This invention relates to polypeptides, particularly immunogenic polypeptides, and associated viral vectors for use in a vaccine against HCV infection.

With 3 - 4 million new infections occurring annually, hepatitis C virus (HCV) is a major global health problem. There is increasing evidence to suggest that HCV will be highly amenable to a vaccine approach, and despite advances in treatment, a vaccine remains the most cost-effective and realistic means to significantly reduce the worldwide mortality and morbidity associated with persistent HCV infection. Currently, there is no licensed vaccine for HCV and treatment is based on pegylated- interferon-a (IFNa) and the nucleoside analogue ribavirin. This is expensive, relatively toxic, prolonged (24 - 48 weeks) and leads to a sustained virological response (SVR) in only 50 - 60% of patients, depending on the infecting genotype .

The characteristic of HCV that will offer the biggest problem for vaccine design is its viral variability. With sequence diversity believed to be 10 times that of human immunodeficiency virus (HIV), HCV strains are classified into 7 genotypes (numbered 1 - 7), which differ at 3 1 - 34% of their nucleotide positions, and which can be further divided into over 100 subtypes. This diversity is largely due to a lack of proofreading capacity of the viral RNA-dependent polymerase (NS5b) used by HCV during replication; therefore, HCV exists within a host as a constantly evolving population of closely related but diverse quasispecies. Attempts are being made to develop vaccines that are based on conserved outer surface features, such as the envelope glycoproteins E l and E2, which are believed to be essential for the infection of liver cells. Alternative strategies have been proposed, which differ from a conventional vaccine by seeking to induce a T cell immune response using viral vectors to express large parts of HCV in a cell for MHC presentation. Synthetic HCV peptides have been used to induce T-cell immunity through direct presentation on antigen-presenting cells. However, peptide vaccines are HLA-specific and target only a selected subset of epitope sequences within HCV, limiting their breadth and coverage within the population. Plasmids encoding HCV NS3/4a (ChronVac-c) or core/E l/E2 (CICGB-230) have shown some efficacy as potential therapeutic vaccines for HCV, but there is no published data on their effectiveness as prophylactic vaccines. Genotypes la and lb account for over 60% of chronic HCV infections worldwide, and much vaccine development to date has concentrated on raising an immune response to these genotypes due to their prevalence. However, a need exists to provide effective pan-genotypic HCV T cell vaccine in humans, which can provide protection against a larger range of HCV genotypes.

Therefore, an aim of the present invention is to provide an improved vaccine for HCV infection.

According to a first aspect of the invention, there is provided a polypeptide comprising a plurality of conserved peptide sequences, or variants thereof, wherein at least one of the conserved sequences is conserved across:

i) HCV genotypes la and lb;

ii) HCV genotypes 1 and 3 ; or

iii) HCV genotypes 1 to 6; and

wherein at least one of the conserved peptide sequences comprises at least part of a sequence of a non-structural protein of the HCV genotypes.

The invention advantageously provides a novel alternative and safer approach to vaccination whereby T cells can be induced to the relatively conserved internal (non- structural) antigens of the virion. The use of specially selected conserved viral segments from the non-structural proteins can provide protection against multiple or all genotypes.

In one embodiment, the polypeptide is a fusion polypeptide. The polypeptide may not be a wild-type polypeptide. The polypeptide may be synthetic/artificial, for example, the polypeptide may not exist in nature. In one embodiment, the polypeptide may not comprise a complete gene sequence. The polypeptide may consist essentially of conserved peptide sequences. In one embodiment, the polypeptide is a recombinant polypeptide, such as a recombinant fusion polypeptide. The term "fusion polypeptide" used herein is understood to mean a polypeptide comprising a combination of sequences from different gene products (for example different HCV non-structural proteins) or combinations of sequences from the same gene product (for example a single HCV non-structural protein), wherein the sequences are from distinct/separate regions of the wild-type gene product. For example the fusion polypeptide may comprise combinations of sequences which are normally separated by other sequence segments in wild-type, and the separating sequence(s) have been removed.

