ANTI-HEPATITIS C ANTIBODIES AND ANTIGEN BINDING FRAGMENTS THEREOF

Abstract

The invention provides an antibody or antigen binding fragment thereof capable of binding to the antigen binding pocket of the AP33 antibody, wherein said antibody or antigen binding fragment thereof comprises VL CDR1 (L1), VL CDR2 (L2), and VL CDR.sub.3 (L.sub.3) consisting of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:23 respectively, and comprises VH CDR1 (H1), VH CDR2 (H2), and VH CDR3 (H3) consisting of the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 respectively. The invention also provides compositions, methods, nucleic acids and uses.

Claims

1. An antibody or antigen binding fragment thereof capable of binding to the antigen binding pocket of the AP33 antibody, wherein said antibody or antigen binding fragment thereof comprises VL CDR1 (L1), VL CDR2 (L2), and VL CDR3 (L3) consisting of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:23 respectively, and comprises VH CDR1 (H1), VH CDR2 (H2), and VH CDR3 (H3) consisting of the amino acid sequences of SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 respectively.

2. The antibody or antigen binding fragment thereof according to claim 1 wherein said antibody or antigen binding fragment thereof comprises VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:20.

3. The antibody or antigen binding fragment thereof according to claim 1 wherein said antibody or antigen binding fragment thereof comprises VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO:22.

4. The antibody or antigen binding fragment thereof according to claim 1 wherein said antibody or antigen binding fragment thereof comprises VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:20 and wherein said antibody or antigen binding fragment thereof comprises VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO:22.

5. The antibody or antigen binding fragment thereof according to claim 1, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv, a Fv, a rIgG, and a diabody.

6. The antibody or antigen binding fragment thereof according to claim 5 wherein said antigen binding fragment is a scFv and wherein said scFv comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:12 or SEQ ID NO:13.

7. A nucleic acid comprising a nucleotide sequence encoding the variable heavy chain domain and/or the variable light chain domain of the antibody or antigen binding fragment according to claim 1 or a complement thereof.

8. The nucleic acid of claim 7, wherein the nucleic acid comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:19 and SEQ ID NO:21.

9. (canceled)

10. A vector comprising the nucleic acid of claim 7, and optionally an expression control sequence operatively linked to the nucleic acid of claim 7.

11. (canceled)

12. A host cell containing the vector of claim 10.

13. The host cell of claim 12, wherein the cell is a eukaryotic cell, a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) cell.

14. (canceled)

15. A method of producing an antibody or antigen binding fragment thereof, comprising incubating a host cell according to claim 12 such that the encoded variable heavy chain domain and/or variable light chain domain is expressed by the cell; recovering the expressed the antibody or antigen binding fragment thereof; and optionally purifying the recovered antibody or antigen binding fragment thereof.

16. (canceled)

17. A composition comprising the antibody or antigen binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient and optionally one or both of a carrier protein and an adjuvant.

18. The A composition according to claim 17, wherein the composition comprises the carrier protein, and the carrier protein is selected from the group consisting of tetanus toxoid and CRM 197 mutant diphtheria toxin.

19. (canceled)

20. The A composition according to claim 17 formulated for use in humans.

21. The antibody or antigen binding fragment of claim 1, wherein said antibody or antigen binding fragment thereof is capable of inducing in a mammal an immune response against the hepatitis C virus E2 protein.

22. (canceled)

23. The antibody or antigen binding fragment thereof of claim 1, wherein said antibody or antigen binding fragment thereof exhibits binding to AP33 antibody mutants FL32A, NL91A, WL96A, YH33A, YH50A, YH58A, IH95A and YH100A of less than 50% of its binding to the AP33 antibody.

24. An antibody that binds to an antibody or antigen binding fragment thereof according to claim 1, which is not an AP33 antibody or a fragment thereof.

25. An antibody according to claim 24 which is obtained by immunisation of a mammal with an antibody or antigen binding fragment thereof according to claim 1.

26. A method of inducing in a mammal an immune response against the hepatitis C virus E2 protein, the method comprising administering to said mammal an antibody according to claim 1, an antibody according to claim 24, a nucleic acid according to claim 7, a vector according to claim 10, or a composition according to claim 17.

27. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0260] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0261] FIG. 1 shows a graph.

[0262] FIG. 2 shows a graph.

[0263] FIG. 3 shows a bar chart.

[0264] FIG. 4 shows a diagram.

[0265] FIG. 5 shows HCV E2 sequence.

[0266] FIG. 6 shows examples of antibodies and antigen binding fragments of the invention.

[0267] FIG. 7 shows The molecular surface of the AP33 binding pocket.

[0268] FIG. 8 shows bar charts.

[0269] FIG. 9 shows graphs.

[0270] FIG. 10 shows bar charts.

[0271] FIG. 11 shows graphs.

[0272] FIG. 12 shows a graph.

[0273] FIG. 13 shows a ribbon diagram.

[0274] FIG. 14 shows that B2.1A docks into the AP33 antigen-binding site. Ribbon and surface representation of AP33 Fab (Ab1; heavy chain: orange, light chain: yellow) in complex with (a) B2.1A scFv (Ab2; heavy chain: purple, light chain: pink), and (b) a peptide corresponding to the HCV E2 epitope (Ag; teal; pdb accession code 4gag).

[0275] FIG. 15 shows Antigen mimicry by B2.1A. Structural alignment of the Ab.sub.1-Ab.sub.2 complex (AP33 heavy chain: orange, light chain: yellow; B2.1A heavy chain: purple, light chain: pink) with the Ab.sub.1-Ag complex (AP33 heavy chain: blue, light chain: teal; peptide: magenta; pdb accession code 4gag). Hydrogen bonds in the Ab.sub.1-Ab.sub.2 complex are shown as black dashed lines and those in the Ab.sub.1-Ag complex as grey dashed lines. Water molecules are shown as red spheres. The panels show the mimicry by B2.1A of E2 residues W420 (a); G418 (b); N415 (c) and L413 (d) and their interactions with AP33.

[0276] FIG. 16 shows site-directed mutagenesis of B2.1A. Purified MBP-B2.1A scFv proteins carrying the indicated mutations were captured on immobilised AP33 and detected by anti-MBP-HRP conjugate in an ELISA assay. (a) WT protein and mutants F98W and N100G, which retained binding; (b) WT protein and mutants that did not bind. Sigmoidal curves were fitted to the absorbance data.

[0277] FIG. 17 shows anti-E2 titre of Rosa26-Fluc mice. Three mice (A, B, C) were given a primary vaccination with B2.1A Fab coupled to KLH, followed by five booster vaccinations. Test bleeds were taken 7-10 days after each booster, with an additional bleed 15 days after the last booster The sera were tested for E2 reactivity by ELISA. The values shown are the mean of two independent titrations

[0278] FIG. 18 shows Ab3 antibodies in vaccinated Rosa26-Fluc mice are specific for the AP33 epitope. Pooled high-titre sera from Rosa26 Flue mice B & C were pre-incubated with a range of peptide concentrations and then transferred to E2-coated microtitre plates. Two peptides were used, one corresponding to the WT AP33 epitope (aa residues 412-424 of E2) and the other containing a W420R substitution. Monoclonal antibodies AP33 and ALP98 served as positive and negative controls, respectively. Bound antibodies were detected with anti-mouse-HRP conjugate followed by TMB substrate.

EXAMPLES

Example 1: Creation of Anti-Idiotypic Antibody

[0279] Antibodies to AP33 were generated using a standard immunisation protocol for antibody production with AP33 as the antigen. Anti-idiotypic (Ab2) antibodies were identified by their ability to inhibit AP33-E2 interaction in a competition ELISA. Nine fusions yielded 122 hybridomas secreting the Ab2 antibodies shown in Table A below:

