# Patent

## Title
Recombinant HCV E2 glycoprotein

## Abstract

The invention provides modified hepatitis C virus (HCV) E2 glycoproteins comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions wherein in at least one of said variable regions at least a part of the variable region is replaced with a flexible linker sequence. The invention also provides vaccine compositions comprising the modified glycoproteins as well as methods of use thereof.

## Background
FIELD OF INVENTION
The present invention relates to a new and improved treatment for viral hepatitis C(HCV) infection. The present invention particularly relates to vaccine compositions for prevention and therapeutic treatment of HCV infection based on administering a recombinant HCV polyprotein.
BACKGROUND OF THE INVENTION
According to the World Health Organisation, hepatitis C virus (HCV) infects approximately 170 million to 200 million people worldwide. While governments have increased education about how HCV is transmitted, and despite prevention programs, HCV continues to proliferate. Approximately 80% of those who are infected with HCV remain carriers of the virus. In Australia about 16,000 new cases of HCV infection are reported each year, the new infections being most prevalent amongst injection drug users. HCV is the most common blood-borne viral infection, causing the death of a substantial proportion of the population.
HCV is known to infect the liver and certain immune cells of a sufferer. As a result, HCV leads to serious liver disease such as fibrosis, cirrhosis, steatosis and heptocellular carcinoma (liver cancer) more frequently than other forms of hepatitis. HCV is a leading cause for the requirement of liver transplants. It is generally believed that the acute phase of the infection is often unrecognised due to the sub-clinical nature of the infection, and 80% of individuals progress to a chronic condition. Chronic infection is a result of the immune system's failure to generate an adequate immune response against the virus.
Currently there is no vaccine for HCV and the only available therapy for treatment of HCV has relied on development of antiviral drugs and drug combinations. The general idea behind antiviral drug design is to identify viral proteins, or parts of proteins, that can be disabled or inhibited. A standard treatment of choice for patients suffering moderate or severe fibrosis includes a combination of alpha-interferon and ribavirin. The antiviral effects of combination alpha-interferon and ribavirin therapy cause a rapid decrease in HCV levels in the blood, even after a single dose. Conventional alpha-interferon treatment for HCV however suffers several drawbacks. For example, (i) when alpha-interferon treatment is stopped after a few weeks or months of treatment, the viral load level is known to re-establish rapidly; (ii) treatment with alpha-interferon/ribavirin is associated with severe side effects, including flu-like symptoms, reduced red or white cell counts, bone marrow suppression, neuropsychiatric effects, particularly depression and anemia; (iii) effective treatment requires patient adherence to a frequent dosing regimen since alpha-interferon is absorbed and eliminated from the body rapidly; and (v) high cost of such treatments.
Some of the above drawbacks, referring particularly to item (iii) above, have been addressed by subjecting alpha-interferon to ‘pegylation’ in which polyethylene glycol molecules are attached to the interferon. The administration of pegylated interferon in combination with ribavirin increases the half-life of interferon and has the advantage of decreasing the frequency of dosing, hence patient compliance. Such treatment however has proven to be efficacious in less than 50% of treated patients. Given the increasing number of chronic sufferers of HCV, there is a need to develop a vaccine for both prophylactic and therapeutic purposes.
Development of a successful vaccine to protect against HCV infection has been elusive. One proposed reason for this difficulty is that HCV, being an RNA virus, is genetically unstable allowing it to achieve a high rate of viral mutation to evade the body's immune response. It is therefore a challenge for researchers to identify a portion of the virus that is conserved.
HCV has been classified in a separate genus (Hepacivirus) of the Flaviviridae family. HCV is non-cytopathic and rather triggers an immune response that either rapidly clears the infection or initiates an inflammatory response leading to chronic infection and liver injury. Spontaneously resolving infections that permanently clear HCV RNA without treatment occur in  ̃30% of acute cases suggesting a natural immunity to HCV and is thus encouraging for the prospect of vaccine development. However, the determinants for this outcome of HCV infection are unknown.
The HCV virion contains a positive-sense single stranded RNA genome of about 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030 amino acids. The structural proteins comprise a core protein forming the viral nucleocapsid and two envelope glycoproteins, E1 and E2. Some recent efforts towards the development of a HCV vaccine have focused on HCV envelope glycoproteins E1 and E2. It has been found that E1 and E2 form non-covalently associated heterodimers on the surface of the virion that mediate both viral attachment and entry and thus present targets for the host immune response.
Recent studies have suggested that envelope glycoprotein E2 binds to CD81 on the surface of CD4+ T cells. During the binding process, E2 undergoes rapid conformational change. To date no research has been able to provide a suitable modified envelope glycoprotein, which can exhibit “wild-type” levels of CD81 binding.
Throughout this specification, including the claims, all numbering of polypeptide residues of the HCV envelope glycoproteins E1 and E2 is based on the prototype HCV-H77 polyprotein sequence, Genbank Accession No. AF 009606. The mature form of glycoprotein E1 is encompassed by polyprotein residues 191 and 383, and the mature form of glycoprotein E2 is encompassed by polyprotein residues 384 and 746.
The receptor-binding domain (RBD) of E2 is encompassed by polyprotein residues 384-661 (E2661). Recombinant forms of E2661 RBD are efficiently secreted from transfected cells and are able to interact with CD81 and other cell surface molecules. The E2 RBD contains two variable regions, HVR1 (384-410) and HVR2 (474-482).
Variable region 1, located at the N-terminus of E2, is the most variable region in the HCV genome, is highly immunogenic and rapidly accumulates neutralization escape mutations. Despite the high level of amino acid variability in HVR1, there is an overall conservation of basic residues that are important for viral entry.
Variable region 2 is located within the region flanked by Cys-459 to Cys-486. Although originally described as a 7-residue sequence, comparison of E2 sequences from different HCV genotypes suggests it may extend from residues 461-481. In comparison to HVR1, the sequence of HVR2 is relatively stable within HCV infected people, although an accumulation of mutations at this location has been shown to correlate with responsiveness to interferon-α treatment.
In work leading to the present invention, the inventors have observed that alignment of E2 sequences representing the six major genotypes of HCV reveals a previously undescribed variable region between polyprotein residues 570-580 that is relatively conserved within a genotype but varies across genotypes due to amino acid insertions and deletions. Accordingly, amino acids 570-580 have been denoted the intergenotypic variable region (igVR). Examination of the corresponding region from all 6 genotypes of HCV, and divergent isolates therein, show that igVR is also flanked by conserved cysteine residues (Cys-569 and Cys-581), suggesting that these sequences form disulfide-constrained loops.
To date no vaccine treatment for HCV using the adaptive immune response route has been successful. Given the drawbacks of current and experimental therapies for treatment of HCV, there is an unmet need for providing a cell-mediated immune response to treat HCV infection.
It is one object of the present invention to provide an immunotherapeutic approach to prevent or treat HCV infection. A further object of the present invention is to provide an immunotherapeutic approach to prevent or treat HCV infectiona. A further object of the present invention is to provide a modified E2 glycoprotein, which approaches ‘wild-type’ binding levels to natural cellular receptors of HCV infection.
International Patent Publication No. WO 02/22155 (Hawaii Biotechnology Group, Inc.) discloses a truncated HCV E2 polypeptide which lacks the HVR1 region and is capable of secretion into growth medium when expressed in recombinant form in a host cell. The polypeptide may also lack its C-terminus after residue 662. International Patent Publication No. WO 03/022880 (XTL Biopharmaceuticals Ltd.) also discloses a truncated version of the E2 protein lacking HVR1 region.
The foregoing discussion is intended to introduce the field of the present invention and should not be construed in any way an admission of the state of common general knowledge in this art. Bibliographic details of publications referred to in this specification are set out at the end of the description. The reference to any prior art document in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the document forms part of the common general knowledge.
SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
In one aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein in at least one of said variable regions at least a part of the variable region is replaced with a flexible linker sequence.
In another aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein at least a part of the HVR2 variable region is removed or is replaced with a flexible linker sequence.
In yet another aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein at least a part of the igVR variable region is removed or is replaced with a flexible linker sequence.
The modified HCV E2 glycoproteins as broadly described above are glycoproteins which substantially approach HCV virion wild-type conformation, and retain the ability to bind to the HCV receptor CD81 and conformation-dependent antibodies.
The present invention also provides a composition comprising a modified HCV E2 glycoprotein as broadly described above, together with a pharmaceutically acceptable carrier or diluent.
Such a composition may be formulated as a vaccine composition, preferably including an adjuvant.
In yet another aspect, the present invention also provides a method of eliciting an immune response in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein as broadly described above.
In this aspect, the invention includes a method for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein as broadly described above.
In a further aspect, this invention provides the use of a modified HCV E2 glycoprotein as broadly described above in, or in the manufacture of a medicament for, eliciting an immune response in a patient.
In this further aspect, this invention includes the use of a modified HCV E2 glycoprotein as broadly described above in, or in the manufacture of a medicament for, prophylactic or therapeutic treatment of HCV infection in a patient.
In yet a further aspect, this invention provides an agent for eliciting an immune response in a patient, which comprises a modified HCV E2 glycoprotein as broadly described above.
In this further aspect, the invention includes an agent for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises a modified HCV E2 glycoprotein as broadly described above.
The present invention also provides an isolated antibody raised against a modified HCV E2 glycoprotein as broadly described above. The antibody may be monoclonal or polyclonal.
In this aspect, the invention also provides a method for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises administration to the patient of an effective amount of an antibody as described above.
The invention also provides the use of an antibody as described above in, or in the manufacture of a medicament for, prophylactic or therapeutic treatment of HCV infection in a patient.
In this aspect also, the invention provides an agent for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises an antibody as described above.
Further, the invention provides a method of detecting HCV infection in a patient, comprising contacting a biological sample from the patient with an antibody as described above under conditions which allow formation of an antibody-antigen complex, and detecting said complex, wherein formation of said complex is indicative of the presence of HCV in the sample.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the expression and heterodimerization of E1E2 containing single variable region deletions in cell lysates. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), pE1E2 containing one variable region deletion, or empty vector were immunoprecipitated with A. anti-E1E2 polyclonal antibody (779), B. anti-E1 monoclonal antibody (A4), C. anti-E2 monoclonal antibody (A11) and D. anti-E2 conformation-dependent monoclonal antibody (H53) under both i) non-reducing and ii) reducing conditions (+β-mercaptopethanol). All samples were run on 10-15% SDS-PAGE gradient gels visualized using a phosphoimager. This data is representative of two independent experiments.
FIG. 2 shows a ClustalX alignment of diverse HCV E2 glycoprotein sequences from genotypes (SEQ ID NOs 88-94, respectively). The two recognized hypervariable regions, HVR1 and HVR2, and the novel variable region igVR are highlighted (grey). The conserved cysteine residues that flank the HVR2 and igVR regions, as well as the first conserved cysteine residue proposed to anchor the N-terminal region to the rest of the E2 glycoprotein, are also indicated (bold type). The positions of the CD81-binding determinants (boxed) and the epitope for the broadly neutralizing antibody AP33 are indicated. N-linked glycosylation sites associated with variable regions are also shown (tree). Predicted transmembrane domain is underlined.
FIG. 3 shows the incorporation of E1E2 glycoproteins containing single variable region deletions into retroviral pseudotyped HIV-1 particles. A. Metabolically labelled HCV glycoprotein pseudotyped HIV-1 particles were pelleted from the tissue culture fluid of 293T cells transfected with wild-type (pE1E2), pE1E2 containing a variable region deletion or empty vector prior to lysis. E1E2 heterodimers were immunoprecipitated with the conformation-dependent anti-E2 monoclonal antibody H53. All samples were separated on a 10-15% SDS-PAGE gradient gel under non-reducing conditions and visualized using a phosphoimager. B. Processing and incorporation of HIV-1 structural proteins into retroviral pseudotyped HIV-1 particles. HIV-1 structural proteins Pr55Gag, p24CA p66RT and p17MA from lysed metabolically-labelled HCV glycoprotein pseudotyped HIV-1 particles were immunoprecipitated using IgG from an HIV-1 infected individual (IgG14). All samples were separated on a 7.5-15% SDS-PAGE gradient gel and visualized using a phosphoimager. This data is representative of two independent experiments.
FIG. 4 shows the ability of HCV glycoprotein pseudotyped HIV-1 particles containing single variable region deletions to enter Huh7 cells. The tissue culture fluid from 293T cells co-transfected with the vector NL4-3.LUC.R-E- and either wild-type (pE1E2), pE1E2 containing a deletion in a variable region, or empty vector was used to infect Huh7 cells in triplicate. The Huh7 cells were lysed and the luciferase activity (relative light units) measured using a Fluostar fitted with luminescence optics. Mean and standard deviation was calculated from triplicate infections. Data is representative of three independent experiments.
FIG. 5 shows the ability of HCV E1E2 containing single variable region deletions to bind CD81-LEL. A. Ability of intracellular forms of E2 to bind to CD81-LEL. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), pE1E2 with a deletion of a variable region, or empty vector were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected using anti-E2 conformation-dependent antibody H53 and rabbit anti-mouse horseradish peroxidase (HRP) conjugate. Absorbance values (optical density) were read at 450 nm and the 620 nm background subtracted. B. Virion-incorporated E2 glycoprotein binding to CD81-LEL. Lysates of metabolically-labelled HCV glycoprotein-pseudotyped HIV-1 particles were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected as described for A. Data is representative of two independent experiments.
FIG. 6 shows the expression and heterodimerization of E1E2 containing modified single variable region deletions in cell lysates. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), or pE1E2 containing single variable region deletions with an extended linker, or empty vector were immunoprecipitated with A. anti-E1E2 polyclonal antibody (779), B. anti-E1 monoclonal antibody (A4), C. anti-E2 monoclonal antibody (A11) and D. anti-E2 conformation-dependent monoclonal antibody (H53) under both i) non-reducing and ii) reducing conditions (+β-mercaptopethanol). All samples were run on 10-15% SDS-PAGE gradient gels and visualized using a phosphorimager. Data is representative of two independent experiments.
FIG. 7 shows the incorporation of E1E2 glycoproteins containing modified single variable region deletions into HCV glycoprotein pseudotyped HIV-1 particles. A. Metabolically labelled E1E2-pseudotyped HIV-1-particles were pelleted from the tissue culture fluid of 293T cells transfected with NL4-3.LUC.R-E- and wild-type (pE1E2), containing single variable region deletions with an extended linker, or empty vector prior to lysis. E1E2 heterodimers were immunoprecipitated with the conformation-dependent anti-E2 monoclonal antibody H53. All samples were separated under non-reducing conditions on a 10-15% SDS-PAGE gradient gel and visualized using a phosphoimager. B. Processing and incorporation of HIV-1 structural proteins into HCV glycoprotein-pseudotyped HIV-1 particles. HIV-1 structural proteins Pr55Gag, p17MA, p24CA and p66RT from lysed metabolically-labelled E1E2-pseudotyped HIV-1 particles were immunoprecipitated using IgG from an HIV-1 infected individual (IgG14). All samples were separated on a 7.5-15% SDS-PAGE gradient gel and visualized using a phosphorimager. Data is representative of two independent experiments.
FIG. 8 shows the ability of HCV glycoprotein-pseudotyped HIV-1 particles containing single modified variable region deletions to enter Huh7 cells. The tissue culture fluid from 293T cells co-transfected with NL4-3.LUC.R-E- and either wild-type (pE1E2), containing single variable region deletions with an extended linker, or empty (pcDNA4) vector was used to infect Huh7 cells in triplicate. The Huh7 cells were lysed and the luciferase activity (relative light units) measured using a Fluostar fitted with luminescence optics. Mean and standard deviation was calculated from triplicate infections. p-value was calculated using the student's t-test. Data is representative of three independent experiments.
FIG. 9 shows the ability of E2 containing single modified deletions of one variable region to bind CD81-LEL. A. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), pE1E2 containing a modified deletion of one variable region, or empty (pcDNA4) were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected using the anti-E2 conformation-dependent monoclonal antibody H53 and rabbit anti-mouse horseradish peroxidase (HRP) conjugate. Absorbance values were read at 450 nm-620 nm (background). Data is representative of two independent experiments. B. Virion-incorporated E2 glycoprotein binding to CD81-LEL in viral lysates. Lysates of metabolically-labelled E1E2-pseudotyped HIV-1 particles were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions and bound E2 was detected as described above. Absorbance values were read at 450 nm-620 nm (background) C. Independent repeat of B.
FIG. 10 shows the expression and heterodimerization of immature forms of E1E2 containing multiple variable region deletions. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), pE1E2 containing multiple variable region deletions, or empty vector were immunoprecipitated with A. anti-E1E2 polyclonal anti-body (779), B. anti-E1 monoclonal antibody (A4), C. anti-E2 monoclonal antibody (A11) and D. anti-E2 conformation-dependent monoclonal antibody (H53) under both i) non-reducing and ii) reducing conditions (+β-mercaptopethanol). All samples were run on 10-15% SDS-PAGE gradient gels and visualized using a phosphorimager. Data is representative of two independent experiments.
FIG. 11 shows the incorporation of E1E2 glycoproteins containing multiple variable region deletions into HCV glycoprotein-pseudotyped HIV-1 particles. Metabolically labelled E1E2-pseudotyped HIV-1 particles were pelleted from the tissue culture fluid of 293T cells co-transfected with NL4-3.LUC.R-E- and either wild-type (pE1E2), pE1E2 containing multiple variable region deletions, or empty vector prior to lysis. E1E2 heterodimers were immunoprecipitated with conformation-dependent anti-E2 monoclonal antibody H53. All samples were separated under non-reducing conditions on a 10-15% SDS-PAGE gradient gel and visualized using a phosphorimager. B. Processing and incorporation of HIV-1 structural proteins into HCV glycoprotein-pseudotyped HIV-1 particles. HIV-1 structural proteins Pr55Gag, p17MA, p24CA and p66RT from lysed metabolically-labelled E1E2-pseudotyped HIV-1 particles were immunoprecipitated using IgG from an HIV-1 infected individual (IgG14). Samples were separated under reducing conditions on a 7.5-15% SDS-PAGE gradient gel and visualized using a phosphorimager. Data is representative of two independent experiments.
FIG. 12 shows the ability of E1E2-pseudotyped HIV-1 particles containing multiple variable region deletions to enter Huh7 cells. The tissue culture fluid from 293T cells co-transfected with the HIV-1 firefly luciferase vector (NL4-3.LUC.R-E-) and either wild-type (pE1E2), pE1E2 containing multiple variable region deletions, or empty (pcDNA4) vector was used to infect Huh7 cells in triplicate. The Huh7 cells were lysed and the luciferase activity measured using a Fluostar fitted with luminescence optics. The mean and standard deviation was calculated from triplicate infections. Data is representative of three independent experiments.
FIG. 13 shows the ability of E2 containing multiple variable region deletions to bind to CD81-LEL. A. Metabolically-labelled cell lysates of 293T cells transfected with wild-type (pE1E2), pE1E2 containing multiple variable region deletions, or empty (pcDNA4) vector were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected using anti-E2 conformation-dependent monoclonal antibody H53 and rabbit anti-mouse horseradish peroxidase (HRP) conjugate. Absorbance values were read at 450 nm-620 nm (background). B. Ability of virion-incorporated forms of E2 containing multiple variable region deletions to bind CD81-LEL. Lysates of metabolically-labelled E1E2-pseudotyped HIV-1 particles were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected as described above. Absorbance values were read at 450 nm-620 nm (background). Data is representative of two independent experiments.
FIG. 14 shows the expression of E2 RBD (residues 384-661) containing single and multiple variable region deletions in cell lysates. A. Metabolically labelled cell lysates of 293T cells transfected with either wild-type (E2-myc), E2-myc containing single or multiple variable region deletions, or empty vector were immunoprecipitated with anti-E2 conformation-dependent antibody H53. B. Secretion of E2 RBD (residues 384-661) containing single and multiple variable region deletions in supernatant fluid. The supernatant fluid from metabolically-labelled 293T cells transfected with either wild-type (E2-myc), E2-myc containing single or multiple variable region deletions, or empty vector were immunoprecipitated with the anti-E2 conformation dependent antibody, H53. All samples were separated on 10-15% SDS-PAGE gradient gels and visualized using a phosphoimager. Data for the single variable region deletion is from one experiment.
FIG. 15 shows the ability of E2 RBD (residues 384-661) containing single variable region deletions to bind CD81-LEL. A. Cell lysates from metabolically-labelled 293T cells transfected with wild-type (E2-myc), E2-myc containing single variable region deletions, or empty vector were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected using anti-E2 conformation-dependent antibody H53 and rabbit anti-mouse horseradish peroxidase (HRP) conjugate. Absorbance values were read at 450 nm-620 nm (background). B. Ability of secreted E2 RBD (residues 384-661) containing single variable region deletions to bind CD81-LEL. Tissue-culture fluid from metabolically-labelled 293T cells transfected with wild-type (E2-myc), E2-myc containing single variable region deletions were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Bound E2 was detected using anti-E2 conformation-dependent antibody H53 and rabbit anti-mouse horseradish peroxidase (HRP) conjugate. Absorbance values were read at 450 nm-620 nm (background). Data is from a single experiment.
FIG. 16 shows the ability of E2 RBD (residues 384-661) containing multiple variable region deletions to bind CD81-LEL. A. Cell lysates from metabolically-labelled 293T cells transfected with wild-type (E2-myc), E2-myc containing multiple variable region deletions, or empty vector were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Absorbance values were read at 450 nm-620 nm (background). B. Ability of secreted E2 RBD (residues 384-661) containing multiple variable region deletions to bind CD81-LEL. Supernatant fluid from metabolically-labelled 293T cells transfected with wild-type (E2-myc), E2-myc containing multiple variable region deletions or empty vector were applied to CD81 MBP-LEL (residues 113-201) coated enzyme immunoassay plates at progressive two-fold dilutions. Absorbance values were read at 450 nm-620 nm (background). Data is from a single experiment.
FIG. 17 shows the binding of E2-myc proteins to full length surface expressed CD81. Equivalent amounts of monomeric secreted E2-myc proteins produced from 293T cells were applied to CHO-K1 cells transfected with vector encoding full length CD81. Bound E2-myc was detected with iodinated 9E10 prior to measurement in a gamma counter. Mean of three independent assays ±standard error. P values derived by comparison with the E2 Δ23-myc E2 construct using the students t test assuming unequal variances.
FIG. 18 shows the ability of E2-myc and E2 Δ123-myc to be detected with human conformation sensitive monoclonal antibodies. Wild-type (E2-myc) and E2 Δ23-myc were metabolically labelled and immunoprecipitated with the “CBH” panel of conformation dependent human monoclonal antibodies specific to three immunogenic domains (A, B and C) of E2. Immunoprecipitated proteins were analysed by SDS-PAGE under non-reducing conditions in 10-15% polyacrylamide gradient gels followed by scanning in a phosphorimager.
FIG. 19 shows a schematic representation of the E1 E2 polyprotein containing single and multiple variable region deletions. The pE1E2 vector encodes the full-length H77c sequence of E1 (dark grey) and E2 (light gray) including their transmembrane domains (horizontal stripes) at the C-terminus of E1 and E2 and the signal peptides with the signal peptidase cleavage sites indicated (arrows). The E2 variable regions (dotted) and the conserved region adjacent to HVR1 (diagonal stripes) are replaced—individually and in combination—with flexible Gly-Ser-Ser-Gly (GSSG) linker motifs (SEQ ID NO: 95) as indicated. FIG. 19 discloses SEQ ID NOS 110-112, 109, 109, 95, 95, 109, 95, 109, 95, 95, 95, 109, 95 and 95, respectively, in order of appearance.
FIG. 20 shows a schematic representation of overlap extension PCR strategy. The variable regions were deleted from the HCV glycoprotein template sequence (diagonal stripes) using two oligonucleotide pairs: each contains one external and one internal primer that amplifies either the 5′ or 3′ fragment adjacent to HVR1 as shown. The internal primer sequence introduces the Gly-Ser-Ser-Gly linker motif (bold) (SEQ ID NO: 95) and a short ‘overlap’ sequence complementary to the respective 5′ or 3′ fragment. These sequences anneal to form the template for the second round of PCR amplification that uses the external primers to amplify the HVR1 deleted glycoprotein sequence. FIG. 20 discloses SEQ ID NOS 113, 114, 113, 114, 113, 114 and 113-115, respectively, in order of appearance.
FIG. 21 shows a schematic representation of HCV E1E2 polyprotein containing modified single variable region deletions with extended linkers. The pE1E2 vector encodes the full-length H77c sequence of E1 (dark gray) and E2 (light gray) including their transmembrane domains (vertical stripes) at the C-terminus of E1 and E2. The signal peptidase cleavage sites are also indicated (arrows). The E2 variable regions and the conserved region adjacent to HVR1 (con) are partially deleted and replaced with flexible Gly-Ser-Ser-Gly linker motifs (SEQ ID NO: 95). FIG. 21 discloses SEQ ID NOS 110-112 and 116-118, respectively, in order of appearance.
FIG. 22 shows a schematic representation of the E2 receptor-binding domain (E2 RBD661) containing single and multiple variable region deletions. The pE2661 vector encodes the E2 RBD encoding residues 384-661 (E2-myc). The variable regions (dotted) and a conserved sequence adjacent to HVR1 (diagonal stripes) were replaced individually and in combination with short flexible Gly-Ser-Ser-Gly (GSSG) linker motifs (SEQ ID NO: 95) as indicated. A myc epitope tag (vertical stripes) was also introduced at the C-terminus of these constructs. FIG. 22 discloses SEQ ID NOS 110-112, 109, 109, 95, 95, 109, 95, 109, 95, 95, 95, 10, 95, and 95, respectively, in order of appearance.
FIG. 23 shows a schematic representation of the strategy used to generate HCV glycoprotein-pseudotyped HIV-1 particles. The HCV glycoprotein expression vector encoding E1E2 (pE1E2) or pE1E2 containing variable region deletions and the retroviral vector (HIV-1 NL4-3.LUC.R−E−) lacking its native envelope gene and containing a luciferase reporter gene are co-transfected into 293T cells. As the retroviral core proteins assemble within the cell they acquire an envelope by budding from the plasma membrane, incorporating the HCV E1E2 glycoproteins. These virions are then collected and used to undergo one round of infection and replication in Huh7 cells. The level of E1E2-mediated entry is then quantified by the resulting luciferase activity within these infected Huh7 cells.
FIG. 24 shows the ability of E2-myc and E2 Δ123-myc proteins to be immunoprecipitated by a panel of human sera obtained from HCV infected individuals. Immunoprecipitated proteins were analysed by SDS-PAGE under non-reducing conditions in 10-15% polyacrylamide gradient gels followed by scanning in a phosphorimager. Below each lane is the HCV RNA status, presence of HCV specific antibody detected using one of BioRad Monolisa, Abbot Murex or Chiron RIBA assays and the 50% neutralizing antibody titre for that serum sample. Molecular weight markers are indicated to the left.
FIG. 25 shows the ability of E2-myc proteins containing variable region deletions to interact with recombinant large extracellular loop of CD81. A. Ability of wild-type E2-myc, E2 Δ12-myc, E2 Δ23-myc and E2-myc protein containing the mutation L441M, that disrupts interaction with CD81, to interact with wild-type recombinant MBP-LEL. E2-myc proteins were serially diluted in MBP-LEL coated enzyme immunoassay plates. Data is the average of two to seven independent experiments and is shown as the mean percentage binding relative to wild-type E2-myc ±standard deviation. B. Ability of WT E2-myc, E2 Δ13-myc and E2 Δ123-myc to interact with wild-type recombinant MBP-LEL (solid lines) or recombinant MBP-LEL containing the F186S mutation that disrupts E2 binding (dashed line). Data is the average of two to seven independent experiments and is shown as the mean percentage binding relative to wild-type E2-myc ±standard deviation.
FIG. 26 shows the ability of E2-myc proteins containing variable region deletions to interact with full length surface expressed CD81. A. Ability of WT E2-myc, E2 Δ12-myc, E2 Δ23-myc and E2-myc protein containing the mutation L441M to interact with full length CD81 transfected CHO-K1 cells. Dilutions of wild-type or variable region deleted E2-myc proteins were applied to ice cold, human CD81 transfected CHO-K1 cells and incubated on ice for 4 hrs. After washing, 1×106 CPM 125I-9E10 was added and plates incubated for 1 h at room temperature, washed and counted in a Packard Auto GammaCounter. Data shown is the mean percentage binding relative to wild-type ±standard error of two to five independent experiments. B. Ability of WT E2-myc, E2 Δ123-myc, and E2 Δ13-myc to interact with full length human CD81 (solid line) or F186S-CD81 (dashed line) transfected CHO-K1 cells. Data shown is the mean percentage binding relative to wild-type ±standard error of two to five independent experiments.
FIG. 27 shows an SDS-PAGE of purified HCV E2 protein variants in 4-20% polyacrylamide gradient gel of the E2-his proteins containing none (wild-type) one or more variable region deletions. The proteins were visualized by staining with Coomassie brilliant blue.
FIG. 28 shows a blue-native PAGE analysis of E2-his proteins. The purified proteins (10 μg) were electrophoresed in 5-15% polyacrylamide gradient gels under native conditions. After electrophoresis, the gels were destained overnight and scanned in a Licor Odyssey scanner at 680 nm. The migration position of protein standards is shown on the right.
FIG. 29 shows a Western blot analysis of purified E2-his proteins. Samples of the purified proteins were subjected to reducing SDS-PAGE followed by electrophoretic transfer to nitrocellulose membrane. E2 proteins were detected with a non-conformation dependent E2 specific monoclonal antibody followed by goat anti-mouse immunoglobulin coupled to Alexafluor 680 nm (Invitrogen). Immunoblots were scanned in an Licor Odyssey scanner. Molecular weight markers are shown on the left (kDa).
FIG. 30 shows the ability of HCV E2-his proteins to bind to a recombinant form of the CD81 large extracellular loop (CD81-LEL). Enzyme immunoassay plates were coated with maltose binding protein fused to (A) wild-type large extracellular loop of CD81 (residues 113-201) (CD81-LEL) or (B) CD81-LEL containing an F186S mutation in the E2 binding site of CD81-LEL. Plates were blocked with bovine serum albumin and then incubated with serial dilutions of E2-his proteins (in 50 μl PBS containing 5 mg/ml bovine serum albumin and 0.05% Tween20) for 2 h. The bound E2-his proteins were detected using an E2 specific monoclonal antibody and rabbit anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako). Plates were developed using tetramethylbenzidine hydrochloride substrate and stopped by the addition of 1M HCl. Absorbance was measured at 450 nm and the background at 620 nm subtracted. Percentage binding was calculated by dividing the absorbance value for each protein by the maximal absorbance obtained for wild-type E2-his and multiplied by 100.
FIG. 31 shows the immunoreactivity towards homologous E2-his antigen of mouse sera obtained after 2 immunizations with E2-his proteins. E2-his proteins were captured on 96 well Maxisorb microtitre plates (Nunc) precoated with galanthis nivalis (GNA) lectin. Serial dilutions of immune mouse sera were incubated with the captured corresponding E2 protein variant used for the immunization and bound immunoglobulins detected with rabbit anti-mouse immunoglobulin coupled to horseradish peroxidase. The assay was developed using tetramethylbenzidine hydrochloride substrate and stopped by the addition of 1M HCl. Absorbance values were measured at 450 nm and the background at 620 nm subtracted in a Fluostar plate reader (BMG technologies). The antibody titres of individual sera were determined as the serum dilution giving 5-times the background absorbance. The maximum (upper error bar), 75th and 25th percentile (upper and lower edges of box, respectively), median (horizontal line within box) and minimum (lower error bar) titres for each immunogen group are shown.
FIG. 32 shows the immunoreactivity towards E2-his (A) and E2 Δ123-his (B) antigens of mouse sera obtained after 2 immunizations with E2 protein variants. E2-his and E2 Δ123-his proteins were captured on 96 well Maxisorb microtitre plates and antibody titres of individual sera determined as described in FIG. 31. The maximum (upper error bar), 75th and 25th percentile (upper and lower edges of box, respectively), median (horizontal line within box) and minimum (lower error bar) titres for each immunogen group are shown.
FIG. 33 shows the immunoreactivity towards homologous E2-his antigen of mouse sera obtained after 3 immunizations with E2-his proteins. E2-his proteins were captured on 96 well Maxisorb microtitre plates and antibody titres of individual sera determined as described in FIG. 31. The maximum (upper error bar), 75th and 25th percentile (upper and lower edges of box, respectively), median (horizontal line within box) and minimum (lower error bar) titres for each immunogen group are shown.
FIG. 34 shows the immunoreactivity towards E2-his (A) and E2 Δ123-his (B) antigens of mouse sera obtained after 3 immunizations with E2 protein variants. E2-his and E2 Δ123-his proteins were captured on 96 well Maxisorb microtitre plates and antibody titres of individual sera determined as described in FIG. 31. The maximum (upper error bar), 75th and 25th percentile (upper and lower edges of box, respectively), median (horizontal line within box) and minimum (lower error bar) titres for each immunogen group are shown.
FIG. 35 shows the immunoreactivity towards Con1 E2RBD-his (A) and JFH1 E2RBD-myc (B) antigens of mouse sera obtained after 3 immunizations with E2 protein variants. Con1 and JFH1 RBD proteins were captured on 96 well Maxisorb microtitre plates and antibody titres determined for individual sera as described in FIG. 31. The maximum (upper error bar), 75th and 25th percentile (upper and lower edges of box, respectively), median (horizontal line within box) and minimum (lower error bar) titres for each immunogen group are shown.
FIG. 36 shows the eighty-percent neutralization titres (log10) of mouse sera obtained after 3 immunizations with E2-his proteins. Serial 5-fold dilutions of heat-inactivated immune mouse sera preincubated for 1 h at 37° C. with H77c HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses (1 h) and then added to quadruplicate Huh7 cell monolayers in 48-well tissue culture plates. Following a 4 h incubation (37° C., 5% CO2) the cells were washed with PBS and the medium replaced. After an additional 3-day incubation (37° C. in 5% CO2), the cells were lysed and luciferase activity assayed in a fluostar fitted with luminescence optics. The neutralization titres of individual sera were determined as the serum dilution giving 80% neutralization compared to HCV glycoprotein-pseudotyped HIV-1 luciferase reporter virus preincubated with medium alone.