The term "conserved peptide sequence" or "conserved segment" used herein is defined as a sequence that is found in more than one genotype or within variant populations of the same genotype, whereby the sequence is identical or highly similar between the genotypes or variants within a genotype. Conserved peptide sequences may be identified using an algorithm which uses a sliding window-based method. In one embodiment, a conserved segment (or otherwise termed conserved peptide sequence) is where the homology of any window of 20 amino acids is at least 90% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 91 % in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 92% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 93% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 94% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 95% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 98% in an alignment of amino acid sequences. In another embodiment, a conserved segment is where the homology of any window of 20 amino acids is at least 99% in an alignment of amino acid sequences. The skilled person will understand that the 20 amino acid window uses an average homology/identity across the 20 amino acid window. Therefore, it is possible that a sequence of less than 20 amino acids may be identified as a conserved peptide sequence within the above definition. The plurality of conserved peptide sequences may comprise 5 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 6 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 7 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 8 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 9 or more conserved peptide sequences. The plurality of conserved sequences may comprise 10 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 1 1 or more conserved peptide sequences. The plurality of conserved peptide sequences may comprise 15 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 20 or more conserved peptide sequences. In another embodiment, the plurality of conserved peptide sequences may comprise 30 or more conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 1 1 conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 12 conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 24 conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 27 conserved peptide sequences. In one embodiment, the plurality of conserved peptide sequences consists of about 30 conserved peptide sequences. In one embodiment at least one conserved peptide sequence is conserved across HCV genotypes la and lb. In one embodiment at least two conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least three conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least four conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least five conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least six conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 7 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 8 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 9 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 10 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 1 1 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 12 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 20 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 25 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 27 conserved peptide sequences are conserved across HCV genotypes la and lb. In another embodiment at least 30 conserved peptide sequences are conserved across HCV genotypes la and lb.

In one embodiment at least one conserved peptide sequence is conserved across HCV genotypes 1 and 3. In one embodiment at least two conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least three conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least four conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least five conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least six conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 7 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 8 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 9 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 10 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 1 1 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 12 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 20 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 25 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 27 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In another embodiment at least 30 conserved peptide sequences are conserved across HCV genotypes 1 and 3. In one embodiment at least one conserved peptide sequence is conserved across all of HCV genotypes 1 to 6. In one embodiment at least two conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least three conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least four conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least five conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least six conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 7 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 8 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 9 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 10 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 1 1 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 12 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 20 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 25 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 27 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6. In another embodiment at least 30 conserved peptide sequences are conserved across all of HCV genotypes 1 to 6.

The plurality of conserved peptide sequences may be derived from distinct regions of sequence relative to each other (i.e. not-naturally concurrent) . For example, in the wild-type genotype the conserved sequences may be separated in the wild-type genotypes by variable/non-conserved sequences. The plurality of conserved peptide sequences may not, or may not significantly, overlap with each other. Two or more, or all, of the plurality of conserved peptide sequences may be directly joined together in the polypeptide, for example not comprising any non-conserved/variable residues there between. The polypeptide sequence may not be found in nature. The polypeptide may not comprise non-conserved sequences or residues. The conserved peptide sequences may not be distanced apart by more than 1 , 2, 3, 4, or 5 residues in the polypeptide sequence, for example in embodiments where there are linker/junction residues between the conserved peptide sequences. Alternatively, the conserved peptide sequences may not be distanced apart by more than 6, 7, 8, 9, or 10 residues in the polypeptide sequence, for example in embodiments where there are linker/junction residues between the conserved peptide sequences. The polypeptide may not comprise non-conserved sequences longer than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In one embodiment, linker residues may be provided between one or more, or all, conserved peptide sequences (e.g. providing junctions between the conserved peptide sequences in the polypeptide). The linker residues may comprise random amino acid sequences, or amino-acids that have been selected to be non-immunogenic based on epitope prediction computer programs or experiments in animal models. For example, a linker may not be considered if it is predicted or known to be an epitope (i.e. in order to avoid an immune response to epitopes, e.g. artificial epitopes, not found in HCV. The linker may be flexible. The linker may comprise or consist of K, G, P or S amino acid residues, or combinations thereof. In one embodiment, the linker may comprise or consist of G and/or P amino acid residues. The linker residues may be between 1 and 10 amino acids in length. In another embodiment, the linker residues may be between 2 and 8 residues in length. In another embodiment, the linker residues may be between 1 and 6 residues in length. The conserved peptide sequences may be distanced apart by between 1 and 10 residues in the polypeptide sequence, for example in embodiments where there are linker/junction residues between the conserved peptide sequences.

In one embodiment, the polypeptide may consist essentially of conserved peptide sequences and one or more linkers, optionally wherein the one or more linkers are disposed between adjacent conserved peptide sequence .