TABLE-US-00003 TABLE A A summary of the Ab2 data obtained from December 2008 to October 2011 Vaccination.sup.a Binding to AP33.sup.b Se- Test- HC quenced.sup.c Ab2 ed Outcome IgG LC hybrid V.sub.L & V.sub.H A1 Yes Negative Positive Negative Negative A1.5 Yes Negative Positive Negative Negative Yes A5 Yes Negative Positive Negative Negative A164 Yes Negative Positive Negative Negative A164.3 Yes Negative Positive Negative Negative A8A No Positive Negative Negative Yes A8B No Positive Negative Negative Yes A8C No Positive Negative Negative Yes A8D No Positive Negative Negative Yes A8E No Positive Negative Negative Yes A8F No Positive Negative Negative Yes A8G No Positive Negative Negative Yes A8H No Positive Negative Negative Yes A8I No Positive Negative Negative Yes A8L No Positive Negative Negative Yes A8M No Positive Negative Negative Yes A11A No Positive Negative Negative Yes A11B Yes Negative Positive Negative Negative Yes A11C No Positive Negative Negative A12.1 No Positive Negative Negative Yes A12.3 No Positive Negative Negative A12.5 No Positive Negative Negative Yes A14.2 No Positive Negative Negative A14.4 No Positive Negative Negative A14.5 No Positive Negative Negative A16A Yes Negative Positive Negative Negative Yes A17.5 No Positive Negative Negative A22A No Positive Negative Negative Yes A22B No Positive Negative Negative A22C No Positive Negative Negative A22D No Positive Negative Negative Yes A22E No Positive Negative Negative A22F No Positive Negative Negative A22G No Positive Negative Negative A22H No Positive Negative Negative A25D Yes Negative Positive Negative Negative Yes A25H No Positive Negative Negative Yes A31A No Positive Negative Negative Yes A31B No Positive Negative Negative Yes A31C No Positive Negative Negative Yes A31D Yes Negative Positive Negative Negative Yes A31E No Positive Negative Negative A31F No Positive Negative Negative A31G No Positive Negative Negative A34A No Positive Negative Negative Yes A34B No Positive Negative Negative A34C No Positive Negative Negative Yes A46A No Positive Negative Negative A46B Yes Negative Positive Negative Negative Yes A46C No Positive Negative Negative Yes A46D No Positive Negative Negative A49A No Positive Negative Negative A49B No Positive Negative Negative A49C No Positive Negative Negative A49D No Positive Negative Negative A49E No Positive Negative Negative A49F No Positive Negative Negative Yes A52B No Positive Negative Negative A52C No Positive Negative Negative Yes A52D No Positive Negative Negative A52E No Positive Negative Negative A52F No Positive Negative Negative A52G No Positive Negative Negative A52H No Positive Negative Negative Yes A52I No Positive Negative Negative A52O No Positive Negative Negative A52P No Positive Negative Negative A53B No Positive Negative Negative A53C No Positive Negative Negative A53D Yes Negative Positive Negative Negative A53E No Positive Negative Negative A53I No Positive Negative Negative A53J No Positive Negative Negative A53K No Positive Negative Negative A53M No Positive Negative Negative Yes A53N No Positive Negative Negative A53O No Positive Negative Negative Yes A53P No Positive Negative Negative A57B No Positive Negative Negative A57C No Positive Negative Negative A57D No Positive Negative Negative Yes A57F No Positive Negative Negative A57G No Positive Negative Negative Yes A57H No Positive Negative Negative A57J No Positive Negative Negative A570 No Positive Negative Negative A71.2 No Positive Negative Negative A71.5 No Positive Negative Negative A71.9 No Positive Negative Negative B2.1A No Positive Negative Negative Yes B2.1B No Positive Negative Negative B4.1A No Positive Negative Negative B4.1D Yes Negative Positive Negative Negative Yes B4.1E Yes Negative Positive Negative Negative Yes B4.1F Yes Negative Positive Negative Negative Yes B4.1G No Positive Negative Negative Yes K201 Yes Negative Positive Negative Negative Yes K271 No Positive Negative Negative K391 Yes Negative Positive Negative Negative Yes 2K19 Yes Negative Positive Negative Negative Yes 2K49 Yes Negative Positive Negative Negative 2K55 Yes Negative Positive Negative Negative Yes 2K56 Yes Negative Positive Negative Negative Yes 2K160 Yes Negative Positive Negative Negative Yes L1.1A No Positive Negative Negative Yes L1.1D Yes Negative Positive Negative Negative L1.2A Yes Negative Positive Negative Negative Yes L1.2B No Positive Negative Negative L1.2C No Positive Negative Negative Yes L1.2D No Positive Negative Negative Yes L1.2E No Positive Negative Negative Yes L1.2F No Positive Negative Negative Yes L1.2H No Positive Negative Negative L1.2I No Positive Negative Negative Yes L1.2K No Positive Negative Negative Yes L1.2L No Positive Negative Negative L1.2M No Positive Negative Negative L1.2N No Positive Negative Negative L1.2O No Positive Negative Negative L1.2P No Positive Negative Negative Yes P1.52 Yes Negative Positive Negative Negative Yes P1.T Yes Negative Positive Negative Negative Yes .sup.aBalb/c mice were vaccinated with purified antibody coupled to KLH and the immune sera were tested for reactivity with E2. A negative result denotes lack of reactivity. An example of negative ELISA data is shown separately .sup.bBinding of the Ab2s to (a) AP33 whole IgG, (b) AP33 light-chain alone and (c) a hybrid comprising AP33 heavy-chain and an irrelevant κ-light-chain. None of the Ab2s bound AP33 LC or HC hybrid. .sup.cSequencing of Ab2 variable regions.

[0280] Over the course of 18 months, twenty-five Ab2s were picked at random and used to vaccinate mice (Table A), in order to identify one or more internal-image antibodies (Ab23) that would be capable of eliciting an immune response to HCV E2. The immune sera were tested by ELISA for:

[0281] 1. Blocking of AP33-Ab2 interaction.

[0282] 2. Binding to E2.

[0283] 3. Inhibition of HCV infection in cell culture

[0284] RESULTS: The immune sera strongly inhibited binding of AP33 to Ab2, indicating that they contained anti-Ab2 antibodies. However, the anti-Ab2 antibodies did not bind to E2, nor did they inhibit HCV in cell culture. This was a significant problem. See FIGS. 2 and 3 for an example of these negative results.

[0285] FIG. 1 shows inhibition of AP33 binding to A164 by immune sera

[0286] Six Balb/c mice were vaccinated with A164 conjugated to KLH. Primary vaccination was followed by 4 boosters at 14-day intervals, and a final bleed taken 5 days after the last booster.

[0287] Serial dilutions of pre-immune and immune sera were co-incubated with biotinylated AP33 (b-AP33) on A164-coated microtitre plates. Binding of b-AP33 was detected with streptavidin-HRP and TMB. A decreased signal indicates blocking of b-AP33-A164 interaction by competing serum antibodies. The graph represents the response of two mice (No 1 & 2) within the group. All other animals showed the same response.

[0288] RESULT: The immune sera contain A164-specific antibodies that block AP33 binding to E2, whereas the pre-immune bleeds have no effect on the interaction.

[0289] FIG. 2 shows binding of serum antibodies to E2—example of negative result Six Balb/c mice were vaccinated with A164 conjugated to KLH. Primary vaccination was followed by 4 boosters at 14-day intervals, and a final bleed taken 5 days after the last booster.

[0290] Serial dilutions of immune sera were incubated on E2-coated microtitre plates. Binding of serum antibodies was detected with anti-mouse-HRP and TMB. An increased signal indicates the presence of E2-specific antibodies. AP33 served as a positive control. The graph represents the response of two mice (No 1 & 2) within the group. All other animals showed the same response.

[0291] RESULT: The immune sera from mice immunized with A164 do not contain antibodies that recognize E2.

[0292] FIG. 3 shows virus neutralization by immune sera—example of negative result Wild type JFH1 virus (WT) and two E2 mutant viruses, G451R and W420Y were incubated with sera (1/100 dilution) obtained from mice immunized with the Ab2 P1T (TB=terminal bleed). Serum was taken from the same mice prior to immunization and served as controls (PB=pre-bleed). After 1 hour at 37° C., the virus/serum mixture was used to infect Huh7-J20 cells. The Huh7-J20 cell-line is engineered to release secreted alkaline phosphatase (SEAP) reporter into the medium following HCV infection, thus enabling a rapid and sensitive quantification of virus infectivity. At 3 hours post-infection, the inoculum was replaced with fresh medium and incubated for 72 hours. The virus infectivity levels were determined by measurement of SEAP released into the medium. The percent infectivity was calculated by quantifying viral infectivity in the presence of mouse immune serum (TB) relative to its respective control non-immune serum (PB). Error bars indicate standard deviation from the mean. A33 is included as a control. The G451R virus is more sensitive than WT to neutralization by AP33. The W420Y virus is resistant to neutralization by AP33.

[0293] RESULT: Infectivity of WT and G451R virus is significantly reduced by pre-incubation with AP33, but not by any of the mouse sera, indicating that the immune sera from mice immunized with P1T do not contain neutralizing antibodies.

[0294] Obtaining B2.1A Antibody

[0295] These results presented a significant challenge: how to identify the Ab2βs? [0296] By immunisation to produce Ab3 [0297] By testing for binding to AP33 light chain and heavy chain

[0298] This is illustrated in FIG. 4.

[0299] Result: all 120 Ab2s behave like Ab2β

[0300] We realised that there were problems in screening 122 antibodies by vaccination, for example time constraints and/or the number of animals that would be required, so we did the following: [0301] 1. We compared the binding of the Ab2s to (a) AP33 whole IgG, (b) AP33 light-chain alone and (c) a hybrid comprising AP33 heavy-chain and an irrelevant κ-light-chain. This approach is illustrated in FIG. 4., and is based on the expectation that an Ab2β would bind to the entire antigen-binding pocket present in (a) but it would not bind to (b) or (c), whereas an Ab2 that did not represent an internal-image of the antigen-binding pocket would bind to either (b) or (c) In fact, all the Ab2s behaved as Ab23s and bound only to (a), so this assay failed to differentiate between them. [0302] 2. We sequenced the variable regions of all the Ab2s, to remove any duplicates. This reduced the panel to 18 unique antibodies.

[0303] Our crystal structure of AP33 Fab complexed with a peptide corresponding to its epitope allowed us to identify the amino acid residues that make up the antigen-binding pocket of AP33. Using a panel of mutant AP33 antibodies in which these residues were individually replaced by alanine, we established which amino acid residues are involved in E2 binding and which are not (Potter et al. 2012 and Table 1 below).

[0304] The same panel of mutant AP33 antibodies was used to differentiate between the Ab2s. This approach proved to be a real breakthrough, because it revealed striking differences between the Ab2s. Some were unaffected by the mutations, whereas others shared binding characteristics with E2. The binding profile of B2.1A most closely resembled that of E2 (Table 1).

TABLE-US-00004 TABLE 1 Binding of Ab2s to wild-type and mutant AP33, data obtained Nov 2011-Jan 2012 [00034] .sup.aThe amino acid residues that comprise the antigen-binding pocket were identified from the crystal structure of AP33 Fab complexed with a peptide corresponding to its epitope. Mutant AP33 antibodies were made in which these residues were individually replaced by alanine. The mutants were named according to the identity and position of the wild type (WT) amino acid, eg Y.sub.L28A has tyrosine at position 28 in the light chain changed to alanine. .sup.bThe reactivity of HCV E2 with each mutant was determined by ELISA and expressed as a percentage of reactivity with WT AP33. .sup.cThe reactivity of each anti-idiotype (anti-Id) with each mutant was determined by ELISA and expressed as a percentage of reactivity with WT AP33. .sup.dThe score is the number of mutants to which binding was reduced by >50% and >80% relative to WT AP33. Values contributing to the score are highlighted. RESULT: AP33 binding to E2 was reduced by >90% by mutation of light chain residues F32, N91 and W96, and of heavy chain residues Y33, Y50, Y58, I95 and Y100 (values highlighted in bold, double underlined). The same eight mutations reduced AP33 binding to anti-Id B2.1A (top line, values highlighted in bold and boxed), whereas binding to other anti-Ids was affected by fewer, or none of the mutations, which shows that B2.1A most closely resembles E2. Binding to some anti-Ids was reduced by mutations that did not affect E2 binding (eg Y.sub.L28A), therefore these reduced values are not highlighted or included in the score.

Example 2.1: Selection of an Anti-Idiotypic Antibody that Represents an Internal Image of the AP33 Paratope

[0305] FIG. 7 shows the molecular surface of the AP33 binding pocket. The positions of eight alanine substitutions that reduced binding by >90% are colored purple, while those that had little or no effect on E2 binding are colored cyan. The epitope peptide is shown as sticks with yellow carbon atoms.

