HCV genotype3 e2 envelope protein for neutralizing infectivity of HCV strains

Abstract

The present disclosure provides compositions comprising E2 envelope glycoprotein from HCV Gt3 strain S52. Surprisingly, this composition provides an immune response effective against heterologous HCV strains, e.g., Gt1 strain H77 and Gt2 strain J6. These compositions may be used for inducing an immune response to HCV Gt1-Gt3, e.g., Gt1 strain H77, Gt2 strain J6, and Gt3 strain S52 in an individual.

Description

HCV GENOTYPES E2 ENVELOPE PROTEIN FOR NEUTRALIZING INFECTIVITY OF HCV STRAINSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 639,381, filed April 26, 2024, which application is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “UALB- 061WO SEQ LIST” created on April 23, 2025, and having a size of 87,955 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.INTRODUCTION

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

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

[0005] There is a need in the art for compositions and methods for inducing immune responses to different genotypes of HCV, e.g., HCV genotypes 1-genotypes 3 (Gtl-Gt3) which are common HCV genotypes around the world.SUMMARY

[0006] The present disclosure provides compositions comprising E2 envelope glycoprotein from HCV Gt3 strain S52. This composition provides an immune response effective at neutralizing the infectivity of multiple HCV genotypes, including Gtl, Gt2 and Gt3. This composition can neutralize infectively of HCV strains that have been difficult to neutralize, e.g., Gt2 strain J6 and Gt3 strain S52. Furthermore, this composition provides an effective immune response against HCV Gtl strain H77. These compositions may be used for inducing a protective immune response to HCV Gtl-Gt3, e.g., Gtlstrain H77, Gt2 strain J6, and Gt3 strain S52 in an individual. These compositions may be used for inducing a protective immune response to additional HCV genotypes, e.g., HCV Gtl-Gt7.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1. Antisera from animals immunized with H77-E2, J6-E2, S52-E2, or a mixture of H77-E2, J6-E2, and S52-E2 were tested for neutralization activity against H77-HCVpp, J6-HCVpp, and S52-HCVpp. Antisera from animals immunized with SLA-SE adjuvant served as control. Antisera from animals immunized with S52-E2 showed broad-neutralization against all three HCVpp. H77-E2 induced neutralizing antibodies against homologous H77-HCVpp. H77-E2 induced antibodies showed reduced neutralization against J6- and S52- HCVpp as compared to neutralization against H77-HCVpp. J6-E2 induced homologous neutralization but showed reduced neutralization against H77-and S52-HCVpp.

[0008] FIGS. 2A-2E provide an amino acid sequence alignment of the core-El-E2 coding region for representative HCV 3A, 3B and 3K genotypes. Genbank database sequences for the coding region core-El-E2 were aligned using Geneious software v5.6.4. Consensus: SEQ ID NO:40; AVI3al77: SEQ ID NO:41; ADF97232(S52): SEQ ID NO:42; YP 0014696: SEQ ID NO:43; CAA54244: SEQ ID NO:44; AAC03058: SEQ ID NO:45; AAY29642: SEQ ID NO:46; ABD85062: SEQ ID NO:47;ABD85063: SEQ ID NO:47; ABD97104: SEQ ID NO:48; BAA06044: SEQ ID NO:49; BAA08372: SEQ ID NO:50; and BAA09890: SEQ ID NO:51.

[0009] FIGS. 3A-3C provide an amino acid sequence alignment of examples of the core-El-E2 coding regions of an HCV genotype 1 virus, specifically representative HCV 1A, IB and 1C genotypes. Genbank database sequences for the coding region core-El-E2 were aligned using Geneious software v5.6.4. Numbering of amino acids is according to strain NP_671941 (H77). Consensus: SEQ ID NO: 1; AVIlal29: SEQ ID NO:2; NP_671491 (H77): SEQ ID NO:3; EU155269: SEQ ID NO:4; EU781810: SEQ ID NO:5; EU781771: SEQ ID NO:6; AB250610: SEQ ID NO:7; EU781752: SEQ ID NO:8;EU781759: SEQ ID NO:9; EF407439: SEQ ID NO: 10; EF407427: SEQ ID NO: 11; EU362905: SEQ ID NO: 11; EF407413: SEQ ID NO: 11; EU781808: SEQ ID NO: 12; EU78170: SEQ ID NO: 13; AJ238799 (Coni): SEQ ID NO: 14; AAK97744: SEQ ID NO: 15; AF139594: SEQ ID NO: 16; AF176573: SEQ ID NO: 17; BAA19625: SEQ ID NO: 18; BAA25076: SEQ ID NO: 19; BAC54896: SEQ ID NO:20;BAD91386: SEQ ID NO:21; BAF46764: SEQ ID NO:22; BAG30950: SEQ ID NO:22; CAB41951: SEQ ID NO:23; AAK95832: SEQ ID NO:24; AAT69968: SEQ ID NO:25; and BAA03581: SEQ ID NO:26.

[0010] FIGS. 4A-4E provide an alignment of amino acid sequences of the core-El-E2 coding region of representative HCV 2A and HCV2B subtypes. Genbank database sequences for the coding region core-El-E2 were aligned using Geneious software v5.6.4. The amino acid numbering depicted isin accordance with the common HCV strains: D00994 (HC-J6) for HCV 2A; ABO47639 (JFH1) and HPCJ8G-J8 (J8) for HCV2A and HCV2B, respectively. Consensus: SEQ ID NO: 27; AB047639 (JFH1): SEQ ID NO:28; AB047645: SEQ ID NO:29; AF238482: SEQ ID NO:30; AF169005: SEQ ID NO:31; AY746460: SEQ ID NO:32; HPCPOLP: SEQ ID NO:33; NC 009823: SEQ ID NO:34; HPCJ8G HC-J8: SEQ ID NO:35; AY232730: SEQ ID NO:36; AY232747: SEQ ID NO:37; AB030907: SEQ ID NO38; and DQ430817: SEQ ID NO:39.DEFINITIONS

[0011] The term “hepatitis C virus” (“HCV”), as used herein, refers to any one of a number of different genotypes and isolates of hepatitis C virus. Thus, “HCV” encompasses any of a number of genotypes, subtypes, or quasispecies, of HCV, including, e.g., Gtl, 2, 3, 4, 5, 6, 7, etc. and subtypes (e.g., Gtla, lb, 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies. Representative HCV genotypes and isolates include: HCV-1, H77, J6, Coni, isolate 1, BK, ECI, EC 10, HC-J2, HC-J5; HC-J6, HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, “Hunan”, “Japanese”, “Taiwan”, TH, type 1, type la, H77 type lb, type 1c, type Id, type le, type If, type 10, type 2, type 2a, type 2b, type 2c, type 2d, type 2f, type 3, type 3a, type 3b, type 3g, type 4, type 4a, type 4c, type 4d, type 4f, type 4h, type 4k, type 5, type 5a, type 6, type 6a, and type 7a.

[0012] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, non-human primates (e.g., simians), equines (e.g., horses), rodents (e.g., rats; mice), and humans.

[0013] As used herein, the term “isolated,” in reference to a polypeptide, refers to a polypeptide that is in an environment different from that in which the polypeptide naturally occurs. An isolated polypeptide can be purified. By “purified” is meant a compound of interest (e.g., a polypeptide) has been separated from components that accompany it in nature. “Purified” can also be used to refer to a polypeptide separated from components that can accompany it during production of the polypeptide (e.g., during synthesis in vitro, etc.). In some embodiments, a polypeptide (or a mixture of polypeptides) is substantially pure when the polypeptide (or mixture of polypeptides) is at least 60% or at least 75% by weight free from organic molecules with which it is naturally associated or with which it is associated during production. In some cases, the polypeptide is from 30% to 60% pure. In some cases, the polypeptide (or mixture of polypeptides) is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. For example, in some cases, an El or an E2 polypeptide (or a mixture of El and E2 polypeptides, e.g., an E1 / E2 heterodimer) is substantially pure when the El or E2 polypeptide (or mixture of El and E2 polypeptides) is at least 60% or at least 75% by weight free from organic molecules with which the polypeptide (s) is naturally associated or with which it is associated during production. In some cases, the El or E2 polypeptide (or mixture of El and E2polypeptides) is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. In some cases, where a composition comprises an E2 polypeptide, the E2 polypeptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. In some cases, where a composition comprises an E1 / E2 heterodimeric complex polypeptide, the E1 / E2 heterodimeric complex polypeptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. In some cases, where a composition comprises a T-cell epitope polypeptide, the T-cell epitope polypeptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure.

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

[0015] The term “heterologous” refers to two components that are defined by structures derived from different sources. For example, where “heterologous” is used in the context of a polypeptide, the polypeptide includes operably linked amino acid sequences that can be derived from one or more different polypeptides, e.g., amino acid sequences that are not operably linked to the polypeptide in nature. As another example, where a composition comprises an HCV E1 / E2 heterodimer and a “heterologous” polypeptide, the “heterologous polypeptide is a polypeptide other than HCV El or HCV E2. As another example, where a fusion polypeptide comprises: a) an E2 polypeptide; and b) a heterologous fusion partner polypeptide, the “heterologous fusion partner polypeptide” is one that is not found associated with the E2 polypeptide in nature, e.g., a purification tag. As another example, where “heterologous” is used in the context of a nucleic acid or nucleotide sequence, the nucleic acid includes operably linked nucleotide sequences that are not normally linked in nature. For example, an IRES can be heterologous to a nucleotide sequence encoding an HCV El and / or E2 polypeptide, where the IRES is from an organism (e.g., a virus) other than HCV.

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

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

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

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

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

[0021] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0022] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.DETAILED DESCRIPTION

[0023] The present disclosure provides compositions comprising HCV Gt3a S52 E2 polypeptide or a nucleic acid encoding the HCV Gt3a S52 E2 polypeptide and use thereof in a method for inducing an immune response to multiple HCV genotypes, e.g., Gts 1, 2, and 3.Overview

[0024] Recombinant E1E2 heterodimer made from CHO cells has many characteristics encouraging its development as a HCV vaccine. These include its ability to bind to many diverse cross- neutralizing monoclonal antibodies (Mabs) and its ability to elicit antibodies that can neutralize the in vitro infectivity of many different HCV strains that occur around the world. This activity is important because the hepacivirus genus is highly variable genetically, even more so than HIV. Despite the ability of E1E2 derived from the HCV Gtla strain H77 to elicit broad cross-neutralizing antibodies, there is a need for a vaccine that can neutralize infection of “difficult to neutralize” strains such as the Gt 3a strain, S52 and the Gt 2a strain J6. The data presented herein shows that S52-E2 has the unusual property of eliciting strong neutralizing antibodies against not only S52 strain but also against J6 and H77 strains. In contrast, E2 antigen derived from H77 or J6 is not as effective at neutralizing all three diverse HCVpp’s (Fig. 1 ). This invention provides the use of the S52-E2 antigen as a vaccine by itself or in enhancing the protection provided by vaccines that include E1E2 and / or another E2.METHODS OF INDUCING AN IMMUNE RESPONSE

[0025] The present disclosure provides a method of inducing antibodies that neutralize the infectivity of at least HCV genotypes 1, 2, and 3 in a subject, where the method comprises administering an effective amount of a composition comprising HCV Gt3a S52 E2 polypeptide or a nucleic acid encoding the HCV Gt3a S52 E2 polypeptide to the subject.