DETAILED DESCRIPTION OF THE INVENTION
The modified E2 core structure of the HCV virion in which at least one of the variable regions, and in particular the variable region igVR identified by the inventors, has been modified by removal or deletion of at least a part of the variable region, and optional insertion of a linker sequence, has practical use as a vaccine that can elicit broadly neutralizing antibodies to diverse strains of HCV. The binding efficiency of the modified E2 glycoprotein exhibits wild-type binding to the HCV receptor CD81, and the modified E2 glycoprotein provides a means of treating HCV infection by mimicking complex conformational changes of the E2 ectodomain required for effective CD81 binding, and thereafter initiating an immune response without cell invasion.
In one aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein in at least one of said variable regions at least a part of the variable region is replaced with a flexible linker sequence.
In another aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein at least a part of the HVR2 variable region is removed or is replaced with a flexible linker sequence.
In yet another aspect, the present invention provides a modified hepatitis C virus (HCV) E2 glycoprotein comprising the HCV-E2 receptor-binding domain (RBD) including the HVR1, HVR2 and igVR variable regions, wherein at least a part of the igVR variable region is removed or is replaced with a flexible linker sequence.
References herein to the “HVR1” and “HVR2” variable regions are to be understood as references to the two variable regions HVR1 (384-410) and HVR2 (461-481), while references herein to the “igVR” variable region are to be understood as references to the intergenotypic variable region igVR (570-580) identified by the inventors.
The term “flexible linker sequence” is used herein to refer to a short, flexible, polypeptide sequence which permits disulfide bond linkages between cysteine residues in the modified glycoprotein leading to retention of the native or “wild-type” disulfide linkages, and in particular retention of the ability to bind to the HCV CD81 receptor and conformation-dependent antibodies. Suitable linker sequences are discussed in review articles by George and Heringa, 2002, and Argos, 1990, and may consist of up to 20 amino acid residues such as Gly and Ser, and include, and comprise amino acids selected from the sequence group consisting of Gly, Ser, Ala, Thr and Arg, more particularly Gly- and Ser-Ser-Gly (GSSG) (SEQ ID NO: 95). Suitable linker sequences include, by way of example, the sequences

    
    
        (Gly)2-Ala-(Gly)2 (SEQ ID NO: 96), (Gly)5 (SEQ ID NO: 97) or (Gly)8 (SEQ ID NO: 98) (see Sabourin et al., 2007),
        (Gly)6 (SEQ ID NO: 99), (Gly)7 (SEQ ID NO: 100) or (Gly)10 (SEQ ID NO: 101) (see Yang and Gruebele, 2006),
        Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 102) (see Dipti et al., 2006),
        (Gly)4 (SEQ ID NO: 103) (see Anandarao et al., 2006),
        Gly-Ala-Gly (see Wyatt et al., 1995),
        (Gly)2-Arg-(Gly)2-Ser (SEQ ID NO: 104) (see Bellamy-McIntyre et al., 2007),
        (Gly-Gly-Gly-Gly-Ser)n=3 ̃4 (SEQ ID NO: 105) (see Arai et al., 2006), and
        Ser-(Gly)2-Ser-Gly (SEQ ID NO: 106) (see Bahrami et al. 2007).
    
    


