Patent Grant
9758794
Hepatitis C virus (HCV) continues to be a global epidemic. In most cases, HCV infection becomes chronic and can persist for decades, leading to cirrhosis, end-stage liver disease and hepatocellular carcinoma. Currently, 2% of the human population—approximately 123 million people—is infected with HCV. In fact, there are 3-4 times more individuals infected with HCV than HIV, making virus transmission a major public health concern. In the United States, HCV infection is the most common cause of liver transplantation and results in 10,000 to 20,000 deaths a year. There is no vaccine, and current HCV therapy, pegylated interferon-alpha in combination with ribavirin, leads to a sustained response in only 50% of genotype 1-infected patients, the prevalent genotype in the United States. The current HCV treatment stimulates the patient's immune system to clear the virus, but numerous side effects cause many patients to prematurely stop treatment. Two new protease inhibitors now used to improve treatment outcomes do not eliminate the need for interferon-alpha and ribavirin. Given the high prevalence of infection and poor response rate, inhibitors that specifically target HCV proteins with fewer side effects are desperately needed. In addition, an effective vaccine would greatly reduce the spread of the virus.
International Patent Publication No. WO 2008/022401 to Mc Caffrey, et. al, describes preparing an HCV E2 polypeptide having internal deletions of the regions within E2. However, this reference does not describe a stable cell line that expresses E2. The cells perform transient expression, which is only good for a few days. The E2 DNA is not incorporated into the genome of the cell and after several days, the cells will remove the gene. This method is inefficient. This publication also does not utilize a construct containing an Fc or protein-A tags.
U.S. Pat. No. 6,326,171 to Chiron describes preparing an HCV E2 polypeptide involving a specific region of E2 that ends at amino acid 715. The construct used does not contain a tag. The cells used for expression are non-human, and included BSC40 (African Green monkey) and F503 (chimpanzee fibroblasts).
U.S. Pat. No. 6,020,122 to Abbott Laboratories describes preparing an HCV E2 polypeptide without the use of a tag. The cells used for expression are CHO (Chinese Hampster Ovary) cells.
However, there still exists a need in the art for more efficient methods of preparing HCV E2 polypeptides, expression of higher levels and higher quality HCV E2 polypeptides, HCV vaccines and inhibitors of HCV infection.
The invention provides methods for preparing HCV E2 ectodomain (eE2) polypeptides, methods of preparing HCV vaccines, methods of preparing HCV cell entrance inhibitors, and models which could be used to assist with, e.g., development of HCV vaccines and new HCV inhibitors. HCV eE2 polypeptides of the invention include full-length ectodomains of HCV envelope 2 surface protein (E2) and fragments of ectodomains of HCV envelope 2 surface protein (E2). Full length ectodomains of HCV envelope 2 surface protein (E2) include aa 384-656 of HCV J6 [2a] genotype and corresponding sequences of other HCV genotypes. Fragments of HCV envelope 2 surface protein include, e.g., eE2 (DHVR1) and E2 core domains. eE2(DHVR1) includes, e.g., aa413-656 of HCV J6 [2a] genotype and corresponding sequences of other HCV genotypes. E2 core domain includes, e.g., aa 456-656 of HCV J6 [2a] genotype and corresponding sequences of other HCV genotypes.
A method of producing a hepatitis C virus (HCV) eE2 polypeptide in accordance with the present invention may comprise: (i) providing a construct comprising: a cytomegalovirus (CMV) promoter, a fragment of HCV envelope 2 surface protein (E2), and a cleavage site (e.g., a PreScission Protease cleavage site); (ii) introducing the construct into HEK293T cells; (iii) selecting cells stably expressing the HCV eE2 polypeptide; (iv) incubating the cells stably expressing the HCV eE2 polypeptide in a supernatant, and recovering and purifying the HCV eE2 polypeptide. A fragment of HCV envelope 2 protein may, e.g., be selected from the group consisting of full length ectodomains of E2 (eE2) and fragments of eE2. Full length eE2 may have a sequence identical to or homologous with a sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, and fragments of any one of the foregoing (e.g., fragments of from about 50 to about 270 or from about 70 to about 250 amino acids in length).
In certain embodiments, the construct may further comprise a C-terminal tag (e.g., a human Fc domain or protein-A (ProtA) tag). If a human Fc domain is used as a C-terminal tag, the recovered and purified HCV eE2 polypeptide in some of the preferred embodiments is substantially free from large disulfide-bonded aggregates and comprises a mixture of dimers and monomers of the HCV eE2 polypeptide, the dimers comprising a greater proportion of the mixture and each having an approximate molecular weight of about 120 kDa. If a protein-A (ProtA) tag is used as a C-terminal tag, in some of the preferred embodiments, the recovered and purified HCV eE2 polypeptide comprises or consists essentially of monomers of the HCV eE2 polypeptide having an approximate molecular weight ranging from about 28 kDa to about 50 kDa (e.g., about 32 kDa, about 42 kDa or about 46 kDa).
In some of the preferred embodiments the recovered and purified HCV eE2 polypeptide is conformationally similar to the E2 present on infectious HCV particles, is recognized by antibodies from chronically infected patients and is capable of one or more of the following: competing with HCVcc, inhibiting HCV cell entry and/or inducing production of HCV antibodies in a mammal (e.g., a human). In some of these embodiments, the recovered and purified HCV eE2 polypeptide can specifically bind the large extracellular loop of human CD81.
Methods for preparing a HCV E2 peptide in accordance with the invention in some of the preferred embodiments yield more than 15 mg, more than 20 mg, more than 25 mg, or more than 29 mg of the hepatitis C virus (HCV) eE2 polypeptide per liter of the supernatant. Thus, in some of the preferred embodiments, yield, e.g., from about 20 mg to about 300 mg, from about 25 mg to about 300 mg, from about 29 mg to about 275 mg, from about 29 mg to about 250 mg, from about 29 mg to about 200 mg, from about 20 mg to about 200 mg, from about 20 mg to about 150 mg, from about 25 mg to about 100 mg, or from about 29 mg to about 50 mg of the hepatitis C virus (HCV) eE2 polypeptide.
The invention also provides a construct comprising the ectodomain of the hepatitis C virus (HCV) E2 sequence and a mammalian expression system. More particularly, the invention relates to a construct comprising the CMV promoter, prolactin signal sequence, the ectodomain of HCV E2 sequence truncated at a position corresponding to aa 664 of J6[2a] genotype, a thrombin cleavage site and a human Fc domain or ProtA tag. The invention also relates to an expression system for the construct, which is stably expressed in human embryonic kidney 293T cells. In some of the embodiments, continuous protein expression in a bioreactor allows for 4 mg, 5 mg, 8 mg, 10 mg, 12 mg 15 mg, 20 mg, 22 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg or 60 mg of purified protein per liter of cell supernatant.
In a first embodiment, the invention is directed to recombinant HCV E2 ectodomain expression without the production of mostly large, disulfide-bonded aggregates. This process is used to make large quantities of the envelope glycoproteins applicable for a variety of commercial applications including but not limited to: 1) Vaccine design—The recombinant protein can be a subunit vaccine to elicit a strong immune response, protecting individuals from future infection; 2) Therapeutic vaccine—The administration of the protein to patients who are chronically infected with HCV to help the individual develop a more robust immune response either by administration alone or in combination with other medications such as IFN and ribavirin; 3) Diagnostics—enzyme-linked immunoassays can be developed using the purified, recombinant protein to screen patient sera for antibodies against these proteins. Although there are commercial screens currently available for this purpose, the proteins used therein were made in yeast or other expression systems and may not be properly folded and would have different post-translational modifications. Since the present protein is produced in human cells, the post-translational modifications are more similar to those seen on the virus; 4) Small molecule inhibitors—The ability to make a properly folded E2 could be an important reagent for finding small molecules that bind to E2. (As shown in
In accordance with the above objects, the invention in some of the embodiments is directed to a construct comprising an ectodomain of the hepatitis C virus (HCV) E2 sequence, or a fragment thereof, and a mammalian expression system. The construct may comprise a CMV promoter, prolactin signal sequence, an ectodomain of J6 HCV E2 truncated at amino acid 664 or a corresponding ectodomain of another HCV genotype, a thrombin cleavage site and a human Fc domain or ProA tag. The construct may be specific for the J6 HCV genotype, or another known HCV genotype. In other embodiments, the construct is a mutant form of eE2 in which C656 was mutated from a cysteine amino acid to a different amino acid. In other embodiments, the amino acid is serine.