[0026] The immune response may include generation of antibodies effective against infection by at least HCV genotypes 1, 2, and 3. The immune response may include generation of antibodies that neutralize at least HCV genotypes 1, 2, and 3.

[0027] In certain embodiments, the immune response to HCV Gtl comprises an immune response to HCV Gtla. In certain embodiments, the immune response to HCV Gt2 comprises an immune response to HCV Gt2a. In certain embodiments, the immune response to HCV Gt3 comprises an immune response to HCV Gt3a.

[0028] In certain embodiments, the immune response to HCV Gtla comprises an immune response to one or more ofHCV Gtla strains H77, 72, 123, 138, UKNP1.11.6, UKNP1.9.1, UKNP1.10.1, and / or UKNP1.16.3. In certain embodiments, the immune response to HCV Gtl comprises an immune response to one or more ofHCV Gtlb. In certain embodiments, the immune response to HCV Gtlb comprises an immune response to HCV Gtlb strain UKNP1.18.1, lb25, lb35, and / or lb58. In certain embodiments, the immune response to HCV Gt2a comprises an immune response to Gt2a J6 and / or UKNP2.4. 1 strain. In certain embodiments, the immune response to HCV Gt3a comprises an immune response to Gt3a S52 and / or UKNP3. 1 .2 strain.

[0029] The immune response may include generation of antibodies effective against infection by at least HCV genotypes 4 and 5. The immune response may include generation of antibodies that neutralize at least HCV genotypes 4 and 5, in addition to Gtl, Gt2, and Gt3. In certain embodiments, the immune response to HCV Gt4 includes an immune response to Gt4a, such as, UKNP4.2.2. In certain embodiments, the immune response to HCV Gt5 includes an immune response to Gt5a, such as, UKNP5.2.1.

[0030] The immune response may include generation of antibodies effective against infection by HCV genotypes 6 and 7. The immune response may include generation of antibodies that neutralize at least HCV genotypes 6 and 7, in addition to Gtl, Gt2, and Gt3. The immune response may include generation of antibodies that neutralize at least HCV genotypes 6 and 7, in addition to Gtl - Gt5.

[0031] The immune response may be measured using an in vitro cell-based neutralization assay involving pseudotyped HCV particles (HCVpp) derived with glycoproteins ofHCV genotypes 1, 2, or 3. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gt3a S52. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least 90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gtla H77. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least 90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gt2a J6. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least 90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gt3a S52 and HCV Gtla H77. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least 90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gt3a S52 and HCV Gt2a J6. In certain embodiments, the immune response may be at least 50% neutralization, e.g., at least 60% neutralization, at least 70% neutralization, at least 80% neutralization, at least 85% neutralization, at least 90% neutralization, at least 95% neutralization, at least 99% neutralization, or 100% neutralization of HCVpp derived with glycoproteins of HCV Gt3a S52, HCV Gt2a J6, and HCV Gtla H77.

[0032] In some cases, a prime-boost vaccine protocol is used. In some cases, a first (priming) immunogenic composition is administered, and, after a time, a second (booster) immunogenic composition is administered. A second immunogenic composition can be administered at a time period of from 1 day to 1 year following administration of the first immunogenic composition. For example, a second immunogenic composition can be administered at a time period of from 1 day to 1 week, from 1 week to 2 weeks, from 2 weeks to 1 month, from 1 month to 2 months, from 2 months to 6 months, or from 6 months to 1 year following administration of the first immunogenic composition.

[0033] In some cases, the first immunogenic composition and the second immunogenic composition are the same. For example, the first immunogenic composition comprises the Gt3a S52 E2 polypeptide; and the second immunogenic composition comprises the Gt3a S52 E2 polypeptide or the first immunogenic composition comprises a nucleic acid encoding the Gt3a S52 E2 polypeptide; and the second immunogenic composition comprises a nucleic acid encoding the Gt3a S52 E2 polypeptide.

[0034] In some cases, the first immunogenic composition and the second immunogenic composition are different. For example, in some cases, the first immunogenic composition comprises the Gt3a S52 E2 polypeptide; and the second immunogenic composition comprises a Gtla H77 E1E2heterodimer or the first immunogenic composition comprises a Gtla H77 E1E2 heterodimer; and the second immunogenic composition comprises the Gt3a S52 E2 polypeptide. In another example, the first immunogenic composition comprises the Gt3a S52 E2 polypeptide; and the second immunogenic composition comprises a nucleic acid encoding the Gt3a S52 E2 polypeptide or the first immunogenic composition comprises nucleic acid encoding the Gt3a S52 E2 polypeptide; and the second immunogenic composition comprises the Gt3a S52 E2 polypeptide.

[0035] In some embodiments, the method of inducing an immune response against HCV Gtl- Gt3 in an individual may involve (i) administering a composition comprising Gtl E1E2 heterodimer to the individual, followed by (ii) administering a composition comprising Gt3a S52 E2 polypeptide to the individual using a prime-boost vaccine protocol such as a prime-boost vaccine protocol provided herein.

[0036] In some embodiments, the method of inducing an immune response against HCV Gtl- Gt3 in an individual may involve (i) administering a composition comprising Gt3a S52 E2 polypeptide to the individual, followed by (ii) administering a composition comprising Gtl E1E2 heterodimer to the individual using a prime-boost vaccine protocol such as a prime-boost vaccine protocol provided herein.

[0037] In some embodiments, the method of inducing an immune response against HCV Gtl- Gt3 in an individual may involve (i) administering a composition comprising a nucleic acid encoding a Gtl E1E2 heterodimer to the individual, followed by (ii) administering a composition comprising a nucleic acid encoding the Gt3a S52 E2 polypeptide to the individual using a prime-boost vaccine protocol such as a prime-boost vaccine protocol provided herein.

[0038] In some embodiments, the method of inducing an immune response against HCV Gtl- Gt3 in an individual may involve (i) administering a composition comprising a nucleic acid encoding Gt3a S52 E2 polypeptide to the individual, followed by (ii) administering a composition comprising a nucleic acid encoding Gtl E1E2 heterodimer to the individual using a prime-boost vaccine protocol such as a prime-boost vaccine protocol provided herein.

[0039] In a further embodiment, the method may further include (iii) administering a composition comprising Gt2 E2 polypeptide to the individual or (iii) administering a composition comprising a nucleic acid encoding Gt2 E2 polypeptide to the individual.

[0040] In certain embodiments, a Gtl E1E2 heterodimer may be a Gtla E1E2 heterodimer. In certain embodiments, a Gtla E1E2 heterodimer may be an H77 E1E2 heterodimer. In certain embodiments, a Gt2 E2 polypeptide may be Gt2a E2 polypeptide. In certain embodiments, a Gt2a E2 polypeptide may be a J6 E polypeptide.

[0041] In a further embodiment, the method may include administering to the individual a composition comprising HCV Gt3 S52 E2 polypeptide and HCV Gtl E1E2 heterodimer. In certainembodiments, Gtl E1E2 heterodimer is Gtla E1E2 heterodimer, and further optionally, the Gtla E1E2 heterodimer is H77 E1E2 heterodimer.

[0042] In a further embodiment, the method may include administering to the individual a composition comprising a nucleic acid encoding a HCV Gt3 S52 E2 polypeptide and a nucleic acid encoding a HCV Gtl E1E2 heterodimer. In certain embodiments, Gtl E1E2 heterodimer is Gtla E1E2 heterodimer, and further optionally, the Gtla E1E2 heterodimer is H77 E1E2 heterodimer.

[0043] In a further embodiment, the method may include administering to the individual a composition comprising HCV Gt3 S52 E2 polypeptide and HCV Gt2 E2 polypeptide. In certain embodiments, Gt2 E2 polypeptide is Gt2a E2 polypeptide, and further optionally, the Gt2a E2 polypeptide is J6 Gt2 E2 polypeptide.

[0044] In a further embodiment, the method may include administering to the individual a composition comprising a nucleic acid encoding a HCV Gt3 S52 E2 polypeptide and a nucleic acid encoding a Gt2 E2 polypeptide. In certain embodiments, Gt2 E2 polypeptide is Gt2a E2 polypeptide, and further optionally, the Gt2a E2 polypeptide is J6 E2 polypeptide.

[0045] In certain embodiments, the composition comprising HCV Gt3 S52 E2 polypeptide may not include an El polypeptide, an E2 polypeptide, and / or E1E2 heterodimer from HCV Gtl. In certain embodiments, the composition comprising a nucleic acid encoding a HCV Gt3 S52 E2 polypeptide may not include a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and / or a nucleic acid encoding E1E2 heterodimer from HCV Gtl.

[0046] In certain embodiments, the composition comprising HCV Gt3 S52 E2 polypeptide may not include an El polypeptide, an E2 polypeptide, and / or E1E2 heterodimer from HCV Gt2. In certain embodiments, the composition comprising a nucleic acid encoding a HCV Gt3 S52 E2 polypeptide may not include a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and / or a nucleic acid encoding E1E2 heterodimer from HCV Gt2.

[0047] In certain embodiments, the composition comprising HCV Gt3 S52 E2 polypeptide may not include an El polypeptide, an E2 polypeptide, and / or E1E2 heterodimer from another HCV Gt2. In certain embodiments, the composition comprising a nucleic acid encoding HCV Gt3 S52 E2 polypeptide may not include a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and / or a nucleic acid encoding E1E2 heterodimer from another HCV Gt2.