A preferred sequence is the sequence Gly-(Ser)2-Gly (SEQ ID NO: 95) disclosed herein. It will be understood that selection of suitable polypeptide linker sequences is a matter of routine experimentation for a person skilled in this field, and the modified HCV E2 glycoproteins of the present invention are not limited to the particular linker sequences disclosed herein. Without wishing to be bound by any theory, by replacing HVR1, HVR2 and igVR with short flexible linkers, disulfide-bond formation between Cys-569 to Cys-581 and between Cys-459 to Cys-486, for example, and the intrinsic folding of conserved E2 core domain, is substantially retained in the modified glycoproteins.
References in this specification to “deletion” of at least part of one of the variable regions of the HCV E2 receptor-binding domain are to be understood as references to deletion or removal of at least part of the sequence of the variable region and optional insertion of a flexible linker sequence to replace the deleted sequence.
The modified HCV E2 glycoproteins of the present invention may be prepared by any suitable method, including in particular preparation of the modified glycoproteins in the form of recombinant products by expression of appropriate DNA deletion constructs as described in the Examples herein.
Preferably at least one of the second and third variable regions of the core E2 receptor-binding domain, HVR2 and igVR, is modified by removal of at least a portion of the residue within the region and inserting a flexible linker sequence. It has been found that deletion of at least part of the HVR2 region, in combination with other variable region deletions, substantially reduces E1E2 heterodimerisation.
In a further embodiment, all three of the variable regions are modified by removal of at least a portion of the residues within these regions and inserting a linker sequence.
Deletion of intergenotypic variable region (igVR) from the E2 glycoprotein that already contains deletions of HVR1 and HVR2 improves binding to CD81 relative to simultaneous deletions of HVR1 and HVR2 alone.
It has been found by the inventors that HVR1, HVR2, and igVR are all required for E1E2-pp mediated viral entry into Huh7 cells. E1E2-pp containing deletions of at least each of HVR1, HVR2, and igVR have been found to retain wild-type levels of recombinant CD81 binding. This represents a significant improvement in the ability to (a) mimic cell binding of the HCV virion, and (b) initiate an immune response without exposing a patient to highly variable immunodominant regions that may be immune decoys.
In a preferred embodiment, all three variable regions can be deleted and retain the core E2 folding domain which is required to assemble at least three discontinuous binding elements involved in the CD81-binding site. A further advantage of various combined deletions of HVR1, HVR2, and igVR from the E2 receptor-binding domain is that a soluble form of the E2 core domain is obtainable, which is suitable for use as an immunogen.
The present invention also provides a composition comprising a modified HCV E2 glycoprotein as broadly described above, together with a pharmaceutically acceptable carrier or diluent.
Such a composition may be formulated as a vaccine composition, preferably including an adjuvant.
Conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may be included in vaccine compositions of this invention. Generally, a vaccine composition in accordance with the present invention will comprise an immunologically effective amount of the modified HCV E2 glycoprotein, and optionally an adjuvant, in conjunction with one or more conventional pharmaceutically acceptable carriers and/or diluents. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter, “Advances in Adjuvant Technology and Application”, in Animal Parasite Control Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press 1992, and in Cox and Coulter, “Adjuvants—A Classification and Review of Their Modes of Action”, Vaccine 15(3), 248-256, 1997. As used herein “pharmaceutically acceptable carriers and/or diluents” include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and is described by way of example in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, U.S.A.
In yet another aspect, the present invention also provides a method of eliciting an immune response in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein as broadly described above.
In this aspect, the invention includes a method for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein as broadly described above.
Reference herein to “treatment” is to be understood in its broadest context. Accordingly, the term “prophylactic treatment” includes treatment to protect the patient against infection or to reduce the likelihood of infection. Similarly, the term “therapeutic treatment” of infection does not necessarily imply that the patient is treated until total recovery from infection, and includes amelioration of the symptoms of infection as well as reducing the severity of, or eliminating, the infection.
The modified HCV E2 glycoprotein of this invention is administered in an effective amount. An “effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of the infection. The amount varies depending upon the health and physical condition of the individual to be treated, the racial background of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. If necessary, the administration of an effective amount may be repeated one or several times. The actual amount administered will be determined both by the nature of the infection which is being treated and by the rate at which the active immunogen is being administered.
Preferably, the patient is a human, however the present invention extends to treatment and/or prophylaxis of other mammalian patients including primates and laboratory test animals (e.g. mice, rabbits, rats, guinea pigs).
In accordance with the present invention, the modified HCV E2 glycoprotein is preferably administered to a patient by a parenteral route of administration. Parenteral administration includes any route of administration that is not through the alimentary canal (that is, not enteral), including administration by injection, infusion and the like. Administration by injection includes, by way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular) and under the skin (subcutaneous). The modified HCV E2 glycoprotein may also be administered in a depot or slow release formulation, for example, subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain the desired pharmacological effect.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in a polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose, trehalose, glucose) and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
In a further aspect, this invention provides the use of a modified HCV E2 glycoprotein as broadly described above in, or in the manufacture of a medicament for, eliciting an immune response in a patient.
In a further aspect, this invention includes the use of a modified HCV E2 glycoprotein as broadly described above in, or in the manufacture of a medicament for, prophylactic or therapeutic treatment of HCV infection in a patient.
In this further aspect, this invention provides an agent for eliciting an immune response in a patient, which comprises a modified HCV E2 glycoprotein as broadly described above.
In this further aspect, the invention includes an agent for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises a modified HCV E2 glycoprotein as broadly described above.
In one embodiment of the invention, it has been found that recombinant E2 glycoprotein in which at least region igVR is subject to deletion of residues in the region and replacement with a flexible linker sequence improves recognition by conformational-dependent antibodies. In addition, it has been found that, in a comparative study with ‘wild-type’ E2 glycoprotein, modification of the region igVR alone and in various combinations with HVR1 and HVR2 show binding to full-length CD81-LEL (large extracellular loop of human tetraspanin CD81) approaching that of the wild-type.
It has been shown by the inventors that alignment of diverse E2 glycoprotein sequences across the six major genotypes identified a novel third variable region, “igVR,” between residues 560 and 581. It is suggested that “igVR” is not strictly a hypervariable region as it exhibits its greatest variability across different genotypes, whereas it is relatively conserved within a genotype and is thus unlikely to be under immune selection pressure. Furthermore, it consists of a series of insertions or deletions that shifts, but always maintains, the glycosylation site located within this region. The inventors have also observed that both variable regions HVR2 and “igVR” are flanked by conserved cysteine residues and proposed that these regions may form solvent-exposed loops stabilised by disulfide-bonding between these residues.
The inventors have proposed that the hypervariable regions form flexible solvent-exposed subdomains that enable the E2 glycoprotein to move between ‘open’ and ‘closed’ conformations, the former being more competent for CD81 binding by exposing the conserved CD81-binding determinants located within the core E2 domain as delineated within this study. Indeed, conformational changes within the E2 glycoprotein have been previously observed upon CD81-binding. In addition, binding of non-neutralizing antibodies to the E2 glycoprotein has been demonstrated to reduce its susceptibility to neutralizing antibodies and is consistent with a model where the non-neutralizing antibodies inhibit the flexibility of these surface-exposed hypervariable regions thus blocking access to the conserved epitopes located within the E2 core domain.
Therefore, this suggests that the modified E2 core domain represents a promising vaccine candidate for eliciting neutralizing antibodies to conserved epitopes within the E2 glycoprotein, including the CD81-binding determinants, that are otherwise occluded by these surface-exposed variable regions that may act as immunological decoys at the surface of the glycoprotein complex during HCV replication.
The present invention also provides an isolated antibody raised against a modified HCV E2 glycoprotein as broadly described above.
The term “antibody” is used broadly herein to include both monoclonal and polyclonal antibodies that specifically bind to the modified HCV E2 glycoprotein, as well as antigen binding fragments of such antibodies including, for example, Fab, F(ab1)2, Fd and Fv fragments of an antibody that retain specific binding activity for the modified HCV E2 glycoprotein.
Antibodies having the desired specificity, both monoclonal and polyclonal, can be obtained using methods which are well known in the art (see, for example, Harlow and Lane, “Antibodies, A laboratory manual”, Cold Spring Harbor Laboratory Press, 1988). Methods for preparing antibodies, and antigen binding fragments thereof, are also described in International Patent Publication WO 02/22155.
The invention also provides a method for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises administration to the patient of an effective amount of an antibody as described above. Accordingly, this method provides passive immunotherapy of the patient.
As previously described, the invention also provides the use of an antibody as described above in, or in the manufacture of a medicament for, prophylactic or therapeutic treatment of HCV infection in a patient.
In this aspect also, the invention provides an agent for prophylactic or therapeutic treatment of HCV infection in a patient, which comprises an antibody as described above.
Further, the invention provides the use of an antibody as broadly described above in diagnosis of HCV infection. In this aspect, the invention provides a method of detecting HCV infection in a patient, comprising contacting a biological sample from the patient with an antibody as described above under conditions which allow formation of an antibody-antigen complex, and detecting said complex, wherein formation of said complex is indicative of the presence of HCV in the sample.
Preferably, the antibody is detectably labelled. Suitable labels are well known in the art and include, for example, enzymes, radioisotopes, fluorescent compounds, colloidal metals, and chemiluminescent, phosphorescent and bioluminescent compounds. Preferably, the biological sample is a sample of a body fluid from the patient, such as a blood sample.
The modified HCV E2 glycoproteins of the present invention may also be used in drug discovery techniques, including for example small molecule screening techniques.
The present invention is further illustrated by the following non-limiting Examples.
Example 1
Materials and methods used in this Example are described below.
1. Role of the Individual Variable Regions in E1E2 Glycoprotein Structure and Function
The results for the various variable deletion mutants are summarized in Table 1.
Intracellular E1E2 Precursor Folding and Heterodimerisation
To examine the role of the individual variable regions in intracellular E1E2 biosynthesis, metabolically-labelled cell lysates of 293T cells transfected with the single variable region deletion constructs were immunoprecipitated with antibodies reactive to E1 and/or E2 epitopes. These preparations were analysed under both non-reducing and reducing conditions to characterise non-covalently associated E1E2 heterodimers existing within the large intracellular population of covalently-linked aggregate as previously observed.
Immunoprecipitation with the polyclonal anti-E1E2 antibody (779) detected the total intracellular population of E1 ( ̃30 kDa) and E2 ( ̃70 kDa) glycoproteins to show efficient expression and cleavage from the polyprotein occurring for each single variable region deletion construct (see FIG. 1A). The mutant E2 glycoproteins exhibited variably lower molecular weights than wild-type: HVR1 con<HVR2 and “igVR”<HVR1<WT. This is consistent with the loss of two glycosylation sites predicted to reside within the HVR1con deletion, one within each of the HVR2 and “igVR” deletions and none within the HVR1 deletion (FIG. 2). However, deglycosylation of these E2 glycoproteins is required to confirm their backbone molecular weights as predicted in Table 2. The band migrating to  ̃100 kDa represents the E1E2 polyprotein that has not been cleaved by host signal peptidases and is more pronounced in both the single HVR2 and “igVR” deletion constructs (FIG. 1A). This is reflected in the reduced levels of total intracellular E1 and E2 observed for the HVR2 and “igVR” mutants suggesting that these deletions may be effecting polyprotein processing.
The non-conformation-dependent anti-E1 monoclonal antibody (A4) precipitated an E1 doublet at  ̃30 kDa and  ̃27 kDa for both the mutant and the wild-type constructs under reducing and non-reducing conditions (FIG. 1B). This doublet represents either alternative E1 glycosylation states or messenger RNA splicing isoforms as previously observed. Furthermore, E1 co-precipitated low levels of the wild-type E2 glycoprotein under non-reducing conditions, whereas it efficiently co-precipitated both wild-type and mutant E2 glycoproteins under reducing conditions. This indicates that A4 recognises an E1 epitope that is more exposed within covalently-linked intracellular E1E2 complexes.
The non-conformation dependent anti-E2 monoclonal antibody (A11) detected intracellular E2 glycoprotein expression for all the individual variable region deletion constructs (FIG. 1C). However, only the mutant E2 glycoproteins containing the HVR1con or HVR1 deletions co-precipitated a detectable amount of E1 under non-reducing and reducing conditions. This indicates that the individual HVR2 and “igVR” deletions are either directly or indirectly disrupting the formation of the intracellular E1E2 heterodimer. Notably, this reduced heterodimerisation is not due to the lower total intracellular E1 and E2 observed for the HVR2 and “igVR” deletions as E2 glycoprotein expression was detected at wild-type levels by A11 in both these mutants. In addition, E2 co-precipitated generally low levels of E1, particularly under reducing conditions, suggesting that the A11 epitope is partly occluded in intracellular E1E2 heterodimer complexes.
The conformation-dependent anti-E2 monoclonal antibody (H53) has been shown to recognise native E2 on the surface of HCV virions and thus the amount of E1 co-precipitating with this E2 species is a good indicator of the proportion of functional non-covalently associated E1E2 heterodimers forming. Immunoprecipitation with H53 detected intracellular E2 glycoprotein expression for all individual variable region deletion constructs (FIG. 1D). This indicates that the inserted Gly-Ser-Ser-Gly linker motif (SEQ ID NO: 95) provides sufficient flexibility within these E2 glycoproteins to retain the intrinsic folding of the native E2 glycoprotein required to present this conformation-dependent epitope. However, the intracellular E2 glycoproteins containing HVR2 or “igVR” deletions failed to co-precipitate E1, suggesting that these regions are directly or indirectly required in the intracellular assembly of the non-covalently associated E1E2 heterodimer.
E1E2 Glycoprotein Maturation and Incorporation into Pseudotyped HIV-1 Particles
To examine the role of the individual variable regions in E1E2 glycoprotein maturation, the small amount of E1 and E2 escaping from the ER to transit through the secretory pathway was enriched for by the incorporation of these complexes into pseudotyped HIV-1 particles as previously described. These metabolically-labelled E1E2-pseudotyped HIV-1 particles were lysed prior to immunoprecipitation with the conformation-dependent anti-E2 monoclonal antibody (H53) and IgG from an HIV-1 infected individual (IgG14).
Immunoprecipitation with IgG14 demonstrated that none of the single variable region deletion constructs had affected processing and virion incorporation of the HIV-1 structural proteins Pr55Gag, p17MA, p24CA or p66RT (FIG. 3B). Immunoprecipitation with H53 detected the virion-incorporated E2 glycoprotein as a diffuse molecular weight band ( ̃70-90 kDa) typical of the mature glycoprotein containing various complex- and hybrid-type carbohydrate modifications as previously described (FIG. 3A). The different mutant E2 glycoproteins exhibited further variation in their relative molecular weights (HVR1con<HVR2 and “igVR”<HVR1<WT) reflecting the loss of glycosylation sites located within these deleted regions as described for the intracellular data. All mutant E2 glycoproteins were incorporated into E1E2-pseudotyped HIV-1 particles at approximately wild-type levels, except for the HVR1con deletion that showed a significantly reduced E2, band. This suggests that the conserved region adjacent to HVR1 is required for the completion of E2 folding, maturation and incorporation into pseudotyped HIV-1 particles.
Immunoprecipitation with H53 also detected a protein species migrating at  ̃33 kDa and another lower molecular weight band at  ̃31 kDa initially suggesting that the virion-incorporated E1 glycoprotein also migrates as a doublet (FIG. 3A). However, across two independent experiments, the lower molecular weight band co-migrated with a non-specific band in the negative control (empty) and thus the higher molecular weight band alone is likely to represent E1. The virion-incorporated E1 glycoprotein ( ̃33 kDa) also migrates more slowly than the intracellular species ( ̃30 kDa) reflecting additional carbohydrate modifications at one or two E1 glycosylation sites acquired during transit through the secretory pathway as previously observed. Furthermore, the virion-incorporated E2 glycoproteins containing the individual HVR2 and “igVR” deletions could not co-precipitate E1 at detectable levels indicating that these variable regions are directly or indirectly required in the formation of the functional E1E2 heterodimer.
E1E2-Mediated Entry into Huh7 Cells
The formation of the functional non-covalently associated E1E2 heterodimer has been shown to be critical for E1E2-mediated entry into Huh-7 cells. In order to investigate the role of the individual variable regions in viral entry, the single deletion constructs were used to generate E1E2-pseudotyped HIV-1 particles to infect Huh7 cells as previously described. As expected, the HVR1con deletion conferred a total loss of entry due to its retarded incorporation of the E2 glycoprotein into pseudotyped HIV-1 virions as observed above (FIG. 4). Similarly, the HVR2 and “igVR” deletions were not entry competent due to the disruption of the functional E1E2 heterodimer complex. However, the HVR1 deletion also conferred a total loss of entry despite retaining both wild-type heterodimerisation levels and incorporation of E1E2 glycoproteins into HIV-1 virions suggesting that this region may have a direct role in E1E2-mediated viral entry.
CD81-LEL Binding
It has been previously demonstrated that the E2 glycoprotein alone is sufficient to mediate binding to the CD81 receptor and therefore, despite exhibiting a loss of heterodimerisation and/or viral entry, both intracellular and virion-incorporated mutant E2 glycoproteins were examined for their ability to bind to the large-extracellular loop (LEL residues 113-201) of CD81. This involved applying both cell and viral lysates to a solid-phase CD81 MBP-LEL (residues 113-201) binding assay as previously described. This data was normalized for the amount of monomeric E2 expressed from each vector as precipitated with the conformation-dependent anti-E2 monoclonal antibody H53 and observed by SDS-PAGE under non-reducing conditions; FIGS. 1D (intracellular) and 3A (virion-incorporated).
All intracellular E2 glycoprotein precursors containing the single variable region deletions exhibited CD81-LEL binding at wild-type levels, except for HVR1 con that demonstrated a total loss of binding (FIG. 5A). This indicates that the inserted Gly-Ser-Ser-Gly linker motifs (SEQ ID NO: 95) are providing sufficient flexibility within the HVR1, HVR2 and “igVR” deleted E2 glycoproteins to form the E2 CD81-binding site and suggests that these individual variable regions are not required in this function. It also indicates that the intracellular E2 glycoprotein precursor lacking the conserved region adjacent to HVR1 (HVR1 con) cannot form the CD81-LEL binding site despite maintaining the conformation-dependent epitope recognised by H53. Intracellular forms of both the HVR2 and “igVR” deletions displayed an additional enhancement of maximum CD 81-LEL binding compared to wild-type (FIG. 5A) although they did not exhibit a significant shift in the overall binding curve and thus additional independent experiments are required to determine whether these variable regions are modulating CD81-LEL binding.
As expected, the virion-incorporated E2 glycoprotein containing the HVR1 con deletion could not mediate CD81-LEL binding, reflecting both its reduced incorporation into E1E2-pseudotyped HIV-1 particles as well as the intracellular data above (FIG. 5B). In contrast, the virion-incorporated E2 glycoproteins containing the HVR1, HVR2 and “igVR” deletions all retained at least wild-type levels of CD81-LEL binding indicating that these mutant E2 glycoproteins retain the CD81-binding site even after maturation. Interestingly, the virion-incorporated E2 glycoprotein containing the HVR1 deletion demonstrated an additional enhancement of maximum CD81-LEL binding compared to wild-type and exhibited an approximately 4-fold increase in the overall binding curve (FIG. 5B). This suggests that HVR1 is negatively modulating CD81-LEL binding within the virion-incorporated E2 glycoprotein despite not being required for this function. The observed absence of this effect in the intracellular E2 glycoprotein precursor containing the same deletion (FIG. 5A) reflects changes in E1E2 folding and the CD81-binding site that occur during glycoprotein maturation through the secretory pathway as previously observed.
2. Effect of the Extended Linker on E1E2 Glycoprotein Structure and Function
Intracellular E1E2 Precursor Folding and Heterodimerisation
In order to enhance the folding of the E2 glycoproteins containing the individual variable region deletions, the Gly-Ser-Ser-Gly linker (SEQ ID NO: 95) was extended by reintroducing several conserved cysteine-proximal residues deleted from the original constructs. Notably, this series of modified deletion constructs does not have a HVR1 counterpart due to the highly variable nature of this region. These constructs were analysed for E1 and E2 glycoprotein expression and heterodimerisation within 293T cells by pulse-chase metabolic-labelling and immunoprecipitation.
Immunoprecipitation with the anti-E1E2 polyclonal antibody (779) detected intracellular expression and cleavage of the E1 ( ̃30 kDa) and E2 ( ̃70 kDa) glycoproteins from the polyprotein occurring for each extended linker deletion construct (FIG. 6A). Polyprotein cleavage appeared to be slightly less efficient in the HVR2link and “igVR link” constructs suggesting that these deletions may be affecting polyprotein processing. The extended linkers did not restore the glycosylation sites deleted from the original constructs and thus the mutant E2 glycoproteins again exhibited variably lower molecular weights (kDa) than the wild-type conferred by the absence of these glycans: HVR1conlink<HVR2link and “igVR link”<WT. However, deglycosylation of these E2 glycoproteins would be required to verify their backbone molecular weights as predicted in Table 2.
The anti-E1 monoclonal antibody (A4) detected the presence of the E1 doublet ( ̃30 kDa and  ̃27 kDa) for all extended linker constructs (FIG. 6B). E1 failed to co-precipitate wild-type E2 glycoprotein under non-reducing conditions, whereas it co-precipitated both mutant and wild-type E2 under reducing conditions again reflecting the low sensitivity of the A4 antibody for non-covalently associated E1. Precipitation with the non-conformation dependent anti-E2 monoclonal antibody (A11) detected wild-type E2 glycoprotein expression for each extended linker construct although only the HVR1conlink deletion co-precipitated detectable amounts of E1 (FIG. 6C). The conformation-dependent monoclonal antibody (H53) also precipitated wild-type levels of the intracellular E2 glycoprotein for each extended linker construct indicating that these mutants retain the intrinsic folding properties of the native E2 glycoprotein required to present this conformation-dependent epitope (FIG. 6D). Furthermore, the extended “igVR” linker recovered co-precipitation with E1 at wild-type levels suggesting that this additional linker region is required, directly or indirectly, for the intracellular assembly of the non-covalently associated E1E2 heterodimer. Notably, however, this effect was not observed for the extended HVR2 linker construct.