In accordance with the above objects, the invention is also directed to an expression system for a construct comprising an ectodomain of HCV E2 sequence, which is stably expressed in human embryonic kidney (HEK) 293T cells.
In accordance with the above objects, the invention is also directed to a method of producing HCV eE2 polypeptide comprising: the ectodomain of the hepatitis C virus (HCV) E2 sequence, or a fragment thereof, and a mammalian expression system. The construct may comprise a CMV promoter, a prolactin signal sequence, an ectodomain of HCV E2 truncated at amino acid 664 of J6 HCV E2 or a corresponding ectodomain of another HCV genotype, a thrombin cleavage site and the human Fc domain, introducing the construct into HEK293T cells, selection of cells stably expressing the polypeptide, incubating the cells stably expressing the polypeptide in a supernatant, and recovering and purifying the polypeptide from the supernatant. In other embodiments, the method produces about 0.5 mg to about 15 mg or from about 10 mg to about 40 mg of polypeptide per liter of supernatant. In other embodiments, the method produces about 0.5 mg to about 4 mg of polypeptide per liter of supernatant. In other embodiments, the method produces about 0.5 to about 2 mg of polypeptide per liter of supernatant. In other embodiments, the method produces the HCV eE2 HCV J6 genotype polypeptide. In other embodiments, the construct is a mutant form of eE2 in which C656 was mutated from a cysteine amino acid to a different amino acid. In other embodiments, the different amino acid is serine. In other embodiments, the incubation occurs in one or more vessels suitable for providing an environment for the cells to express the polypeptide. In other embodiments, the vessels are in a rotating bottle apparatus. In other embodiments, the vessel is a bioreactor.
In accordance with any of the above objects, the invention is also directed to a method wherein the produced polypeptide is folded and sequesters human HCV receptor sites (i.e., blocks HCV binding to these sites). In other embodiments, the amount of polypeptide in monomer and dimer form exceeds the amount of polypeptide existing in higher orders, and is recognized by antibodies in the sera of patients infected with HCV. In other embodiments, the polypeptide contains 16 preserved cysteine residues.
In accordance with any of the above objects, the invention is also directed to a method of vaccinating a patient comprising administering to a patient in need thereof, a sufficient amount of a polypeptide produced by a method of the embodiments above to produce a strong immune response protecting the patient from future HCV infection.
In accordance with any of the above objects, the invention is also directed to a method of vaccinating a patient chronically infected with HCV comprising administering to a patient in need thereof, a sufficient amount of a polypeptide produced by a method of the embodiments above to produce a more robust immune response. In other embodiments, an additional therapeutic agent that provides a more robust immune response.
In accordance with any of the above objects, the invention is also directed to a method of inhibiting HCV infection in a human patient comprising administering to a patient in need thereof, a sufficient amount of a polypeptide produced by a method of the embodiments above to effectively block entry into the entry site of human cells. In other embodiments, the sufficient amount of polypeptide is non-toxic to a human patient. In other embodiments, a sufficient amount of an active fragment of the polypeptide is administered.
In accordance with any of the above objects, the invention is also directed to a method for detection of antibodies to HCV in human sera comprising contacting the sera with the polypeptide as prepared herein.
In accordance with any of the above objects, the invention is also directed to a method of producing antibodies comprising introducing the polypeptide as prepared herein to achieve a response that leads to production of antibodies to said polypeptide.
The invention is also directed to methods of preparing HCV cell entrance inhibitors. Cell entrance inhibitors may comprise a hepatitis C virus (HCV) eE2 polypeptide according to the present invention or have a structure conformationally similar to the hepatitis C virus (HCV) eE2 polypeptide.
The invention is also directed to a model or crystal of an E2 core comprising a structure of an E2 core domain of HCV J6 genotype, the E2 core domain comprising: (i) a four-stranded antiparallel N-terminal β-sheet comprising an N-terminal loop, a first strand, a second strand, a third strand, a fourth strand, and a first loop between the second and third strands, the four strands of the N-terminal β-sheet stabilized by two disulfide bonds between the first strand and the third strand (C7 (510) and C8 (554)) and the N-terminal loop with the fourth strand (C5 (496) and C9 (566)), the first loop between the second and the third strands is flexible and comprises amino acid sequences implicated in CD81 binding; (ii) a four-stranded antiparallel β-sheet comprising a fifth strand, a sixth strand, a seventh strand and an eighth strand and an α-helix (H2) loop between the sixth strand and the seventh strand, the four-stranded antiparallel β-sheet B stabilized by a disulfide bond (C14 (611) and C16 (648)) between the sixth strand and the eighth strand, the seventh and the eighth stands comprising approximately nine amino acids each and together encompassing an epitope of 2A12 antibody; and (iii) a second loop after the fourth strand, the second loop followed by a first short helix loop (H1); wherein the first short helix is followed by the four-stranded β-sheet B; a plane of the four-stranded antiparallel N-terminal β-sheet and a plane of the four-stranded antiparallel β-sheet are approximately perpendicular to each other; and the two β-sheets held together by two disulfide bonds, connecting the N-terminal loop with the α-helix loop (H2) (C4 (488) with C15 (624)) and the second loop with the first short helix (H1) (C10 (571) and C13 (601)). In some of the preferred embodiments, the model is used to identify polypeptides, which are conformationally similar to the structure of the E2 core domain of HCV J6 genotype, or a different known genotype of HCV. The identified polypeptides may then be tested as potential inhibitors of HCV cell entry.
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Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide101.
Since its identification in 1989, phylogenetic analysis of various isolates has resulted in the classification of seven distinct genotypes that are further divided into a number of subtypes (e.g. 1a, 1b, 1c, etc.). The HCV virion consists of an enveloped nucleocapsid containing the viral genome, a single-stranded, positive sense RNA that encodes a single, open-reading frame (
HCV belongs to the genus Hepacivirus of the Flaviviridae family. Other members of the family include the flavivirus and pestivirus genera, which consist of arthropod-borne viruses and important livestock pathogens, respectively116.
Once the virus penetrates a permissive cell, the HCV genome is released into the cytosol where the viral RNA is translated in a cap-independent manner by an internal ribosome entry site (IRES) located within the 5′ nontranslated region (NTR). Translation generates a viral polyprotein that is proteolytically processed by cellular and viral encoded proteases into ten proteins (
HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. No vaccine is available and current therapies are effective against some, but not all, genotypes.
HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies102-4.
Described below, e.g., is the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4A° resolution.
HCV Envelope Glycoprotein 2 (E2)
The flavivirus envelope glycoprotein (E) is a class II fusion protein and HCV E2 was expected to have a similar fold113,117,118. All class II fusion proteins have a common elongated structure, consisting of predominantly β-sheets, and exist as homo- or heterodimers with the membrane-fusion, hydrophobic peptide buried at the dimer interface at neutral pH. On receptor binding and/or exposure to low pH, these proteins undergo self-rearrangement into stable trimers, exposing the fusion peptide and resulting in viral and host membrane fusion. Despite containing a similar extended organization, the recent structure of the pestivirus bovine viral diarrhoea virus (BVDV) E2 glycoprotein does not represent a typical class II fusion protein fold and lacks an apparent fusion peptide, indicating that it is unlikely to be a class II fusion protein119,120.
HCV envelope glycoprotein 2 (E2) is a type I transmembrane protein with an amino-terminal ectodomain connected to a carboxy-terminal transmembrane helix through an amphipathic, α-helical stem (
E2 is highly modified post-translationally with 9-11 N-linked glycosylation sites and 18 cysteine residues that are conserved across all genotypes. For ease of comparison with other genotypes, we refer to the cysteines and N-linked glycosylation sites as C1 to C18 and N1 to N11, respectively, with residue numbers from the J6 genotype (2a) given in parentheses.