[0048] In certain embodiments, the composition comprising HCV Gt3 S52 E2 polypeptide may not include an El polypeptide and / or E1E2 heterodimer from HCV Gt3 S52. In certain embodiments, the composition comprising a nucleic acid encoding a HCV Gt3 S52 E2 polypeptide may not include a nucleic acid encoding an El polypeptide and / or a nucleic acid encoding E1E2 heterodimer from HCV Gt3 S52.

[0049] In certain embodiments, the composition comprising HCV Gt3 S52 E2 polypeptide may not include another HCV antigen. In certain embodiments, the composition comprising a nucleic acid encoding HCV Gt3 S52 E2 polypeptide may not include a nucleic acid encoding another HCV antigen.

[0050] In certain embodiments, the S52-E2 is produced in a mammalian cell. In certain embodiments, the S52-E2 is produced in a mammalian cell line. In certain embodiments, the S52-E2 is produced in CHO cells or HEK293 cells. Production of S52-E2 in mammalian cells can provide several advantages. For example, the S52-E2 production can be scaled up by using a mammalian cell line that can be grown to higher cell density in bioreactors.General considerations

[0051] A composition of the present disclosure is generally administered to a human subject who: i) has an HCV infection; or ii) is at risk of acquiring an HCV infection (e.g., is at greater risk than the general population of acquiring an HCV infection); or iii) is naive with respect to HCV infection. The administering may prevent or at least partially arrest the development of disease and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose” or a “therapeutically effective amount.” “Prophylactic” use of a subject immunogenic composition generally refers to administration to an individual who has not been infected with HCV (e.g., a “naive” individual or at risk individual). “Therapeutic” use of a subject immunogenic composition can refer to “prophylactic” use (administration to an individual who has not been infected with HCV) and / or to administration to an individual who has an HCV infection. A “therapeutically effective amount” of an immunogenic composition of the present disclosure, can be an amount that, when administered in one or more doses to an individual who is not infected with HCV, is effective to induce an immune response in the individual to HCV. A “therapeutically effective amount” of an immunogenic composition of the present disclosure, can be an amount that, when administered in one or more doses to an individual who is infected with HCV, is effective to enhance an immune response in the individual to HCV. The vaccine is also useful in vaccinating HCV patients who have been cured through antiviral therapy but who remain susceptible to HCV re-infection.

[0052] Amounts effective for prophylactic or therapeutic use will depend on, e.g., the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. Single or multiple doses of a subject immunogenic composition can be administered depending on the dosage and frequency required and tolerated by the patient, and route of administration. Typically for prophylactic use, doses containing between l-100ug vaccine antigen are administered intramuscularly (i / m) with an appropriate adjuvant on weeks 0,4,24 or weeks 0,8,24 and sometimes with a later 4thboost.

[0053] In some cases, an effective amount of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce an antibody response (e.g., a neutralizing antibody response) to HCV in the individual. For example, antibody to HCV (e.g., extracellular HCV), and / or to an HCV-infected cell, can be induced.

[0054] An effective amount of an immunogenic composition of the present disclosure can be an amount that, when administered to an individual in one or more doses, is effective to induce a neutralizing antibody response to HCV of a variety of genotypes (e.g., Gtl; Gt2; Gt3; etc.). A neutralizing antibody response may reduce binding of HCV to one or more host receptors for HCV and inhibits entry of HCV into a cell.

[0055] In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce a cytotoxic T lymphocyte (CTL) response to HCV (e.g., a CD8+T cell response). For example, a CTL response to an HCV-infected cell can be induced.

[0056] In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce a helper T lymphocyte (e.g., CD4+T cell) to HCV in an individual.

[0057] In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce an antibody response (e.g., a neutralizing antibody response) and / or a CTL response and / or a helper T cell response to HCV Gtl . In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce an antibody response (e.g., a neutralizing antibody response) and / or a CTL response and / or a helper T cell response to HCV Gt3. In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce an antibody response (e.g., a neutralizing antibody response) and / or a CTL response and / or a helper T cell response to HCV Gtl, Gt2 and HCV Gt3. In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce an antibody response (e.g., a neutralizing antibody response) and / or a CTL response and / or a helper T cell response to HCV of any genotype. In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective toinduce a CD8+T cell response to HCV genotypes 1-6. In some cases, an effective amount (e.g., a therapeutically effective amount) of an immunogenic composition of the present disclosure is an amount that, when administered to an individual in one or more doses, is effective to induce a CD4+T cell response to HCV genotypes 1-6.

[0058] An immunogenic composition of the present disclosure is generally administered in an amount effective to elicit an immune response, e.g., a humoral immune response (e.g., an antibody response) and / or a CTL response, in the mammalian subject. Effective amounts of HCV antigen, e.g., E1 / E2, El, or E2 polypeptides for immunization will vary, and can generally range from about 1 pg to 100 pg per 70 kg patient, e.g., from about 5 pg / 70 kg to about 50 pg / 70 kg. Substantially higher dosages (e.g. 10 mg to 100 mg or more) of an HCV E1 / E2, El, or E2 polypeptide may be suitable in oral, nasal, or topical administration routes. In some cases, a dose of an immunogenic composition of the present disclosure comprises an HCV E2 in an amount of from 4 pg to 100 pg. For example, in some cases, a dose of an immunogenic composition of the present disclosure comprises an HCV E2 in an amount of from 4 pg to 5 pg, from 5 pg to 10 pg, from 10 pg to 15 pg, from 15 pg to 20 pg, from 20 pg to 25 pg, from 25 pg to 30 pg, from 30 pg to 40 pg, from 40 pg to 50 pg, from 50 pg to 60 pg, from 60 pg to 70 pg, from 70 pg to 80 pg, from 80 pg to 90 pg, or from 90 pg to 100 pg. In some cases, a dose of an immunogenic composition of the present disclosure comprises an HCV E2 in an amount of from about 100 pg to about 200 pg. El and E1E2 heterodimer if administered may also be in an amount similar to that specified for E2.

[0059] The initial administration can be followed by booster immunization of the same composition or a different composition. In some instances, a subject method of inducing an immune response involves an initial administration of a composition of the present disclosure, followed by at least one booster, and in some instances involves two or more (e.g., three, four, or five) boosters. The interval between an initial administration and a booster, or between a give booster and a subsequent booster, can be from about 1 week to about 12 weeks, e.g., from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 10 weeks, or from about 10 weeks to about 12 weeks. The interval between an initial administration and a booster, or between a give booster and a subsequent booster, can be from 4 months to 6 months, or from 6 months to 1 year.

[0060] In some cases, a first dose of an composition of the present disclosure is administered at a first time, where the administration is intramuscular; a second dose of an composition of the present disclosure is administered at a second time, where the administration is intranasal administration; a third dose of an composition of the present disclosure is administered at a third time, where the administration is intranasal administration. In some cases, the second dose (a “first booster”) is administered from about 1 week to about 12 weeks, e.g., from about 1 week to about 2 weeks, from about 2 weeks to about 4weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 10 weeks, from about 10 weeks to about 12 weeks, from 4 months to 6 months, or from 6 months to 1 year, after the first dose. In some cases, the third dose (a “second booster”) is administered from about 1 week to about 12 weeks, e.g., from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about 8 weeks to about 10 weeks, from about 10 weeks to about 12 weeks, from 4 months to 6 months, or from 6 months to 1 year, after the second dose.

[0061] In general, immunization can be accomplished by administration of a composition of the present disclosure by any suitable route, including administration of the composition orally, nasally (e.g., intranasally), nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, or intradermally. In some cases, a composition of the present disclosure is administered intramuscularly. In some cases, a composition of the present disclosure is administered subcutaneously. Immunization can be accomplished by administration of a composition of the present disclosure in tablet, solid, powdered, liquid, or aerosol form, locally or systemically, with or without added excipients. A composition of the present disclosure is in many cases in liquid form. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980). In some instances, immunization is accomplished by intramuscular injection of an immunogenic composition of the present disclosure.INDIVIDUALS SUITABLE FOR ADMINISTRATION

[0062] Individuals who are suitable for administration with a composition of the present disclosure include immunologically naive individuals (e.g., individuals who have not been infected with HCV and / or who have not been administered with an HCV vaccine). Individuals suitable for administration include humans.

[0063] Individuals who are suitable for administration with a composition of the present disclosure composition of the present disclosure include individuals who are at greater risk than the general population of becoming infected with HCV, where such individuals include, e.g., intravenous drug users; individuals who are the recipients, or the prospective recipients, of blood or blood products from another (donor) individual(s); individuals who are the recipients, or the prospective recipients, of non-autologous cells, tissues, or organs from another (donor) individual; health care workers; emergency medical and non-medical personnel (e.g., first responders; fire fighters; emergency medical team personnel; etc.) and the like.

[0064] Individuals who are suitable for administration with a composition of the present disclosure composition of the present disclosure include individuals who recently became exposed toHCV or who recently became infected with HCV. For example, a subject immunogenic composition can be administered to an individual within from about 24 hours to about 48 hours, from about 48 hours to about 1 week, or from about 1 week to about 4 weeks, following possible or suspected exposure to HCV or following infection with HCV.

[0065] Individuals who are suitable for administration with a composition of the present disclosure composition of the present disclosure include individuals who were previously infected with HCV, who were treated for HCV, and who were cured.

[0066] Individuals who are suitable for administration with a composition of the present disclosure include individuals who have been diagnosed as having an HCV infection, and include chronically infected individuals. In some cases, an individual who has been diagnosed as having an HCV infection is treated with an anti-viral agent and a composition of the present disclosure. Suitable antiviral agents for treating HCV infection include, e.g., sofosbuvir, daclatasvir or derivatives thereof, HCV protease inhibitors, ribavirin (l-P-D-ribofuranosyl-lH-l,2,4-triazole-3-carboxamide); interferon-alpha (IFN-a) (where “IFN-a” includes IFN-a2a; IFN-a2b; IFN-a that is conjugated with polyethylene glycol) (“pegylated IFN-a), where the pegylated IFN-a can be pegylated IFN-a2a or pegylated IFN-a 2b); an HCV NS3 protease inhibitor (e.g., boceprevir; telaprevir); and an HCV NS5a inhibitor such as daclatasvir or a derivative thereof, or of a HCV NS5b polymerase inhibitor such as sofosbuvir..