E1E2 Glycoprotein Maturation and Incorporation into Pseudotyped HIV-1 Particles
To examine whether the extended linkers altered E1E2 glycoprotein maturation and virion incorporation, the modified single deletions were introduced into E1E2-pseudotyped HIV-1 particles prior to lysis and immunoprecipitation with the conformation-dependent anti-E2 monoclonal antibody (H53) and IgG from an HIV-1 infected individual (IgG14) as previously described.
Immunoprecipitation with IgG14 indicated that none of the modified single deletion constructs affected processing and virion incorporation of the HIV-1 structural proteins (FIG. 7B). Precipitation with H53 again detected a diffuse molecular weight band ( ̃70-kDa) typical of the mature virion-incorporated E2 glycoprotein (FIG. 7A). The virion-incorporated mutant E2 glycoproteins also exhibited marginally lower molecular weights (kDa) than the wild-type species conferred by the loss of one or two glycosylation sites as described for the intracellular data. Precipitation with H53 again demonstrated reduced virion-incorporation of the E2 glycoprotein containing the HVR1conlink deletion despite the reintroduction of four conserved cysteine-proximal residues absent in the original HVR1con deletion construct. The HVR2link deletion demonstrated wild-type levels of E2 glycoprotein incorporated into pseudotyped HIV-1 particles, yet still failed to co-precipitate a detectable amount of E1 reflective of the intracellular data. In contrast, the virion-incorporated E2 glycoprotein containing the extended “igVR” linker co-precipitated wild-type levels of E1 suggesting that this region is directly or indirectly required in the formation of the functional E1E2 heterodimer.
E1E2-Mediated Entry into Huh7 Cells
To examine whether these extended linkers conferred E1E2-mediated entry into Huh-7 cells, these modified single deletion constructs were used to generate E1E2-pseudotyped HIV-1 particles to infect Huh7 cells as previously described. As expected, the extended linkers in the HVR1conlink and HVR2link deletion constructs did not recover any entry activity consistent with the observed failure of these mutant E2 glycoproteins to be incorporated into virions or heterodimerise, respectively (FIG. 8). However, the virion-incorporated E2 glycoprotein containing the extended “igVR” linker demonstrated a significant return of entry activity ( ̃8-fold) compared to the negative control (empty) consistent with its recovered heterodimerisation with E1. Notably, however, “igVR link” still exhibited reduced entry activity compared to wild-type ( ̃10-fold).
CD81-LEL Binding
To examine whether the intracellular or virion-incorporated E2 glycoproteins containing these extended linkers altered CD81-LEL binding, both cell and viral lysates were applied to a solid phase CD81 MBP-LEL (residues 113-201) binding assay as previously described. This data was normalized for the amount of monomeric E2 expressed from each vector as precipitated with H53 and observed by SDS-PAGE under non-reducing conditions; FIGS. 6D (intracellular) and 7A (virion incorporated). The extended linkers did not appear to alter the CD81-LEL binding ability of the intracellular E2 glycoproteins containing the HVR2link or “igVR link” deletions as they still demonstrated wild-type levels, or even slightly enhanced, binding as exhibited by the original HVR2 and “igVR” deletion constructs (FIG. 9A). Notably, the intracellular E2 glycoprotein containing the HVR1conlink deletion demonstrated a marginal recovery in CD81-LEL binding compared to the negative control (empty) suggesting that the conserved cysteine-proximal residues reintroduced into this construct may contribute directly or indirectly to the formation of the CD81-binding site. The virion-incorporated E2 glycoprotein containing HVR1conlink deletion, however, lost even this reduced CD81-LEL binding ability (FIG. 9B) consistent with its retarded incorporation into HIV-1 virions. The virion-incorporated E2 glycoproteins containing the HVR2link and “igVR link” deletion constructs maintained CD81-LEL binding in viral lysates, but generated inconsistent results in regard to their relative CD81-LEL binding ability across two independent assays (FIGS. 9B and 9C) and thus a third independent assay is required to verify these results.
3. Effect of Multiple Variable Region Deletions on E1E2 Glycoprotein Structure and Function
Intracellular E1E2 Precursor Folding and Heterodimerisation
Despite a loss of heterodimerisation with E1 and/or viral entry, the above findings indicate that HVR1, HVR2 and “igVR” can be individually deleted from both the intracellular and virion-incorporated E2 glycoprotein without disrupting its intrinsic folding properties as recognised by the conformation-dependent monoclonal antibody H53 and binding to the large extracellular loop (LEL) of the CD81 receptor. In order to further characterise these variable regions and delineate a conserved E2 core domain, multiple deletions were introduced into the E1E2 polyprotein and analysed within 293T cells by pulse-chase metabolic-labelling and immunoprecipitation. Precipitation with the anti-E1E2 polyclonal antibody (779) showed efficient expression and cleavage of E1 ( ̃30 kDa) and E2 ( ̃70 kDa) glycoproteins from the polyprotein occurring for each of the multiple variable region deletion constructs (FIG. 10 A). These mutant E2 glycoproteins also migrated faster than the wild-type species corresponding to the loss of a glycosylation site within each HVR2 and/or “igVR” deletion: HVR1+2+igVR and HVR2+igVR<HVR1+2 and HVR1+igVR<WT. However, deglycosylation of these glycoproteins is required to confirm their backbone molecular weights as predicted in Table 3. The anti-E1 monoclonal antibody (A4) again detected an E1 doublet ( ̃30 kDa and  ̃27 kDa) present for each of the variable region deletion constructs (FIG. 10B). E1 co-precipitated with both wild-type and mutant E2 glycoproteins under reducing conditions demonstrating the formation of covalently-linked E1E2 heterodimers. The failure to detect the wild-type E1E2 heterodimer under non-reducing conditions again reflects the low sensitivity of A4 for the non-covalently associated E1 glycoprotein.
In contrast, the non-conformation dependent anti-E2 monoclonal antibody (A 11) did not detect the E2 glycoproteins containing these multiple variable deletions coprecipitating E1 under reducing or non-reducing conditions despite demonstrating wild-type levels of intracellular E2 glycoprotein expression for each of these deletion constructs (FIG. 10C). The conformation-dependent anti-E2 monoclonal antibody H53 also precipitated wild-type levels of the mutants E2 glycoproteins (FIG. 10D). This indicates that the inserted Gly-Ser-Ser-Gly linker motifs (SEQ ID NO: 95) provided sufficient flexibility within these mutant E2 glycoproteins to present this conformation-dependent epitope despite the introduction of multiple variable region deletions (FIG. 10D). These mutant E2 glycoproteins also demonstrated a loss of heterodimerisation with E1 consistent with the absence of HVR2 and/or “igVR” required in the assembly of the E1E2 heterodimer as observed above.
E1E2 Glycoprotein Maturation and Incorporation into Pseudotyped HIV-1 Particles
To examine whether the multiple variable region deletions altered E1E2 glycoprotein maturation, these deletions were introduced into E1E2-pseudotyped HIV-1 particles and lysed prior to immunoprecipitation with the conformation-dependent anti-E2 monoclonal antibody H53 and IgG from an HIV-1 infected individual (IgG14) as previously described.
IgG14 precipitation indicated that none of the multiple variable deletions had affected processing and virion incorporation of the HIV-1 structural proteins (FIG. 11B). Immunoprecipitation with H53 again detected E2 as a diffuse band ( ̃70-90 kDa) typical of the mature virion-incorporated E2 glycoprotein (FIG. 11A) and demonstrated the E2 glycoproteins containing multiple variable region deletions to be incorporated into HIV-1 virions at wild-type levels. These mutant E2 glycoproteins also migrated marginally faster than the wild-type species reflecting the loss of one or two glycosylation sites within the HVR2 and/or “igVR” deletions as described for the intracellular data. This suggests that the virion-incorporated E2 glycoproteins containing multiple variable region deletions retain the conformation-dependent H53 epitope even after maturation. Again, none of these mutant E2 glycoproteins co-precipitated detectable amounts of E1 as observed in the intracellular data.
E1E2-Mediated Entry into Huh7 Cells
As expected, E1E2-pseudotyped HIV-1 particles containing multiple variable region deletions were not competent for entry into Huh7 cells (FIG. 12).
CD81-LEL Binding
Despite a loss of heterodimerisation and E1E2-mediated viral entry, both the intracellular and virion-incorporated E2 glycoproteins containing multiple variable region deletions were examined for their ability to bind CD81-LEL by applying both cell and viral lysates to a solid-phase CD81 MBP-LEL (residues 113-201) binding assay as previously described. This data was normalized for the amount of monomeric E2 expressed from each vector as precipitated with H53 and observed by SDS-PAGE under non-reducing conditions; FIG. 10D (intracellular) and 11A (virion-derived).
The intracellular E2 glycoproteins containing multiple variable region deletions all demonstrated wild-type levels of CD81-LEL binding (FIG. 13A). This indicates that the Gly-Ser-Ser-Gly linker motifs (SEQ ID NO: 95) provide sufficient flexibility within these mutant E2 glycoproteins to form the precursor CD81 receptor-binding site. In addition, the E2 glycoprotein containing the HVR2 and “igVR” double deletion (pE1E2 Δ23) demonstrated a further enhancement of maximum CD81-binding across two independent experiments. This is consistent with this same effect observed in the individual HVR2 and “igVR” mutants although, similarly, the overall binding curve was not increased and thus additional independent assays are required to determine the statistical significance of this effect.
The virion-incorporated E2 glycoproteins containing multiple variable region deletions all maintained CD81-LEL binding (FIG. 13B) again indicating that the inserted linker motifs provide sufficient flexibility within these glycoproteins to present the CD81-binding site even after maturation. This again suggests that HVR1, HVR2 and “igVR” are not required for this function reflecting both the intracellular data and that obtained for the individual variable region deletions. Importantly, this demonstrates that the E2 glycoprotein containing the triple variable deletion encompasses all the structural and functional determinants required in CD81-LEL binding and thus constitutes a minimal E2 core domain. These virion-derived E2 glycoproteins containing double and triple variable region deletions also demonstrated slightly reduced or enhanced maximum CD81-LEL binding activity compared to wild-type, respectively, although again none of these mutants exhibited a significant increase in the overall binding curve.
4. Role of the Variable Regions in E2 Receptor-Binding Domain (E2 RBD661myc) Structure and Function
Folding and Secretion
The above results demonstrate that the simultaneous deletion of all three variable regions does not disrupt the intrinsic folding of the E2 glycoprotein as recognised by the conformation-dependent monoclonal antibody H53 or binding to the CD81-LEL strongly suggesting that this construct constitutes an E2 core domain. However, in the context of the full-length E2 glycoprotein, the presence of both the transmembrane and membrane-proximal regions would make it difficult to crystallize this core E2 structure. Therefore, both single and multiple variable region deletions were introduced into E2 receptor-binding domain (E2 RBD661myc) that is both soluble and amenable to high level expression. The E2 RBD661myc glycoproteins containing single and multiple variable region deletions were metabolically labelled within 293T cells and both the intracellular and secreted protein analysed by immunoprecipitation using the conformation-dependent anti-E2 monoclonal antibody (H53).
H53 precipitated wild-type levels of intracellular E2 RBD661myc ( ̃50 kDa) for all single and multiple deletion constructs indicating that each of these mutant glycoproteins is efficiently expressed (FIG. 14A). These intracellular E2 RBD661myc glycoproteins also exhibited variably lower molecular weights than wild-type consistent with the loss of glycosylation sites located within the deleted regions as observed in the context of the full-length E1E2 heterodimer. However, deglycosylation of these truncated glycoproteins is required to confirm their molecular weights as predicted in Table 4. Furthermore, 1153 precipitated a diffuse molecular weight band ( ̃55-65 kDa) corresponding to the secreted form of the E2 RBD661myc (FIG. 14B) reflecting the various complex- and hybrid-type carbohydrate modifications acquired during transit through the secretory pathway. All E2 RBD661myc glycoproteins containing single and multiple variable region deletions were shown to be efficiently secreted and again displayed variable molecular weights (kDa) as described for the intracellular data. Together these results confirm that the variable regions can be deleted—individually and in combination—from the E2 RBD661myc and retain the intrinsic folding of both the intracellular and secreted receptor-binding domain as detected by the conformation-dependent anti-E2 monoclonal antibody H53.
CD81-LEL Binding
To determine whether the E2 receptor-binding domain containing single and multiple variable region deletions bind to CD81-LEL, both intracellular and secreted E2 RBD661myc lysates were applied to a solid-phase CD81 MBP-LEL (residues 113-201) binding assay as previously described. This data was normalized for the amount of monomeric E2 expressed from each vector as precipitated with H53 and observed by SDS-PAGE; FIG. 14 A (intracellular) and 14B (secreted).
Both intracellular and secreted forms of the E2 RBD661myc containing the single variable region deletions demonstrated CD81-LEL binding although, as observed in the context of the full-length E2 glycoprotein, the HVR1con deletion conferred a total loss of CD81-LEL binding (FIGS. 15A and 15B). The secreted form of the E2 glycoprotein containing the single HVR1 deletion also exhibited an additional enhancement of CD81-LEL binding compared to wild-type consistent with the data obtained for the mature virion-derived forms of the full-length E2 glycoprotein. Importantly, both intracellular and secreted forms of the E2 RBD661myc containing the triple variable region deletion (HVR1+2+igVR) retained CD81-LEL binding at wild-type levels (FIGS. 16A and 16B) confirming that this glycoprotein species encompasses all the structural and functional CD81-binding determinants to constitute a minimal or near-minimal E2 core receptor-binding domain.
5. Binding of E2 RBD661 Bearing Simultaneous HVR Deletions to Cell Surface Expressed Full-Length CD81
The abilities of secreted E2 RBD661 proteins with multiple HVR deletions to bind cell surface expressed full length CD81 were determined. CHO-K1 cells were transfected with a vector encoding human CD81. Forty-eight hours after transfection, cells were placed on ice, and equivalent amounts of E2 added to cells. Four hours later, bound E2 was detected with radioiodinated MAb 9E10 and the amount of bound E2 quantitated. The results (FIG. 17) show that wild type and ΔHVR2+igVR-deleted E2 RBD661 bound to full length CD81 at similar levels. By contrast, the binding of ΔHVR1+2-E2 RBD661 to CD81 was significantly decreased (p=0.038); further deletion of igVR in construct E2 Δ123-myc restored wild type CD81 binding activity (p=0.11). These data indicate that the 3 variable regions can be deleted simultaneously without disruption to the CD81 binding ability of E2. Interestingly, simultaneous deletion of HVR1 and HVR2 from E2 RBD661 led to decreased CD81 binding however this defect was rectified by further deletion of igVR.
6. Ability of E2 RBD661 Bearing Simultaneous HVR Deletions to be Recognised by Human Conformation Sensitive Monoclonal Antibodies
Wildtype E2-myc and E2 Δ123-myc was radiolabelled with 35S-Met/Cys and immunoprecipitated with a panel of human conformation sensitive monoclonal antibodies (Keck et al., 2004) and analysed by non-reducing SDS-PAGE. This panel of conformation-dependent MAbs are specific to conformational epitopes representing three distinct immunogenic domains of E2-A, B and C (Keck et al., 2004). The E2 Δ123-myc protein demonstrated a similar immunogenic profile to the wild-type E2-myc protein (FIG. 18) indicating that the E2-myc, lacking all three variable regions, retains the conformational epitopes specific to these three distinct structural and functional domains of E2. This data is consistent with previously published data by Keck et al. that also demonstrated a lack of HVR1 involvement in these domains (Keck et al., 2004). This data extends these observations to suggest that HVR2 and igVR also do not participate in these domains.
7. Discussion
In the course of this study, the two recognized E2 variable regions, HVR1 and HVR2, and the novel variable region “igVR” have been individually deleted to further investigate the role of these regions in E1E2 biosynthesis, heterodimerisation, CD81-binding and viral entry. All E2 glycoproteins containing these variable region deletions were shown to retain the intrinsic folding properties of the wild-type glycoprotein as recognized by the conformation-dependent monoclonal antibody H53. HVR2 and “igVR” were further shown to be required for heterodimerisation and all three variable regions were required in pre- or post-CD81-binding stages in viral entry. It has also been demonstrated that none of the individual variable regions are required for CD81-binding and that all three can be simultaneously deleted from the E2 glycoprotein with the retention of CD81-binding properties.
The envelope glycoproteins E1 and E2 exhibit the greatest genetic heterogeneity in the HCV genome, especially in hypervariable region 1 (HVR1)—a highly variable  ̃27 amino-acid sequence located at the N-terminus of the E2 glycoprotein. Here, it has been demonstrated that deletion of HVR1 (polyprotein residues 387-408) results in a loss of E1E2-mediated entry despite retaining both wild-type levels of heterodimerisation and incorporation of E1E2 into pseudotyped HIV-1 particles suggesting that this region has a direct role in viral entry. However, it has also been shown that HVR1 is not required for CD81-LEL binding in the context of either the functional E1E2 heterodimer or the E2 receptor-binding domain (E2 RBD661myc) indicating that HVR1 is involved in another pre- or post-CD81-binding stage in viral entry. This is consistent with a previous study that has reported the deletion of HVR1 (polyprotein residues 384-410) to ablate E2 binding to the SR-B1 receptor suggesting that this region may be required for essential SR-B1 contacts in viral entry.
However, the present findings contradict several other studies that have demonstrated HCV pseudotyped particles (HCVpp) lacking HVR1 to mediate entry into Huh7 cells, although at reduced levels, inconsistent with an essential role for this region in viral entry. It has also been reported that the enhancement of HCVpp infectivity observed in the presence of the high density lipoprotein (HDL), a natural ligand of SR-B1, is lost upon the deletion of HVR1. This suggests that HVR1 facilitates infectivity rather than being essential for this function, although whether this effect is due to the association of HCV particles with HDL remains undetermined. In addition, a recent study has found that the substitution of all the conserved basic residues within HVR1 significantly reduces HCVpp entry although these residues were not found to be involved in either CD81-LEL binding or to correlate with an HDL enhancement of SR-B1 binding. Therefore the exact role of HVR1 in viral entry remains unclear.
It has also been shown that the deletion of HVR1 significantly enhances CD81-binding (approximately 4-fold) in agreement with previous studies. This suggests that HVR1 negatively modulates the accessibility of the conserved CD81-binding sites within the E2 glycoprotein despite not being required in this function. This is consistent with the recent identification of a conserved CD81-binding determinant G436WLAGLFY (SEQ ID NO: 107) located between HVR1 and HVR2. In addition, the broadly neutralizing antibody AP33 has been demonstrated to recognise a conserved epitope directly adjacent to HVR1 (polyprotein residues 412-423) and to inhibit interactions between CD81 and a range of presentations of the E2 glycoprotein. The extension of the HVR1 deletion to include the conserved I411 residue has also been shown to confer a reduction, rather than an enhancement, in CD81-binding further implicating this region in this function. Similarly, it has been demonstrated that the extension of the HVR1 deletion to include this adjacent conserved region (HVR1con, polyprotein residues 387-428) disrupts E2 glycoprotein folding and maturation critical in the formation of the CD81-binding site. Furthermore it has been demonstrated that residue W420 located in this conserved region is also involved in CD81 binding
The location of these highly conserved regions adjacent to HVR1, suggests that this highly variable region performs the dual function of both mediating viral entry and modulating the accessibility of these conserved regions to the host immune system to escape recognition by broadly neutralizing antibodies such as AP33. Indeed, a previous study has found that deletion of HVR1 results in an increased sensitivity of the envelope glycoprotein complex to neutralizing antibodies and sera. Therefore, it has been proposed that HVR1 forms a solvent-exposed subdomain external to a conserved core domain of the E2 glycoprotein consistent with both its modulating role in receptor-binding and its elicitation of an immunodominant response. In order to further characterise this proposed conserved core domain, the remaining E2 variable regions, HVR2 and “igVR′′” have also been deleted. As observed for the HVR1 deletion, both E2 glycoproteins lacking the individual HVR2 and “igVR” regions retained the intrinsic folding properties of the wild-type glycoprotein as detected by the conformation-dependent monoclonal antibody H53.
Interestingly, however, both HVR2 and “igVR” were shown to be required for heterodimerisation and, accordingly, the deletion of these regions demonstrated a total loss of E1E2-mediated entry in to Huh7 cells. This suggests that these regions within the E2 glycoprotein are involved in direct heterodimer contacts or, alternatively, modulate allosteric effects that are indirectly required for this function. Therefore to further investigate the role of these regions, several conserved cysteine-proximal residues were reintroduced into the HVR2 and “igVR” deletion constructs affecting to extend their linker motifs and thus enhance E2 glycoprotein folding. The extension of the “igVR” linker was found to recover wild-type levels of heterodimerisation with E1 and a significant level of E1E2-mediated entry ( ̃8-fold) although this effect was not observed for HVR2. This finding may reflect the relatively short length of the “igVR” deletion where two of the five glycine residues lost from this region are further compensated for by the introduction of the Gly-Ser-Ser-Gly linker motif (SEQ ID NO: 95) resulting in only a five amino-acid total deletion within this region. The role of the “igVR” linker in heterodimerisation is also consistent with its observed conservation within genotypes. Notably, the extended “igVR” linker construct still exhibited a reduction in entry activity compared to wild-type ( ̃10-fold) and is perhaps due to absence of a conserved N-linked glycosylation site within this region that has been previously observed to reduce HCVpp entry.
Furthermore, it has been demonstrated that the E2 glycoprotein containing either the HVR2 or “igVR” deletions retains wild-type levels of CD81-binding indicating that these regions, as observed in HVR1, are not required in this function. Previous studies have shown that monoclonal antibodies targeting HVR2 inhibit E2 glycoprotein binding to CD81 and proposed that this region forms a CD81-binding determinant although, based on the present findings, it is more likely that these antibodies are creating a steric effect that occludes the G436WLAGLFY (SEQ ID NO: 107) CD81-binding determinant adjacent to this region. Therefore, despite a loss of heterodimerisation and/or viral entry, HVR1, HVR2 and “igVR” can all be individually deleted from the E2 glycoprotein without disrupting its intrinsic folding properties and, indeed, it has been further shown that all three variable regions can be simultaneous deleted with a retention of CD81-binding. This strongly supports the hypothesis that these variable regions form solvent-exposed subdomains external to an E2 core domain encompassing the conserved structural and functional determinants required for binding to the confirmed HCV receptor, CD81.
Interestingly, it has also been observed that the E2 glycoproteins containing the HVR1 deletion in combination with either HVR2 or “igVR” deletions did not exhibit the dramatic enhancement in CD81-LEL binding observed for the individual HVR1 deletion. A previous study has demonstrated that the substitution of HVR1 and HVR2 with corresponding sequences from a different subtype in combination achieves either increased or decreased CD81 binding (depending on the introduced and backbone strains) that is not observed for either of the individual HVR1 or HVR2 substitutions. Together this data suggests that intramolecular interactions are occurring between these regions to modulate CD81-binding and that they are likely to adopt intrinsically flexible structures to entertain these contacts. This is further supported by both the recent identification of a conserved CD81-binding determinant G436WLAGLFY443 (SEQ ID NO: 107) located directly between HVR1 and HVR2 as well as the immunodominant responses elicited by both HVR1, and to a lesser extent, HVR2 consistent with their position as surface-exposed subdomains that may perform modulating functions in cell attachment. This same effect observed for the HVR1 and “igVR” double deletion suggests that the “igVR” region may also contribute to these cooperative interactions.