HCV E2 is modified by N-linked glycosylation, which is necessary for proper folding and immune invasion.
E2 from the J6 genotype has 11 glycosylation sites.
In certain embodiment, four of the glycosylation sites are in the flexible N-terminal region, which were deleted, and seven are in the core domain (N5-N11). The location of N7, N8, N10 and N11 are modelled in the final E2 core structure. All of these glycans are present in loop areas, indicating that these sites are solvent exposed and flexible. Mutagenesis studies in HCVcc have shown that N6, N8 and N10 are integral for virus infectivity. Removal of the N6 site results in improved CD81 binding, whereas N8 and N10 mutations destabilize the protein and cause defective particle production122. Both sheets have one critical glycosylation site: N8 in sheet A and N10 in sheet B. All four of the observed glycosylation sites are on the periphery of the core and are located on a highly basic surface (
Full-length, E2 ectodomain (eE2) (384-656) was produced in N-acetylglucosaminyltransferase I-negative (GnTI2)HEK293T cells by a lentiviral expression system and grown in an adherent cell bioreactor. The resulting eE2 protein is monomeric as determined by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (
Solution-based studies using limited proteolysis and hydrogen deuterium exchange demonstrated that approximately 80 amino acids on the N terminus (384-463) from hypervariable region (HVR) 1 through to HVR2 are exposed and flexible. This region includes conserved sequences implicated in binding to the cellular receptors (SR-BI and CD81) as well as several epitopes for neutralizing antibodies (
HCV E2 Core
Similar to the flavivirus and pestivirus glycoproteins, the HCV E2 core secondary structure consists of predominantly β-sheets and random coil. However, E2 core is a monomer with a compact globular shape, in contrast to the extended structures reported in other viruses.
The E2 core has a compact, globular domain structure, consisting mostly of β-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulfide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold.
Monoclonal antibodies were generated against recombinant eE2 and crystals of deglycosylated E2 core were produced in complex with a Fab (2A12) and diffracted to 2.4A° resolution (
E2 core (456-656) is soluble, monomeric and maintains similar secondary structure content when compared with eE2 as determined by reactivity towards HCV infected patient sera (
The ab initio SAXS envelopes of E2 core and eE2 are similar, with approximately the same radius of gyration (Rg) (
Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and assists, e.g., with developing an HCV vaccine and new HCV inhibitors.
Structural comparison of the HCV E2 core domain with all known folds in the Protein Data Bank using the Dali server 25 identified proteins with IgG-like folds similar to the N-terminal sheet A, none of which is a class II fusion protein, although IgG-like folds are common in these proteins. The server failed to identify any statistically significant structures to sheet B, suggesting a novel fold.
Subsequent to the present invention, a structure for HCV E2 from genotype 1a was reported126. The core domain of reported structure and the structure described herein is highly similar with a root mean squared deviation of 0.8 Å for similar carbon atoms.
Biochemical and structural data disclosed herein provide valuable information towards defining the role of E2 and establish a foundation for further studies in understanding HCV entry and infection.
CD81 Binding Region
SAXS was used to investigate the CD81 binding region on the E2 ectodomain. To simplify data interpretation, eE2 (DHVR1) was used, as HCV lacking HVR1 remains infectious121. The binding site of CD81 was identified by superimposing the SAXS envelopes of eE2(DHVR1) alone and in complex with CD81-LEL (
Construct Design, Expression and Purification of HCV eE2
HCV E2 is a type I transmembrane protein with an amino-terminal ectodomain and a carboxy-terminal membrane-associating region. The protein is composed of 333 residues. E2 is glycosylated and contains intramolecular disulfide bonds, making it extremely challenging for structural, biochemical, and biophysical studies. However, we use E2 ectodomain (eE2) (amino acids 384-660 of the HCV polyprotein genotype 1a starting from position 1 in the HCV core and/or amino acids 384-664 of the HCV polyprotein genotype 2a) that is lacking the C-terminal membrane anchor, since this has been demonstrated to express well11 and retain interactions with CD81 and SR-BI.
Biochemical, biophysical, and structural studies rely on the production of purified eE2. Studies on recombinant E1 and E2 expression have yielded two different forms of the molecules: a glycosylated protein with intramolecular disulfide bonds that is believed to be the active form and high molecular weight aggregates caused by intermolecular disulfide bonds. The formation of disulfide bonded aggregates and misfolded protein has hampered structural and biophysical studies on the HCV glycoproteins. Our approach was to explore three different variables (cell type, genotype and affinity tag) to determine which construct produced high levels of properly folded eE2.
Cell Type
The cell lines used for expression can have a pronounced effect on stability. Expression of viral glycoproteins in bacteria has proven to be a challenge and several reports document that expression of eE2 in E. coli leads to the formation of insoluble, inclusion bodies. This is not surprising given the large number of potential glycosylation sites and intramolecular disulfide bonds. Proteins with these posttranslational modifications are known to be difficult to express in bacteria. The unique properties of the mammalian cellular environment make this the ideal choice for homologous expression of eE2. HEK293T and Huh7 cells were the first cell lines tested, since these have been shown to produce active E1 and E2 in the form of HCVpp and HCVcc. Initially, HEK293T were chosen over Huh7 owing to ease of handling, robust growth rate, excellent transfectability, and high capacity for recombinant protein expression.
Genotype
Given that E2 forms the virus particle, the surface of the glycoproteins would be expected to undergo mutation to escape immune pressure; therefore the behavior of E2 from different genotypes might be markedly different. In fact, it has been shown that preparations of E2 from different genotypes can vary in binding to CD81 and the aggregation state of the glycoproteins. This approach of expressing the same protein from different genotypes was essential to defining constructs that would yield the highest amount of folded protein for functional studies. In fact the same protein from different genotypes behaved differently in terms of amount expressed, solubility and protein stability.
Affinity Tag
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or intein splicing.
Tags are attached to proteins for various purposes. For example, affinity tags may be appended to proteins so that they can be purified from their crude biological source using an affinity technique.
The methods of the invention may or may not include an affinity tag. The affinity tag may, e.g., comprise Fc domain of human IgG or a protein-A.
In certain embodiments, protein-A is used as an affinity tag. Protein A is a surface protein of S. aureus which binds IgG molecules through the Fc region.
In certain embodiments, the addition of an affinity tag can permit rapid purification and added protein stability. In previous work relating to the expression and purification of eE2, a construct consisting of the E1 signal sequence followed by the ectodomain of E2 and a C-terminal, six histidine tag was made and expressed in HEK293T cells. The signal sequence would target eE2 to the ER where glycosylation and disulfide formation occur naturally and permit the protein to be secreted into the media. Expression levels were very low and supernatants stripped the Ni from the resin, resulting in poor retention of the eE2. Buffer exchange of the media inhibited the Ni stripping while promoting eE2 binding, however this approach would not be applicable for large-scale expression. Therefore, a better tag was needed which had high affinity and specificity to a resin, and could be used in the presence of media.
We sought to use the Fc domain of human IgG as an affinity tag. The Fc domain is glycosylated, contains an intermolecular disulfide bond, is very soluble, binds with high affinity to protein A resin, and can bind to the resin in the presence of tissue culture media. Our hypothesis was that since the Fc domain is glycosylated and disulfide bonded it might assist similar modifications found on eE2 and only a properly folded Fc domain is competent for secretion and protein A binding. These unique properties of the Fc domain seemed ideal for this situation.
The HCV eE2 was cloned as a fusion with the Fc domain into a modified pcDNA3.1 vector for constitutive expression in mammalian cell lines. A schematic representation of the expression construct is shown in
Encouraged by our preliminary expression results, we attempted to produce a stable cell line that constitutively secreted eE2. HEK293T cells were transfected with eE2 constructs from both genotypes and were placed under hygromycin B selection. We developed a quantitative enzyme-linked immunosorbent assay (ELISA) against the Fc domain to quickly identify and quantitate which drug resistant cells were expressing the most eE2. We have isolated HEK293T cells that constitutively express eE2 from J6 and H77 at levels comparable to transient transfection.