[0067] In some cases, an individual who has been diagnosed as having an HCV infection is treated with, e.g.: 1) IFN-a + ribavirin; and a composition of the present disclosure; 2) IFN-a + ribavirin + an HCV protease inhibitor (e.g., boceprevir or telaprevir); and a composition of the present disclosure; 3) Harvoni; and a composition of the present disclosure; 4) an inhibitor of HCV NS5B; and a composition of the present disclosure; 5) an inhibitor of HCV NS5A; and a composition of the present disclosure; or 6) an inhibitor of HCV NS5B + an inhibitor of HCV NS5A; and a composition of the present disclosure. Suitable anti-viral agents for treating HCV infection include Sovaldi (Sofosbuvir; a nucleotide analog that functions as an NS5B polymerase inhibitor), alone or in combination with pegylated IFN-a and ribavirin; and Harvoni. Harvoni is a formulation comprising 90 mg ledipasvir and 400 mg sofosbuvir. Ledipasvir is an inhibitor of HCV NS5A.El polypeptides

[0068] An HCV El polypeptide can have a length of from about 100 amino acids (aa) to about 150 aa, from about 150 aato about 175 aa, from about 175 aa to about 195 aa, from about 131 aa to about 175 aa, or from about 175 aa to about 193 aa. In some cases, an HCV El polypeptide is an HCV El ectodomain polypeptide. In some cases, an HCV El polypeptide is a full-length HCV El polypeptide.

[0069] In FIG. 3A-3E, the amino acid sequence of El is amino acid 192 to amino acid 383. In FIG. 4A- 4E, the amino acid sequence of El is amino acid 192 to amino acid 383. In FIG. 2A-2E, the amino acidsequence of El is amino acid 192 to amino acid 383. As used herein, "El polypeptide" includes a precursor El protein, including the signal sequence; includes a mature El polypeptide which lacks this sequence; and includes an El polypeptide with a heterologous signal sequence. An El polypeptide can include a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 360-383 (see, e.g., WO 96 / 04301). In some cases, a suitable El polypeptide lacks a C-terminal portion that includes a transmembrane region. For example, in some cases, a suitable El polypeptide lacks the C- terminal portion from amino acid 330 to amino acid 383, or from amino acid 360 to amino acid 383. El polypeptides can be an El polypeptide of any genotype, subtype or isolate of HCV. El polypeptides of genotype 1 and El polypeptides of genotype 3 are included in an E1 / E2 heterodimer of the present disclosure.

[0070] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 2A-4E.

[0071] For example, an El polypeptide of genotype 1A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 1A and depicted in FIG. 3A-3E. For example, an El polypeptide of genotype IB can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as IB and depicted in FIG. 3A-3E. For example, an El polypeptide of genotype 1C can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 1C and depicted in FIG. 3A-3E.

[0072] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 4A-4E. For example, an El polypeptide of genotype 2A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 2A and depicted in FIG. 4A-4E. For example, an El polypeptide of genotype 2B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of an amino acid sequence identified as 2B and depicted in FIG. 4A-4E.

[0073] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the consensus El polypeptide amino acid sequence depicted in FIG. 2A-2E.

[0074] An El polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an El polypeptide depicted in FIG. 2A-2E. For example, an El polypeptide of genotype 3A, 3B, 3K can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192-383 of the amino acid sequence depicted in FIG. 2A- 2E.E2 polypeptides

[0075] An E2 polypeptide can have a length of from about 200 amino acids (aa) to about 250 aa, from about 250 aa to about 275 aa, from about 275 aa to about 300 aa, from about 300 aa to about 325 aa, from about 325 aa to about 350 aa, or from about 350 aa to about 380 aa. In some cases, an HCV E2 polypeptide is an HCV E2 ectodomain polypeptide. In some cases, an HCV E2 polypeptide is a full- length HCV E2 polypeptide.

[0076] In FIG. 3A-3E, the amino acid sequence of E2 is amino acid 384 to amino acid 746. In FIG. 4A- 4E, the amino acid sequence of E2 is amino acid 384 to amino acid 751. In FIG. 2A-2E, the amino acid sequence of E2 is amino acid 385 to amino acid 754. As used herein, an "E2 polypeptide" includes a precursor E2 protein, including the signal sequence; includes a mature E2 polypeptide which lacks this sequence; and includes an E2 polypeptide with a heterologous signal sequence. An E2 polypeptide can include a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 715-730 and may extend as far as approximately amino acid residue 746 (see, Lin et al., J. Virol. (1994) 68:5063-5073).

[0077] In some cases, a E2 polypeptide lacks a portion of its C-terminal region, e.g., from about amino acid 715 to the C-terminus; from about amino acid 625 to the C-terminus; from about amino acid 661 to the C-terminus; from about amino acid 655 to the C-terminus; from about amino acid 500 to the C- terminus, where the amino acid numbering is with reference to the numbering in FIG. 3A-3E. See, e.g., U.S. Patent No. 6,521,423.

[0078] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 2A-4E. For example, an E2 polypeptide of genotype 1, e.g., 1A, IB, or 1C can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-746 of an amino acid sequence depicted in FIG. 3A-3E.

[0079] For example, an E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-751 of an amino acid sequence depicted in FIG. 4A-4E. For example, an E2 polypeptide of genotype 2A or 2B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 384-751 of the “consensus” amino acid sequence depicted in FIG. 4C-4E.

[0080] An E2 polypeptide can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an E2 polypeptide depicted in FIG. 2A-2E. For example, an E2 polypeptide of genotype 3 can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of an amino acid sequence depicted in FIG. 2A-2E. For example, an E2 polypeptide of genotype 3A can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of an amino acid sequence identified as 3A and depicted in FIG. 2A-2E. For example, an E2 polypeptide of genotype 3B can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of the amino acid sequence identified as 3B and depicted in FIG. 2A-2E. For example, an E2 polypeptide of genotype 3K can comprise an amino acid sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385-754 of the amino acid sequence identified as 3K and depicted in FIG. 2A-2E.HCV E1 / E2 heterodimers

[0081] HCV E1 / E2 heterodimers El and E2 polypeptides associated with each other and can include wild-type HCV El polypeptides; wild-type HCV E2 polypeptides; variant HCV El polypeptides; and variant HCV E2 polypeptides. An HCV E1 / E2 heterodimer can include any of the HCV El polypeptides discussed above and any of the HCV E2 polypeptides discussed herein.Genotype 3 - S52 E2

[0082] An S52 E2 polypeptide of the present disclosure can have a length of from about 200 amino acids (aa) to about 250 aa, from about 250 aa to about 275 aa, from about 275 aa to about 300 aa, from about 300 aa to about 325 aa, from about 325 aa to about 350 aa, or from about 350 aa to about 365aa. In some cases, the S52 E2 polypeptide of the present disclosure is an HCV S52 E2 ectodomain polypeptide (e.g., lacking the transmembrane domain of a naturally-occurring S52 E2 polypeptide). In some cases, the S52 E2 polypeptide of the present disclosure is a full-length HCV S52 E2 polypeptide. In some cases, the S52 E2 polypeptide of the present disclosure is a soluble HCV S52 E2 polypeptide.

[0083] As used herein, an "S52 E2 polypeptide" includes: a precursor S52 E2 protein, including the signal sequence; a mature S52 E2 polypeptide which lacks this sequence; and a S52 E2 polypeptide with a heterologous signal sequence. A S52 E2 polypeptide can include a C-terminal membrane anchor sequence which occurs at approximately amino acid positions 715-730 and may extend as far as approximately amino acid residue 746 (see, Lin et al., J. Virol. (1994) 68:5063-5073).

[0084] In some cases, the genotype 3a S52 E2 polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 385 to 750 of the S52 amino acid sequence identified as ADF97232 (S52):

[0085] MSTLPKPQRKTKRNTIRRPQDVKFPGGGQIVGGVYVLPRRGPRLGVRATRKTSERSQ PRGRRQPIPKARRSEGRSWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPNDPRRRSRNLGKVI DTLTCGFADLMGYIPLVGAPVGGVARALAHGVRALEDGINFATGNLPGCSFSIFLLALFSCLVHPAASLEWRNTSGLYVLTNDCSNSSIVYEADDVILHTPGCVPCVQDDNTSTCWTPVTPTVAVRYVGAT TASIRSHVDLLVGAATLCSALYVGDMCGAVFLVGOAFTFRPRRHQTVOTCNCSLYPGHVSGHR MAWDMMMNWSPAVGMVVAHILRLPQTLFDILAGAHWGILAGLAYYSMOGNWAKVAIVMIM FSGVDAETYVTGGSVAHSARGLTSLFSMGAKQKLQLVNTNGSWHINSTALNCNESINTGFI AGLFYYHKFNSTGCPQRLSSCKPIISFRQGWGPLTDANITGPSDDRPYCWHYAPRPCSVVP ASSVCGPVYCFTPSPVVVGTTDIKGKPTYNWGENETDVFLLESLRPPSGRWFGCAWMNST GFLKTCGAPPCNIYGGEGDPENETDLFCPTDCFRKHPEATYSRCGAGPWLTPRCMVDYPY RLWHYPCTVNFTLFKVRMFVGGFEHRFTAACNWTRGERCNIEDRDRSEQHPLLHSTTEL AILPCSFTPMPALSTGLIHLHQNIVDVQYLYGVGSDMVGWALKWEFVILVFLLLADARVC VALWLMLMVSQAEA (SEQ ID NO 42).

[0086] SEQ ID NO:42 is annotated to recite different domains: the italicized sequence forms the core region, the underlined sequence is the El domain, and the bold sequence is the E2 domain.

[0087] In certain embodiments, the S52 E2 polypeptide can have a length of about 366 amino acids (e.g., from about 363 amino acids to about 370 amino acids) and can have at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acid sequence:

[0088] ETYVTGGSVAHSARGLTSLFSMGAKQKLQLVNTNGSWHINSTALNCNESINTGF IAGLFYYHKFNSTGCPQRLSSCKPIISFRQGWGPLTDANITGPSDDRPYCWHYAPRPCSVVPASS VCGPVYCFTPSPVVVGTTDIKGKPTYNWGENETDVFLLESLRPPSGRWFGCAWMNSTGFLKTC GAPPCNIYGGEGDPENETDLFCPTDCFRKHPEATYSRCGAGPWLTPRCMVDYPYRLWHYPCTVNFTLFKVRMFVGGFEHRFTAACNWTRGERCNIEDRDRSEQHPLLHSTTELAILPCSFTPMPALSTGLIHLHQNIVDVQYLYGVGSDMVGWALKWEFVILVFLLLADARVCVALWLMLMVSQAEA (SEQ ID NO:52)

[0089] In some cases, the S52 E2 polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 385 to 695 of the S52 amino acid sequence depicted in SEQ ID NO:42; and can have a length of about 300 amino acids (e.g., from about 275 amino acids to about 320 amino acids, from about 280 amino acids to about 315 amino acids, from about 285 amino acids to about 310 amino acids, or from about 300 amino acids to about 320 amino acids), where such an E2 polypeptide is referred to as a “soluble” S52 E2 polypeptide.