[0306] FIG. 7 also shows a schematic diagram to illustrate the principles of the anti-idiotype network theory. Exposure to antigen induces the production of antibodies, termed Ab1. The specificity of an Ab1 antibody is determined by the sequence and structure of its hypervariable regions, and this unique antigen-binding site is also recognised as a set of idiotypic epitopes, or idiotopes, by the immune system. Anti-idiotypic (anti-Id) antibodies generated against the Ab1 are termed Ab2, and a subset of these, termed Ab2β, fit into the antigen-binding site (paratope) of the Ab1 precisely enough to be an “internal image” of it, and, by the same token, an effective mimic of the original antigen. An Ab2β antibody can therefore be used as a surrogate antigen to elicit anti-anti-Id antibodies (Ab3), which have the same binding properties as the Ab1.

[0307] Balb/c mice were vaccinated with AP33 to generate a large number of hybridomas. These were screened for the production of Ab2 antibodies that were able to block the AP33-E2 interaction by binding to the hypervariable region of AP33.

[0308] To identify, from this panel of various anti-idiotypes, the desired Ab2β that represents an “internal image” of the AP33 paratope, we used a panel of AP33 antibody mutants, in which each residue within the antigen-binding pocket was individually mutated to alanine. Eight residues in the centre of the pocket were essential for E2 recognition, and the same eight residues were also required for binding of one of the Ab2s, designated B2.1A. This indicates that the molecular surface of B2.1A closely resembles that of the AP33 epitope on E2.

Example 2.2: Vaccination with B2.1A Elicits Ab3 Antibodies that Recognise HCV E2

[0309] Balb/c mice were vaccinated with B2.1A conjugated to KLH. A different adjuvant was used for each group of four mice: (A) Complete Freunds/Incomplete Freunds (CFA/IFA); (B) Alum; (C) Alum & lipopolysaccharide (LPS); (D) Quil-A. The immune and pre-immune sera were tested by ELISA for [0310] 1. Blocking of AP33-B2.1A interaction: Sera at 1:300 dilution were co-incubated with biotinylated AP33 (b-AP33) on B2.1A-coated microtitre plates. Decreased binding of b-AP33 to B2.1A indicates blocking of the interaction by competing serum antibodies. [0311] 2. Binding to E2: Sera at 1:300 dilution were incubated on E2-coated microtitre plates. Binding of serum antibodies indicates the presence of E2-specific Ab3 antibodies.

[0312] Result

[0313] All the immune sera strongly inhibited binding of b-AP33 to B2.1A, indicating that they contain B2.1A-specific antibodies. However, not all of them contain E2-specific antibodies. Immune sera A2 and D3 show the strongest E2 reactivity, with an anti-E2 titre of over 300. As expected, the pre-immune sera are uniformly negative. These results show that B2.1A is able to elicit an E2-specific response. See FIG. 8.

Example 2.3: Vaccination with B2.1A Elicits Ab3 Antibodies that Bind to the Same Epitope as AP33

[0314] A) Peptide Inhibition

[0315] Immune sera A2 and D3 and anti-E2 monoclonal antibodies (MAbs) AP33 and ALP98 were pre-incubated with peptide, transferred to E2-coated microtitre plates and bound antibodies were detected with anti-mouse-HRP.

[0316] Result

[0317] The binding to E2 of Ab1 (AP33) and of Ab3 in the immune sera is specifically inhibited by a peptide that corresponds to the AP33 epitope. There is no inhibition by a peptide in which W420, an essential contact residue for AP33, has been replaced by R, nor by an unrelated control sequence. As expected, ALP98, which binds to a different linear epitope on E2, is not inhibited.

[0318] See FIG. 9.

[0319] B) Alanine Scanning Across AP33 the Epitope

[0320] ELISA was used to test the reactivity of Ab3 antibodies in immune sera A2 and D3 with a panel of E2 mutants, in which each residue across the AP33 epitope was individually replaced by alanine. MAbs AP33 and ALP98 served as positive and negative controls, respectively.

[0321] Result

[0322] The binding of AP33 to E2 was reduced by alanine substitution of L413, N415, G418 or W420. This agrees with our previous data.sup.2 and with the crystal structure of the AP33-peptide complex, in which these four residues are buried at the molecular interface.sup.1. The binding profile of the Ab3 antibodies was very similar to that of AP33: Their binding to E2 was reduced or abrogated by the same four mutations, and also by alanine substitution of I414. As expected, the binding of ALP98 was not affected by any of the substitutions.

[0323] This is compelling evidence that vaccination with B2.1A elicits AP33-like antibodies. See FIG. 10

Example 2.4. The Titre of E2-Specific Ab3 Antibodies in Immune Sera

[0324] FIG. 11A shows Serial dilutions of purified total IgG from immune sera A2 and D3, from non-immune mouse serum (NIM) and from a mouse vaccinated with anti-Id A164 were tested for E2 binding by ELISA. MAbs AP33 and ALP98 served as positive controls.

[0325] FIG. 11B shows E2-specific Ab3 antibodies from immune sera A2 and D3 were affinity-purified on immobilized E2. Serial dilutions of the purified Ab3 antibodies and of AP33 were tested for E2 binding by ELISA.

[0326] Result

[0327] The anti-E2 titre of total IgG from sera A2 and D3 was about 1000-fold lower than that of AP33, whereas the anti-E2 titre of the E2-specific affinity-purified IgG was only 2- to 3-fold lower than that of AP33. Taken together, these data indicate that the proportion of E2-specific antibody to total IgG in the immune sera is in the range of 1/500-1/2000.

Example 2.5. Vaccination with B2.1A Elicits Ab3 Antibodies that Neutralize Virus

[0328] HCVcc were pre-incubated for 1 h with serial dilutions of E2-specific IgG affinity-purified from the serum of a mouse vaccinated with B2.1A. The virus-IgG mix was used to infect Huh7-J20 reporter cells.sup.3. Virus growth was measured by the level of secreted alkaline phosphatase (SEAP) reporter present in the cell culture medium after 3 days. MAb AP33 and IgG purified from a mouse vaccinated with another anti-Id served as positive and negative controls, respectively.

[0329] Result

[0330] The Ab3 antibodies elicited by B2.1A neutralize virus infectivity very effectively, with an IC.sub.50 that is about twice that of AP33.

[0331] Summary

[0332] We have used a broadly neutralizing antibody, AP33, as a template to reverse engineer an immunogen that induces similar antibodies upon vaccination. This has been achieved by isolating an anti-idiotypic antibody that represents the internal image of the AP33 binding pocket and thus mimics the protective epitope. We demonstrate, for the first time in the HCV vaccine field, the success of such a focused, structure-based approach.

REFERENCES TO EXAMPLE 2

[0333] 1. Potter, J. A. et. al (2012). Towards a hepatitis C virus vaccine: the structural basis of hepatitis C virus neutralization by AP33, a broadly neutralizing antibody. J. Virol. 86, 12923-12932. [0334] 2. Tarr, A. W. et. al (2006). Characterization of the hepatitis C virus E2 epitope defined by the broadly neutralizing monoclonal antibody AP33. Hepatology 43, 592-601. [0335] 3. Iro, M. et. al (2009). A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication. Antivir. Res. 83, 148-155.

Example 3: scFv's

[0336] scFv's were produced from B2.1A. scFv amino acid sequences for eukaryotic such as mammalian expression and for prokaryotic such as bacterial expression are shown below.

[0337] Mammalian Expression Construct.

[0338] A mammalian expression construct containing the B2.1A scFv sequence was generated. This sequence was expressed in CHO cells and purified. The purified product was shown to interact with AP33 in ELISA. The B2.1A scFv protein sequence is shown below.

TABLE-US-00005 (SEQ ID NO: 11) [00035] [00036] [00037] [00038] HHHHHH [00039]

[0339] Bacterial Expression Construct.

[0340] The above B2.1A scFv mammalian expression construct was used as a template to provide the scFv-encoding sequence and this was sub-cloned in-frame to the maltose-binding protein (MBP) into the bacterial expression vector pMBP. The MBP-B2.1A scFv amino acid sequence is shown below. The scFv was expressed in bacteria and purified following cleavage of the MBP domain and tested in mouse immunization experiments. The bacterial scFv was effective in eliciting AP33-like antibodies, but less effective than the mammalian scFv.

[0341] The MBP-B2.1A scFv fusion protein sequence is shown below:

TABLE-US-00006 (SEQ ID NO: 12) [00040] [00041] [00042] [00043] [00044] [00045] [00046] [00047] [00048]

[0342] Cleaved Sequence:

TABLE-US-00007 (SEQ ID NO:  13) [00049] [00050] [00051] [00052] [00053] / = proteolytic cleavage site to remove MBP from the MBP-scFv fusion protein [00054] [00055] [00056]

[0343] Nucleic Acid Constructs

[0344] In the exemplary sequences presented below, the coding sequence may be separately taken and placed into the vector of choice if the skilled worker desires.