With the creation of a stable cell line expressing eE2, we set out to determine purification conditions. The stable eE2 cell lines were expanded into 10 roller bottles with media containing fetal bovine serum to assist in cell attachment and growth. Once the cells become confluent, the supernatant is harvested, replaced with media without serum, and left for another two days. The media from both harvests is pooled together (approximately 1 L total volume) and incubated in the presence of protein A resin with agitation overnight in a cold room. The next morning the resin is harvested and washed extensively with buffer. The fusion protein can be eluted off the resin by either the addition of thrombin to cleave between eE2 and the Fc or by lowering the pH of elution buffer to disrupt the Fc/protein-A interaction. Since HCV and other viruses undergo a low pH triggered membrane fusion, eluting eE2 by low pH may cause a structural rearrangement in the glycoproteins. The resin can be washed with buffer to collect eE2, leaving the contaminants bound to the resin. Samples (5 μL) of each step of the purification are analyzed by SDS-PAGE and stained with Coomassie blue protein dye (
Properties of J6 eE2 Glycosylation
The addition and modification of glycans onto proteins is one of the major biosynthetic pathways found in the lumen of the ER. During translation of a glycoprotein, an oligosaccharide composed of two N-acetylglucosamine, nine mannose, and three glucose molecules are transferred en bloc to a sequon of Asn-X-Ser/Thr (where X is any amino acid except Pro). Oligosaccharyl transferase (also known as N-acetylglucosaminyltransferase), is an ER membrane bound enzyme that characterizes the transfer to the NH2 group of the Asn side chain. However, not every sequon is modified and the efficiency of transfer can vary for the same sequon, leading to a mixture of modified and unmodified protein. Once the oligosaccharide is on the protein, it can be trimmed and further glycans added as the protein travels through the ER and Golgi apparatus. The processing pathway is highly ordered and begins in the ER with the removal of all the glucose and certain mannose molecules. The remaining steps occur in the Golgi apparatus, where three more mannose molecules are removed and various sugars are added. Although the steps of processing and subsequent sugar addition are rigidly ordered, complex oligosaccharides can be heterogeneous. The end result is two broad classes of N-linked oligosaccharides, referred to as complex and high mannose oligosaccharides. Whether a given oligosaccharide remains high-mannose or is processed is largely determined by its configuration on the protein and if the site is accessible to the modifying enzymes. High mannose and complex oligosaccharides can be differentiated by endoglycosidase H (Endo H) sensitivity, since Endo H will only cleave high mannose glycans. Peptide-N-glycosidase F (PNGase F) will remove all types of N-linked glycosylation. eE2 appears as a smeary band by reducing SDS-PAGE (
Disulfide Bond Formation and Aggregation
As mentioned previously, expression of HCV eE2 has resulted in the formation of high molecular weight aggregates caused by the presence of intermolecular disulfide bonds that are considered to be misfolded.
The following examples are meant to illustrate, but not limit the scope of the invention.
HEK293T cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS (DMEM10). A 6-well plate was seeded with 0.5×106 cells per well and the pPro-eE2-Fc vector (from J6 strain) was transfected the following day using FuGene-HD (Roche Diagnostics, Basel, Switzerland). After three days, the cells were placed under hygromycin (Calbiochem, San Diego, Calif.) selection at 75 μg/ml. Individual colonies were selected, expanded, and tested for eE2-Fc expression using an anti-Fc ELISA.
MaxiSorp plates (Nunc, Thermo Fisher Scientific, Rochester, N.Y.) were coated with 100 μL of supernatant for two hours at room temperature. The wells were washed 3 times with 200 μL of PBS+0.05% Tween-20 (PBS-T), then blocked with 200 μL of 2% BSA in PBS-T for one hour at room temperature. After three more washes with PBS-T, 100 μL of goat anti-Fc antibody (Pierce, Thermo Fisher Scientific, Rochester, N.Y.) at 1:15,000 dilution (in PBS-T) was incubated for one hour at room temperature. The ELISA was developed with TMB substrate (Pierce) and quantified using the SpectraMAX 250 plate reader and SOFTMax 2.6 software.
The supernatant from stable cell lines of Example 1 was harvested, centrifuged to remove cellular debris, and filtered through a 0.22 μm membrane. The eE2-Fc protein was applied to protein A-conjugated resin (GE Healthcare, Piscataway, N.J.) overnight with gentle rocking. The resin was pooled together the next day, washed with buffer (50 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol), and incubated with thrombin protease (GE Healthcare, Piscataway, N.J.) to release the protein from the Fc tag. After cleavage, the protein eluate was consolidated and the concentration determined by Bio-Rad Protein Assay. The protein was analyzed by SDS-PAGE and Coomassie staining Yields are found to be about 2 mg of eE2 per liter of supernatant.
eE2 was deglycosylated using either Endo H or PNGase F according to manufacturer's protocol (New England Biolabs, Ipswich, Mass.). 20 μg of eE2 was denatured and 10 Units of Endo H or PNGase F was added. The reaction was incubated at 37° C. for one hour and analyzed by SDS-PAGE followed by Coomassie staining.
The eE2 protein sample was denatured in 6M urea, then reduced with 10 mM DTT for 30 min at 60° C. After denaturation, 20 mM iodoacetamide was added to alkylate sulfhydryl groups and incubated in the dark for one hour at room temperature. Following this treatment, the sample was buffer-exchanged into 50 mM NH4HCO3. The sample was digested using either sequencing grade trypsin (Promega, Madison, Wis.) or chymotrypsin (Roche Diagnostics) according to manufacturers' protocol. Digested samples were dried via speed vacuum and reconstituted in 50 mM NH4HCO3. Deglycosylation with 50 U of PNGase F (New England Biolabs, MA) was incubated for 1 hour at 37° C. and the reaction was stopped with 0.1% trifluoroacetic acid (TFA).
All LC-MSMS experiments were performed using the U3000 (Dionex, Sunnyvale, Calif.) in nano-LC mode on line with LTQ (Thermo Fisher Scientific). Samples were first solubilized in 0.1% TFA and loaded onto a 75 μm×12 cm emitter column self-packed with Magic C18AQ, 3 μm 200 Å. (Michrom Bioresources Inc, Auburn, Calif.). The sample was eluted using a linear gradient from 98% of 0.1% formic acid in water to 45% of 0.1% formic acid in acetonitrile over 30 min. Mass spectrometry data was acquired using a data-dependent acquisition procedure with a full scan cyclic series. This was followed by zoom scans and MSMS scans of the five most intense ions with a repeat count of two and a dynamic exclusion duration of 60 sec.
The LC-MSMS data was searched against a human database using a local version of the Global Proteome Machine (GPM USB, Beavis Informatics Ltd, Winnipeg, Canada). Carbamidomethylation of cysteine was used as the fixed modification, while oxidation of methionine and deamination of asparagine were used as potential modifications. Manual interpretation and peak integration was performed on all peptide peaks covering potential glycosylation sites (NXT/S).
Purified eE2 protein was loaded onto a Superdex 200 10/300 size exclusion column (GE Healthcare) equilibrated with either HEPES buffer (50 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol) or phosphate buffer (25 mM sodium phosphate pH 5.0 or 7.0, 50 mM KCl).
To label free cysteines, the protein sample was incubated with a 20-fold molar excess of N-ethylmaleimide (NEM) and 6M guanidine-HCl at room temperature for one hour in the dark. The sample was then buffer exchanged to 6M guanidine-HCl using a spin filter and washed three times with 400 μl of 6M guanidine-HCl to remove the NEM. Disulfide bonds were reduced by adding 10 mM DTT at 60° C. for 30 min. The newly generated free sulfhydryl groups were alkylated with 20 mM iodoacetamide (JAM) at room temperature for one hour in the dark. After buffer exchange into 50 mM NH4HCO3, the samples were digested with trypsin protease at 37° C. overnight. The samples were then deglycosylated with PNGaseF (100 U) at 37° C. for three hours and acidified prior to LC-MSMS analysis.
