Recombinant hepatitis c virus glycoproteins, composition and application(s) thereof

Abstract

The present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of E1 and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the E1 and E2 glycoproteins partially or completely lack their respective transmembrane domain(s). Further provided herein are nucleic acid(s) encoding the said recombinant Hepatitis C Virus glycoprotein, nanoparticle conjugate(s) comprising the said recombinant Hepatitis C Virus glycoprotein and compositions comprising the recombinant Hepatitis C Virus glycoprotein, the said nucleic acid(s) or the nanoparticle conjugate(s). Also envisaged herein are applications of the recombinant Hepatitis C Virus glycoprotein or the composition comprising the same.

Description

[0001] “RECOMBINANT HEPATITIS C VIRUS GLYCOPROTEINS, COMPOSITION AND APPLICATION(S) THEREOF”

[0002] TECHNICAL FIELD

[0003] The present disclosure relates generally to recombinant Hepatitis C Virus (HCV) glycoproteins and compositions, products and applications related thereto. More particularly, the present disclosure describes the structural and functional features of the recombinant Hepatitis C Virus (HepC) glycoprotein, its protein sequences and composition(s) derived therefrom, for example, in the form of vaccines against HepC.

[0004] BACKGROUND

[0005] Hepatitis C virus (HCV) continues to be a global health burden with an estimated 58 million people living with chronic HCV infection (“WHO” n.d.). Despite the high efficacy of direct- acting antivirals (DAAs), with cure rates up to 95%, several factors hamper HCV eradication programs. First, most people are unaware of their infection status, because the infection causes little or no symptoms initially. Second, DAA treatment does not protect against reinfection and, thirdly, most low-income countries have only restricted access to DAAs. Therefore, HCV causes around 1.5 million new infections and -290,000 deaths each year worldwide. A vaccine that effectively prevents HCV (re)infection is therefore a critical requirement to overcome these factors and is still a major unmet need.

[0006] HCV is an enveloped positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Its genome encodes for nonstructural proteins (NS), responsible for mediating viral processing inside the host cell, as well El and E2, its envelope glycoproteins that form heterodimers and are involved in viral entry. E2 is the receptor binding-subunit and interacts with scavenger-receptor class 1 B (SR-B1) and tetraspanin CD81 on the host cell to mediate cell entry. The precise mechanism on how E1E2 interacts with the host cell membrane and causes fusion and subsequent viral entry remains unknown. E1E2 probably undergoes several conformational changes after E2 binds to its receptors (first SR-B1, then CD81) and El probably facilitates fusion by engaging its putative fusion peptide.

[0007] E1E2 is the only target for neutralizing antibodies (NAbs) and therefore crucial for the generation of an effective HCV vaccine. However, E1E2 displays extraordinarily high genetic diversity, it is heavily glycosylated and it is an intrinsically flexible complex. Developing a vaccine that can overcome these obstacles is a major challenge. The recent elucidation of the structures of the E1E2 heterodimer in its membrane-bound and as a soluble protein provides opportunities to do so. Most, if not all, viral glycoproteins exhibit different conformational states following binding to the putative host cell receptor and membrane fusion. The prefusion state of a viral glycoprotein, i.e. its conformation before membrane fusion and receptor engagement, is most relevant for a vaccine because it most optimally presents the epitopes that can be targeted by neutralizing antibodies. As a result, viral vaccines that contain prefusion viral glycoprotein immunogens more effectively elicit neutralizing antibodies compared to vaccines that contain post fusion or non-native forms of those antigens. Similarly, developing immunogens that represent the presumed prefusion state of E1E2 is crucial for an effective HCV vaccine. Versions of E1E2 described in the art have been found to have a tendency to induce strong antibody responses that might be autoreactive in humans, indicating that sE1E2.LZ is probably not suitable for vaccination in humans. Thus, next-generation designs are necessary to improve the immunogenicity of recombinant E1E2 antigens. BRIEF DESCRIPTION OF THE FIGURES In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where: FIGURE 1 depicts A) the design of HCV glycoproteins sE1E2.v1- sE1E2.v4 of the present disclosure, B) binding of HCV glycoproteins sE1E2.v1-sE1E2.v4 to antibodies AR4A, AT1618, AT1209, AP33 and CBH-4B. FIGURE 2 depicts A) the design of HCV glycoproteins sE1E2.v4- sE1E2.v6 of the present disclosure, B) antigenicity of HCV glycoproteins sE1E2.v4- sE1E2.v6. FIGURE 3 depicts interaction of HepCon sE1E2.v3 with several inferred germline broadly neutralizing antibodies (bNAbs). FIGURE 4 depicts the sE1E2.v1- sE1E2.v4 design template. FIGURE 5 depicts comparison of antigenicity of sE1E2.v1 and sE1E2.v2 by ELISA. FIGURE 6 depicts results of reducing and non-reducing SDS confirming the presence of an interprotomer disulfide bond in SE1E2.V4.

[0008] FIGURE 7 depicts results of a correlation between ELISA based antigenicity measurement of the AMS0232 SE1E2.V1-SE1E2.V4 versus neutralization of the AMS0232 HCV pseudoparticle using an extended panel of 26 HCV mAbs against known neutralizing and non-neutralizing epitopes.

[0009] FIGURE 8 depicts conformational ElE2-dependent binding of SE1E2.V1-SE1E2.V4 to bNAbs AR4A, AT1618, HEPC111 and E2-dependent conformational antibodies AR3C, AT1209, HEPC108 and AP33 as loading control.

[0010] FIGURE 9 depicts results of thermostability ELISA assays using sElE2.vl- SE1E2.V4.

[0011] FIGURE 10 depicts binding of HCV glycoproteins SE1E2.V1-SE1E2.V4 based on the UKNP4.1.1 strain to antibodies AR4A, AT1618, AP33.

[0012] FIGURE 11 depicts design of HCV glycoproteins sElE2.v5-I53-50NP and SE1E2.V5-TMD (SAR-CoV2) of the present disclosure.

[0013] FIGURE 12 depicts a heatmap representing the EC50 when versions 2 to 4 designs were applied to strains H77 (genotype la), AMS0230 (genotype la), AMS3a (genotype 3a) and UKNP4.1.1 (genotype 4a) and tested against a reduced panel of 17 bNAbs and CD81-Fc.

[0014] FIGURE 13 depicts the EC50 when versions 1 to 4 designs were applied to strains H77 (genotype la), AMS0230 (genotype la), AMS3a (genotype 3a) and UKNP4.1.1 (genotype 4a) and tested against AR4A.

[0015] FIGURE 14 depicts EC50 values for SE1E2.V2-V4-SZ vs. sElE2.v2-v4-Jun / Fos (strain AMS0232) to compare the effect of the heterodimerization domain.

[0016] FIGURE 15 depicts the effect of removal of the dimerization domains on EC50 values obtained for vl-v5 designs, wherein the antibodies tested include those targeting AR4 and AR3.

[0017] FIGURE 16 depicts images of reducing and non-reducing SDS gels that confirm formation of the disulfide bond and the protein.

[0018] FIGURE 17 depicts results of thermostability ELISA using SE1E2.V5 with the sequence of AMS0232. FIGURE 18 depicts size exclusion chromatography (SEC) profiles of AMS0232 ElE2.v5- I53-50NP, Mosaic ElE2.v5-I53-50NP displaying AMS0232 ElE2.v5-I53-50A, H77 ElE2.v5- I53-50A, UKNP4.1.1 ElE2.v5-I53-50A and AMS3a ElE2.v5-I53-50A simultaneously and HepCon ElE2.v5-I53-50NP on a Superose 6 increase 10 / 300 column.

[0019] FIGURE 19 depicts results of ELISA based antigenicity measurements of AMS0232 SE1E2.V5-I53-50NP, Mosaic ElE2.v5-I53-50NP displaying AMS0232 SE1E2.V5-I53-50A, H77 SE1E2.V5-I53-50A, UKNP4.1.1 SE1E2.V5-I53-50A and AMS3a SE1E2.V5-I53-50A simultaneously and HepCon ElE2.v5-I53-50NP against several (conformational) bNAbs.

[0020] FIGURE 20 depicts results of ELISA based antigenicity measurement of UKNP4.1.1 E2E1- I53-50A, UKNP4.1.1 SE1E2.V3, HepCon SE1E2.V3-V5, HepCon sElE2.v5-I53-50A and HepCon ElE2.v5-I53-50NP in a heatmap depicting half maximum effective concentration (EC50) values against 17 bNAbs, CD81-Fc and AP33. Additionally, results are shown of biolayer interferometry (BLI) analyses of HepCon SE1E2.V5 and HepCon sElE2.v5-I53-50NP in a bar graph depicting AUC-values.

[0021] FIGURE 21 depicts results of ELISA based antigenicity measurement of UKNP4.1.1 E2E1- I53-50A, UKNP4.1.1 SE1E2.V3, HepCon sElE2.v3-v5, HepCon sElE2.v5-I53-50A and HepCon ElE2.v5-I53-50NP in a heatmap depicting half maximum effective concentration (EC50) values against 17 gl-bNAbs. Additionally, results are shown of BLI analyses of HepCon SE1E2.V5 and HepCon sElE2.v5-I53-50NP in a bar graph depicting AUC-values.

[0022] FIGURE 22 depicts (A) schedule of immunization study (B) results of ELISA indicating high immunogenicity from the heterodimerization domains.

[0023] FIGURE 23 depicts (A) schedule of immunization study (B) the achieved heterologous neutralization (left) and the heterologous and autologous neutralization (right) of the HepCon group and the Mosaic group at week 39.

[0024] FIGURE 24 depicts flow cytometry analysis of AR4A binding to native AMS0232 E1E2 and AMS0232 sElE2.v5 fused to the SARS-CoV-2 transmembrane domain (TMD) expressed on mammalian 293T cells. SUMMARY OF THE INVENTION

[0025] Addressing the aforesaid need in the art, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0026] In some embodiments, the furin cleavage site replaces a natural protease site between the El and E2 glycoproteins.

[0027] In an exemplary embodiment, the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0028] In some embodiments, the transmembrane domain(s) of El and E2 are replaced by leucine zipper(s).

[0029] In some embodiments, the leucine zipper(s) is human-derived Jun / Fos leucine zippers, wherein Fos and Jun chains of the Jun / Fos leucine zipper(s) partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins.

[0030] In some embodiments, the recombinant Hepatitis C Virus glycoprotein further comprises at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3 (Kuiken et al. 2006, Hepatology 44(5) :p 1355-1361, November 2006. | DOI: 10.1002 / hep.21377).

[0031] In some embodiments, the present disclosure provides an antigenic cocktail comprising the recombinant Hepatitis C Virus glycoprotein as described above, based on at least two HCV strain(s) that demonstrate at least about 60% homology between their E1 / E2 sequences.

[0032] Further provided in the present disclosure is a nucleic acid encoding the recombinant Hepatitis C Virus glycoprotein as described above. In a preferred embodiment, the nucleic acid is mRNA.

[0033] The present disclosure further provides nanoparticle and membrane-anchored conjugates comprising the recombinant Hepatitis C Virus glycoprotein(s) or the nucleic acid encoding the said glycoprotein(s) as described in any of the above embodiments. Also provided in the present disclosure is a vector comprising the nucleic acid as described above.

[0034] The present disclosure further provides an isolated or recombinant eukaryotic or prokaryotic host cell comprising the vector as described above.

[0035] Further envisaged herein is a pharmaceutical composition comprising the recombinant Hepatitis C Virus glycoprotein or the antigenic cocktail as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

[0036] Also provided in the present disclosure is a recombinant Hepatitis C Virus glycoprotein as described above, for use in the treatment or prophylaxis of HCV infection.

[0037] DETAILED DESCRIPTION

[0038] Addressing the aforesaid need pertaining to improved designs for HCV E1 / E2 antigens, the present disclosure provides a recombinant HCV glycoprotein design that may be suitably adapted to any HCV strain of interest. Said recombinant HCV glycoprotein of the present disclosure lacks E1ZE2 dimerization domains from different HCV strains and genotypes and displays enhanced antigenicity, thermostability and immunogenicity as compared to previous designs. However, before describing the recombinant HCV glycoprotein of the present disclosure in further detail, definitions of some terms / phrases used throughout the present disclosure are provided below for purposes of clarity.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. A number of terms are defined and used throughout the specification with the following definitions provided for convenience.

[0040] Definitions

[0041] “HCV” is an abbreviation representing Hepatitis C virus which is a major cause of liver disease and cancer. HCV comprises a core protein and envelope glycoproteins, El and E2. El and E2 form a heterodimer that facilitates infection. The only target for neutralizing antibodies against HCV is the E1 / E2 heterodimeric glycoprotein on the virion surface. “Recombinant Hepatitis C Virus glycoprotein” in the context of the present disclosure refers to a Hepatitis C Virus glycoprotein composed of the E1 / E2 glycoproteins engineered to comprise the structural features as defined in the below embodiments and appended claims

[0042] The terms “sequence homology” and “sequence identity” have been used interchangeably in the context of the present disclosure to depict the sequence similarity as observed from an alignment of two or more sequences.

[0043] Reference to “features” or “structural features” of the Hepatitis C Virus glycoprotein encompasses the different structural components of the recombinant Hepatitis C Virus glycoprotein as defined in the below embodiments and appended claims.

