation of residues proximal to the epitope.

[00151] One example of this is antigenic domain E (aa 412-423), which based on x-ray structural characterization adopts several widely varying conformations depending on the mAb to which it is bound (Figure 4). In particular, mAb HC33.1 recognizes the antigen domain E epitope in a specific extended conformation (Figure 4A), and is not susceptible to the glycan shift to residue N415 which allows viral escape from antibodies that target the β-hairpin conformation of this epitope (Figure 4B). This is evident from the alanine scanning data which indicate that N415 is not a hotspot residue for this antibody (Figure 2), while for other antibodies, such as HCV1, N415 is a key binding site residue [8]. Given its apparent mobility, conformational analysis of the HC33.1-bound epitope conformation was utilized to identify potential proline substitution sites to favor that conformation (Table 3; Figure 5), without altering key hotspot residues L413, G418, W420 (Figure 2). Mutations N415P and N417P were selected based on this analysis. Analogous analysis of the antigenic domain D epitope structure (Table 4) led to identification of mutants A439P and H445P. [00152] We determined the crystal structure to 2.0 Å resolution of a broadly neutralizing domain D human mAb (HC84.26AM) bound to a 13-mer peptide spanning residues 434–446 of the HCV E2 glycoprotein (E2_434–446) (PDB code 4Z0X). In the HC84.26AM–E2_434–446 complex, the N-terminal portion of peptide adopts an ^-helical conformation, with Phe442 and Tyr443 forming a hydrophobic protrusion that inserts into the antibody binding site, while the C-terminal portion assumes an extended conformation. The structure of E2_434–446 bound to HC84.26AM is similar overall to the structure of these residues in the native E2 core protein, except for a significant deviation in C-terminal residues 443–446 that leads to a displacement of 7.7 Å in the C ^ position of Lys446. Thus, binding of HC84.26AM to the E2 core may induce conformational changes in residues 443–446 of the native protein. Based on these results, we reasoned that mutations in the E2 protein that lock residues 443–446 in the conformation seen in the HC84.26AM–E2_434–446 complex would improve domain D antibody binding, thereby enhancing immunogenicity.

[00153] Structural models of mutant proline residues were generated to confirm that they would not significantly disrupt antibody recognition or epitope structure (Figure 6). Experimental characterization of mutants N415P and N417P by isothermal calorimetry binding assays of mutant peptides (Table 5) and surface plasmon resonance of soluble E2∆HVR1₄₁₀ mutants (Table 6) confirms that these mutants maintain or exceed wild-type levels of binding to the HC33.1 mAb while disrupting binding to the AP33 mAb, which is a murine antibody that targets the neutralization escape-associated β hairpin form of the epitope. (Kong et al. J Virol 2012, 86(23):13085-13088). Further characterization using ELISA confirmed that AP33 antibody binding by the N415P was ablated (Figure 7).

[00154] A series of ELISA assays using a panel of mAbs from antigenic domains A-E were carried out to confirm that antibody binding is maintained for the Conformation and∆HVR1 mutants (Figures 8-11). While∆HVR1 showed reduced binding to the HC33.4 mAb,∆HVR1₄₀₈ restored HC33.4 binding (Figure 8), likely due to the presence of residue 408 which is a hotspot residue for HC33.4 (Figure 2). Thus we believe that∆HVR1₄₀₈ is the preferred HVR1 modification, which is a non-obvious observation and differs from previously used constructs proposed by us and others with full HVR1 removal (i.e.∆HVR1₄₁₀,∆HVR1₄₁₁). N415P maintained wild-type levels binding across the panel of mAbs (Figure 9), while H445P exceeded wild-type binding levels for the panel of mAbs (Figure 11). For comparison, other mutants near domain D had little (A439P, N448Q) or negative effects (A440C/W616C) on mAb binding (Figure 10), in the context of a genotype 1b E2 protein.

[00155] A set of mutants designed to insert glycans at specific sites in antigenic domain A were also produced. They were selected based on alanine scanning data (Figure 12), which provided the key antibody hotspot residues in this region. In addition, the two crystal structures of E2 core contain this region, and were analyzed for residue surface exposure and potential to mutate residue pairs to N-glycan codons (NxT/NxS) without disrupting the E2 core fold (Figure 13).

156] The rationally designed glycan insertion mutants were cloned and expressed as soluble E2 constructs, and tested for antibody binding using the panel of domain A-E mAbs (Figures 14-16). These assays identified the R630N/Y632T and Y632N/G634S in particular for their disruption of antigenic domain A mAb binding while maintaining binding of other mAbs, while F627N/V629T and K628N/R630T were less successful as they did not block domain A mAb binding and/or led to reduced binding for other mAbs.