[0345] pDisMod2-B2.1A-scFv—Example Sequence

TABLE-US-00008 A modified pDisplay vector carrying the B2.1A scFv sequence (the coding sequence is highlighted) scFv coding sequence key as follows: [00057] (SEQ ID NO:  16)    1 GCGCGCGTTG ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG   61 TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT  121 GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC  181 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGACTATTT ACGGTAAACT GCCCACTTGG  241 CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT  301 GGCCCGCCTG GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA  361 TCTACGTATT AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC  421 GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA  481 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT  541 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCTCTGGC  601 TAACTAGAGA ACCCACTGCT TACTGGCTTA TCGAAATTAA TACGACTCAC TATAGGGAGA  661 CCCAAGCTTG GTACCGAGCT CGGATCTACT AGTAACGGCC GCCAGTGTGC TGGATTTCGG  721 CTTGGGGATA TCCACCATGG AGACAGACAC ACTCCTGCTA TGGGTACTGC TGCTCTGGGT [00058] [00059] [00060] [00061] [00062] [00063] [00064] [00065] [00066] [00067] [00068] [00069] [00070] [00071] 1621 TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 1681 CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG 1741 GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT 1801 GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGTGGCGG TAATACGGTT 1861 ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC 1921 CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA 1981 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA 2041 CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC 2101 CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG 2161 TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC 2221 CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 2281 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT 2341 AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT 2401 ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG 2461 ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC 2521 GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 2581 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 2641 CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAACC 2701 TGAGGCTATG GCAGGGCCTG CCGCCCCGAC GTTGGCTGCG AGCCCTGGGC CTTCACCCGA 2761 ACTTGGGGGG TGGGGTGGGG AAAAGGAAGA AACGCGGGCG TATTGGCCCC AATGGGGTCT 2821 CGGTGGGGTA TCGACAGAGT GCCAGCCCTG GGACCGAACC CCGCGTTTAT GAACAAACGA 2881 CCCAACACCG TGCGTTTTAT TCTGTCTTTT TATTGCCGTC ATAGCGCGGG TTCCTTCCGG 2941 TATTGTCTCC TTCCGTGTTT CAGTTAGCCT CCCCCTAGGG TGGGCGAAGA ACTCCAGCAT 3001 GAGATCCCCG CGCTGGAGGA TCATCCAGCC GGCGTCCCGG AAAACGATTC CGAAGCCCAA 3061 CCTTTCATAG AAGGCGGCGG TGGAATCGAA ATCTCGTGAT GGCAGGTTGG GCGTCGCTTG 3121 GTCGGTCATT TCGAACCCCA GAGTCCCGCT CAGAAGAACT CGTCAAGAAG GCGATAGAAG 3181 GCGATGCGCT GCGAATCGGG AGCGGCGATA CCGTAAAGCA CGAGGAAGCG GTCAGCCCAT 3241 TTGAGCCTGG CGAACAGTTC GGCTGGCGCG AGCCCCTGAT GCTCTTGATC ATCCTGATCG 3301 GCCACACCCA GCCGGCCACA GTCGATGAAT CCAGAAAAGC GGCCATTTTC CACCATGATA 3361 TTCGGCAAGC AGGCATCGCC ATGGGTCACG ACGAGATCCT CGCCGTCGGG CATGCTCGCC 3421 TTGAGCCTGG CGAACAGTTC GGCTGGCGCG AGCCCCTGAT GCTCTTGATC ATCCTGATCG 3481 ACAAGACCGG CTTCCATCCG AGTACGTGCT CGCTCGATGC GATGTTTCGC TTGGTGGTCG 3541 AATGGGCAGG TAGCCGGATC AAGCGTATGC AGCCGCCGCA TTGCATCAGC CATGATGGAT 3601 ACTTTCTCGG CAGGAGCAAG GTGAGATGAC AGGAGATCCT GCCCCGGCAC TTCGCCCAAT 3661 AGCAGCCAGT CCCTTCCCGC TTCAGTGACA ACGTCGAGCA CAGCTGCGCA AGGAACGCCC 3721 GTCGTGGCCA GCCACGATAG CCGCGCTGCC TCGTCTTGCA GTTCATTCAG GGCACCGGAC 3781 AGGTCGGTCT TGACAAAAAG AACCGGGCGC CCCTGCGCTG ACAGCCGGAA CACGGCGGCA 3841 TCAGAGCAGC CGATTGTCTG TTGTGCCCAG TCATAGCCGA ATAGCCTCTC CACCCAAGCG 3901 GCCGGAGAAC CTGCGTGCAA TCCATCTTGT TCAATCATGC GAAACGATCC TCATCCTGTC 4021 TAGCTCAGAG GCCGAGGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA TTAGTCAGCC 4081 ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC GGGATGGGCG GAGTTAGGGG 4141 CGGGACTATG GTTGCTGACT AATTGAGATG CATGCTTTGC ATACTTCTGC CTGCTGGGGA 4201 GCCTGGGGAC TTTCCACACC TGGTTGCTGA CTAATTGAGA TGCATGCTTT GCATACTTCT 4261 GCCTGCTGGG GAGCCTGGGG ACTTTCCACA CCCTAACTGA CACACATTCC ACAGCTGGTT 4321 CTTTCCGCCT CAGGACTCTT CCTTTTTCAA TAAATCAATC TAAAGTATAT ATGAGTAAAC 4381 TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT 4441 TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 4501 ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 4561 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC 4621 CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA 4681 TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG 4741 TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT 4801 GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 4861 AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT 4921 AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG 4981 GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC 5041 TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC 5101 GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT 5161 TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 5221 AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG 5281 CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA 5341 ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGCGC CCTGTAGCGG 5401 CGCATTAAGC GCGGCGGGTG TGGTGGTTAC GCGCAGCGTG ACCGCTACAC TTGCCAGCGC 5461 CCTAGCGCCC GCTCCTTTCG CTTTCTTCCC TTCCTTTCTC GCCACGTTCG CCGGCTTTCC 5521 CCGTCAAGCT CTAAATCGGG GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT TACGGCACCT 5581 CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC CCTGATAGAC 5641 GGTTTTTCGC CCTTTGACGT TGGAGTCCAC GTTCTTTAAT AGTGGACTCT TGTTCCAAAC 5701 TGGAACAACA CTCAACCCTA TCTCGGTCTA TTCTTTTGAT TTATAAGGGA TTTTGCCGAT 5761 TTCGGCCTAT TGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA ATTTTAACAA 5821 AATATTAACG CTTACAATTT AC

[0346] pDisMod2-B2.1A-scFv—Preferred Sequence

TABLE-US-00009 A modified pDisplay vector carrying the B2.1A scFv sequence (the coding sequence is highlighted). There are TWO changes relative to Example Sequence (SEQ ID NO: 16) above - these are in line 781 and are marked in bold. scFv coding sequence key as follows: [00072] (SEQ ID NO: 27)    1 GCGCGCGTTG ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG   61 TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT  121 GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC  181 CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGACTATTT ACGGTAAACT GCCCACTTGG  241 CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGTAAAT  301 GGCCCGCCTG GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA  361 TCTACGTATT AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC  421 GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA  481 GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT  541 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCTCTGGC  601 TAACTAGAGA ACCCACTGCT TACTGGCTTA TCGAAATTAA TACGACTCAC TATAGGGAGA  661 CCCAAGCTTG GTACCGAGCT CGGATCTACT AGTAACGGCC GCCAGTGTGC TGGATTTCGG  721 CTTGGGGATA TCCACCATGG AGACAGACAC ACTCCTGCTA TGGGTACTGC TGCTCTGGGT [00073] [00074] [00075] [00076] [00077] [00078] [00079] [00080] [00081] [00082] [00083] [00084] [00085] [00086] 1621 TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC 1681 CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG 1741 GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT 1801 GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGTGGCGG TAATACGGTT 1861 ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC 1921 CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA 1981 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA 2041 CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC 2101 CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA GCTCACGCTG 2161 TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC 2221 CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 2281 ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT 2341 AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA GAAGGACAGT 2401 ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG 2461 ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC 2521 GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 2581 GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 2641 CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAACC 2701 TGAGGCTATG GCAGGGCCTG CCGCCCCGAC GTTGGCTGCG AGCCCTGGGC CTTCACCCGA 2761 ACTTGGGGGG TGGGGTGGGG AAAAGGAAGA AACGCGGGCG TATTGGCCCC AATGGGGTCT 2821 CGGTGGGGTA TCGACAGAGT GCCAGCCCTG GGACCGAACC CCGCGTTTAT GAACAAACGA 2881 CCCAACACCG TGCGTTTTAT TCTGTCTTTT TATTGCCGTC ATAGCGCGGG TTCCTTCCGG 2941 TATTGTCTCC TTCCGTGTTT CAGTTAGCCT CCCCCTAGGG TGGGCGAAGA ACTCCAGCAT 3001 GAGATCCCCG CGCTGGAGGA TCATCCAGCC GGCGTCCCGG AAAACGATTC CGAAGCCCAA 3061 CCTTTCATAG AAGGCGGCGG TGGAATCGAA ATCTCGTGAT GGCAGGTTGG GCGTCGCTTG 3121 GTCGGTCATT TCGAACCCCA GAGTCCCGCT CAGAAGAACT CGTCAAGAAG GCGATAGAAG 3181 GCGATGCGCT GCGAATCGGG AGCGGCGATA CCGTAAAGCA CGAGGAAGCG GTCAGCCCAT 3241 TCGCCGCCAA GCTCTTCAGC AATATCACGG GTAGCCAACG CTATGTCCTG ATAGCGGTCC 3301 GCCACACCCA GCCGGCCACA GTCGATGAAT CCAGAAAAGC GGCCATTTTC CACCATGATA 3361 TTCGGCAAGC AGGCATCGCC ATGGGTCACG ACGAGATCCT CGCCGTCGGG CATGCTCGCC 3421 TTGAGCCTGG CGAACAGTTC GGCTGGCGCG AGCCCCTGAT GCTCTTGATC ATCCTGATCG 3481 ACAAGACCGG CTTCCATCCG AGTACGTGCT CGCTCGATGC GATGTTTCGC TTGGTGGTCG 3541 AATGGGCAGG TAGCCGGATC AAGCGTATGC AGCCGCCGCA TTGCATCAGC CATGATGGAT 3601 ACTTTCTCGG CAGGAGCAAG GTGAGATGAC AGGAGATCCT GCCCCGGCAC TTCGCCCAAT 3661 AGCAGCCAGT CCCTTCCCGC TTCAGTGACA ACGTCGAGCA CAGCTGCGCA AGGAACGCCC 3721 GTCGTGGCCA GCCACGATAG CCGCGCTGCC TCGTCTTGCA GTTCATTCAG GGCACCGGAC 3781 AGGTCGGTCT TGACAAAAAG AACCGGGCGC CCCTGCGCTG ACAGCCGGAA CACGGCGGCA 3841 TCAGAGCAGC CGATTGTCTG TTGTGCCCAG TCATAGCCGA ATAGCCTCTC CACCCAAGCG 3901 GCCGGAGAAC CTGCGTGCAA TCCATCTTGT TCAATCATGC GAAACGATCC TCATCCTGTC 3961 TCTTGATCGA TCTTTGCAAA AGCCTAGGCC TCCAAAAAAG CCTCCTCACT ACTTCTGGAA 4021 TAGCTCAGAG GCCGAGGAGG CGGCCTCGGC CTCTGCATAA ATAAAAAAAA TTAGTCAGCC 4081 ATGGGGCGGA GAATGGGCGG AACTGGGCGG AGTTAGGGGC GGGATGGGCG GAGTTAGGGG 4141 CGGGACTATG GTTGCTGACT AATTGAGATG CATGCTTTGC ATACTTCTGC CTGCTGGGGA 4201 GCCTGGGGAC TTTCCACACC TGGTTGCTGA CTAATTGAGA TGCATGCTTT GCATACTTCT 4261 GCCTGCTGGG GAGCCTGGGG ACTTTCCACA CCCTAACTGA CACACATTCC ACAGCTGGTT 4321 CTTTCCGCCT CAGGACTCTT CCTTTTTCAA TAAATCAATC TAAAGTATAT ATGAGTAAAC 4381 TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT 4441 TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 4501 ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 4561 ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATC 4621 CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA 4681 TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG 4741 TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT 4801 GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 4861 AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT 4921 AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCG 4981 GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC 5041 TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC 5101 GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT 5161 TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 5221 AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG 5281 CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA 5341 ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGCGC CCTGTAGCGG 5401 CGCATTAAGC GCGGCGGGTG TGGTGGTTAC GCGCAGCGTG ACCGCTACAC TTGCCAGCGC 5461 CCTAGCGCCC GCTCCTTTCG CTTTCTTCCC TTCCTTTCTC GCCACGTTCG CCGGCTTTCC 5521 CCGTCAAGCT CTAAATCGGG GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT TACGGCACCT 5581 CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC CCTGATAGAC 5641 GGTTTTTCGC CCTTTGACGT TGGAGTCCAC GTTCTTTAAT AGTGGACTCT TGTTCCAAAC 5701 TGGAACAACA CTCAACCCTA TCTCGGTCTA TTCTTTTGAT TTATAAGGGA TTTTGCCGAT 5761 TTCGGCCTAT TGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA ATTTTAACAA 5821 AATATTAACG CTTACAATTT AC