The conserved peptide sequences may be selected from any of the group comprising SEQ ID NO : 1 to 1 17; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO : 1 to 38; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO : 1 to 6; 7 or 8; 9; 10 or 1 1 ; 12; 13 or 14; 15 or 16; 17; 18 or 19; 20; 21 or 22; 23 to 26; 27 or 28; 29 to 34; 35 or 36; 37; and 38; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 1; 7 or 8; 13 or 14; 15 or 16; 17; 18 or 19; 20; 23; 33; 35 or 36; and 37; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 39 to 80; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 39 or 40; 41 or 42; 43 to 47; 48 or 49; 50 or 51; 52 or 53; 54 or 55; 56 or 57; 58 or 59; 60; 61 or 62; 63; 64; 65 or 66; 67 or 68; 69 or 70; 71; 72; 73 or 74; 75; 76 or 77; 78 or 79; and 80; variants thereof or combinations thereof.

In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 39 or 40; 47; 48 or 49; 50 or 51; 52 or 53; 54 or 55; 56 or 57; 61 or 62; 69 or 70; 76 or 77; and 78 or 79; variants thereof or combinations thereof.

In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 44; and 81-117; variants thereof or combinations thereof.

In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 44; 81 or 82; 83-85; 86 or 87; 88 or 89; 90 or 91; 92 or 93; 94 or 95; 96; 97 or 98; 99 or 100; 101 or 102; 103 or 104; 105; 106 or 107; 108; 109; 110 or 111; 112; 113; 114 or 115; and 116 or 117; variants thereof or combinations thereof.

In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NO: 81 or 82; 85; 86 or 87; 90 or 91; 92 or 93; 94 or 95; 97 or 98; 99 or 100; 101 or 102; 103 or 104; 106 or 107; and 116 or 117; variants thereof or combinations thereof.

In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NOs: 126 to 149; variants thereof or combinations thereof. In another embodiment, the conserved peptide sequences may be selected from any of the group comprising SEQ ID NOs : 150 to 193 ; variants thereof or combinations thereof.

Some or all of the conserved peptide sequences may be derived from non-structural HCV proteins (i.e. comprising a sequence identical to, or substantially similar to a sequence of a non-structural HCV protein). The non-structural proteins may comprise any of NS2, NS3, NS4A, NS4B, NS5A, and NS5B; or combinations thereof. In another embodiment, the non-structural proteins may comprise any of NS3, NS4B, and NS5B; or combinations thereof. One or more of the conserved peptide sequences may also be derived from the HCV core protein (i.e . comprising a sequence identical to, or substantially similar to a sequence of the core HCV protein). One or more of the conserved peptide sequences may also be derived from the HCV E l and E2 protein. For example, comprising a sequence identical to, or substantially similar to a sequence of the E l and, or E2 HCV protein or fragments thereof. Fragments may be at least the minimum number of residues for specific T cell recognition.

The polypeptide may comprise or consist of the sequence of SEQ ID NO: 1 18; or variants thereof. In one embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 1 19; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 120; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 121 or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 122; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 123 ; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 124; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 125 ; or variants thereof.

The polypeptide may comprise or consist of the sequence of SEQ ID NO : 1 18 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In one embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO : 1 19 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 120 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO : 121 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 122 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 123 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 124 without the TPA peptide adjuvant, or with an alternative peptide adjuvant such as the shark invariant chain; or variants thereof. In another embodiment, the polypeptide may comprise or consist of the sequence of SEQ ID NO: 125 without the TPA peptide adjuvant, or with an alternative peptide adjuvant, such as the shark invariant chain; or variants thereof. Embodiments of the polypeptide without a peptide adjuvant may also not comprise the associated first (N-terminal) linker sequence.

The polypeptide may comprise any one of GTl short A TPA described herein. In another embodiment, the polypeptide may comprise GTl long D TPA described herein. In another embodiment, the polypeptide may comprise GTl &3_short_A_TPA described herein.

In another embodiment, the polypeptide may comprise GT l &3_long_D_TPA described herein. In another embodiment, the polypeptide may comprise GT1 - 6_short_A_TPA described herein. In another embodiment, the polypeptide may comprise GTl -6_long_D_TPA described herein. In another embodiment, the polypeptide may comprise GT l -6_long_D_TPA_no linkers described herein. In another embodiment, the polypeptide may comprise GT l -6_long_D_Non-TPA_linkers described herein. Variants of the above polypeptides may also be provided with or without the TPA peptide adjuvant, or with an alternative peptide adjuvant. In one embodiment, the polypeptide may consist essentially of conserved peptide sequences and a peptide adjuvant. In one embodiment, the polypeptide may consist essentially of conserved peptide sequences, one or more linkers, and a peptide adjuvant. The one or more linkers may be disposed between adjacent conserved peptide sequence. The peptide adjuvant may be N-terminal.