SEQ ID NO: 1 : E2 polypeptide. H77 (genotype 1 a). aa 384-746 (NCBI refseq ID NP 751921 .1 )

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHK

FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY

CFTPSPVWGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP

CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM

YVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ

NIVDVQYLYGVGSSIASWAIKWEYWLLFLLLADARVCSCLWMMLLISQAEA

SEQ ID NO:2, N 15P substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLIPTNGSWH INSTALNCNESLNTGWLAGLFYQHK

FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY

CFTPSPVWGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP

CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM

YVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ

NIVDVQYLYGVGSSIASWAIKWEYWLLFLLLADARVCSCLWMMLLISQAEA

SEQ ID NO:3, N 17P substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTPGSWH INSTALNCNESLNTGWLAGLFYQHK

FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY

CFTPSPVWGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP

CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM

YVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ

NIVDVQYLYGVGSSIASWAIKWEYWLLFLLLADARVCSCLWMMLLISQAEA

SEQ IDNO:4, A439P substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLPGLFYQHK

FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY

CFTPSPVWGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP

CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM

YVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ

N IVDVQYLYGVG SSIASWAI KWEYWLLF LLLADARVCSCLWM M LLISQAEA

SEQ ID NO:5, H445P substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQPK

FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY

CFTPSPVWGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM YVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ NIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLLISQAEA SEQ ID NO:7, R360N; T-632T substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHK FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY CFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVNM TVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ NIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLLISQAEA SEQ ID NO:8; Y632N; G634S substitution

ETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHK FNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPAKSVCGPVY CFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRM NVSGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLIHLHQ NIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWMMLLISQAEA Table 3. Computational analysis of domain E epitope proline substitutions, using the HC33.1- bound epitope structure (PDB code 4XVJ).

Table 4. Computational analysis of domain D epitope conformation and proline substitutions, using the HC84-26AM-bound epitope structure (PDB code 4Z0X).

Table 5. Isothermal calorimetry affinity data for E2 domain E peptide (aa 412-423) and proline mutants binding to the HC33.1 antibody.

Table 6. Surface plasmon resonance binding affinity data for soluble E2∆HVR1₄₁₀ protein (residues 410-661) and mutants binding to HC33.1 and AP33 mAbs.

Claims

WHAT IS CLAIMED IS:

1 . A modified Hepatitis C Virus (HCV) E2 polypeptide or fragment thereof comprising one or more of the following modifications:

a) substitution of one or more of (a) amino acid substitution N415P; (b) amino acid substitution N417P; (c) amino acid substitution A439P; (d) amino acid substitution H445P; b) amino acid substitution to generate a motif for N-glycosylation of either (a) Y632N and G634S or G634T; or (b) R630N and Y632S or Y632T.

2. The modified HCV E2 polypeptide of claim 1 , wherein the modifications comprise substitution of one or both of N415P and H445P.

3. The modified HCV E2 polypeptide of claim 1 or claim 2, further comprising a deletion of all or part of amino acid residues 384-410.

4. The modified HCV E2 polypeptide of claim 3, wherein the deletion comprises residues 384-407, or residues 384-410.

5. The modified HCV E2 polypeptide of any one of claims 1 -4, further comprising C-terminal truncation of amino acid residues 662-746.

6. The modified HCV E2 polypeptide of any one of claims 1 -5, further comprising elimination of specific N-glycans at one or more of N417, N423, N448, and N532.

7. A polynucleotide comprising a modified HCV E2 polypeptide or fragment thereof as set forth in any one of claims 1 -6.

8. A vector comprising the polynucleotide of claim 7.

9. The vector of claim 8, wherein the vector is a viral vector suitable for administration as a vaccine.

10. An immunogenic composition comprising:

a modified HCV E2 polypeptide or fragment thereof as set forth in any one of claims 1 -6, or a polynucleotide as set forth in any one of claims 7-9; and

a pharmaceutically acceptable excipient.

1 1 An immunogenic composition of claim 10, further comprising an adjuvant.

12. The immunogenic composition of claim 1 1 , further comprising a virus-like particle.

13. A method of inducing a protective immune response to HCV in an individual, the method comprising:

administering to the individual an immunogenic composition according to any one of claims 10-12, in a dose and frequency sufficient to induce a protective immune response.