Example 4: Production of B2.1A Antibody

[0347] B2.1A Antibody Chains are produced using conventional antibody expression systems incorporating the CDRs of the B2.1A as disclosed herein.

[0348] In this example the conventional expression system used is the ‘antibody generation’ system which is commercially available from InvivoGen at 5, rue Jean Rodier, F-31400 Toulouse, France.

TABLE-US-00010 pFUSEss-CHIg-mG1-B2.1a-vH - Example Sequence B2.1A vH sequence cloned into pFUSEss-CHIg-Mg1 to generate a full heavy chain. Coding sequences highlighted: [00087] (SEQ ID NO: 17)    1 GGATCTGCGA TCGCTCCGGT GCCCGTCAGT GGGCAGAGCG CACATCGCCC ACAGTCCCCG   61 AGAAGTTGGG GGGAGGGGTC GGCAATTGAA CGGGTGCCTA GAGAAGGTGG CGCGGGGTAA  121 ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC CGAGGGTGGG GGAGAACCGT  181 ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA CGGGTTTGCC GCCAGAACAC  241 AGCTGAAGCT TCGAGGGGCT CGCATCTCTC CTTCACGCGC CCGCCGCCCT ACCTGAGGCC  301 GCCATCCACG CCGGTTGAGT CGCGTTCTGC CGCCTCCCGC CTGTGGTGCC TCCTGAACTG  361 CGTCCGCCGT CTAGGTAAGT TTAAAGCTCA GGTCGAGACC GGGCCTTTGT CCGGCGCTCC  421 CTTGGAGCCT ACCTAGACTC AGCCGGCTCT CCACGCTTTG CCTGACCCTG CTTGCTCAAC  481 TCTACGTCTT TGTTTCGTTT TCTGTTCTGC GCCGTTACAG ATCCAAGCTG TGACCGGCGC  541 CTACCTGAGA TCACCGGCGA AGGAGGGCCA CCATGTACAG GATGCAACTC CTGTCTTGCA [00088] [00089] [00090] [00091] [00092] [00093] [00094] [00095] [00096] [00097] [00098] [00099] [00100] [00101] [00102] [00103] [00104] [00105] [00106] [00107] [00108] [00109] [00110] 1981 GTCCCTAGCT GGCCAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA 2041 ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT ATTTGTAACC 2101 ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT GTTTCAGGTT 2161 CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTATGGAA 2221 TTAATTCTAA AATACAGCAT AGCAAAACTT TAACCTCCAA ATCAAGCCTC TACTTGAATC 2281 CTTTTCTGAG GGATGAATAA GGCATAGGCA TCAGGGGCTG TTGCCAATGT GCATTAGCTG 2341 TTTGCAGCCT CACCTTCTTT CATGGAGTTT AAGATATAGT GTATTTTCCC AAGGTTTGAA 2401 CTAGCTCTTC ATTTCTTTAT GTTTTAAATG CACTGACCTC CCACATTCCC TTTTTAGTAA 2461 AATATTCAGA AATAATTTAA ATACATCATT GCAATGAAAA TAAATGTTTT TTATTAGGCA 2521 GAATCCAGAT GCTCAAGGCC CTTCATAATA TCCCCCAGTT TAGTAGTTGG ACTTAGGGAA 2581 CAAAGGAACC TTTAATAGAA ATTGGACAGC AAGAAAGCGA GCTTCTAGCT TATCCTCAGT 2641 CCTGCTCCTC TGCCACAAAG TGCACGCAGT TGCCGGCCGG GTCGCGCAGG GCGAACTCCC 2701 GCCCCCACGG CTGCTCGCCG ATCTCGGTCA TGGCCGGCCC GGAGGCGTCC CGGAAGTTCG 2761 TGGACACGAC CTCCGACCAC TCGGCGTACA GCTCGTCCAG GCCGCGCACC CACACCCAGG 2821 CCAGGGTGTT GTCCGGCACC ACCTGGTCCT GGACCGCGCT GATGAACAGG GTCACGTCGT 2881 CCCGGACCAC ACCGGCGAAG TCGTCCTCCA CGAAGTCCCG GGAGAACCCG AGCCGGTCGG 2941 TCCAGAACTC GACCGCTCCG GCGACGTCGC GCGCGGTGAG CACCGGAACG GCACTGGTCA 3001 ACTTGGCCAT CATGGCTCCT Cctgtcagga gaggaaagag aagaaggtta gtacaattgC 3061 TATAGTGAGT TGTATTATAC TATGCAGATA TACTATGCCA ATGATTAATT GTCAAACTAG 3121 GGCTGCAggg ttcatagtgc cacttttcct gcactgcccc atctcctgcc caccctttcc 3181 caggcataga cagtcagtga cttacCAAAC TCACAGGAGG GAGAAGGCAG AAGCTTGAGA 3241 CAGACCCGCG GGACCGCCGA ACTGCGAGGG GACGTGGCTA GGGCGGCTTC TTTTATGGTG 3301 CGCCGGCCCT CGGAGGCAGG GCGCTCGGGG AGGCCTAGCG GCCAATCTGC GGTGGCAGGA 3361 GGCGGGGCCG AAGGCCGTGC CTGACCAATC CGGAGCACAT AGGAGTCTCA GCCCCCCGCC 3421 CCAAAGCAAG GGGAAGTCAC GCGCCTGTAG CGCCAGCGTG TTGTGAAATG GGGGCTTGGG 3481 GGGGTTGGGG CCCTGACTAG TCAAAACAAA CTCCCATTGA CGTCAATGGG GTGGAGACTT 3541 GGAAATCCCC GTGAGTCAAA CCGCTATCCA CGCCCATTGA TGTACTGCCA AAACCGCATC 3601 ATCATGGTAA TAGCGATGAC TAATACGTAG ATGTACTGCC AAGTAGGAAA GTCCCATAAG 3661 GTCATGTACT GGGCATAATG CCAGGCGGGG CATTTACCGT CATTGACGTC AATAGGGGGC 3721 GTACTTGGCA TATGATACAC TTGATGTACT GCCAAGTGGG CAGTTTACCG TAAATACTCC 3781 ACCCATTGAC GTCAATGGAA AGTCCCTATT GGCGTTACTA TGGGAACATA CGTCATTATT 3841 GACGTCAATG GGCGGGGGTC GTTGGGCGGT CAGCCAGGCG GGCCATTTAC CGTAAGTTAT 3901 GTAACGCCTG CAGGTTAATT AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC 3961 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA 4021 AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT 4081 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC 4141 TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC 4201 TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC 4261 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT 4321 TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG 4381 CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA 4441 TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA 4501 AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA 4561 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG 4621 AAAACTCACG TTAAGGGATT TTGGTCATGG CTAGTTAATT AACATTTAAA TCAGCGGCCG 4681 CAATAAAATA TCTTTATTTT CATTACATCT GTGTGTTGGT TTTTTGTGTG AATCGTAACT 4741 AACATACGCT CTCCATCAAA ACAAAACGAA ACAAAACAAA CTAGCAAAAT AGGCTGTCCC 4801 CAGTGCAAGT GCAGGTGCCA GAACATTTCT CTATCGAA