Variants of the polypeptide may comprise or consist of a sequence having at least 80% identity with the polypeptide of the invention, for example any one of SEQ ID NO : 1 18 to 125. Alternatively, variants of the polypeptide may comprise or consist of a sequence having at least 85 % identity with the polypeptide of the invention. Variants of the polypeptide may comprise or consist of a sequence having at least 90% identity with the conserved sequence . Variants of the polypeptide may comprise or consist of a sequence having at least 95 % identity with the polypeptide of the invention. Variants of the polypeptide may comprise or consist of a sequence having at least 98% identity with the polypeptide of the invention. Variants of the polypeptide may comprise or consist of a sequence having at least 99% identity with the polypeptide of the invention. Variants of the polypeptides of SEQ ID NO: 1 18 to 125 may include the consensus sequence of one or more conserved peptide sequences instead of the specific patient sequence, or vice versa.

Variants of conserved peptide sequences may comprise or consist of a sequence having at least 80% identity with the conserved peptide sequence. Alternatively, variants of conserved peptide sequences may comprise or consist of a sequence having at least 85% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 90% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 95% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 98% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a sequence having at least 99% identity with the conserved peptide sequence. Variants of conserved peptide sequences may comprise or consist of a truncated sequence of the conserved peptide sequences. For example any one or more of the sequences of SEQ ID NOs: 1 - 1 17, herein may be truncated and still provide immunogenicity in the polypeptide. The truncated sequence may comprise a sufficient number of amino acids to form a recognisable epitope (e.g. at least the minimum number of residues for specific T cell recognition) from a sequence within any one of the sequences of SEQ ID NOs: 1 - 1 17. The truncated sequence may comprise at least 7 amino acids of the sequences of SEQ ID NOs: 1 - 1 17. Alternatively, the truncated sequence may comprise at least 8 amino acids of the sequences of SEQ ID NOs: 1 - 1 17. Alternatively, the truncated sequence may comprise at least 9, 10, 1 1 or 12 amino acids of the sequences of SEQ ID NOs: 1 - 1 17. Multiple truncated sequences may be provided within one of the conserved peptide sequences of SEQ ID NOs: 1 - 1 17. In one embodiment, any one of the conserved peptide sequences of SEQ ID NOs: 1 - 1 17 may be varied, for example by residue substitution, addition or deletion. The variant conserved peptide sequences may still function to provide recognisable HCV epitopes. The skilled person will understand that natural variation exists in any given population and that these variants may have some sequence variation with the consensus sequence, or example patient sequences provided in SEQ ID NOs: 1 - 1 17. Therefore, the variant conserved peptide sequences may have at least 70% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 74% sequence identity with any one of SEQ ID NOs : 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 75% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 79% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 80% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 82% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 83% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 85% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 88% sequence identity with any one of SEQ ID NOs : 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 90% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 92% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 95 % sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 98% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 99% sequence identity with any one of SEQ ID NOs: 1 - 1 17. In another embodiment, the variant conserved peptide sequences may have at least 99.5% sequence identity with any one of SEQ ID NOs: 1 - 1 17.

Reference to sequence "identity" used herein may refer to the percentage identity between two aligned sequences using standard NCBI BLASTp parameters (http://blast.ncbi.nlm.nih.gov).

The conserved peptide sequences may vary in length, with the minimum length being defined as the minimum number of residues required to form a recognisable epitope. Therefore the conserved peptide sequence may be from about 7 to 250 amino acids in length, or more . For example, at least one conserved peptide sequence may be at least about 7 amino acids in length. In another embodiment, at least one conserved peptide sequence may be at least about 8 amino acids in length. In another embodiment, at least one conserved peptide sequence may be at least about 10 amino acids in length. In another embodiment, at least one conserved peptide sequence may be at least about 15 amino acids in length. In another embodiment, at least one conserved peptide sequence may be at least about 20 amino acids in length. In another embodiment, at least one conserved peptide sequence may be at least about 30 amino acids in length. In one embodiment, at least one conserved peptide sequence may be between about 20 and about 220 amino acids in length. In one embodiment, at least one conserved peptide sequence may be no more than about 300 amino acids in length. In another embodiment, at least one conserved peptide sequence may be no more than about 250 amino acids in length. In another embodiment, at least one conserved peptide sequence may be no more than about 200 amino acids in length. In another embodiment, at least one conserved peptide sequence may be no more than about 150 amino acids in length. In another embodiment, at least one conserved peptide sequence may be no more than about 100 amino acids in length. In another embodiment, at least one conserved peptide sequence may be no more than about 50 amino acids in length. The conserved peptide sequences may be an average length of between about 20 and about 80 amino acids in a population of conserved peptide sequences.