The LC-MSMS data was searched using Sequest against an E. coli genome database (a common contaminant of in-gel digest) and added sequences of the target protein. +57 Da (alkylation by IAM) and +125 Da (alkylation by NEM) on cysteine, oxidation of methionine (+16 Da), and deamination of asparagine (+1 Da) were used as potential modifications. The identification was confirmed manually.
All sedimentation experiments were performed with a Beckman Optima XL-I at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies at the University of Texas Health Science Center at San Antonio. Sedimentation velocity data were analyzed with UltraScan version 9.9. All measurements were made at 230 nm in intensity mode, at 20° C., and at 37 krpm, using standard upon 2-channel centerpieces. All samples were measured in 25 mM sodium phosphate buffer containing 50 mM KCl, adjusted to either pH 5.0 or 7.0. Concentration dependency of the sedimentation data was assessed by sedimenting the sample at both high concentration (˜0.8 optical density (OD) at 230 nm) and at low concentration (˜0.25 OD at 230 nm). Hydrodynamic corrections for buffer density and viscosity were made according to methods outlined in Laue et al. and as implemented in UltraScan. The data were analyzed by 2-dimensional spectrum analysis (2DSA) using the ASTFEM-RA solution with simultaneous removal of time-invariant noise. Molecular weight and shape distributions obtained in the 2DSA were further refined by Monte Carlo analysis. Composition comparisons were made by overlaying sedimentation coefficient distributions derived from the van Holde-Weischet analysis. The calculations were performed on the Lonestar cluster at the Texas Advanced Computing Center at the University of Texas at Austin, and at the Bioinformatics Core Facility at the University of Texas Health Science Center at San Antonio.
The protein sample was buffer-exchanged into 25 mM sodium phosphate pH 5.0 or 7.0, and 50 mM KCl. The CD spectra in the wavelength range of 195-260 nm were measured at 0.5 nm intervals on an Aviv spectropolarimeter model 400 (Lakewood, N.J.) at 25° C. A quartz cell with a path length of 0.1 cm was used. The CD spectra were analyzed for secondary structure using multilinear regression as described previously.
96-well EIA/RIA plates (Corning, Lowell, Mass.) were coated with 100 μl of a 1 μg/ml solution of eE2 in NaHCO3 overnight at 4° C. The plates were washed twice with 200 μl/well PBS-T, then blocked with a 10% solution of normal goat serum in PBS-T (Jackson ImmunoResearch, West Grove, Pa.) for one hour at 37° C. Human serum was isolated from whole blood samples (Emory University School of Medicine, PI Arash Grakoui, IRB#1358-2004) collected in SST tubes (Becton Dickenson, Franklin Lakes, N.J.) via centrifugation and frozen in aliquots at −80° C. Ten-fold serial dilutions were made for each serum sample using binding buffer composed of 0.1% normal goat serum in PBS-T. 100 μl of the dilutions was added to each well of the plates and incubated for 90 minutes at room temperature. The plates were washed eight times with PBS. 100 μl of goat anti-human IgG-Biotin conjugate (Biosource, Camarillo, Calif.) diluted 1:20,000 in binding buffer was added and allowed to incubate for 90 minutes at room temperature. Finally, 100 μl streptavidin-HRP conjugate (Biosource) was added to each well at a 1:2,000 dilution and incubated for 45 minutes at room temperature. Using TMB substrate solution (Ebioscience, San Diego, Calif.), absorbance was measured using a VersaMax Microplate reader and SoftMax Pro software (Molecular Devices, Sunnyvale, Calif.).
Approximately 100 TCID50 of Cp7 viruses were incubated with two-fold dilutions of the purified eE2, eE2-C656S, GST, GST-CD81LEL or GST-mCD81 starting at 200 μg/ml. 6.0×103 cells were seeded into a collagen-coated 96-well plate. The virus-protein mixture was incubated with the cells for three days at 37° C. Cells were stained by immmunohistochemistry as previously described.
Huh-7.5 cells were incubated with various dilutions of the purified proteins as described above. Three days later, cells were washed twice with PBS, harvested by trypsinization, and resuspended in 100 μl of PBS. Cells were stained with BD Via-Probe™ (BD Biosciences, San Jose, Calif.) according to the manufacturer's instructions and counted using FACSCalibur (BD Biosciences) equipment and FlowJo (v8) analysis software.
CD81-LEL was expressed with an amino-terminal GST tag and carboxy-terminal histidine tag. The protein was expressed and purified as described previously. The GST tag alone was expressed and purified using the same method.
Expression of eE2 in E. coli, yeast, insect cells, CHO cells, and various other eukaryotic and viral recombinant systems, has consistently resulted in the formation of insoluble, misfolded and aggregrated protein. We sought to develop a system for the expression of HCV eE2 that would yield large amounts of highly purified, active protein for functional studies. Our approach was to utilize cell lines that have been shown to produce functional E2, while adding an affinity tag to increase eE2 stability and facilitate purification. HEK293T cells were chosen owing to a) their ability to produce functional E2 in the form of HCV pseudoparticles (HCVpp), b) their ease of handling and robust growth rate and, c) their excellent transfectability and high capacity for recombinant protein expression. We expressed the J6 (genotype 2a) E2 ectodomain (aa384-664) because this fragment of E2 has been shown to be the minimal functional unit for binding and entry (
Glycosylation of viral envelope proteins is critical for folding, structural integrity, and immune evasion. The number and conservation of glycosylation sites varies across different HCV genotypes. J6-E2 contains 11 potential N-linked glycosylation sites (N-X-T/S) along with three potential O-linkage consensus sites (
To investigate the glycosylation pattern in further detail, we employed high-resolution mass spectrometry. eE2 was digested with either trypsin or chymotrypsin and samples of the protein fragments were deglycosylated with either PNGase F or Endo H. PNGase F deaminates the asparagine residue to which the N-linked glycan is attached and converts it to aspartic acid. If the glycan is of the high mannose form, it will be sensitive to Endo H, which leaves one N-acetylglucosamine (GlcNAc) bound to the Asn. Thus, the gain of 1 Da (Asn to Asp; nitrogen to oxygen) by PNGase F or 203 Da by the GlcNAc residue left by Endo H cleavage can be resolved by MS of the peptides. From the resulting three spectra (untreated, Endo H and PNGase F treated) we are able to map all the glycosylation sites, estimate the approximate usage of each site, and determine whether the glycan at a particular site was complex or high mannose. We achieved 100% coverage of the eE2 sequence using trypsin and chymotrypsin. Peptides from the 11 predicted N-linked glycosylation sites were shown to be fully glycosylated, since we were unable to detect unglycosylated peptides with Asn residues in them (
Since previous reports have shown that E2 tends to aggregate, we set out to define the oligomeric state of eE2, using nonreducing SDS-PAGE, size exclusion chromatography and analytical ultracentrifugation. SDS-PAGE analysis of eE2 under non-reducing conditions demonstrated that the eE2 consisted of a mixture of two components with approximate molecular weights of ˜120 kDa (dimer) and ˜60 kDa (monomer) without any large, disulfide-linked aggregates (
Since, there are 18 conserved cysteines in E2, this could result in the formation of nine disulfide bonds. The eE2 fragment contains only 17 cysteines, leaving at least one unpaired. We therefore sought to determine which cysteine residue was responsible for the intermolecular disulfide bond demonstrated in
Consequently, we generated a mutant form of eE2 in which C656 was mutated to a serine (eE2-C656S). Serine was chosen to conserve the biochemical properties at this position. We generated a HEK293T cell line that stably expresses eE2-C656S-Fc and the protein was purified as before. eE2-C656S was analyzed by size exclusion chromatography under conditions identical to the eE2 wild type experiment and the results demonstrated that the mutant was predominantly monomeric at pH 7.5 (
Entry of HCV and other flaviviruses is a pH-triggered event. The single envelope protein (E) of flaviviruses undergoes a slight conformation shift upon incubation at low pH, resulting in a change in the oliogomeric state from dimer at neutral pH to trimer at low pH. Therefore we were interested in determining if HCV eE2 or eE2-C656S undergoes any structural or oligomeric changes when the pH is lowered. Gel filtration results for eE2 at pH 5 revealed a slight increase in the amount of monomer relative to pH 7 (
Sedimentation velocity experiments by analytical ultracentrifugation can be used to determine frictional properties (mass and shape) of proteins without the use of standards or interactions with sieving matrix. Sedimentation of eE2 at pH 7 indicates the presence of two dominant species, a monomeric form (60-70 kDa) and a dimeric form (80-130 kDa) (
The HCV glycoproteins are predicted to be class II fusion proteins, due to their similarity to alphaviruses and flaviviruses. These proteins are composed of mostly β sheet structure and do not undergo major structural rearrangements upon exposure to low pH. We investigated this possibility by measuring circular dichroism of eE2 and eE2-C656S at pH 7 and pH 5. The resulting spectra revealed that both eE2 and eE2-C656S contain predominantly β-sheet structure and random coil with a small amount of α-helix (
J6 eE2 is recognized by antibodies from patients chronically infected with different genotypes of HCV. The presence of high levels of anti-E2 antibodies in HCV-infected human serum has been reported. In order to further examine if purified eE2 is conformationally similar to the E2 present on infectious HCV particles, we tested whether eE2 was recognized by antibodies from infected patient sera. An enzyme-linked immunosorbent assay (ELISA) plate was coated with eE2 and probed with serum from patients chronically infected with either genotype 1, 2 or 3. Serum from a healthy donor was tested in parallel as a negative control. Anti-human HRP was used to quantify the result. The serum of infected patients bound to eE2 at similar titers regardless of genotype, while the serum of the uninfected donor responded at background levels (
The HCV eE2 blocks HCVcc entry. In order to confirm correct folding and function of HCV eE2, we performed a similar assay using cell culture derived HCV (HCVcc). ˜100 TCID50 of HCVcc was incubated with serial 2-fold dilutions of purified eE2, eE2-C656S, GST-human-CD81LEL (hCD81), GST-mouse-CD81LEL (mCD81) or GST. eE2, eE2-C656S, and hCD81 reduced the number of focus forming units (FFU) in a concentration dependent manner, while mCD81 and GST protein had no effect. This experiment yielded a 50% blocking efficiency in the range of 25-150 μg/ml for eE2 and eE2-C656S (
Inhibition of HCVcc entry by eE2 is thought to occur by sequestering cellular receptors. To confirm this, we analyzed the ability of eE2 to bind hCD81 in vitro. We adapted an ELISA first described by Flint et al. for the detection of properly folded E2 based on hCD81 binding. We have obtained identical results using wild type and mutant eE2-Fc supernatants (
HCV E1 and E2 are primary determinants of entry and pathogenicity. Deletion mutagenesis has defined the ectodomain of HCV E2 to comprise amino acids 384-664 of E2. Functional and biophysical studies of HCV E2 have been hindered by the formation of mostly aggregated, misfolded material. The eE2 protein produced as described here maintains many of the functionalities associated with E2 found on virions. eE2 can compete with HCVcc to inhibit infection, is recognized by antibodies from chronically infected patients, and can specifically bind the large extracellular loop of human CD81.
All eleven of the predicted N-linked glycosylation sites in E2 are utilized with high efficiency. This is consistent with previous data using HCVpp. Ten of the eleven sites have complex glycans attached, while only the most C-terminal is of the high mannose form. This observation suggests that the glycan at this position is concealed from the modification enzymes in the secretory pathway. This may occur as a result of the dimeric interface formed between two eE2-Fc molecules, or because of steric effects of the Fc tag attached to the C-terminus. As reported by Goffard et al, mutation of the C-terminal glycosylation site does not affect folding, secretion, or E1/E2 heterodimer formation, but does result in less than 50% infectivity when incorporated into HCVpp. Peptides containing the putative O-linked glycosylation sites (
The HCV glycoproteins, like those from related alphaviruses and flaviviruses, are predicted to be class II fusion proteins. This class of proteins is characterized as mostly beta sheet structures that do not undergo changes in secondary structure upon exposure to low pH. The flavivirus E protein (comprised of three domains) is responsible for receptor binding and membrane fusion. Flaviviruses have icosahedral symmetry, with E arranged as 90 dimers. Cryoelectron microscopy has demonstrated that the E protein lies flat on the surface of the viral lipid bilayer. Upon exposure to low pH there is a slight rotation of domain III, resulting in dissociation of the E dimers and rearrangement into trimers via a monomeric intermediate stage. eE2 appears mostly as a monomer and dimer with a much lower amount of trimer at pH7. Upon exposure to low pH there is an increase in the amount of monomer relative to dimer and trimer. This result represents the first observation of a shift in oligomeric arrangement of the HCV glycoproteins. However, the oligomeric state of E2 may be influenced by the deleted C-terminal portion, the high concentration found on virus particles, and/or heterodimerization with E1. CD spectroscopic analysis of HCV eE2 demonstrated a pronounced minimum at about 203 nm, consistent with a mostly n-sheet protein. Exposure to low pH does not result in a major rearrangement of secondary structure as determined by CD. However, CD cannot rule out the possibility of structural rearrangements that preserve the overall proportion of β-sheet and random coil. These data support the categorization of HCV E2 as a member of the class II fusion proteins.
HCV E2 has 18 highly conserved cysteine residues, although the eE2 construct as defined by Michalak et al. has the first 17. The 18th is located between the ectodomain and the C-terminal membrane anchor. Differential labeling of free and disulfide-bonded cysteines has demonstrated the presence of eight disulfide bonds and one free cysteine (C656) in eE2. Mutating C656S did not affect inhibition of HCV entry (
Based on the results shown in this study and in accordance with previous studies on the HCV envelope proteins, it is highly possible that E2 may provide an excellent vaccine candidate. Chimpanzees immunized with E1/E2 heterodimeric proteins are protected from infection with low doses of homologous hepatitis C virus. In 1991, Weiner et al. identified hypervariable regions in E2, while it was later shown that deletion of hypervariable region 1 attenuated infection in chimpanzees. Youn et al. did further work in chimpanzees to show that an E2 antibody response correlates with lower viral titres. Most recently, rodents injected with HCV envelope glycoproteins have been shown to produce antibodies that are broadly cross-reactive in their neutralization properties. The production of large quantities of functional and properly folded E2 ectodomain will aid in our understanding of E2 function as well as assist in designing a vaccine and entry inhibitors.
Recombinant, full-length E2 ectodomain (384-656, genotype J6), eE2(DHVR1) (413-656) and E2 core (456-656) were produced in HEK293T GnTI2cells27 by a lentiviral expression system with a C-terminal, protein-A tag. Stable cell lines were grown in an adherent cell bioreactor. Collected supernatants were purified over IgG sepharose and the E2 proteins were eluted by PreScission Protease digestion. The eluted protein was deglycosylated with endoglycosidase H (EndoH) and purified to homogeneity by heparin affinity and size-exclusion chromatography. Monoclonal antibody 2A12 secreted by hybridoma cells maintained in suspension culture was purified over protein G resin. Fab was generated using papain digestion and subtractive protein A chromatography. E2 and 2A12 Fab complex was purified by size-exclusion chromatography and crystals were obtained in 18% (w/v) PEG 3350, 0.5M MgCl2, 0.1M HEPES (pH7.5), 15% dioxane and 2% formamide at 20° C. by the hanging-drop vapour diffusion method. The crystals belong to space group P21212 with cell parameters a=85.96 A°, b=194.57 A° and c=37.92 A°. The structure was determined by molecular replacement to 2.4 A° resolution using a mouse Fab (Protein Data Bank accession 2GSI). The final model has an Rwork and Rfree of 0.217 and 0.269, respectively. SAXS measurements were performed on eE2, E2 core, eE2(DHVR1) and eE2(DHVR1)/CD81-LEL. Data were analysed using BioXTAS RAW28 and applications from the ATSAS29 program suite.