[0349] pFUSEss-CHIg-mG1-B2.1a-vH—Preferred Sequence

TABLE-US-00011 B2.1A vH sequence cloned into pFUSEss-CHIg-Mg1 to generate a full heavy chain. There is one change relative to Example Sequence (SEQ ID NO: 17) above - this is in line 601 and is marked in bold. Coding sequences highlighted: [00111] (SEQ ID NO: 28)    1 GGATCTGCGA TCGCTCCGGT GCCCGTCAGT GGGCAGAGCG CACATCGCCC ACAGTCCCCG   61 AGGAGTTGGG GGGAGGGGTC GGCAATTGAA CGGGTGCCTA GAGAAGGTGG CGCGGGGTAA  121 ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC CGAGGGTGGG GGAGAACCGT  181 ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA CGGGTTTGCC GCCAGAACAC  241 AGCTGAAGCT TCGAGGGGCT CGCATCTCTC CTTCACGCGC CCGCCGCCCT ACCTGAGGCC  301 GCCATCCACG CCGGTTGAGT CGCGTTCTGC CGCCTCCCGC CTGTGGTGCC TCCTGAACTG  361 CGTCCGCCGT CTAGGTAAGT TTAAAGCTCA GGTCGAGACC GGGCCTTTGT CCGGCGCTCC  421 CTTGGAGCCT ACCTAGACTC AGCCGGCTCT CCACGCTTTG CCTGACCCTG CTTGCTCAAC  481 TCTACGTCTT TGTTTCGTTT TCTGTTCTGC GCCGTTACAG ATCCAAGCTG TGACCGGCGC  541 CTACCTGAGA TCACCGGCGA AGGAGGGCCA CCATGTACAG GATGCAACTC CTGTCTTGCA [00112] [00113] [00114] [00115] [00116] [00117] [00118] [00119] [00120] [00121] [00122] [00123] [00124] [00125] [00126] [00127] [00128] [00129] [00130] [00131] [00132] [00133] [00134] 1981 GTCCCTAGCT GGCCAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA 2041 ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT ATTTGTAACC 2101 ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT GTTTCAGGTT 2161 CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG TGGTATGGAA 2221 TTAATTCTAA AATACAGCAT AGCAAAACTT TAACCTCCAA ATCAAGCCTC TACTTGAATC 2281 CTTTTCTGAG GGATGAATAA GGCATAGGCA TCAGGGGCTG TTGCCAATGT GCATTAGCTG 2341 TTTGCAGCCT CACCTTCTTT CATGGAGTTT AAGATATAGT GTATTTTCCC AAGGTTTGAA 2401 CTAGCTCTTC ATTTCTTTAT GTTTTAAATG CACTGACCTC CCACATTCCC TTTTTAGTAA 2461 AATATTCAGA AATAATTTAA ATACATCATT GCAATGAAAA TAAATGTTTT TTATTAGGCA 2521 GAATCCAGAT GCTCAAGGCC CTTCATAATA TCCCCCAGTT TAGTAGTTGG ACTTAGGGAA 2581 CAAAGGAACC TTTAATAGAA ATTGGACAGC AAGAAAGCGA GCTTCTAGCT TATCCTCAGT 2641 CCTGCTCCTC TGCCACAAAG TGCACGCAGT TGCCGGCCGG GTCGCGCAGG GCGAACTCCC 2701 GCCCCCACGG CTGCTCGCCG ATCTCGGTCA TGGCCGGCCC GGAGGCGTCC CGGAAGTTCG 2761 TGGACACGAC CTCCGACCAC TCGGCGTACA GCTCGTCCAG GCCGCGCACC CACACCCAGG 2821 CCAGGGTGTT GTCCGGCACC ACCTGGTCCT GGACCGCGCT GATGAACAGG GTCACGTCGT 2881 CCCGGACCAC ACCGGCGAAG TCGTCCTCCA CGAAGTCCCG GGAGAACCCG AGCCGGTCGG 2941 TCCAGAACTC GACCGCTCCG GCGACGTCGC GCGCGGTGAG CACCGGAACG GCACTGGTCA 3001 ACTTGGCCAT GATGGCTCCT Cctgtcagga gaggaaagag aagaaggtta gtacaattgC 3061 TATAGTGAGT TGTATTATAC TATGCAGATA TACTATGCCA ATGATTAATT GTCAAACTAG 3121 GGCTGCAggg ttcatagtgc cacttttcct gcactgcccc atctcctgcc caccctttcc 3181 caggcataga cagtcagtga cttacCAAAC TCACAGGAGG GAGAAGGCAG AAGCTTGAGA 3241 CAGACCCGCG GGACCGCCGA ACTGCGAGGG GACGTGGCTA GGGCGGCTTC TTTTATGGTG 3301 CGCCGGCCCT CGGAGGCAGG GCGCTCGGGG AGGCCTAGCG GCCAATCTGC GGTGGCAGGA 3361 CCCCCCCCCC AAGGCCGTGC CTGACCAATC CGGAGCACAT AGGAGTCTCA GCCCCCCGCC 3421 CCAAAGCAAG GGGAAGTCAC GCGCCTGTAG CGCCAGCGTG TTGTGAAATG GGGGCTTGGG 3481 GGGGTTGGGG CCCTGACTAG TCAAAACAAA CTCCCATTGA CGTCAATGGG GTGGAGACTT 3541 GGAAATCCCC GTGAGTCAAA CCGCTATCCA CGCCCATTGA TGTACTGCCA AAACCGCATC 3601 ATCATGGTAA TAGCGATGAC TAATACGTAG ATGTACTGCC AAGTAGGAAA GTCCCATAAG 3661 GTCATGTACT GGGCATAATG CCAGGCGGGC CATTTACCGT CATTGACGTC AATAGGGGGC 3721 GTACTTGGCA TATGATACAC TTGATGTACT GCCAAGTGGG CAGTTTACCG TAAATACTCC 3781 ACCCATTGAC GTCAATGGAA AGTCCCTATT GGCGTTACTA TGGGAACATA CGTCATTATT 3841 GACGTCAATG GGCGGGGGTC GTTGGGCGGT CAGCCAGGCG GGCCATTTAC CGTAAGTTAT 3901 GTAACGCCTG CAGGTTAATT AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC 3961 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA 4021 AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT 4081 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC 4141 TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC 4201 TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC 4261 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT 4321 TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG 4381 CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGAACA GTATTTGGTA 4441 TCTGCGCTCT GCTGAAGCCA GTTACCTTGG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA 4501 AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA 4561 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG 4621 AAAACTCACG TTAAGGGATT TTGGTCATGG CTAGTTAATT AACATTTAAA TCAGCGGCCG 4681 CAATAAAATA TCTTTATTTT CATTACATCT GTGTGTTGGT TTTTTGTGTG AATCGTAACT 4741 AACATACGCT CTCCATCAAA ACAAAACGAA ACAAAACAAA CTAGCAAAAT AGGCTGTCCC 4801 CAGTGCAAGT GCAGGTGCCA GAACATTTCT CTATCGAA

[0350] pFUSE2ss-CLIg-mk-B2.1a-vL

TABLE-US-00012 B2.1A vL sequence cloned into pFUSEss-CLIg-MK to generate a full light chain. Coding sequences highlighted: [00135] (SEQ ID NO:  18)    1 GGATCTGCGA TCGCTCCGGT GCCCGTCAGT GGGCAGAGCG CACATCGCCC AGAGTCCCCG   61 AGAAGTTGGG GGGAGGGGTC GGCAATTGAA CGGGTGCCTA GAGAAGGTGG CGCGGGGTAA  121 ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC CGAGGGTGGG GGAGAACCGT  181 ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA CGGGTTTGCC GCCAGAACAC  241 AGCTGAAGCT TCGAGGGGCT CGCATCTCTC CTTCACGCGC CCGCCGCCCT ACCTGAGGCC  301 GCCATCCACG CCGGTTGAGT CGCGTTCTGC CGCCTCCCGC CTGTGGTGCC TCCTGAACTG  361 CGTCCGCCGT CTAGGTAAGT TTAAAGCTCA GGTCGAGACC GGGCCTTTGT CCGGCGCTCC  421 CTTGGAGCCT ACCTAGACTC AGCCGGCTCT CCACGCTTTG CCTGACCCTG CTTGCTCAAC  481 TCTACGTCTT TGTTTCGTTT TCTGTTCTGC GCCGTTACAG ATCCAAGCTG TGACCGGCGC  541 CTACCTGAGA TCAACATGTA CAGGATGCAA CTCCTGTCTT GCATTGCACT AAGTCTTGCA [00136] [00137] [00138] [00139] [00140] [00141] [00142] [00143] [00144] [00145] [00146] 1261 AGACAAAGGT CCTGAGAGCT AGCTGGCCAG ACATGATAAG ATACATTGAT GAGTTTGGAC 1321 AAACCACAAC TAGAATGCAG TGAAAAAAAT GCTTTATTTG TGAAATTTGT GATGCTATTG 1381 CTTTATTTGT AACCATTATA AGCTGCAATA AACAAGTTAA CAACAACAAT TGCATTCATT 1441 TTATGTTTCA GGTTCAGGGG GAGGTGTGGG AGGTTTTTTA AAGCAAGTAA AACCTCTACA 1501 AATGTGGTAT GGAATTAATT CTAAAATACA GCATAGCAAA ACTTTAACCT CCAAATCAAG 1621 ATGTGCATTA GCTGTTTGCA GCCTCACCTT CTTTCATGGA GTTTAAGATA TAGTGTATTT 1681 TCCCAAGGTT TGAACTAGCT CTTCATTTCT TTATGTTTTA AATGCACTGA CCTCCCACAT 1741 TCCCTTTTTA GTAAAATATT CAGAAATAAT TTAAATACAT CATTGCAATG AAAATAAATG 1801 TTTTTTATTA GGCAGAATCC AGATGCTCAA GGCCCTTCAT AATATCCCCC AGTTTAGTAG 1861 TTGGACTTAG GGAACAAAGG AACCTTTAAT AGAAATTGGA CAGCAAGAAA GCGAGCTTCT 1921 AGCTTTAGTT CCTGGTGTAC TTGAGGGGGA TGAGTTCCTC AATGGTGGTT TTGACCAGCT 1981 TGCCATTCAT CTCAATGAGC ACAAAGCAGT CAGGAGCATA GTCAGAGATG AGCTCTCTGC 2041 ACATGCCACA GGGGCTGACC ACCCTGATGG ATCTGTCCAC CTCATCAGAG TAGGGGTGCC 2101 TGACAGCCAC AATGGTGTCA AAGTCCTTCT GCCCGTTGCT CACAGCAGAC CCAATGGCAA 2161 TGGCTTCAGC ACAGACAGTG ACCCTGCCAA TGTAGGCCTC AATGTGGACA GCAGAGATGA 2221 TCTCCCCAGT CTTGGTCCTG ATGGCCGCCC CGACATGGTG CTTGTTGTCC TCATAGAGCA 2281 TGGTGATCTT CTCAGTGGCG ACCTCCACCA GCTCCAGATC CTGCTGAGAG ATGTTGAAGG 2341 TCTTCATGAT GGCTCCTCct gtcaggagag gaaagagaag aaggttagta caattgCTAT 2401 AGTGAGTTGT ATTATACTAT GCTTATGATT AATTGTCAAA CTAGGGCTGC Agggttcata 2461 gtgccacttt tcctgcactg ccccatctcc tgcccaccct ttcccaggca tagacagtca 2521 gtgacttacC AAACTCACAG GAGGGAGAAG GCAGAAGCTT GAGACAGACC CGCGGGACCG 2581 CCGAACTGCG AGGGGACGTG GCTAGGGCGG CTTCTTTTAT GGTGCGCCGG CCCTCGGAGG 2641 CAGGGCGCTC GGGGAGGCCT AGCGGCCAAT CTGCGGTGGC AGGAGGCGGG GCCGAAGGCC 2701 GTGCCTGACC AATCCGGAGC ACATAGGAGT CTCAGCCCCC CGCCCCAAAG CAAGGGGAAG 2761 TCACGCGCCT GTAGCGCCAG CGTGTTGTGA AATGGGGGCT TGGGGGGGTT GGGGCCCTGA 2821 CTAGTCAAAA CAAACTCCCA TTGACGTCAA TGGGGTGGAG ACTTGGAAAT CCCCGTGAGT 2881 CAAACCGCTA TCCACGCCCA TTGATGTACT GCCAAAACCG CATCATCATG GTAATAGCGA 2941 TGACTAATAC GTAGATGTAC TGCCAAGTAG GAAAGTCCCA TAAGGTCATG TACTGGGCAT 3001 AATGCCAGGC GGGCCATTTA CCGTCATTGA CGTCAATAGG GGGCGTACTT GGCATATGAT 3061 ACACTTGATG TACTGCCAAG TGGGCAGTTT ACCGTAAATA CTCCACCCAT TGACGTCAAT 3121 GGAAAGTCCC TATTGGCGTT ACTATGGGAA CATACGTCAT TATTGACGTC AATGGGCGGG 3181 GGTCGTTGGG CGGTCAGCCA GGCGGGCCAT TTACCGTAAG TTATGTAACG CCTGCAGGTT 3241 AATTAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT 3301 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG 3361 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC 3421 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC 3481 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT 3541 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT 3601 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC 3661 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA 3721 GTGGTGGCCT AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA 3781 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG 3841 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA 3901 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG 3961 GATTTTGGTC ATGGCTAGTT AATTAACATT TAAATCAGCG GCCGCAATAA AATATCTTTA 4021 TTTTCATTAC ATCTGTGTGT TGGTTTTTTG TGTGAATCGT AACTAACATA CGCTCTCCAT 4081 CAAAACAAAA CGAAACAAAA CAAACTAGCA AAATAGGCTG TCCCCAGTGC AAGTGCAGGT 4141 GCCAGAACAT TTCTCTATCG AA