[0044] “Transmembrane Domain” or the abbreviation “TMD” refers to the transmembrane domains (TMDs) of (virus-derived) protein domains that facilitate anchoring of the El and E2 ectodomains in the membrane.

[0045] “R6” is the abbreviation used to refer to the furin cleavage site RRRRRR in the recombinant Hepatitis C Virus glycoprotein of the present disclosure.

[0046] “3 xGGS” is the abbreviation used to refer to the Glycine-Serine hydrophilic amino acid linker GGSGGSGGS in the recombinant Hepatitis C Virus glycoprotein of the present disclosure. Due to native sequence and to minimize mutations of the sequences of AMS3a and UKNP4.1.1, the linker is SGGSGGSGG. Similar are the “2xGGS” and “5xGGS”.

[0047] The term “mutation” as used herein refers to a residue in SEQ ID NO:1 (unless otherwise indicated) which is replaced by another residue. For instance, P244C means that a proline on position 244 of SEQ ID NO: 1 is replaced by a cysteine. In other Hepatitis C Virus glycoproteins there may be another residue on the same position, but in that case said another residue is replaced by a cysteine.

[0048] Terms such as “E1 / E2 heterodimer”, “E1 / E2” or “E1E2” as used interchangeably in the present disclosure encompass a heterodimer formed between the ectodomains of the El and E2 glycoproteins of HCV, wherein said heterodimer has been engineered to comprise the structural features as defined in the below embodiments and appended claims.

[0049] “I53-50A” refers to component A of the two-component protein nanoparticle 153-50 (Bale, J. 2016). “Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an HCV glycoprotein, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In the context of the present disclosure, the HCV E1E2 amino acids are numbered according to the reference H77 sequence having SEQ ID No. 3 (GenBank AF009606) (Kuiken, C., Simmonds, P. (2006).

[0050] As used herein, reference to a “composition” or an “immunogenic composition” envisages a composition comprising the recombinant HCV glycoprotein of the present disclosure, as per any embodiment of the description, wherein said composition may comprise further components such as but not limited to supplementary proteins, DNA sequences, excipients, vehicles, adjuvants and carriers which enhance or boost the effect of the HCV glycoprotein.

[0051] As used herein, the term “vaccine” refers to a composition or a formulation which comprises one or more recombinant HCV glycoproteins of the present disclosure, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and / or ameliorate an infection and / or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of the recombinant HCV glycoprotein or other vaccines. Vaccines can be used conveniently to prevent, ameliorate, or otherwise treat an infection. Upon introduction into a subject, the vaccine induces an immune response including, but not limited to, the production of antibodies and / or cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses.

[0052] As used herein the terms "effective amount” or “immunologically effective amounts” generally refer to that amount of the recombinant HCV glycoproteins of the one or more embodiments of the present disclosure sufficient to induce immunity, to prevent and / or ameliorate an infection or to reduce at least one symptom of an infection and / or to enhance the efficacy of another dose of the recombinant HCV glycoproteins. An effective dose may refer to the amount of the recombinant HCV glycoproteins sufficient to delay or minimize the onset of an infection. An effective dose may also refer to the amount of the recombinant HCV glycoproteins that provides a therapeutic benefit in the treatment or management of an infection. The effective amount may vary depending upon the manner of administration, the age, body weight, and general health of the subj ect.

[0053] A "vector" refers to any nucleic acid vector known in the art. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors and bacteriophage vectors. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as animal papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTC or MoMLV) , Semliki Forest virus or SV40 virus. However, the vectors employable in the present disclosure are not limited to the said examples and may include other plasmids known to those skilled in the art.

[0054] "Host cells" include, but are not limited to, prokaryotic cells, e.g., bacterial cells (including gram-positive cells), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to HEK293, HEK293F, HeLa cells, COS cells, CV1 cells and various primary mammalian cells. Numerous mammalian cells can be used as hosts, including, but not limited to, mouse embryonic fibroblast NIH-3T3 cells, CHO cells, HeLa cells, L(tk-) cells, PER.C6 and COS cells. Mammalian cells can be transfected by methods well known in the art, such as calcium phosphate precipitation, electroporation and microinjection. Electroporation can also be performed in vivo.

[0055] As used herein, the term "subject" is a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, livestock, athletic animals, pets and the like.

[0056] As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

[0057] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

[0058] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. For example, the term “inserted at a position” as used herein in reference to a polypeptide sequence refers to insertion at one or more (such as one, two, three, etc.) amino acid positions in the polypeptide sequence. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0059] Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0060] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / -5% or less, + / -!% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0061] As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps

[0062] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I;

[0063] B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H;

[0064] C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0065] Disclosure

[0066] Recombinant Hepatitis C Virus glycoprotein

[0067] The present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0068] Before describing the recombinant Hepatitis C Virus glycoprotein of the present disclosure in further detail, it is clarified that the order of the different features of the Hepatitis C Virus glycoprotein as described in the above and subsequent embodiments is not restrictive. The present disclosure envisages a Hepatitis C Virus glycoprotein comprising the features as described herein in any order. A Hepatitis C Virus glycoprotein shall satisfy the requirements of the present disclosure as long as the features described in the embodiments are present, irrespective of their order.

[0069] Furthermore, reference to “at least 60% homology to SEQ ID No. 1” in the above embodiment and embodiments that follow envisages a recombinant Hepatitis C Virus glycoprotein of the present disclosure bearing a homology of 60% or more, 70% or more, 80% or more, 90% or more with SEQ ID No. 1.

[0070] In a preferred embodiment, the recombinant Hepatitis C Virus glycoprotein of the present invention has at least 60%, more preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with SEQ ID No. 1.

[0071] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure bears about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, about 60%, about 61%, about 62%, about 63%, about 64%, about 64.59%, about 65%, about 65.32%, about 65.50%, about 65.54%, about 65.91%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 72.41%, about 72.78%, about 73%, about 73.15%, about 73.42%, about 73.70%, about 73.80%, about 74%, about 74.8%, about 75%, about 75.28%, about 75.37%, about 75.74%, about 76%, about 76.10%, about 76.48%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 85.53%, about 85.90%, about 86%, about 86.64%, about 87%, about 87.02%, about 88%, about 89%, about 90%, about 91% about 92% about 93% about 94% about 95% about 96%, about 97% about 98% about 99% or about 100% homology to SEQ ID No. 1.

[0072] In some embodiments, the definition of the recombinant Hepatitis C Virus glycoprotein by its sequence homology to SEQ ID No. 1 is also extrapolatable to any of SEQ ID Nos. 30 -33, 45 and / or 46. Accordingly, the reference to % homology to SEQ ID No. 1 in the above embodiment and embodiments that follow encompass recombinant Hepatitis C Virus glycoproteins bearing the same sequence identity to any of SEQ ID Nos. 30-33, 45 and / or 46. Thus, in some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure bears about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% homology to any of SEQ ID Nos. 30-33, 45 and / or 46 .

[0073] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure bears about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, about 60%, about 61%, about 62%, about 63%, about 64%, about 64.59%, about 65%, about 65.32%, about 65.50%, about 65.54%, about 65.91%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 72.41%, about 72.78%, about 73%, about 73.15%, about 73.42%, about 73.70%, about 73.80%, about 74%, about 74.8%, about 75%, about 75.28%, about 75.37%, about 75.74%, about 76%, about 76.10%, about 76.48%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 85.53%, about 85.90%, about 86%, about 86.64%, about 87%, about 87.02%, about 88%, about 89%, about 90%, about 91% about 92% about 93% about 94% about 95% about 96%, about 97% about 98% about 99% or about 100% homology to any of SEQ ID Nos. 30-33, 45 and / or 46.

[0074] Embodiments envisaging the definition of the recombinant Hepatitis C Virus glycoprotein by % identity to any of SEQ ID Nos. 30-33, 45 and / or 46 are not re-iterated for reasons of brevity.

[0075] Further defining the recombinant Hepatitis C Virus glycoprotein as described above, in an exemplary embodiment, the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0076] Accordingly, in some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0077] In some embodiments, the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins of the recombinant Hepatitis C Virus glycoprotein. Without intending to be limited by theory, the furin cleavage site facilitates native-like E1 / E2 assembly.

[0078] In some embodiments, the furin cleavage site comprises a polyarginine sequence.

[0079] In an exemplary embodiment, the furin cleavage site comprises the sequence RRRKRR (i.e.R6).

[0080] In some embodiments, envisaged herein is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site that replaces the natural protease site between the El and E2 glycoproteins and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0081] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site RRRRRR that replaces the natural protease site between the El and E2 glycoproteins and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0082] As defined herein the term “hydrophilic amino acid linker” as used herein refers to a polypeptide sequence comprising one or more hydrophilic amino acids, selected from polar uncharged residues and charged residues, wherein the amino acids are selected from the group consisting of Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gin), Aspartic Acid (Asp), Glutamic Acid (Glu), Lysine (Lys), Arginine (Arg), and Histidine (His). The hydrophilic amino acid linker is designed to provide solubility in aqueous environments, flexibility between protein domains, reduce steric hindrance, facilitating the proper folding, stability, and / or activity of recombinant fusion proteins. In a preferred embodiment, the length of said hydrophilic amino acid linker is between 2 and 20 amino acids. Preferably, said hydrophilic amino acid linker does not contain any hydrophobic amino acid. In a preferred embodiment, said hydrophilic amino acid linker is a GS linker. The length can be optimized by adjusting the repetition of Gly-Ser units (e.g., (GGGGS)n or (GGS) / / to achieve appropriate separation of functional domains.

[0083] In some embodiments, the hydrophilic amino acid linker has a length of about 6 to about 15 amino acids.

[0084] In some embodiments, the hydrophilic amino acid linker has a length of about 6, about 7, about 8, about 10, about 11, about 12, about 13, about 14 or about 15 amino acids.

[0085] In some embodiments, the hydrophilic amino acid linker has a length of about 7 to about 12 amino acids. In some embodiments, the hydrophilic amino acid linker is a Glycine-Serine sequence.

[0086] In some embodiments, the hydrophilic amino acid linker comprises at least one Glycine residue and at least one Serine residue.

[0087] In some embodiments, the hydrophilic amino acid linker comprises at least two Glycine residues and at least one Serine residue.

[0088] In some embodiments, the hydrophilic amino acid linker comprises about 2 to 10 Glycine residues and about 1-5 Serine residues. In some embodiments, the hydrophilic amino acid linker is GGSGGS (i.e.2*GGS), GGSGGSGGS (i.e. 3*GGS), GGSGGSGGSGGSGGS (i.e. 5*GGS) or SGGSGGSGG.

[0089] In some embodiments, the hydrophilic amino acid linker is GGSGGS (i.e.2*GGS), GGSGGSGGS (i.e. 3*GGS) or GGSGGSGGSGGSGGS (i.e. 5*GGS)

[0090] In an exemplary embodiment, the hydrophilic amino acid linker comprises the sequence GGSGGSGGS (i.e. 3xGGS)

[0091] In some embodiments, the hydrophilic amino acid linker contains additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.

[0092] In some embodiments, the hydrophilic amino acid linker replaces about 34 amino acids of the putative fusion peptide (pFP) in the El glycoprotein, wherein the pFP represents a transmembrane region of the El glycoprotein.

[0093] In some embodiments, the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3. The removal of the hydrophobic pFP in improves folding of the E1 / E2 heterodimer and decreases aggregation of the recombinant Hepatitis C Virus glycoprotein.

[0094] Thus, in some embodiments, the incorporation of the hydrophilic amino acid linker in the recombinant Hepatitis C Virus glycoprotein such that it replaces a hydrophobic portion of the glycoprotein improves antigenicity of the recombinant Hepatitis C Virus glycoprotein. In some embodiments, the said modification also renders the glycoprotein non-infectious while ensuring that proper antigenic folding is retained.

[0095] Accordingly, in some embodiments, envisaged herein is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s). Said design of the recombinant Hepatitis C Virus glycoprotein, in a non-limiting embodiment, has improved antigenicity as compared to a glycoprotein lacking the hydrophilic amino acid linker. In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker comprising the sequence GGSGGSGGS; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; the hydrophilic amino acid linker replaces residues 261- 294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0096] In some embodiments, the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s).

[0097] In some embodiments, the leucine zipper(s) is selected from a group comprising human- derived Jun / Fos leucine zippers and SYNZIP leucine zippers.

[0098] In an exemplary embodiment, the leucine zipper is a human-derived Jun / Fos leucine zipper.

[0099] Jun / Fos leucine zippers are composed of Fos and Jun chains. In some embodiments, the Fos and the Jun chains of the Jun / Fos leucine zipper partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins.

[0100] In some embodiments, the Jun chain partially or completely replaces the transmembrane membrane domain of the El glycoprotein and the Fos chain partially or completely replaces the transmembrane membrane domain of the E2 glycoprotein.

[0101] In some embodiments, the Fos chain partially or completely replaces the transmembrane membrane domain of the El glycoprotein and the Jun chain partially or completely replaces the transmembrane membrane domain of the E2 glycoprotein.

[0102] In an exemplary embodiment, the Jun chain completely replaces the transmembrane membrane domain of the El glycoprotein and the Fos chain completely replaces the transmembrane membrane domain of the E2 glycoprotein.

[0103] Accordingly, in some embodiments, envisaged herein is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s). In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by human-derived Jun / Fos leucine zippers.

[0104] In an exemplary embodiment, the leucine zipper is a SYNZIP leucine zipper. These are synthetic computationally designed leucine zippers that allow for the heterodimerization of molecules.

[0105] Accordingly, in some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by SYNZIP leucine zipper.

[0106] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and human-derived Jun / Fos leucine zippers; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the human-derived Jun / Fos leucine zippers partially or completely replaces transmembrane membrane domains of the El and E2 glycoproteins.

[0107] In some embodiments, the recombinant glycoproteins of the present invention may be further stabilized by introducing mutations. In some embodiments, the mutations may be introduced in the direct vicinity of antibody epitopes, with the aim to improve stability and folding.

[0108] In some embodiments, further envisaged in the present disclosure is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins, comprising at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3. In some embodiments, the said recombinant Hepatitis C Virus glycoprotein bears at least 60% homology to SEQ ID No. 1. In some embodiments, the said recombinant Hepatitis C Virus glycoprotein comprises mutations P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

[0109] In some embodiments, the recombinant Hepatitis C Virus glycoprotein as described in any of the earlier embodiments is designed to further comprise at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

[0110] Without intending to be limited by theory, in some embodiments, the said mutations increase the number of target epitopes the recombinant Hepatitis C Virus glycoprotein, thus enhancing the binding of antibodies.

[0111] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure comprises at least one mutation selected from a group comprising W326P, V622A, L682P, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3. The proline substitutions are located near or within predicted heptad repeat regions of El and E2 and it is hypothesized that these substitutions prevent conformational changes in the El and E2 heptad repeats and thereby the formation of a postfusion state.

[0112] Accordingly, in some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0113] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0114] In some embodiments, envisaged herein is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s).

[0115] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, human-derived Jun / Fos leucine zippers and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered forin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the human-derived Jun / Fos leucine zippers partially or completely replaces transmembrane membrane domains of the El and E2 glycoproteins.

[0116] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, SYNZIP leucine zippers and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered forin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the SYNZIP leucine zippers partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins. In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure comprises mutations W326P, V622A and L682P, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3

[0117] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure further comprises mutation(s) selected from a group comprising P244C, I340C, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

[0118] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure comprises mutations selected from a group comprising P244C / T687Cand / or I340C / W716C, wherein said mutations result in one or more engineered disulphide bonds, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3. Without intending to be limited by theory, in some embodiments, the design variants of the Hepatitis C Virus glycoprotein of the present disclosure comprising such engineered disulphide bond(s) shows improved antigenicity profile as well as thermostability as compared to designs lacking such engineered disulphide bond(s).

[0119] Accordingly, in some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0120] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0121] In some embodiments, envisaged herein is a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s).

[0122] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, human-derived Jun / Fos leucine zippers and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the human-derived lun / Fos leucine zippers partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins.

[0123] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, SYNZIP leucine zippers and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261- 294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the SYNZIP leucine zippers partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins. In some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein as per any of the above embodiments, completely lacking the transmembrane domain(s) of the El and E2 glycoproteins.

[0124] Accordingly, in some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0125] In some embodiments, the present disclosure provides a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0126] In some embodiments, the recombinant Hepatitis C Virus glycoprotein comprises a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0127] Without wishing to be limited by theory, in some embodiments, said design variants of the recombinant Hepatitis C Virus glycoprotein of the present disclosure that completely lack the transmembrane domain(s) of the El and E2 glycoproteins are antigenically thermostable i.e. the binding capacity is not influenced by protein degradation at higher temperatures. In some embodiments, the recombinant Hepatitis C Virus glycoprotein may further comprise a signal sequence and / or purification tag to enable efficient expression and purification of the recombinant Hepatitis C Virus glycoprotein.

[0128] In some embodiments, the glycoprotein design of the present disclosure may be adapted suitably to the EIZE2 heterodimer of any HCV strain.

[0129] In some embodiments, the recombinant Hepatitis C Virus glycoprotein may be based on HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a and UKNP4.1.1 or HCV consensus sequence.

[0130] Table 1 below depicts the percentage identity matrix obtained from Clustal Omega (Madeira et al., 2024) after aligning the full length E1E2 sequences for HepCon (HCV consensus),

[0131] AMS3a (Genbank ID: KR094964), AMS0230 (Genbank ID: OL855835), AMS0232 (Genbank ID: OL855837), UKNP4.1.1 (Genbank ID: KU285220) and H77 (Genbank ID: AF009606).

[0132] Table 1;

[0133]

[0134] The percentage identity matrix indicates the similarity in sequence identity between the HepCon reference strain and different strains for which the version 1-5 (vl-5 - see Figures 1A and 2A) stabilizing mutations have been successfully implemented. The sequence identity varies from 89.19% to as low as 76.26%, indicating that the stabilizing designs could benefit a range of diverse E1E2 sequences to at least as low as a 76.26% sequence identity compared to the HepCon E1E2 sequence. The lowest percentage identity observed is between the strains UKNP4.1.1 and AMS3a which share a sequence identity of 68.47%. This indicates that for the stabilizing designs to be successfully implemented strains could potentially share a sequence identity of as little as at least 68.47%. While the above embodiments describe the different variants of the glycoprotein of the present disclosure, the recombinant Hepatitis C Virus glycoprotein of the present disclosure as per some exemplary embodiments are represented in the below table by way of reference to their sequence identifier as used in the working examples, combination of characterizing features and their respective SEQ ID Nos. Table 2: Accordingly, in some embodiments, envisaged herein are recombinant Hepatitis C Virus glycoproteins having sequence bearing at least about 60% homology to sequence(s) selected from a group comprising sequences represented by SEQ ID Nos. 8-11, 14-17, 20-23, 26-32.

[0135] In some embodiments, the recombinant Hepatitis C Virus glycoprotein of the present disclosure has sequence selected from a group comprising sequences represented by SEQ ID Nos. 8-11, 14-17, 20-23, 26-32.

[0136] The present disclosure further provides an antigenic cocktail comprising the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least two HCV strain(s) that demonstrate at least about 60% homology between their E1 / E2 sequences.

[0137] In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least two HCV strain(s) that demonstrate at least about 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology between their E1 / E2 sequences.

[0138] In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least two HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a, UKNP4.1.1 and / or the HCV consensus sequence. The antigenic cocktail can induce cross-neutralizing responses against HCV. In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least three HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a, UKNP4.1.1 and / or the HCV consensus sequence.

[0139] In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least four HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a, UKNP4.1.1 and / or the HCV consensus sequence.

[0140] In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on at least five HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a, UKNP4.1.1 and / or the HCV consensus sequence.

[0141] In some embodiments, the antigenic cocktail comprises the recombinant Hepatitis C Virus glycoprotein as described in any of the above embodiments, based on HCV strain(s) H77, AMS0232, AMS0230, AMS3a, UKNP4.1.1 and / or the HCV consensus sequence.

[0142] In some embodiments, the antigenic cocktail comprises two or more recombinant Hepatitis C Virus glycoproteins selected from a group comprising sequences represented by SEQ ID Nos. 8-11, 14-17, 20-23, 26-32. mRNA encoding the recombinant Hepatitis C Virus glycoprotein of the present disclosure

[0143] The present disclosure further provides a nucleic acid encoding the recombinant Hepatitis C Virus glycoprotein as described above.

[0144] In some embodiments, the nucleic acid is selected from mRNA and DNA.

[0145] In some embodiments, the nucleic acid is mRNA.

[0146] In a preferred embodiment, the present disclosure provides mRNA encoding the recombinant Hepatitis C Virus glycoprotein as described above.

[0147] Accordingly, in some embodiments, envisaged herein is an mRNA encoding a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0148] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker comprising the sequence GGSGGSGGS, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0149] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s).

[0150] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0151] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising W326P, V622A, L682P wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0152] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0153] In some embodiments, the mRNA encodes a recombinant Hepatitis C Virus glycoprotein comprising a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the transmembrane domain(s) of the El and E2 glycoproteins are replaced by leucine zipper(s).

[0154] Further envisaged herein is an antigenic cocktail comprising mRNA sequence(s) encoding the recombinant Hepatitis C Virus glycoprotein(s) as described in any of the above embodiments, based on at least two HCV strain(s) that demonstrate at least about 60% homology between their E1 / E2 sequences.

[0155] In some embodiments, the present disclosure provides an antigenic cocktail comprising mRNA sequence(s) encoding the recombinant Hepatitis C Virus glycoprotein(s) as described in any of the above embodiments, based on at least two HCV strain(s) selected from a group comprising H77, AMS0232, AMS0230, AMS3a and UKNP4.1.1 or HCV consensus sequence. The antigenic cocktail can induce cross-neutralizing responses against HCV.

[0156] Although the above embodiments focus on an mRNA encoding the recombinant Hepatitis C Virus glycoprotein(s) of the present disclosure, the features and characteristics of the recombinant HepC glycoprotein(s), including the components and / or characteristics as such are as described by any of the embodiments above that characterize the said glycoprotein(s). For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again with respect to the method. However, each of the said embodiments completely fall within the purview of the mRNA described above.

[0157] Vector and Cell carrying nucleotide (s) encoding the recombinant Hepatitis C Virus glycoproteinfs) of the present disclosure

[0158] The present disclosure further envisages a vector comprising the nucleic acid as described above.

[0159] Also provided herein is an isolated or recombinant eukaryotic or prokaryotic host cell comprising the vector as described above.

[0160] Nanoparticle or membrane-anchored conjugatefs) comprising the recombinant Hepatitis C Virus glgcoproteinfs) of the present disclosure or mRNA encoding the said glycoproteinfs)

[0161] The present disclosure further provides nanoparticle or membrane-anchored conjugates comprising the recombinant Hepatitis C Virus glycoprotein(s) or nucleic acid encoding the said glycoprotein(s) as described in any of the above embodiments. Thus, in some embodiments, provided herein is a nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane anchoring TMD and the recombinant Hepatitis C Virus glycoprotein or the nucleic acid encoding the said glycoprotein(s) as described in any of the above embodiments.

[0162] Accordingly, in some embodiments, the present disclosure provides a nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane anchoring TMD a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0163] In some embodiments, the present disclosure provides a nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane anchoring TMD a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0164] In some embodiments, the present disclosure provides a nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane anchoring TMD and a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0165] In an exemplary embodiment, the nanoparticle or membrane-anchored conjugate comprises a nanoparticle or membrane anchoring TMD and a recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site, a hydrophilic amino acid linker, and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0166] In some embodiments, the present disclosure provides a nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane anchoring TMD and a recombinant Hepatitis C Virus glycoprotein comprising heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261- 294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

[0167] In an exemplary embodiment, the nanoparticle or membrane-anchored conjugate comprises a nanoparticle or membrane anchoring TMD and a recombinant Hepatitis C Virus glycoprotein comprising heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; and wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

[0168] In some embodiments, the nanoparticle is selected from a group comprising I53-50A, ferritin (Kanekiyo et al. Nature 2013, doi: 10.1038 / naturel2202, alum adjuvant (Moyer et al. Nat. Med (doi: 10.1038 / s41591-020-0753-3)) or liposomes (Moon et al. PNAS 2012 (doi: 10.1073 / pnas.1112648109).

[0169] In some embodiments, the membrane anchored conjugates are conjugated to membraneanchoring transmembrane domain(s). In some embodiments, non-limiting examples of membrane-anchoring transmembrane domain(s) (TMD) include the TMD of SARS-CoV-2. Two versions of such TMDs are employable in the context of the present disclosure - both with and without the cytoplasmic tail (CT). In some embodiments, the membrane-anchoring TMD is encapsulated in a lipid nanoparticle.

[0170] In some embodiments, the nanoparticle is a mosaic nanoparticle comprising two or more Hepatitis C Virus glycoprotein(s) or nucleic acid encoding the said glycoprotein(s) as described in any of the above embodiments.

[0171] In some embodiments, the recombinant Hepatitis C Virus glycoprotein(s) or nucleic acid encoding the said glycoprotein(s) are conjugated to the nanoparticle or membrane-anchoring TMD by way of a hydrophilic amino acid linker or by conjugation via a free C-terminal cysteine to conjugate alum.

[0172] In an exemplary embodiment, the recombinant Hepatitis C Virus glycoprotein(s) or nucleic acid encoding the said glycoprotein(s) are conjugated to the nanoparticle by way of a hydrophilic amino acid linker.

[0173] In some embodiments, the hydrophilic amino acid linker is a Glycine- Serine sequence.

[0174] In some embodiments, the hydrophilic amino acid linker comprises at least one Glycine residue and at least one Serine residue.

[0175] In some embodiments, the hydrophilic amino acid linker comprises at least two Glycine residues and at least one Serine residue.

[0176] In some embodiments, the hydrophilic amino acid linker comprises at least one Glycine residues and at least two Serine residue.

[0177] In some embodiments, the hydrophilic amino acid linker comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Glycine residues and about 1, 2, 3, 4 or 5 Serine residue.

[0178] In some embodiments, the hydrophilic amino acid linker comprises about 2 to 10 Glycine residues and about 1-5 Serine residues.

[0179] In some embodiments, the hydrophilic amino acid linker is GGSGGS (i.e.2xGGS), GGSGGSGGS (i.e. 3*GGS) or GGSGGS GGSGGS GGS (i.e. 5*GGS). In an exemplary embodiment, the hydrophilic amino acid linker comprises the sequence GGSGGSGGS (i.e. 3*GGS) or GGSGGS (i.e. 2*GGS).

[0180] In an exemplary embodiment, provided herein is a membrane anchored conjugate comprising the TMD of SARS-CoV-2 conjugated to a recombinant Hepatitis C Virus glycoprotein comprising heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR. a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s); and wherein the TMD of SARS- CoV-2 is conjugated to the recombinant Hepatitis C Virus glycoprotein by way of a hydrophilic amino acid linker.

[0181] In another exemplary embodiment, provided herein is a nanoparticle conjugate comprising 153- 50A conjugated to a recombinant Hepatitis C Virus glycoprotein comprising heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site comprising the sequence RRRRRR, a hydrophilic amino acid linker comprising the sequence GGSGGSGGS and at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3; wherein the engineered furin cleavage site replaces the natural protease site between the El and E2 glycoproteins; wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein; wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s); and wherein the nanoparticle is conjugated to the recombinant Hepatitis C Virus glycoprotein by way of a hydrophilic amino acid linker.

[0182] In a non-limiting embodiment, some examples of such nanoparticle or membrane-anchored conjugates of the present disclosure are represented in the below table by way of reference to their sequence identifier as used in the working examples, combination of characterizing features and their respective SEQ ID Nos. Table 3:

[0183] Accordingly, in some embodiments, envisaged herein are nanoparticle or membrane anchored Hepatitis C Vims glycoprotein conjugates having sequence bearing at least about 60% homology to sequence(s) selected from a group comprising sequences represented by SEQ ID Nos. 18, 24, 33 and 43-46.

[0184] In some embodiments, the nanoparticle or membrane anchored Hepatitis C Vims glycoprotein conjugate of the present disclosure has sequence selected from a group comprising sequences represented by SEQ ID Nos. 18, 24, 33 and 43-46.

[0185] In some embodiments, the conjugation of the anchored Hepatitis C Vims glycoprotein to nanoparticle(s) or membrane-anchoring TMDs as described above improves binding strength of the glycoprotein to targeted antibodies.

[0186] Composition comprising the recombinant Hepatitis C Virus glycoprotein(s) of the present disclosure or mRNA encoding the said glycoprotein(s)

[0187] The present disclosure further provides a pharmaceutical composition comprising the recombinant Hepatitis C Vims glycoprotein(s), the antigenic cocktail, the nucleic acid encoding the said glycoprotein(s) or the nanoparticle or membrane-anchored conjugate(s) as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

[0188] In some embodiments, the pharmaceutical composition comprises the recombinant Hepatitis C Vims glycoprotein(s) or the antigenic cocktail as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

[0189] In some embodiments, the pharmaceutical composition comprises the mRNA encoding the recombinant Hepatitis C Vims glycoprotein(s) or the antigenic cocktail as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

[0190] In some embodiments, the pharmaceutical composition comprises the nanoparticle or membrane-anchored conjugates comprising the recombinant Hepatitis C Vims glycoprotein(s) or the mRNA encoding the same as described above and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

[0191] In some embodiments, the pharmaceutical composition is a vaccine.

[0192] Kit

[0193] The present disclosure further provides a kit comprising the pharmaceutical composition of the present disclosure along with means for administration of the same and optionally a manual comprising instructions for administration.

[0194] In some embodiments, the kit of the present disclosure may comprise, preferably in separate containers, one or more of the recombinant Hepatitis C Virus glycoprotein(s) or the mRNA encoding the same as described above, the nanoparticle(s) for conjugation, means to affect the conjugation, the excipient(s) and the pharmaceutically acceptable carrier, excipient, or diluent, and the adjuvant, along with means for administration of the same and optionally a manual comprising instructions for administration.

[0195] Application(s)

[0196] Further envisaged herein is the recombinant Hepatitis C Virus glycoprotein(s) of the present disclosure, the antigenic cocktail comprising the said glycoprotein(s), the composition comprising the said glycoprotein(s) and / or the nanoparticle or membrane-anchored conjugate(s) comprising the said glycoprotein(s), for use in in the treatment or prophylaxis of HCV infection.

[0197] The present disclosure also provides a method for preventing or treating HCV, comprising administering an immunologically effective amount of the composition as described above to a subject in need thereof.

[0198] Further provided herein is a method for eliciting an immune response against HCV in a subject comprising administering an immunologically effective amount of the composition as described above to the subject in need thereof.

[0199] Also provided in the present disclosure is a method for screening of natural, isolated or synthetic antibodies capable of neutralizing HCV, comprising contacting the antibodies with the recombinant HCV glycoprotein(s) or the nanoparticle or membrane-anchored conjugate(s) of the present disclosure as described above; and identifying the antibodies that demonstrate neutralization of the HCV glycoprotein above a pre-determined threshold.

[0200] The subject matter of embodiments of the present invention is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the disclosure. The disclosed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described.

[0201] EXAMPLES

[0202] Materials and Methods

[0203] Antibody constructs were human codon-optimized and cloned into a human IgGl expression vector (Genscript Biotech). Most mature and germline inferred antibody sequences were retrieved from previously published work. Sequences for inferred germline AT1209 were a king gift of Tim Beaumont and Sabrina Merat (AIMM therapeutics).

[0204] Constructs were based on the HCV glycoprotein sequences from the following strains (GenBank ID; mutation(s)): AMS0232 (OL855837.1; I442F), H77 (AAB67037; R564C, V566A, G650E), AMS3a (KR094964.1), UKNP4.1.1 (ALV85530.1).

[0205] Mutagenesis

[0206] Unless stated otherwise, all mutations were incorporated into ElE2-encoding plasmids through conventional site-directed mutagenesis. Mutagenesis was performed using the QuikChange II (XL) Site-Directed Mutagenesis Kit (Cat# 200522-5) following manufacturer’s protocol. DMSO (3.7% final concentration) was added to the PCR reaction to optimize results. After mutagenesis, 2 pL of PCR product was added to 20 pL of XLl-Blue supercompetent cells. A 45 s heat shock was performed at 42 °C and immediately after 200 pL of SOC medium was added and cells were incubated in ice for 2 min. Following an hour of incubation at 37 °C, 200 pL were plated on antibiotic selection plates. Single colonies were picked and grown, from which DNA was stracted using NucleoSpin Plasmid Mini kit for plasmid DNA (MACHEREY- NAGEL). All mutations were confirmed by sequencing (Macrogen Europe). Mutant screen in HEK293T cells

[0207] Plasmids encoding all mutated proteins were expressed in HEK293T cells. These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) and 0.1% penicillin-streptomycin (PS) and incubated at 37°C and 5% CO2. A day prior to transfection cells were seeded in 6-well plates to achieve 60-70% confluency at day of transfection. ElE2-expressing plasmids were co-transfected with furin in a 1:1 ratio using PEI MAX (polysciences) transfection reagent (1 :3 DNA to PEI MAX ratio) in Opti-MEM (Gibco). Supernatants containing secreted protein were harvested three days post-transfection and diluted 1 :4 in Tris-buffered saline (TBS) before use for analysi by Enzyme-linked immunosorbent assay (ELISA).

[0208] 100 pL of diluted supernatants containing E1E2 were coated on 96-well Strep-TactinXT microplates (IBA LifeSciences) and incubated for two hours at room temperature (RT). Plates were washed with TBS twice before incubating with serially diluted mAbs in casein blocking buffer (Thermo Fisher Scientific) for 90 min. After three washes with TBS, a 1 :3000 dilution of HRP-labeled goat anti-human IgG (Jackson Immunoresearch) in casein blocking buffer was added for 45 min. After washing the plates five times with TBS + 0.05% Tween-20, plates were developed by adding develop solution [1% 3,3',5,5'-tetraethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate, 100 mM citric acid] and the reaction was stopped after 3:30 min by adding 0.8 M H2SO4. Absorbance was measured at 450 nm and area under the curve (AUC) values were calculated using Graphpad Prism. All data was normalized against the binding signal of the AP33 bNAb.

[0209] Antibody expression and purification

[0210] Antibodies were transiently expressed in HEK293F cells (Invitrogen, cat no. R79009) maintained in Freestyle medium (Life Technologies). Cells were co-transfected at a density of 0.8-1.2 million cells / mL with two plasmids expressing IgGl heavy chain (HC) and light chain (LC) in a 1:1 ratio. PEI MAX (Polysciences) was used as transfection agent at a 1 :3 ratio (plasmid DNA PEI MAX). Supernatants containing the antibodies were harvested five days post-transfection, centrifuged for 30 min at 4000 x g and filtered using 0.22 pm Steritop filters (Merck Millipore). The filtered supernatant was run over a 1 mL protein A / G column (Pierce) followed by two column volumes of PBS wash. The antibodies were eluted with 18 mL 0.1 M glycine pH 2.5 directly captured in 2 mL neutralization buffer (1 M TRIS pH 8.7). The purified antibodies were buffer exchanged to PBS using 100 kDa VivaSpin20 columns (Sartorius). The IgG concentration was determined using a NanoDrop (Thermo Fisher Scientific) and the antibodies were stored at 4 °C until further analyses.

[0211] HCV glycoprotein expression and purification

[0212] StrepII-tagged E1E2 were produced in HEK293F cells (Invitrogen, cat no. R79009) similarly to antibodies. Cells were seeded for transfection at 0.8- 1.2 million cells / mL and transfected using a mix of PEI MAX (Img / mL) with expression plasmids (312.5 pg DNA / L of 293F cells) in a 1: 1 ratio (ElE2:furin) in Opti-MEM. Six days post transfection, supernatants were harvested, centrifuged for 30 min at 4000 x g and filtered using 0.22 pm Steritop filters (Merck Millipore). The glycoproteins were purified using Strep-TactinXT columns (IBA Life Sciences) by gravity flow (-0.5-1.0 ml supematant / min). The eluted protein was collected in a 30 kDa Vivaspin® (Vivaspin® 20, 30 kDa MWCO Polyethersulfone, Sartorius) and buffer- exchanged to TBS. Samples were collected and concentration was determined using Nanodrop (Thermo Fisher Scientific). Proteins were then used for size exclusion chromatography (SEC). Protein purification process was performed at 4 °C.

[0213] Purified and concentrated samples were fractionated using a Superdex 200 Increase column in TBS (GE Healthcare). Fractions corresponding to E1E2 dimers were collected and pooled. Protein concentration was determined by Nanodrop (Thermo Fisher Scientific) using theoretical molecular weight and extinction coefficient. Proteins were then aliquoted and stored at -80 °C until further use. Fractions containing aggregated protein were also kept for further characterization.

[0214] SDS-PAGE

[0215] SDS-PAGE analyses were performed as described elsewhere (de Taeye et al. 2015). with some modifications. Briefly, 5 pg of purified SE1E2 were mixed with loading dye (25 mM Tris, 192 mM Glycine, 20% v / v glycerol, 4% m / v SDS, 0.1% v / v bromophenol blue in milli-Q water), and incubated at 95 °C for 10 min prior to loading on a 10-20% Tris-Glycine gel (Invitrogen). For reducing SDS-PAGE, dithiothreitol (DTT; 100 mM) was included in the loading dye and loaded on a Novex 10-20% Tris-Glycine gel (Thermo Fisher Scientific). Gels were run in a buffer containing 25 mM Tris, 192 mM glycine, and 0.5% SDS for 1 h at 200 V at 4 °C. Coomassie blue staining of SDS-PAGE gels was performed using the PageBlue Protein Staining Solution (Thermo Fisher Scientific). Neutralization assays

[0216] For generating the HCVpps, 1.5 x 10A6 HEK293T cells were seeded on a 10 cm dish a day prior to transfection. E1E2, a MLV Gag-Pol packaging construct and firefly luciferase were co-transfected in optimized ratios (1 : 1 : 1 or 1 :2.6:3.4 or 1 :26:34) with a total amount of 6 pg of DNA and 12 pl of Lipofectamine 2000 (Invitrogen) in Opti-MEM (ThermoFisher). Opti-MEM was replaced by DMEM (Gibco) / 10% fetal bovine serum (FCS) / 0.1% Penicillin-Streptomycin (PS) the next day. Two days later, the supernatant containing the HCVpps was passed through a 0.45 pm filter and frozen at -80 °C for long-term storage or 4 °C when used within a week. One day prior to the neutralization assay, 15 x 10A3 Huh-7 cells per well in a 96-well plate were seeded in 100 pL DMEM / 10%FCS / 0.1%PS / 1% nonessential amino acids / 1% HEPES buffer (Huh-7 medium). HCVpps were incubated with monoclonal antibodies in duplicate at 37 °C in 5% CO2 which were serially 3 -fold diluted starting with a 25 pg / ml dilution. After 1 h, the HCVpp / serum mixture was added to the Huh-7 cells and incubated for 4h. Next, 200 pL Huh-7 medium was added and incubated for 72 h. Cells were lysed and luciferase signal was measured using the SteadyGlo luciferase reagent kit and a GloMax luminometer (Promega, USA). Neutralization data was analyzed with Graphpad Prism (version 8.3).

[0217] I53-50B.4PT1 expression and purification

[0218] Lemo21 (DE3) (NEB) cells expressing I53-50B.4PT1 were grown in a 10 L BioFlo 320 Fermenter (Eppendorf) or a 2 L shake flask. Cells were grown in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) at 37 °C to an OD600 of 0.8. After the cells were induced with 1 mM of IPTG, temperature was reduced to 18 °C and cells were grown for 16 h. The cells were then lysed in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS using a Microfluidics Ml 10P at 18,000 psi. Lysate was centrifuged at 24,000 x g for 30 min. Clarified lysate was next applied to aNi Sepharose 6 FF column (Cytiva) linked to an AKTA Avantl50 FPLC system for immobilized metal affinity chromatography. I53-50B.4PT1 was eluted using a 30-500 mM imidazole linear gradient in 50 mM Tris pH 8, 500 mMNaCl and 0.75% CHAPS. Fractions containing I53-50B.4PT1 were pooled, concentrated using centrifugal filters with a 10,000 kDa cutoff (Millipore), sterilized and applied to a Superdex 200 Increase 10 / 300 (Cytiva) for further purification. Batches were tested to ensure low levels of endotoxin before use. Nanoparticle assembly

[0219] SE1E2.V5-I53-50 nanoparticles (sE2Elv5-I53-50NP) were assembled essentially as described (Brouwer et al., 2019) with some adjustments. To remove aggregated protein, E1E2-I53-50A fusion proteins were passed through a Superose 6 Increase 10 / 300 SEC column (GE Healthcare) in TBS / 5% glycerol, pH 7.5. The column fractions containing non-aggregated sElE2.v5-I53-50A trimers were pooled and concentrated at 4000 x g using vivaspin filters with a lOkDa molecular weight cutoff (Sartorius) and mixed in an equimolar ratio with 153- 50B.4PT1 (produced as described above) for an overnight (-16 h) incubation at 4 °C. The assembly mix was then passed through a Superose 6 Increase 30 / 100 column in TBS / 5% glycerol, pH 7.5. The fractions corresponding to the assembled NPs were pooled and nanoparticle concentrations were determined with a Nanodrop using the peptidic molecular weight and extinction coefficient.

[0220] Bio-layer interferometry (BLI)

[0221] BLI assays were performed as described previously. (Capella-Pujol et al., 2023) BLI assays were performed using an Octet K2 instrument (ForteBio). All assays were performed at 30 °C and with agitation speed of 1000 rpm. Antibodies, SE1E2, SE1E2-I53-50A trimer and sE!E2- 153-50 nanoparticle samples were dissolved in running buffer (PBS, 0.02% Tween, 0.1% bovine serum albumin (BSA)) in a volume of 250 pl / well. Antibodies (1 pg / mL) were immobilized onto protein A biosensors (ForteBio, cat no. 18-5010) until a loading threshold of 1.0 nm was reached, followed by a 30 s baseline measurement in the running buffer. Purified proteins were diluted to 250 nM and association and dissociation were measured for 300 s each. A well containing running buffer without protein was used for background correction. Data was analyzed and visualized in GraphPad Prism 9.5.1.

[0222] Addition to ELISA method

[0223] ELISA analysis of SE1E2.V5-I53-50NP was performed similarly as described, with some adjustments. 50 pL 20 pg / mL of GNL (Galanthus Nivalis Lectin, Vector Laboratories) was coated overnight on high binding half area plates (Greiner Bio-One). The plates were washed twice with TBS after which residual binding was blocked with 50 pL casein (Thermo Fisher Scientific) for 30 minutes at room temperature. The plates were washed with TBS three times after which the plates were incubated with 50 pL of purified SE1E2-I53-50NP (2 pg / mL in TBS + 5% glycerol) for two hours at room temperature. The plates were washed twice with TBS before incubating with serially diluted mAbs in casein blocking buffer (Thermo Fisher Scientific) for two hours. After three washes with TBS, a 1 :3000 dilution of HRP -labeled goat anti-human IgG (Jackson Immunoresearch) in casein blocking buffer was added for 1 hour. After washing the plates five times with TBS + 0.05% Tween-20, plates were developed by adding develop solution [1% 3,3 ',5,5 '-tetraethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate, 100 mM citric acid] and the reaction was stopped after 3.5 min by adding 0.8 M H2SO4. Absorbance was measured at 450 nm, EC50 values were calculated using Graphpad Prism 9.5.1.

[0224] For the mbElE2 binding ELISA HEK293T cells in a T-75 flask (-70-80% confluency) were transfected with AMS3a E1E2 plasmid using PEI MAX. After 72 h, the cells were harvested and lysed with 2.0 mL of 1.0% Triton X-100 in TBS (-10 x 106 cells / mL). After 30 min, the lysate was clarified by centrifugation (1500 x g);filtered (0.22 pm filter), aliquoted and the ElE2-containing lysates were stored at -80 °C. Half-well 96-well plates were coated with Galanthus nivalis lectin (Vector Laboratories) at 20 pg / mL in 0.1 M NaHCO3 pH 8.6. The next day, the plates were blocked with casein blocking buffer and then E1E2 lysate (1 :25 diluted in TBS) was added to the lectin-coated plates. After 2 h, the plates were washed (TBS) and rabbit sera was added at a 1 :50 dilution in casein and serially diluted in 4-fold steps. After 2 h incubation, plates were washed three times with TBS and a 1 :3000 dilution of HRP -lab eled goat anti-rabbit IgG (Jackson Immunoresearch) in blocking buffer (TBS 2% milk) was added for 1 hour. After washing the plates five times with TBS + 0.05% Tween-20, plates were developed by adding develop solution [1% 3,3',5,5'-tetraethylbenzidine (Sigma-Aldrich), 0.01% H2O2, 100 mM sodium acetate, 100 mM citric acid] and the reaction was stopped after 3.5 min by adding 0.8 M H2SO4. Absorbance was measured at 450 nm, EC50 values were calculated using Graphpad Prism 9.5.1.

[0225] For the JunFos binding ELISA, Jun and Fos peptides were obtained from Genscript. Both were used for coating of half area 96-wells plates in a 1 pg / mL concentration for a final concentration of 2pg / mL Jun-Fos in NaHCO3. The next day these were washed in TBS and subsequently blocked with PBS-TB (PBS, 0.05% Tween-20, 2% BSA) with 3% fetal calf serum (FCS, PBS- TFB). The sera was then added in a 1:50 ratio of serum to PBS-TB, serially diluted, and incubated for 2 hours. The following steps were the same as for the lysate ELISA. Rabbit immunizations

[0226] In a first immunization study, 12 rabbits (New Zealand White, female, 2 groups, 6 animals / group) were immunized under subcontract at Covance (Denver, USA) with either 18 pg AMS0232 sElE2.vl or 16 pg of AMS0232 SE1E2.V5). Antigens were mixed 1 : 1 with squalene o / w emulsion (SE) (i.e., 250 pL of antigen in PBS combined with 250 pL SE) (Polymun, Klosterneuburg, Austria) and administered by three intramuscular immunizations. Rabbits were bled at weeks 0, 4, 6, 12, 20 and 22. All immunization procedures complied with all relevant ethical regulations and protocols of the Covance Institutional Animal Care and Use Committee (IACUC, study C0201-23).

[0227] In parallel two extra groups of six rabbits were immunized. One group, HepCon group, was immunized thrice with 18 pg HepCon ElE2.v4 and once with 25 pg HepCon ElE2.v5-I53- 50NP. The other group, Mosaic group, was immunized thrice with a cocktail of 4.5 pg AMS0232 sElE2.v4, 4.5 pg H77 SE1E2.V4, 4.5 pg UKNP4.1.1 SE1E2.V4 and 4.5 pg AMS3a SE1E2.V4 and once with 25 pg of a mosaic NP displaying AMS0232 SE1E2.V5-I53-50A, H77 SE1E2.V5-I53-50A, UKNP4.1.1 SE1E2.V5-I53-50A and AMS3a SE1E2.V5-I53-50A simultaneously. The immunizations were administered at weeks 0, 4, 20 and 37, respectively, and the rabbits were bled at weeks 0, 4, 6, 12, 20, 22, 37 and 39.

[0228] Flow cytometry

[0229] Plasmids encoding native AMS0232 E1E2 (as described in Torrents et al. Science 2022; doi: 10.1126 / science.abn9884) or AMS0232 ElE2.v5 fused to SARS-CoV-2 TMD were transiently expressed in CD81KO HEK293T cells (Kalemera et al. IGV; doi: 10.1099 / jgv.0.001512). After 48 hours, cells were harvested and 2.0 X 106cells were incubated in 200 ml FACS buffer (PBS / 2% FCS / 2.5 mM HEPES / lmM EDTA) with 10 mg / ml AR4A antibody and the LIVE / DEAD Fixable Violet Dead Cell Stain kit (according to manufacturer instructions) for 30 minutes at room temperature. Subsequently, cells were washed twice with FACS buffer and incubated with PE-labelled goat anti-human Fab2 for 30 minutes at room temperature. Following two washes with FACS buffer and the cells were subsequently analyzed on a Sony SH800. The live cells were gated for singlets and the live single cells were subsequently analyzed for PE signal. Example 1: Replacing the putative fusion in soluble E1E2 improves native-like folding sElE2.vl comprising a E1 / E2 heterodimer, with an R6 sequence replacing the natural protease site and the two transmembrane domains replaced by human-derived Jun / Fos leucine zippers (LZ) (Figure la) was used as the starting point for the design of the recombinant Hepatitis C Virus glycoprotein of the present disclosure.

[0230] The sElE2.vl design template was combined with the genotype la AMS0232 E1E2 sequence (Figure 4). This structure revealed the majority of conserved regions of the E1E2 ectodomain except the putative fusion peptide (pFP) in El .

[0231] It was hypothesized that the hydrophobic and membrane-associating pFP hampers folding of soluble recombinant E1E2 in sElE2.vl . Therefore, SE1E2 version 2 (hereinafter referred to as SE1E2.V2) was designed in which 34 amino acids of pFP i.e. residues 261-294, as per standard H77-numbering were replaced with a short hydrophilic 9 amino acid flexible linker (GGSGGSGGS) (Figure la, Figure 4). sElE2.vl and SE1E2.V2 were produced in human embryonic kidney (HEK)-293F cells and purified the proteins using StrepTactinXT at similar yields of about 4 mg / L. The size exclusion chromatography (SEC) curves of SE1E2.V2 showed a lower higher molecular weight peak indicating it produced less aggregates than sElE2.vl. Additionally, the non-reducing sodium dodecyl sulfate (SDS) gels of the purified E1E2 dimers revealed that sElE2.v2 contains less disulphide scrambled E1E2, which is probably explained by the removal of the pFP that contains free cysteines (Figure 4).

[0232] Next, the antigenicity of sElE2.vl and SE1E2.V2 were compared by ELISA. Overall, binding to conformational bNAbs that target the metastable ElE2-dependent antigenic region AR4 and AR3 (which overlaps with the CD81 binding site) was markedly enhanced for sElE2.v2. Almost a 14-fold and about 10-fold lower half-maximal effective binding concentrations (EC50) and almost 2-fold higher maximum binding plateau for AR4-targeting AR4A and AT1618 was observed for SE1E2.V2 (Figure 5). Furthermore, SE1E2.V2 showed detectable binding to AR3-specific bNAbs AR3C and HEPC74. Notably, HCV pseudoparticles (HCVpp) in which the pFP was replaced by the same 9-amino acid linker failed to infect susceptible Huh-7 cells, but retained binding to bNAbs. This indicated that the virion-associated E1E2 retained proper antigenic folding but was rendered non-infectious. In summary, it was observed that by replacing the pFP by a short linker antigenicity of the recombinant secreted E1E2 was improved.

[0233] Example 2: Stabilization of sE!E2,v2 by iterative structure-based design

[0234] A structure-based design was adopted to stabilize sElE2.v2. Two rationally designed mutational libraries were constructed. In the first mutational library more than 30 individual amino acids in sElE2.v2 were substituted by either proline or alanine substitutions. These amino acid substitutions were designed to be in the core of the E1E2 heterodimer and not in the direct vicinity of antibody epitopes, since the aim was to improve stability and folding and not antibody binding directly.

[0235] In the second mutational library, disulfide bond(s) were introduced between the El and E2 glycoproteins to enhance stability of the complex. Cysteine residues were introduced at four positions in El and at five positions in E2 for a total of five designed disulfide bonds. Single cysteines were also introduced at the same positions to serve as negative controls.

[0236] The constructs of both libraries were transfected in HEK293T cells and tested unpurified proteins in the supernatant for binding to AR4A and AT1618 bNAbs, CD81-Fc to probe receptor binding and used AP33 bNAb as a control for expression. In the first library, substitutions W326P and L682P were identified to show markedly improved AR4A and AT1618 binding compared to sE!E2.v2. Additionally, it was observed that V622A (I622A for AMS0232) increased binding to CD81-Fc. Combining W326P, V622A (I622A for AMS0232) and L682P in sElE2.v2 further augmented AR4A and AT1618 binding compared to only the single mutations. sE!E2.v2 with W326P, V622A (I622A for AMS0232) and L682P is hereinafter referred to as sE!E2.v3.

