Novel hepatitis c virus (HCV) immunogens and related uses

Abstract

Provided are Hepatitis Virus C (HCV) soluble fusion immunogens (SE1E2) that are derived from engineered or redesigned HCV sE1 and sE2 polypeptides and are configured to form native-like E1E2 interface that may be used in vaccine formulations. Also provided are related vaccine compositions that display the engineered SE1E2 immunogens after cleavage into an SE1E2 protein with native E1E2 conformation on a self-assembling nanoparticle scaffold. Also provided are methods of using the immunogens and vaccine compositions in methods for prophylaxis or therapy for HCV infections.

Description

NOVEL HEPATITIS C VIRUS (HCV) IMMUNOGENS AND RELATED USESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application no. 63 / 580,740, filed September 6, 2023, the entire disclosure of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under AI168251 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “2213_lPC_Sequence Listing”, was created on September 4, 2024, and is 43.6 KB bytes in size.BACKGROUND

[0004] HCV is a small, enveloped, single-stranded, positive-sense RNA virus in the genus Hepacivirus of the family Flaviviridae. HCV includes seven major genotypes, which can be further divided into subtypes with the number of subtypes depending on the genotype. The RNA genome of HCV encodes a single polyprotein that is processed by host and viral proteases into three structural and seven nonstructural proteins. This RNA genome is single stranded with positive polarity and is packaged by core protein and enveloped by a lipid bilayer containing two glycoproteins (GPs), E1 and E2, to form the virion. E1 and E2 envelope glycoproteins form a heterodimer that mediates HCV infection, during which E2 attaches to the CD81 and SR-B1 coreceptors to initiate a multistep entry process into hepatocytes. Although HCV is classified as a flavivirus, E1 and E2, do not appear to share sequence or structural features with other flavivirus type II fusion glycoproteins. Both E1 and E2 are single-pass transmembrane proteinswith N-terminal ectodomains of 160 and 330 residues, respectively, and C-terminal transmembrane domains of roughly 30 residues. Similar to HIV-1, the HCV glycoproteins carry immunogenic variable regions (VRs) and a dense glycan shield to evade the immune response. Over 60% of the E2 core domain (E2c) are either loops or disordered, while N-linked glycans constitute 50% of the E1 and E2 molecular mass, with 5-6 and 9-11 N-linked glycosylation sites on E1 and E2, respectively. HCV infects ~1% of the world population with an estimated 58 million people living with HCV. Approximately 75% of HCV infections become chronic and can lead to liver cirrhosis, hepatocellular carcinoma, and other severe liver diseases. Direct-acting antivirals (DAAs) provide a possible cure for infection but cannot prevent reinfection. Additionally, DAA treatment is only accessible for less than 10% of the HCV-infected population, due to healthcare and financial constraints. Given these limitations, vaccination is still the most cost-effective method for controlling HCV infections and protecting the blood supply from virus contamination in countries where HCV nucleic acid test (NAT) is not readily accessible. However, development of a prophylactic HCV vaccine remains a challenge due to the significant diversity and hypervariability of this RNA virus.

[0005] How to elicit a broadly neutralizing antibody (bNAb) response poses a significant challenge for HCV vaccines. The genetic diversity, glycan shielding, and preparation of a homogeneous E1E2 antigen all contribute to the difficulty in generating protective, cross-genotype neutralizing antibodies (NAbs). NAb and bNAb responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer. A panel of bNAbs has been identified, marking several major sites of vulnerability on HCV glycoproteins. Of these bNAbs, AR3 series, HEPC3 / 74 series, HC84 series, HCV1, and HC33 series target the neutralizing face (NF) on E2, while AR4A and AR5 A target the quaternary structure near the E1E2 interface. In passive transfer experiments, these bNAbs protected against heterologous HCV infection in humanized mouse models, highlighting the importance of eliciting a bNAb response for HCV vaccines. However, membrane-bound E1E2 formulations failed to induce a bNAb response in chimpanzees and humans, only showing strain-specific NAb responses to the immunodominant VR loops in E2.

[0006] There is a need in the art for more effective and potent HCV vaccine immunogens based on the E1E2 heterodimer, e.g., for preventing HCV infection. The present disclosure addresses this and other unmet needs in the art.SUMMARY

[0007] In one aspect, the disclosure provides engineered HCV soluble E1E2 (sE1E2) fusion immunogen polypeptides. These fusion immunogen polypeptides each contain (1) a first fusion partner containing an engineered E1 ectodomain (sE1) with a deletion of the C-terminal membrane proximal external region (MPER), (2) a peptide spacer containing a cleavage site for a host cell protease, and (3) a second fusion partner containing an engineered soluble E2 ectodomain (sE2) with a deletion of the C- terminal membrane proximal external region (MPER). In these fusion polypeptides, the engineered E1 ectodomain and the engineered E1 ectodomain are respectively fused to two interacting motifs of a protein heterodimerization system with a configuration to allow the sE1 and sE2 domains to form native-like E1E2 heterodimer interface upon expression and protease cleavage of the fusion polypeptides in the host cell. In some of these fusion polypeptides, the engineered E1 ectodomain further contains a modification of the unstructured hydrophobic loop motif in E1 that encompasses the putative fusion peptide. This unstructured hydrophobic loop motif in E1 corresponds to residues 264-293 of HCV H77 isolate polyprotein sequence shown in SEQ ID NO: 1. In some examples, the modification is deletion of the unstructured E1 loop motif. In some other examples, the unstructured E1 loop motif is replaced with a spacer that contains from one to about three non-polar, hydrophilic, or charged amino acid residues. For example, this unstructured E1 sequence motif can be replaced with a spacer that comprises or consists of a glycine residue. In some examples, in addition to modification of the unstructured E1 loop motif, an additional unstructured motif containing 7 residues at the N-terminus (i.e., residues corresponding to residues 257- 263 of HCV H77 isolate polyprotein) can be replaced with a peptide sequence containing 7 or more residues that do not contain cystines or any stretch (e.g., 2 or more) hydrophobic residues.

[0008] In some engineered HCV sE1E2 fusion immunogen polypeptides, the protease cleave site between the two fusion partners is a furin cleavage site. In some examples, the furin cleavage site contains RRRRRR (SEQ ID NO:6). In someexamples, the engineered E1 ectodomain and its fused interacting motif are located N- terminal to the engineered E2 ectodomain and its fused interacting motif In some engineered HCV sE1E2 fusion immunogen polypeptides, the employed protein heterodimerization system is a coiled coil system. In some of these examples, each engineered ectodomain is fused at its C-terminus to a coiled coil via a PGG tripeptide linker. In some examples, the two interacting motifs of the coiled coil system respectively contain SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:24 and 25, or SEQ ID NOs:26 and 27. In some other engineered HCV sE1E2 fusion immunogen polypeptides, the employed protein heterodimerization system is a SpyTag / SpyCatcher system. In some of these examples, the SpyCatcher contains a truncation of the unstructured N-terminal sequence. In some examples, the SpyTag motif and the SpyCatcher motif respectively contain (1) SEQ ID NO: 7 and SEQ ID NO:8 or (2) SEQ ID NO:7 and SEQ ID NO:43. In some examples, the engineered E1 ectodomain is fused at its C-terminus to the SpyTag via a GSGS linker (SEQ ID NO:34) linker. In some examples, the engineered E2 ectodomain is fused at its C- terminus to the SpyCatcher via an AS dipeptide linker.

[0009] In some engineered HCV sE1E2 immunogen polypeptides of the disclosure, the deletion of the MPER in E1 contains a truncation of E1 C-terminal sequence after residue H312, and the deletion of the MPER in E2 contains a truncation of E2 C-terminal sequence after residue Y701, with the amino acid numbering based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1). In some engineered HCV sE1E2 immunogen polypeptides, the deletion of the MPER in E1 contains a truncation of E1 C-terminal sequence after residue 310, 311, 313, or 314, and the deletion of the MPER in E2 contains a truncation of E2 C-terminal sequence after residue 702, 703, 704, or 705, with the amino acid numbering based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1). In some engineered HCV sE1E2 immunogen polypeptides, the deletion of the MPER in E1 contains a truncation of E1 C-terminal sequence after residue 310, 311, 313, or 314, and the deletion of the MPER in E2 comprises a truncation of E2 C-terminal sequence after residue Y701, with the amino acid numbering based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO:1). In some engineered HCV sE1E2 immunogen polypeptides, the deletion of the MPER in E1 contains a truncation of E1 C-terminal sequence after residue 312, and the deletion of the MPER in E2 contains a truncation of E2 C-terminal sequence afterresidue 702, 703, 704, or 705, with the amino acid numbering based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1).

[0010] Some engineered HCV sE1E2 immunogen polypeptides of the disclosure contain from the N-terminus to the C-terminus an engineered E1 ectodomain shown in SEQ ID NO: 9, a first PGG tripeptide spacer, a first coiled coil of a coiled coil dimerization system, a furin clearage site, an engineered E2 ectodomain shown in SEQ ID NO: 10, a second PGG tripeptide spacer, and a second coiled coil of the coiled coil dimerization system. Some engineered HCV sE1E2 immunogen polypeptide of the disclosure contain from the N-terminus to the C-terminus an engineered E1 ectodomain shown in SEQ ID NO:9, a GSGS (SEQ ID NO:34) linker, a SpyTag motif comprising SEQ ID NO: 7, a furin clearage site, an engineered E2 ectodomain shown in SEQ ID NO: 10, an AS dipeptide linker, and a SpyCatcher motif comprising SEQ ID NO:8 or SEQ ID NO:43. Some engineered HCV sE1E2 immunogen polypeptides of the disclosure contain from the N-terminus to the C-terminus an engineered E1 ectodomain (e.g., SEQ ID NO:9), a flexible linker containing about 4-12 glycine and / or serine residues, a SpyTag motif comprising SEQ ID NO:7, a furin clearage site, an engineered E2 ectodomain (e.g., SEQ ID NO: 10), a spacer comprising AS dipeptide, and a SpyCatcher motif comprising SEQ ID NO:8 or SEQ ID NO:43. In some of these examples, the flexible linker contains a sequence set forth in any one of SEQ ID NOs:34-40. Some engineered HCV sE1E2 immunogen polypeptides of the disclosure can optionally further contain at the C-terminus a GS dipeptide linker and a His tag HHHHHH (SEQ ID NO:29).

[0011] In some related aspects, the disclosure provides (1) polynucleotides that encode the engineered HCV sE1E2 immunogen polypeptides described herein, (2) pharmaceutical compositions that contain one described engineered HCV sE1E2 immunogen polypeptide that has been cleaved at the host cell protease cleavage site or one of the polynucleotides encoding the described engineered HCV sE1E2 immunogen polypeptides, as well as a pharmaceutically acceptable carrier, and (3) nanoparticle vaccine compositions that contain a described engineered HCV sE1E2 immunogen polypeptide that has been cleaved at the protease cleavage site and is displayed on the surface of a self-assembling nanoparticle. In some examples of these nanoparticle vaccine compositions, C-terminus of the HCV sE1E2 immunogen polypeptide is fused to the N-terminus of the subunit of the self-assembling nanoparticle via a linkersequence. In some examples, the subunit of the self-assembling nanoparticle contains the polypeptide sequence shown in SEQ ID NO:16 (I3-01v9a), SEQ ID NO:15 (ferritin) or SEQ ID NO:31 (E2p), a conservatively modified variant or a substantially identical sequence thereof In another aspect, the disclosure provides polynucleotides that encode fusion proteins containing (1) a described HCV sE1E2 immunogen polypeptide and (2) a self-assembling nanoparticle subunit. In the fusion proteins encoded by the polynucleotides, the HCV sE1E2 immunogen polypeptide is fused at its C-terminus to the N-terminus of the self-assembling nanoparticle subunit. In still another aspect, the disclosure provides pharmaceutical compositions that contain a described nanoparticle vaccine composition or a polynucleotide encoding the same, and a pharmaceutically acceptable carrier.

[0012] In another aspect, the disclosure provides methods of treating or preventing HCV infection in a subject. These methods entail administering to a subject in need of treatment a pharmaceutical composition described herein. In another aspect, the disclosure provides methods of producing an sE1sE2 fusion protein (a monomer) or sE1sE2 heterodimer that forms native-like E1E2 interface. These methods entail (1) expressing an engineered HCV soluble E1E2 (sE1E2) fusion immunogen polypeptide described herein in a eukaryotic host cell, and (2) separating from the eukaryotic host cell the sE1-sE2 fusion protein or heterodimer that is connected or associated by the two interacting motifs of the protein heterodimerization system after cleavage at the protease cleavage site.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows biochemical and antigenic characterization of four sE1E2 truncation variants based on the H77 strain and the LZ scaffold. (A) SEC profile and non-reducing SDS-PAGE gel. (B) ELISA binding to AR3C and AR4A.