J6 eE2 Expression
eE2, eE2(ΔHVR1) and E2 core domain encompasses residues 384-656, 413-656 and 456-656 from the HCV J6 genome, respectively. Owing to incomplete deglycosylation at N7 (542) with Endo H, the crystallization construct contained an asparagine to glutamine mutation at this position. The expression constructs consisted of a CMV promoter, a prolactin signal sequence, E2 fragment, PreScission Protease cleavage site and a C-terminal protein-A (ProtA) tag. The entire prolactin-E2-ProtA sequence was PCR amplified and cloned into the pJG lentiviral vector (J. Shires).
Wild-type and GnTI-HEK293T cells127 (provided by D. Comoletti) were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37° C. with 5% CO2. One day before the planned transfection, a single T-225 monolayer flask was seeded with 6.0×106 HEK293T cells. 90 μg pJG-E2, 60 μg psPAX2 (HIV Gag-Pol packaging vector), 30 μg pMD2.G (VSV glycoprotein vector) and 450 μl of 2 M CaCl2 were mixed and brought to a final volume of 4.5 ml with ddH2O. 4.5 ml of 2×HEPES buffered saline was added at room temperature. After a 2-min incubation, the mixture was added directly to HEK293T cells. After 6-8 h, the media was replaced with DMEM with 10% FBS and 1% antibiotic/antimycotic (A/A) media and incubated for another 70 h.
Two days after transfection, 10,000 GnTI-HEK293T cells were seeded into a single well of a 96-well plate. The supernatant from the transfection, containing the recombinant lentiviruses, was collected and centrifuged for 30 min at 4,000 g at 4° C. to pellet large cellular debris. Clarified supernatant was transferred to a Beckman Ultracentrifuge tube and virus was pelleted for 1.5 h at 25,000 r.p.m. (80,000 g) at 4° C. in an SW28 rotor. Supernatant was discarded and the pellet re-suspended in 120 μl of DMEM containing 20% FBS, 1% A/A, and 8 μg ml−1 of polybrene. 60 μl−1 of virus suspension was added to the prepared GnTI-HEK293T cells and incubated overnight. Infected cells were expanded and ultimately seeded into an adherent cell bioreactor (Cesco Bioengineering) for long-term growth and protein production.
eE2, eE2(ΔHVR1) and E2 Core Purification
Cell supernatant containing E2-ProtA was centrifuged for 10 min at 7,000 g, filtered through a 0.22-μm membrane, and loaded onto an IgG FF column (GE Healthcare). The column was extensively washed with 20 mM sodium phosphate pH 7.0 and then equilibrated with 20 mM HEPES pH 7.5, 250 mM NaCl and 5% glycerol. PreScission Protease was added into the column at approximately 400 μg 1−1 of supernatant and incubated overnight at 4° C. For deglycosylation, the pH of the protein solution was adjusted using 1 M sodium citrate pH 5.5 to a final concentration of 100 mM. Endo H was added at a ratio of 1 mg per 2 mg of E2 and the reaction was incubated at room temperature for 3-4 h. The deglycosylated proteins were desalted into 20 mM HEPES pH 7.5, 50 mM NaCl, and 5% glycerol and purified by heparin affinity followed by size-exclusion chromatography over a Superdex 200 column. Final yields for all E2 proteins averaged 30 mg 1−1 of supernatant.
Crystallization
A 1.1:1 molar ratio of E2 core domain to Fab was incubated for 1-2 h at 4° C. The complex was purified over a Superdex200 column equilibrated with 20 mM HEPES pH 7.5 and 100 mM NaCl. The complex was concentrated to 5-7 mg ml−1 and crystals were grown by the hanging-drop vapor diffusion method. Briefly, 2.5 μl of complex was mixed with an equal volume of reservoir solution, comprising of 22% (w/v) PEG 3350, 0.5 M MgCl2, 0.1 M HEPES pH 7.5, and 15% (v/v) dioxane. Initially, clusters of plate-like crystals grew in 3-4 days. Single, plate-like crystals were obtained via microseeding using a similar reservoir solution supplemented with 2% (v/v) formamide. Crystals were cryoprotected using reservoir solution with 24% (v/v) ethylene glycol and flash cooled in liquid nitrogen. Data were collected at a wavelength of 0.979 A using beamline X25 of the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory.
Structure Determination and Refinement
The crystals belong to space group P21212 with cell parameters a=85.96 Å, b=194.57 Å, c=37.92 Å. Phases were determined by the molecular replacement method using PHENIX130 and the coordinates from chains A and B from PDB entry 2GSI. Unambiguous placement of the Fab heavy and light chains provided the necessary phases to extend the map to cover E2 core domain using iterative rounds of model building and density modification by COOT131, PHENIX, REFMAC132 and PARROT133. The final model was built to a resolution of 2.40 A, comprising residues 492-522, 538-571 and 596-649 of E2 from the J6 genome, 1-217 of 2A12 light chain, and 1-133 and 136-218 of 2A12 heavy chain with two N-linked, N-acetylglucosamine, six molecules of formamide and 141 solvents molecules. The model coordinates were refined to Rwork 0.217 and Rfree 0.269. Model validation demonstrated 95.0% of the residues located in the most favorable region of the Ramachandran plot with 4.8% in the generously allowed regions30. Statistics of the data processing and structure refinement are summarized in
Small Angle X-Ray Scattering (SAXS)
Glycosylated E2 proteins were purified over IgG and anion exchange columns. The proteins were equilibrated with either pH 7.5 buffer (50 mM HEPES pH 7.5, 250 mM NaCl and 1% glycerol) or pH 5.0 buffer (50 mM sodium citrate pH 5.0, 250 mM NaCl and 1% glycerol) by Superdex200 gel filtration column. Glycosylated eE2(AHVR1) alone or complex with CD81-LEL (1:2 molar ratios) was purified using pH 7.5 buffer by gel filtration chromatography. Three concentrations of each protein were prepared along with their respective buffers as background control. SAXS data was collected on the SIBYLS beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory. Sample analysis and processing was performed using BioSAXS RAW128, ATSAS129, and GNOM134. The ab initio models were calculated using the application DAMMIF135. Consensus models and the normalized spatial discrepancy (NSD) values were calculated by averaging 10 ab initio models using the application DAMAVER136. X-ray structures of CD81 (PDB ID 1G8Q), HCV E2 core and ab initio models were aligned using the application SUPCOMB137.
Hydrogen Deuterium Exchange
HD exchange experiments were conducted as described previouslyl38. Briefly, 5 μl of deglycosylated eE2 (1.5 mg ml−1), in 200 mM NaCl, 20 mM HEPES pH 7.5, was incubated with 15 μl of the same buffer made with 99.96% 2H2O (Cambridge Isotope Laboratories) for 10, 100, or 1,000 s and quenched in 30 μl of 2 M urea, 0.8% formic acid and 50 mM tris(2-carboxyethyl)phosphine (TCEP). The reaction mixture was immediately frozen on dry ice until injection. For the zero time point experiment, the protein was incubated in the buffer made with 1H2O and then quenched and frozen. To correct background exchange, a completely deuterated sample was produced by incubating the protein with 100 mM TCEP in 99.96% 2H2O overnight before being quenched and frozen. Dionex RSLC with a C18 column (2.1×50 mm, 3 μm, Q-C18, 150A, CMP Scientific) and LTQ Velos Orbitrap pro were used for LC-MS analysis. The mass was measured using Orbitrap with resolution of 60,000 and mass range from 300 to 2,000 m/Z. The LC-MS data were analysed using HDexaminer 1.2.0 (Serra Analytics) with manual checking of each peptide afterwards.
Limited Proteolysis
8-10 μg of deglycosylated eE2 protein was mixed with trypsin, chymotrypsin or GluC at 1:120 (w/w) ratio (endopeptidase:E2) and incubated at room temperature. Samples were taken at noted time points and analysed by reducing SDS-PAGE, mass spectrometry and N-terminal sequencing.
Production of Monoclonal Antibody 2A12
Six to eight week old female BALB/c mice were immunized intraperitoneally with 50 μg eE2 in either complete Freund's adjuvant (first immunization only), or incomplete Freund's adujvant bi-weekly for 8 weeks. A final immunization with 50 μg of eE2 was given intravenously 4 days before collection of splenocytes. Hybridomas were generated using a cloned HAT-sensitive mouse myeloma cell line as a fusion partner. Proliferating hybridomas were screened for their ability to bind eE2 via ELISA, at which point 2A12 was positively identified. Monoclonal antibodies were generated in the laboratory of A. Grakoui (IACUC protocol number YER-2002369-070816GN).
Generation and Purification of 2A12 Fab
Hybridoma cells were expanded to a final volume of 21 in spinner flasks at 100 r.p.m. using Iscove's Modified Dulbecco's Medium, 10% ultra-low IgG FBS, 1% A/A, and 10 mM HEPES (Life Technologies). Cells were collected at 2-3×106 cells per ml, centrifuged for 10 min at 7,000 g, filtered through a 0.22 μM membrane, and loaded onto a Protein G column (GE Healthcare Life Sciences). After completion, the column was washed with 20 mM sodium phosphate (pH 7.0) followed by phosphate buffered saline (PBS). The antibody was eluted with 0.05% TFA in 2 ml fractions into tubes containing 100 μl of 1 M Tris pH 7.5 for immediate pH neutralization. The eluted antibody was dialysed into 20 mM sodium phosphate pH 7.0 and 10 mM EDTA. Insoluble papain was added to the antibody at 0.15 mg per 1 mg of antibody. Freshly prepared L-cysteine was added to the reaction to a final concentration of 20 mM and mixed at 37° C. for 2 h. The papain was removed by centrifugation at 3,500 g for 2 min and filtration through a 0.22 μM membrane. Fab was purified by subtractive chromatography over Protein A FF column and desalted into 20 mM Tris pH 8.0.
Sequencing Ig H and L Chain Gene Segments of 2A12 Antibody
Total RNA isolated from 2A12 hybridoma cells was reverse transcribed into cDNA using random hexamers. Expressed heavy (H) and light (L) chains were amplified using standard primers that are complimentary to all murine H and L chain gene segments39. The PCR products were sequenced either directly or following cloning into pCR 2.1-TOPO vector (Life Technologies).
CD81 Purification and Binding Assays
Human CD81-LEL (residues 112-202) was produced as a fusion with C-terminal ProtA tag in HEK293T cells using the same lentiviral expression system described for eE2. Cell culture supernatants were loaded onto an IgG FF column, washed with 20 mM sodium phosphate pH 7.0, eluted with 100 mM sodium citrate pH 3.0 containing 20 mM KCl and immediately neutralized with 1 M Tris pH 9.0. The ProtA tag was cleaved by PreScission Protease (GE Healthcare Life Sciences) in a ratio of 1:50 (w/w) followed by overnight dialysis in 20 mM HEPES pH 7.5, 250 mM NaCl, and 5% glycerol. High-purity CD81 protein was obtained by anion exchange and size-exclusion chromatography.
For binding studies, a 96-well plate (Nalgene Nunc, Thermo Fisher Scientific) was coated with 50 μg of CD81-LEL overnight at 4° C. All experiments were duplicated against BSA as a negative control. Plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 3% (w/v) BSA in PBS-T for 1 h at room temperature. 50 μl of eE2 or E2 core at different concentrations was added to appropriate wells and incubated overnight at 4° C. On day 3, the wells were washed three times with PBS-T and incubated with monoclonal antibody 2A12 cell supernatant for 1 h at room temperature. Plates were washed three times with PBS-T and incubated with anti-mouse-HRP conjugated antibody for 1 h at room temperature. Finally, the plate was washed five times with PBS-T. 50 μl of TMB substrate (ThermoFisher Scientific) was added to each well and incubated for 5 min, followed by the addition of 50 μl of 2 M sulphuric acid to stop the reaction. Absorbance readings were acquired at 450 nm using Softmax Pro software on a Spectra Max 250 (Molecular Devices).
Neutralization Assay
Huh-7.5 cells were maintained in DMEM containing 10% FBS (Hyclone) and 100 μg ml−1 of penicillin and streptomycin (Cellgro) at 37° C. in 5% CO2. Naive Huh-7.5 cells were seeded at 6,000 cells per well in a 96-well plate. The following day, 100 μl of 2C1, 2A12, or H113 serially diluted in complete media were added per well at various concentrations. In parallel, 100 μl of eE2, E2 core, gp140, or CD81-LEL serially diluted in complete DMEM were added at varying concentrations beginning at 100 μg ml−1. Cells were then infected with 100 μl of genotype 2a virus Cp7 encoding the Renilla luciferase gene140. 72 h after infection, relative light units were measured on a Clarity 4.0 luminometer (Biotek) using the Renilla Luciferase Assay System (Promega).
Assessment of Cellular Cytotoxicity
Huh-7.5 cells were incubated with varying concentrations of protein as described above, beginning at 100 μg ml−1. After 72 h, cells were washed once with PBS, treated with trypsin, and collected in 100 μl PBS. Cells were stained with 7-AAD according to the manufacturer's instructions (BD Biosciences) and analysed using a BD LSR II and FlowJo software (Tree Star).
Human Plasma ELISA
96-well enzyme immunoassay plates (ThermoFisher Scientific) were coated overnight at 4° C. with 50 μl of a 1 μg ml−1 solution of eE2 or E2 core diluted in 0.1 M Na2CO3. Plates were washed twice with PBS-T and then blocked for 1 h at 37° C. in PBS-T containing 10% fetal calf serum (HyClone). Blood samples were collected in heparin tubes (Becton Dickinson) and plasma was isolated and frozen at −80° C. Plasma was serially diluted in a binding solution composed of 0.1% (v/v) normal goat serum in PBS-T (Jackson ImmunoResearch Laboratories). 100 μl of sample were added per well and incubated at room temperature for 90 min. After eight washes with PBS, 100 μl of mouse anti-human IgG biotin antibody (Mabtech) diluted 1:20,000 in binding solution were added per well and incubated 1 h at room temperature. Following five additional washes with PBS, 100 μl streptavidin-horseradish peroxidase (HRP) conjugate was added to each well at a 1:2,000 dilution in binding buffer and incubated for 45 min at room temperature (Mabtech). Absorbance was measured and analysed using a VersaMax microplate reader and SoftMax Pro software (Molecular Devices) following five washes and the addition of tetramethylbenzidine substrate solution (Ebioscience). Human sera were isolated from whole-blood samples, and informed consent was obtained for all subjects (IRB no. 1358-2004, Emory University School of Medicine, principal investigator A. Grakoui).
Alignment
Secondary structures were assigned using the program DSSP141. Sequences were obtained from the National Center for Biotechnology Information (NCBI) using the following accession numbers: J6 ADV40003.1, H77 ACA53555.1, J8 P26661.3, S52 AEB71616.2, ED43 AEB71617.2, SA13 AEB71618.2, HK6a AEB71619.2, QC69 ACM69041.1. The E2 sequences were aligned with multiple alignment using fast Fourier transform (MAFFT)143 and edited for figure generation using JalView version 2143 (ref 43).
The disclosures of all references cited herein are hereby incorporated by reference in their entireties.
Other modifications and variations of the specific embodiments of the invention as set forth herein will be apparent to those skilled in the art.
This application is a continuation-in-part of U.S. patent application Ser. No. 12/988,864, which is a National Stage Entry of International Application No. PCT/US09/02502, filed on Apr. 22, 2009, which claims priority to U.S. Provisional Patent Application No. 61/046,944, filed on Apr. 22, 2008, the disclosures of which are hereby incorporated by reference in their entireties.
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| Parent | 12988864 | US | |
| Child | 14267012 | US |