One hypothesis to explain this data, proposes that the variable regions form flexible solvent-exposed subdomains that enable the E2 glycoprotein to move between ‘open’ and ‘closed’ conformations, the former being more competent for CD81 binding by exposing the conserved CD81-binding determinants located within the core E2 domain as delineated within this study. Indeed, conformational changes within the E2 glycoprotein have been previously observed upon CD81-binding. In addition, binding of non-neutralizing antibodies to the E2 glycoprotein has been demonstrated to reduce its susceptibility to neutralizing antibodies and is consistent with a model where the non-neutralizing antibodies inhibit the flexibility of these surface-exposed variable regions thus blocking access to the conserved epitopes located within the E2 core domain. Therefore, this suggests that the modified E2 core domain represents a promising vaccine candidate for eliciting neutralizing antibodies to conserved epitopes within the E2 glycoprotein, including the CD81-binding determinants, that are otherwise occluded by these surface-exposed variable regions that may act as immunological decoys at the surface of the glycoprotein complex during HCV replication.
The observed loss of heterodimerisation in the E2 glycoproteins containing HVR2 and “igVR” deletions suggests that conformational changes are occurring within these glycoproteins that cannot be recognised by the single conformation-dependent monoclonal antibody H53. Therefore, a panel of conformation-dependent antibodies that have been previously used to identify the three major immunogenic regions within the E2 glycoprotein were obtained to examine more subtle alterations in glycoprotein structure and function mediated by these variable regions. The results show that in the context of E2 RBD661myc monoclonal antibodies to domain A, B and C retain wild-type levels of reactivities to the RBD containing simultaneous deletion of all three variable regions. This further suggests that the E2 Δ123-mycΔ123RBD construct retains the intrinsic core domain folding properties of the native E2 RBD.
8. Materials and Methods
Construction of the Variable Region Deletion Mutants
Vectors
In order to introduce the E2 variable region deletions into the E1E2 heterodimer, the HCV-based expression vector pE1E2H77c was obtained (Drummer et al., 2003). The pE1E2H77c vector contains a DNA sequence from the full length pCV-H77c (genotype 1a) infectious clone (Yanagi et al., 1997) encoding the E1E2 polyprotein residues 165-746 as described (Drummer et al., 2003). To ensure efficient ER targeting and glycoprotein cleavage, this sequence includes the E1 signal peptide located at the C-terminus of the immature core protein and both the full-length E1 and E2 polyprotein sequences (FIG. 19). This DNA fragment was cloned into the backbone of the pcDNA4HisMax vector (Invitrogen, Carlsbad, Calif., USA) that contains both a translational enhancer sequence and a cytomegalovirus (CMV) promoter for expression in mammalian systems. It also contains an ampicillin resistance gene for selection in bacterial cells.
The CD81 open reading frame was amplified by PCR from pcDM8-TAPA-1 (Levy et al., 1998)30) using the primers, 5′-CCGAAGCTTCCACCATGGGAGTGGAGGGCTGC-3′ (SEQ ID No:1) and 5′-GGCTCTAGATTAGTACACGGAGCTGTTCCG-3′ (SEQ ID NO: 2). The PCR product was cloned into pcDNA3 using HindIII and XbaI (shown in bold type) to generate the plasmid pcDNA3-CD81.
To enable characterisation of the variable regions within the soluble E2 receptor-binding domain (E2 RBD661), the HCV-based expression vector pE2661 was also obtained (Drummer et al., 2002). The pE2661 vector contains a pCV-H77c DNA sequence encoding HCV polyprotein residues 384-661 as described (Drummer et al., 2006) 2002). This sequence encodes an independently folding subdomain of the E2 glycoprotein that has been shown to retain both CD81 and SR-B1 receptor-binding (Pileri et al., 1998, Scarselli et al., 2002, Pileri et al., 1998). This DNA product was cloned into a pcDNA3 vector (Invitrogen) backbone at the C-terminus of a tissue plasminogen-activator (tpa) leader sequence designed to ensure efficient ER targeting and signal cleavage of the E2 RBD661 in the absence of E1 (E2-myc). This vector also contains a CMV promoter for expression in mammalian cells and an ampicillin resistance gene to facilitate selection in bacterial cells.
Overlap Extension PCR
Overlap extension PCR is a two step strategy that facilitates the deletion of large segments of DNA (FIG. 20) (Horton et al., 1989). In round one, the pE1E2H77c vector was used as a template to introduce individual variable region deletion into the full-length E1E2 polyprotein. This step required two oligonucleotide pairs (Geneworks, Ann Arbor, Mich., USA); each pair containing one external and one internal primer responsible for amplifying the sequence either upstream (5′) or downstream (3′) of the variable region. The internal oligonucleotide primers were designed to introduce the Gly-Ser-Ser-Gly linker (SEQ ID NO: 95) as well as a short overlap sequence complementary to the corresponding 5′ or 3′ fragment (Table 5). Once generated, these first round 5′ 3′ PCR products were isolated by agarose-gel electrophoresis and purified prior to being added to the second round PCR reaction. Round two allowed annealing of the 5′ and 3′ fragments via their overlapping, complementary sequences to form a template for extensions and amplification by the relevant external primers. The external primers also contained unique EcoR1/XbaI restriction endonuclease sites to facilitate cloning back into the template vector.
E1E2 Variable Region Deletion Constructs
The overlap extension PCR strategy was used to generate the single variable region deletions as summarized in Table 5 and represented in FIG. 20. Notably, two alternative HVR1 deletion constructs, HVR1 and HVR1con, were designed to further investigate this region. The HVR1 deletion lacks the highly variable segment between polyprotein residues 387 and 408, whereas HVR1con extends this deletion to encompass the adjacent region up to the first conserved cysteine residue in E2 (polyprotein residues 387-428) proposed to anchor this N-terminal region to the rest of the glycoprotein. The first three amino-acids (E384TH) of HVR1 were also retained in these constructs to ensure efficient cleavage between E1 and E2 during glycoprotein biosynthesis. HVR2 and “igVR” deletions encompassed polyprotein residues 460-485 and 570-580, respectively.
To enhance E2 glycoprotein folding, a second series of modified single variable region deletion constructs were generated using overlap extension PCR (FIG. 21). This strategy utilized a set of modified internal oligonucleotide primers to extend Gly-Ser-Ser-Gly linker motif (SEQ ID NO: 95) by reintroducing several conserved cysteine-proximal residues deleted from the original constructs and summarized in Table 2 and represented in FIG. 21. Notably, this modified ΔHVRlink E1 E2 series lacks a HVR1 deletion construct due to the lack of any conserved residues located within this region.
In order to delineate a minimal E2 core domain, multiple HVR1, HVR2 and “igVR” deletions were introduced into the context of the E1E2 polyprotein as represented in FIG. 19. These multiple deletion constructs were again generated using overlap extension PCR, but utilized the relevant single or double variable region deletion constructs a template instead of the wild-type pE1E2H77c vector as outlined in Table 3.
E2 RBD661myc Variable Region Deletion Constructs (E2-myc)
In order to characterise the single and multiple variable region deletions in the context of the soluble E2 receptor-binding domain (E2 RBD661myc), standard PCR was used to amplify polyprotein residues 384 to 661 from the full-length E1E2 constructs containing the single and multiple variable region deletions as outlined in Table 4 and represented in FIG. 22. The oligonucleotide primers were designed to introduce a C-terminal myc epitope tag and unique NheI/XbaI restriction endonuclease sites to facilitate the cloning of these PCR products into the pE2661vector.
All PCR reactions were carried out in the Biometra Thermocycler (Biometra, Goettingen, Germany) using the Expand High Fidelity PCR system (Invitrogen) under the conditions summarized in Table 6.
Agarose-Gel Electrophoresis
DNA products were isolated by agarose-gel electrophoresis using Bio-Rad gel tanks (Bio-Rad Laboratories, Hercules, Calif., USA) containing TAE (0.001M EDTA and 0.04M Tris-Acetate). 1% (w/v) DNA-grade agarose in TAE was made up with ethidium bromide (0.5 μg/mL) to enable DNA visualization under UV light (Sambrook and Russel, 2001). DNA samples were made up at 20% (v/v) with Orange G gel-loading dye (1% (w/v) Orange G (Sigma, St Louis, Mo., USA), 50% (v/v) glycerol). Agarose gels were run at 100V, 23 mA for 30-40 min and DNA fragment size was confirmed and quantified using the GelDOC system (Bio-Rad) in reference to a 1 kb Plus DNA Marker (Invitrogen). Confirmed DNA products were subsequently purified from PCR reactions using the Mo Bio PCR Clean-up kit (Mo Bio, Carlsbad, Calif., USA) or from agarose gels with the Mo Bio Gel-Spin kit (Mo Bio) according to the manufacturer's instructions.
Restriction Endonuclease Digestion of DNA
All DNA was digested with New England Biolabs restriction endonucleases according to the manufacturer's instructions (New England Biolabs, Ipswich, Mass., USA). Any digested vectors were de-phosphorylated with Shrimp Alkaline-phosphatase (Invitrogen) for 30 min at 37° C. to reduce self-annealing.
Ligation and Transformation of Plasmid Constructs
Digested insert and vector DNA were ligated together at approximately a 4:1 ratio using the T4 DNA-ligase system (Invitrogen) and incubated at 4° C. for 16 hrs. DNA ligation products were isolated using the sodium-acetate precipitation method (3M Na-Acetate pH 5 and 100% Ethanol) (Sambrook and Russel, 2001) and resuspended for 1 hr in sterile distilled water. Ligation products were transformed into 20 uL of electroporation-competent DH10B E. coli cells (New England Biolabs) using 1 mm electroporation cuvettes (BTX, Holliston, Mass., USA) and a Gene Pulser Electroporator (BioRad) set at 2.0V, 200Ω and 25 μF. The electro-treated DH10B cells were then transferred into Luria Bertani Medium (LB) (1% (w/v) Tryptone, 0.5% (w/v) Yeast Extract, 0.5% (w/v) NaCl) to recover for 1 hr at 37° C. The cells were then spread-plated out onto LB-Agar plates (LB, 5% (w/v) Agar) containing 50 ug/mL ampicillin and incubated at 37° C. for 16 hrs to select for transformed cells (Sambrook and Russel, 2001).
Colony PCR
In order to rapidly screen for transformed colonies containing the desired DNA insert, approximately 20 colonies were selected from each LB-Agar plate for colony PCR (Sambrook and Russel, 2001). Colony PCR was performed using the Taq polymerase system (Invitrogen) and the appropriate external primers in the Biometra Thermocycler (Biometra) under the conditions specified in Table 6. The colonies containing positive inserts were identified by agarose-gel electrophoresis as described above.
Small-Scale Preparation of Plasmid DNA
Small-scale preparations of plasmid DNA from positive colonies were then generated using the Mo Bio Mini-prep kit (Mo Bio) using the alkaline lysis method in accordance with the manufacturer's specifications. Inserts were confirmed by restriction endonuclease digest and agarose-gel electrophoresis as described above.
DNA Sequencing
The sequences of all plasmid DNA inserts were confirmed using the relevant sequencing primers and the PRISM BigDye Terminator Mix (version 3.1) (Applied Biosystems, Foster City, Calif., USA) according to the Micromon Reaction Set-Up Protocol (Micromon, Victoria, Australia). All sequencing reactions were run in a Biometra Thermocycler (Biometra) under the conditions outlined in Table 6 and the resulting DNA prepared according to the Micromon Reaction Clean-Up Protocol (Micromon). Sequence analysis was performed in a 3730S Genetic Analyser (Applied Biosystems) at the Micromon sequencing facility.
Large-Scale Preparation and Quantification of Plasmid DNA
Plasmid DNA from confirmed clones was then isolated on a larger scale using the Qiagen Midi-prep Kit (Qiagen, Hilden, Germany) based on the alkaline lysis method in accordance with the manufacturer's specifications. These large scale plasmid DNA preparations were purified using phenol-chloroform (1:1) and centrifuged at 10000×g for 3 min to obtain an upper phase containing the DNA and repeated with chloroform (50% (v/v)). Purified DNA was concentrated using sodium-acetate (10% (v/v) and 2.5 volumes of 100% ethanol at 70° C. for 30 min. DNA was pelleted at 10000×g for 15 mins prior to washing twice in 70% precipitation method and resuspended in 100 uL of TE. All DNA concentrations were determined using a Biophotometer at 260 nm (Eppendorf, Hamburg, Germany).
During this study, all bacterial work utilised an orbital-shaker (Ratek Instruments, Victoria, Australia) or an incubator (Memmet, Schwabach, Germany) set at 37° C.
Biochemical and Functional Assays
Cell Culture
293T human embryo kidney cells (HEK 293T) from a human fibroblast cell line was used in this study to ensure high transfection efficiency and to achieve good levels of cellular protein expression. The human liver hepatocyte is the primary target cell for HCV and thus the human hepatocellular carcinoma cell line, Huh7, was used as a model liver cell system in this study. Furthermore, the Huh7 cell line has been demonstrated to support subgenomic HCV replicons, is highly permissive to HCVpp entry and can support cell culture grown HCV (HCVcc) suggesting that it contains all the cellular factors required for HCV tropism in vivo (ZhongBartosch et al., 2005, Wakita et al., 20052003a, Lindenbach et al., 2005b, Lohmann et al., 1999, LindenbachWakita et al., 2005a, Zhong et al., 2005, Bartosch et al., 2003a).
All cells were maintained in DMF10: Dulbecco's minimal essential medium (Invitrogen), 10% (v/v) heat-inactivated foetal bovine serum (Invitrogen), 2 mM L-glutamine (GE Healthcare, Bukinghamshire, UK), 1M HEPES buffer solution (Invitrogen), Gentamycin (GE Healthcare) and 2 ug/mL minocycline-hydrochloride salt (Sigma). The cells were subcultured every 3-4 days in 75 cm2 or 150 cm2 Falcon flasks (Becton Dickenson, Franklin Lakes, N.J., USA) using 0.025% (v/v) Trypsin in PBS-EDTA (phosphate buffered saline (PBS)—ethylenediaminetetraacetic acid (EDTA)) to detach the monolayer. All cells were incubated in a Thermo Direct Heat CO2 incubator at 37° C. with 5% CO2 (Thermo, Waltham, Mass., USA). During this study, all vectors were transfected into HEK 293T cells seeded in 6-well culture dishes (Nalge Nunc, Rochester, N.Y., USA) using the FuGene6 transfection reagent (Roche) as previously described (Drummer et al., 2003).
Antibodies
The non-conformational anti-E1 monoclonal antibody A4 and non-conformational anti-E2 monoclonal antibody A11 were gifts from Drs. Jean Dubuisson and Harry Greenberg (Dubuisson et al., 1994). The anti-E2 conformation-dependent monoclonal antibody, H53, was also a gift from Dr. Jean Dubuisson (Deleersnyder et al., 1997). The anti-E1E2 polyclonal antibody, 779, purified from the plasma of an HCV genotype 1a-infected individual was obtained (Drummer and Poumbourios, unpublished). Immunoglobulin G from an HIV-1 infected individual (IgG14) and the anti-myc monoclonal antibody 9E10 were also obtained (Drummer et al., 2002, Drummer et al., 2003). A panel of conformation-dependent human MAbs, specific to conformational epitopes representing three distinct immunogenic domains of E2 A, B and C, were obtained from Dr. Steven Foung (Keck et al., 2004).
E1E2 Pseudotyped HIV-1 Particle Entry Assay
The incorporation and display of functional HCV envelope glycoproteins by heterologous retro- or lentiviral core particles, called pseudotyping, provides a relatively rapid and simple method for characterising mutant E1E2 glycoproteins without introducing these mutations into the full-length HCV genome (Drummer et al., 2003, Bartosch et al., 2003b) 2003a, Drummer et al., 2003). This strategy involves the co-transfection of both an E1E2 expression vector and a retroviral or lentiviral expression vector lacking its native envelope gene and containing a reporter construct (FIG. 23). As the viral core proteins assemble within the cell they acquire an envelope by budding from the plasma membrane and incorporate the HCV envelope glycoproteins present at the cell surface in place of their native envelope complex. These E1E2-pseudotyped particles (HCVpp) can undergo a single round of E1E2-mediated infection and replication that can be quantified by measuring the activity of the reporter gene within the infected cell. During this study, the human immunodeficiency virus (HIV-1) fire-fly luciferase vector, HIV-1 NL4-3.LUC.R−E− (He and Landauet al., 1995) was used to generate E1E2-pseudotyped virus for infection of Huh-7 cells as previously described (Drummer et al., 2003).
293T cells were seeded at 350000 cells/well in 6-well culture dishes (Nalge Nunc) and co-transfected with 1 ug of NL4-3.LUC.R−E− and 1 ug of either pE1E2H77c (wild-type), pΔHVR E1E2 or the empty pcDNA4HisMax vector (negative control). After three days incubation, the tissue culture fluid containing E1E2-pseudotyped HIV-1 particles was collected and filtered using Minisart 0.45 um sterile syringe filters (Sartorius, Goettingen Germany). The filtered product was then used to infect Huh-7 cells seeded at 30000 cells/well in 48-well culture plates (Nalge Nunc) in triplicate. Following 4 hr incubation at 37° C., the innoculum was removed and the cells cultured in DMF10 for a further 3 days before being lysed with cell culture lysis reagent (Promega, Madison, Wis., USA). Cell lysates were clarified of cellular debris prior to being transferred into white 96-well plates (BMG Labtech, Offenburg, Germany) to be analysed for luciferase activity using the Steady-Glo luciferase reagent system (Promega) and a Fluostar (BMG Labtech) fitted with luminescence optics. The average luciferase activity (relative light units) was calculated from triplicate infections and the standard deviation calculated accordingly.
Radioimmunoprecipitation (RIP)
Radiolabelling
In order to analyse the role of the variable regions in intracellular E1E2 biosynthesis, 293T cells seeded at 500000 cells/well in 6-well culture dishes (Nalge Nunc) were transfected with 2 ug of either pE1E2 (wild-type), pE1E2 containing one or more variable region deletions or empty pcDNA4 vector (negative control). 24 hours post-transfection, cells were pulse-chase metabolically labelled for 30 min at 37° C. with 150 μCi Trans-35S-label/well (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) in L-cysteine and L-methionine deficient DMF 10: DMEM (MP Biomedicals), 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 2 mM L-glutamine (GE Healthcare). The cells were then chased for 4 hrs in DMF10 at 37° C. prior to being washed in PBS and lysed in RIP lysis buffer (0.6 M KCl, 0.05 M Tris pH 7.4, 1 mM EDTA, 0.02% sodium azide, 1% Triton X-100). The cell lysates were clarified of any remaining cell debris by centrifugation for 10 min at 4° C. in a refrigerated bench-top centrifuge (Heraeus, Hanau, Germany).
To enrich for mature E1E2 heterodimers as incorporated into pseudotyped HIV-1 particles, 293T cells seeded at 350000 cells/well in 6 well-culture dishes were transfected with 1 ug of pNL4-3.LUC.R-E- plus 1 ug of either pE1E2 (wild-type), pE1E2 containing one or more variable region deletions or empty pcDNA4 vector (negative control). 24 hrs hours post-transfection, the cells were metabolically labelled with 75 μCi Trans-35S label/well (Santa Cruz Biotechnology) in L-cysteine and L-methionine deficient DMF10 for 16 hrs at 37° C. The tissue culture fluid was then collected and filtered through a Minisart 0.2 um syringe filter (Sartorius) prior to being enriched for virions through a sucrose gradient (25% sucrose (v/v) in PBS) using the Beckman L-90 ultracentrifuge (SW41 rotor, Beckman Coulter, Fullerton, Calif., USA) at 25000×g for 2 hrs at 4° C. The supernatant was removed prior to lysis of the virions in RIP lysis buffer.
To characterise the role of the variable regions in the context of the E2 receptor-binding domain (E2 RBD661myc), metabolically labelled 293T cells seeded at 350000 cells/well in 6-well culture dishes (Nalge Nunc) were transfected with 2 ug of either the E2-myc (wild-type), E2-myc containing one or more variable region deletions por empty pcDNA3 vector (negative control). 6 hrs post-transfection, cells were pulse-chase metabolically labelled with 75 μCi Trans-35S-label/well (Santa Cruz Biotechnology) for 1 hr at 37° C. and chased in OptiMEM serum reduced media (Invitrogen) for 16 hrs to accumulate secreted protein. This tissue culture fluid was then collected and lysed in RIP lysis buffer prior to clarification by centrifugation at 10,000×g at 4° C. The cell monolayer was also washed in PBS, lysed in RIP lysis buffer and clarified as described above.
Immunoprecipitation
Radioimmunoprecipitation was used as a conformational-sensitive strategy to analyse protein expression. All protein preparations above were pre-cleared with Sepharose (GE Healthcare) coupled to BSA (Sigma) in the presence of the relevant antibody for 16 hrs at 4° C. The BSA-Sepharose was pelleted at 8000×g for 10 min to remove any non-specific protein species. The supernatant containing antibody-bound proteins was then precipitated using 30% (v/v) Protein-G Sepharose (GE Healthcare) in RIP lysis buffer for 1 hr at room temperature and isolated by centrifugation as above prior to washing three times in RIP wash buffer (0.5 M NaCl, 0.05 M Tris pH 7.4, 1 mM EDTA, 0.02% sodium azide and 1% Triton-X 100) and once in PBS.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Protein Separation and Analysis
All immunoprecipitates were resuspended in sample loading buffer (0.5 M Tris pH 6.8, 5% (v/v) SDS, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue). Intracellular lysates were run under either reducing (+3% (v/v) (3-mercaptoethanol) or non-reducing conditions (no β-mercaptoethanol) to identify non-covalently associated E1E2 species within the large amounts of intracellular covalently-linked E1E2 aggregate as previously observed (Dubuisson et al., 1994). Metabolically-labelled virion-incorporated E1E2 in viral lysates were run under non-reducing conditions, while the HIV-1 structural proteins were run under reducing conditions. All samples were denatured at 100° C. for 5 min prior to separation on 10-15% SDS-PAGE polyacrylamide gradient gels (except IgG14 samples that were separated on a 7.5-15% gradient) using the Miniprotean II SDS-PAGE system (BioRad) in reference to a pre-stained broad-range protein marker (BioRad). Electrophoresis was conducted at 100V, 23 mA for 1.5 hrs in 1× electrode buffer (0.2M Tris-HCl and 2M Glycine). The SDS-PAGE gels were then submerged in 10% (v/v) acetic acid and 10% (v/v) methanol to fix the samples prior to drying at 80° C. using a vacuum slab-gel dryer (Hoefer Scientific, San Francisco, Calif., USA). The broad-range marker positions were marked with 35S-Trans label (Santa Cruz Biotechnology) prior to protein analysis and quantification using the FLA-2000 Phosphoimager and Software (Fuji Film, Tokyo, Japan).
Solid-phase CD81-LEL Binding Assay
The large extracellular loop of CD81 has been demonstrated to be sufficient to mediate E2 glycoprotein binding (Pileri et al., 1998, Petracca et al., 2000, Pileri et al., 1998) and contains all the residues that form the E2 interaction site and contains all the residues that form the E2 interaction site (Drummer et al., 2002). Therefore, to rapidly screen for the CD81-LEL binding ability in the variable region deletion mutants, we used a solid-phase binding assay constructed using a chimera composed of the maltose-binding protein (MBP) linked to the CD81 large-extracellular loop residues 113-201 (MBP-LEL) as previously described (Drummer et al., 2002).
Briefly, 96-well maxisorb enzyme linked immunosorbant plates (Nalge Nunc) were coated with 5 ug/mL of dimeric CD81 MBP-LEL in PBS and incubated for 16 hrs at 4° C. The MBP-LEL was removed prior to blocking with BSA10PBS (10 mg/mL BSA (Sigma) in PBS) for 2 hrs at 37° C. to reduce non-specific binding. The plates were then washed four times in PBST (0.05% Tween-20 (Sigma) in PBS) prior to the addition of all the protein lysate preparations at twelve two-fold serial dilutions in BSA5PBST (5 mg/mL BSA in PBST). Plates were incubated for 2 hrs at room temperature, washed again, and probed for bound E2 with the conformation-dependent anti-E2 monoclonal antibody H53 for 1 hr (1:1000 dilution in BSA5PBST). After further washing in PBST, the antibody-bound E2 complexes were detected using a rabbit anti-mouse immunoglobulin-horseradish peroxidise conjugate (DAKO) (1:1000 dilution in BSA5PBS/Tween) and developed with a tetra-methylbenzidine substrate (Sigma) according to the manufacturer's instructions. The resulting absorbance values (optical density) were read at 450 nm-620 nm (background) on the Fluostar (BMG technologies). This data was then normalized against monomeric E2 as detected by conformation-dependent antibody H53, visualized and quantified in the previous sections.
E2−myc-CD81 Cell Surface-Binding Assays.
CHO-K1 cells were seeded in 12-well culture plates at 1.25×105 cells/well and transfected with 2 μg pcDNA3-CD81 24 h later. At 48-h post transfection the CD81 transfected CHO-K1 cells were chilled on ice and incubated with serial dilutions of wild-type or E2-myc protein containing variable region deletions in BSA10PBS for 4 h on ice. The cells then were washed twice in BSA10PBS prior to a 1 h incubation with 125I-MAb 9E10 (106 cpm) that had been precleared with 107 CHO-K1 cells for 2 h on ice. After 4 further washes with BSA10PBS, the cells were lysed in 1% SDS in PBS and counted in a Packard Auto-Gamma counter.