[0090] In some cases, the S52 E2 polypeptide is a soluble E2 polypeptide comprising an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acid sequence:

[0091] ETYVTGGSVAHSARGLTSLFSMGAKQKLQLVNTNGSWHINSTALNCNESINTGF IAGLFYYHKFNSTGCPQRLSSCKPIISFRQGWGPLTDANITGPSDDRPYCWHYAPRPCSVVPASS VCGPVYCFTPSPVVVGTTDIKGKPTYNWGENETDVFLLESLRPPSGRWFGCAWMNSTGFLKTC GAPPCNIYGGEGDPENETDLFCPTDCFRKHPEATYSRCGAGPWLTPRCMVDYPYRLWHYPCTV NFTLFKVRMFVGGFEHRFTAACNWTRGERCNIEDRDRSEQHPLLHSTTELAILPCSFTPMPALS (SEQ ID NO:53).Genotype 1 -H77

[0092] In some cases, a H77 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 383 of the amino acid sequence identified as “NP_671491 (H77)” (also referred to herein as “H77”) and set forth below:

[0093] MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERS QPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKV IDTLTCGFADEMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA YQVRNSSGLYHVTNDCPNSSIVYEAADAILHTPGCVPCVREGNASRCWVAVTPTVATRDGKLP TTOLRRHIDLLVGSATLCSALYVGDLCGSVFLVGOLFTFSPRRHWTTQDCNCSIYPGHITGHRM AWDMMMNWSPTAALVVAQLLRIPQAIMDMIAGAHWGVLAGIAYFSMVGNWAKVLVVLLLF AGVDAETHVTGGSAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCNESLNTGWL AGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVP AKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWH YPCTINYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPC SFTTLPALSTGLIHLHQNIVDVQYLYGVGSSIASWAIKWEYVVLLFLLLADARVCSCLWM MLLISQAEA (SEQ ID NO: 3)

[0094] SEQ ID NO:3 is annotated to recite different domains: the italicized sequence forms the core region, the underlined sequence is the El domain, and the bold sequence is the E2 domain.

[0095] H77 El polypeptide can have a length of about 192 amino acids (e.g., from 190 amino acids to 195 amino acids). In some cases, an H77 El polypeptide can have a length of about 161 amino acids (e.g., from about 158 amino acids to about 165 amino acids) and comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 352 of SEQ ID NO:3; and where such an El polypeptide is referred to as a “soluble” H77 El polypeptide. In some cases, a H77 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 367 of the H77 amino acid sequence of SEQ ID NO:3; and can have a length of about 176 amino acids (e.g., from about 174 amino acids to about 180 amino acids), where such an El polypeptide is referred to as a “soluble” H77 El polypeptide. In some cases, a H77 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 359 of the H77 amino acid sequence of SEQ ID NO:3; and can have a length of about 168 amino acids (e.g., from about 165 amino acids to about 172 amino acids), where such an El polypeptide is referred to as a “soluble” H77 El polypeptide. In some cases, a H77 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 331 of the H77 amino acid sequence of SEQ ID NO:3; and can have a length of about 140 amino acids (e.g., from about 135 amino acids to about 145 amino acids), where such an El polypeptide is referred to as a “soluble” H77 El polypeptide.Genotype 2 - J 6

[0096] An amino acid sequence of D00944 (HC-J6) is as follows:

[0097] MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRR QPIPKDRRSTGKSWGKPGYPWPLYGNEGLGWAGWLLSPRGSRPSWGPNDPRHRSRNVGKVIDTLTC GFADLMGYIPWGAPLGGVARALAHGVRVLEDGVNFATGNLPGCSFSIFLLALLSCITTPVSA^V^Y STGYMVTNDCTNDSITWQLQAAVLHVPGCVPCEKVGNTSRCWIPVSPNVAVQQPGALTQGLRT HIDMVVMSATLCSALYVGDLCGGVMLAAQMFIVSPQHHWFVQDCNCSIYPGTITGHRMAWD MMMNWSPTATMILAYAMRVPEVIIDIIGGAHWGVMFGLAYFSMQGAWAKVVVILLLAAGVD AQTHTVGGSTAHNARTLTGMFSLGARQKIQLINTNGSWHINRTALNCNDSLHTGFLASLF YTHSFNSSGCPERMSACRSIEAFRVGWGALQYEDNVTNPEDMRPYCWHYPPRQCGVVSASSVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTRPPQGSWFGCTWMNSTG YTKTCGAPPCRIRADFNASMDLLCPTDCFRKHPDTTYIKCGSGPWLTPRCLIDYPYRLWH YPCTVNYTIFKIRMYVGGVEHRLTAACNFTRGDRCNLEDRDRSQLSPLLHSTTEWAILPCT YSDLPALSTGLLHLHQNIVDVQFMYGLSPALTKYIVRWEWVVLLFLLLADARVCACLWM LILLGQAEA (SEQ ID NO 33)

[0098] SEQ ID NO:33 is annotated to recite different domains: the italicized sequence forms the core region, the underlined sequence is the El domain, and the bold sequence is the E2 domain.

[0099] In some cases, genotype 2 J6 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 383 of the amino acid sequence of SEQ ID NO:33 and can have a length of about 192 amino acids (e.g., from 190 amino acids to 195 amino acids); where such a polypeptide is referred to as a “J6 El polypeptide.” In some cases, J6 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 352 of the J6 amino acid sequence of SEQ ID NO:33; and can have a length of about 161 amino acids (e.g., from about 158 amino acids to about 165 amino acids), where such an El polypeptide is referred to as a “soluble” J6 El polypeptide. In some cases, a J6 El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 367 of the J6 amino acid sequence of SEQ ID NO:33; and can have a length of about 176 amino acids (e.g., from about 174 amino acids to about 180 amino acids), where such an El polypeptide is referred to as a “soluble” J6 El polypeptide. In some cases, an El polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 359 of the J6 amino acid sequence of SEQ ID NO:33; and can have a length of about 168 amino acids (e.g., from about 165 amino acids to about 172 amino acids), where such an El polypeptide is referred to as a “soluble” J6 El polypeptide. In some cases, an El polypeptide that is comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 192 to 331 of the J6 amino acid sequence of SEQ ID NO:33; and can have a length of about 140 amino acids (e.g., from about 135 amino acids to about 145 amino acids), where such an El polypeptide is referred to as a “soluble” J6 El polypeptide.

[0100] The soluble E2 sequences disclosed herein may include an optional Methionine residue at the N-terminus. The soluble E2 sequences disclosed herein may be expressed with a purification tag at the N- or C-terminus. The soluble E2 sequences disclosed herein may include remnants of a protease recognition site remaining after cleavage by a protease. For example, a soluble E2 protein expressed as a E2 -protease recognition site-Fc may retain the sequence LEVLFQ (SEQ ID NO:56) following cleavage after residue Q if the protease recognition site.NUCLEIC ACID COMPOSITIONS

[0101] The present disclosure provides compositions (e.g., immunogenic compositions) comprising a nucleic acid encoding a HCV Gt3 S52 E2. The composition may include additional nucleic acids, e.g., a nucleic acid encoding a HCV Gtl E1E2 heterodimer. In some cases, the nucleic acids are DNA molecules. In some cases, the nucleic acids are RNA (e.g., mRNA) molecules.

[0102] In some cases, a nucleic acid composition of the present disclosure comprises: a) one or more RNA molecules; and b) one or more of: i) a lipid; ii) a polymer; iii) a peptide; iv) a viral-like particle; and v) a cationic nanoemulsion; where the one or more RNA molecules comprises a nucleotide sequence encoding the S52 E2 polypeptide. In some cases, a nucleic acid composition of the present disclosure comprises: a) one or more RNA molecules; and b) one or more of: i) a lipid; ii) a polymer; iii) a peptide; iv) a viral -like particle; and v) a cationic nanoemulsion; where the one or more RNA molecules comprises: i) a 5’ untranslated region (5’ UTR), e.g., a 5’ IRES; and ii) a nucleotide sequence encoding the S52 E2 polypeptide.

[0103] In some cases, the one or more RNA molecules are mRNA. In some cases, the one or more RNA molecules are self-amplifying or self-replicating RNA molecules

[0104] A self-amplifying or self-replicating RNA molecule (also referred to as a “replicon”) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself). A self- replicating RNA molecule is thus in certain cases, a (+) strand molecule that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase (RdRP) which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as co-linear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded immunogen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the immunogen. The overall result of this sequence of transcriptions is a large amplification in the number of the introduced replicon RNAs and so the encoded immunogen becomes a major polypeptide product of the host cells.

[0105] In some cases, the RNA molecules of the present disclosure include a 5’ cap structure. The 5' cap structure of an mRNA is involved in initiation of protein translation, nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0106] RNA molecules (e.g., mRNA molecules) may be 5'-end capped, generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7- methyl -guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated.

[0107] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and / or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'- hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

[0108] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wildtype or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule. For example, the Anti -Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5'- 5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m.sup.7G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA or mmRNA). The N7- and 3'-O-methlyated guanine provides the terminal moiety of the capped RNA molecule.

[0109] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m.sup.7Gm-ppp-G).

[0110] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5'-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0111] An RNA molecule of the present disclosure may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of moreauthentic 5 'cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5 'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N, pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0112] A 5' terminal cap suitable for inclusion in an RNA molecule of the present disclosure can include endogenous caps or cap analogs. A 5' terminal cap suitable for inclusion in an RNA molecule of the present disclosure can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0113] In some cases, the 5’ cap structure present in an RNA molecule of the present disclosure is selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine.5’ UTR

[0114] An RNA molecule of the present disclosure can include a 5’ UTR. Natural 5'UTRs bear features which play roles in for translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G' . 5 'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0115] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides, primary constructs or mRNA of the disclosure. For example, introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of an RNA molecule, in hepatic cell lines or liver. Likewise, use of 5' UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), for leukocytes(CD45, CD 18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A / B / C / D).