[0237] In the second library, four out of five designed disulfide bonds decreased or abolished detectable binding to AR4A and AT1618. Only sElE2.v2 with cysteines at positions 244 and 687 (P244C-T687C) retained binding to AR4A and AT1618. To confirm that an inter-subunit disulfide bond was formed, sElE2.v2 with P244C-T687C was purified, which showed a single band in non-reducing SDS PAGE at the expected size of an E1E2. The P244C-T687C disulfide bond was introduced in sElE2.v3 and the said construct is hereinafter referred to as SE1E2.V4 (Figures 1A, 2 A, 4). Example 3: Antigenicity of engineered sE!E2,v3 and sE!E2,v4

[0238] Soluble E1E2 versions 1-4 (sElE2.vl-v4) were compared by producing them in HEK-293F cells followed by StrepTactinXT affinity chromatography. Purification yields between the versions were similar, in the range of about 2-4 mg / L. The SEC profiles indicated that sElE2.vl contained the most aggregates, while the profile of SE1E2.V4 showed a more narrow and thus possibly more homogeneous population of dimers. The SDS PAGE revealed that all four versions of SE1E2 were efficiently cleaved between El and E2 as intended. The nonreducing SDS confirmed the presence of an interprotomer disulfide bond in SE1E2.V4 as expected from the previous experiment which combined sElE2.v2 with P244C-T687C (Figure 6).

[0239] Next, the antigenicity of the purified proteins was probed using an extended panel of 26 HCV mAbs against known neutralizing and non-neutralizing epitopes in ELISA. It was observed that both SE1E2.V3 and SE1E2.V4 showed markedly enhanced binding to most tested conformational bNAbs compared to sElE2.vl, confirming the results of the initial supernatant screening. Conformational ElE2-dependent bNAbs AR4A, AT1618 and HEPC111 displayed much stronger binding to SE1E2.V3 and SE1E2.V4 with EC50 values that were at least 200-, 200- and 80-fold higher compared to sElE2.vl and at least 15-, 20- and 2-fold higher compared to SE1E2.V2. Additionally, the higher binding plateaus also indicated that the maximum number of target epitopes on SE1E2.V2-V4 is higher than on sElE2.vl (Figure 7). Binding to several AR3 bNAbs was also (strongly) enhanced for sE!E2.v3 and sE!E2.v4 compared to sElE2.vl, but also compared to SE1E2.V2 (Figure 7). Additionally, binding to CD81 and conformational bNAbs, such as HEPC108, was also enhanced for SE1E2.V3 and SE1E2.V4 (Figure 7). Finally, binding of non-neutralizing CBH-4B, CBH-4D and CBH-4G mAbs was decreased for SE1E2.V4 compared to sE!E2.v2 and SE1E2.V3 (Figure IB).

[0240] Next, to determine if the engineered soluble E1E2 versions are antigenic mimics of virion- associated E1E2, the binding ELISA AUC values were plotted against the neutralization midpoint inhibitory concentration (IC50) values of the sequence-matched AMS0232 HCV pseudoparticle (HCVpp). The neutralization IC50 values of AMS0232 HCVpp correlated significantly (p < 0.0001) and strongly to the binding AUC values of AMS0232 SE1E2.V3 and SE1E2.V4 (Spearman r = 0.77; p < 0.0001 and r = 0.81; p < 0.0001, respectively) (Figure 8). The correlations were weaker for sElE2.vl (Spearman r = 0.61; p = 0.001) (Figure 8), in which AR4A and AT 1618 are two major outliers in the correlation plot, and somewhat weaker for SE1E2.V2 (Spearman r = 0.70: p <0.0001). In summary, SE1E2.V3 and SE1E2.V4 displayed enhanced binding to conformational antibodies and were understood to be excellent antigenic mimics of virion-associated E1E2.

[0241] Example 4: Thermal stability and site-specific glycan analysis of engineered secreted E1E2

[0242] To compare antigenic stability, thermostability ELISA assays were performed wherein sElE2.vl-v4 were incubated at temperatures of 21°C, 37°C, 60°C and 90°C for one hour and then tested binding to conformation-sensitive bNAbs AR4A and AT1618 and non- conformational bNAb AP33. As expected, AP33 binding was unaffected by the increased temperatures (Figure 9). It was observed that after incubation at 60°C and 90°C, sElE2.vl and sElE2.v2 lost all ormostofthe detectable binding to AR4A and AT 1618. In contrast, sElE2.v3 and SE1E2.V4 binding to AR4A and AT1618 seemed unaffected by the 60°C incubation. Remarkably, both SE1E2.V3 and sElE2.v4 retained most of their binding to AR4A and AT1618 after incubation at 90°C (Figure 9). Freeze / thawing did not significantly affect the antigenicity of any of the antigen versions.

[0243] Glycans play a major role in the folding of E1E2 and in particular the glycans at positions 196 and 305 in the interface of E1 / E2. To compare the glycan occupancy of the engineered E1E2 versions, site-specific analysis was performed. Most potential N-linked glycosylation sites (PNGS) were occupied by complex-type glycans for the four soluble E1E2 versions, while notable differences between the versions were also observed. Low occupancy was observed at sites N209 and N305 in El and at sites N623 and N645 in E2 for ElE2.vl . For SE1E2.V2, low occupancy was also observed forN305, N623 and N645, along with improved occupancy for N209. For SE1E2.V3 and SE1E2.V4, enhanced occupancy was observed for N305, N623 and N645, but somewhat lower occupancy of N209 as compared to SE1E2.V2. Glycosylation at 695, which contains the non-canonical NxV motif, was low overall, with maximum occupancy of about 20% for SE1E2.V3. It was hypothesized that the increased occupancy at N623 for SE1E2.V3 and SE1E2.V4 was caused by the V622A mutation, directly upstream of N623. To investigate this, glycan analysis was performed on SE1E2.V2 with V622A and this confirmed that V622A increases glycosylation at N623. Overall, occupancy was higher for SE1E2.V3 and SE1E2.V4 compared to sElE2.vl, which probably is associated with their more desirable antigenicity profile. Example 5: Compatibility of stabilized E1E2 designs with other strains

[0244] To determine if the E1E2 designs are compatible with other dimerization domains, El and E2 of the ElE2.vl-v4 designs were fused to the synthetic coiled-coil SYNZIP dimerization domain (sElE2.vl-v4-SZ). These are synthetic computationally designed leucine zippers that allow for the heterodimerization of molecules. The four versions of SYNZIP -fused E1E2 were tested for their antigenicity in ELISA. The same accumulative improvement in AR4A, AT 1618 and other conformational antibodies was observed as before. Additionally, non-reducing SDS PAGE indicated that the 244-687 disulfide bond formed efficiently in SE1E2.V4-SZ.

[0245] ELISA was performed for side-by-side comparison of v2-v4 (using strain AMS0232). The EC50 values (in pg / mL) of the resulting experiments were plotted in the graph as depicted in Figure 14 wherein Spearman correlation coefficient is shown as r. No significant difference was found between the two types of heterodimerization domain indicating that the use of heterodimerization domains did not influence the binding of antibodies to the glycoproteins of the present disclosure. Instead, the effect can solely be associated with the stabilizing mutations. The maximum concentration of antibody used was 5 pg / mL.

[0246] The data also shows that the engineered modifications in El and E2 are compatible with other coiled-coil heterodimerization domains.

[0247] Next, the sElE2.vl-v4 templates were applied to other HCV strains to produce a variety of recombinant E1E2 glycoproteins. Genotype la strain H77, genotype 3 strain AMS3a and genotype 4 strain UKNP4.1.1 were used. These twelve constructs were produced efficiently with sElE2.vl producing the highest amount of aggregates and a significant amount of disulfide scrambled dimers were observed in non-reducing SDS gel. The three SE1E2.V4 constructs again produced a single band in non-reducing SDS PAGE gel, indicating that the C244-C687 disulfide bond formed efficiently.

[0248] The antigenic profiles of the twelve proteins were compared by ELISA. Similar to the constructs based on AMS0232, the antigenicity profiles of the SE1E2.V3 and SE1E2.V4 constructs from the other strains were superior: binding affinities for AR4A were at least 100- fold and 10-fold higher compared to sElE2.vl and SE1E2.V2, respectively, and similar gains were observed for many of the other conformational bNAbs (example for UKNP4.1.1 -based sElE2.vl-v4 in Figure 10). Additionally, binding of several inferred germline bNAbs to UKNP4.1.1 improved considerably in SE1E2.V3 and SE1E2.V4, also compared to a previous E2E1-I53-50A design by the inventors.

[0249] Lastly, an antigen was constructed based on an HCV consensus sequence (HepCon) using sElE2.v3 as a design template (Figure 4). This sequence was made by generating a consensus sequence of the prototypic sequence H77 with the consensus of genotypes la, lb, 1c, 2a, 2b, 3a, 4a, 5a, 6a, 6k and (Figure 4). This consensus is expected to contain the most common amino acid at each position, which might translate to a favorable antigenic profile. Therefore, it was also tested if this antigen engaged different inferred germline (gl) precursors of HCV bNAbs. It was found that HepCon SE1E2.V3 engaged several VH1-69 derived inferred germline bNAbs, including HEPC3, HEPC74, AR3A, AR3C and AR3D (Figure 3). Compared to the previous germline-targeting candidate immunogen, binding to VH1-69 derived gl -bNAbs was stronger by about 100-fold for gl-AR3C and gl-HEPC74 and broader: 7 / 8 with detectable binding versus 2 / 8 gl-bNAbs with detectable binding. Moreover, it was found that HepCon SE1E2.V4 also engaged inferred germline versions of AR4-targeting AT1618 (derived from VH3-23) and AR4A (derived from VH5-51) (Figure 3). In summary, HepCon sElE2.v3 was identified as a promising candidate for germline-targeting vaccination strategies.

[0250] Example 6: Stabilizing mutations are broadly applicable to other strains

[0251] The set of mutations from versions 2 to 4 designs (i.e. sElE2.vl to SE1E2.V4) were applied to strains H77 (genotype la), AMS0230 (genotype la), AMS3a (genotype 3a) and UKNP4.1.1 (genotype 4a) and tested against a reduced panel of 17 bNAbs and CD81-Fc. The EC50 for each bNAb was calculated from duplo experiments. The logarithm of the fold change in EC50 (version 1 / version X) is depicted and summarized for all results in a heatmap in Figure 12. In the figure, darker color represents more binding (i.e. lower EC50), white shows less binding than version 1. Crossed cells indicate no detectable binding at the maximum concentration tested (5 pg / mL).

[0252] The binding experiments performed were ELISA starting at concentration of 5 pg / mL. A significant improvement in binding of most conformational antibodies in versions 3 and 4 containing the stabilizing mutations (removal of the pFP + point mutations + cysteine bond) was observed. Notably vl of UKNP4.1.1 and AMS0230 did not show any binding to AR4- targeting antibodies (AR4A, AT 1618 and HECP111) but exhibited binding in the stabilized versions (figure 13). Another supporting result that was found is that CBH-4G, a non- neutralizing antibody, did not bind SE1E2.V4, which could have a crucial role in immunogenicity if the non-neutralizing epitopes are not engaged.

[0253] To provide a different representation, Figure 13 depicts binding of AR4A to the different strains described above. The figure represents EC50 values obtained. As can be observed from the figure UKNP4.1.1 vl and AMS0230 vl and v2 did not exhibit binding to AR4A, while the stable v3 and v4 showed strong(er) binding.

[0254] Example 7: Generation of a soluble native-like E1E2 without dimerization domains

[0255] Heterologous dimerization domains, such as JunFos or SYNZIP, are considered necessary to ensure heterodimerization of El and E2. However, a rabbit immunization study and a mouse immunogenicity study demonstrated that heterologous dimerization domains can be highly immunogenic. These anti-scaffold responses possibly distract from more productive on-target responses towards E1E2. Therefore, the inventors engineered a recombinant native-like E1E2 without fused dimerization domains to prevent unwanted anti-scaffold responses and to enable novel E1E2 designs (Figure 11).

[0256] It was hypothesized that the C244-C687 disulfide bridge between El and E2 in the SE1E2.V4 design is sufficient for heterodimerization. To test this, soluble E1E2 version 5 (SE1E2.V5) was engineered by removing the Jun / Fos dimerization domains from sElE2.v4 (Figure 2A).

[0257] Additionally, a soluble E1E2 version 6 (sElE2.v6) was also engineered by designing another disulfide bond between El and E2 at positions 340 and 716 in SE1E2.V5 to stabilize the C- terminal ends (Figure 2A). It was observed that both constructs produced proteins efficiently with yields of about ~3-4 mg / L HEK293F cells. The SEC profiles of SE1E2.V5 showed a broad aggregate peak and distinct dimer peak, while the SE1E2.V6 showed two peaks of relatively the same height. Reducing SDS PAGE and Western Blot revealed that ElE2.v5 and SE1E2.V6 were completely cleaved and non-reducing SDS PAGE confirmed the presence of an inter- E1 / E2 disulfide bridge. The ElE2.v5 and ElE2.v6 heterodimers without dimerization domains were also found to display binding to AR4A and AT1618 that was almost comparable to that of sElE2.v3 and sElE2.v4, which contain stabilizing dimerization domains (Figure 2B)

[0258] When tested in rabbits, as expected, no anti-scaffold responses were detected in the rabbits receiving SE1E2.V5. Finally, a variety of SE1E2.V5 and SE1E2.V6 antigens were produced based on different HCV strains (H77, AMS3a and UKNP4.1.1). These constructs produced efficiently and were cleaved efficiently. The sElE2.v5 and SE1E2.V6 designs were thus considered suitable starting points for the designs of next-generation native-like E1E2 heterodimer vaccines.

[0259] Example 8: Structural and Functional characterization of the sE!E2,v5 design

[0260] Initially, it was tested if sElE2.v5 retained binding to conformational antibodies despite removal of the dimerization domains. The panel of antibodies tested for binding with this new version included those targeting AR4 and AR3, the latter being a very well characterized antigenic region that overlaps with the natural receptor CD81 (Figure 15). Overall, it was seen that sElE2.v5 retained binding to most conformational antibodies and most notably it showed binding to AR4A, AT1618 and HEPC111 (AR4-targeting) which had never been achieved before (i.e. soluble E1E2 without any dimerization domain). These experiments were all done with constructs using the AMS0232 sequence. The maximum concentration of antibody used was 5 pg / mL.

[0261] Further to the above, for structural characterization, the formation of the disulfide bond and the protein was confirmed by reducing and non-reducing SDS gels (Figure 16). The non-reducing gels showed higher bands than the reducing gels, indicating that in non-reducing conditions (i.e. with the cysteines available to form bonds) the proteins sElE2.v4 and sElE2.v5 showed a higher molecular weight that is consequence of the El and E2 interprotomeric cysteine bond P244C-T687C. The difference in size between SE1E2.V4 and sElE2.v5 as seen in both gels is representative of the removal of the heterodimerization domains JunFos.

[0262] In addition to the above, thermostability of the SE1E2.V5 design was also tested. Figure 17 depicts the results of thermostability ELISA using SE1E2.V5 with the sequence of AMS0232. Proteins were incubated for one hour at different temperatures as indicated (31°C, 37°C, 60°C and 90°C). After incubation, ELISA protocol was followed as usual and residual binding was tested. Binding of conformational antibodies AR4A and AT1618 until 60 °C was found to be the same independent of the temperature. After incubating the protein for one hour at 90 °C, sElE2.v5 retained most of the binding to conformational antibodies. The maximum concentration of antibody tested was 5 pg / mL. AP33 is a linear antibody, and its binding capacity should not be influenced by protein degradation (higher temperatures). In parallel, it was also tested and observed that vl and v2 of the same sequence (AMS0232) did not show, or showed very low, binding to AR4A and AT1618 at 60 and 90°C (Figure 10). It was thus concluded that the thermostability can be associated with the combination of mutations. The mutations alone provide substantial antigenic stability, since SE1E2.V5, which does not contain dimerization domains is antigenically thermostable (Figure 17), while SE1E2 vl and SE1E2.V2, which contain stabilizing dimerization domains, but lack the stabilizing mutations, are significantly less antigenically thermostable (compare Figure 10 and Figure 17).

[0263] Example 9: Glycoprotein-nanoparticle conjugates

[0264] The glycoprotein-nanoparticle conjugates were prepared as described under materials and methods.

[0265] Figure 18 depicts the size exclusion chromatography (SEC) profiles of AMS0232 ElE2.v5- I53-50NP, Mosaic ElE2.v5-I53-50NP displaying AMS0232 ElE2.v5-I53-50A, H77 ElE2.v5- I53-50A, UKNP4.1.1 ElE2.v5-I53-50A and AMS3a ElE2.v5-I53-50A simultaneously and HepCon ElE2.v5-I53-50NP on a Superose 6 increase 10 / 300 column.

[0266] The SEC profiles confirmed the successful assembly of the 153-50 nanoparticles (NPs). The first large peak corresponds to the formed NPs and the small second peak corresponds to excess I53-50A component, which was still present in the assembly mix. The SEC profiles are similar for all three formed NPs.

[0267] Figure 19 depicts results of ELISA based antigenicity measurements of AMS0232 sElE2.v5- I53-50NP, Mosaic E1E2 v5-I53-50NP displaying AMS0232 SE1E2.V5-I53-50A, H77 SE1E2.V5-I53-50A, UKNP4.1.1 SE1E2.V5-I53-50A and AMS3a SE1E2.V5-I53-50A simultaneously and HepCon E1E2.V5-I53-50NP against several (conformational) bNAbs. These results show that the v5 design allows for two-component nanoparticle display of SE1E2. All tested nanoparticles, displaying either AMS0232 SE1E2.V5-I53-50A, AMS0232 SE1E2.V5- I53-50A, H77 E1E2.V5-I53-50A, UKNP4.1.1 ElE2.v5-I53-50A and AMS3a sElE2.v5-I53- 50A simultaneously or HepCon SE1E2.V5-I53-50A, were able to bind conformational bNAbs AR4A and AT1618, indicating display of properly folded E1E2 on the NPs. Additionally, the NPs were found to be able to bind the El targeting bNAb IGH505, indicating that this epitope is still accessible by bNAbs, even though El is located more internally on the NPs. Example 10: Antigenicity of UKNP4.L1 E2E1-I53-50A, UKNP4.L 1 sE!E2,v3, HepCon sElE2,v3-v5. HepCon SE1E2,V5-I53-50A and HepCon ElE2,v5-I53-50NP

[0268] Figure 20 depicts results of ELISA based antigenicity measurement of UKNP4.1.1 E2E1-I53- 50A, UKNP4.1.1 SE1E2.V3, HepCon sElE2.v3-v5, HepCon SE1E2.V5-I53-50A and HepCon ElE2.v5-I53-50NP in a heatmap depicting half maximum effective concentration (EC50) values against 17 bNAbs, CD81-Fc and AP33. Additionally, results are shown of bio-layer interferometry analyses of HepCon SE1E2.V5 and HepCon SE1E2.V5-I53-50NP in a bar graph depicting AUC-values.

[0269] Previously an antigen capable of binding several HCV bNAbs was identified, UKNP4.1.1 E2E1-I53-50A. (Capella-Pujol et al., 2023) However, this antigen did not bind conformational bNAbs AR4A and ATI 618, which indicates that this protein did not display proper native-like folding. Here, on the other hand it was seen that introduction of the v3 design into the UKNP4.1.1 sequence results in a native-like folded protein exhibiting a more potent and broader bNAb binding profile and strong binding to the conformational bNAbs AR4A and AT1618. Incorporation of this design in the designed HepCon sequence further increased binding strength which was maintained in the further stabilized v4 and v5 designs. Similar results were observed for the v5 designs for H77, UKNP4.1.1, AMS0230, and AMS3a. Additionally, bNAb binding was maintained when HepCon sElE2.v5 was displayed as an 153- 50A trimer and when these I53-50A trimers were displayed on an I53-50NP. In addition to ELISA characterization, bio-layer interferometry (BLI) was used to assess bNAb binding strength of HepCon sElE2.v5, HepCon ElE2.v5-I53-50A and HepCon ElE2.v5-I53-50NP. Here it was found that, while the ELISA EC50 remained relatively unchanged, the binding strength on BLI was increased by the NP display of the protein. This effect is likely due to the avidity effect achieved by NP display of an antigen, while one NP is able to bind multiple bNAbs simultaneously.

[0270] Figure 21 depicts results of ELISA based antigenicity measurement of UKNP4.1.1 E2E1-I53- 50A, UKNP4.1.1 SE1E2.V3, HepCon sElE2.v3-v5, HepCon sElE2.v5-I53-50A and HepCon ElE2.v5-I53-50NP in a heatmap depicting half maximum effective concentration (EC50) values against 17 gl-bNAbs. Additionally, results are shown of BLI analyses of HepCon sElE2.v5 and HepCon sElE2.v5-I53-50NP in a bar graph depicting AUC-values.

[0271] It was found that HepCon sElE2.v3 engaged several inferred gl-bNAbs. To assess its germline binding potential, the potency and breadth of binding were compared to a previous germline- targeting candidate immunogen UKNP4.1.1 E2E1-I53-50A (Capella-Pujol et al., 2023) and UKNP4.1.1 SE1E2.V3. It was found that UKNP4.1.1 SE1E2.V3 greatly improves potency and breadth of gl-bNAb binding compared to UKNP4.1.1 E2E1-I53-50A. Incorporation of the v3 design into the designed HepCon sequence yields HepCon SE1E2.V3, which greatly outperforms UKNP4.1.1 E2E1-I53-50A in potency and breadth of gl-bNAb binding (Figure 21). The gl-bNAb binding potential was also found to be maintained for the HepCon SE1E2 v4 and v5 designs. The HepCon SE1E2.V5 design can be applied for I53-50NP display of I53-50A trimers of SE1E2.V5. It was shown that gl-bNAb binding is maintained as an I53-50A trimer and as an I53-50NP on ELISA. When comparing gl-bNAb binding of HepCon SE1E2.V5 and HepCon SE1E2.V5-I53-50NP on BLI, HepCon SE1E2.V5-NP showed an avidity effect in binding, shown by a large increase in binding strength compared to HepCon SE1E2.V5. Additionally, the avidity effect was shown by binding of the NP to 1382_01_H05-gl and 1334_03_A04-gl on BLI, which bound neither the NP nor HepCon sElE2.v5 on ELISA.

[0272] Interestingly, the difference in binding strength on BLI was more significant for gl-bNAb binding compared to mature bNAb binding. These BLI results indicate that the observed avidity effect of the NP could be increasingly crucial in binding to gl-bNAb, and therefore a very important characteristic of an immunogen for a germline targeting vaccination strategy.

[0273] Example 11: Immunization study for AMS0232 sE!E2

[0274] Immunogenicity was tested in New Zealand White rabbits. To assess the importance of HCV antigen stability AMS0232 sElE2.vl was compared with sElE2.v5. Groups of six rabbits were immunized with 18 pg of AMS0232 sElE2.vl or 16 pg of AMS0232 SE1E2.V5 (equimolar SE1E2 amounts) at weeks 0,4 and 20 in squalene emulsion adjuvant. The schedule, adjuvant and antigen dosage (in molar amounts) were the same as in previous vaccine study in rabbits as reported in Sliepen et al. 2022 (Figure 22 A).

[0275] Rabbit sera was tested against membrane bound E1E2 (mbElE2). In short, HEK 293T were transfected with native AMS0232 sequence and cells were harvested and lysed after 72h. The ElE2-containing lysates were plated on ELISA plates previously coated with Galanthus nivalis lectin (GNL, Vector Laboratories) at 20 pg / mL. After blocking and washing, heat-inactivated sera was added in serial dilutions and binding was measured using a secondary antibody targeting mouse IgG. All animals bound native mbElE2 with an increase in binding observed after boosts from week 4 and week 20 (Fig 22 B). The same sera was tested against JunFos- coated plates. In short, previously GNL-coated ELISA plates were coated with a solution containing equimolar amounts of Jun and Fos peptides (Genscript). The ELISA followed similarly to the lysates. JunFos binding was strongly detected for the rabbits immunized with sElE2.vl, indicating high immunogenicity from the heterodimerization domains (Fig B). The sera from rabbits immunized with sElE2.v5 did not show any signal for JunFos detection. This finding highlights the need for removal of JunFos from the constructs, which can only be achieved by the set of stabilizing mutations described in the present disclosure as discussed and demonstrated in Example 7.

[0276] The neutralization activity of the sera was then tested against the sequence-matched HCVpp AMS0232 in neutralization assays. The neutralization capacity was compared from the rabbits in week 6 and week 22 (two-weeks post boost). After the second boost with SE1E2.V5 higher neutralization activity was seen (ID50 sera dilution being greater) as compared to sElE2.vl (Figure 22B, right panel). No cross-neutralization was observed, highlighting the need to display SE1E2.V5 in a multivalent display such as nanoparticle or mRNA.

[0277] Example 12: Immunization study in Hepcon and Mosaic groups

[0278] To assess breadth inducing potential of the HCV immunogens of the present disclosure, two extra groups of six rabbits were immunized in addition to the immunogenicity study described above. The first group, HepCon group, was immunized thrice with 18 pg HepCon SE1E2.V4 and once with 25 pg HepCon SE1E2.V5-I53-50NP at weeks 0, 4, 20 and 37, respectively. The second group, Mosaic group, was immunized thrice with a cocktail of 4.5 pg AMS0232 E1E2 v4, 4.5 pg H77 SE1E2.V4, 4.5 pg UKNP4.L1 sEIE2.v4 and 4.5 pg AMS3a SE1E2 v4 and once with 25 pg of a mosaic NP displaying AMS0232 SE1E2.V5-I53-50A, H77 SE1E2.V5- I53-50A, UKNP4.1.1 SE1E2.V5-I53-50A and AMS3a SE1E2.V5-I53-50A simultaneously at weeks 0, 4, 20 and 37, respectively.

[0279] Figure 23 depicts the achieved heterologous neutralization (left) and the heterologous and autologous neutralization (right) of the HepCon group and the Mosaic group at week 39.

[0280] Neutralization activity of the sera against was tested against 13 sequence-matched HCVpps. These 13 also contained sequence matched HCVpps for the strains AMS0232, H77, AMS3a and UKNP4.1.1, leaving a total of 9 HCVpps to be tested for heterologous neutralization activity for the Mosaic group and 13 HCVpps for the HepCon group. When comparing the geometric means of the neutralization ID50s, it was found that there was no significant difference in neutralization between the groups. When the sequence matched HCVpps were included for the strains AMS0232, H77, AMS3a and UKNP4.1.1, thus introducing autologous neutralization for the Mosaic group, no significant difference was observed in overall HCVpp neutralization for the complete panel of 13 HCVpps. These results indicate that HepCon is able to induce similar neutralization breadth as a cocktail and mosaic NP of four different strain specific immunogens covering genotypes 1, 3 and 4.

[0281] Being able to induce a similar neutralization breadth with only one immunogen compared to four different immunogens could greatly benefit the cost-effectiveness aspect of vaccine production. In large scale vaccine production, whether used as a protein subunit vaccine or as an mRNA vaccine, it would be less costly to produce one type of immunogen than it would be to produce and combine four types of immunogens. This would benefit the implementation of these vaccines in low-income environments, where there is a predominant need for an HCV vaccine.

[0282] Example 13: Expression of sE!E2,v5 on the membrane of mammalian cells

[0283] Efficient extracellular membrane expression is important for optimal immunogenicity of nucleic acid-based vaccines, such as mRNA vaccines. However, native E1E2 is mostly retained in the endoplasmic reticulum, leading to poor expression on the membrane of cells. In contrast, most viral glycoprotein spikes, including those of SARS-CoV-2, are efficiently expressed on the surface of infected cells. The multivalent presentation provided by efficient extracellular expression of the SARS-CoV-2 spike probably explains why the mRNA SARS-CoV-2 vaccines, which express the membrane-anchored SARS-CoV-2 spike, induce a strong humoral immune response. Therefore, it was tested if fusing SE1E2.V5 to the transmembrane domain (TMD) of the SARS-CoV-2 spike could enhance presentation of E1E2 on the extracellular membrane.

[0284] Native AMS0232-derived E1E2 and AMS0232-derived SE1E2.V5 were expressed fused to the SARS-CoV-2 TMD (SEQ ID No. 43). AR4A antibody binding was then measured using flow cytometry to determine the expression of native-like E1E2 on the outer membrane of the transfected cells.

[0285] Figure 24 depicts the flow cytometry analysis of mammalian cells expressing either native E1E2 or SE1E2.V5-TMD. The binding signal of AR4A was found to be strongly enhanced for SE1E2.V5-TMD, indicating more efficient expression of native-like folded E1E2 on the extracellular membrane of these cells. This enhanced expression shows that SE1E2.V5-TMD is a promising candidate for nucleic acid-based vaccines and mRNA vaccines in particular.

[0286] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0287] The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure.

[0288] Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[0289] SEQUENCE LISTING

[0290] ALIGNMENT 1

[0291] H77. E1E2. AF009606 YQVRNSSGLYHVTNDCPNSSIVYEAADAILHTPGCVPCVREGNASRCWVAVTPTVATRDG

[0292] AMSO232_E1E2 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTPTVATRDG

[0293] AMS0232_sElE2 . vl YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTPTVATRDG

[0294] AMS0232_sElE2 . v2 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTPTVATRDG

[0295] AMS0232__sElE2 . v3 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTPTVATRDG

[0296] AMS0232_sElE2 . v4 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTCTVATRDG AMS0232_sElE2 , v5 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTCTVATRDG AMS0232_sElE2 . v6 YQVRNSTGLYHVTNDCPNSSIVYETADAILHTPGCVPCVREGNASRCWVPMTCTVATRDG

[0297] HepCon sE!E2 . v3 VEVRNSSGLYHVTNDCPNSSIVYEADDAILHLPGCVPCVREGNASRCWVAVTPTVAARDA

[0298] HepCon sElE2 . v4 VEVRNSSGLYHVTNDCPNSSIVYEADDAILHLPGCVPCVREGNASRCWVAVTCTVAARDA

[0299] HepCon_ElE2 GAPTTGLRRHVDLLVGAATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTVQDCNCSIYPG

[0300] H77. ElE2_AF009606 KLPTTQLRRHIDLLVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTTQDCNCSIYPG

[0301] AMSO232_E1E2 KLPATQLRRHIDLLVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTTQDCNCSIYPG

[0302] AMS0232_sElE2 . vl KLPATQLRRHIDLLVGSATLCSALYVGDLCGSVFLVGQLFTFSPRRHWTTQDCNCSIYPG __

[0303] HepCon E1E2 HITGHRMAWDMMMNWSPTTALWAQLLRI PQAILDI IAGAHWGVLAGLAYFSMVGNWAKV

[0304] H77_ElE2_AF009606 HITGHRMAWDMMMNWSPTAALWAQLLRIPQAIMDMIAGAHWGVLAGIAYFSMVGNWAKV

[0305] AMSO232_E1E2 HVTGHRMAWDMMMNWS PTTALWAQLLRI PQAILDMIAGAHWGVLAGLAYFSMVGNWAKV

[0306] AMS0232_sElE2 . vl HVTGHRMAWDMMMNWSPTTALWAQLLRIPQAILDMIAPGGRIARLEEKVKTLKAQNSEL

[0307] AMS0232_sElE2 . v2 HVTGHRMAWDMMMNWSPTTALWAQLLRIPQAILDMIAPGGRIARLEEKVKTLKAQNSEL

[0308] AMS0232_sElE2 . v3 HVTGHRMAWDMMMNPSPTTALWAQLLRIPQAILDMIAPGGRIARLEEKVKTLKAQNSEL

[0309] AMS0232_sElE2 . v4 HVTGHRMAWDMMMNPSPTTALWAQLLRIPQAILDMIAPGGRIARLEEKVKTLKAQNSEL

[0310] AMS0232_sElE2 . v5 HVTGHRMAWDMMMNPS PTTALWAQLLRI PQAILDMIAPGG -

[0311] AMS0232_sElE2 , v6 HVTGHRMAWDMMMNPSPTTALWAQLLRCPQAILDMIAPGG - HepCon SE1E2 . V3 HITGHRMAWDMMMNPSPTTALWAQLLRI PQAILDI IAPGGRIARLEEKVKTLKAQNSEL

[0312] HepCon_sElE2 . v4 HITGHRMAWDMMMNPSPTTALWAQLLRI PQAILDI IAPGGRIARLEEKVKTLKAQNSEL

[0313]

[0314] HepCon_ElE2 NGSWHINRTALNCNDSLNTGFIAGLFYTHKFNSSGCPERLASCRPLTAFDQGWGPITYAN

[0315] H77_E1E2_AF009606 NGSWHINSTALNCNESLNTGWLAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYAN

[0316] AMS O232_E1E2 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0317] AMS0232_sElE2 . vl NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0318] AMS 0232_sElE2 . v2 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0319] AMS0232_sElE2. v3 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0320] AMS 0232_sElE2 , v4 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0321] AMS 0232_sElE2 , v5 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0322] AMS0232_sElE2 . v6 NGSWHINRTALNCNTSLETGWIAGLIYLNKFNSSGCPERMASCRPLADFAQGWGPISYAN

[0323] HepCon_sElE2 . v3 NGSWHINRTALNCNDSLNTGFIAGLFYTHKFNSSGCPERLASCRPLTAFDQGWGPITYAN

[0324] HepCon_sElE2 . v4 NGSWHINRTALNCNDSLNTGFIAGLFYTHKFNSSGCPERLASCRPLTAFDQGWGPITYAN HepCon_ElE2 I SGPS DDRP YCWHYPPRPCGI VPARSVCGPVYCFT PSPVWGTTDRSGVPT YTWGENET D

[0325] H77_E1E2_AF009606 GSGL- DERP YCWHYPPRPCGI VPAKSVCGPVYC FT PSPVWGTTDRSGAPT YSWGANDT D

[0326] AMS O232_E1E2 GSGP-DHRPYCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0327] AMS 0232_sElE2 . vl GSGP-DHRPYCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0328] AMS 0232_sElE2 . v2 GSGP-DHRPYCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0329] AMS 0232_sElE2. v3 GSGP-DHRPYCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0330] AMS 0232_sElE2. v4 GSGP- DHRP YCWHYPPRPCGI VSAKSVCGPVYCFT PSPVWGTTNKLGAPT YSWGENET D

[0331] AMS 0232_sElE2 .v5 GSGP-DHRPYCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0332] AMS 0232_sElE2 . v6 GSGP- DHRP YCWHYPPKPCGIVSAKSVCGPVYCFTPSPVWGTTNKLGAPTYSWGENETD

[0333] HepCon_sElE2 . v3 I SGPSDDRP YCWHYPPRPCGI VPARSVCGPVYCFT PSPVWGTTDRSGVPT YTWGENET D

[0334] HepCon_sElE2 . v4 I SGPS DDRP YCWHYPPRPCGI VPARSVCGPVYCFT PSPVWGTTDRSGVPT YTWGENET D

[0335]

[0336] H77 numbering : 720 730 740 746 features : &H# I ###### ##♦ I #### ##### I ##### I ##♦##### ###

[0337] HepCon_ElE2 WEYWLLFLLLADARVCACLWMMLLISQAEAA - -

[0338] H77_E1E2_AF009606 WE Y WLLFLLLADARVC SCLWMMLLISQAEA- - - - -- -- - - -

[0339] AMSO232__E1E2 WE YWLLFLLLADARVC SCLWMMLLISQAEA- -

[0340] AMS0232_sElE2 . vl GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0341] AMS0232__sElE2 . v2 GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0342] AMS0232_sElE2 . v3 GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0343] AMS0232_jsElE2 . v4 GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0344] AMS0232_sElE2 . v5

[0345] AMS0232_sElE2 .v6 c -

[0346] HepCon_sElE2 . v3 GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0347] HepCon_sElE2 .v4 GGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY

[0348]

[0349] 68

[0350] HecUn jESft . S ........ .......... --RPKBS.BET HiTQlUAGHt TSQFM.FTF iAJ.Qt.ZC.X’J I HeptpQtt*: .s-:y-w. . -PRRRSRET HV-rtKUfil- TSCFAUF-F aAf QNTQ.XN T

[0351]

[0352]

[0353]

Claims

CLAIMS:

1. A recombinant Hepatitis C Virus glycoprotein comprising a heterodimer of El and E2 glycoproteins bearing at least 60% homology to SEQ ID No. 1, an engineered furin cleavage site and a hydrophilic amino acid linker, wherein the El and E2 glycoproteins partially or completely lack their respective transmembrane domain(s).

2. The recombinant Hepatitis C Virus glycoprotein of claim 1, wherein the furin cleavage site replaces a natural protease site between the El and E2 glycoproteins.

3. The recombinant Hepatitis C Virus glycoprotein of any of claim 1 or 2, wherein the furin cleavage site comprises the sequence RRRRRR.

4. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-3, wherein the hydrophilic amino acid linker replaces residues 261-294 of the putative fusion peptide (pFP) in the El glycoprotein, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

5. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-4, wherein the hydrophilic amino acid linker comprises the sequence GGSGGSGGS.

6. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-5, wherein the El and E2 glycoproteins completely lack their respective transmembrane domain(s).

7. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-6, wherein the transmembrane domain(s) of El and E2 are replaced by leucine zipper(s).

8. The recombinant Hepatitis C Virus glycoprotein of claim 7, wherein the leucine zipper(s) is selected from a group comprising human-derived Jun / Fos leucine zippers and SYNZIP leucine zippers.

9. The recombinant Hepatitis C Virus glycoprotein of claim 8, wherein the leucine zipper(s) is human-derived Jun / Fos leucine zippers, wherein Fos and Jun chains of the Jun / Fos leucine zipper(s) partially or completely replace transmembrane membrane domains of the El and E2 glycoproteins.

10. The recombinant Hepatitis C Virus glycoprotein as claimed any of claims 1-9, further comprising at least one mutation selected from a group comprising P244C, W326P, I340C, V622A, L682P, T687C and W716C, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

11. The recombinant Hepatitis C Virus glycoprotein of claim 1-10, comprising mutations W326P, V622A, L682P, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

12. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-11, comprising mutations P244C / T687C and / or I340C / W716C, wherein said mutations result in one or more engineered disulphide bonds, wherein the numbering is according to the reference H77 sequence having SEQ ID No. 3.

13. The recombinant Hepatitis C Virus glycoprotein of any of claims 1-12, based on HCV strain(s) selected from a group comprising H77, AMS0230, AMS0232, AMS3a and UKNP4.1.1 or HCV consensus sequence.

14. An antigenic cocktail comprising the recombinant Hepatitis C Virus glycoprotein of any of claims 1-13, based on at least two HCV strain(s) that demonstrate at least about 60% homology between their E1 / E2 sequences.

15. A nucleic acid encoding the recombinant Hepatitis C Virus glycoprotein(s) of any of claims 1-14.

16. A nanoparticle or membrane-anchored conjugate comprising a nanoparticle or membrane-anchoring transmembrane domain(s) and the recombinant Hepatitis C Virus glycoprotein(s) of any of claims 1-14 or the nucleic acid of claim 15.

17. The nanoparticle or membrane-anchored conjugate of claim 16, wherein the nanoparticle is selected from a group comprising I53-50A, ferritin, liposomes; and / or wherein the membrane-anchoring TMD is TMD of SARS-CoV-2.

18. A vector comprising the nucleic acid of claim 15.

19. An isolated or recombinant eukaryotic or prokaryotic host cell comprising the vector of claim 18.

20. A pharmaceutical composition comprising the recombinant Hepatitis C Virus glycoprotein of any one of claims 1-13 or the antigenic cocktail of claim 14 and a pharmaceutically acceptable carrier, excipient, or diluent, and optionally, an adjuvant.

21. The recombinant Hepatitis C Virus glycoprotein(s) of any of claims 1-14, for use in the treatment or prophylaxis of HCV infection.

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