TABLE 1







Summary of results obtained for variable region deletion constructs in both the context of the E1E2


polyprotein and the E2 receptor-binding domain constructs containing a myc epitope tag (E2 -myc).

















Heterodimeriza-
H53
CD81-LEL
Heterodimeriza-
H53
Viral
CD81-LEL



Deletion
tion 1
Recognition
Binding 2
tion 1
Recognition
Entry 3
binding 2



















Polyprotein


















Mutant

Intracellular E1E2 precursors
Mature virion-incorporated E1E2


















pE1E2
Wild-type
+
+
+
+
+
+
+


pE1E2 Δ1
HVR1
+
+
++
+
+
−
++


pE1E2 Δ1con
HVR1 + conserved
+
+
−
−
reduced
−
−



region


pE1E2 Δ2
HVR2
−
+
++
−
+
−
+


pE1E2 Δ3
igVR
−
+
++
−
+
−
+


pE1E2 Δ1
HVR1 + conserved
+
+
+/−
−
reduced
−
−


conlink
region with



extended linker


pE1E2 Δ2
HVR2 with
−
+
++
−
+
−
+


link
extended linker


pE1E2 Δ3
igVR with extended
+
+
++
+
+
+/−
+


link
linker


pE1E2 Δ12
HVR1 and 2
−
+
+
−
+
−
+


pE1E2 Δ13
HVR1 and igVR
−
+
+
−
+
−
+


pE1E2 Δ23
HVR2 + igVR
−
+
++
−
+
−
+


pE1E2 Δ123
HVR1, HVR2 +
−
+
+
−
+
−
++



igVR





E2RBD










Mutant

Intracellular E2 RBD661myc
Secreted E2 RBD661myc


















E2-myc
Wild-type

+
+

+

+


E2 Δ1-myc
HVR1

+
++

+

++


E2 Δ1con-
HVR1 + conserved

+
−

+

−


myc
region


E2 Δ2-myc
HVR2

+
+/−

+

+


E2 Δ3-myc
igVR

+
+/−

+

+


E2 Δ12-myc
HVR1 and 2

+
+/−

+

−


E2 Δ13-myc
HVR1 and igVR

+
+

+

++


E2 Δ23-myc
HVR2 + igVR

+
+

+

+


E2 Δ123-myc
HVR1, HVR2 +

+
++

+

+



igVR





1 + represents heterodimerization levels similar to wild-type E1E2. − represents no heterodimerization detected. +/− represents reduced heterodimerization relative to wild-type E1E2.


2 + represents CD81-LEL binding levels similar to wild-type E1E2. − represents no CD81-LEL binding detected. +/− represents reduced CD81-LEL binding relative to wild-type E1E2. ++ enhanced binding relative to wild-type.


3 + represents viral entry at levels similar to wild-type E1E2. − represents no detectable viral entry. +/− represents partially entry competent.













TABLE 2







Summary of the overlap extension PCR strategy used to introduce modified single


variable region deletions with linker sequences into the HCV E1E2 polyprotein.
















pE1E2

Round 




PCR
Insert



con-
Template
1 PCR


RE
Epitope
product
size
MW


struct
Vector
Product
External Primer
Internal Primer
Site
Tag
(bp)
(bp)
(Da)



















pE1E2
pE1E2H77c
5′
5′ ggt gga att ctg    
5′ gct ttc att gca att   
EcoRI
none
716
1563
36 


Δ1con


gca aca ggg aac ctt 
caa ggc cgt GCC GCT ACT    




482


link


cct gg 3′
ACC gtg ggt ttc cgc gtc










(SEQ ID NO: 3)
gac 3′ (SEQ ID NO: 4)








pE1E2H77c
3′
5′ ccg tct aga tta   
5′ GGT AGT AGC GGC acg 
XbaI
none
993







cgc ctc cgc ttg gga 
gcc ttg aat tgc aat gaa 










tat gag 3′
agc ctt aac acc 3′










(SEQ ID NO: 5)
(SEQ ID NO: 6)







 


pE1E2 
pE1E2H77c
5′
5′ ggt gga att ctg   
5′ gta gtg cca gca gta  
EcoRI
none
938
1614
38 


Δ2 link


gca aca ggg aac ctt 
ggg GCC GCT ACT ACC aag    




071





cct gg 3′
gcg tcg gca gct ggc caa 










(SEQ ID NO: 7)
cct ctc 3′











(SEQ ID NO: 8)








pE1E2H77c
3′
5′ ccg tct aga tta   
5′ GGT AGT AGC GGC ccc  
XbaI
none
816







cgc ctc cgc ttg gga 
tac tgc tgg cac tac cct  










tat gag 3′
cca aga cct tgt ggc 3′










(SEQ ID NO: 9)
(SEQ ID NO: 10)







 


pE1E2 
pE1E2H77c
3′
5′ ggt gga att ctg   
5′ gca atc agt ggg gca 
XbaI
none
1268
1656
39 


Δ3 link


gca aca ggg aac ctt 
gag caa GCC GCT ACT ACC    




693





cct gg 3′
gat gac aca agg ggg cgc  










(SEQ ID NO: 11)
tcc gca cac 3′











(SEQ ID NO: 12)








pE1E2H77c
3′
5′ ccg tct aga tta   
5′ GGT AGT AGC GGC ttg 
XbaI
none
531







cgc ctc cgc ttg gga 
ctc tgc ccc act gat tgc 










tat gag 3′
ttc cgc 3′










(SEQ ID NO: 13)
(SEQ ID NO: 14)





Capitalised and underlined primer sequences represent the introduced


Gly-Ser-Ser-Gly linker motif and restriction endonuclease sites, respectively.













TABLE 3







Summary of the overlap extension PCR strategy used to introduce


multiple variable region deletions into the HCV E1E2 polyprotein.
















pE1E2

Round1




PCR
Insert



con-
Template
PCR


RE
Epitope
product
size
MW


struct
Vector
Product
External Primer
Internal Primer
Site
Tag
(bp)
(bp)
(Da)



















pE1E2 
pE1E2 
5′
5′ ggt gga att ctg  
5′ ttg gat gtt ctg  
EcoRI
none
704
1545
35 


Δ12
Δ2

gca aca ggg aac ctt 
GCC GCT ACT ACC gtg   




697





cct gg 3′
ggt ttc cgc gtc gac 3′










(SEQ ID NO: 15)
(SEQ ID NO: 16)







 



pE1E2 
3′
5′ ccg tct aga tta  
5′ GGT AGT AGC GGC  
XbaI
none
981





Δ2

cgc ctc cgc ttg gga 
cag aac atc caa ctg 










tat gag 3′
atc aac acc 3′










(SEQ ID NO: 17)
(SEQ ID NO: 18)







 


pE1E2 
pE1E2 
5′
5′ ggt gga att ctg  
5′ ttg gat gtt ctg  
EcoRI
none
704
1590
37 


Δ13
Δ3

gca aca ggg aac ctt 
GCC GCT ACT ACC gtg  




566





cct gg 3′
ggt ttc cgc gtc gac 3′










(SEQ ID NO: 19)
(SEQ ID NO: 20)







 



pE1E2 
3′
5′ ccg tct aga tta  
5′ GGT AGT AGC GGC  
XbaI
none
1041





Δ3

cgc ctc cgc ttg gga 
cag aac atc caa ctg










tat gag 3′
atc aac acc 3′










(SEQ ID NO: 21)
(SEQ ID NO: 22)







 


pE1E2 
pE1E2 
5′
5′ ggt gga att ctg  
5′ gta gtg cca gca  
EcoRI
none
932
1578
36 


Δ23
Δ3

gca aca ggg aac ctt 
GCC GCT ACT ACC gca 




651





cct gg 3′
gct ggc caa cct ctc 3′










(SEQ ID NO: 23)
(SEQ ID NO: 24)







 



pE1E2 
3′
5′ ccg tct aga tta  
5′ GGT AGT AGC GGC  
XbaI
none
810





Δ3

cgc ctc cgc ttg gga 
tgc tgg cac tac cct 










tat gag 3′
cca aga cct tgt ggc 3′










(SEQ ID NO: 25)
(SEQ ID NO: 26)







 


pE1E2 
pE1E2 
5′
5′ ggt gga attt ctg  
5′ gca atc agt ggg  
EcoRI
none
1256
1524
34 


Δ123
Δ12

gca aca ggg aac ctt 
gca GCC GCT ACT ACC  




962





cct gg 3′
aca agg ggg cgc tcc










(SEQ ID NO: 27)
gca cac 3′











(SEQ ID NO: 28)







 



pE1E2 
3′
5′ ccg tct aga tta  
5′ GGT AGT AGC GGC  
XbaI
none
525





Δ12

cgc ctc cgc ttg gga 
tgc ccc act gat tgc 










tat gag 3′
ttc cgc 3′










(SEQ ID NO: 29)
(SEQ ID NO: 30)





Capitalized and underlined primer sequences represent the introduced


Gly-Ser-Ser-Gly linker motif and restriction endonuclease sites, respectively.













TABLE 4







Summary of the standard PCR strategy used to construct single and


multiple variable region deletions into the E2 receptor-binding domain (residues 384-661).




















Insert



E2-myc
Template


RE
Epitope
 Size
MW


construct
Vector
External sense primer
External anti-sense primer
Sites
Tag
(bp)
(Da)





E2 Δ1con-
pE1E2 
5′ caa gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
783
29,385


myc
Δ1con
cac ggt agt agc ggc 3′
ctc ttc tga gat gag ttt ttg ttc 
XbaI







(SEQ ID NO: 31)
agt act ctc gga cct gtc cct gtc 3′









(SEQ ID NO: 32)






 


E2 Δ1-myc
pE1E2 
5′ caa gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
843
31,623



Δ1
cac ggt agt agc ggc 3′
ctc ttc tga gat gag ttt ttg ttc 
XbaI







(SEQ ID NO: 33)
agt act ctc gga cct gtc cct gtc 3′









(SEQ ID NO: 34)






 


E2 Δ2-myc
pE1E2 
5′ cca gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
831
31,282



Δ2
cac gtc acc ggg gga  
ctc ttc tga gat gag ttt ttg ttc 
XbaI







aat gc 3′
agt act ctc gga cct gtc cct gtc 3′








(SEQ ID NO: 35)
(SEQ ID NO: 36)






 


E2 Δ-myc
pE1E2 
5′ cca gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
876
33,139



Δ3
cac gtc acc ggg gga  
ctc ttc tga gat gag ttt ttg ttc 
XbaI







aat gc 3′
agt act ctc gga cct gtc cct gtc 3′








(SEQ ID NO: 37)
(SEQ ID NO: 38)






 


E2 M2-myc
pE1E2 
5′ caa gct agc gaa acc 
5′ ccg tct aga cta att cag atc
NheI/
myc
777
28,865



Δ12
cac ggt agt agc ggc 3′
ctc ttc tga gat gag ttt ttg ttc 
XbaI







(SEQ ID NO: 39)
agt act ctc gga cct gtc cct gtc 3′









(SEQ ID NO: 40)






 


E2 Δ13-myc
pE1E2 
5′ caa gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
822
30,624



Δ13
cac ggt agt agc ggc 3′
ctc ttc tga gat gag ttt ttg ttc 
XbaI







(SEQ ID NO: 41)
agt act ctc gga cct gtc cct gtc 3′









(SEQ ID NO: 42)






 


E2 Δ23-myc
pE1E2 
5′ cca gct agc gaa acc 
5′ ccg tct aga cta att cag atc  
NheI/
myc
811
30,381



Δ23
cac gtc acc ggg gga  
ctc ttc tga gat gag ttt ttg ttc  
XbaI







aat gc 3′
aag tct ctc gga cct gtc cct gtc 3′








(SEQ ID NO: 43)
(SEQ ID NO: 44)






 


E2 Δ123-
pE1E2 
5′ caa gct agc gaa acc 
5′ ccg tct aga cta att cag atc 
NheI/
myc
756
27,964


myc
Δ123
cac ggt agt agc ggc 3′
ctc ttc tga gat gag ttt ttg ttc  
XbaI







(SEQ ID NO: 45)
agt act ctc gga cct gtc cct gtc 3′









(SEQ ID NO: 46)





Underlined primer sequences represent introduced restriction endonuclease sites.













TABLE 5







Summary of the overlap extension PCR strategy used to introduce


single variable region deletions into the HCV E1E2 polyprotein.
















pE1E2

Round




PCR
Insert



con-
Template
1 pCR


RE
Epitope
product
size
MW


struct
Vector
Product
External Primer
Internal Primer
Site
Tag
(bp)
(bp)
(Da)



















pE1E2
pE1E2H77c
5′
5′ ggt gga att ctg 
5′ gct ttc att gca GCC  
EcoRI
none
704
1551
36 


Δ1con


gca aca ggg aac at 
GCT ACT ACC gtg ggt ttc 




083





cct gg 3′
cgc gtc gac 3′










(SEQ ID NO: 47)
(SEQ ID NO: 48)







 



pE1E2H77c
3′
5′ ccg tct aga tta 
5′ GGT AGT AGC GGC tgc  
XbaI
none
981







cgc ctc cgc ttg gga 
aa tgaa agc ctt aac 










tat gag 3′
acc 3′










(SEQ ID NO: 49)
(SEQ ID NO: 50)







 


pE1E2 
pE1E2H77c
5′
5′ ggt gga att ctg 
5′ ttg gat gtt ctg GCC  
EcoRI
none
704
1611
38 


Δ1


gca aca ggg aac ctt 
GCT ACT ACC gtg ggt ttc 




301





cct gg 3′
cgc gtc gac 3′










(SEQ ID NO: 51)
(SEQ ID NO: 52)







 



pE1E2H77c
3′
5′ ccg tct aga tta 
5′ GGT AGT AGC GGC cag  
XbaI
none
1041







cgc ctc cgc ttg gga 
aac atc caa ctg atc aac 










tat gag 3′
acc 3′










(SEQ ID NO: 53)
(SEQ ID NO: 54)







 


pE1E2 
pE1E2H77c
5′
5′ ggt gga att ctg 
5′ gta gtg cca gca GCC 
EcoRI
none
932
1599
37 


Δ2


gca aca ggg aac ctt 
GCT ACT ACC gca gct ggc 




386





cct gg 3′
caa cct ctc 3′










(SEQ ID NO: 55)
(SEQ ID NO: 56)







 



pE1E2H77c
3′
5′ ccg tct aga tta 
5′ GGT AGT AGC GGC tgc 
XbaI
none
810







cgc ctc cgc ttg gga 
tgg cac tac cct cca aga 










tat gag 3′
cct tgt ggc 3′










(SEQ ID NO: 57)
(SEQ ID NO: 58)







 


pE1E2 
pE1E2H77c
5′
5′ ggt gga att ctg 
5′ gca atc agt ggg gca 
EcoRI
none
1256
1644
39 


Δ3


gca aca ggg aac ctt 
GCC GCT ACT ACC aca agg  




255





cct gg 3′
ggg cgc tcc gca cac 3′










(SEQ ID NO: 59)
(SEQ ID NO: 60)







 



pE1E2H77c
3′
5′ ccg tct aga tta 
5′ GGT AGT AGC GGC tgc  
XbaI
none
525







cgc ctc cgc ttg gga 
ccc act gat tgc ttc 










tat gag 3′
cgc 3′










(SEQ ID NO: 61)
(SEQ ID NO: 62)





Capitalized and underlined oligonucleotide sequences represent the introduced


Gly-Ser-Ser-Gly linker motif and restriction endonuclease sites, respectively.













TABLE 6





Polymerase chain reaction (PCR) amplification reaction conditions.