In some embodiments of the invention, the polypeptide of the invention may further comprise a peptide adjuvant, such as a TPA (tissue plasminogen activator) sequence, or functional variants thereof. The TPA may comprise or consist of the sequence: MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR (SEQ ID NO: 194), or a functional variant thereof. In one embodiment, the peptide adjuvant may comprise a Shark invariant chain, for example of the sequence SLLWGGVTVLAAMLIAGQVASSVVFLV (SEQ ID NO: 195), or a functional variant thereof. The peptide adjuvant may be N-terminal on the polypeptide of the invention. A functional variant of a peptide adjuvant may be a truncated or mutated peptide variant, which can still function as an adjuvant, for example a truncated or mutated variant of the TPA or shark invariant chain, which still function as an adjuvant. The skilled person will appreciate that 1 , 2, 3, 4, 5 or more amino acid residues may be substituted, added or removed without affecting function. For example, conservative substitutions may be considered.

In one embodiment the polypeptide is an isolated polypeptide. In another embodiment, the polypeptide may be encoded in nucleic acid or in a viral vector.

Combinations of different polypeptides according to the invention may be provided as a vaccine. For example, a prime and/or boost vaccine formulation may comprise nucleic acid or viral vector encoding two or more polypeptides of the invention, which may be different relative to each other.

The polypeptide may be used in a vaccine in combination with another therapeutically or prophylactically active ingredient. The polypeptide may be used in a vaccine in combination with an adjuvant.

The polypeptide, nucleic acid encoding the polypeptide, or associated viral particle may be provided in a pharmaceutically acceptable carrier. According to another aspect of the invention there is provided a composition comprising a plurality of different polypeptides according to the invention, optionally wherein the composition is a pharmaceutically acceptable composition. According to another aspect of the invention there is provided a nucleic acid comprising a sequence encoding a polypeptide according to the invention herein.

The nucleic acid may be a plasmid vector for vaccination. The nucleic acid may comprise viral vector sequences.

According to another aspect of the invention there is provided a viral vector comprising the nucleic acid according to the invention herein.

The viral vector may comprise a virus. The viral vector may comprise an adenovirus, such as a human or simian adenovirus. The viral vector may comprise an adenovirus when used in a prime vaccine of a prime boost regime. The viral vector may comprise ChAdOxl (a group E simian adenovirus, like the AdCh63 vector used safely in malaria trials) or ChAdOx2 (as described in Morris et al 2016. Future Virol 1 1 (9), pp. 649-659) The viral vector may comprise AdCh63. The viral vector may comprise AdC3 or AdH6. The viral vector may be a human serotype. The viral vector may comprise Modified Vaccinia Ankara (MVA). The viral vector may comprise MVA when used as a vaccine boost in a prime boost regime . The viral vector may comprise Adeno-associated virus (AAV) or lentivirus. The viral vector may be an attenuated viral vector. The polypeptide sequence of the invention may be cloned into any suitable viral vector that is known to elicit good immune response . Suitable viral vectors have been described in Dicks et al (Vaccine. 2015 Feb 25;33(9): 1121-8. doi: 10.1016/j .vaccine.2015.01.042. Epub 2015 Jan 25), Antrobus et al (Mol Ther. 2014 Mar;22(3):668-74. doi: 10.1038/mt.2013.284. Epub 2013 Dec 30.), and (Warimwe et al. (Virol J. 2013 Dec 5; 10:349. doi: 10.1186/1743-422X-10-349), which are incorporated herein by reference.

According to another aspect of the invention there is provided a composition comprising one or more of:

-the polypeptide according to the invention;

-the nucleic acid according to the invention; and -the viral vector according to the invention.

The composition may be immunogenic, for example in a mammal, such as a human. The composition may comprise a pharmaceutically acceptable carrier. The composition may be a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be for use in the prophylaxis or treatment of HCV infection.

According to another aspect of the invention there is provided a method of treatment or prophylaxis of HCV infection comprising the administration of:

-the polypeptide according to the invention;

-the nucleic acid according to the invention;

-the composition according to the invention or

-the viral vector according to the invention.

The method of treatment or prophylaxis of HCV infection may be a method of vaccination.

According to another aspect of the invention there is provided an agent for use in the prophylaxis or treatment of HCV infection, the agent comprising or consisting of:

-the polypeptide according to the invention;

-the composition according to the invention;

-the nucleic acid according to the invention; or

-the viral vector according to the invention.