Example 5: Challenge Studies in Mice

[0351] Mouse Model

[0352] We use the immunocompetent mouse model developed by Dorner et al (Dorner et al 2011 Hepatology Vol 54 No 5 pages 1873-1875; Dorner et al 2011 Nature Vol 474 pages 208-211; Dorner et al 2013 Methods Vol 59 pages 249-257; Zeisel et al 2011). This is the most appropriate model for testing HCV vaccines.

[0353] Commercially available transgenic Gt(ROSA)26Sortml(Luc)Kaelin mice (Rosa26-Fluc) contain a LoxP-flanked STOP cassette restricting firefly luciferase expression. They are made permissive for HCV entry by infection with adenoviruses encoding essential cell surface receptors (human CD81, occludin, claudin 1 and SR-BI), and then infected with recombinant bicistronic HCVcc expressing cyclization recombination (CRE) recombinase. Upon HCV entry into mouse hepatocytes, the recombinant viral genome is translated and the CRE protein is expressed. The CRE recombinase excises the STOP cassette and activates the luciferase reporter, leading to bioluminescence that can be measured using a using a whole body bioluminescence imager.

[0354] Experimental Details

[0355] 1. Establish a small colony (˜30) of the commercially-available transgenic (Rosa26-Fluc CRE reporter mice.

[0356] 2. Carry out a small-scale vaccination (6-8 animals) with B2.1A Fab-KLH and check the anti-E2 serum titre after each vaccination by ELISA. (Primary vaccination with immunogen in Freund's Complete Adjuvant, followed by 5 boosts with immunogen in Freund's Incomplete Adjuvant).

[0357] 3. If adequate anti-E2 serum titres are obtained, vaccinate a larger number (24) as above.

[0358] 4. Genetically humanise the immunised mice by administering adenovirus vectors encoding human CD81 and OCLN, and human or murine SR-BI and CLDN1.

[0359] 5. After 24 hours administer 2×10.sup.7 TCID50 of HCV-CRE. Use 4 different HCV viruses representing a range of genotypes.

[0360] 6. After 72 hours measure bioluminescence using a whole body imager, and correlate anti-E2 titre with HCV infection. An inverse correlation indicates that the vaccine protects against HCV challenge

Example 6: B2.1A Structure

[0361] A Fab fragment of AP33 was co-crystallised in complex with a single-chain variable fragment (scFv) of B2.1A, and the structure determined to a resolution of 1.8 Å, which unambiguously shows the positions of all the amino acid side-chains and of water molecules at the interface between the two antibodies. The asymmetric unit of this Ab.sub.1-Ab.sub.2 complex was composed of one molecule of AP33 Fab and one molecule of B2.1A scFv. The structural coordinates were determined.

[0362] The structure (FIG. 13) shows that the CDR loops of B2.1A correspond more closely to the definition of IgG regions described by Chothia et al. than by Kabat et al.

[0363] The combining site of B2.1A has an overall concave surface from which the CDR-L1 and CDR-H3 loops protrude outwards, towards the groove formed between the CDR-L2, CDR-L3 and CDR-H3 loops of AP33. The groove on AP33 has an overall negative charge, while the L1 loop on B2.1A has a complementary positive charge. Overall, both combining sites have a hydrophobic nature, due to the presence of numerous aromatic residues. All the heavy and light chain CDRs of B2.1A are involved in interactions with AP33 via hydrogen bonds and other hydrophilic interactions, hydrophobic interactions and van der Waals contacts. The area of the interface is 1069 Å, which is approximately 9% of the total surface of the B2.1a scFv.

Example 7: Antigen Mimicry by B2.1A

[0364] A comparison of this Ab.sub.1-Ab.sub.2 complex with the Ab.sub.1-Ag complex (i.e. the structure of AP33 in complex with a peptide corresponding to its E2 epitope (Potter et al. 2012; pdb accession code 4gag)) shows that B2.1A docks into the AP33 antigen-binding site (FIG. 14). It reveals that CDR-H3 of B2.1A mimics the shape and character of the E2 epitope, even though there is no sequence similarity. The critical E2 residue W420, which is deeply buried in the Ab.sub.1-Ag complex, is mimicked by F.sub.H98 of B2.1A in the Ab.sub.1-Ab.sub.2 complex (FIG. 15a).

[0365] The other important E2 residues at the Ab.sub.1-Ag interface are G418, N415 and L413. The shape of the antigen around G418 is preserved by the side chain of B2.1A Y.sub.H100A, which forms extensive contacts with W.sub.L96 of AP33 (FIG. 15b). The polar character of E2 residue N415, which is deeply buried in the Ab.sub.1-Ag complex, is conferred by N.sub.H100 of B2.1A, while the neighbouring Y.sub.H100A provides a hydrogen bond to Y.sub.H50 of AP33 (FIG. 15c). Interestingly, the interactions of L413 with AP33 are mimicked not by an amino acid residue but by five water molecules in the Ab.sub.1-Ab.sub.2 complex (FIG. 15d). In keeping with our biochemical and immunisation data (shown in Table 1 and FIGS. 9-12), this structural analysis confirms that B2.1A is an Ab.sub.2β, i.e. an anti-idiotypic antibody that fits into the antigen-binding site (paratope) of the Ab.sub.1 precisely enough to be an “internal image” of it, and, by the same token, an effective mimic of the original antigen.

Example 8: B2.1A Binding Affinity

[0366] We measured the binding affinity of B2.1A for AP33 by Surface Plasmon Resonance (SPR). B2.1A scFv was immobilised in three different ways: (a) amine coupling to a CM5 chip; (b) amine coupling to a CM4 chip; (c) capture via a histidine tag to a NTA chip. AP33 was then injected over the surface, using single-cycle kinetics. All the data sets were high quality and the three experiments yielded affinity constants of 29 nm, 20 nm and 8 nm, respectively:

TABLE-US-00013 TABLE Binding affinity of B2.1A for AP33 Expt Ka (1/Ms) Kd (1/s) KD (M) a) 1.12*10.sup.4 3.21*10.sup.−4 2.86*10.sup.−8 b) 1.18*10.sup.4 2.43*10.sup.−4 2.07*10.sup.−8 c) 4.87*10.sup.4  3.9*10.sup.−4  8.0*10.sup.−9

[0367] These values are comparable to the affinity constants of 5.5-6.6 nm, measured by SPR, for binding of antibody MRCT10 (humanised AP33-WO2009/081285) to soluble E2.sub.661 (Pantua et al 2013).

Example 9: B2.1A Mutagenesis

[0368] The crystallographic structure of B2.1A scFv, together with protein-protein interaction prediction servers, inspired the inventors to design point mutations aimed at increasing its binding affinity for AP33. The inventors reasoned that this might translate into an increased affinity for HCV E2 of Ab3 antibodies elicited by vaccination with B2.1A. The following mutations were introduced into the heavy chain sequence of B2.1A: W33V, E50F, E50Y, F98Y, F98W, N100G, N100del and G100BF, in a wild-type (WT) protein comprising a fusion of B2.1A scFv with maltose binding protein (MBP). The affinity of the mutant proteins for AP33 was assessed by AP33-capture ELISA, using MBP as a detection tag. As shown in FIG. 16b, most of the mutants showed little or no binding to AP33. Only two of the mutants, F98W and N100G, retained binding, but it was weaker than WT (FIG. 16a).

[0369] The EC.sub.50 values, estimated by fitting a sigmoidal curve to the data, were 1.48 μg/ml for WT and 4.6 μg/ml for F98W.

[0370] Thus it seems that it is not possible to improve the affinity of B2.1A for AP33 by mutagenesis.