Overlapping: Round 1













95° C.
2
min





92° C.
30
sec



65° C.
30
sec
 {close oversize brace} 
X 29



72° C.
2
min



72° C.
10
min







Overlapping: Round 2













95° C.
2
min





92° C.
30
sec



55° C.
30
sec
 {close oversize brace} 
X 29



72° C.
4
min



72° C.
10
min







Standard













95° C.
2
min





92° C.
30
sec



55° C.
30
sec
 {close oversize brace} 
X 29



72° C.
2
min



72° C.
10
min







Colony













94° C.
2
min





94° C.
30
sec



55° C.
30
sec
 {close oversize brace} 
X 29



72° C.
1
min



72° C.
10
min







Sequencing













96° C.
1
min





96° C.
20
sec



50° C.
15
sec
 {close oversize brace} 
X 29



60° C.
4
min



 4° C.
10
min










Example 2
1. Antigenic Structure of E2-myc and E2 Δ123-myc Probed with a Panel of Sera Obtained from HCV-Infected Individuals
A panel of sera obtained from HCV-infected individuals was used to compare the global antigenic profiles of biosynthetically labeled E2-myc and E2 Δ123-myc by immunoprecipitation. The sera were screened for the presence of neutralizing antibodies towards H77c E1E2 pseudotyped retroviral particles as described previously (Grollo et al., 2006), with 50% neutralization titers ranging from 0 to 1,600 observed (FIG. 24, lower panel). The antibody reactivity pattern of E2-myc (FIG. 24, upper panel) was almost identical to that of E2 Δ123-myc (FIG. 24, middle panel), indicating that the gross antigenic structure of the two proteins is similar.
2. CD81 Binding Properties of E2-myc Wild Type and Variant Proteins
The abilities of the secreted E2-myc proteins to interact with the large extracellular loop (LEL) of the HCV cellular receptor CD81 was examined. In this assay, CD81-E2-myc binding was detected using a conformation-dependent E2 monoclonal antibody (H53) in an ELISA employing solid-phase maltose binding protein fused to the large extracellular loop of CD81 (residues 113-201; “CD81-LEL”). E2-myc proteins containing one or more deletions of the variable regions displayed wild-type levels of CD81-LEL binding, again indicating that the E2 global fold was not detectably affected by the deletions (FIG. 25A,B). By contrast, binding between the E2 L441M-myc protein, containing an LEL binding site mutation (L441M), and CD81-LEL was not observed, confirming the specificity of the binding assay (FIG. 25A).
3. Abilities of E2-myc Wild Type and Variant Proteins to Interact with Full-Length CD81 Expressed in CHO-K1 Cells
The abilities of E2-myc proteins to interact with full-length CD81 receptor were determined by a cell surface binding assay using CHO-K1 cells transfected with a full-length CD81 expression vector as previously described (Drummer et al., 2002). The secreted E2-myc wild type and variant proteins were serially diluted and incubated with CD81-transfected CHO-K1 cells on ice. After washing, the bound E2-myc proteins were detected by using 125I-labeled monoclonal antibody 9E10, directed to the C-terminal c-myc epitope tag. The specificity of the assay was confirmed by the lack of binding by E2 L441M-myc, containing the L441M CD81 binding site mutation, to wild-type-CD81-transfected CHO-K1 cells (FIG. 26A).
The results shown in FIGS. 26A and B indicate that E2-myc, E2 Δ23-myc, E2 Δ13-myc and E2 Δ123-myc have similar CD81 binding properties, whereas the deletion of HVR1 plus HVR2 (E2 Δ12-myc) caused an approximately 50% reduction in CD81 binding compared to binding by E2 Δ23-myc (p<0.035). By contrast, the CD81 binding abilities of E2 Δ13-myc (which exhibited a binding curve identical to that of E2-myc) and E2 Δ123-myc were not significantly different (p=0.62), indicating that the presence of igVR compromises the CD81 binding function when HVR1 and HVR2 are absent in E2 Δ12-myc. Although HVR1, HVR2, and igVR are not required for the core folding properties of E2 RBD661, these data point to a functional interaction between igVR and one or both of HVR1 and HVR2 such that the CD81 binding site is properly formed or becomes fully accessible to the receptor.
Although variations in binding to recombinant CD81-LEL for singly and multiply deleted E2-myc constructs were not detected in FIGS. 25A and 25B, differences in binding to surface-expressed CD81 were observed in FIGS. 26A and 26B, perhaps reflecting subtle differences in LEL structure when it is expressed in isolation versus in native tetraspanin.
4. Materials and Methods
Neutralization Assays
The abilities of immune and control human sera to neutralize a single cycle of infection by HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses were determined as follows. HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses were prepared by cotransfection of HEK-293T monolayers (350,000 cells per well of 6-well culture dishes) with pE1E2 and pNL4.3LUCR-E- plasmids (Drummer et al., 2003). After 3 days incubation at 37° C. in a humidified atmosphere containing 5% CO2, the culture supernatants were filtered through 0.45 μm sterile syringe filters (Sartorius). Serial dilutions of heat-inactivated immune and control human sera were preincubated with HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses (1 h) and then added to quadruplicate Huh7 cell monolayers in 48-well tissue culture plates. Following a 4 h incubation (37° C., 5% CO2) the cells were washed with PBS and fresh medium replaced. After an additional 3-day incubation (37° C. in 5% CO2), the cells were lysed, the lysates clarified by centrifugation and then assayed for luciferase activity (Promega) in a Fluostar (BMG) fitted with luminescence optics. The neutralization titres of individual sera were determined as the serum dilution giving 50% neutralization compared to HCV glycoprotein-pseudotyped HIV-1 luciferase reporter virus preincubated with medium alone.
Radioimmunoprecipitation.
293T cells seeded at 500,000 cells/well in 6-well culture dishes were transfected with E2-myc expression vectors using Fugene 6 (Roche). At 24 hours post-transfection, the cells were labelled with 150 μCi Trans-35S-label/well (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) in L-cysteine and L-methionine deficient DMF10 (DMEM [MP Biomedicals], 10% (v/v) heat-inactivated fetal bovine serum [Invitrogen] and 2 mM L-glutamine [GE Healthcare]). After harvesting the cell supernatent, the labelled secreted proteins were adjusted to 0.6 M KCl, 0.05 M Tris pH 7.4, 1 mM EDTA, 0.02% sodium azide, 1% Triton X-100 and pre-cleared with CNBr-activated Sepharose (GE Healthcare) coupled to BSA (Sigma) in the presence of the relevant antibody for 16 h at 4° C. Antibody-antigen complexes within the clarified supernatants were then immunoprecipitated using 30% (v/v) Protein-G Sepharose (GE Healthcare) prior to washing three times in RIP wash buffer (0.5 M NaCl, 0.05 M Tris pH 7.4, 1 mM EDTA, 0.02% sodium azide and 1% Triton-X 100) and once in PBS. The immunoprecipitated proteins were subjected to SDS-PAGE in 10-15% polyacrylamide gradient gels under nonreducing conditions and visualized by scanning in a phosphorimager.
Transient Expression of E2-myc Proteins
HEK 293T cells (350,000 cells per well of 6-well culture plates) were transfected with E2-myc expression vectors using Fugene 6 (Roche). At 8-h post transfection, the transfection medium was replaced with Optimem (Invitrogen) and the cells incubated for 3 days at 37° C. in a humidified atmosphere containing 5% CO2. The tissue culture fluid was clarified through 0.45-μm-pore-size filters and then concentrated by approximately 10-fold in Centricon YM30 concentrators (Amersham).
Recombinant CD81 Large Extracellular Loop (CD81-LEL) Binding Properties.
The abilities of the E2-myc proteins to interact with the HCV cellular receptor CD81 were examined using a solid phase enzyme immunoassay. Enzyme immunoassay plates (Nunc Maxisorb®) were coated with maltose binding protein fused to the recombinant large extracellular loop of CD81 (residues 113-201) at 5 μg/ml in PBS overnight at 4° C. Coating solution was removed and unoccupied sites blocked with bovine serum albumin (10 mg/ml) in PBS (BSA10PBS) for 1 h at room temperature. Plates were washed 4 times with PBS containing 0.05% Tween 20 (PBST). The secreted E2-myc proteins were serially diluted in a 50 μl PBS containing 5 mg/ml bovine serum albumin (BSA5PBST) and incubated for 2 h. Bound E2-myc proteins were detected using an E2 specific monoclonal antibody followed by rabbit anti-mouse immunoglobulins coupled to horseradish peroxidase (Dako). Plates were developed using tetramethylbenzidine hydrochloride substrate and stopped by the addition of 1M HCl. Absorbance values were measured at 450 nm and the background at 620 nm subtracted in a Fluostar plate reader (BMG technologies).
CD81 Binding Properties.
CHO-K1 cells were seeded in 12-well culture plates at 1.25×105 cells/well and transfected with 2 μg pcDNA3-CD81 24 h later. At 48-h post transfection the CD81 transfected CHO-K1 cells were chilled on ice and incubated with serial dilutions of wild-type E2-myc or E2-myc proteins containing variable region deletions in BSA10PBS for 4 h on ice. The cells then were washed twice in BSA10PBS prior to a 1-h incubation with 125I-MAb 9E10 (106 cpm) that had been precleared with 107 CHO-K1 cells for 2 h on ice. After 4 further washes with BSA10PBS, the cells were lysed in 1% SDS in PBS and counted in a Packard Auto-Gamma counter.
Example 3
1. SDS-PAGE Analysis of Purified E2-his Wild Type and Variant Proteins
The purity of E2-his wild type and variant proteins was assessed by SDS-PAGE under reducing conditions. FIG. 27 indicates a single band for each purified protein species. The migration of each species was consistent with the number of variable regions deleted. For example, E2 Δ123-his, lacking 3 variable regions, exhibited the fastest migration. The diffuse nature of the protein bands is consistent with glycosylation of E2.
2. Blue-Native PAGE of Purified E2-his Proteins
The oligomerization status of the purified E2-his wild type and variant proteins was analysed using blue native polycrylamide gel electrophoresis. FIG. 28 shows two major bands for each E2-his protein corresponding in molecular weight to monomer and dimer.
3. Immunodetection of E2-his Proteins
The purified E2-his wild type and variant proteins were detected by a non-conformation dependent E2 specific monoclonal antibody (H52) in a western blot. A major protein species migrating at the expected molecular mass was revealed for each E2-his protein variant (FIG. 29).
4. CD81 Binding Properties of E2-his Wild Type and Variant Proteins
The ability of the purified E2-his proteins to interact with the large extracellular loop (LEL) of the HCV cellular receptor CD81 was examined. In this assay, CD81-E2-his binding was detected using a conformation-dependent E2 monoclonal antibody (H53) in an ELISA employing solid-phase maltose binding protein fused to the large extracellular loop of CD81 (residues 113-201; “CD81-LEL”). E2-his proteins containing one or more deletions of the variable regions displayed wild-type levels of CD81-LEL binding, indicating that the E2 global fold was not detectably affected by the deletions (FIG. 30A). By contrast, binding between the E2-his proteins and CD81-LEL containing an E2 binding site mutation (F186S) was not observed, confirming the specificity of the binding assay (FIG. 30B).
5. Immunoreactivity Towards Homologous E2-his Antigen of Mouse Sera Obtained after 2 Immunizations with E2-his Protein Variants
The immunoreactivity of mouse sera obtained after 2 immunizations with the E2-his wild type and variant proteins towards solid-phase “homologous antigen” (i.e. that antigen used to immunize a particular mouse group) was examined by ELISA. Substantial antibody binding activity towards the homologous solid-phase antigen was demonstrated in each of the vaccinated animals (FIG. 31). By contrast, antibody reactivity towards solid-phase E2-his proteins was not detected in the mice vaccinated with adjuvant alone, nor in two representative control sera obtained prior to immunization (prebleed) from each vaccination group (data not shown), confirming that the animals receiving E2-his protein elicited antibodies specific to the immunogen.
6. Immunoreactivity Towards E2-his and E2Δ123-his Antigens of Mouse Sera Obtained after 2 Immunizations with E2-his Protein Variants
The ability of the antibodies in immune sera from each vaccination group to bind to solid-phase E2-his (containing the three variable regions) was compared to the antibody reactivity towards solid-phase E2 Δ123-his (lacking the three variable regions) in ELISA. Substantial binding titres against solid phase E2-his (FIG. 32A) and E2 Δ123-his (FIG. 32B) were observed for all immunization groups (except adjuvant alone; data not shown).
Pair-wise statistical analyses of the antibody binding titres obtained for the various immunization groups were performed in order to determine whether apparent differences in binding titre were significant. Table 7 indicates that the antibody titres against solid-phase E2-his calculated for the E2-his-immunization group were not significantly different to those of the other immunization groups (p>0.06). However, statistically significant differences in antibody reactivity against solid-phase E2-his were observed between the E2 Δ1-his immunization group versus E2 Δ13-his, E2 Δ23-his and E2 Δ123-his (p≦0.04).
A pair-wise statistical analysis of antibody binding titres against solid-phase E2 Δ123-his for the various immunization groups revealed a highly significant increase in antibody reactivity for the E2 Δ123-his immunization group compared with the E2-his immunization group (p=0.003, Table 8). Statistically significant increases in antibody reactivity for the E2 Δ1-his, E2 Δ3-his, and E2 Δ12-his immunization groups versus E2-his were observed, and also for E2 Δ1-his relative to E2 Δ2-his immunization groups (p≦0.04).
7. Immunoreactivity Towards Homologous E2-his Antigen of Mouse Sera Obtained after 3 Immunizations with E2-his Protein Variants
The immunoreactivity of mouse sera obtained after 3 immunizations with the E2-his wild type and variant proteins towards solid-phase “homologous antigen” (i.e. that antigen used to immunize a particular mouse group) was examined by ELISA. Substantial antibody binding activity towards the homologous solid-phase antigen was demonstrated in each of the vaccinated animals (FIG. 33), but not in two representative control sera obtained prior to immunization (prebleed) from each vaccination group (data not shown). Furthermore, antibody reactivity towards solid-phase E2-his proteins was not detected in the mice vaccinated with adjuvant alone (data not shown), confirming that the animals receiving E2-his protein elicited antibodies specific to the immunogen.
8. Immunoreactivity Towards E2-his and E2Δ123-his Antigens of Mouse Sera Obtained after 3 Immunizations with E2-his Protein Variants
The ability of the antibodies in immune sera from each vaccination group to bind to solid-phase E2-his (containing the three variable regions) was compared to the antibody reactivity towards solid-phase E2 Δ123-his (lacking the three variable regions) in ELISA. Substantial binding titres against solid phase E2-his (FIG. 34A) and E2 Δ123-his (FIG. 34B) were observed for all immunization groups (except adjuvant alone, data not shown).
Pair-wise statistical comparisons of the antibody binding titres obtained for the various immunization groups were performed in order to determine whether apparent differences in binding titre were significant. In contrast to the data obtained after the second immunization, Table 9 indicates that the antibody titres against solid-phase E2-his calculated for the E2-his-immunization group were significantly higher than those of 6 of 7 other immunization groups (p=0.003 to 0.04), excepting the E2 Δ123-his (p=0.11).
A pair-wise statistical analysis of antibody binding titres against solid-phase E2 Δ123-his for the various immunization groups revealed a highly significant increase in antibody reactivity for the E2 Δ123-his immunization group compared with the E2-his immunization group (p=0.0007, Table 10). Statistically significant differences were also observed between the E2-his versus E2 Δ3-his, E2 Δ12-his, and E2 Δ23-his immunization groups, and also between the E2 Δ1-his versus E2 Δ3-his, E2 Δ12-his, E2 Δ23-his and E2 Δ123-his immunization groups (p=0.009 to 0.022).
Finally, the mean binding titres obtained for each immunization group against solid-phase wild type E2-his antigen (i.e. containing HVR1, HVR2 and IgVR) were compared with the binding titres towards solid-phase E2 Δ123-his antigen (i.e. lacking HVR1, HVR2 and igVR and representing the conserved glycoprotein core). This analysis was performed in order to gauge the relative abilities of the immunogens to elicit antibodies reactive with the conserved core of the E2RBD. Table 11 shows significantly lower antibody titres to solid-phase E2 Δ123-his antigen relative to solid-phase E2-his antigen for antisera elicited by E2-his immunogen ( ̃4.4-fold reduction; p=0.0009) and E2 Δ1-his immunogen ( ̃2-fold reduction; p=0.005).
9. Immunoreactivity Towards Solid-Phase Con1 E2RBD-his and JFH1 E2RBD-myc Antigens of Mouse Sera Obtained after 3 Immunizations with E2-his Protein Variants
The abilities of the E2-his wild type and variant proteins, which are derived from the genotype 1a H77c isolate, to elicit cross-genotype reactive antibodies was determined by comparing the immunoreactivity of immune sera with solid-phase Con1 E2RBD-his (genotype 1b) and JFH1 E2RBD-myc (genotype 2a) antigens in ELISA. Substantial binding titres against solid phase Con1 E2RBD-his (FIG. 35A) and JFH1 E2RBD-myc (FIG. 35B) were observed for all immunization groups (except adjuvant alone, data not shown).
A pair-wise statistical analysis of antibody binding titres against solid-phase Con1 E2RBD-his for the various immunization groups revealed significantly higher binding titres for E2 Δ2-his, E2 Δ3-his, E2 Δ12-his, and E2 Δ123-his immunization groups compared with the E2-his immunization group (p<0.05, Table 12). The Con1 E2RBD-his-binding titre elicited in the E2 Δ123-his immunization group was also significantly higher than the Con1 E2RBD-his binding titres elicited by E2 Δ1-his and E2 Δ23-his (p<0.05).
By contrast, a similar pair-wise statistical analysis of antibody binding titres against solid-phase JFH1 E2RBD-myc for the various immunization groups did not reveal significantly different binding titres between groups (Table 13). Despite this lack of significance, the trends observed above were also apparent in these analyses.
10. Neutralization
The abilities of immune and control mouse sera to neutralize a single cycle of infection by homologous H77c strain E1E2-pseudotyped HIV-1 luciferase reporter viruses were determined. Serial 5-fold dilutions of heat-inactivated immune and control mouse sera were preincubated with E1E2-pseudotyped HIV-1 luciferase reporter viruses (1 h) and then incubated for 4 h with Huh7 cell monolayers. The cells were washed with PBS, fresh culture medium added, and after an additional 3-day incubation (37° C. in 5% CO2), the cells were lysed and assayed for luciferase activity. FIG. 36 shows the 80%-neutralization titres of individual sera, which were determined as the serum dilution giving 80% neutralization compared to HCV glycoprotein-pseudotyped HIV-1 luciferase reporter virus preincubated with medium alone. At least 80% of mice immunised with E2-his, E2 Δ123-his and E2 Δ23-his produced neutralizing antibodies against the E1E2-HIV-1 pseudotypes with mean 80% neutralization titres of 908, 413 and 140, respectively. By contrast, at least 80% of mice immunized with E2 Δ1-his or adjuvant alone lacked 80%-neutralization activity with mean 80% neutralization titres of 7 and 26, respectively (Table 14).
11. Discussion
The E2-his proteins containing one or more deletions of the three variable regions were capable of eliciting antibody reactive towards homologous antigens. The specificity of the antibody response was examined by comparing the ability of the immune sera to react towards intact wild-type E2-his protein containing three variable regions to E2 Δ123-his protein which represents the core E2 folding unit lacking all three variable regions but retaining CD81 binding. This analysis revealed that mice immunized with E2-his protein or E2 lacking HVR1 (E2 Δ1-his) elicited significantly less antibody reactive towards the core domain of E2. By contrast antibodies elicited in mice immunized with E2-his proteins lacking HVR1 and 2 (E2 Δ12-his), HVR1 and igVR (E2 Δ13-his), HVR2 and igVR (E2 Δ23-his) or lacking all three variable regions (E2 Δ123-his) reacted similarly towards wild-type E2-his and E2 Δ123-his antigens. This suggests that HVR2 and the igVR may hinder antibody access to epitopes present in the underlying conserved core domain of E2. To test this hypothesis, E2RBD constructs were synthesised representing heterologous isolates of HCV from genotype 1b (Con1) and genotype 2a (JF-H1). The immunoreactivity of mouse serum obtained after the third vaccination to these heterologous E2RBD constructs revealed that mice immunized with E2 Δ123-his, E2 Δ12-his, E2 Δ3-his, E2 Δ2-his elicited significantly higher levels of cross-reactive antibody to epitopes present in the genotype 1b isolate Con1. Although cross-reactive antibody to the E2RBD of genotype 2a isolate JF-H1 were also elicited, the differences in binding titres between the immunogen groups were not statistically significant although trends similar to those above were observed. These data suggest that deletion of at least one variable region, preferably either HVR2 and/or igVR, may improve the ability of E2-his proteins to elicit cross-reactive antibodies with broader neutralization capacity.
The ability of the immune serum to neutralize homologous virus was examined using HCV E1E2 pseudotyped retroviral particles. The data showed that mice vaccinated with E2 Δ123-his or E2 Δ23-his possessed on average  ̃59 and 20 fold higher neutralizing antibody titres compared with E2-his lacking HVR1 alone (p=0.11 and 0.002, respectively). HVR1 has previously been shown to be an immunodominant region of the E1E2 glycoprotein complex. Deletion of HVR1 region alone may result in the removal of an important type-specific immunodominant epitope. However, the presence of HVR2 and/or the igVR regions in the HVR1 deleted construct may shield underlying cryptic neutralizing epitopes. It is therefore likely that the antibody response elicited by wild-type E2-his protein may be largely directed towards the HVR1 region and not the conserved core. This is in part reflected by (i) the lower level of antibody reactive to the E2 core domain (FIG. 34B) and (ii) the lower levels of cross-reactive antibody (FIG. 35A).
Together the data suggest that deletion of HVR2 and/or the igVR region is a significant improvement on the use of E2 RBD constructs lacking none of the variable regions or HVR1 alone. It is likely that E2 RBD proteins lacking at least HVR2 and/or igVR and possibly all three variable regions will elicit antibody capable of cross-neutralizing divergent HCV strains from within a genotype as well as cross-genotype neutralization. E2 RBD proteins lacking one or more variable regions may be useful tools for both therapeutic or prophylactic vaccination strategies for the prevention or treatment of HCV infection. In addition these variable region deleted E2 RBD constructs may also be useful to elicit novel specificities of antibodies directed to the conserved E2 core region that neutralize HCV for therapeutic and prophylactic use.
12. Materials and Methods
Construction of Wild Type and Variant HCV E2-his cDNAs and Secreted Expression of the Encoded Proteins in Mammalian Cells.
Generation of cDNA Expression Plasmids Encoding HCV E2-his Variants
A synthetic gene encoding a wild type E2 protein fragment (residues 384-661; strain H77c) was constructed by Geneart AG (Regensburg, Germany). The human trypsinogen signal peptide (MNPLLILTFVAAALA) (SEQ ID NO: 63) was appended in-frame to the N-terminus of the wild type E2 mature protein in order to facilitate secretion of the mature polypeptide into the expression medium. A Kozak sequence was introduced just before the N-terminus to increase translational initiation and a (His)6 sequence (SEQ ID NO: 108) was added in-frame to enable subsequent purification of the secreted proteins by immobilised metal affinity chromatography. Two stop codons were added after the His-tag at the C-terminus to ensure efficient translational termination. This construct is referred to as E2-his. The codon usage of the E2-his cDNA was adapted to the codon bias of Homo sapiens genes. An Nhe I restriction site at the 5′ end of the cDNA and a Xho I restriction site was introduced at the 3′ end in order to ligate the Geneart cDNA into Nhe I-Xho I digested pcDNA3.1 (Invitrogen).
The cDNAs encoding seven variants of the E2-his mature protein where one or more of the variable regions were substituted by linker sequences, were constructed using standard PCR mutagenesis procedures with Accuprime Pfx DNA Polymerase (Invitrogen) according to the manufacturer's instructions and the primers shown in Table 15. The combinations of primers used to construct cDNAs of the E2-his are shown in Table 16.
The specific PCR parameters were as follows: 2 minutes of denaturation at 95° C. was followed by 18 cycles of 95° C. for 15 seconds, 64° C. for 30 seconds and 68° C. for 2 minutes with a final 2 minute incubation at 68° C.
The Geneart wild type E2-his cDNA was used as a PCR template for all the E2-his variant cDNAs. Each deletion variant was constructed using sequential overlap PCR of multiple fragments. Once each cDNA was complete, it was digested with Nhe I and Xho I and ligated into pcDNA3.1. Large scale preparations of plasmid DNA were carried out using a Qiagen Maxi Kit. The nucleotide sequences of all the plasmid constructs were verified by sequencing both strands using Big Dye Terminator v3.1 Cycle Sequencing and an Applied Biosystems Automated Sequencer.
The sequences of the mature wild type and variant E2 proteins are as follows:







E2-his mature protein


(SEQ ID NO: 64)


ETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCN


 


ESLNTGWLAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANG


 


SGLDERPYCWHYPPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGA


 


PTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPPCVI


 


GGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHY


 


PCTINYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSE


 


E2Δ1-his mature protein


(SEQ ID NO: 65)


ETHQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSSGC


 


PERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPA


 


KSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPL


 


GNWFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEAT


 


YSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRL


 


EAACNWTRGERCDLEDRDRSE


 


E2Δ2-his mature protein


(SEQ ID NO: 66)


ETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCN


 


ESLNTGWLAGLFYQHKFNSSGCPERLASCGSSGCWHYPPRPCGIVPA


 


KSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPL


 


GNWFGCTWMNSTGFTKVCGAPPCVIGGVGNNTLLCPTDCFRKHPEAT


 


YSRCGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRL


 


EAACNWTRGERCDLEDRDRSE


 


E2Δ3-his mature protein


(SEQ ID NO: 67)


ETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCN


 


ESLNTGWLAGLFYQHKFNSSGCPERLASCRRLTDFAQGWGPISYANG


 


SGLDERPYCWHYPPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGA


 


PTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPPCGS


 


SGCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTINYT


 


IFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSE


 


E2Δ12-his mature protein


(SEQ ID NO: 68)


ETHQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSSGC


 


PERLASCGSSGCWHYPPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDR


 


SGAPTYSWGANDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPP


 


CVIGGVGNNTLLCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRL


 


WHYPCTINYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSE


 


E2Δ13-his mature protein


(SEQ ID NO: 69)


ETHQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSSGC


 


PERLASCRRLTDFAQGWGPISYANGSGLDERPYCWHYPPRPCGIVPA


 


KSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVEVLNNTRPPL


 


GNWEGCTWMNSTGFTKVCGAPPCGSSGCPTDCFRKHPEATYSRCGSG


 


PWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACNWT


 


RGERCDLEDRDRSE


 


E2Δ23-his mature protein


(SEQ ID NO: 70)


ETHVTGGNAGRTTAGLVGLLTPGAKQNIQLINTNGSWHINSTALNCN


 


ESLNTGWLAGLFYQHKFNSSGCPERLASCGSSGCWHYPPRPCGIVPA


 


KSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTRPPL


 


GNWFGCTWMNSTGFTKVCGAPPCGSSGCPTDCFRKHPEATYSRCGSG


 


PWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACNWT


 


RGERCDLEDRDRSE


 


E2Δ123-his mature protein


(SEQ ID NO: 71)


ETHQNIQLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFNSSGC


 


PERLASCGSSGCWHYPPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDR


 


SGAPTYSWGANDTDVEVLNNTRPPLGNWEGCTWMNSTGETKVCGAPP


 


CGSSGCPTDCFRKHPEATYSRCGSGPWITPRCMVDYPYRLWHYPCTI


 


NYTIFKVRMYVGGVEHRLEAACNWTRGERCDLEDRDRSE






Cell Culture

FreeStyleTM 293-F cells (Invitrogen) were cultured in FreeStyleTM Expression Medium (Invitrogen) supplemented with penicillin/streptomycin/fungizone (Invitrogen). All cells were maintained at 37° C. in humidified incubators with an atmosphere of 8% CO2.
Transient Protein Expression
Transient expression of each of the E2-his proteins was carried out in FreeStyleTM 293-F cells by transfection with the pcDNA3.1-based expression plasmids and 293fectin transfection reagent (Invitrogen) according to the manufacturer's instructions. The cells in a total volume of 180 ml were transfected at a final concentration of 1×106 viable cells/ml and incubated in a sterile shaker flask (Corning) for 5 days on an orbital shaker (IKA) rotating at 150 rpm in a 37° C. humidified incubator with an atmosphere of 8% CO2. Twenty-four hours after transfection the cell cultures were supplemented with Tryptone N1 (Organotechnie, France) to a final concentration of 0.5% v/v. Typically the cell cultures were harvested 5 days after transfection. Protein expression was examined by electrophoresis of a sample of cell culture supernatant using 4-20% Tris-Glycine SDS polyacrylamide gel and the proteins visualised by staining with Coomassie Blue reagent. For protein purification, cell culture supernatants were harvested by centrifugation at 2500 rpm and then passed through a 0.45 μM filter (Nalgene) prior to chromatography.
Purification of Expressed Wild Type and Variant HCV E2-his Proteins.
Following filtration the cell culture supernatants were subjected to immobilised metal affinity chromatography (IMAC) using Nickel sepharose to purify the wild type and variant E2-his proteins.
The purification procedure is described below:
1. Buffers:
Ni-MAC Buffer A
50 mM NaH2PO4 pH 8.0
300 mM NaCl
10 mM imidazole
Ni-MAC Buffer B (Elution)
50 mM NaH2PO4 pH 8.0
300 mM NaCl-500
500 mM imidazole
2. Protocol:


    
    
        1. Procedures 2 to 6 were carried out at 4-8° C.
        2. 1 ml Ni Sepharose 6 Fast Flow resins (GE Healthcare) in a 10 ml Poly-Prep columns (Bio-Rad) were washed with 5 volumes of ddH2O.
        3. The columns were equilibrated with 10 ml Ni-MAC Buffer A.
        4. Samples were loaded onto the columns, and break-through (B/T) collected.
        5. The columns were washed with 10 ml Ni-MAC Buffer A.
        6. The proteins were eluted with 5 ml Ni-MAC Buffer B (Elution) and 1 ml fractions collected.
        7. Peak fractions were identified using 96-well plate format Bradford Assay and Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels.
        8. Peak fractions were pooled and dialysed in 1×PBS overnight at 4° C.
        9. Following dialysis protein concentrations were determined using 96-well plate format Bradford Assay and 1 mg/ml BSA for a standard curve.