According to another aspect of the invention there is provided the polypeptide according to the invention; the composition according to the invention; the nucleic acid according to the invention; or the viral vector according to the invention; for use in, or as, a vaccine.

According to another aspect of the invention there is provided a vaccine comprising the polypeptide of the invention comprising or consisting of:

-the polypeptide according to the invention;

-the composition according to the invention;

-the nucleic acid according to the invention; or -the viral vector according to the invention.

The vaccine may be a prime vaccine. The vaccine may be a boost vaccine . Where a boost vaccine is provided following a prime vaccine, the polypeptide may be different. For example, the polypeptide may comprise a re-ordered sequence of conserved peptide sequences. The conserved peptide sequences may be identical, but the order in which they are provided in the polypeptide may be changed. Therefore, the invention herein provides any of the sequences/embodiments of the invention wherein the order in which conserved peptide sequences are provided may be changed. Such embodiments may also include re-ordered or differed linker/junction sequences.

Advantageously, the re-ordering of the conserved peptide sequences of the polypeptide between prime and boost vaccines can avoid the provision of "false" epitopes formed across junctions of one conserved peptide sequence with another conserved peptide sequence, i.e. the same junction may not occur in the re-ordered polypeptide.

According to another aspect of the invention, there is provided a polypeptide according to the invention for use in, or as, a vaccine .

According to another aspect of the invention, there is provided a prime boost vaccination kit comprising

-a prime vaccination according to the invention;

-a boost vaccination according to the invention.

The prime and boost vaccinations may be different. The prime and boost vaccination may differ in the polypeptide sequence. The prime and boost vaccination may comprise different viral vectors. The term "immunogenic", when applied to the polypeptide or composition of the present invention means capable of eliciting an immune response in a human or animal body. The immune response may be protective .

The term "isolated", when applied to the polypeptide of the present invention means a polypeptide: (i) encoded by nucleic acids using recombinant DNA methods; or (ii); synthesized by, for example, chemical synthetic methods; or (iii) separated from naturally-occurring biological materials, and then purified using polypeptide analytical procedures; or (iv) associated with chemical moieties (e.g. peptides, carbohydrates, fatty acids, and the like) other than those associated with the antigenic peptide in its naturally-occurring state; or (v) that do not occur in nature . An isolated polypeptide of the invention includes a polypeptide expressed from a nucleotide sequence encoding the polypeptide, or from a recombinant vector containing a nucleotide sequence encoding the polypeptide. An isolated polypeptide of the invention may include a polypeptide expressed from a virus-like particle.

The term "protective" means prevention of a disease, a reduced risk of disease infection, transmission and/or progression, reduced severity of disease, a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.

The term "prophylaxis" means prevention of or protective treatment for a disease . The prophylaxis may include a reduced risk of disease infection, transmission and/or progression, or reduced severity of disease . The term "treatment", means a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.

According to another aspect of the invention, there is provided a composition comprising a polypeptide according to the invention herein, and a pharmaceutically acceptable carrier.

The composition may not comprise wild-type HCV. The composition may not comprise full length/complete structural or non-structural HCV protein sequence. The use may be with a pharmaceutically acceptable carrier. Additionally or alternatively, the use may be with an adjuvant.

According to another aspect of the invention, there is provided a nucleic acid encoding essentially or at least the polypeptide according to the invention herein. According to another aspect of the invention, there is provided a viral vector encoding the polypeptide according to the invention herein.

The viral vector or nucleic acid may be provided in a composition, wherein composition may comprise a pharmaceutically acceptable carrier. The viral vector or nucleic acid may not encode wild-type HCV or full length/complete HCV NS protein. The viral vector or nucleic acid may not encode structural protein sequence of HCV. The viral vector or nucleic acid may not encode non-conserved protein/peptide sequence of HCV.

The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention. Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

Figure 1 : Identification of conserved HCV peptide segments. Sequence diversity plot of the full HCV genome with defined conserved HCV segments. (A) The calculated sequence diversity for an example sequence dataset is shown for the full HCV genome (sequence dataset HCV gtl/3a, containing 72 sequences) using a window size of k=20. For conserved vaccine design, segments with a variability <25% (lowest quartile, marked blue) were defined as conserved and selected for conserved immunogen design. (B) Consensus sequences for selected conserved segments for three different immunogen analyses HCV genotype 1 (a, blue), HCV genotype 1/3 (b, green) and HCV genotype 1 -6 (c, orange) are depicted. Conserved segments are numbered after position on the HCV genome, with viral regions specified. Figure 2: Patient sequence selection for final immunogen design. (A)

Similarity of subtype consensus sequences (depicted as coloured spots) to overall consensus sequences at each conserved segment, shown for analyses HCV gtl (a, left), HCV gtl/3 (b, middle) and HCV gtl -6 (c, right) immunogens. (B) Number of patient sequences selected of each genotype for the final conserved immunogens HCV gtl (left), HCV gtl/3 (middle) and HCV gtl -6 (right).