[0116] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs. For example, introns or portions of introns sequences may be incorporated into the flanking regions of an RNA molecule of the present disclosure. Incorporation of intronic sequences may increase protein production as well as mRNA levels.3’ UTR

[0117] An RNA molecule of the present disclosure can include a 3 ’ untranslated region (3 ’ UTR). 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well- studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. Examples of suitable 3’ UTRs include those described in USPN 9,827,332.Poly-A tails

[0118] An RNA molecule of the present disclosure can include a poly (adenine) (poly-A) tail. The poly-A tail can be from 30 nucleotides to 300 nucleotides in length.

[0119] A nucleic acid (e.g., a DNA molecule) comprising a nucleotide sequence encoding RNA of the present disclosure can include a polyadenylation signal. Suitable polyadenylation signals are known in the art; and any such polyadenylation signals can be suitable for use. As is known in the art, a polyadenylation signal can include an AATAAA motif.RNA modifications

[0120] An RNA molecule of the present disclosure can include one or more of: a) a modified nucleoside comprising a modified nucleoside base; b) a modified nucleoside comprising a modified sugar; c) a modified intemucleoside linkage; and d) a modified backbone linking two or more nucleosides.

[0121] A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofiiranosyl sugar, the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double -stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the intemucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

[0122] Suitable nucleic acid modifications include, but are not limited to: 2’0-methyl modified nucleotides, 2’ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5’ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.

[0123] A 2'-O-Methyl modified nucleoside (also referred to as 2'-O-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. RNA can be directly synthesized so that it contains one or more 2'-O-Methyl nucleosides. Such an RNA is stable with respect to attack by single-stranded ribonucleases.

[0124] 2’ Fluoro modified nucleosides (e.g., 2' Fluoro bases) have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA.

[0125] LNA bases have a modification to the ribose backbone that locks the base in the C3'- endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an RNA at any position except the 3'-end.

[0126] The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). This modification renders the intemucleoside linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleosides at the 5'- or 3'-end of an RNA molecule to inhibit exonuclease degradation. Including phosphorothioate bonds within an RNA molecule can help reduce attack by endonucleases as well.

[0127] In some cases, a subject nucleic acid has one or more nucleotides that are 2'-O-Methyl modified nucleosides. In some cases, a subject nucleic acid has one or more 2’ Fluoro modified nucleosides. In some cases, a subject nucleic acid has one or more LNA bases. In some cases, a subject nucleic acid has one or more nucleosides that are linked by a phosphorothioate bond (i.e., the subject nucleic acid has one or more phosphorothioate linkages). Adjuvants

[0128] In some cases, a composition of the present disclosure comprises an adjuvant (e.g., an immunostimulating amount of an adjuvant). An immunogenic composition of the present disclosure can include an immune-stimulating amount of an adjuvant. Examples of known suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w / v squalene, 0.5% w / v Tween 80™, 0.5% w / v Span 85), CpG-containing nucleic acid (where the cytosine is unmethylated), QS21, monophosphoryl lipid A (MPL), 3-Q-desacyl- 4'-monophosphoryl lipid A (3DMPL), extracts from Aquilla, immune -stimulating complexes (ISCOMS; complexes of cholesterol, phospholipids, and Quillaja saponins), LT / CT mutants, poly(D,L-lactide-co- glycolide) (PLG) microparticles, Quil A, interleukins, and the like. For experimental animals, one can use Freund's incomplete adjuvant, or Freund’s complete adjuvant. Also suitable for use are N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion. The effectiveness of an adjuvant may be determined by one or more of: i) measuring the amount of antibodies directed against the immunogenic antigen or antigenic epitope thereof; ii) measuring a cytotoxic T lymphocyte response to the antigen; and iii) measuring a helper T cell response to the antigen.

[0129] Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (see, e.g., WO 90 / 14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), e.g.,MPL+CWS (Detox TM); (2) saponin adjuvants, such as QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.; a purified extract of Quillaja saponaria) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO 00 / 07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99 / 44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO 00 / 56358; (6) combinations of 3dMPL with, for example, QS21 and / or oil-in-water emulsions (see, e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231); (7) oligonucleotides comprising a CpG motif containing at least one CG dinucleotide, where the cytosine is unmethylated (see, e.g., WO 96 / 02555, WO 98 / 16247, WO 98 / 18810, WO 98 / 40100, WO 98 / 55495, WO 98 / 37919 and WO 98 / 52581); (8) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g. WO 99 / 52549); (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (WO 01 / 21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01 / 21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO 00 / 62800); (11) an immunostimulant and a particle of metal salt (see, e.g. WO 00 / 23105); (12) a saponin and an oil-in-water emulsion (see e.g. WO 99 / 11241); (13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally including a sterol) (see, e.g. WO 98 / 57659); (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl- normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc. Also suitable for use is Matrix-M™; Matrix-M™ is an adjuvant that comprises 40 nm nanoparticles comprising Quillaja saponins, cholesterol, and phospholipid. Adjuvants suitable for administration to a human are of particular interest. In some cases, the adjuvant is one that enhances a CD4+T helper response to the immunogen. Also suitable for use is a poly inosine :cytosine (poly I: C) nucleic acid. Poly I:C is a synthetic double -stranded RNA Also suitable for use is a cyclic dinucleotide activator of the STING pathway. Examples of suitable cyclic dinucleotide adjuvants include, but are not limited to: 1) bis-(3’,5’)-cyclic dimeric adenosine monophosphate (c-di-AMP); 2) bis-(3’,5’)-cyclic dimeric guanosine monophosphate (c-di-GMP); and bis-(3’,5’)-cyclic dimeric inosine monosphosphate (c-di-IMP). Also suitable for use is poly(LC).

[0130] QS21 has the following structure:

[0131] MPL has the following structure:

[0132] In some instances, the adjuvant is MF59, with or without a CpG-containing oligonucleotide. In other instances, the adjuvant is alum, with or without a CpG-containing oligonucleotide. In other instances, the adjuvant is poly(D,L-lactide-co-glycolide), with or without a CpG-containing oligonucleotide. In other instances, the adjuvant is MPL, with or without a CpG- containing oligonucleotide. In some cases, the adjuvant is Matrix-M™, with or without a CpG- containing oligonucleotide. In some cases, the adjuvant is keyhole limpet hemocyanin. In some cases, the adjuvant is alum. In some cases, the adjuvant is aluminum phosphate. In some cases, the adjuvant is aluminum hydroxide. In some cases, the adjuvant is alum + MPL. In some cases, the adjuvant is MF59. In some cases, the adjuvant is alum + MF59. In some cases, the adjuvant is AS01. AS01 contains QS-21Stimulon® adjuvant, MPL, and liposomes. In some cases, the adjuvant comprises QS21 and MPL in a liposomal formulation. In some cases, the adjuvant is AS03. A dose of AS03 contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In some cases, the adjuvant comprises aluminum hydroxide and MPL. In some cases, the adjuvant is AS04. AS04 comprises aluminum hydroxide and MPL. In some cases, the adjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence 5'- TCGTCGTTTTGTCGTTTTGTCGTT-3'; (SEQ ID NO:54), in a liposomal formulation. In some instances, the adjuvant is a cyclic dinucleotide (CDN). Suitable CDNs are described below.

[0133] In some cases, the adjuvant is selected from the group consisting of an aluminum salt, RIBI, atoll-like receptor agonist, AS01, AS02, AS03, AS04, AS05, a CpG-oligodeoxynucleotide, MF- 59, Montanide ISA-51 VG, Montanide ISA-720, Quil A, QS21, a synthetic saponin, an immunostimulatory complex, stearyl tyrosine, a virus-like particle, a reconstituted influenza virosome, a cytokine, mast cell activator compound 48 / 80, a liposome, a muramyl dipeptide, SAF-1, and combinations thereof. In some cases, the adjuvant is selected from the group consisting of an aluminum salt, alum, PHAD, a CDN, AS01, AS04, a CpG oligodeoxynucleotide, MF59, and combinations of two or more of the foregoing.

[0134] In some cases, the adjuvant is a disaccharide synthetic lipid compound, e.g., as described in U.S. Patent No. 9,518,078. A disaccharide synthetic lipid compound can be a phosphorylated hexaacyl disaccharide (PHAD).Liposomal compositions comprising a saponin (SLA-liposomal compositions)

[0135] In some cases, a composition of the present disclosure comprises SLA or GLA in a liposomal composition comprising a saponin. See, e.g., US 2020 / 0276299. SLA in a liposomal composition comprising a saponin is also referred to herein as “SLA-LSQ”.

[0136] In some cases, the saponin is present in the liposomal composition at a concentration of from about 1 pg per unit dose to about 10 pg per unit dose, or from about 1 pg per dose to about 8 pg per unit dose. In some cases, a unit dose is 0.5 mb In some cases, the weight of ratio of SLA to saponin is about 2.5 to 1. In some cases, a liposomal composition comprising a saponin is complexed to a sterol. In some cases, the weight: weight ratio of saponin to sterol is from about 1 : 110 to about 1 :200, from about 1: 110 to 1: 150, from about 1: 120 to about 1: 150, or about 1: 125. In some cases, the weightweight ratio of saponin to sterol is 1: 125. In some cases, the sterol is cholesterol. In some cases, the saponin is QS21 and the sterol is cholesterol. The saponin can be present in the formulation in a concentration of from about 2 pg per mL to about 8 pg per mL. In some cases, a unit dose of an SLA-liposomal composition comprises from 2 pg to 4 pg saponin. In some cases, a unit dose is 0.5 mL. The SLA can be present in the formulation in a concentration of from about 5 pg per unit dose to about 10 pg per unit dose. In some cases, a unit dose is 0.5 mL.

[0137] In some cases, an SLA or GLA-liposomal composition comprises: i) 20 pg / mL SLA; ii) 8 pg / mL QS-21; iii) cholesterol; and iv) dioleoyl phosphatidylcholine; where the QS21 and the cholesterol are present in a weightweight (w:w) ratio of 1: 125. The dioleoyl phosphatidylcholine and the cholesterol can be present in a 4: 1 w:w ratio.

[0138] The liposomes can have an average diameter (i.e., the number average diameter) of 1 micrometer or less. In some cases, the average particle size (i.e., the number average diameter) of the liposome particles is about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 400 nm or less, 300 nm or less, or 200 nm or less, for example, from about 50 nm to about 900 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 175 nm, from about 50 nm to about 150 nm, from about 50 nm to about 125 nm, from about 50 nm to about 100 nm. The size of the liposomes is about 80 nm, is about 85 nm, is about 90 nm, is about 95 nm, is about 100 nm, is about 105 nm, is about 110 nm, is about 115 nm, is about 120 nm, is about 125 nm, is about 130 nm, is about 135 nm, is about 140 nm, is about 145 nm, is about 150 nm, is about 155 nm, is about 160 nm, is about 165 nm, is about 170 nm, is about 175 nm, is about 180 nm, is about 185 nm, is about 190 nm, is about 195 nm, or is about 200 nm.Phospholipids

[0139] The SLA-liposomal composition can comprise a phospholipid. Suitable phospholipids include l,2-dilauroyl-5«-glycero-3 -phosphocholine (DLPC); l,2-Dimyristoyl-sn-Glycero-3- phosphatidylcholine (DMPC); l,2-Dipalmitoyl-sn-Glycero-3 -phosphatidylcholine (DPPC); 1,2- Distearoyl-sn-Glycero-3-phosphatidylcholine (DSPC); l,2-Dioleoyl-sn-Glycero-3 -phosphatidylcholine (DOPC); 1 -Palmitoyl, 2-oleoyl-sn-Glycero-3 -phosphatidylcholine (POPC); distearoyltrimethylammonium propane (DSTAP); dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP); l,2-Diostearoyl-sn-Glycero-3 -phosphatidylethanolamine (DSPE); 1,2-Dipalmitoyl-sn- Glycero-3-phosphatidylethanolamine (DPPE); l,2-Dimyristoyl-sn-Glycero-3 -phosphatidylethanolamine (DMPE); l,2-Dilauroyl-sn-glycero-3 -phosphorylglycerol) (DLPG); l,2-Dimyristoyl-sn-glycero-3- phosphoglycerol (DMPG); l,2-Dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG); 1,2-Distearoyl-sn- glycero-3 -phosphoglycerol (DSPG); l,2-Dioleoyl-sn-glycero-3 -phosphoglycerol (DOPG); 1,2-dilauroyl- sn-Glycero-3-phosphatidylethanolamine (DLPE); l,2-Dilauroyl-sn-Glycero-3 -phosphatidylserine (DLPS); l,2-dilauroyl-sn-glycero-3-phospho-L-serine; DMPS: l,2-myristoyl-sn-glycero-3-phospho-L- serine; DPPS: l,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, DSPS l,2-distearoyl-sn-glycero-3- phospho-L-serine; DOPS: l,2-dioleoyl-sn-glycero-3-phospho-L-serine; POPS: 1 -palmitoyl -2 -oleoyl-sn- glycero-3-phospho-L-serine; DLPI: l,2-dilauroyl-sn-glycero-3-phospho-(l'-myo-inositol); DMPI: 1,2- myri stoyl-sn-glycero-3-phospho-(l '-myo-inositol); DPPI: l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-myo-inositol); DSPI: l,2-distearoyl-sn-glycero-3-phosphoinositol; DOPI: l,2-dioleoyl-sn-glycero-3- phospho-(l'-myo-inositol); and POPI: l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoinositol. In some cases, the phospholipid is selected from DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DLPG, DMPG, DPPG, DSPG, DOPG, DSTAP, DPTAP, DSPE, DPPE, DMPE, and DLPE.Saponins

[0140] In some cases, the saponin is an immunologically active saponin fraction derived from the bark of Quillaja saponaria Molina. In some cases, the saponin is QS21.

[0141] QS21 has the following structure:

[0142] In some cases, the saponin is a synthetic saponin, such as synthetic QS21 (SQS21), QS21-Api, or QS21-Xyl. See, e.g., Ragupathi et al. (2011) Expert Rev. Vaccines 10:463.Stable emulsions

[0143] As noted above, in some cases, an immunogenic composition of the present disclosure comprises SLA or GLA in a stable oil-in-water emulsion (“SE”). SLA in a stable oil-in-water emulsion is also referred to herein as “SLA-SE”. US 2015 / 0017191.

[0144] In some cases, an SLA-SE is an emulsion comprising: a) an aqueous phase comprising: i) an ammonium phosphate buffer; ii) a detergent (e.g., Pluronic F68 or Tween 80); and iii) glycerol; and b) an oil phase comprising: i) SLA; ii) phosphatidylcholine (PC); and iii) squalene. Miglyol 810 can be used instead of squalene. In some cases, the SLA-SE comprises, as the PC, l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC). In some cases, the SLA-SE does not comprise an antioxidant. In some cases, the SLA-SE does comprise an antioxidant, e.g., where the antioxidant is a tocopherol (vitamin E). In some cases, the detergent is Pluronic F68. SLA-SE or GLA-SE functions as oil / water emulsion (SE) containing an agonist of TLR4 providing immune activation ( SLA or GLA ). Shweta- we need to include the structure of the SLA & GLA TLR4-activating molecules of which the IDRI / AAHI formulation contains a semi-synthetic version of MPL. MPL could also be used with Alum or inoil / water emulsion formulations. This needs some expansion please since our clinical vaccine currently includes SLA-SE adjuvant from AAHI.

[0145] In some cases, the squalene is present in a concentration of from about 0.01% v / v to about 1% v / v. The hydrophobic: lipophilic balance of the emulsion is generally greater than about 9 (e,g., greater than 10, or between 9-12). In some cases, the squalene is present in a concentration of from about 0.01% v / v to 0.5% v / v. In some cases, the emulsion comprises, as the PC, l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC). In some cases, the emulsion comprises, as the PC, DOPC.

[0146] The PC can be egg yolk PC, soy PC, l,2-dioleoyl-sw-glycero-3 -phosphocholine (DOPC), l,2-dipahnitoyl-sw-glycero-3 -phosphocholine (DPPC), l .2-distcaroyl-s77-glyccro-3-phosphocholinc. 1,2- dilinoleoyl-5«-glycero-3 -phosphocholine, or l,2-diarachidonoyl-5«-glycero-3-phosphocholine. In some cases, the PC is DOPC.

[0147] In some cases, an SLA-SE comprises: i) 10% weight / volume squalene; ii) 0.25 mg / mL SLA; iii) 0.5% weight / volume vitamin E; iv) glycerol; and v) PC.EXAMPLES

[0148] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.METHODS & MATERIALS

[0149] Production of E2:

[0150] Amino acid residues used according to the numbering of polyprotein of each HCV strain:

[0151] A) H77 E2 (a.a. 384-688); B) J6 E2 (a.a. 384-693); C) S52 E2 (a.a. 384-695).

[0152] Plasmid encoding E2 was transfected in Expi293T cells. To aid the secretion of the protein, the E2 coding sequence is preceded by tissue plasminogen activator (tPA)-signal peptide. At the carboxyl terminus of E2, there is a cleavage site recognized by human rhinovirus protease 3C (LEVLFQGP; SEQ ID NO: 55) and followed by a human IgGl Fc tag to aid purification. Thesupernatant of transfected cells was collected on day 3 and day 6 post-transfection and used for protein purification. Lysate was incubated with Mab select resin to bind E2 protein. Subsequently, the resin was washed with PBS and treated with protease to release E2. The flow-through fraction was then concentrated with 30 kDa MWCO Amicon filter units. The amount of E2 in the concentrated fraction was determined and the purity of E2 was >95%.

[0153] Immunization protocol:

[0154] E2 antigen (7.5pg) were mixed at a 1: 1 equal volume ratio with SLA-SE. The final antigenic preparation (100 pl) of E2 was administered via intramuscular injection to each guinea pig on days 0, 30 and 90. Pre-vaccination blood samples were collected at day 0, and post-vaccination blood samples (terminal bleeds) were obtained 14 days after the final immunization. After clotting, whole blood samples were centrifuged at 5,000 X g for 15 min, and sera were collected, and heat inactivated at 56°C for 30 min. Serum samples were stored in aliquots at -80°C until use.Example 1: E2 -immunized antisera effectively neutralizes pseudotyped HCV particles (HCVpp) derived with glycoproteins of Gtla. Gt2a (J6) or Gt3a (S52)RESULTS

[0155] FIG. 1. Guinea pigs were immunized with recombinant E2 protein (amino acid 384-688) derived from Gtla (H77), Gt2a (J6), Gt3a (S52) or combination of all three. Antisera after 3 immunizations were tested at 1 :200 dilution for neutralization against pseudotyped HCV particles (HCVpp) derived with glycoproteins of Gtla (H77), Gt2a (J6) or Gt3a (S52). While H77-E2 induced homologous protection against H77-HCVpp, it showed reduced neutralization against J6- and S52- HCVpp. Similarly, J6-E2 induced homologous protection, but showed reduced neutralization against H77-and S52-HCVpp. Surprisingly, antisera from S52 E2 or combined E2 immunized animals showed broad-neutralization against all three HCVpp.

[0156] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method of inducing an immune response in an individual to hepatitis C virus (HCV) Gtl, Gt2 and Gt3, the method comprising: administering to the individual an effective amount of a composition comprising: HCV Gt3 (Gt3) S52 E2 polypeptide, or a nucleic acid encoding the HCV Gt3 S52 E2 polypeptide.

2. The method of claim 1, wherein the immune response comprises production of antibodies that neutralize the infectivity of HCV Gtl a (Gtl a) and / or HCV Gtlb (Gtlb).

3. The method of claim 1 or 2, wherein the immune response comprises production of antibodies that neutralize the infectivity of HCV Gt2a (Gt2a).

4. The method of any one of claims 1-3, wherein the immune response comprises production of antibodies that neutralize the infectivity of HCV Gt3a (Gt3a).

5. The method of any one of claims 1-4, wherein the immune response comprises production of antibodies that neutralize the infectivity of Gtla, Gt2a, and Gt3a.

6. The method of any one of claims 1-5, wherein the immune response comprises production of antibodies that neutralize the infectivity of HCV Gt4a (Gt4a) and / or HCV Gt5a (Gt5a).

7. The method of any one of claims 1-6, wherein the HCV Gtl is Gtla strain H77, 72, 123, 138, UKNP 1.11.6, UKNP 1.9.1, UKNP 1.10.1, and / or UKNP1.16.3 and / or wherein the HCV Gtl is Gtlb strain UKNP 1.18.1, lb25, lb35, or lb58.

8. The method of any one of claims 1-7, wherein the HCV Gt2 is Gt2a strain J6 or UKNP2.4.1.

9. The method of any one of claims 1-8, wherein the HCV Gt3 is Gt3a strain S52 or UKNP3. I .2.

10. The method of any one of claims 1-9, wherein the HCV Gt4 is Gt4a strain UKNP4.2.2 and .

11. The method of any one of claims 1-10, wherein the HCV Gt5 is Gt5a strain UKNP5.2.1.

12. The method of any one of claims 1-11, wherein the composition comprises an adjuvant selected from SLA-SE, MF59, alum, a CpG oligonucleotide, a cyclic dinucleotide, 3’-O- desacyl-4’-monophosphoryl lipid A (MPL), aluminum hydroxide, aluminum phosphate, AS01, AS01B, AS02, AS03, AS04, 3M-052, Alum / 3M-052, GLA lipid, a second-generation lipid adjuvant (SLA or GLA), GLA-SE, GLA-LSQ, SLA-LSQ, and combinations thereof.

13. The method of any one of claims 1-11, wherein the composition comprises the adjuvant SLA-SE.

14. The method of any one of claims 1-13, comprising: a) administering a first dose of the composition; and b) administering a second dose of the composition.

15. The method of claim 14, comprising administering a third dose of the composition.

16. The method of claim 15, wherein the second and third dose are administered over a period of time of about 6 months from the first time.

17. The method of any one of claims 1-16, wherein the administering is after or before administering a composition comprising Gtl E1E2 heterodimeric polypeptide or a nucleic acid encoding the Gtl E1E2 heterodimeric polypeptide, optionally, wherein Gtl E1E2 heterodimeric polypeptide is Gtla E1E2 heterodimeric polypeptide, and further optionally, wherein Gtl E1E2 heterodimeric polypeptide is Gtla H77 E1E2 heterodimeric polypeptide.

18. The method of any one of claims 1-17, wherein the method comprises first administering to the individual a composition comprising Gtl E1E2 or a nucleic acid encoding the Gtl E1E2 heterodimeric polypeptide prior to administering the composition comprising HCV Gt3 S52 E2 polypeptide or a nucleic acid encoding the HCV Gt3 S52 E2 polypeptide.

19. The method of any one of claims 1-17, wherein the method comprises first administering to the individual the composition comprising HCV Gt3 S52 E2 polypeptide or a nucleic acid encoding the HCV Gt3 S52 E2 polypeptide and then administering to the individual a composition comprising Gtl E1E2 heterodimer or a nucleic acid encoding the Gtl E1E2 heterodimer.

20. The method of any one of claims 1-19, further comprising administering to the individual a composition comprising HCV Gt3 E2 polypeptide or a nucleic acid encoding the HCV Gt3 E2 polypeptide, optionally, wherein the HCV Gt3 E2 polypeptide is HCV Gt3 J6 E2 polypeptide.

21. The method of any one of claims 1-20, wherein the composition comprising HCV Gt3 S52 E2 polypeptide or the nucleic acid encoding the HCV Gt3 S52 E2 polypeptide further comprises HCV Gtl E1E2 heterodimer or the nucleic acid encoding the HCV Gtl E1E2 heterodimer, optionally, wherein Gtl E1E2 heterodimeric polypeptide is Gtla E1E2 heterodimer, and further optionally, wherein Gtl E1E2 heterodimer is Gtla H77 E1E2 heterodimer.

22. The method of any one of claims 1-16 or claim 21, wherein the composition comprising HCV Gt3 S52 E2 polypeptide further comprises HCV Gt2 E2 polypeptide, HCV Gtl E2 polypeptide, or HCV Gt2 E2 polypeptide and HCV Gtl E2 polypeptide or the composition comprising the nucleic acid encoding the HCV Gt3 S52 E2 polypeptide further comprises a nucleic acid encoding the HCV Gt2 E2 polypeptide, a nucleic acid encoding the HCV Gtl E2 polypeptide, or one or two nucleic acids encoding HCV Gt2 E2 polypeptide and HCV Gtl E2 polypeptide, optionally, wherein the HCV Gt2 E2 polypeptide is HCV Gt2 J6 E2 polypeptide and HCV Gtl E2 polypeptide is HCV Gtl H77 E2 polypeptide.

23. The method of any one of claims 1-16, wherein the composition comprising HCV Gt3 S52 E2 polypeptide does not include an El polypeptide, an E2 polypeptide, and / or E1E2 heterodimer from HCV Gtl or HCV Gt2 and / or (ii) an El polypeptide, an E2 polypeptide, and / or E1E2 heterodimer from another HCV Gt3; and / or (iii) an El polypeptide and / or E1E2 heterodimer from HCV Gt3a S52 and wherein the composition induces production of antibodies that neutralize the infectivity of the HCV strains S52, J6, and H77.

24. The method of any one of claims 1-16, wherein the composition comprising a nucleic acid encoding the HCV Gt3 S52 E2 polypeptide does not include (i) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and / or a nucleic acid encoding the E1E2 heterodimer from HCV Gtl or Gt2; and / or (ii) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and a nucleic acid encoding E1E2 heterodimer from another HCV Gt3; and / or (iii) a nucleic acid encoding an El polypeptide and E1E2 heterodimer from HCV Gt3a S52.

25. The method of any one of claims 1-16, wherein the composition comprising HCV Gt3 S52 E2 polypeptide or the nucleic acid encoding HCV Gt3 S52 E2 polypeptide does not include another HCV antigen or a nucleic acid encoding another HCV antigen.

26. The method of any one of claims 1-25, wherein the administering is intramuscular administration.

27. The method of any one of claims 1-25, wherein the administering is subcutaneous administration.

28. The method of any one of claims 1-27, wherein the individual has HCV infection.

29. The method of any one of claims 1-27, wherein the individual is at risk of acquiringHCV infection.

30. The method of claim 28 or 29, wherein the HCV infection is infection with one or both of HCV Gtl and Gt3.

31. The method of claim 28 or 29, wherein the HCV infection is infection with one, two, or all three of HCV Gtl, Gt2, and Gt3.

32. The method of any one of claims 1-31, wherein the individual is an individual who injects drugs intravenously.

33. A composition comprising: a HCV Gt3 S52 (Gt3 S52) E2 polypeptide or a nucleic acid encoding the Gt3 S52 E2 polypeptide; and a pharmaceutically acceptable excipient.

34. The composition of claim 33, wherein the pharmaceutically acceptable excipient comprises an adjuvant.

35. The composition of claim 34, wherein the adjuvant is selected from MF59, alum, a CpG oligonucleotide, a cyclic dinucleotide, 3 ’-O-desacyl-4’ -monophosphoryl lipid A (MPL), aluminum hydroxide, aluminum phosphate, AS01, AS02, AS03, AS04, 3M-052, a second- generation lipid adjuvant (SLA), and combinations thereof. MF59, alum, a CpG oligonucleotide, a cyclic dinucleotide, 3 ’-O-desacyl-4’ -monophosphoryl lipid A (MPL), aluminum hydroxide, aluminum phosphate, AS01, ASOlb, AS02, AS03, AS04, 3M-052, Alum / 3M-052, GLA lipid, a second-generation lipid adjuvant (SLA), GLA-SE, SLA-SE, and combinations thereof.

36. The composition of claim 34, wherein the adjuvant is SLA-SE.

37. The composition of any one of claims 33-36, comprising:(i) the Gt3 S52E2 polypeptide and further comprising HCV Gtl (Gtl) E2 or E1E2 heterodimer; or(ii) the nucleic acid encoding the Gt3 S52E2 polypeptide and further comprising a nucleic acid encoding a HCV Gtl (Gtl) E2 or E1E2 heterodimer.

38. The composition of claim 37, wherein the HCV Gtl E1E2 heterodimer is Gtla (Gtla) E1E2 heterodimer.

39. The composition of claim 38, wherein the HCV Gtla E1E2 heterodimer is Gtla H77 E1E2 heterodimer.

40. The composition of any one of claims 33-39, comprising(i) the Gt3 S52E2 polypeptide and further comprising HCV Gt2 (Gt2) E2 polypeptide; or(ii) the nucleic acid encoding the Gt3 S52E2 polypeptide and further comprising a nucleic acid encoding a HCV Gt2 E2 polypeptide;(iii) the Gt3 S52E2 polypeptide, the Gtl E1E2 heterodimer, and Gt2 E2 polypeptide; or(iv) the nucleic acid encoding the Gt3 S52E2 polypeptide, the nucleic acid encoding Gtl E1E2 heterodimer, and the nucleic acid encoding Gt2 E2 polypeptide.

41. The composition of claim 40, wherein the HCV Gt2 E2 polypeptide is HCV Gt2 J6 E2 polypeptide.

42. The composition of any one of claims 33-36, wherein the immunogenic composition does not comprise:(i) an El polypeptide, an E2 polypeptide, and E1E2 heterodimer from HCV Gtl and HCV Gt2 and / or (ii) an El polypeptide, an E2 polypeptide, and E1E2 heterodimer from another HCV Gt2; and / or (iii) an El polypeptide and E1E2 heterodimer from another HCV Gt2a S52; or(i) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and a nucleic acid encoding E1E2 heterodimer from Gtl and Gt2 and / or (ii) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and a nucleic acid encoding E1E2 heterodimer from another HCV Gt2; and / or (iii) a nucleic acid encoding an El polypeptide and a nucleic acid encoding E1E2 heterodimer from another HCV Gt2a S52.

43. The composition of any one of claims 33-36 and 42, wherein the composition comprising HCV Gt3 S52 E2 polypeptide does not include:(i) an El polypeptide, an E2 polypeptide, and E1E2 heterodimer from HCV Gt3; and / or (ii) an El polypeptide, an E2 polypeptide, and E1E2 heterodimer from another HCV Gt2; and / or (iii) an El polypeptide and E1E2 heterodimer from another HCV Gt2a S52; or the composition comprising the nucleic acid encoding the Gt3 S52 E2 polypeptide does not include:(i) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and a nucleic acid encoding E1E2 heterodimer from HCV Gt3; and / or (ii) a nucleic acid encoding an El polypeptide, a nucleic acid encoding an E2 polypeptide, and a nucleic acid encoding E1E2 heterodimer from another HCV Gt2; and / or (iii) a nucleic acid encoding an El polypeptide and a nucleic acid encoding E1E2 heterodimer from another HCV.

44. The composition of any one of claims 33-43, wherein the composition does not include another HCV antigen or wherein the composition does not include a nucleic acid encoding another HCV antigen.

45. A container comprising the composition of any one of claims 33-44.

46. The container of claim 45, wherein the container and the composition are sterile.

47. The container of claim 45 or claim 46, wherein the container is a syringe.

48. Use of the composition of any one of claims 33-44 or the container of any one of claims 41-43 in a method for inducing an immune response to HCV Gtl, Gt2, and Gt3 in an individual.

49. The use of claim 48, wherein the individual has an HCV infection or is at risk of acquiring an HCV infection.

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