Construction of Con1 E2RBD-his and JFH1 E2RBD-myc cDNAs and Secreted Expression of the Encoded Proteins in Mammalian Cells.

Generation of cDNA Expression Plasmids Encoding Con1 E2RBD-his and JFH1 E2RBD-myc.

    
    


cDNAs encoding Con1 E2 (HCV Con1 polyprotein residues 384 to 661; Genbank accession number AJ238799) and JFH1 E2 (HCV JFH1 polyprotein residues 384 to 665; Genbank accession number AB047369), were prepared by PCR using Expand-Hifi polymerase (Roche) and the primers indicated in Table 17. The forward primers encoded an NheI restriction site for incorporation in-frame at the 5′ ends of the cDNAs. The reverse primers encoded (His)6 (SEQ ID NO: 108) and c-myc epitope tags for addition in-frame to the 3′ ends of Con1 and JFH1 cDNAs, respectively, and for both cases were followed by a TAG translation termination codon and an XbaI restriction site. The specific PCR parameters were as follows: 5 minutes of denaturation at 95° C. was followed by 30 cycles of 92° C. for 1 min, 55° C. for 1 min and 72° C. for 2 minutes with a final 10 minute incubation at 72° C. The cDNAs were cloned downstream of a tissue plasminogen activator signal peptide in vector pcDNA3 to facilitate secretion of the expressed proteins. The nucleotide sequences of the cloned cDNAs were verified by using Big Dye Terminator v3.1 Cycle Sequencing and an Applied Biosystems Automated Sequencer.
Transient Expression of Con1 E2RBD-his and JFH1 E2RBD-myc
HEK 293T cells (350,000 cells per well of 6-well culture plates) were transfected with Con1 E2RBD-his and JFH1 E2RBD-myc expression vectors using Fugene 6 (Roche). At 8-h post transfection, the transfection medium was replaced with Optimem (Invitrogen) and the cells incubated for 3 days at 37° C. in a humidified atmosphere containing 5% CO2. The tissue culture fluid was clarified by centrifugation at 1500 rpm for 10 min followed by filtration through 0.45-μm-pore-size syringe filters.
Biochemical and Functional Analysis of Wild Type and Variant E2-his Proteins.
Blue-Native PAGE
The oligomerization status of the purified E2-his proteins was analysed using blue native polycrylamide gel electrophoresis (BN-PAGE). Ten μg of each protein was added to 10 μl of solubilization buffer and 1.5 μl of sample buffer and loaded onto 5-15% gradient separating gels containing a 4% stacking gel. The gels were electrophoresed at 4° C. at 100 volts until the dye front entered the stacking gel. The voltage was then increased to 200 volts until the dye front migrated to the bottom of the gel. Electrophoresis was conducted for 1.5 h with 1× cathode buffer containing 0.01% Serva G in the upper reservoir, followed by electrophoresis for 1-1.5 h in 1× cathode buffer without Serva G. The lower reservoir contained 1× anode buffer. After electrophoresis, the gels were destained overnight and scanned in an Odyssey scanner at 680 nm.
The solutions used for BN-PAGE are as follows:
Polyacrylamide Gels:















4% stacker
5% separating gel
15% separating gel






















40% acrylamide
0.299
ml
0.262
ml
0.787
ml


3x gel buffer
1
ml
0.7
ml
0.7
ml


75% Glycerol


0.14
ml
0.62
ml


water
1.72
ml
0.978
ml
0


TEMED
6
μl
2
μl
2
μl


5% ammonium
32
μl
11
μl
11
μl


persulphate









Other Solutions:

    
    
        3× Gel Buffer. 150 mM BisTris-HCl, 0.5M 6-amino caproic acid, pH 7.0
        10× Cathode buffer. 0.5M Tricine, 150 mM BisTris
        5× Anode buffer. 0.25M BisTris-HCl, pH 7.0
        2× BisTrisACA. 200 mM BisTris-HCl, 1M 6-amino-caproic acid, pH 7.0.
        Sample buffer. 50 mg ServaG, 500 μl 2× BisTrisACA, 400 μl 75% sucrose and 100 μl water.
        Solubilization buffer. 0.5M 6-amino-caproic acid, 20 mM BisTris, 2 mM EDTA, pH 7.0, 1% Triton X-100, 10% glycerol.
        Destain. 10% acetic acid, 10% methanol, 80% water.

Immunodetection of E2-his Proteins.

    
    


Samples of the purified E2-his proteins were subjected to reducing SDS-PAGE followed by electrophoretic transfer to nitrocellulose membrane. E2-his proteins were detected with a non-conformation dependent E2 specific monoclonal antibody followed by goat anti-mouse immunoglobulin coupled to Alexa fluor 680 nm (Invitrogen). Immunoblots were scanned in an Odyssey detection system.
CD81 Binding Properties.
The ability of the E2-his proteins to interact with the HCV cellular receptor CD81 was examined using a solid phase enzyme immunoassay. Enzyme immunoassay plates (Nunc Maxisorb®) were coated with maltose binding protein fused to the recombinant large extracellular loop of CD81 (residues 113-201) at 5 μg/ml in PBS overnight at 4° C. Coating solution was removed and unoccupied sites blocked with bovine serum albumin (10 mg/ml) in PBS (BSA10PBS) for 1 h at room temperature. Plates were washed 4 times with PBS containing 0.05% Tween 20 (PBST). 50 ng E2-his proteins were serially diluted in a 50 μl PBS containing 5 mg/ml bovine serum albumin (BSA5PBST) and incubated for 2 h. Bound E2-his protein was detected using an E2 specific monoclonal antibody followed by rabbit anti-mouse immunoglobulins coupled to horse radish peroxidase (Dako). Plates were developed using tetramethylbenzidine hydrochloride substrate and stopped by the addition of 1M HCl. Absorbance values were measured at 450 nm and the background at 620 nm subtracted in a Fluostar plate reader (BMG technologies).
Immunogenicity in Mice of Wild-Type and Variant E2-his Proteins.
Immunization Protocol.
Groups of 10 7-8 week old female Balb/c mice were immunised with purified E2-his wild type and variant proteins formulated with ISCOMATRIX® adjuvant (Pearse and Drane, 2005) (Table 18). Each mouse dose contained 10 μg specific protein and 5 μg ISCOMATRIX® adjuvant in a 0.1 ml volume. Mice were dosed subcutaneously three times at three-weekly intervals and bleeds taken 1 day prior to the first dose and at one week after both the second and third doses.
Enzyme-Linked Immunosorbent Assay (ELISA)
The immune mouse sera were examined for the presence of anti-E2 antibodies in ELISA. The E2-his, Con1 E2RBD-myc or JFH1 E2RBD-myc proteins were captured on 96 well Maxisorb microtitre plates (Nunc) using Galanthis nivalis (GNA) lectin. Plates were coated with GNA lectin at 5 μg/ml in PBS overnight at 4° C. Unoccupied sites were blocked with 100 μl BSA10PBS for 1 h at room temperature. After washing the plates 4 times with PBST, the E2 proteins were applied in 50 μl BSA5PBST and incubated for 1 h at room temperature. After washing the plates 4 times with PBST, serial dilutions of mouse sera were applied in a 50 μl volume of BSA5PBST and incubated for 1 h room temperature. Bound immunoglobulins were detected with rabbit anti-mouse immunoglobulin coupled to horseradish peroxidase and developed using tetramethylbenzidine hydrochloride substrate and stopped by the addition of 1M HCl. A Fluostar plate reader (BMG technologies) was used to measure the absorbance at 450 nm and the background at 620 nm. The background was subtracted from the absorbance to generate the binding curves.
Neutralization Assays
The abilities of immune and control mouse sera to neutralize a single cycle of infection by HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses were determined as follows. HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses were prepared by cotransfection of HEK-293T monolayers (350,000 cells per well of 6-well culture dishes) with pE1E2 and pNL4.3LUCR-E- plasmids (Drummer et al., 2003). After 3 days incubation at 37° C. in a humidified atmosphere containing 5% CO2, the culture supernatants were filtered through 0.45 μm sterile syringe filters (Sartorius). Serial 5-fold dilutions of heat-inactivated immune and control mouse sera were preincubated with HCV glycoprotein-pseudotyped HIV-1 luciferase reporter viruses (1 h) and then added to quadruplicate Huh7 cell monolayers in 48-well tissue culture plates. Following a 4 h incubation (37° C., 5% CO2) the cells were washed with PBS and the medium replaced. After an additional 3-day incubation (37° C. in 5% CO2), the cells were lysed, the lysates clarified by centrifugation and then assayed for luciferase activity (Promega) in a Fluostar (BMG) fitted with luminescence optics. The neutralization titres of individual sera were determined as the serum dilution giving 80% neutralization compared to HCV glycoprotein-pseudotyped HIV-1 luciferase reporter virus preincubated with medium alone.







TABLE 7







Pair-wise statistical comparison of antibody titres reactive


to solid-phase E2-his antigen obtained after 2nd vaccination.









Immunogen Group















E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his


















E2-his
0.06
0.96
0.25
0.26
0.37
0.6
0.08


E2 Δ1-his

0.06
0.27
0.09
0.034
0.014
0.04





p values calculated using student's t test.













TABLE 8







Pair-wise statistical comparison of antibody titres reactive to solid


phase E2 Δ123-his protein antigen obtained after 2nd vaccination.









Immunogen Group















E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his


















E2-his
0.04
0.96
0.02
0.02
0.09
0.077
0.003


E2 Δ1-his

0.04
0.09
0.12
0.06
0.06
0.17





p values calculated using student's t test.













TABLE 9







Pair-wise statistical comparison of antibody titres reactive to solid-


phase E2-his protein antigen obtained after 3rd vaccination.









Immunogen Group















E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his


















E2-his
0.02
0.003
0.002
0.04
0.002
0.02
0.11


E2 Δ1-his

0.1
0.021
0.58
0.44
0.9
0.17





p values calculated using student's t test assuming unequal variances.













TABLE 10







Pair-wise statistical comparison of antibody titres reactive to solid-


phase E2 Δ123-his protein antigen obtained after 3rd vaccination.









Immunogen















E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his


















E2-his
0.64
1
0.016
0.004
0.31
0.003
0.0007


E2 Δ1-his

0.62
0.022
0.007
0.44
0.005
0.009





p values calculated using student's t test assuming unequal variances.













TABLE 11







Statistical comparison of relative antibody titres obtained after


3rd vaccination against E2-his antigen and E2 Δ123-his antigen.









Immunogen
















E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his
E2-his



















Mean antibody
1.16
0.81
0.68
1.31
.71
1.19
1.54
2.26


titre against


E2-his (×105)


Mean antibody
0.56
0.51
1.15
1.11
0.71
1.19
1.54
0.51


titre against


E2 Δ123-his (×105)


p value
0.005
0.045
0.06
0.46
1
1
1
0.0009





p values calculated using student's t test assuming unequal variances.













TABLE 12







Pair-wise statistical comparison of antibody titres reactive to solid-


phase Con1 E2RBD-his protein antigen obtained after 3rd vaccination.
















E2-his
E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his



















E2-his










E2 Δ1-his
0.526


E2 Δ2-his
0.0453
0.072


E2 Δ3-his
0.043
0.061
0.883


E2 Δ12-his
0.0123
0.091
0.926
0.797


E2 Δ13-his
0.185
0.343
0.847
0.756
0.888


E2 Δ23-his
0.173
0.568
0.284
0.238
0.217
0.534


E2 Δ123-his
0.0123
0.028
0.259
0.325
0.197
0.251
0.0499





p values calculated using student's t test assuming unequal variances.













TABLE 13







Pair-wise statistical comparison of antibody titres reactive to solid-


phase JFH1 E2RBD-myc protein antigen obtained after 3rd vaccination.
















E2-his
E2 Δ1-his
E2 Δ2-his
E2 Δ3-his
E2 Δ12-his
E2 Δ13-his
E2 Δ23-his
E2 Δ123-his



















E2-his










E2 Δ1-his
0.609


E2 Δ2-his
0.148
0.511


E2 Δ3-his
0.159
0.253
0.371


E2 Δ12-his
0.305
0.549
0.861
0.48


E2 Δ13-his
0.392
0.624
0.916
0.484
0.970


E2 Δ23-his
0.123
0.287
0.492
0.652
0.705
0.702


E2 Δ123-his
0.058
0.084
0.117
0.406
0.154
0.156
0.209





p values calculated using student's t test assuming unequal variances.













TABLE 14







Mean 80% Neutralizing antibody titres and statistical comparison.









Immunogen group













E2-
E2 Δ1-
E2 Δ23-
E2 Δ123-
No



his
his
his
his
antigen
















Mean neutralizing
908
7
140
413
26


antibody titre


p value relative

0.044
0.08
0.29
0.048


to E2-his


p value relative


0.002
0.11
0.31


to E2 Δ1-his





p values were calculated using student's t test assuming unequal variances.













TABLE 15







Primers used for preparation of E2-his


protein variant DNA sequences.








Primer



designation
Primer Sequence





HCV-1
5′ TATAGCTAGCGCCACCATGAACCCCCTGC 3′



(SEQ ID NO: 72)


 


HCV-2
5′ CTGGATGTTCTGGTGGGTCTCGGCCAG 3′



(SEQ ID NO: 73)


 


HCV-3
5′ GCCGAGACCCACCAGAACATCCAGCTG 3′



(SEQ ID NO: 74)


 


HCV-4
5′ CGCTGCTGCCGCAGGAGGCGAG 3′



(SEQ ID NO: 75)


 


HCV-4a
5′ GTAGTGCCAGCAGCCGCTGCTGCCGCAGGAGGCGAGC



CTCTCGGG 3′ (SEQ ID NO: 76)


 


HCV-5
5′ GGCAGCAGCGGCTGCTGGCACTAC 3



(SEQ ID NO: 77)


 


HCV-5a
5′ CTCGCCTCCTGCGGCAGCAGCGGCTGCTGGCACTACC



CCCCCAGA 3′ (SEQ ID NO: 78)


 


HCV-6a
5′ GTCGGTGGGGCAGCCGCTGCTGCCGCAGGGAGGGGCG



CCACACAC 3′ (SEQ ID NO: 79)


 


HCV-7a
5′ GCCCCTCCCTGCGGCAGCAGCGGCTGCCCCACCGACT



GCTTTAGG 3′ (SEQ ID NO: 80)


 


HCV-8
5′ TCTGCTCGAGTTATCAGTGGTGATGGTGGTGG 3′



(SEQ ID NO: 81)


 


HCV-11
5′ CTCTCGTTGCAGTTCAGGGCGGTGCTG 3′



(SEQ ID NO: 82)


 


HCV-12
5′ CAGCACCGCCCTGAACTGCAACGAGAG 3′



(SEQ ID NO: 83)
















TABLE 16







Primer combinations used for preparation


of E2-his protein variant DNA sequences.








E2 protein
Primer combination





E2Δ1-his
HCV-1 HCV-3 HCV-8


E2Δ2-his
HCV-1 HCV-11 HCV-12 HCV-8


E2Δ3-his
HCV-1 HCV-6a HCV-7a HCV-8


E2Δ12-his
HCV-1 HCV-2 HCV-3 HCV-4 HCV-5


E2Δ13-his
HCV-1 HCV-6a HCV-7a HCV-8


E2Δ23-his
HCV-1 HCV-5a HCV-4A HCV-6A HCV-7A HCV-8


E2Δ123-his
HCV-1 HCV-6a HCV-7a HCV-8
















TABLE 17







Primers used for preparation of Con1 E2RBD-his and JFH1 E2RBD-myc DNA sequences











E2RBD


RE
Epitope


construct

Primer Sequence
site
tag





Con1 E2RBD-his
Forward primer
5′-CAAGCTAGCggaacctatgtgacaggg (SEQ ID NO: 84)
NheI



 



Reverse primer
5′-CCCTCTAGATTAGTGGTGGTGGTGGTGGTGGCCGCCCTCTGATCTGTCCCTGTC
XbaI
His6




(SEQ ID NO: 85)




 


JFH1 E2RBD-myc
Forward primer
5′CAAGCTAGCggcaccaccaccgttggaggc (SEQ ID NO: 86)
NheI



 



Reverse primer
5′CCGTCTAGActaattcagatcctcttctgagatgagtttttgttcagtactCTGA 
XbaI
myc




CTCCTGTCCCTGTC (SEQ ID NO: 87)
















TABLE 18







Immunisation of mice with E2-his wild type and variant proteins


formulated with ISCOMATRIX ® adjuvant Antigens









Group
Protein
ISCOMATRIX ® adjuvant





1
E2Δ1-his
+


2
E2Δ2-his
+


3
E2Δ3-his
+


4
E2Δ123-his
+


5
E2Δ12-his
+


6
E2Δ13-his
+


7
E2Δ23-his
+


8
E2-his
+


9
—
+









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## Claims

1. A modified hepatitis C virus (HCV) E2 glycoprotein comprising a modified HCV-E2 receptor-binding domain (RBD), wherein the intergenotypic variable region (igVR) of the HCV-E2 receptor-binding domain is deleted and is replaced with a flexible linker sequence, and optionally, one or both of the hypervariable region 1 (HVR1) of said domain and the hypervariable region 2 (HVR2) of said domain are deleted and replaced with a flexible linker sequence.

2. A modified glycoprotein according to claim 1, wherein the HVR1 variable region also is deleted and is optionally replaced with a flexible linker sequence.

3. A modified glycoprotein according to claim 1, wherein the HVR2 variable region also is deleted and is optionally replaced with a flexible linker sequence.

4. A modified glycoprotein according to claim 1, wherein the variable regions HVR1 and HVR2 also are deleted and each optionally is replaced with a flexible linker sequence.

5. A modified glycoprotein according to claim 1, wherein the variable region igVR is replaced with a flexible linker sequence.

6. A modified glycoprotein according to claim 2, wherein the HVR1 variable region is replaced with a flexible linker sequence.

7. A modified glycoprotein according to claim 3, wherein the HVR2 variable region is replaced with a flexible linker sequence.

8. A modified glycoprotein according to claim 4, wherein the variable regions igVR, HVR1 and HVR2 are replaced with flexible linker sequences.

9. A modified glycoprotein according to claim 1, wherein the modified glycoprotein binds to the HCV receptor CD81.

10. A modified glycoprotein according to claim 5, wherein the flexible linker sequence comprises a peptide comprised of up to 20 amino acid residues and comprises residues selected from the group consisting of Gly, Ser, Ala, Thr and Arg.

11. A modified glycoprotein according to claim 6, wherein the flexible linker sequence comprises a Gly-Ser-Ser-Gly (GSSG) sequence (SEQ ID NO: 95).

12. A modified glycoprotein according to claim 6, wherein the flexible linker sequence, comprises a Glu-Thr-His-Gly-Ser-Ser-Gly (ETHGSSG) sequence (SEQ ID NO: 109).

13. A composition comprising a modified HCV E2 glycoprotein according to claim 1, together with a pharmaceutically acceptable carrier or diluent.

14. A composition according to claim 13, further comprising an adjuvant.

15. A method of eliciting an immune response in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein according to claim 1.

16. A method according to claim 15 wherein the patient is a human.

17. A method for therapeutic treatment of HCV infection in a patient, which comprises administration to the patient of an effective amount of a modified HCV E2 glycoprotein according to claim 1.

18. A method according to claim 17 wherein the patient is a human.

19. An agent for eliciting an immune response in a patient, which comprises a modified HCV E2 glycoprotein according to claim 1.

20. An agent for therapeutic treatment of HCV infection in a patient, which comprises a modified HCV E2 glycoprotein according to claim 1.