Figure 3 : The Total magnitude of HCV specific T cell responses to conserved segment vaccines in mouse models. BALB/c mice (4/group) are vaccinated with each vaccine at 10 ⁸ IU intramuscularly. Splenocytes are harvested 2 weeks later. The total magnitude of HCV specific T cell responses using pools of HCV genotype lb peptides in ex vivo IFN-γ ELISpot assays. Bars represent the mean.

Figure 4: The breadth of HCV specific T cell responses to conserved segment vaccines. BALB/c mice receive 10 ⁸ IU vaccine intramuscularly. Splenocytes are harvested 2 weeks later. The magnitude of HCV specific T cell responses to individual pools (A-M) of HCV genotype lb peptides spanning the viral genome and concavalin A (positive control) are assessed in ex vivo

IFN-γ ELISpot assays. Bars represent the mean +/-SD. (A) Indivual data in four mice receiving Gtl -6D-TPA vaccine is shown. (B) HCV specific T cell responses to gtl -6D, GT1/3D and ChAdOxl GFP control vaccines are shown. Figure 5: Inter-genotypic T cell cross-reactivity of total HCV specific T cell responses to conserved segment vaccines. C57BL/6 mice receive 10 ⁸ IU Gtl -6D-TPA vaccine intramuscularly. Splenocytes are harvested 2 weeks later. The total magnitude of HCV specific T cell responses to HCV genotype l a, ab and 3a peptides spanning the entire immunogen are shown. Bars represent the mean.

Figure 6: Inter-genotypic T cell cross-reactivity in peptide pools to conserved segment vaccines. C57BL/6 mice receive 10 ⁸ IU of gtl -6D-TPA vaccine intramuscularly. Splenocytes are harvested 2 weeks later. The magnitude of HCV specific T cell responses to individual pools of HCV genotype la, lb and 3b peptides spanning the viral genome are assessed in ex vivo IFN-γ ELISpot assays. Bars represent the mean +/-SD.

Figure 7: Breadth, magnitude and T cell cross-reactivity of conserved segment compared to an NS genotype lb immunogen. BALB/c mice receive 10 ⁸ IU of gtl -6D-TPA, Gtl/3D ChAdOxl or NS lb ChAdOxl vaccine intramuscularly. Splenocytes are harvested 2 weeks later and stimulated with HCV peptides or the positive control concavalin A in in ex vivo IFN-γ ELISpot assays. (A) The magnitude of HCV specific T cell responses to individual peptide pools (genotype lb; A-M) is shown for each vaccine . (B) and (C) total

T cell cross-reactivity to HCV la, lb and 3b peptides spanning the viral genome are assessed.. Bars represent the mean +/- SD.

Figure 8 : A2-restricted HCV-specific T cell responses in C57BL/6-Tg (HLA-A2.1) transgenic mice. Ex vivo IFNg ELISpot responses from transgenic C57BL/6-Tg (HLA A2.1) mice when vaccinated, intramuscularly, with conserved segment HCV vaccines, Gtl/3D-TPA and Gtl -6D-TPA, and a Gtlb NS-TPA control. At 14 days post-vaccination, splenocytes were harvest and stimulated with 15- 18mer peptides matching known HCV A2 epitopes. Of the 10- 15 A2-specific epitopes present in the conserved segment vaccines, only the statistically significant responses are shown (unpaired T-test). Note, the significant A2-specific T cell response in Gtl -6D was stimulated by the Gt-3 variant of E2614, despite the recalled T cell population initially primed with a Gt- la sequence during vaccination. Bars represent the geometric mean.

Figure 9: Effect of shark invariant chain (sli) on the immunogenicity of conserved segment vaccine, Gtl-6D. T cell magnitude of conserved segment vaccine, Gtl -6L, is shown with different genetic adjuvants tethered to the immunogen cassette. Outbred CD- I mice (8/group) received various dosages (IU) of gtl -6D-TPA vaccine, in a ChAdOxl vector, intramuscularly. Splenocytes were harvested 3 -weeks post-vaccination and stimulated with HCV lb peptides. Bars represent the mean.