[0371] These results demonstrate that AP33 appears to represent the best possible antibody and additionally show that it is demonstrably superior to rationally designed alternatives and therefore possesses significant technical advantages over other antibody species having different amino acid sequences.

Example 10: Vaccination with B2.1A/Protection from HCV Infection

[0372] The immunocompetent mouse model developed by Marcus Dorner (Dorner et al 2011; Dorner et al 2013) is used to test whether vaccination with B2.1A can protect against infection by HCV. This is the most appropriate model for testing HCV vaccines. Commercially available transgenic Rosa26-Fluc mice contain a LoxP-flanked STOP cassette restricting firefly luciferase expression. They are made permissive for HCV entry by infection with adenoviruses encoding essential cell-surface receptors (human CD81, occludin, claudin 1 and SR-BI), and then infected with recombinant bicistronic HCVcc expressing cyclisation recombination (CRE) recombinase. Upon HCV entry into mouse hepatocytes, the recombinant viral genome is translated and the CRE protein is expressed. The CRE recombinase excises the STOP cassette and activates the luciferase reporter, leading to bioluminescence that can be measured using a using a whole body bioluminescence imager.

[0373] Detailed Protocols of Immunisation & Challenge Experiments in Mice

[0374] Mice Strain FVB.129S6(B6)-Gt(ROSA)26Sor.sup.tm1(Luc)Kael/J, (abbreviate to Rosa26-Fluc; Jackson Laboratories stock no 005125). Purchase 2-3 mating pairs and breed the mice to obtain sufficient numbers for immunisation.

[0375] Immunisation Protocol 1

[0376] Immunogens: (A) B2.1A Fab conjugated to KLH, 1 mg/ml [0377] (B) Peptide IQLINTNGSWHINS conjugated to KLH, 1 mg/ml [0378] (The peptide corresponds to the AP33 epitope, ie aa 412-423 of HCV E2)

[0379] For primary vaccination make up a 1:1 emulsion of immunogen (A) with Freund's Complete Adjuvant (FCA). The final protein concentration is 0.5 mg/ml.

[0380] For all booster vaccinations make up a 1:1 emulsion of immunogen (A) or (B), as appropriate, with Freund's Incomplete Adjuvant (IFA). [0381] Day 0 Pre-immune bleed. [0382] Day 7 Primary vaccination. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen (A) in CFA [0383] Day 28 Booster 1. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen (A) in IFA. [0384] Day 35 Test bleed 1. [0385] Day 42 Booster 2. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen (B) in IFA. [0386] Day 49 Test bleed 2. [0387] Day 56 Booster 3. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen (A) in IFA. [0388] Day 63 Test bleed 3. [0389] Day 70 Booster 4. Subcutaneous injection of 50 μg in h100 μl per mouse of immunogen (B) in IFA. [0390] Day 77 Test bleed 4. [0391] Day 84 Booster 5. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen (A) in IFA. [0392] Day 91 Test bleed 5.

[0393] The timing does not have to be exactly as above. The first boost should be at least three weeks after the primary immunisation, and the subsequent boosters should be at least two weeks apart. A test bleed should be taken 7-10 days after the booster.

[0394] Immunisation Protocol 2

[0395] Immunogen: B2.1A Fab conjugated to KLH, 1 mg/ml

[0396] For primary vaccination make up a 1:1 emulsion of immunogen with Freund's Complete Adjuvant (FCA). The final protein concentration is 0.5 mg/ml.

[0397] For all booster vaccinations make up a 1:1 emulsion of immunogen with Freund's Incomplete Adjuvant (IFA). [0398] Day 0 Pre-immune bleed. [0399] Day 7 Primary vaccination. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in CFA [0400] Day 28 Booster 1. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in IFA. [0401] Day 35 Test bleed 1. [0402] Day 42 Booster 2. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in IFA. [0403] Day 49 Test bleed 2. [0404] Day 56 Booster 3. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in IFA. [0405] Day 63 Test bleed 3. [0406] Day 70 Booster 4. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in IFA. [0407] Day 77 Test bleed 4. [0408] Day 84 Booster 5. Subcutaneous injection of 50 μg in 100 μl per mouse of immunogen in IFA. [0409] Day 91 Test bleed 5.

[0410] The timing does not have to be exactly as above. The first boost should be at least three weeks after the primary immunisation, and the subsequent boosters should be at least two weeks apart. A test bleed should be taken 7-10 days after the booster.

[0411] If the test bleeds show that the mice have developed HCV E2-specific antibodies, proceed with genetic humanisation and challenge according the protocol below. If the test bleeds show that the mice have developed a high titre (>1:10,000) of HCV E2-specific antibodies after two or three boosters, there is no need to give all the boosters.

[0412] We have described two immunisation protocols. The first protocol includes boosters with a peptide corresponding to the E2 epitope that is mimicked by the CDRs of B2.1A. This aims to focus the immune response on the desired region of B2.1A. The second protocol boosts with B2.1A Fab alone. Our data show that we can definitely elicit E2-specific antibodies using B2.1A Fab alone. Boosting with peptide may or may not confer an advantage. The skilled worker may choose the protocol according to their needs.

[0413] Test bleeds are processed as is known in the art, i.e. by taking the test bleed, clotting it, taking the supernatant, centrifuging it to pellet any cells not removed with the clot, adding 1 mM sodium azide and storing it at 4 degrees Celsius until needed.

[0414] Titration.sup.1 of mouse serum by sE2 by ELISA .sup.1 The titre of a serum is defined as the lowest concentration that gives a positive antigen-specific signal. In this assay, a positive signal is defined as an A.sub.450 reading that is three times higher than that produced by non-immune, control serum at the same dilution. The mean signal from several non-immune sera is used as the control. [0415] 1. Coat the wells of a 96-well Immulon 2 HB plate with 0.2 μg/well of purified soluble HCV E2.sup.2 in 100 ul PBS. Incubate overnight at RT. .sup.2 soluble E2 (sE2) expressed and purified from insect cells. It comprises aa 384-661 of the HCV polyprotein, ie the ectodomain without the membrane-proximal and trans-membrane regions. [0416] 2. Discard sE2 and block with 2% skimmed milk powder in PBST.sup.3, 200 μl/well. Incubate for 2 hours at RT. .sup.3 PBST=PBS+0.05% Tween 20 [0417] 3. Wash 3× with PBST. The plate can be stored at this stage at −20° C. or 4° C. [0418] 4. Add two-fold dilutions of serum in 100 μl of PBST. Incubate for 2 hours at RT. [0419] 5. Wash 3× with PBST. [0420] 6. Add 100 μl/well of anti-mouse HRP conjugate (Sigma A4416) diluted 1/3000 in PBST. Incubate for 1 hour at RT. [0421] 7. Wash 4×PBST. [0422] 8. Add 100 μl/well of TMB substrate. Incubate at RT for 30 mins. [0423] 9. Stop the reaction by adding 50l/well of 0.5M H.sub.2SO.sub.4. [0424] 10. Read the absorbance at 450 nm in a microplate reader.

[0425] Infection of Genetically Humanised Rosa26-Fluc Mice with HCV-CRE

[0426] The preparation of adenoviruses and recombinant HCV-CRE and the analysis of HCV entry by in vivo bioluminescence imaging are carried out exactly as known in the art, for example as described in sections 2.2.1, 2.2.2 and 2.3.2 of Dorner et al, 2013 which is incorporated herein by reference specifically for the detailed description of performing this technique.

[0427] In this example we show data for six mice. Three mice were given a primary vaccination with B2.1A Fab coupled to KLH, followed by five booster vaccinations. This elicited robust anti-E2 titres of 1:12,800 in two mice and 1:1,600 in the third mouse (FIG. 17).

[0428] The E2 reactivity is inhibited by a peptide containing the AP33 epitope, showing that the Ab3 antibodies elicited in the Rosa26-Fluc mice have the same specificity as AP33 (FIG. 18). This shows that the binding to E2 of AP33 and of Ab3 in the Rosa26 Flue immune sera is specifically inhibited in a concentration-dependent manner by the WT peptide that contains the AP33 epitope. There is no inhibition by the peptide in which W420, an essential contact residue for AP33, has been replaced by R. As expected, ALP98, which binds to a different linear epitope on E2 (aa residues 644-651), is not inhibited by either of the peptides.

[0429] Thus the Rosa26Fluc mice showed a good immune response.

[0430] The vaccinated and unvaccinated mice are made permissive for HCV infection as described above, and then challenged with 2×10.sup.7 TCID.sub.50 of HCV-CRE.

REFERENCES TO EXAMPLES

[0431] 1. Chothia, C., A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip et al. (1989). Conformations of immunoglobulin hypervariable regions. Nature 342, 877--883. [0432] 2. Dorner, M., Horwitz, J. A., Robbins, J. B., Barry, W. T., Feng, Q., Mu, K., Jones, C. T., Schoggins, J. W., Catanese, M. T., Burton, D. R., Law, M., Rice, C. M. & Ploss, A. (2011). A genetically humanized mouse model for hepatitis C virus infection. Nature 474, 208-211. [0433] 3. Dorner, M., Rice, C. M. & Ploss, A. (2013). Study of hepatitis C virus entry in genetically humanized mice. Methods 59, 249-257. [0434] 4. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. (1991). Sequences of proteins of immunological interest. 5.sup.th Edition ed. U.S. Department of Health and Human Services/NIH, Bethesda, Md. [0435] 5. Potter, J. A., Owsianka, A. M., Jeffery, N., Matthews, D, J, Keck, Z.-Y., Lau, P. L., Foung, S. K. H., Taylor, G. L. & Patel, A. H. (2012). Towards a hepatitis C virus vaccine: the structural basis of hepatitis C virus neutralization by AP33, a broadly neutralizing antibody. J. Virol. 86, 12923-12932. [0436] 6. Pantua, H., Diao, J., Ultsch, M., Hazen, M., Mathieu, M., McCutcheon, K., Takeda, K., Date, S., Cheung, T. K., Phung, Q., Hass, P., Arnott, D., Hongo, J-A., Matthews, D. J., Brown, A., Patel, A. H., Kelley, R. F., Eigenbrot, C. and Kapadia, S. B. (2013). Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies. J. Mol. Biol. 425, 1899-1914.

[0437] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.