[0015] Figure 2 shows structure-based rational design and biochemical characterization of next-generation sE1E2 antigens. (A) Structural alignment of two cryo-EM models for E1E2 in the membrane-bound form (PDB ID: 7T6X) and in the soluble form (PDB ID: 8FSJ). (B) Two truncation variants of H77 sE1E2, with theunstructured C-termini in both sE1 ectodomain sequence (SEQ ID NO:4) and sE2 ectodomain sequence (SEQ ID NO:5) removed (Cut 1) and a long loop in sE1 removed (Cut 2). (C) Five heterodimeric scaffolds used to stabilize sE1E2 (LZ, SZ, GCP, IAAL, and SpyTag002 / SpyCatcher002dN or SPYdN), with the structural models (top) and sE1E2. scaffold design schemes (bottom). (D) SEC profiles of sE1E2-v0.LZ-His6and 10 newly designed sE1E2-Cut.scaffold antigens. A: aggregates; DD: dimer of dimer; D: dimer. (E) Non-reducing SDS-PAGE of sE1E2-v0.LZ-His6and 10 newly designed sE1E2-Cut.scaffold antigens. For sE1E2-Cut1+2. scaffold antigens, dissociated sE1 and sE2 subunits are circled in red boxes. (F) SEC profiles of sE1E2-Cut1+2.SPYdN-His6constructs designed for HCV1 (genotype 1), 312 (genotype 3), and 521 (genotype 5).

[0016] Figure 3 shows activities of rationally designed sE1E2 scaffold antigens.(A) ELISA binding to bNAbs AR3C (E2 neutralizing face, NF) and AR4A (E1E2 interface) for sE1E2-v0.LZ-HIS6 and 10 newly designed sE1E2-cut.scaffold antigens.(B) Negative-stain EM analysis and model building of HCV1, 312, and 521 sE1E2- Cut1+2.SPYdN-HIS6 antigens.

[0017] Figure 4 shows activities of rationally designed sE1E2 scaffold antigens on nanoparticles. (A) Rational design of sE1E2-displaying protein nanoparticle vaccines. Left: schematic representation of sE1E2-Cut1+2. SPYdN antigen and sE1E2- Cut1+2. SPYdN on protein nanoparticles. Right: Surface models of sE1E2-Cut1+2. SPYdN displayed on ferritin (FR) 24-mer and I3-01v9a 60-mer. (B) and (C) SEC profiles and negative-stain EM images of HCV1 sE1E2-Cut1+2.SPYdN-10GS-FR purified by ARC3 and AR4A columns. (D) Negative-stain EM images of ARC3 and AR4A-purified HCV1 sE1E2-Cut1+2.SPYdN-5GS-I3-01v9a-LD7-PADRE nanoparticles.

[0018] Figure 5 shows sequence logo plot of HCV envelope protein E1 sequences (residues from viral isolates of Genotypes 1-6 that span the unstructured E1 loop sequence motif.DETAILED DESCRIPTIONI Overview

[0016] Glycoproteins E1 and E2 form a heterodimer on the HCV envelope that mediates viral entry into host hepatocytes. Optimization of E1E2 to improve its structural stability and immunogenicity through rational design may lead to an effective B cell-based HCV vaccine that elicits a bNAb response. Native membrane-bound E1E2antigens have to be extracted from cell surface for biochemical and structural analyses, limiting their use as vaccine antigens. See de la Pena et al., Science 2022, 378:263-269. Currently available soluble E1E2 (sE1E2) antigens require co-expression of a neutralizing antibody to stabilize the E1E2 interface and can only be expressed in HEK293 cells with low yield, making it impossible for industrial production and further development of nanoparticle vaccines. See, e.g., Guest et al., PNAS 2021, 118:e2015149118; and Metcalf et al., Nat. Commun. 2023, 14:3980.

[0019] The disclosure includes next-generation sE1E2 antigens that exhibit superior stability, antigenicity, and immunogenicity. As detailed herein, the improved sE1E2 antigens can be applied to diverse HCV genotypes, expressed in various cell types suitable for industrial production, and displayed on single-component selfassembling protein nanoparticles (1c-SApNPs) as virus-like particle (VLP) vaccines.

[0020] The disclosure includes analysis of four H77 sE1E2 constructs containing a heterodimeric coiled coil (LZ) and an His6-tag. Following HEK293 F expression, these engineered antigens were tested for binding to bNAbs AR3C and AR4A. All four sE1E2 designs showed poor expression and no binding to AR4A, which only recognizes the native-like E1E2 interface. The C termini of sE1 and sE2 was truncated to remove membrane-proximal external regions (MPERs), termed “Cut 1”, and fused them to five heterodimeric scaffolds, including previously reported LZ and SZ, newly identified coiled coils GCP and IAAL, and a redesigned SpyTag002 / SpyCatcher002 (the unstructured N terminus of SpyCatcher002 was removed, termed “SPYdN”), to exert structural control in its sE1E2 fusion form. An unstructured, hydrophobic, cystine-containing loop (aa 264-293) in E1, termed “Cut 2” was then removed. In nonlimiting examples, 10 sE1E2 constructs based on two truncation variants (Cut 1 and Cut 1+2) and five heterodimeric scaffolds (LZ, SZ, GCP, IAAL, and SPYdN) were analyzed in this disclosure. Following expression in HEK293 F cells, binding to bNAbs AR3C and AR4A was analyzed. Surprisingly, all sE1E2-Cut1. scaffold antigens bound to AR4A with similar or better binding affinities, measured by half maximal effective concentration (EC50), compared to the sE1E2.SZ previously described protein (Metcalf et al., Nat. Commun. 2023, 14:3980). Even more surprisingly, all sE1E2-Cut1+2. scaffold antigens showed greater dimer purity with substantially higher binding affinities (~100- fold) for AR4A, suggesting a highly stable, native-like E1E2 interface. Not to be bound in theory, it is believed that this E1 loop may form random disulfide bonds with E1 orE2 of other sE1E2 antigens, thus causing misfolding, aggregation, and aberrant E1E2 forms. Additionally, the possibility of displaying sE1E2-Cut1+2.SPYdN on two 1c- SApNPs, ferritin (FR) 24-mer and “multilayered” I3-01v9a (I3-01v9a-LD7-PADRE) 60-mer. After transient expression in ExpiCHO cells, sE1E2 1c-SApNPs were purified by AR3C and AR4A-based immunoaffinity chromatography (IAC) columns and characterized structurally by negative-stain EM. In summary, the sE1E2-Cut1+2. scaffold antigens can be developed as HCV dimer vaccines or displayed on various 1c-SApNPs as HCV VLP vaccines. The disclosure demonstrates that the same engineering strategy can be applied to diverse HCV strains and genotypes, including extended to other HCV strains, including HC VI (genotype 1), 312 (genotype 3), and 521 (genotype 5).

[0021] In accordance with these studies, the disclosure provides novel HCV soluble immunogen polypeptides that are derived from the engineered or redesigned HCV sE1E2 polypeptides described herein. Also provided are HCV vaccine compositions containing a displaying platform, including a self-assembling nanoparticle that displays one or more of the engineered HCV sE1E2 polypeptides after expression and protease cleavage in a host cell. The disclosure includes uses of the engineered HCV sE1E2 polypeptides and the related nanoparticle vaccine compositions, e.g., for treating or preventing HCV infections. As demonstrated herein, the novel engineered SH1H2 immunogen polypeptides, such as the sE1E2-Cut1and sE1E2-Cut1+2designs, can be applied to diverse HCV strains and genotypes to create next-generation soluble E1E2 antigens with a “native-like” E1E2 interface that can be recognized by AR4A and other neutralizing antibodies. The newly identified heterodimeric coiled coils (e.g., GCP and IAAL) and redesigned SpyTag / SpyCatcher variant (e.g., SpyTag002 / SpyCatcher002dN, or SPYdN) can be used as heterodimeric scaffolds to create scaffolded antigens (e.g., the exemplified sE1E2-Cut1 / 1+2format) as next-generation HCV sE1E2 vaccine antigens. Further, the engineered sE1E2 immunogens (e.g., the sE1E2-Cut1 / 1+2. scaffold antigens) can be displayed on selfassembling platforms, such as ferritin 24-mer and multilayered 60-mer (I3-01v9a-LD7- PADRE) described herein, to create effective HCV nanoparticle vaccines.

[0022] Without intending to be constrained by any particular interpretation, it is considered that there are a number of technological advantages of the sE1E2 HCV immunogens and vaccine compositions described herein in comparison to HCV immunogenic compositions currently known in the art. For example, the exemplifiedsE1E2-Cut1 / 1+2.scaffold antigens showed ~100-fold higher AR4A binding affinity than sE1E2.LZ and sE1E2.SZ reported in the literature (See, e.g., Guest et al., PNAS 2021, 118:e2015149118; and Metcalf et al., Nat. Commun. 2023, 14:3980), making them what are believed to be the most effective HCV antigens to induce interface-specific bNAb responses in vaccination described to date. In addition, the exemplified sE1E2- Cut1 / 1+2. scaffold antigens can be readily expressed in eukaryotic cells, including but not necessarily limited to HEK293 F cells, and purified to any desired degree of purity using commonly used Nickel columns, whereas sE1E2.LZ and sE1E2.SZ antigens - the only existing antigens that bind AR4A - require AR4A co-expression and Avi-tag purification. Easy production and purification, as well as the high homogeneity and high yield, make sE1E2-Cut1 / 1+2. scaffold antigens suitable for industrial production. Further, the exemplified sE1E2-Cut1 / 1+2.SPYdN Ic-SApNPs can be expressed in GMP- compatible CHO cells with a “native-like” E1-E1E2 interface, thus providing the protein-based HCV nanoparticle vaccines.

[0023] Unless otherwise specified herein, the HCV vaccine immunogens of the disclosure, the encoding polynucleotides, expression vectors and host cells, as well as the related therapeutic applications, can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., (3rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention.II. Definitions

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4thed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0025] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, "an Env-derived trimer" can refer to both single or plural Env-derived trimer molecules, and can be considered equivalent to the phrase "at least one Env-derived trimer."

[0026] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and / or cellular type directed against that protein. Unless otherwise noted, the term “vaccine immunogen” is used interchangeably with “protein antigen” or “immunogen polypeptide”.

[0027] As used herein, a coiled coil dimer or coiled coil system refers to protein structural motifs that consist of two helices (e.g., α-helices) in parallel or antiparallelorientation. These helices wind around each other to form a supercoil and can be used as versatile modules for the construction of designed proteins. Coiled coils are frequently present in natural proteins. Examples of proteins with coiled coil regions include oncoproteins c-Fos / c-Jun, myosins, tropomyosins and intermediate filaments.

[0028] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0029] Epitope refers to an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0030] Effective amount of a vaccine or other agent that is sufficient to generate a desired response, such as to reduce or eliminate a sign or symptom of a condition or disease, such as a viral infection, can be determined by those skilled in the art when given the benefit of the present disclosure. For instance, an effective amount can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection. In general, this amount will be sufficient to measurably inhibit virus (for example, HCV) replication or infectivity. When administered to a subject, a dosage will generally be used that will achieve a target concentration that has beenshown to be sufficient for in vitro inhibition of viral replication. In some examples, an "effective amount" is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease, for example to treat HCV infection. In some examples, an effective amount is a therapeutically effective amount. In some examples, an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with HCV infection.

[0031] As used herein, a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein. The unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence. As used herein, proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment s) (e.g., inside a cell). For example, the amino acid sequences of bacterial enzymes such as B. stearothermophilus dihydrolipoyl acyltransferase (E2p) and the amino acid sequences of HCV E2 glycoproteins are not normally found joined together via a peptide bond.

[0032] As used herein, “E1” and “E2” each refers to an envelope glycoprotein on hepatitis C virus. In HCV-infected cells, E1 and E2 interact to form a noncovalent heterodimer. The E1 polypeptide is located in the HCV polyprotein immediately after the core protein at amino acid residues corresponding to residues 192-383 of the HCV genotype la, H77 isolate polyprotein. The sequence of this HCV isolate is described in, e.g., Kolykhalov et al., Science 1997; 277:570-574. The full length amino acid sequence of its polyprotein is set forth in SEQ ID NO: 1 (Accession No. AAB66324). As used herein, the ectodomain of E1 of this HCV isolate contains residues 192-353 (SEQ ID NO:2). In some examples, an extra residue at the C-terminus, residue 354, can also be included in the starting E1 ectodomain sequence for redesign, as exemplified herein. The E2 polypeptide immediately follows E1 in the HCV polyprotein sequence. For HCV genotype la, H77 isolate, E2 contains residues 384-746. As used herein, the ectodomain of E2 of this HCV isolate encompasses residues 384-716 (SEQ ID NO:3). In some examples, an extra residue at the C-terminus, residue 717, can also be included in the E1 ectodomain sequence for redesign, as exemplified herein. Unless otherwise noted, reference to an amino acid position in any of the mature, processed HCVproteins, such as E1 or E2, is made with numbering relative to the full length HCV polypeptide, and not with reference to the mature polypeptide.

[0033] Any database entry reference, such as reference to a sequence database, includes the sequence in the database and is incorporated herein as it exists on the effective filing date of this application.

[0034] The disclosure includes all polynucleotide and amino acid sequences described herein expressly and by reference, and every polynucleotide sequence referred to herein includes its complementary sequence, and its reverse complement

[0035] Sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1):1301 ADGGCSGGAY DIIICDECHS TDATSILGIG TVLDQAETAG ARLVVLATAT1351 PPGSVTVSHP NIEEVALSTT GEIPFYGKAI PLEVIKGGRH LIFCHSKKKC1401 DELAAKLVAL GINAVAYYRG LDVSVIPTSG DVVVVSTDAL MTGFTGDFDS1451 VIDCNTCVTQ TVDFSLDPTF TIETTTLPQD AVSRTQRRGR TGRGKPGIYR1501 FVAPGERPSG MFDSSVLCEC YDAGCAWYEL TPAETTVRLR AYMNTPGLPV1551 CQDHLEFWEG VFTGLTHIDA HFLSQTKQSG ENFPYLVAYQ ATVCARAQAP1601 PPSWDQMWKC LIRLKPTLHG PTPLLYRLGA VQNEVTLTHP ITKYIMTCMS1651 ADLEVVTSTW VLVGGVLAAL AAYCLSTGCV VIVGRIVLSG KPAIIPDREV1701 LYQEFDEMEE CSQHLPYIEQ GMMLAEQFKQ KALGLLQTAS RQAEVITPAV1751 QTNWQKLEVF WAKHMWNFIS GIQYLAGLST LPGNPAIASL MAFTAAVTSP1801 LTTGQTLLFN ILGGWVAAQL AAPGAATAFV GAGLAGAAIG SVGLGKVLVD1851 ILAGYGAGVA GALVAFKIMS GEVPSTEDLV NLLPAILSPG ALVVGVVCAA1901 ILRRHVGPGE GAVQWMNRLI AFASRGNHVS PTHYVPESDA AARVTAILSS1951 LTVTQLLRRL HQWISSECTT PCSGSWLRDI WDWICEVLSD FKTWLKAKLM2001 PQLPGIPFVS CQRGYRGVWR GDGIMHTRCH CGAEITGHVK NGTMRIVGPR2051 TCRNMWSGTF PINAYTTGPC TPLPAPNYKF ALWRVSAEEY VEIRRVGDFH2101 YVSGMTTDNL KCPCQIPSPE FFTELDGVRL HRFAPPCKPL LREEVSFRVG2151 LHEYPVGSQL PCEPEPDVAV LTSMLTDPSH ITAEAAGRRL ARGSPPSMAS2201 SSASQLSAPS LKATCTANHD SPDAELIEAN LLWRQEMGGN ITRVESENKV2251 VILDSFDPLV AEEDEREVSV PAEILRKSRR FARALPVWAR PDYNPPLVET2301 WKKPDYEPPV VHGCPLPPPR SPPVPPPRKK RTVVLTESTL STALAELATK2351 SFGSSSTSGI TGDNTTTSSE PAPSGCPPDS DVESYSSMPP LEGEPGDPDL2401 SDGSWSTVSS GADTEDVVCC SMSYSWTGAL VTPCAAEEQK LPINALSNSL2451 LRHHNLVYST TSRSACQRQK KVTFDRLQVL DSHYQDVLKE VKAAASKVKA2501 NLLSVEEACS LTPPHSAKSK FGYGAKDVRC HARKAVAHIN SVWKDLLEDS2551 VTPIDTTIMA KNEVFCVQPE KGGRKPARLI VFPDLGVRVC EKMALYDVVS2601 KLPLAVMGSS YGFQYSPGQR VEFLVQAWKS KKTPMGFSYD TRCFDSTVTE2651 SDIRTEEAIY QCCDLDPQAR VAIKSLTERL YVGGPLTNSR GENCGYRRCR2701 ASGVLTTSCG NTLTCYIKAR AACRAAGLQD CTMLVCGDDL VVICESAGVQ2751 EDAASLRAFT EAMTRYSAPP GDPPQPEYDL ELITSCSSNV SVAHDGAGKR2801 VYYLTRDPTT PLARAAWETA RHTPVNSWLG NIIMFAPTLW ARMILMTHFF2851 SVLIARDQLE QALNCEIYGA CYSIEPLDLP PIIQRLHGLS AFSLHSYSPG2901 EINRVAACLR KLGVPPLRAW RHRARSVRAR LLSRGGRAAI CGKYLFNWAV2951 RTKLKLTPIA AAGRLDLSGW FTAGYSGGDI YHSVSHARPR WFWFCLLLLA3001 AGVGIYLLPN R

[0036] In the above HCV polyprotein sequence, sE1 sequence is underlined (SEQ ID NO: 12), including the italicized MPER motif (SEQ ID NO:41). Residues of the E1 unstructured hydrophobic loop sequence (residues 264-293) are double underlined (SEQ ID NO:32). The sE2 sequence is dash underlined (SEQ ID NO: 13), with the C- terminal MPER motif (SEQ ID NO:42) italicized.

[0037] Unless otherwise noted, reference to an E1 or E2 polypeptide herein also includes variants or truncated forms thereof, such as soluble forms that are truncated at their C-terminus to remove the transmembrane domain, e.g., sE1 and sE2 shown in SEQ ID NO:4 and SEQ ID NO:5, respectively. In some examples, the variants a property, such as a binding property, and variants, such as those from different HCV isolates and quasispecies, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the E2 polypeptide from HCV genotype la, isolate H77.

[0038] In examples, a vaccine of this disclosure comprises modified HCV E1 and E2 proteins, which may include additional protein components as described herein. The modified HCV E1 and E2 proteins, when used to stimulate an HCV immune response in an individual, are typically used in a configuration that contains the engineered sE1 and sE2 domains that are connected by the interacting motifs of a protein heterodimerization system and form native-like E1E2 interface. The disclosure includes all intact (e.g., single) contiguous polypeptides described herein, and their products which result from proteolytic cleavage of the contiguous proteins. In examples, engineered sE1E2 proteins are provided as self-assembled nanoparticles, as further described herein. In examples, the disclosure provides vaccine formulations comprising pre-formed heterodimers. In examples, an mRNA encoding a described polypeptide can be delivered to cells, and may combined with a delivery agent. In examples, an mRNA may comprise modifications that improve one or more immune responses. The modifications may be such that the mRNA is resistant to enzyme degradation and therefore the amount of protein translated is increased, relative to use of unmodified mRNA.

[0039] Unless otherwise noted, variants of E1 and E2 polypeptides include synthetic molecules prepared by translation of nucleic acids, proteins generated by chemical synthesis, such as syntheses that include ligation of shorter polypeptides, through recombinant methods, proteins isolated from HCV-infected human and non-human tissue and cells, chimeric E1 or E2 polypeptides and modified forms thereof. Variants of E1 or E2 polypeptides also include fragments or portions of E1 or E2 that are of sufficient length or include appropriate regions to retain at least one property of a full-length E1 or E2 polypeptide, such as the ability to bind to anti-E1 or anti-E2 antibodies. Variants of E1 or E2 polypeptides also include those that contain chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, famysylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0040] As used herein, the HCV E1 ectodomain (E1 ECTO or E1 ATM) refers to the soluble portion of the E1 polypeptide that is outside the viral membrane, i.e., the N- terminal portion of E1 protein that does not include the transmembrane helix. In the prototype H77 isolate, E1 ATM or EIECTO can consist of amino acids 192-353 (SEQ ID NO:2) or 192-354 (SEQ ID NO:4). Similarly, the HCV E2 ectodomain (E2ECTO or E2ATM) refers to the soluble portion of the E2 polypeptide that is outside the viral membrane, i.e., the N-terminal portion of E2 protein that does not include the transmembrane helix. In the prototype H77 isolate, E2ATM or E2ECTO can consist of amino acids 384-716 (SEQ ID NO:3) or 384-717 (SEQ ID NO:5). The C-terminus of each of E1 ectodomain and E2 ectodomain is a membrane-proximal external region (MPER).

[0041] As used herein, corresponding residues refers to residues that occur at aligned loci. Related or variant polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches, and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTP) and others known to those of skill in the art. By aligning the sequences of polypeptides, one skilled in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. As demonstrated in Figure 5 and Example 5 herein, by aligning the sequences of E1 (or E2) polypeptides from different HCV isolates and strains, one of skill in the art can identify corresponding residues, using conserved and identical amino acid residues as guides. Corresponding positions also can be based on structural alignments, for example by using computer simulated alignments of protein structure.

[0042] As used herein, the term “hepatitis C virus,” “HCV,” or “HCVs” includes different viral genotypes, subtypes, quasispecies and isolates and include any noncytopathic RNA virus that has a single and positive-stranded RNA genome belonging to the Hepacivirus genus of the Flaviviridae family. The term includes different isolates of HCV such as, without limitation, those having polyprotein sequences and accession numbers shown above, as well as any others in the NCBI database. Examples of different genotypes encompassed by this term include, without limitation, genotype 1, 2, 3, 4, 5 and 6, as described in Simmonds et al. (Hepatology 42:962-973, 2005). Reference to HCV also includes those of any additional genotypes that are established. Examples of different subtypes include, without limitation, 1a, 1b, 1c, 2a, 2b, 2c, 2i, 2k, 3a, 3b, 3k, 4a, 4d, 4f, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6q, 6p and 6t . The term also includes cell culture HCVs (HCVcc) and pseudotype HCVs (HCVpp), as well as HCV quasispecies. Various HCVs are described by Simmonds P. in Genetic diversity and evolution of hepatitis C virus— 15 years on, J Gen Virol 85:3173-3188 (2004) and Simmonds et al. in Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes, Hepatology 42:962-973 (2005). HCV nucleotide sequences are known in the art. For example, see Viral Bioinfomatics Research Center (hcvdb.org) and the Hepatitis C Virus database (hcv.lanl.gov).

[0043] Immunogen as used herein refers to a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen can lead to protective immunity and / or proactive immunity against a pathogen of interest.

[0044] Immune response refers to a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some examples, the response is specific for a particular antigen (an "antigen-specific response"). In some examples, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In some other examples, the response is a B cell response, and results in the production of virus-specific antibodies.

[0045] Immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against virus expressing the immunogenic polypeptide, or induces a measurable B cell response (such as production of antibodies) against the immunogenic polypeptide.

[0046] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0047] Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity / similarity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237- 44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0048] SpyCatcher-SpyTag refers to a protein ligation system that is based on based on the internal isopeptide bond of the CnaB2 domain of FbaB, a fibronectin- binding MSCRAMM and virulence factor of Streptococcus pyogenes. See, e.g., Terao et al., J. Biol. Chem. 2002;277:47428-47435; and Zakeri et al., Proc. Natl. Acad. Sci. USA. 2012;109:E690-E697. It utilizes a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technologyhas been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags.

[0049] The term "subject" refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. In an example, the subject is a human.

[0050] The term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an HCV infection), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0051] Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In some examples of the invention, vaccines or vaccine immunogens or vaccine compositions are expressed from fusion constructs and self-assemble into nanoparticles displaying an immunogen polypeptide or protein on the surface. In examples, an mRNA encoding a described sE1E2 fusion immunogen polypeptide is administered to cells of an individual, after which the fusion polypeptide is cleaved by a host protease and the cleaved sE1E2 protein may be secreted from the cells in order to promote a prophylactic or therapeutic immune response.

[0052] Virus-like particle (VLP) refers to a non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

[0053] A self-assembling nanoparticle refers to a ball-shape protein shell with a diameter of tens of nanometers and well-defined surface geometry that is formed by identical copies of a non-viral protein capable of automatically assembling into a nanoparticle with a similar appearance to VLPs. Known examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B. stearothermophilus dihydrolipoyl acyltransferase (E2p), Aquifex aeolicus lumazine synthase (LS), and Thermotoga maritima encapsulin, which all form 60-mers. Self-assembling nanoparticles can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for nanoparticle production, detection, and characterization can be conducted using the same techniques developed for VLPs. ni. Engineered soluble E1E2 immunogen polypeptides

[0054] The disclosure provides engineered or redesigned HCV soluble E1E2 (sE1E2) polypeptides or proteins for producing HCV vaccines. These HCV glycoprotein-based immunogens contain a modified (or engineered) E1 ectodomain sequence and a modified (or engineered) E2 ectodomain sequence. Also, the two modified ectodomain sequences are respectively fused, independently at the N-terminus or the C-terminus, to the two interacting motifs (association motifs or binding motifs) of a protein heterodimerization system (e.g., a SpyCatcher-SpyTag system or a coiledcoil system). This results in two fusion partners or components (sE1 fusion partner and sE2 fusion partner) for generating the final sE1E2 immunogen polypeptides. Typically, a spacer motif is inserted between the sE1 fusion partner (which includes the linked protein heterodimerization motif) and the sE2 fusion partner (which includes the linked protein heterodimerization motif). In some of these examples, the spacer motif is a peptide cleavage site for a host protease (e.g., furin as exemplified herein), which allows digestion of the sE1E2 fusion immunogen polypeptides by the host protease after their expression / folding in a host cell (e.g., HEK293 F cell as exemplified herein). In these examples, fusions between the sE1 / sE2 sequences and the interacting motifs of the protein heterodimerization system are configured to produce engineered sE1 ectodomain and engineered E2 ectodomain that will form the native-like heterodimer E1E2 interface upon expression and protease cleavage of the fusion immunogen polypeptide in a host cell. Specifically, once digested at the cleavage site, the sE1 and sE2 domains remain connected or associated by the interacting motifs of the protein heterodimerization system and form the native-like E1E2 interface.

[0055] Depending on the employed protein heterodimerization system, the connection between the sE1 and sE2 domains in the cleaved proteins upon expression and protease digestion of the engineered fusion sE1E2 polypeptides in a host cell can be covalent or non-covalent. Due to connection or association between the interacting motifs of the protein heterodimerization system, the otherwise separated sE1 and sE2 ectodomains can interact with each other to form the correct native-like E1E2 interface.In some examples, the inserted spacer contains a furin cleavage site, such as RRRRRR (SEQ ID NO: 6) as exemplified herein. Other than a furin cleavage site, many other peptide cleavage sites for host endo-proteases known in the art may also be used in the spacer motif in the engineered HCV soluble E1E2 (sE1E2) polypeptides described herein. These include, e.g., cleavage sites for TTSPs such as TMPRSS2, matrix metalloproteases (MMPs) such as MMP-1, cathepsins such as cathepsin B, and Factor Xla / FVIIa. See, e.g., Chen et al., Adv Drug Deliv Rev. 2013; 65(10): 1357-69; Kuhnel et al., Protein Eng. Des. Sei. 2003; 16: 777-83; and Saadat et al., Front. Mol. Biosci. 2021; 8: 725528.

[0056] As noted above, the modified E1 and E2 ectodomain sequences in the sE1E2 immunogen polypeptides typically each contain a deletion of the membrane- proximal external region (MPER) at the C-terminus. As is understood in the art, MPERof HCV refers to the extracellular sequence motif that is immediately next to the N- terminus of the transmembrane domain that anchors the E1 or E2 glycoprotein to the virus membrane. Using the polyprotein sequence of the prototype HCV isolate H77 (SEQ ID NO: 1; Accession No. AAB66324) as exemplification, deletion of MPER in E1 ectodomain involves truncation of the sequence at the C-terminus after residue H312 or a residue that is about 1 to 2 residues upstream or downstream of residue H312 (e.g., truncation after residue P310, G311, I313, or T314), and deletion of MPER in E2 ectodomain encompasses truncation of C-terminal sequence after residue Y701 or a residue that is about 1 to 4 residues downstream of residue Y701 (e.g., truncation after residue L702, Y703, G704, or V705).

[0057] In addition to the MPER deletion, the modified E1 ectodomain sequence in some sE1E2 immunogen polypeptides of the disclosure also contain modification (deletion or substitution) of an unstructured loop sequence. As used herein, this unstructured E1 loop sequence or motif refers to a sequence fragment in E1 that contains about 30 or more amino acid residues and encompasses the putative fusion peptide. The putative fusion peptide corresponds to residues 272-285 of the polyprotein ofHCV H77 isolate. See, e.g., Tong et al., Front. Immunol. 2018; 9: 1411. This unstructured E1 loop is a hydrophobic and cystine-containing (C272 and C281) sequence motif that is unstructured in cryo-EM structure of the E1E2 glycoprotein complex. As revealed herein, the unstructured E1 loop may form random disulfide bonds with E1 or E2 of other sE1E2 molecules, thus causing misfolding, aggregation, and aberrant E1E2 forms (e.g., dimer-of-dimer). It is to be removed to achieve a stable, native-like E1E2 interface in the engineered sE1E2 immunogen polypeptides.

[0058] In some examples, the unstructured E1 loop that is modified in the sE1E2 immunogens of the invention contains a shorter 30-aa sequence motif that corresponds to residues 264-293 (LLVGSATLCSALYVGDLCGSVFLVGQLFTF; SEQ ID NO:32) of the polyprotein of HCV H77 isolate, as exemplified herein. In some examples, modification of this unstructured E1 loop involves a complete deletion of this unstructured, hydrophobic, and cystine-containing loop sequence. In some examples, the unstructured E1 loop is substituted with a short spacer of about 1 to about 3 amino acids that are not hydrophobic and cysteines. In some of these examples, the substituting amino acid residues are non-polar, e.g., glycine or alanine. In someexamples, the substituting amino acid residues are hydrophilic. In some examples, the substituting residues are charged.

[0059] In addition to the deletion or substitution of the unstructured sequence motif corresponding to residues 264-293 of HCV H77 polyprotein, modification of the unstructured E1 loop or motif can also include alteration of additional residues at the N- terminus. As described herein (e.g., Example 5), residues 257-263 at the N-terminus (QLRRHID; SEQ ID NO:33) beyond the deleted or substituted residues 264-293 contain 3-5 charged or polar residues. In some sE1E2 immunogen polypeptides of the invention, these N terminus residues are retained because they are suitable for use as a surface loop after the truncation of the unstructured E1 loop containing residues 264- 293). In some other sE1E2 immunogen polypeptides of the invention, this 7-aa N- terminal fragment can be replaced with a loop sequence or motif of 7 or more hydrophilic or charged residues, which are capable of forming a flexible surface loop.

[0060] In some examples, the modified HCV E1 and E2 ectodomain sequences are fused respectively at their C-terminus to the N-terminus of the two protein heterodimerization motifs. Typically, a short linker is used to connect the sE1 and sE2 sequences to the protein heterodimerization motifs, e.g., a PGG or a GS linker exemplified herein. In some of these embodiments, the linker connecting the sE1 or sE2 sequence to the heterodimerization motif (e.g., SpyTag or SpyCatcher) is a flexible GS- rich peptide that contains about 4 -12 glycine and / or serine residues. Examples of these linkers include, e.g., GSGS (SEQ ID NO:34), GGGS (SEQ ID NO:35), SGGG (SEQ ID NO:36), GSGSGSGS (SEQ ID NO:37), GGGSGGGS (SEQ ID NO:38), GSGSGSGSGSGS (SEQ ID NO:39), and GGGSGGGSGGGS (SEQ ID NO:40).

[0061] While exemplified herein with E1 and E2 sequences of the HCV H77 isolate, sE1E2 immunogenic polypeptides of the disclosure can be readily generated with E1 and E2 glycoprotein sequences from other HCV isolates or strains by those skilled in the art when given the benefit of the present description. HCV isolates have been grouped into seven genotypes and a number of subtypes, which have different geographical distributions. E1 and E2 sequences of many of these isolates are already known. For example, HCV related sequence information can be obtained from the Hepatitis C Virus Databases maintained by the National Institute of Allergy and Infectious Diseases (NIAID). While there is a considerable degree of variability among E1 and E2 sequences of different HCV isolates, a certain number of conserved motifsor neutralizing epitopes have been identified in the E1 and E2 sequences. In engineering sE1E2 polypeptides of the invention with a given HCV polyprotein sequence in accordance with the strategy described herein, one can readily determine the appropriate residues for modifications via sequence alignment and also considering the conserved motifs known in the art. See, e.g., Simmonds et al., Hepatology 42:962- 973, 2005; Wang et al., Viruses 3:2127-2145, 2011; and Krey et al., PLoS Pathog. 6:el000762, 2010; Bhattarai et al., PLoS Pathog. 11: e1005183, 2015; and Kachko et al., Vaccine 30:69-77, 2011.

[0062] Unless otherwise noted, the amino acid residue numbering of the soluble E1E2 immunogen polypeptides and the E1 and E2 components, as well as derivative or variant sequences described herein, is based on HCV genotype la, H77 isolate. Due to conserved sequence motifs or epitopes in the E1 and E2 sequences among the different HCV genotypes and isolates, corresponding amino acid residues bordering the various domains, regions and loops of an E1 or E2 protein from any other HCV genotype and isolate can be ascertained (e.g., by sequence alignment) or otherwisse known in the art. For example, the unstructured E1 loop sequence to be deleted from the sE1E2 immunogen polypeptides spans residues 264-293 for the H77 isolate. As demonstrated herein with multiple E1 sequences from diverse HCV genotypes, this unstructured E1 loop sequence from other HCV isolates can be readily determined via a sequence alignment. Specifically, the unstructured E1 loop or motif is exemplified herein by sequence conservation plot of the encompassing E1 sequences from genotypes 1-6 using that of H77 isolate as a template in sequence alignment and numbering (Figure 5). Similarly, the MPER motifs in E1 and E2 glycoproteins from other HCV isolates or stains can also be ascertained by a sequence alignment with the exemplified H77 isolate sequence, as well as structure analysis of the respective glycoprotein. See, e.g., Idrees et al., Theor Biol Med Model. 10: 24, 2013; Kong et al., Science. 342:1090-1094, 2013; and Khan et al., Nature 509:381-384, 2014.

[0063] In some examples, the sE1E2 immunogen polypeptides of the invention can contain a soluble E1 and a soluble E2 ectodomain sequences as described herein from two different HCV isolates or strains. For example, the sE1 ectodomain sequence can be derived from the H77 isolate, and the sE1 ectodomain sequence can be derived from another HCV isolate or strain. In some examples, the engineered HCV sE1E2 immunogen polypeptides from other isolates can be obtained by directly adopting theH77 sequence designs without further modification. This includes deletion of the MPERs from the C-terminus of each of the E1 and E2 ectodomain sequences, and replacing the corresponding unstructured hydrophobic loop in E1 with the same nonpolar residue or a short hydrophilic motif as in the engineered H77 sequences exemplified herein.

[0064] To promote a stable and native-like E1E2 interface, a protein heterodimerization system is incorporated into the sE1E2 immunogen polypeptides of the invention. A protein heterodimerization system refers to any interacting protein or peptide motifs that differ in sequence (e.g., motif A and motif B) and can promote dimerization or association of two distinct polypeptide sequences that are respectively linked to the two motifs. Protein heterodimerization systems well known in the art may be employed and / or modified in the practice of the invention when given the benefit of this description. Examples of suitable systems include leucine zipper proteins which are well known in the art. For example, several leucine zipper motifs such as c-Jun and c- Fos have been developed to induce protein dimerization in bioengineering. See, e.g., Junius et al., J. Neurochem., 1996; 63, 2014-2020; Mason and Arndt, Chembiochem. 2004; 5 (2), 170-176; and Gazon et al., Front. Microbiol., 2017; 8, 2686. Other protein heterodimerization systems that can be employed in the invention include, e.g., other coiled coil zipper motifs and the SpyTag / SpyCatcher protein superglue system as exemplified herein.

[0065] In some examples, a SpyTag / SpyCatcher dimerization system can be incorporated into the HCV sE1E2 immunogenic polypeptides of the invention. Once the sE1E2 fusion immunogen polypeptides of the disclosure are expressed and cleaved by a host protease (e.g., furin) at the cleavage site, the SpyTag and SpyCatcher motifs can form an isopeptide bond. This results in an sE1sE2 fusion monomer that has the sE1 and sE2 sequences indirectly but covalently connected by the bonded SpyCatcher / SpyTag motifs. As detailed in the Examples herein, the engineered soluble E1 polypeptide can be fused to a SpyTag motif, and the engineered soluble E2 polypeptide can be fused to a SpyCatcher motif. Alternatively, the soluble E1 sequence can be fused to a SpyCatcher motif, and the soluble E2 sequence can be fused to a SpyTag motif. In some of these examples, the original SpyTag and Spy Catcher motifs are modified and optimized to promote E1E2 dimerization and homogeneity. For example, as exemplified herein, an unstructured, 21-aa or 24-aa N-terminus can beremoved from the SpyCatcher. In addition, a short AS linker (e.g., AS dipeptide) can be inserted at the N-terminus of SpyCatcher. In some exemplified examples, a modified SpyTag motif shown in SEQ ID NO:7 and a modified SpyCatcher motif shown in SEQ ID NO:8 or SEQ ID NO:43 can be used in the sE1E2 fusion sequence to promote formation of the native-like E1E2 interface.

[0066] In some examples, a coiled-coil based dimerization system is used to provide stabling scaffolds for the HCV sE1E2 immunogenic polypeptides of the invention. Coiled-coil (CC) motifs, ubiquitous in natural proteins, are characterized by the relatively well-defined assembly and specificity rules and are therefore suitable as building blocks for designing and developing novel polypeptide scaffolds, functional proteins, and as a regulator of protein-protein interactions. The specificity of coiled- coil dimer pairing is mainly based on hydrophobic and electrostatic interactions between residues at positions a, d, e, and g of the heptad repeat. Binding affinity, on the other hand, can also be affected by surface residues that face away from the dimerization interface. For dimerization of two polypeptides of interest (e.g., sE1 and sE2), two interacting coils are respectively fused to the two polypeptides.

[0067] A number of coiled-coil dimer motifs can be employed in the sE1E2 immunogen polypeptides of the invention. In some examples, a modified C-Jun / C-Fos based dimerization system can be used, e.g., the “LZ” motifs (SEQ ID NOs:20 and 21) as exemplified herein. In some examples, sE1 and sE2 can be dimerized via the “SZ” coiled coil motifs (SEQ ID NOs:22 and 23) as exemplified herein. In some other examples, the sE1E2 polypeptides of the invention can contain a heterodimeric leucine zipper coils as its dimerization scaffold, e.g., the “GCP” leucine zipper motif (SEQ ID NOs:24 and 25) exemplified herein. In still some other examples, the sE1E2 polypeptides can utilize the “IAAL” heterodimeric coiled coils (SEQ ID NOs:26 and 27) as exemplified herein. In these examples, sE1 can be fused to either one of the two interacting coil motifs, while sE2 is fused to the other. For example, with the “LZ” coiled coils, sE1 can be fused to the C-Jun motif (SEQ ID NO:20) while sE2 is fused to the C-Fos motif (SEQ ID NO:21). Alternatively, sE1 can be fused to the C-Fos motif (SEQ ID NO:21) while sE2 is fused to the C-Jun motif (SEQ ID NO:20).

[0068] In addition to the specific SpyTag / SpyCatcher and coiled-coil dimerization motifs exemplified herein, many other heterodimeric peptide systems known in the art may also be used in the practice of the invention. See, e.g., Lebar et al., Nat. Chem.Biol. 16, 513-519, 2020; Fink et al., Nat. Chem. Biol. 15, 115-122, 2018; Chen et al., Nature 565, 106-111, 2019; O’Shea et al., Curr. Biol. 3, 658-667, 1993; Reinke et al., J. Am. Chem. Soc. 132, 6025-6031, 2010; Litowski et al., J. Biol. Chem. 277, 37272- 37279, 2002; Drobnak et al., J. Am. Chem. Soc. 139, 8229-8236, 2017; Gradisar et al., J. Pept. Sci. 17, 100-106, 2011; Moll et al., M. Protein Sci. 10, 649-655, 2001. In various examples, a His tag, e.g., HHHHHH (SEQ ID NO:29) as exemplified herein, may be added to the C-terminus of the sE1E2 fusion polypeptides of the invention to facilitate purification. In these examples, a short peptide spacer or linker (e.g., GS as exemplified herein) can be inserted between the His tag and the second motif of the protein heterodimerization system.

[0069] The various engineered HCV sE1E2 polypeptides of the disclosure can be obtained or generated in accordance with the protocols exemplified herein or methods well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). Upon recombinant expression (e.g., in HEK293 F cells as detailed herein), the proteins can be purified by any of the routinely practiced procedures. See, e.g., Guide to Protein Purification, Ed. Deutscher, Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Once purified, antigenicity and other properties of the engineered HCV sE1E2 polypeptides can also be readily examined with standard methods, e.g., antigenic profiling using known bNAbs and non-Nabs, differential scanning calorimetry (DSC), electron microscopy, binding analysis via ELISA, Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and co-crystallography analysis as exemplified herein.IV. Scaffolded HCV sE1E2 vaccine compositions

[0070] Other than the engineered or redesigned HCV sE1E2 immunogen polypeptides described above, the disclosure also provides HCV sE1E2 immunogen polypeptide-based vaccine compositions that contain a heterologous scaffold that presents or incorporates an engineered HCV sE1E2 polypeptide. The vaccinecompositions are expressed from polynucleotide constructs that encode fusion proteins containing (1) a soluble sE1E2 fusion immunogen polypeptide described above, and (2) the subunit sequence of a heterologous protein scaffold (e.g., a self-assembling nanoparticle protein sequence). Upon expression of the polynucleotide constructs and protease digestion of the encoded fusion proteins in a host cell, the engineered HCV sE1E2 polypeptide displayed on the heterologous scaffold contains sE1 and sE2 domains that form the correct E1E2 interface via the association of the interacting motifs of the protein heterodimerization system as described above.

[0071] Any heterologous scaffold can be used to present the engineered HCV sE1E2 polypeptide in the construction of the vaccines of the invention. These include nanoparticles, virus-like particles, protein carriers (e.g., immunoglobulin chains or domains such as Fc, KLH, BSA, tetanus toxoid, and diphtheria toxoid), as well as various chemical scaffolds. In some examples, a virus-like particle (VLP) such as bacteriophage Qp VLP and nanoparticles can be used. In some examples, the heterologous scaffold for presenting or displaying the engineered HCV sE1E2 polypeptide is a self-assembling nanoparticle. Various nanoparticle platforms can be employed in generating the vaccine compositions of the invention. In general, the nanoparticles employed in the invention need to be formed by multiple copies of a single subunit, or distinct subunits. The nanoparticles are typically ball-like shaped, and / or have rotational symmetry (e.g., with 3-fold and 5-fold axes), e.g., with an icosahedral structure exemplified herein. Additionally or alternatively, the aminoterminus of the particle subunit is exposed and in close proximity to the 3-fold axis, and the spacing of three amino-termini has to closely match the spacing of the carboxyltermini of the HCV sE1E2 polypeptides. In some examples, the immunogens comprise self-assembling naoparticles with a diameter of about 20nm or less (usually assembled from 12, 24, or 60 sububits) and 3 -fold axes on the particle surface.

[0072] In some examples, the immunogen protein or polypeptide (e.g., an engineered soluble HCV sE1E2 polypeptide described herein) is presented on selfassembling nanoparticles such as self-assembling nanoparticles derived from 13-01 and ferritin (FR) as exemplified herein. 13-01 is an engineered protein that can selfassemble into hyperstable nanoparticles. See, e.g., Hsia et al., Nature 535, 136-139, 2016. Sequences of 13-01 and a number of variants with improved properties are known in the art. See, e.g., WO2017 / 192434 and WO2019 / 089817. Ferritin is a globularprotein found in all animals, bacteria, and plants. The globular form of ferritin is made up of monomeric subunit proteins (also referred to as monomeric ferritin subunits), which are polypeptides having a molecule weight of approximately 17-20 kDa. Amino acid sequences of an 13-01 variant (I3-01v9a) and ferritin nanoparticle subunits as exemplified herein are shown in SEQ ID NOs:15 and 16, respectively. In various examples, the HCV sE1E2 nanoparticle vaccines of the invention can employ any of these known nanoparticles, as well as their conservatively modified variants or variants with substantially identical (e.g., at least 90%, 95% or 99% identical) sequences.

[0073] Ferritin sequence (SEQ ID NO: 15)

[0074] I3-01v9a (for monomeric antigen display) (SEQ ID NO: 16)

[0075] E2p subunit sequence (SEQ ID NO:31)

[0076] In addition to these exemplified nanoparticle sequences, many other nanoparticles or VLPs known in the art may also be used in the practice of the invention. These include the well-known self-assembling E2p nanoparticle. E2p is a redesigned variant of dihydrolipoyl acyltransferase from Bacillus stearothermophilus that has been shown to self-assemble into thermostable 60-meric nanoparticle. See, e.g., He et al., Nat. Commun. 7:12041, 2016. Additional scaffolds that may be employed in the nanoparticle vaccines of the invention also include, e.g., Aquifex aeolicus lumazine synthase, Thermotoga Maritima encapsulin, Myxococcus xanthus encapsulin, bacteriophage Qbeta virus particle, Flock House Virus (FHV) particle, ORSAY virus particle, and infectious bursal disease virus (IBDV) particle. Other molecules that maybe used as the presenting platform of the nanoparticle vaccines of the invention include, e.g., molecules with the following PDB IDs: 1 JIG (12-mer Dlp-2 from Bacillus anthracis). 1UVH (12-mer DPS from Mycrobacterium smegmatis), 2YGD (24-mer eye lens chaperone aB-crystallin), 3CS0 (24-mer DegP24), 3MH6 and 3MH7 (24-mer HtrA proteases), 3PV2 (12-mer HtrA homolog DegQ WT), 4A8C (12-mer DegQ from E. Coli.), 4A9G (24-mer DegQ from E. Coli.), 4EVE (12-mer HP -NAP from Helicobacter pylori strain YS29), and 4GQU (24-mer Hi sB from Mycobacterium tuberculosis').

[0077] Some HCV sE1E2 nanoparticle vaccine compositions can additionally contain other structural components that function to further enhance stability and antigenicity of the displayed immunogen. In some examples, a locking protein domain can be inserted into the nanoparticle construct, e.g., by covalently fused to the C- terminus of the nanoparticle subunit. The locking domain can be any dimeric protein that is capable of forming an interface through specific interactions such as hydrophobic (van der Waals) contacts, hydrogen bonds, and / or salt bridges. General guidance on selecting locking domains and specific examples (e.g., LD7 as exemplified herein and LD4) are described in the art, e.g., PCT / 2019 / 036917. In some examples, HCV sE1E2 nanoparticle vaccines of the invention can also contain a T-cell epitope to promote robust T-cell responses and to steer B cell development towards bNAbs. The T-cell epitope can be located at any position in relation to the other structural components as long as it does not impact presentation of the immunogen polypeptides on the nanoparticle surface. Any T-cell epitope sequences or peptides known in the art may be employed in the practice of the present invention. They include any polypeptide sequence that contain MHC class-II epitopes and can effectively activate CD4+ and CD8+ T cells upon immunization, e.g., T-helper epitope that activates CD4+ T helper cells. See, e.g., Alexander et al., Immunity 1, 751-761,1994; Ahlers et al., J. Clin. Invest. 108: 1677-1685, 2001; Fraser et al., Vaccine 32: 2896-2903, 2014; De Groot et al., Immunol. Cell Biol. 8:255-269, 2002; and Gene Ther. 21 : 225-232, 2014. In some examples, the universal pan DR epitope peptide (PADRE) T-cell epitope (AKFVAAWTLKAAA; SEQ ID NO: 30) can be used, as exemplified herein.

[0078] The scaffolded HCV sE1E2 vaccine compositions of the invention can be constructed in accordance with standard recombinant techniques and the methods described herein and / or other methods that have been described in the art, e.g., He et al., Nat. Comm. 7, 12041, 2016; Kong et al., Nat. Comm. 7, 12040, 2016; and He et al.,Sci Adv. 4(11):eaau6769, 2018. In various examples, nanparticle displaying any of the engineered HCV sE1E2 polypeptides can be constructed by fusing the E2 polypeptide to the subunit of the nanoparticle (e.g., I3-01v9a subunit). Preferably, C-terminus of the HCV sE1E2 polypeptide is fused to the N-terminus of the nanoparticle subunit. In some examples, a short peptide linked can be used to connect the sE1E2 polypeptide and the nanoparticle. For example, as exemplified herein, a 10GS linker GGGGSGGGGS (SEQID NO: 17) can be used in the ferritin scaffolded vaccines, and a 5GS linker GGGGS (SEQ ID NO: 18) can be used to fuse the sE1E2 immunogen to the 13-01 displaying scaffold. Once constructed, the antigeniciy and structural integrity of the nanoparticle displayed HCV sE1E2 polypeptides can be readily analyzed via standard assays, e.g., antibody binding assays, biolayer interferometry, and negative-stain electron microscopy (EM). As exemplified herein, the various fusion molecules can all self-assemble into nanoparticles that display immunogenic epitopes of the HCV sE1E2 polypeptides. By eliciting a robust neutralizing antibody response, these nanoparticles are useful for vaccinating individuals against a broad range of HCV viruses.V. Polynucleotides and expression constructs

[0079] The engineered HCV sE1E2 polypeptides and the nanoparticle vaccine compositions of the disclosure are typically produced by first generating expression constructs (i.e., expression vectors) that contain operably linked coding sequences of the various structural components described herein. Accordingly, in some related aspects, the invention provides substantially purified polynucleotides (DNA or RNA) that encode the engineered HCV sE1E2 polypeptides and nanoparticle displayed HCV sE1E2 immunogens as described herein, as well as expression vectors that harbor such polynucleotides and host cells for producing the HCV sE1E2 immunogen polypeptides and the vaccine compositions (e.g., HEK293 F cells and ExpiCHO cells exemplified herein). The fusion polypeptides encoded by the polynucleotides or expressed from the vectors are also included in the invention. These include sE1sE2 fusion monomers covalently linked by or sE1sE2 heterodimers non-covalently associated by the interacting motifs of a protein heterodimerization system, with or without a separate nanoparticle component.

[0080] The polynucleotides and related vectors can be readily generated with standard molecular biology techniques or the protocols exemplified herein. Forexample, general protocols for cloning, transfecting, transient gene expression and obtaining stable transfected cell lines are described in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., (3rded., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou edition, 2003). Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1 : 17, 1991.

[0081] The selection of a particular vector depends upon the intended use of the fusion polypeptides. For example, the selected vector must be capable of driving expression of the fusion polypeptide in the desired cell type, whether that cell type be prokaryotic or eukaryotic. Many vectors contain sequences allowing both prokaryotic vector replication and eukaryotic expression of operably linked gene sequences. Vectors useful for the invention may be autonomously replicating, that is, the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome. Alternatively, the replication of the vector may be linked to the replication of the host's chromosomal DNA, for example, the vector may be integrated into the chromosome of the host cell as achieved by retroviral vectors and in stably transfected cell lines. Both viral-based and nonviral expression vectors can be used to produce the immunogens in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adenoassociated viruses, cytomegalovirus, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0082] Depending on the specific vector used for expressing the fusion polypeptide, various known cells or cell lines can be employed in the practice of the invention. The host cell can be any cell into which recombinant vectors carrying asequence encoding a described fusion polypeptide may be introduced and wherein the vectors are permitted to drive the expression of the fusion polypeptide is useful for the invention. The cells in which described polypeptides are expressed may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells. Cells expressing the fusion polypeptides of the invention may be primary cultured cells or may be an established cell line. Thus, in addition to the cell lines exemplified herein (e.g., HEK293 F cells), a number of other host cell lines capable well known in the art may also be used in the practice of the invention. These include, e.g., various Cos cell lines, HeLa cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B-cells and hybridomas.

[0083] The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N. Y., N.Y., 1987. The fusion polypeptide-expressing vectors may be introduced to the selected host cells by any of a number of suitable methods known to those skilled in the art. For the introduction of fusion polypeptide-encoding vectors to mammalian cells, the method used will depend upon the form of the vector. For plasmid vectors, DNA encoding the fusion polypeptide sequences may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. These methods are detailed, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINE™ (Life Technologies) or LipoTaxi™ (Stratagene) kits are available. Other companies offering reagents and methods for lipofection include Bio-Rad Laboratories, Clontech, Glen Research, Life Technologies, JBL Scientific, MBI Fermentas, PanVera, Promega, Quantum Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA.

[0084] For long-term, high-yield production of recombinant fusion polypeptides, stable expression may be used. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the fusion polypeptide- encoding sequences controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. The selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into their chromosomes. Commonly used selectable markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30: 147, 1984). Through appropriate selections, the transfected cells can contain integrated copies of the fusion polypeptide encoding sequence. In examples, the disclosure includes expressing the described fusion polypeptides within any suitable cells using any suitable expression system, allowing described sE1sE2 monomers or heterodimers to form, and separating the expressed sE1sE2 proteins which are optionally in nanoparticle form from the cells. The sE1sE2 proteins and nanoparticles displaying the same that are separated from the cells may be purified to any desired degree of purity. The separated and / or purified sE1sE2 proteins and nanoparticles may be used in any described vaccine formulation, which may further include any suitable adjuvant, including for example, an immunogenically effective amount of an adjuvant. In examples, an adjuvant increases the immunogenicity of a described vaccine formulation, relative to the same vaccine formulation but without the adjuvant.VI. Pharmaceutical compositions and therapeutic applications

[0085] The disclosure provides pharmaceutical or immunogenic compositions and related methods of using the engineered HCV sE1E2 polypeptides and nanoparticles displaying the polypeptides described herein for preventing and treating HCV infections. In some examples, an engineered HCV sE1E2 polypeptide after expression and protease digestion in a host cell, or a nanoparticle displaying the same, is included in a pharmaceutical composition. The pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation. Typically, the composition additionally includes one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antibiotics or antiviral drugs). Various pharmaceutically acceptable additives can also be used in the compositions.

[0086] Some of the pharmaceutical compositions of the invention are vaccines. For vaccine compositions, appropriate adjuvants can be additionally included. Examples of suitable adjuvants include, e.g., aluminum hydroxide, lecithin, Freund's adjuvant,MPL™ and IL-12. In some examples, the engineered HCV sE1E2 polypeptides disclosed herein can be formulated as a controlled-release or time-release formulation.This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel. The various pharmaceutical compositions can be prepared in accordance with standard procedures well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 19.sup.th Ed., Mack Publishing Company, Easton, Pa., 1995; Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978); U.S. Pat. Nos. 4,652,441 and 4,917,893; U.S. Pat. Nos. 4,677,191 and 4,728,721; and U.S. Pat. No. 4,675,189.

[0087] Therapeutic methods of the invention involve administering an engineered HCV sE1E2 polypeptide of the invention or a pharmaceutical composition containing the polypeptide to a subject having or at risk of developing HCV infection. In some examples, a pharmaceutical compositions of the invention is employed in therapeutic or prophylactic applications for treating HCV infection or eliciting an protective immune response against HCV in a subject. For example, the composition can be administered to a subject to induce an immune response to HCV, e.g., to induce production of broadly neutralizing antibodies to HCV. For subjects at risk of developing an HCV infection, a vaccine composition of the invention can be administered to provide prophylactic protection against viral infection. Depending on the specific subject and conditions, the pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intraarticular, intraperitoneal, or parenteral routes. In general, the pharmaceutical composition is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected disease or condition or one or more symptom(s) thereof. Symptoms of HCV exposure or infection include, e.g., inflammation of the liver, decreased appetite, fatigue, abdominal pain, jaundice, flu-like symptoms, itching, muscle pain, joint pain, intermittent low-grade fevers, sleep disturbances, nausea, dyspepsia, cognitive changes, depression headaches and mood changes.

[0088] Typically, the immunogenic composition of the disclosure is administered in an amount sufficient to induce an immune response against HCV. For therapeuticapplications, the compositions should contain a therapeutically effective amount of an engineered HCV sE1E2 polypeptide or nanoparticle vaccine composition described herein. For prophylactic applications, the compositions should contain a prophylactically effective amount of the HCV sE1E2 polypeptide or a nanoparticle displaying the polypeptide. The appropriate amount of the polypeptide immunogen or the nanoparticle composition can be determined based on the specific disease or condition to be treated or prevented, severity, age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the robustness of the subject's immune system). Determination of effective dosages is additionally guided with animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.

[0089] For prophylactic applications, the immunogenic composition is provided in advance of any symptom, for example in advance of infection. The prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection. Thus, in some examples, a subject to be treated is one who has, or is at risk for developing, an HCV infection, for example because of exposure or the possibility of exposure to HCV. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for HCV infection, symptoms associated with HCV infection, or both.

[0090] For therapeutic applications, the immunogenic composition is provided at or after the onset of a symptom of disease or infection, for example after development of a symptom of HCV infection, or after diagnosis of HCV infection. The immunogenic composition can thus be provided prior to the anticipated exposure to HCV virus so as to attenuate the anticipated severity, duration or extent of an infection and / or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection.

[0091] The pharmaceutical composition of the disclosure can be combined with other agents known in the art for treating or preventing HCV infections. These include, e.g., (1) Daclatasvir, (2) E1basvir and grazoprevir, (3) Glecaprevir and pibrentasvir, (4) Ledipasvir and sofosbuvir, (5) Ombitasvir, paritaprevir, and ritonavir, (6) Simeprevir (Olysio) and sofosbuvir (Sovaldi), (7) Sofosbuvir and velpatasvir (Epclusa), and (8) Sofosbuvir, velpatasvir, and voxilaprevir (Vosevi). See, e.g., Kish et al., P T. 42:316-329, 2017; and Dahiya et al., BCMJ 61 :72-77, 2019. Administration of the pharmaceutical compositions and the known anti-HCV agents can be either concurrently or sequentially.

[0092] Pharmaceutical compositions containing an engineered HCV sE1E2 polypeptide or nanoparticle vaccine of the invention can be provided as components of a kit. Optionally, such a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents. An optional instruction sheet can be additionally provided in the kits.EXAMPLES

[0093] The following examples are offered to illustrate, but not to limit the present invention.Example 1 Design of four HCV sE1E2 truncation variants based on the H77 strain and the LZ scaffold

[0094] This example is demonstrated using used HCV strain H77 (genotype la, GenBank: AF009606.1) as an example to evaluate representative sE1E2 construct designs. In an example, it is considered that a construct that is effective against H77 can be used against other HCV strains and genotypes. The sequences of the E1 ectodomain including extra residue 354 (sE1, 192-354; SEQ ID NO:4) and E2 ectodomain including extra residue 717 (sE2, 384-717; SEQ ID NO: 5) used in the constructs are shown below. They respectively contain an extra residue beyond the E1 and E2 ectodomain sequences. G354 in E1 is the first residue of the “GxxxG” motif, which is known to be involved in intramembrane protein-protein interactions. Single or double mutations at Gly354 and Gly358 can impair HCV infectivity. E717 in E2 marks the first residue of the transmembrane domain. Without structural information, four truncation variants, v0-v4 (v0: sE1-Y192-A349 / sE2-E384-I714; v1: sE1-Y192- M318 / sE2-E384-Y703; v2: sE1- Y192-S327 / sE2-E384-1709; v3: sE1-Y192-I344 / sE2- E384-W716), were made to examine the impact of the cutting point on sE1E2.LZ expression and antigenicity. LZ is the C-terminal region of a human heterodimeric transcription factor (c-FOS:c-JUN), which has been used by Guest et al. to stabilize sE1E2 (Guest et al., PNAS 2021, 118:e2015149118). Following a similar strategy, c-JUN (R285-Y324) (SEQ ID NO:20) and c-FOS (L161-Y200) (SEQ ID NO:21) were used to stabilize sE1E2-v0, v1, v2, and v3 with a “PGG” linker added between sE1 and c-JUN and between sE2 and c-FOS, an RRRRRR (SEQ ID NO:6) furin cleavage motif added between sE1-PGG-c-JUN and sE2-PGG-c-FOS, and a His6-tag added to the C terminus, as exemplified for sE1E2-v0 below.

[0095] >H77-sE1 (H77, residues 192-354; SEQ ID NO:4)

[0096] >H77-sE2 (H77, residues 384-717; SEQ ID NO: 5)

[0097] >Modified C-JUN motif (LZ, PDB ID: 1FOS; H324Y; derived from Guest et al . , PNAS 2021, 118: e2015149118) (SEQ ID NO : 20)RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY

[0098] >Modified C-FOS motif (LZ, PDB ID: 1FOS; H200Y; derived from Guest et al., PNAS 2021, 118:e2015149118) (SEQ ID NO:21)LTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAY

[0099] >sE1E2-v0 and LZ fusion (SEQ ID NO: 19)N-terminal leader sequence -

[0100] These four sE1E2-VX.LZ constructs were transiently expressed in 250ml HEK293 F cells and purified using a Nickel column followed by size-exclusion chromatography (SEC) on a Superdex 200 Increase 100 / 300 GL column (Fig. 1). Purification by the Nickel column resulted in a low yield for all four constructs, as shown by the SEC profiles, with an aggregate peak at 8.5-9ml and a barely recognizable peak at 10.5-11ml, potentially corresponding to the “dimer of dimer” (DD) fraction (Fig. 1, A). SEC fractions were further analyzed by Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. Consistent with the SEC profiles, SDS-PAGE showed two high bands at 150 and 250kD that may correspond to E1E2 or E2 multimers, whereas SEC fractions 19 and 20 appeared to contain E1E2 dimers and E2 monomers. Enzyme-linked immunosorbent assay (ELISA) was performed to characterize the binding of pooled SEC fractions 19 and 20 to AR3C, which targets the E2 NF, and AR4A, which recognizes the “native-like” E1E2 interface (Fig. 1, B). While sE1E2-v0, v2, and V3.LZ-His6constructs showed AR3C binding signals, none of the four sE1E2 constructs could be recognized by the E1E2 quaternary epitope-specific bNAb, AR4A, thus highlighting the challenges facing rational design of sE1E2 antigens.Example 2 Structure-based rational design of sE1E2 scaffolds with superior protein yield and homogeneity

[0101] Two reported cryo-EM models of E1E2 provide structural information for rational design of next-generation sE1E2 antigens. Although the two E1E2 constructs were from different genotype-1 isolates and in different forms - one in the membranebound form (PDB ID: 7T6X; de la Pena et al., Science 2022, 378:263-269) and the other in the soluble form (PDB ID: 8FSJ; Metcalf et al., Nat Commun 2023, 14:3980), they shared highly similar structures, with an overall Cα-RMSD of 1.34 A and individual Cα-RMSDs of 1.01 Å and 0.91 Å for E1 and E2 subunits, respectively (Fig. 2, A). In the cryo-EM analysis of the sE1E2.SZ construct (PDB ID: 8FSJ), structures cannot be built for sE1 beyond residue H312, for sE2 beyond residue Y701, and for the SZ scaffold (Fig. 2, A). sE1-H312 and sE2-Y701 appeared to sandwich the E2 C- terminal loop (688-694) from both sides, providing improved anchoring sites for linking to a dimeric scaffold (Fig. 2, A). Based on this structural observation, we analyzed whether cutting E1 C-terminus beyond H312 and E2 C-terminus beyondY701 (termed “Cut 1”, Fig. 2, B) and fusing a “tight” heterodimeric scaffold to sE1- H312 and sE2-Y701 would create a highly stable sE1E2 dimer with a native-like E1E2 interface. In both cryo-EM models, a long E1 loop (between T256 and S294 in 7T6X and between T255 and P295 in 8FSJ) cannot be resolved structurally. This unstructured E1 loop contains 2 cystines and 17 hydrophobic residues, which may form random disulfide bonds and hydrophobic contacts with other E1 or E2 to cause misfolding, aggregation, and aberrant E1E2 forms. Based on this observation, we analyzed whether replacing a 30 aa-stretch of this E1 loop (aa 264-293) with a Glycine (termed “Cut 2”, Fig. 2, B) would improve sE1E2 folding, homogeneity, stability, and antigenicity. To summarize, in this example, two sE1E2 truncation variants were produced for further evaluation, with the sequences shown below for H77 and in Fig. 2, B.

[0102] Two heterodimeric coiled coils, LZ and SZ, have been used as scaffolds to stabilize sE1E2 (Guest et al., PNAS 2021, 118:e2015149118; Metcalf et al., Nat. Comm. 2023, 14:3980). LZ and SZ are long coiled coils with 40 and 49-52 residues, respectively. Such long coiled coils may form non-specific interactions and aggregation in solution. We analyzed whether short heterodimeric coiled coils with improved specificity may provide more suitable scaffolds for sE1E2 stabilization. A protein database (PDB) was analyzed and identified two short heterodimeric coiled coils, GCP and IAAL. GCP is a designed heterodimeric leucine zipper (Marti et al., Biochemistry, 2004, 43: 12436-12447), comprising an acidic A chain (32-aa) and a basic B chain (32- aa). IAAL is a de novo heterodimeric coiled coil (Lindhout et al., Biopolymers 2004, 75: 367-375) also comprising an acidic A chain (21-aa) and a basic B chain (21-aa). In examples, the unstructured N-terminus is removed so that these scaffolds can exert a tight structural control when they are fused to sE1E2. In previous work, the SpyTag / SpyCatcher system was used to display the SARS-CoV-2 receptor-binding domain (RBD) on protein nanoparticles (He et al., Sci Adv 2021, 7(12):eabfl591). The SpyTag / SpyCatcher system utilizes an isopeptide bond within a bacterial adhesin to covalently link two proteins (Zakeri et al., PNAS 2012, 109 (12): E690-7). This disclosure includes improving the SpyTag002 / SpyCatcher002 system for use as a covalently linked heterodimeric scaffold to stabilize the sE1E2 conformation. We analyzed whether removing an unstructured, 21-aa N-terminus of SpyCatcher002 (see the sequence below) and by adding a “GSGS” linker to the N-terminus of SpyTag002 and an “AS” linker to the N-terminus of SpyCatcher002dN, would achieve a precisestructural control of the fused sE1 and sE2 subunits. To summarize, this example provides five heterodimeric scaffolds (LZ, SZ, GCP, IAAL, andSpyTag002 / SpyCatcher002DN or SPYdN) for sE1E2 stabilization (Fig. 2, C, top), with the sequences and scaffold-specific design schemes shown below.

[0103] >H77-sE1 (Cut 1 : removed MPER; unstructured loop underlined) (SEQ IDNO:28):

[0104] >H77-sE1 “Cut 1+2” (removed MPER, replaced unstructured E1 loop with a “G”) (SEQ ID NO:9):

[0105] >H77-sE2 “Cut 1” and “Cut 1+2” (removed MPER) (SEQ ID NO: 10)

[0106] >C-JUN (LZ, PDB ID: 1FOS; H324Y; used in Guest et al., PNAS 2021, 118:e2015149118) (SEQ ID NO:20)RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY

[0107] >C-FOS (LZ, PDB ID: 1FOS; H200Y; used in Guest et al., PNAS 2021, 118:e2015149118) (SEQ ID N0:21)LTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAY

[0108] >SYNZIP2 (SZ, PDB ID: 3HE5; used in Metcalf et al., Nat Commun. 2023, 14:3980) (SEQ ID NO:22)RNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVA

[0109] >SYNZIP1 (SZ, PDB ID: 3HE5; used in Metcalf et al., Nat. Commun. 2023, 14:3980) (SEQ ID NO:23)LVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIE

[0110] >GCP-A E9 (GCP, PDB ID: 1U2U) (SEQ ID NO:24):EVAQLEKEVAQLEAENYQLEQEVAQLEHEG

[0111] >GCP-B K9 (GCP, PDB ID: 1U2U) (SEQ ID NO:25):EVQALKKRVQALKARNYALKQKVQALRHKG

[0112] >IAAL-A E3 (IAAL, PDB ID: 1U0I) (SEQ ID NO:26):EIAALEKEIAALEKEIAALEK

[0113] >IAAL-B K3 (IAAL, PDB ID: 1U0I) (SEQ ID NO:27):KIAALKEKIAALKEKIAALKE

[0114] >SpyTag002 (SEQ ID NO:7):VPTIVMVDAYKRYK

[0115] >SpyCatcher002dN (SEQ ID NO:8):DSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT

[0116] In the above SpyCatcher002dN sequence, C-terminal unstructured residues are underlined. N-terminal unstructured residues of the original SpyCatcher sequence, VTTLSGLSGEQGPSGDMTTEE (SEQ ID NO: 11), are deleted.

[0117] Further modifications to SpyCatcher sequence can be made to generate variant SpyCatcher motifs that are suitable for the invention. One example of the SpyCatcher variants is SpyCatcher002dN v2 (SEQ ID NO:43). Compared to SpyCatcher002dN, 3 additional N-terminal unstructured residues, DSA, are deleted in SpyCatcher002dN v2.

[0118] SpyCatcher002dN v2 (SEQ ID NO:43): C-terminal unstructured residues are underlined.THIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT

[0119] The structural scheme for the engineered sE1E2 polypeptides constructed via the different heterodimeric coiled coils are shown in Fig. 2, C (bottom, left 4): [sE1-Cut1 / 1+2]-PGG-[coiled coil 1]-RRRRRR (SEQ ID NO:6)-[sE2-Cut1]-PGG-[coiled coil 2]-GSHHHHHH (SEQ ID NO: 14). The structural scheme for the engineered sE1E2 polypeptides constructed via SpyTag002 / SpyCatcher002dN motifs are shown in Fig. 2, C (bottom, rightmost): [sE1-Cut1 / 1+2]-GSGS (SEQ ID NO:34)-[SpyTag002]-RRRRRR (SEQ ID NO:6)-[sE2-Cut1]-AS-[SpyCatcher002dN]-GSHHHHHH (SEQ ID NO: 14). All ten sE1E2-Cut1 / 1+2.scaffold-His6constructs were transiently expressed in 500 ml HEK293 F cells and purified using a Nickel column followed by SEC on a Superdex200 Increase 100 / 300 GL column (Fig. 2, D). Compared with sE1E2-v0.LZ, the newly designed sE1E2-Cut.scaffold antigens showed small to significant improvement in yield with clearly visible dimer of dimer (DD) and dimer (D) peaks. All sE1E2-Cut1+2constructs showed lower dimer-of-dimer (DD) but higher dimer (D) peaks than their sE1E2-Cut1counterparts in SEC profiles, suggesting that that the long, unstructured E1 loop (residues 264-293) may be the major cause of dimer-of-dimer (DD) formation. GCP and IAAL produced higher dimer yield than LZ and SZ, supporting our hypothesis that short heterodimeric coiled coils with high specificity may provide more suitable scaffolds for sE1E2. GCP appeared to be the best performer among the four heterodimeric coiled coils, whereas SPYdN showed the highest dimer yield among all five scaffolds tested. Overall, sE1E2-Cut1+2.SPYDN-His6appeared to be the best construct design in terms of dimer yield and homogeneity.

[0120] SEC fractions were then analyzed by SDS-PAGE under non-reducing conditions (Fig. 2, E). Compared with sE1E2-v0.LZ, which displayed abnormal bands on the gel, the newly designed sE1E2-Cut1 / 1+2. scaffold antigens showed clear bands corresponding to dimer of dimer (DD, ~150kD) and dimer (D, 75-100kD). All sE1E2- Cut1+2antigens using the coiled coils as scaffolds displayed low-molecular-weight (MW) bands corresponding to dissociated sE1 and sE2 subunits, whereas the sE1E2- Cut1+2. SPYdN antigen showed a clean gel with mainly dimer bands. The appearance of dissociated sE1 and sE2 bands on the SDS-PAGE gels was accompanied with higher right shoulder / slope in the SEC profiles for the coiled coil-containing sE1E2-Cut1+2antigens, further confirming that the long, unstructured E1 loop (residues 264-293) is the cause of dimer-of-dimer (DD) formation. Lastly, the sE1E2-Cut1+2.SPYDN-His6construct design was extended to other HCV strains, including HCV1 (genotype 1), 312 (genotype 3), and 521 (genotype 5). Following transient expression in 500ml HEK293 F cells and purification on a Nickel column, the three constructs were analyzed by SEC (Fig. 2, F). All three constructs produced similar SEC profiles with high yield, confirming that “sE1E2-Cut1+2. SPYDN” is a general construct design applicable to diverse HCV strains and genotypes.Example 3 Antibody binding and structure of rationally designed sE1E2 scaffold antigens

[0121] The dimer-of-dimer and dimer fractions obtained from the SEC analysis of ten sE1E2-Cut1 / 1+2.scaffold-His6antigens were assessed by ELISA for their binding to AR3C and AR4A, which target the E2 neutralizing face (NF) and a quaternary epitope only present on the functional E1E2 heterodimer, respectively (Fig. 3, A). In this ELISA analysis, four antibody dilutions were tested without duplicate. The half maximal effective concentration (EC50) values were calculated to estimate the apparent binding affinity (Fig. 3A). Two sE1E2 antigens were used as controls (Fig. 3, A, leftmost), sE1E2-v0.LZ-His6that was expressed in HEK293 F and purified by a Nickel column (in-house production) and sE1E2.SZ-Avi that was co-expressed with AR4A in HEK293 F and purified using the Avi-tag. Surprisingly, although sE1E2-v0.LZ-His6is nearly identical to the recently reported sE1E2.LZ antigen (Guest et al., PNAS 2021, 118:e2015149118), it showed no binding to AR4A. In contrast, sE1E2.SZ-Avi bound both ARC3 and AR4A, with a 12-fold higher affinity observed for AR3C. This result highlights a problem with previously available sE1E2 designs: they must be coexpressed with AR4A to facilitate correct E1E2 folding and ensure a native-like E1E2 interface, suggesting that their construct design is suboptimal and intrinsically unstable. Notably, all sE1E2-Cut1. scaffold antigens showed similar or stronger AR4A binding compared to sE1E2.SZ-Avi (Guest et al., PNAS 2021, 118:e2015149118) in ELISA (Fig. 3, A, right, top 5).

[0122] For all sE1E2-Cut1. scaffold antigens, the DD fraction showed higher AR4A binding affinities than the D fraction, although DD and D fractions appeared to bind AR3C with similar EC50values. Deletion of the long E1 loop (residues 264-293) appeared to have a marked impact on the sE1E2-antibody binding profiles (Fig. 3, A, right, bottom 5). For all sE1E2-Cut1+2. scaffold antigens, the D fraction bound to both bNAbs with significantly enhanced affinities, with ~5-fold and ~100-fold lower EC50values for AR3C and AR4A, respectively. Surprisingly, for all sE1E2-Cut1+2. scaffold antigens, the DD fraction showed lower AR3C and AR4A binding affinities than the D fraction - an opposite pattern compared to their sE1E2-Cut1counterparts. In summary, the described ELISA data demonstrated importance of this long, unstructured E1 loop to the overall antigenicity of sE1E2 antigens and suggests that this E1 loop should be removed to achieve a stable, native-like E1E2 interface. The D fraction of HCV1, 312, and 521 sE1E2-Cut1+2.SPYdN-His6antigens were also analyzed by negative-stain EM (Fig. 3, B-D). CryoSPARC was used to process images to cluster molecules into 2Dclasses (Figs. 3, B-D, left, bottom), in some of which the globular SPYdN domain was visible. The 3D volumes were constructed from 2D classes and used to fit the sE1E2 (PDB ID: 8FSJ) and SpyTag / SpyCatcher (PDB ID: 4MLI) structures. For HCV1 sE1E2-Cut1+2.SPYdN-His6, which had the highest image quality, the fitted model rendered a perfect match to the design, with SPYdN holding sE1-H312 and sE2-Y701 from underneath (Fig. 3, B).Example 4 Display of rationally designed s E1E2 scaffold antigens on protein nanoparticles

[0123] The previous difficulties in obtaining properly folded sE1E2 with a nativelike E1E2 interface have hindered the development of protein nanoparticle or VLP- based HCV vaccines. In a recent study, Sliepen et al. reported HCV E1E2 nanoparticle vaccine designs based on the two-component nanoparticle (2c-NP) platform (Sliepen et al., 2022 Nat. Commun., 13(1):7271). However, this permuted HCV sE1E2 antigen does not represent the native conformation of viral E1E2, because it does not bind E1E2 interface-directed bNAbs such as AR4A (Sliepen et al., 2022 Nat. Commun., 13(1) :7271 ). Given the markedly increased AR4A binding observed for the described sE1E2-Cut1+2. SPYdN antigens, we analyzed whether multivalent display of this sE1E2 antigen on 1c-SApNP platforms would generate effective VLP-type HCV vaccines (Fig. 4, A, left). In this example, we displayed HCV1 sE1E2-Cut1+2. SPYdN on ferritin (FR) 24-mer with a 10GS linker and on a multilayered I3-01v9a 60-mer with a 5GS linker, resulting in particles of 36.9 nm and 47.0 nm, respectively (Fig. 4, A, right). Following a previous protocol, both sE1E2 1c-SApNP constructs were expressed in 25ml ExpiCHO cells and purified using an AR3C antibody column or an AR4A antibody column. AR3C purification of the ExpiCHO-expressed HCV1 sE1E2- Cut1+2. SPYdN- 10GS-FR generated three well-separated peaks corresponding to aggregates, individual particles, and unassembled sE1E2 subunits in the SEC profile (Fig. 4, B, left). Consistently, negative-stain EM analysis revealed that fraction 4 (F4) contained large aggregates whereas fraction 11 (F11) contained well-formed particles with recognizable sE1E2 antigens on the surface (Fig. 4, B, right).

[0124] AR4A purification of the ExpiCHO-expressed HC VI sE1E2- Cut1+2. SPYdN- 10GS-FR resulted in a distinct SEC profile with higher particle yield (by two-fold) and lower amount of unassembled sE1E2 subunits (Fig. 4, C, left). Negative-stain EM analysis revealed that fraction 4 (F4) contained both aggregates and particle clusters, while fraction 12 (F12) contained high-quality sE1E21c-SApNPs with homogeneous surface decorations (Fig. 4, C, right). For HCVl sE1E2-Cut1+2.SPYdN- 5GS-I3-01v9a-LD7-PADRE, due to the relatively low yield, the AR3C and AR4A- purified materials were directly visualized by negative-stain EM without SEC (Fig. 4, D). Consistent with pattern observed for the FR samples, AR4A purification resulted in greater particle purity. Thus, this example demonstrates that the described sE1E2- Cut1+2.SPYdN antigen can be displayed on two nanoparticle platforms, which can be purified by interface-specific AR4A column for laboratory-scale production, highlighting the presence of “native-like” E1E2 interface on these nanoparticles.Example 5 Sequence analysis of E1 unstructured loops for diverse HCV genotypes

[0125] Given the significance of the Cut2 design to AR4A binding, we sought to determine the sequence conservation around this region. We analyzed diverse HCV E1E2 sequences within the ViPR database (bv-brc.org) downloaded on April 21, 2020, including Genotype 1 (1488), Genotype 2 (636), Genotype 3 (596), Genotype 4 (77), Genotype 5 (19), and Genotype 6 (251). After removing incomplete E1E2 sequences or those with undetermined amino acids, the remaining data sets were subjected to multiple sequence alignment (MSA) using CLUSTALW2. The sequence logo plot is shown for residues 252-298 of E1 within each genotype to measure sequence conservation (Fig. 5). Of note, the structure of residues 257-293 is invisible in two recently reported cryo-EM models of E1E2 (PDB ID: 7T6X and PDB ID: 8FSJ), suggesting that this region is highly flexible (Fig. 5, outer black line box). In the described “Cut2” design, residues 257-263 were kept whereas residues 264-293 (Fig. 5, inner black line box) were replaced with a single glycine “G”. Two conserved patterns are observed in the sequence logo analysis. First, residues 257-263, which were kept intact in the Cut2 design, contain 3 to 5 charged or hydrophilic residues, thus making it suitable for being used as a “bridging” surface loop after the truncation of residues 264- 293. In examples, other charged or hydrophilic loops of 7 residues or longer can be used to replace residues 257-293 and likely render similar results. Second, residues 264-293 contain two conserved cysteines, C272 and C281, along with several hydrophobic stretches, supporting the notion that deletion of this region will reduce the formation of random disulfide bonds and aggregation.***

[0126] The disclosure has been disclosed broadly and illustrated in reference to representative examples described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof.

[0127] It is further noted that all publications, sequence accession numbers, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety and for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. An HCV soluble E1E2 (sE1E2) immunogen polypeptide, comprising (1) an engineered E1 ectodomain with a deletion of the C-terminal membrane proximal external region (MPER), (2) a cleavage site for a protease, and (3) an engineered E2 ectodomain with a deletion of the C-terminal membrane proximal external region (MPER); wherein the engineered E1 ectodomain and the engineered E1 ectodomain are respectively fused to two interacting motifs of a protein heterodimerization system.

2. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the engineered E1 ectodomain further comprises (1) a deletion of the unstructured hydrophobic loop motif or (2) a replacement of said loop motif with a spacer consisting of from one to about three non-polar, hydrophilic, or charged amino acid residues, and wherein the loop motif corresponds to residues 264-293 (SEQ ID NO:32) of HCV H77 isolate polyprotein sequence shown in SEQ ID NO:1.

3. The HCV sE1E2 immunogen polypeptide of claim 2, further comprising a substitution of a sequence motif corresponding to residues 257-263 of HCV H77 isolate polyprotein with a peptide sequence containing 7 or more residues that do not contain cystines or any stretch of 2 or more hydrophobic residues.

4. The HCV sE1E2 immunogen polypeptide of claim 2, wherein the spacer is present and comprises or consists of a Glycine residue.

5. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the cleave site for a protease is a furin cleavage site.

6. The HCV sE1E2 immunogen polypeptide of claim 5, wherein the furin cleavage site comprises RRRRRR (SEQ ID NO:6).

7. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the engineered E1 ectodomain and its fused interacting motif are located N-terminal to the engineered E2 ectodomain and its fused interacting motif.

8. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the protein heterodimerization system is a coiled coil system.

9. The HCV sE1E2 immunogen polypeptide of claim 8, wherein each engineered ectodomain is fused at its C-terminus to a coiled coil via a PGG tripeptide linker.

10. The HCV sE1E2 immunogen polypeptide of claim 8, wherein the two interacting motifs of the coiled coil system respectively comprises SEQ ID NOs:20 and 21, SEQ ID NOs:22 and 23, SEQ ID NOs:24 and 25, or SEQ ID NOs:26 and 27.

11. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the protein heterodimerization system is a SpyTag / SpyCatcher system.

12. The HCV sE1E2 immunogen polypeptide of claim 11, wherein the SpyCatcher comprises a truncation of the unstructured N-terminal sequence.

13. The HCV sE1E2 immunogen polypeptide of claim 11, wherein the SpyTag motif and the SpyCatcher motif respectively comprise (1) SEQ ID NO:7 and SEQ ID NO:8, or (2) SEQ ID NO:7 and SEQ ID NO:43.

14. The HCV sE1E2 immunogen polypeptide of claim 13, wherein the engineered E1 ectodomain is fused at its C-terminus to the SpyTag via a GSGS (SEQ ID NO:34) linker.

15. The HCV sE1E2 immunogen polypeptide of claim 13, wherein the engineered E2 ectodomain is fused at its C-terminus to the SpyCatcher via an AS dipeptide linker.

16. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the deletion of the MPER in E1 comprises a truncation of E1 C-terminal sequence after residue H312, and the deletion of the MPER in E2 comprises a truncation of E2 C- terminal sequence after residue Y701; and wherein the amino acid numbering is based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO:1).

17. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the deletion of the MPER in E1 comprises a truncation of E1 C-terminal sequence after residue P310, G311, I313, or T314, and the deletion of the MPER in E2 comprises atruncation of E2 C-terminal sequence after residue L702, Y703, G704, or V705; and wherein the amino acid numbering is based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO:1).

18. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the deletion of the MPER in E1 comprises a truncation of E1 C-terminal sequence after residue P310, G311, I313, or T314, and the deletion of the MPER in E2 comprises a truncation of E2 C-terminal sequence after residue Y701; and wherein the amino acid numbering is based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1).

19. The HCV sE1E2 immunogen polypeptide of claim 1, wherein the deletion of the MPER in E1 comprises a truncation of E1 C-terminal sequence after residue H312, and the deletion of the MPER in E2 comprises a truncation of E2 C- terminal sequence after residue L702, Y703, G704, or V705; and wherein the amino acid numbering is based on the sequence of HCV H77 isolate polyprotein (SEQ ID NO: 1).

20. The HCV sE1E2 immunogen polypeptide of claim 1, comprising from the N-terminus to the C-terminus an engineered E1 ectodomain shown in SEQ ID NO: 9, a first PGG tripeptide spacer, a first coiled coil of a coiled coil dimerization system, a furin clearage site, an engineered E2 ectodomain shown in SEQ ID NO: 10, a second PGG tripeptide spacer, and a second coiled coil of the coiled coil dimerization system.

21. The HCV sE1E2 immunogen polypeptide of claim 1, comprising from the N-terminus to the C-terminus an engineered E1 ectodomain shown in SEQ ID NO: 9, a GSGS (SEQ ID NO:34) linker, a SpyTag motif comprising SEQ ID NO:7, a furin clearage site, an engineered E2 ectodomain shown in SEQ ID NO: 10, an AS dipeptide linker, and a SpyCatcher motif comprising SEQ ID NO:8 or SEQ ID NO:43.

22. The HCV sE1E2 immunogen polypeptide of any one of claims 17-19, comprising from the N-terminus to the C-terminus the engineered E1 ectodomain, a linker comprising 4-12 glycine and / or serine residues, a SpyTag motif comprising SEQ ID NO:7, a furin clearage site, the engineered E2 ectodomain, a spacer comprising AS dipeptide, and a SpyCatcher motif comprising SEQ ID NO:8 or SEQ ID NO:43.

23. The HCV sE1E2 immunogen polypeptide of claim 22, wherein the linker comprises or consists of a sequence set forth in any one of SEQ ID NOs:34-40.

24. The HCV sE1E2 immunogen polypeptide of any one of claims 1-21, further comprising at the C-terminus a GS dipeptide linker and a His tag HHHHHH (SEQ ID NO:29).

25. A polynucleotide encoding the HCV sE1E2 immunogen polypeptide of claim 1.

26. A pharmaceutical composition, comprising (1) the HCV sE1E2 immunogen polypeptide of claim 1 that has been cleaved by the protease or the polynucleotide of claim 25, and (2) a pharmaceutically acceptable carrier.

27. A vaccine composition, comprising the sE1E2 immunogen polypeptide of claim 1 that has been cleaved at the protease cleavage site and is displayed on the surface of a self-assembling nanoparticle.

28. The vaccine composition of claim 27, wherein C-terminus of the HCV sE1E2 immunogen polypeptide is fused to the N-terminus of subunit of the selfassembling nanoparticle via a linker sequence.

29. The vaccine composition of claim 28, wherein subunit of the selfassembling nanoparticle comprises the polypeptide shown in SEQ ID NO: 16 (I3-01v9a), SEQ ID NO: 15 (ferritin) or SEQ ID NO:31 (E2p), a conservatively modified variant or a substantially identical sequence thereof.

30. A polynucleotide, encoding a fusion protein comprising (1) a HCV sE1E2 immunogen polypeptide of claim 1 and (2) a self-assembling nanoparticle subunit, wherein the HCV sE1E2 immunogen polypeptide is fused at its C-terminus to the N- terminus of the self-assembling nanoparticle subunit.

31. A pharmaceutical composition, comprising the vaccine composition of claim 27 or the polynucleotide of claim 30, and a pharmaceutically acceptable carrier.

32. A method of treating or preventing HCV infection in a subject, comprising administering to the subject a pharmaceutical composition of claim 26 or claim 31, thereby treating or preventing HCV infection in the subject.

33. A method of producing an HCV sE1-sE2 protein with native-like E1E2 interface, comprising (1) expressing the HCV soluble E1E2 immunogen polypeptide ofclaim 1 in eukaryotic cells, and (2) separating from the eukaryotic cells the sE1-sE2 protein that is linked or associated by the two interacting motifs of the protein heterodimerization system after cleavage at the protease cleavage site.

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