1. We developed a computer algorithm to identify HCV genomic segments from open resource databases and in-house sequences that were conserved between viral subtypes. Conserved segments below a pre-defined threshold spanning the entire HCV coding genome were selected (Figure 1A) and combined to create novel immunogens of either approximately 1000 amino acids (denoted in vaccine name by letter A), or 1500 AA (denoted in vaccine name by letter D) for each of HCV genotypes la and lb, genotypes 1 and 3, and genotypes 1 to 6. In the vaccine constructs following homology was observed - Genotype 1 : 94.8%, Genotypes 1 - 3 : 95.5%, genotypes 1 -6: 90.3%.

We note 1 141 amino-acids are 100% conserved between the GTl , GT l/3 and GTl -6 vaccines, which equates to 74% for GTl ( 1 141/1544), 79% for GTl/3 ( 1 141/1444) and 83% for GTl -6 ( 1 141/1377).

2. The exact sequence to be included in each conserved segment was identified in 2 steps-(i) assessing the HCV subtype or genotype consensus sequence that was most homologous with a consensus of all HCV sequences in the algorithm, and (ii) identifying a real patient sequence that was most homologous with the HCV subtype consensus sequence (Figure 2).

3. Putative artificial epitopes restricted by common HLA super-types in junction regions were abrogated through the insertion of amino-acid linkers and analysed using BLAST to exclude potential cross-reactivity with human self-peptides.

4. Plasmid DNA, encoding HCV conserved genomic segments with linkers, tissue plasminogen activator (TPA) leader sequence and Kozak sequence were chemically synthesised using ThermoFisher Scientific GeneArt™ Gene Synthesis service. Gene constructs were cloned into a pENTR4 vector downsteam to the human cytomegalovirus immediate early promoter and tetracycline operator sequences. The entire coding cassette was then moved into the ChAdOxl destination vector using Thermo Fisher Scientific LR gateway cloning procedure. Simian adenoviral vaccines (ChAdOxl) encoding the new HCV immunogens were derived by transfecting ChAdOxl -HCV _cons plasmids into T-REx™-293 cells (Thermo Fisher Scientific). All the ChAdOxl -HCVcons vaccines were generated at the viral vector core facility at the Jenner Institute, University of Oxford, Oxford, UK.

5. We show that conserved immunogens administered using the adenoviral vector ChAdOxl prime potent T-cell response in mice . In BALB/c mice, total responses for

Gtl/3D ChAdOxl and Gtl -6D ChAdOxl had an average SFU/10 ⁶ splenocytes of 594 and 25 14 (Figure 3). These immunogens gave significant responses to most individual HCV peptides pools when compared to the DMSO control (figure 4a; gtl -6D-TPA shown and to the control eGFP ChAdOxl vaccine (figure 4b). 6. Vaccination with conserved immunogen vaccines induced T cell responses that were highly cross-reactive with different HCV genotypes. C57BL6 mice were vaccinated with Gtl -6D ChAdOxl . Splenocytes were harvested 2 weeks later and stimulated with peptides from HCV genotypes la, lb and 3a giving mean total magnitude responses of 935, 1474 and 1 1 12 SFU/10 ⁶ splenocytes respectively that were significantly higher than the negative DMSO control (figure 5) . T cell responses that were cross-reactive were also observed at the level of the individual HCV peptide pools (Figure 6). The novel conserved immunogen vaccines were equally immunogenic and more cross-reactive with multiple HCV genotypes than a vaccine encoding a single HCV genotype- lb genome (NS lb ChAdOxl) encoded by the same ChAdOxl vector (Figure 7).

Conclusions: Novel pan-genotypic HCV simian adenoviral vectored vaccines encoding conserved segments from all major HCV genotypes are highly immunogenic target multiple areas of the HCV genome and are cross-reactive between HCV genotypes, in mouse models. These studies pave the way for the assessment of pan- genotypic HCV T cell vaccines in humans.

Overview on I mmunogens designed, generated and tested in mice

Table 1: Experimental stages of designed HCV conserved vaccine constructs. Constructs are marked in grey if they have moved forward to the next experimental stage.

Key for sequences above:

TPA leader sequence (underlined bold):

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR

With reference to SEQ ID NOs : 126- 149, the residues placed in parentheses are intended to be provided as options, such that one residue within the parentheses is selected. In one embodiment where the option is between two residues, the first option is selected in any given sequence. In another embodiment where the option is between two residues the second option is selected in any given sequence .

44 conserved peptide sequences that are 100% conserved across HCVl-6 and useful as an epitope: