Patent Application
20050272029
The invention relates to hepatitis C virus-like particles, a method for purifying the particles, methods of screening for the presence of hepatitis C virus, methods for screening compounds that interfere with binding and/or internalization of the virus-like particles to/into host cells, cell lines used for screening of the compounds, methods for detecting and identifying cellular receptors for hepatitis C virus and use of the hepatitis C virus-like particles to induce an immune reaction in an animal.
Hepatitis C Virology
Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus belonging to the genus Hepacivirus and family Flaviviridae. HCV is classified into six major genotypes and ˜100 subtypes. The viral genome (˜9.6 kb) is translated into a single polyprotein of ˜3,000 amino acids. A combination of host and viral proteases are involved in polyprotein processing to give at least nine different proteins. This precursor is processed during and after translation to yield the mature structural (core, E1 and E2-p7) and non-structural NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. The structural proteins of HCV are believed to comprise the core protein (˜21 kDa), and two envelope glycoproteins, E1 (˜31 kDa) and E2 (˜70 kDa).
E1 and E2 proteins are thought to play a role in the HCV life cycle, both in the assembly of infectious particles and in the initiation of viral infection by binding to its cellular receptor(s). Expression of recombinant E1 and E2 proteins in mammalian cells has shown that they associate into heterodimers. Both proteins are glycosylated and lack sialic acid at the termini of their carbohydrate domain in mammalian cells and probably in insect cells. Yet, it is not known whether these proteins form heterodimers at the surface of viral particles. In other enveloped viruses, a major role of envelope proteins is to bind to cellular receptor(s) and facilitate virus entry, thereby contributing in determining viral tropism.
E2 protein has also been implicated in the viral evasion from the immune system. Sequence analyses of different HCV isolates and sequential studies of virus isolates from infected patients suggest that the highly variable region 1 (HVR-1) in the amino-terminus of E2 protein is under immune selective pressure resulting in the selection of variants within the HVR-1. Previous studies have shown that antibodies specific for HVR-1 are neutralizing. However, these antibodies tend to be isolate-specific and over time drive the selection of new viral variants that are not recognized by the preexisting antibodies. Likewise, E2 protein may contribute to HCV resistance to interferon and impair natural killer (NK) cell function. The carboxy-terminal part of E2, p7, is generally cleaved, but only partially in some strains of genotype 1a. Although recent studies suggested that p7 might assist virion assembly and secretion from infected cells, its function remains unknown.
Studies have shown that HCV particles vary in size, between 30 to 60 nm in diameter. In addition, HCV particles display significant heterogeneity in buoyant density on sucrose density-gradient centrifugation, ranging from low (<1.07 g/ml) to high (1.25 g/ml) density. The heterogeneity of particle density has been attributed to the variability in size, non-enveloped nucleocapsid particles, and association with antibodies or β-lipoproteins.
Disease
HCV is the major etiology of non-A, non-B hepatitis that infects an estimated 170 millions people worldwide. One of its major characteristics is the high incidence of persistent infection, which may lead to autoimmune disorders and severe liver damage ranging from chronic hepatitis to liver cirrhosis and even hepatocellular carcinoma. Approximately 70-80% of patients develop chronic hepatitis, of which 20-30% progress onto liver cirrhosis.
As hepatocytes represent the primary site of HCV replication in vivo, the HCV genome has also been found in lymphoid cells. Infection of the lymphoid cells has been implicated in extra-hepatic manifestations of HCV infection such as mixed cryoglobulinemia and B-lymphocyte proliferative disorders.
Cellular Receptors for HCV
To date, the cellular receptor(s) for HCV remains controversial. The observations that HCV can infect both hepatic and lymphoid cells suggest that HCV may use different cellular receptors to access different cell types. However, the absence of an in vitro system that supports HCV replication and particle assembly has hampered studies to elucidate the early steps of HCV infection, i.e. virus binding and entry. Association of HCV virions with β-lipoproteins in plasma has raised the possibility that HCV may use the LDL receptor (LDL-R) for viral entry. Others have proposed that CD81, a cellular surface protein belonging to the tetraspanin protein superfamily, is the putative receptor for HCV, based on the interaction of CD81 with recombinant truncated E2 protein of HCV 1a. Nevertheless, several studies have shown that using the truncated E2 protein alone may not accurately reflect interaction of the HCV virion with cells. Both E1 and E2 glycoproteins are known to associate in two types of complexes: (i) heterodimers stabilized by non-covalent bonds, which presumably represents the pre-budding form of the viral envelope, and (ii) high molecular mass disulfide-bonded aggregates representing the misfolded proteins. Indeed, using a pseudotype vesicular stomatitis virus (VSV) expressing either HCV E1 or E2 protein, it has been shown that both proteins are required for efficient infection and fusion into target cells. Furthermore, the HCV virion binds to mononuclear cell lines regardless of their CD81 expression, while recombinant E2 protein binds poorly to cells that lack CD81.
Deficiencies
The structure of HCV virions has not yet been elucidated. This is in part due to the difficulties to obtain sufficient amounts of free, purified virion. So far, modeling of HCV ultrastructure is based on data obtained from other members of the Flaviviridae family (dengue and tick-borne encephalitis viruses). Several studies have shown that the genome of HCV is detected in association with other components in the serum: immunoglobulins and β-lipoproteins. Although antibodies recognizing envelope proteins have been detected in the serum, no demonstration is available on the presence of circulating envelope proteins. A recent report suggests the presence of core containing particles in the serum.
No HCV vaccine is yet available and the current treatment of chronic hepatitis (interferon in combination with ribavirin) is at best only effective in 61% of cases. Efficacy in fact depends in part on the genotype of the infecting HCV strain. The initial steps of HCV infection (binding and entry) that are critical for tissue tropism and hence pathogenesis, is poorly understood. Studies to elucidate this process have been hampered by the lack of robust cell culture systems or convenient small animal models that can support HCV infection. Therefore, there is a need for systems for producing and isolating HCV or HCV-like particles.
The present invention relates to new methods for obtaining HCV complexes and HCV-like particles from cells, particularly insect cells, infected with recombinant baculoviruses encoding HCV structural genes. In one method, cells are lysed, preferably with digitonin in the presence of protease inhibitors. Polyethylene glycol is slowly added to the lysate, to provide a precipitate that comprises complexes of the HCV structural proteins associated with lipid vesicles or micelles and complexes comprising viral structural proteins in the form of insoluble aggregates. Preferably, the cells are thoroughly washed prior to lysis to remove recombinant baculoviruses in suspension in the culture medium. In another method, the lysate is centrifuged through a sucrose cushion, preferably a 20% sucrose cushion. Preferably, the pellet is then subjected to equilibrium ultracentrifugation, to provide a preparation of HCV-like particles. Preferably, the infected cells are thoroughly washed prior to lysis to remove baculovirus in suspension in the culture medium. The particles obtained by this method are heterogenous in size. Fractions containing viral structural proteins typically comprise three subpopulations of particles whose average diameters are about 35, 42, and 49 nm. The third method comprises subjecting the infected cells to hypertonic/hypotonic shock, and then lysing the cells with digitonin in the presence of protease inhibitors. Preferably, lysis and hypotonic shock are performed simultaneously. The lysate is pelleted, fractionated, preferably by equilibrium ultracentrifugation, to provide a population of homogenous HCV-like particles having an average diameter of about 50 nm. As used herein the term “homogenous” means that both the size and the shape of the particles are similar. Preferably the cells are washed thoroughly prior to hypertonic/hypotonic shock to remove recombinant baculoviruses in suspension in the culture medium. The present invention also relates to the preparations of HCV structural protein complexes and HCV-like particles obtained by the present isolation methods.
The invention also relates methods of using the HCV complexes and HCV-like particles as screening tools, diagnostic tools, and immunogenic compositions. In one embodiment, the present preparations, particularly the preparations of HCV-like particles, are used to detect specific anti-HCV antibodies in HCV-infected patients. In another embodiment, the preparations, particularly the preparations comprising HCV-like particles are used with cultured cells expressing receptors for HCV, to screen for compounds or substances that interfere with binding of the HCV-like particles to the cells and/or interfere with internalization of the HCV-like particles by the cells. In another embodiment, the HCV-like particles are used to identify cellular receptors for binding of the virus to cells. In another embodiment the preparations, including the HCV structural protein complexes and HCV-like particles, are used as immunogenic compositions to induce production of anti-HCV antibodies in a mammal, including humans.
Methods of treating HCV using the compounds or substances that interfere with binding or internalization, especially those that interfere with binding of the present HCV-like particles to asialoglycoprotein receptors, are also part of the invention.
The present invention may be more readily understood by reference to the following drawings wherein:
Because of the lack of in vitro systems for HCV replication and the inability to obtain the purified virus in sufficient quantity, virologists have attempted to express HCV genes in various expression systems with the idea that expressed HCV structural proteins would assemble into virion-like structures. It is well known for some viruses that expression of recombinant virus structural proteins in eukaryotic cells leads to the spontaneous-formation of pseudo-viral particles, so called viral- or virus-like particles.
In 1998, Baumert et al. reported that expression of recombinant structural proteins of HCV in insect cells led to the formation of virus-like particles, so-called hepatitis C viral-like particles (HCV-LP). The structural genes of HCV derived from 1b genotype were cloned into baculovirus allowing their expression under control of the polyhedrin promoter. These investigators also described a method of purifying HCV-LP from the infected insect cells. Insect cells were infected with an inoculum of recombinant baculovirus, in general, at a multiplicity of infection per cell of 1. Four days after infection, insect cells were harvested and lysed by sonication and homogenized in 50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl, 0.5 mM EDTA, 0.1% NP40 and a cocktail of protease inhibitors. Cell lysates containing HCV-LP were centrifuged through a 30% sucrose cushion at 100,000-×g for 6 h. The pellet was homogenized and sonicated; the resuspended pellet then subjected to ultracentrifugation on a 20-60% sucrose gradient at 150,000×g for 22 h. Ten fractions were collected from the top of the tube and analyzed for the presence of HCV structural proteins by Western Blot.
The Baumert et al. method results in a low yield of HCV-LP. In addition, the HCV-LP resulting from this method is heterogeneous and contains significant amount of contaminating baculoviruses. This is a disadvantage, especially if the particles are to be used to immunize individuals, as the impurities present in the preparation might cause adverse immune reactions. The HCV-LP preparations that are obtained showed poor binding to target cells and significant death of those cells. This is unfortunate since a purified and biologically functional HCV-LP, theoretically, is a useful tool to identify cellular receptor(s) for HCV. Therefore, although HCV-LP are produced in cells infected with baculoviruses encoding HCV genes, isolation and purification of the particles from the infected cells has not yielded pure HCV-LP, and the quantities of HCV-LP obtained is not sufficient for significant further biological studies.
Purification and Characterization of Recombinant HCV Complexes and Particles From Infected Cells
The present invention describes new methods for purifying Hepatitis C recombinant material from insect cells infected with recombinant baculoviruses encoding HCV structural proteins. In one aspect the material is a complex of HCV structural proteins, referred to in the examples as “HCV-SP”. In another aspect, the material is HCV-like particles, referred to in the examples as “HCV-LP”.
In these methods, a protein expression system is used to express HCV structural proteins in eukaryotic cells. Preferably, the protein expression system used is a baculovirus expression system. One highly preferred expression system is a recombinant baculovirus whose genome comprises the structural genes of HCV. Preferably, the baculoviruses encode all of the structural proteins of HCV. In one embodiment, the recombinant baculovirus expresses core, E1 and E2-p7 proteins of HCV. In another embodiment, the recombinant baculovirus expresses core, E1 and E2, without p7. Transcription of the genes encoding the HCV proteins is driven by powerful promoters that initiate transcription of the HCV genes within the baculovirus after host cells are infected. The expression system may also comprise one baculovirus that encodes some of the structural proteins of HCV and that a second baculovirus that encodes the remaining HCV structural proteins. Methods for incorporating genes into the genome of baculoviruses are well known in the art. Such methods involve recombinant DNA technology and are well described in the art. Many such methods are described in U.S. Pat. No. 6,387,662 of Liang and Baumert.
Although it is possible to use any eukaryotic cell that has a glycosylation system, such as mammalian cells as a host cell for the expression system, it is highly preferred to use insect cells that are natural hosts or that are engineered to be hosts for baculovirus. There are a variety of methods known in the art for growing insect cells in culture and for infecting such cells with the baculoviruses. Any of these methods can be used.
Once insect cells are infected with the HCV structural protein encoding baculoviruses, the HCV proteins assemble into virus-like particles. Such particles can be detected within the baculovirus-infected cells by various methods, one being immunofluorescence using one or more antibodies specific for HCV proteins. Other methods for detecting the particles are available. The preferred method of choice is electron microscopy, which allows visualization of viral-like particles in the infected cells, preferably in combination with immunolabeling method.
Three different methods for purification of the HCV material from the host cells are described herein. For convenience, the methods described herein provide details of purification as they relate to baculovirus infected cells. The methods involve lysis of the infected cells in order to release viral protein complexes and/or virus-like particles from within the cells. The methods used for lysis preferably lyse the cells without damaging or by minimally damaging the virus-like particles within the cells. Preferably the cells are thoroughly washed prior to lysis or hypertonic shock, as described below, to remove recombinant baculoviruses in suspension in the culture medium.
In the first method of purification, cells containing HCV-like particles, e.g., baculovirus infected cells (Example 2) are lysed in a buffer comprising digitonin and protease inhibitors. Preferably, the concentration of digitonin used is less than 0.25%. Preferably insoluble debris is removed from the lysate, for example by centrifugation, and the resulting supernatant precipitated with the addition of polyethylene glycol (PEG). Various concentrations of PEG can be used, at various pHs and in various buffers, depending on the time and temperature of treatment. Good results have been obtained using PEG 8000 in 0.15 M NaCl at a concentration of 10%. Layering the precipitate onto a sucrose gradient, and subjecting the gradient to ultracentrifugation can achieve further purification. After a suitable time of centrifugation, fractions are collected from the sucrose gradient and tested for the presence of virus-like particles. This testing can be done in a variety of ways. One way is analysis of the proteins within the collected fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation technique may be used as a prelude to Western blotting, where HCV proteins are detected using one or more antibodies specific for the proteins. Such methods are well known in the art. The preparation that results from this first method comprises complexes of the HCV structural proteins associated with lipid vesicles or micelles and complexes comprising viral structural proteins in the form of insoluble aggregates.
In the second method for virus-like particle purification (Example 11), cells containing HCV-like particles, e.g., baculovirus-infected cells are lysed with digitonin. Insoluble debris is removed by centrifugation and the supernatant is centrifuged over a sucrose cushion, e.g. a 20% or 30% sucrose cushion. The pellet is resuspended and, preferably layered onto a gradient and ultracentrifligation is performed. The gradient can be a sucrose gradient. Alternatively, the gradient can be of various types known in the art. For example, the gradient can comprise cesium chloride or other iodinated compounds, nycodenz or iodixanol for example. After a suitable time of centrifugation, fractions are collected from the gradient and tested for the presence of virus-like particles, as described above.
The third method for purification of virus-like particles (Example 18) involves hypertonic/hypotonic of cells containing HCV-like particles, e.g., baculovirus infected cells. The method suspending the cells in a hypertonic buffer (e.g., Hepes plus glycerol), then in a hypotonic buffer (e.g., Hepes). It is also possible to use other components or steps to achieve successive treatment in a hypertonic buffer and a hypotonic buffer. For example, sucrose or hypertonic saline solution can be followed by hypotonic shock. Preferably, lysis and hypotonic shock are performed simultaneously. Insoluble debris is removed from the lysate preferably by centrifugation and the supernatant is centrifuged over a sucrose cushion, e.g. a 20% or 30% sucrose cushion to provide a preparation of HCV-like particles that are approximately 50 nm in size.
The virus-like particles obtained from the methods of purification described above are characterized. Such particles contain one or more, preferably all, of the HCV proteins expressed in the baculovirus-infected insect cells. Such particles also contain lipids. Such particles may or may not contain nucleic acid. Nucleic acid contained in the particles is preferably RNA.
There are a variety of methods well known in the art of virology for characterizing virus particles. SDS-PAGE with or without Western blotting has already been described. Other immunological methods, ELISA for example, can also be used to detect and analyze proteins present within or associated with the virus-like particles. Electron microscopy can be used to visualize and to measure the size of the particles. Cryoelectron microscopy can be used. Ultracentrifugation can be used to determine buoyant density of the particles. Other methods can be used to detect and analyze a viral genome that may be present within the particles. Such a method, for example, consists in extracting RNA from virus-like particles and subjecting the extract to a step of reverse transcription to synthesize cDNA. The HCV specific DNA fragment is then amplified using specific primers and thermoresistant DNA polymerase (polymerase chain reaction, or PCR), followed by agarose gel electrophoresis and ethidium bromide staining to visualize the size of viral specific DNA fragment.
Other assays may be used to ascertain various functions of the particles. For example, assays to determine whether the virus-like particles bind to host or target cells can be used. The same or other assays can be used to determine whether the particles enter host or target cells. Some such assays are described in various Examples of this application.
In addition there are many techniques and methods that exist in the art of virology that can be used to detect and measure various aspects of viruses or virus particles or their functioning or interaction with cells. Such methods are well known in the art and can be found in numerous textbooks or laboratory manuals of virology. Such methods can be used to analyze and test the virus-like particles of the present invention and their functioning.
Briefly, the characteristics of the HCV recombinant material obtained by the three different methods of purification described above are as follows. The material resulting from the first purification method described above are complexes of HCV structural proteins associated with lipid vesicles or micelles or complexes that are aggregates of the HCV structural proteins. The material resulting from the second purification method described above are irregular particles containing E1-E2 envelope proteins representing three subpopulations of particles that are more apparent. The material resulting from the third purification method described above is a preparation of particles that are substantially homogeneous. As used herein the term “substantially homogenous means that the particles are similar in shape and vary in size by ±10% or less. The HCV-like particles prepared by this third purification method are approximately 50 nm (±10%) in diameter with an apparent structure resembling other known viruses of the family Flaviviridae. This latter method of purification preserves the structure of the virus-like particles during the purification process. Other characteristics of the HCV-like particles obtained from the three different methods of purification are described in the Examples of this application.
In those cases where the cells are thoroughly washed prior to lysis or hypertonic shock, the preparations contain very low levels of baculovirus particles. In the prior art methods discussed above, the % of baculovirus in the preparation is greater than 50% and can be as much as 80%. In the present methods the percent of baculovirus in the preparation is less than 30%. For example, method 1 results in the production of preparations that contain less than 30% and in some cases no baculovirus. Method 2 results in preparations that are expected to contain less than 10% baculovirus. Method 3 results in preparations that are highly purified and that typically contain 1% or less of contaminating baculovirus.
Assays
The HCV-like particles are used in variety of assays. In one type of assay, the particles are used to detect HCV in a sample, in the blood of a patient for example. In another type of assay, the HCV-like particles are used in assays to screen for compounds or substances that interfere or prevent binding of the particles to cells and/or internalization of the particles into the cell. In another type of assay, the present HCV-like particles can be used to detect and identify receptors or co-receptors for HCV.
A. Diagnostic Assays
Assays to detect HCV in a sample or to determine if an individual is or has been exposed or infected with the virus can be of a variety of types. One type of involves detecting antibodies in a subject that are cross-reactive with the HCV-like particles produced by the present invention. Many such assays are well known in the art. For example, such assays include competitive binding assays, direct and indirect sandwich-type immunoassays, agglutination assays and precipitation assays.
Because the HCV-like particles structurally mimic hepatitis C virions, the particles can be used to capture anti-HCV antibodies and antibodies that recognize the HCV-like particles can also recognize HCV. Generally, diagnostic kits using immunoassay formats use the HCV-like particles to assay for anti-HCV antibodies in a human infected with HCV, or use antibodies that bind to HCV-like particles to detect HCV in human tissue (such as blood or serum) obtained from an HCV-infected individual. The detection can be direct or indirect as is well known in the art.
Cell-free assays can be used to measure the binding of human antibodies in serum to HCV-like particles. For example, the particles can be attached to a solid support such as a plate or sheet-like material and binding of anti-HCV antibodies to the immobilized HCV-like particles can be detected by using a labeled anti-human immunoglobulin to visualize the bound anti-HCV antibodies attached to the HCV-like particles on the support. Similarly, the virus-like particles can be attached to inert particles such as latex beads, which can be used to detect human anti-HCV antibodies by detecting agglutination or capture of the particles at a discrete position.
In another type of assay, which can be used to detect either antibodies against HCV, or HCV particles in a sample, binding of the HCV-like particles to a cell to which HCV or HCV-like particles normally bind is used as the endpoint. In one embodiment, cultured cells to which HCV-like particles are capable of binding are used. Serum from a patient suspected of having antibodies specific for HCV is incubated with the HCV-like particles. The serum-incubated particles are then incubated with the cultured cells and it is determined whether the virus-like particles are able to bind to the cells. If the patient serum contains antibodies specific for HCV, the antibodies bind to or inactivate the HCV-like particles. In such case, no binding of the HCV-like particles to the cells is detected. In the control study, where the HCV-like particles were not pre-incubated with patient serum, the particles bind to the cells.
In another embodiment of this assay, a patient sample suspected of containing HCV is incubated with the cultured cells to which HCV-like particles are capable of binding. Subsequently, HCV-like particles are incubated with the cells and it is determined whether the particles bind to the cells. In the case where the patient sample contains HCV, the HCV binds to the cells and inhibits binding of the HCV-like particles.
Binding of the HCV-like particles to the cells in assays as described above can be detected and quantified in a variety of ways. In one technique, the particles may be labeled using radioactive or nonradioactive labels. The label may be directly or indirectly coupled to the particles using methods well known in the art. For example, HCV-like particles may be radioactively labeled with 3H, 125I, 35S, 14C or 32P using standard in vivo or in vitro labeling methods and the binding of HCV-like particles to cells may be detected using autoradiography or scintillation counting. The particles may also be labeled with labels that are non-radioactive. One such non-radioactive label attaches to the lipids of the virus envelope. The CellTracker dyes from Molecular Probes are of this type. Another type of dye binds to the nucleic acid of the particle. Examples of dyes of this type are the SYTO dyes, also available commercially from Molecular Probes.
B. Screening Tools.
Assays to screen for compounds or substances that interfere or prevent binding of HCV and HCV-like particles to cells can be of a variety of types. The HCV-like particles can be used to assay for proteins, antibodies or other compounds capable of inhibiting interaction between HCV and mammalian cells. For example, compounds that interfere with the ability of HCV to effectively infect human cells can be detected by measuring the ability of labeled HCV-like particles to bind to human cells, in vivo or in vitro, in the presence of the compound compared to control conditions where the compound is not present. Cell lines used in such assays have receptors for binding of HCV. Exemplary cell lines for detecting such interference with HCV-like particles include Hep 3B, HepG2, Chang liver, Daudi and MOLT-4, all available from the American Type Culture Collection (Rockville, Md.), and HuH7 cells, available from many research laboratories. Other such cell lines are primary human hepatocytes.
Cells that do not have receptors for HCV can also be used if the cells are manipulated in such a way that the cells express the receptors. Such cells that do not have receptors are 3T3-L1 cells, for example. One method for manipulating cells that do not express receptors is to transfect or otherwise introduce into the cells and express therein a nucleotide or nucleotide sequences that encode such receptors. Such nucleotide sequences are, for example, cDNAs from human liver encoding the hH1 and hH2 of the asialoglycoprotein receptor (ASGP-R).
Purified HCV-like particles are incubated with the cells and it is determined if the particles bind to the cells. Binding can be determined in a variety of ways. One way to determine binding is to label the HCV-like particles. The particles can be radioactively labeled or can be non-radioactively. In the case of radioactively labeled particles, binding of the particles to cells can be detected by autoradiography or scintillation counting of the cells. In the case of non-radioactively labeled particles, for example in the case where the particles have been labeled with a fluorochrome, fluorescence imaging of the cells will detect the attached particles. Alternatively, the fluorochrome-labeled particles can be detected after flow cytometry analysis of the cells. Binding of the HCV-like particles to the cells can also be detected in the case that the particles are not labeled. In this case, particles bound to cells can be detected by incubating the cells and attached particles with an antibody reactive with the virus. If the antibody is labeled with a fluorochrome or reacted with a second antibody that is labeled with a fluorochrome, attached particles can be detected after imaging or flow cytometry analysis of the cells.
Using such a cell binding assay, compounds or substances suspected of interfering with the binding of HCV-like particles and HCV to cells, is detected by first incubating the substance with either or both of the cells or the HCV-like particles, then incubating the cells with the particles and assaying for binding of the particles to the cells. Compounds or substances that interfere with particle binding will cause a reduction in the measurement of particles bound to the cells as compared to controls where no compound or substance was added before the cells and particles were contacted with one another.
Cell lines have been created that are readily used as assay targets for HCV infection. To do this, a nucleotide sequence encoding a receptor for HCV is introduced into cultured cells. For example, nucleotide sequences encoding subunits of ASGP-R are transfected into cells. The cells can be cells that have no receptors (e.g., 3T3-L1 fibroblasts) or can be cells that express receptors (e.g., HepG2 cells). In the latter instance, the level of receptors on the cell surface is much higher in transfected cells than in non-transfected cells. Preferably, the cells are also expressing a marker gene, which is linked to a promoter that is inducible upon virus entry or HCV-like particles internalization. Such cells are called indicator cells.
For example, HCV that bind the viral receptors on the surface of the indicator cells will cause induction of transcription of the marker gene, luciferase or green fluorescent protein (GFP). Induction of the marker gene is conveniently detected. Prior incubation of indicator cells with HCV-like particles will prevent the induction of transcription of the marker gene by the virus. Such indicator cells can be used to assay for HCV in fluid samples from a patient.
Such cells can also be used to assay for antibodies reactive with HCV in fluid samples of a patient. For example, HCV-like particles are contacted incubated with the fluid sample. Antibodies therein that are reactive with HCV, bind to the HCV-like particles and inactivate them. Subsequent contact incubation of the fluid-treated HCV-like particles with the indicator cells do not cause induction of expression of the marker gene to the same extent as contact with the cells of HCV-like particles that have not been contacted incubated by patient fluid not containing HCV-reactive antibodies.
Similarly, the indicator cells, along with the HCV-like particles, can be used to screen various substances and compounds for the ability to inhibit binding of HCV to a cell and/or to inhibit internalization of HCV into a cell. To do this, the indicator cells and/or HCV-like particles are incubated with a desired substance or compound. Subsequently, the HCV-like particles are incubated with the indicator cells and the level of induction of the marker gene is measured. Substances or compounds that inhibit HCV binding to the cells and/or internalization of HCV by the cells, will cause a reduction in the expression level of the marker gene of the indicator cells as compared to a similar control experiment where no substance or compound was used.
Similarly, antibodies that interfere with HCV infection of human cells can be detected and their ability to block infection can be measured by assaying the level of interaction between HCV-like particles and human cells (such as hepatocytes and HuH-7 cells) in the presence of the antibodies compared to the level of interaction achieved when the antibodies are absent.
Another type of assay can be used to measure internalization of virus by cells. In such an assay, virus is detected within cells. One method of doing this is by labeling HCV-like particles. The particles may be labeled by any of the methods described above. The labeled particles are incubated with cells and, at some later time, the cells are examined to determine if labeled virus or virus components can be detected within the cell. For example, in the case where radioactively labeled HCV-like particles are used, autoradiography of intact cells can be used to detect internalization. Another method is fractionation of various cell components or compartments using cell biological and/or biochemical techniques that are well known in the art. After the cell components are fractionated, scintillation counting is used to detect the radioactive label and determine if the virus has been internalized by the cell and where within the cell the HCV-like particles is located. In the case where HCV-like particles are not radioactively labeled, but, for example, are labeled with some type of fluorochrome as described earlier, cells can be fixed and then examined using methods such as confocal microscopy and flow cytometry.
The invention also encompasses methods of treating HCV infection in a patient using compounds or substances identified through use of the above assays, that inhibit binding of HCV to cells and/or inhibit internalization of HCV into target cells. Some such compounds or substances bind to ASGP-R or prevent binding of HCV to ASGP-R. Some such substances that have been identified include asialo-orosomucoid, thyroglobulin, asialo-thyroglobulin and antibodies reactive against peptides in the ASGP-R, such antibodies are preferably humanized antibodies. One specific antibody reactive against ASGP-R is a polyclonal antibody specific for a peptide of the CRD of hH1 subunit of the ASGP-R. Such compounds and substances can be used therapeutically to treat individuals infected with HCV or even prophylactically to prevent infection of individuals by HCV. The compounds and substances used in these methods are prepared into pharmaceutically acceptable compositions and easily administered to individuals at dosages that are therapeutically effective.
Compositions Containing HCV Structural Protein Complexes and HCV-Like Particles for Induction of an Immune Response
It should also be recognized that the HCV-like particles and the HCV structural protein complexes of the present invention could be used as an immunogenic composition to induce production of antibodies reactive with HCV in an animal. Such antibodies can be used in a variety of ways. One such use is to detect HCV in a sample from a patient in a diagnostic assay, many of which are known in the art. The anti-HCV antibodies can be made by a variety of methods that are well known in the art. In one such method, the HCV-like particles are injected into an animal, a rabbit, mouse, rat, rabbit, goat, sheep or horse, for example, to cause the animal to have a humoral immune response. In such animals, the serum contains antibodies specific for HCV. Antibodies can be used to detect HCV in patient samples.
In another method, HCV-like particles are used to make monoclonal antibodies, using methods well known in the art. Monoclonal antibodies that bind to HCV-like particles can readily be produced by fusing lymphatic cells isolated from an immunized animal using well-known techniques. Polyclonal or monoclonal antibodies that bind to HCV-like particles may be bound to a variety of solid supports such as polysaccharide polymers, filter paper, nitrocellulose membranes or beads made of polyethylene, polystyrene, polypropylene or other suitable plastics.
Vaccination against and treatment of HCV infection may be accomplished using pharmaceutical compositions, including HCV-like particles and HCV structural protein complexes. Suitable formulations for delivery of HCV-like particles are found in Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Co., Philadelphia, Pa., 1985). These pharmaceutical compositions are suitable for use in a variety of drug delivery systems (Langer, Science 249:1527-1533, 1990).
HCV-like particles in compositions are suitable for single administration or in a series of inoculations (e.g., an initial immunization followed by subsequent inoculations to boost the anti-HCV immune response). The pharmaceutical compositions are intended for parenteral, or oral administration. Parenteral administration is preferably by intravenous, subcutaneous, intradermal, intraperitoneal or intramuscular administration. Parenteral administration may be preferentially directed to the patient's liver such as by catheterization to hepatic arteries or into a bile duct. For parenteral administration, the compositions can include HCV-like particles suspended in a suitable sterile carrier such as water, aqueous buffer, 0.4% saline solution, 0.3% glycine, hyaluronic acid or emulsions of nontoxic nonionic surfactants as is well known in the art. The compositions may further include substances to approximate physiological conditions such a buffering agents and wetting agents such as NaCl, KCl, CaCl2, sodium acetate and sodium lactate. Aqueous suspensions of HCV-like particles can be lyophilized for storage and can be suitably recombined with sterile water before administration.
Solid compositions including HCV-like particles in conventional nontoxic solid carriers such as, for example, glucose, sucrose mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For oral administration of solid compositions, the HCV-like particles preferably comprise 10% to 95%, and more preferably 25% to 75% of the composition.
HCV-Hike particles can also be administered in an aerosol such as for pulmonary and/or intranasal delivery. The HCV-like particles are preferably formulated with a nontoxic surfactant (e.g., esters or partial esters of C6 to C22 fatty acids or natural glycerides) and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery.
HCV-like particles can be used prophylactically as a vaccine to prevent HCV infection. A vaccine containing HCV-like particles contains an immunogenically effective amount of the particles in a pharmaceutically acceptable carrier such as those described above. The vaccine may further include carriers known in the art such as, for example, thyroglobulin, albumin, tetanus toxoid, polyamino acids such as polymers of D-lysine and D-glutamate, inactivated influenza virus and hepatitis B recombinant protein(s). The vaccine may also include any well-known adjuvant such as alum, aluminum phosphate and aluminum hydroxide. Double-stranded nucleotide or polynucleotides can also be used as adjuvants. When double-stranded polynucleotides are used as antigens, the vaccine preparation is preferably administered to the individual by intramuscular injection. The immune response generated to the HCV-like particles may include generation of anti-HCV antibodies and/or generation of a cellular immune response (e.g., activation of cytotoxic T lymphocytes or CTL) against cells that present peptides derived from HCV.
Vaccine compositions containing HCV-like particles are administered to a patient to elicit protective immune response against HCV, which is defined as an immune response that prevents infection or inhibits the spread of infection from cell to cell after an initial exposure to the virus. An amount of HCV-like particles sufficient to elicit a protective immune response is defined as an immunogenically effective dose. An immunogenically effective dose will vary depending on the composition of the vaccine (e.g., containing adjuvant or not), the route of administration, the weight and general health of the patient and the judgment of the prescribing health care provider. For initial vaccination, the general range of HCV-like particles in the administered vaccine is about 100 μg to about 1 gm per 70 kg patient; subsequent inoculations to boost the immune response include HCV-like particles in the range of 100 μg to about 1 gm per 70 kg patient. A single or multiple boosting immunizations are administered over a period of about two weeks to about six months from the initial vaccination. The prescribing health care provider may determine the number and timing of booster immunizations based on well known immunization protocols and the individual patient's response to the immunizations (e.g., as monitored by assaying for anti-HCV antibodies or to avoid hyperimmune responses).
For treatment of a patient infected with HCV, the amount of HCV-like particles to be delivered will vary with the method of delivery, the number of administrations and the state of the person receiving the composition (e.g., age, weight, severity of HCV infection, active or chronic status of HCV infection and general state of health). Before therapeutic administration, the patient will already have been diagnosed as HCV-infected and may or may not be symptomatic. A therapeutically effective dose of HCV-like particles is defined as the amount of HCV-like particles needed to inhibit spread of HCV (e.g., to limit a chronic infection) and thus partially cure or arrest symptoms or prevent further deterioration of liver tissue.
In one embodiment, HCV-like particles are used to immunize animal generally using a procedure where about 10 to 100 μg, preferably about 50 μg of the particles are initially administered to the animal to induce a primary immune response followed by one to about five booster injections of about 10 to 100 μg of HCV-like particles over a period of about two weeks to twelve months. Depending on the size of the animal to which the particles are administered, the dosage may vary, as will be readily determined by those skilled in the art. The timing and dosage of the booster injections in particular are determined based on the immune response detected in the animal, using methods well known to those skilled in the art. The virus-like particles are preferably administered subcutaneously as a suspension that includes an adjuvant such as Freund's complete or incomplete adjuvant, although a wide variety of available adjuvants are also suitable.
Another type of pharmaceutical composition that can be administered for the purpose of stimulating a protective immune response against HCV is a composition comprising HCV-like particles and cells, preferably cells that are antigen presenting cells. In one embodiment, dendritic cells are isolated from an individual and incubated with HCV-like particles. The dendritic cells internalize the HCV-like particles. The dendritic cells that have been incubated with HCV-like particles are then administered to an individual as part of a pharmaceutical composition, for the purpose of stimulating an immune response in the individual that is protective or therapeutic for HCV infection. Although dendritic cells can be used in this procedure, other types of antigen presenting cells can be used. It is also possible to take cells that are not antigen presenting cells, and express within those cells, increased levels of MHC class I and/or MHC class II molecules. Such cells are also made to express, on the cell surface, molecules to which an immune response is desired, HCV proteins for example. Such cells, expressing both MHC and the desired antigen, are used as a component of the pharmaceutical composition comprising the vaccine. This procedure is advantageous in that the previously described immunization procedures, in which HCV-like particles alone (no cells) comprise the vaccine, is that such procedures usually induce immune responses to dominant antigens, which are not always the protective antigens important for host defense.
In another embodiment, immunization is performed using a pharmaceutical composition made as follows: monocytes are isolated from an individual, transfected with double-stranded DNA and one or more genes encoding HCV proteins. The cells are then treated with mitomycin C, or other treatment to kμl the cells, and administered back into the individual, preferably by intramuscular or intraperitoneal injection.
In another embodiment, immunization is performed using a pharmaceutical composition made as follows: monocytes are isolated from an individual, transfected with a polynucleotide sequence encoding ASGP-R. These cells are then exposed to HCV proteins, which bind to and are internalized by the cells. These cells are then treated with mitomycin C and administered back into the individual as above.
In any of the embodiments where the pharmaceutical composition used for the vaccine comprises cells and HCV proteins, the cells can be incubated with one or more cytokines before administration into the individual for the purpose of providing cells that are better able to stimulate an immune response when administered to the individual.
In addition, one or more of the above compositions may be combined to provide an effective pharmaceutical composition to be used for immunization against HCV.
The invention may be better understood by reference to the following examples, which serve to illustrate but not to limit the present invention.
Two recombinant baculoviruses expressing the structural proteins of HCV derived from 1a genotype (H77 strain) were generated (
A plasmid containing an infectious HCV clone of the 1a genotype H77 strain, p90/HCV.FL-long pU (gift of M. E. Major & S. M. Feinstone; FDA; Bethesda, Md.), was used as a template to generate two recombinant baculoviruses coding for the structural HCV proteins: core, E1 and either E2/p7+ (Bac.HCV-S) or E2/p7− (Bac.HCV-S/p7−). The Bac.HCV.S has an additional 63 nt of the amino terminal part of NS2. This plasmid was digested with Stu I and Tth111 I, releasing a DNA fragment (nt 278-2831) corresponding to core, E1 and E2/p7+ proteins, which was subcloned between the Stu I-Xba I sites of a pFastBac plasmid, allowing its expression under the control of a polyhedrin promoter (pFB90S). A second DNA fragment (nt 1814-2579) was generated from p90/HCV.FL-long pU; PCR was performed with Pfu DNA polymerase and the two following primers 5′-AAG ACC TTG TGG CAT TGT GC-3′ (sense) and 5′-TCG AAA GCT TAC GCC TCC GCT TGG GAT ATG AGT-3′ (anti-sense); for construct purpose, a Hind III site (underlined) was introduced in this amplimer. The 775-bp PCR product was subcloned into the Sma I site (blunt-end) of pUC19 vector (pUC775). pUC775 and pFB90S plasmids were digested with Asc I and Hind III, respectively, to obtain a 671-bp DNA fragment (nt 1909-2579) and to remove a fragment (nt 1909-2831) of pFB90S. The 671-bp fragment was then ligated with the truncated plasmid (pFB90S/p7−) that encodes for an E2/p7− protein. The schematic diagram of the cloning procedures is shown in
Plasmids pFB90S and pFB90S/p7− were used to generate recombinant baculoviruses, Bac.HCV-S and Bac.HCV-S/p7−, respectively, using BAC-to-BAC Baculovirus Expression System (Gibco-BRL/Life Technologies, Gaithersburg, Md.) according to the manufacturer's protocols. Virus titer was determined by BacPAK Baculovirus Rapid titer kit (Clontech, Palo Alto, Calif.).
Expression of core, E1, and E2 proteins of the recombinant baculoviruses in Sf9 cells (from Spodoptera fugiperda) was analyzed by indirect immunofluorescence. Indirect immunofluorescence was performed as follows: cells were seeded in a flat bottom 96-well plate (Sf9 cells attach after 1 h at 27° C. without shaking). When attached, the culture medium was removed and washed once with ice-cold PBS×1. Cells were fixed on ice with freshly prepared ice-cold methanol/acetone (50:50) for 2 min; fixation solution was then removed and washed 3 times with ice-cold PBS×1. Cells were incubated with PBS×1 containing 0.25% Igepal CA-630 (or NP-40) for 15 min on ice; detergent solution was removed and washed 3 times with ice-cold PBS×1. Cells were incubated with PBS×1 containing primary antibody ( 1/100) plus 0.1% Tween-1% BSA and 0.02% sodium azide for 1 h at room temperature with gentle shaking. Cells were washed 3 times with PBS×1 and incubated in the dark with FITC-coupled goat anti-mouse, antibody ( 1/250) in the same buffer for 45 min. The cells were washed 3 times with PBS×1 and analyzed with a fluorescence microscope.
Sf9 cells were grown at 27° C. in Sf900 medium (Gibco-BRL/Life Technologies, Gaithersburg, Md.) and were infected with recombinant baculovirus at multiplicity of infection (MOI) of 5 in a 500-ml Erlenmeyer flask, and cells were harvested at 3 days post-infection. All purification steps were carried out at 4° C. on ice. Cells were harvested (3,000 rpm for 15 min), washed once in 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2 (TNC) buffer containing 1 mM Pefabloc SC and a cocktail of EDTA-free protease inhibitors (Roche, Indianapolis, Ind.), and finally resuspended at 1×107 cells/ml in TNC buffer containing 0.25% digitonin and protease inhibitors (cf. above). Cells were homogenized, and placed on ice for 4 hr with gentle agitation, and centrifuged at 30,000×g for 45 min. The supernatant was collected, precipitated with 10% PEG 8000 and 0.15 M NaCl for 2 hr, and pelleted at 10,000 rpm for 30 min at 4° C. The pellet was resuspended in TNC buffer and briefly homogenized. 100-200 μl of homogenized suspension was applied onto a 10.5 ml of 20-60% sucrose gradient and centrifuged at 156,000×g for 16 hr. Fractions, 1 ml, were collected from the top of the tube and were tested for E1, E2 and core proteins by ELISA and western blot. Fractions containing Bac.HCV-S proteins (HCV-SP) were pooled, diluted with TNC buffer and pelleted at 100,000×g for 3 hr. Pellets containing HCV-SP were resuspended in TNC buffer and stored at −70° C. Protein concentration was determined using Coomassie Plus protein assay reagent (Pierce, Rockford, Ill.) with BSA as the protein standard. Similar methods were used to express and purify proteins produced with Bac.HCV-S/p7− (HCV-SP/p7−).
The fractions collected from the sucrose gradients, as described in Example 3, were analyzed for the presence of E1, E2 and core proteins by both ELISA and Western blot. E2 ELISA was performed as described: a 96-well plate was coated with 100 μl (20 μg/ml in PBS) of GNA (lectin from Galanthus nivalis) at 37° C. for 3 hr. To prevent non-specific binding, 150 μl of 4% goat serum (in 5% skim milk-PBS) was added and incubated for 3 hr at room temperature. Samples containg HCV-SPs were diluted in 5% skim milk-PBS, added to each well and incubated at 4° C., overnight. Anti-E2 monoclonal antibody (mAb AP33, 100 μl, 6 μg/ml) was added and plate was incubated for 3 hr at 37° C. Peroxidase labeled goat anti-mouse IgG (at a dilution of 1/1000) was then added and incubated for 1 hr at 37° C. Bound antibodies were detected by adding ABTS Microwell Peroxidase Substrate System and measured on an ELISA reader at an optical density of 405 nm (OD 405 nm). Plate was washed six times with PBS between each step and, after addition of anti-E2 mAb, with PBS-0.05% Tween 20. All dilutions were made in PBS containing 5% skim milk.
ELISA results (
Binding of the HCV-SP preparations to HepG2 cells was performed as follows: the assays were performed in a U-bottom 96-well plate. All the incubation (on a rocking platform) and centrifugation/washing steps (800 rpm, 5 min) were carried out at 4° C. All dilutions were made in ice-cold binding buffer (TNC buffer containing 1% BSA and a cocktail of EDTA-free protease inhibitors). Adherent cells (HepG2) were washed twice with PBS and detached with 2.5 mM EDTA (in PBS) at 37° C. for 10 min prior to use. Cells were rinsed once, resuspended in TNC buffer at 2×106 cells/ml and 100 μl were added to each well. Bac.HCV-SP binding was measured by indirect labeling. 0.125-2.5 μg of HCV-SPs were incubated with cells for 2 hr, and cells were washed twice to remove unbound proteins. Anti-E2 mAb (AP33) was added and cells were incubated for 1 hr, washed twice, and further incubated for 1 hr with FITC goat anti-mouse IgG (4 μg/ml). Cells were washed twice, resuspended in 150 μl of binding buffer, and bound HCV-SP was analyzed by flow cytometry. Nonspecific fluorescence was measured by adding primary and secondary antibodies in the absence of HCV-SPs to cells. The mean fluorescence intensity (MFI) of bound HCV-SPs was determined after subtracting the nonspecific fluorescence value.
As shown in
The ability of HCV-SP to bind various target cells was analyzed by flow cytometry (
It was tested whether asialation of HCV envelope glycoproteins plays a role in binding of HCV-SP to hepatic cells. The asialo-glycoprotein receptor (ASGP-R) is a C-type (calcium-dependent) lectin that is most commonly found in the liver, although it is also expressed in other tissues. It has been implicated in the clearance of asialo-glycoproteins, i.e. desialated or galactose-terminal glycoproteins, from the circulation by receptor-mediated endocytosis. This receptor consists of a hetero-multimer of two homologous subunits, hH1 and hH2. Each subunit is subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk, and the carbohydrate recognition domain (CRD). The CRD of hH1 requires three calcium ions for proper binding conformation and sugar binding.
The cell binding assay described in Example 4 was used, but modified as described below: cells were pre-incubated with various ASGP-R ligands prior to the addition of HCV-SP. 19S-Tg fraction (Tg=thyroglobulin) contains Tg-dimers (apparent molecular weight of 660 kDa) that have a sedimentation coefficient of 19S by ultracentrifugation. Crude Tg was extracted from bovine thyroid gland and 19S-Tg was purified by column chromatography, as previously described. Orosomucoid and 19S-Tg were incubated with agarose bead-linked neuraminidase, as recommended by the manufacturer (Sigma). After centrifugation, protein concentration of the supernatants containing asialo-orosomucoid and asialo-Tg was determined. All pre-incubation steps were performed for 2 hr at 4° C.
Since ASGP-R binding is calcium-sensitive, it was first asked whether HCV-SP binding to cells occurred in a calcium-dependent manner. The simultaneous removal of calcium from the binding medium together with the addition of 5 mM of the calcium chelator EGTA reduced HCV-SP binding to Molt-4 and HepG2 cells (
Thyroglobulin (Tg) has been previously reported to bind the ASGP-R. 19S-Tg and its desialated form (asialo-Tg) both inhibited HCV-SP binding to HepG2 cells. At lower concentration, asialo-Tg (0.4 mg/ml) showed the similar or higher inhibitory effect on HCV-SP binding as of 19S-Tg (at 1 mg/ml); desialated Tg is indeed known to have a higher affinity to the ASGP-R than 19S-Tg. Inhibition of binding was not stronger than 60-70%. It is therefore possible that additional binding site of HCV-SP exists that is neither competed by ASGP-R ligands nor sensitive to calcium.
The question was then asked, after binding to cell surface receptor, HCV-SP could be internalized into human hepatic cells? To do this, Sf9 cells (5×108 cells) were infected with Bac-HCV 1a.S (MOI 5) in Sf900 medium containing 0.5% FBS at 27° C. for 4 hr. Cells were pelleted, washed once with starvation medium (Sf900 medium minus cysteine and methionine), and then cells were grown in this medium for 24 hr. Then, 2 mCi of Redivue Pro-Mix [35S]-methionine and cysteine mix were added to the medium and cells were further incubated for 24 hr. The labeling medium was discarded; cells were washed once with Sf900 medium and resuspended in Sf900 medium. HCV-SP (now radiolabeled) were harvested at 3 days post infection. The internalization experiment was then performed as follows: 100 μg [35S]-HCV-SP was used/2×108 cells/well in a 6-well plate. Cells were directly incubated at 37° C. for 15, 30, and 60 min. Cells were then harvested, disrupted and submitted to cell fractionation with sucrose gradient ultracentrifugation, resulting in four fractions corresponding to four membrane-enriched cell compartments.
It was then asked whether ASGP-R was involved in internalization. For this purpose, a clone of stable transfected HepG2 cells expressing a fusion protein between the hH1 subunit of ASGP-R and the green fluorescent protein (GFP-hH1/HepG2 cells) was established. To establish such cells, a GFP-ASGP-R construct was obtained by cloning the PCR amplimer coding for ASGP-R hH1 subunit into pcDNA3.1/NT-GFP-Topo vector (Invitrogen Corporation; Carlsbad, Calif.). Briefly, cytoplasmic RNA extracted from HepG2 cells was subjected to reverse transcription, then PCR with specific primers to obtain DNA fragments coding for hH1. The pcDNA3.1/NT-GFP-hH1 construct was verified by sequencing for correct sequence and alignment. Transient transfection experiments were performed to confirm the expression of green fluorescent protein (GFP)-hH1 fusion protein. By laser scanning confocal microscopy (LSCM) analysis, a green fluorescent signal was detected in few cells, predominating at the levels of Golgi apparatus and plasma membrane, but was also detected in other cell structures, such as vesicles (not shown). HepG2 cells were then transfected with this plasmid construct using lipofectamine-Plus and after a few days, selection antibiotic was added into the culture medium. Stable transfectants were obtained and the most positive cells were sorted using a Beckman-Coulter system.
Also used were HCV-SP that were labeled with dye. HCV-SP was labeled with 4 μM CellTracker CM-DiI (Molecular Probes; Eugene, Oreg.) in TNC buffer for 1 hr at 4° C. in the dark. Dye-labeled HCV-SP was purified through a 30% sucrose cushion at 100,000×g for 3 hr; the pellet was resuspended in TNC buffer containing 1% BSA and protease inhibitors. HepG2 cells were seeded into sterile glass chamber slides one day before the assay. Cells were incubated with labeled HCV-SPs in serum-free DMEM at 4° C. for 30 min, followed by incubation at 37° C. for 5, 15, or 30 min. Cells were rinsed once with ice-cold PBS and fixed with 4% paraformaldehyde in PEM buffer (80 mM PIPES-KOH, pH 6.8, 5 mM EGTA, 2 mM MgCl2) for 30 min on ice. Cells were then rinsed three times with PEM buffer and slides were mounted with DAPI/antifade system and kept at dark at 4° C. until LSCM analysis was performed. Cells were analyzed with a LSCM (Leica, TCS SP) coupled with a DMIRBE inverted epifluorescent microscope. Wavelengths used to analyze GFP and CM-DiI staining were 499 and 553 nm for excitation, and 519 and 570 nm for emission, respectively.
In the transfected HepG2, without added virus, some GFP signal was visible in the endoplasmic reticulum area, but mostly in the Golgi apparatus area, suggesting that GFP-hH1 subunit was properly glycosylated before targeting to the plasma membrane. Following incubation of cells with CM-DiI-labeled HCV-SP (red), co-localization was analyzed by LSCM. It was observed that, after uptake, this material accumulated in the cell area surrounding the nucleus. Moreover, by superimposing the pictures obtained in green and red channels, it was observed that there was a clear co-localization with recombinant GFP-hH1 (
Furthermore, as shown in
3T3-L1 cells, a cell line of mouse fibroblasts that do not bind HCV-SPs (
The cells were then tested for HCV-SP binding. As shown in
Parental 3T3-L1 cells, and cell clones 3T3-22Z and 3T3-24X were used to study whether the expression of ASGP-R, not only allowed non-permissive cells to bind HCV-SP, but also rendered them permissive for HCV-SP internalization.
Sf9 cells, grown at 27° C. in Sf900 medium (Gibco-BRL/Life Technologies, Gaithersburg, Md.) were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 5-10, and cells were harvested at day 3 post-infection. All purification steps were carried out on ice. Cells were washed once with ice-cold 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM CaCl2 (TNC) buffer containing 1 mM Pefabloc SC and a cocktail of EDTA-free protease inhibitors (Roche, Indianapolis, Ind.), and resuspended in TNC buffer containing 0.25% digitonin and protease inhibitors. Cells were homogenized and let sit on ice with gentle agitation and monitored for cell lysis by trypan blue exclusion. Cell lysate was centrifuged to remove nuclei debris and plasma membrane, and the supernatant was pelleted over 30% sucrose cushion. The pellet was resuspended in TNC buffer, and applied onto a 10.5 ml of 20-60% sucrose gradient in SW41 tubes (Beckman) and centrifuged at 100,000×g for 16 hours. One-milliliter fractions were collected from the top of the tube and tested for E1, E2 and core proteins by ELISA and Western blot. Fractions containing HCV-LPs were stored at −70° C. Protein concentration was determined using Coomassie Plus protein assay reagent (Pierce, Rockford, Ill.) with BSA as the protein standard. The ultrastructural morphology of HCV-LPs was analyzed by cryoelectron microscopy.
The fractions collected from the sucrose gradients, as described in Example 11, were analyzed for the presence of E1, E2 and core proteins by both ELISA and Western blot, as described in Example 3.
ELISA results (
Using HCV-LPs, as isolated in Example 11, a cell-based binding assay in two formats has been developed. Both binding assays were performed at 4° C. in 100 μl of TNC buffer containing 1% BSA. For the first, indirect binding method, anti-E2 mAb was used to detect HCV-LP binding to cells. In this method, cells were incubated with various amounts of HCV-LPs for 2 h, washed twice, and cells were incubated with anti-E2 mAb (AP33) (15 μg/ml) followed by FITC goat anti-mouse IgG (4 μg/ml). Cell-bound HCV-LPs was analyzed by flow cytometry. Nonspecific fluorescence was measured by adding primary and secondary antibodies in the absence of HCV-LP to cells. The mean fluorescence intensity (MFI) of bound HCV-LP was determined after subtracting the nonspecific fluorescence value.
In the second method, the HCV-LPs were labeled with a lipophilic (CM-DiI) or nucleic acid dye (SYTO 12) and used for direct binding assay. To label, HCV-LPs were incubated with 5 μM of SYTO-12 or 1-5 μM of CM-DiI in TNC buffer at 4° C. for 15 min and re-purified through a 30% sucrose cushion to remove free dye. Cells were incubated with increasing concentrations of labeled HCV-LPs for 1 h at 4° C., washed twice, and bound (B) HCV-LPs was analyzed directly by flow cytometry. As a control for the direct binding assay, fraction prepared identically from control Bac-GUS-infected cells was labeled with the dye and used for binding assay. The MFI values of total binding (T) were based on the MFI of 100 μg/ml HCV-LPs in the absence of cells. Scatchard plot was analyzed as described.
The ability of HCV-LPs to bind various target cells was analyzed by flow cytometry first using the indirect method. As shown in
Pretreatment of cells with 0.25% trypsin abolished HCV-LPs binding (data not shown), suggesting that binding of HCV-LPs to cells is mediated by cellular surface protein(s). HCV-LPs binding to cells occurred, at least partially, in a calcium-dependent manner as addition of 5 mM EGTA reduced this binding (
To estimate the affinity of HCV-LP binding to hepatic and lymphoid cells, Scatchard plot analysis was performed. Using the direct binding assay with SYTO 12-labeled HCV-LPs, it was demonstrated the presence of a biphasic binding with high and low affinities to NKNT-3 and Molt-4 cells. The high affinity binding site has a dissociation constants (Kd) of ˜1 μg/ml, while the lower affinity binding site has a Kd of ˜50-60 μg/ml (
To test whether binding of HCV-LPs to cells is mediated through the envelope proteins, E1 and E2, the following study was done. SYTO 12-labeled HCV-LPs were pre-incubated with increasing amounts of anti-E2 (AP33, ALP98), anti-E1 (A4), or isotype (control) IgG for 2 h at 4° C. The HCV-LPs-antibody mixtures were then incubated with cells for 1 h. After washing, cell-bound HCV-LPs were analyzed. The results (
While HepG2, HuH7, NKNT-3 and Molt-4 cells all bound to HCV-LPs, significant differences in their CD81 expression existed. As assessed by RT-PCR, the strain of HepG2 cells used lacks CD81 expression, while others express CD81 (data not shown). Hence, HCV-LPs bound to HepG2 cells in a CD81-independent manner. Recombinant CD81 failed to inhibit HCV-LP binding to HuH7 cells, although it partially inhibited HCV-LPs binding to Molt-4 and NKNT-3 cells (
Molt-4 cells which express LDL receptors and have been used previously to characterize HCV-cell interaction were used in this study. HCV-LPs were pre-incubated with the lipoproteins before being added to cells in the indirect binding assay. It was found that LDL inhibited HCV-LPs binding when added simultaneously to cells (
Previous study has proposed that association of HCV virions and β-lipoproteins in the plasma may mask the virions from circulating antibodies, and at the same time, represent one mechanism of HCV entry into cells, i.e. through the LDL receptor. There are two explanations for this finding. LDL may bind to the HCV-LPs and inhibit their binding to cells; alternatively, LDL binding to HCV-LPs may hinder the accessibility of HCV-LPs to anti-E2 mAb used in this indirect binding method. To distinguish between these two possibilities, the direct binding method was used. Cells were incubated with SYTO 12-labeled HCV-LPs. As shown in
It was examined whether binding of HCV-LPs to cells can be followed by entry. HuH7 and NKNT-3 cells were incubated with CM-DiI or SYTO-labeled HCV-LPs at 4° C. for 30 min, followed by incubation at 37° C. for various time points. The specificity of internalization process was determined by pre-incubating dye-labeled HCV-LPs with anti-E1 and anti-E2 antibodies before added to cells. As a negative control, cells were incubated with CM DiI- or SYTO-labeled preparation from cells infected with Bac-GUS. Alternatively, Aro cells were incubated with dye-labeled HCV-LPs. Cells were fixed with 4% paraformaldehyde, washed and mounted with DAPI/antifade system. Cells were imaged on a Leica TCS SP laser-scanning confocal microscope mounted on a DMIRBE inverted epifluorescent microscope. SYTO and CM-DiI fluorescent dyes were excited by a 499 nm and 553 nm, respectively, laser lines from a water-cooled argon laser (Coherent Laser, CA). SYTO and CM-DiI fluorescence emissions were monitored at 519 and 570 nm, respectively.
The ability of NKNT-3 Cells to internalize SYTO lableled-HCV-LPs was shown in
Sf9 insect cells were grown in Sf900 II medium containing antibiotics-antimycotics at 27° C. (125 rpm) in sterile Erlenmeyer flasks with a volume ratio <1/3. To amplify HCV recombinant baculovirus stock, insect cells were infected at an MOI 0.1 (Virus titer was determined by BAC-Pak Rapid Titer kit) and harvested at 3 days post-infection. Supernatant containing baculovirus was concentrated by centrifugation at 48,000×g for 2 h at 4° C. (SW28 rotor; Beckman). The virus pellet was resuspended in Sf900 medium and stored in small aliquots at −70° C.
The infection protocol for small-scale preparation was as follows: Sf9 cells were infected with recombinant baculovirus at an MOI of 1 or 10/cell. To ensure that cells were infected simultaneously, cells were resuspended in a small volume of medium containing the inoculum (˜108 cells/5 ml) for 1 h in 125 ml sterile Erlenmeyer flask. After 1 h, without removing the inoculum, fresh Sf900 II medium (containing 0.5% fetal bovine serum and antibiotics-antimycotics solution) was added to reach a density of 2.5-5×106 cells/ml. Cells were grown at 27° C. (125 rpm) and harvested after 2, 3 or 4 days incubation.
The following steps in the cell lysis protocol were performed either on ice or at 4° C.: Sf9 cells were centrifuged into a pellet by rapid centrifugation (3,500 rpm for 1-2 min, without brake) and culture medium was removed. The volume of the pellet was measured and the term “volume” in the following steps refers to pellet volume. Cells were rinsed by suspending them once in 20 volumes of ice-cold PBS×1, and then pelleted by rapid centrifugation (cf. above) and supernatant was removed. The cells were resuspended by brief vortexing or gentle pipetting in 10 volumes of ice-cold glycerol buffer (50 mM Hepes-NaOH, pH 7.4, containing 5% glycerol, 2 mM EGTA and 2 mM EDTA) and incubated on ice for 30 min; gently swirling the solution by inverting the tube once or twice every 5 to 10 min.
Cells were centrifuged at high speed to pellet the cells and if to remove the excess glycerol. The supernatants were removed and the tube walls were carefully rinsed with 2 volumes of ice-cold hypotonic buffer (10 mM Hepes-NaOH, pH 7.4, containing 1× protease inhibitor cocktail, 2 mM EGTA and 2 mM EDTA) without resuspending the pellet. Then the liquid used to rinse the tubes was removed (if necessary centrifugation was briefly done again). Cells were resuspended (no vortex, no pipetting) in 2 to 6 volumes (depending on percentage of glycerol used above) of ice-cold lysis buffer (hypotonic buffer containing 0.25% digitonin) and incubated on ice for 15 min; gently swirling the solution every 5 min. The cell lysate was centrifuged at 1,500×g for 5 min to remove cell nuclei and debris. The lysate from this step was centrifuged at 30,000×g (15,000 rpm, SW28, Beckman) for 30 min to remove membranes. The lysate from this step was then centrifuged at 100,000×g for 3 h (28,000 rpm, SW28) through 10 ml of 30% sucrose cushion to pellet VLP; [rate zonal gradient: make continuous sucrose gradient: 0.75 ml of each 20, 30, 40, 50, 60 and 66% sucrose and incubate at 37° C. for 1 h, then cool on ice]. The pellet was gently resuspended in TNC buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 1 mM CaCl2) plus protease inhibitor cocktail with potter (0.5 ml glass/teflon homogenizer [1 ml for maxipreps]) without foaming.
The resuspended pellet was then subjected to equilibrium centrifugation as follows: less than 0.3 ml of sample was loaded on the top of a 20-60% sucrose gradient: 0.75 ml of each 20, 30, 40 and 50% sucrose, and 1.5 ml of 60% sucrose (for 5 ml tubes of SW55, Beckman). Centrifugation was at 100,000×g (slow acceleration, without brake) for 18 h. One-half ml fractions were collected from the top of the gradient. Bands are visible from fraction 5 to 7. Protein concentration was determined using Coomassie Plus protein assay reagent with BSA as the protein standard.
Alternatively, equilibrium centrifugation was performed by centrifuging on the top of a preformed sucrose gradient (cf. above) at 100,000×g for 2 h 30 min (slow acceleration without brake) or using a SW41 rotor (Beckman), the 20-60% sucrose gradient is as follows: 1.5 ml of each 20, 30, 40 and 50% sucrose, and 2.5 ml of 60% sucrose (10.5 ml tubes). Less than 0.5 ml sample was loaded and centrifuged at 100,000×g (slow acceleration, without brake) for 18 h. Collect 1 ml fractions from the top.
The virus was then collected from the collected gradient fractions by centrifuging the fractions at 100,000×g (33,000 rpm, SW55 with brake) through 1.5 ml of 30% sucrose cushion to pellet purified VLP for 90 min at 4° C.
Several aspects of the HCV-LP obtained with this method were analyzed: yield of HCV-LP containing fractions (total protein concentration/ml culture), biophysical properties, immunoreactivity of HCV-LP (Western Blot) and its ultrastructure (by cryoelectron microscopy analysis).
Yield: With 30 ml culture (108 cells), a maximum protein concentration of 1.2 mg/ml was obtained in the fractions with a total of≈2.2 mg protein containing core, E1 and E2 proteins.
Biophysical properties: Following sucrose gradient centrifugation, HCV-LP was found at buoyant densities of 1.15-1.18 g/ml (
Immunoreactivity: The fractions collected after sucrose gradient ultracentrifugation were analyzed by Western Blot using specific anti-core, anti-E1, and anti-E2 monoclonal antibodies. The result showed that fractions 5-7 exhibited very strong reactivity to all anti-structural protein antibodies tested (
Cryoelectron microscopy: The HCV-LP preparation was so examined and homogenous double-shelled particles of ˜50 nm in diameter were observed. In addition, this preparation was ‘clean’ from impurities.
HCV-LP have been tested for its ability to bind to target cells. Human hepatic cells (HuH7) and kidney cells (293) were obtained from American Type Culture Collection. An immortalized human hepatocyte cell line (NKNT-3) and a replication-deficient recombinant adenovirus (Ad) that express the Cre recombinase tagged with a nuclear localization signal (AdCANCre) was a gift from I. J. Fox (Omaha, Nebr.). Differentiation of NKNT-3 cells to mimic normal primary hepatocytes was achieved by transduction with AdCANCre followed by selection with G418 (Ad-NKNT-3) with a slight modification. Cells were grown in Chee's Modified MEM containing 5% fetal bovine serum and were analyzed for HCV-LP binding at 3 days post-transduction. HCV-LP was directly labeled with SYTO-12 (nucleic acid dye) according to the manufacturer's protocol. Briefly, HCV-LP were incubated with 5 μM of SYTO-12 in TNC buffer at 4° C. for 15 min and re-purified through a 30% sucrose cushion to remove free dye. 2×105 cells were incubated with 2.5 μg of SYTO 12-labeled HCV-LP in 50 μl TNC buffer containing 1% BSA and a cocktail of EDTA-free protease inhibitors, for 1 hr at 4° C. Cells were washed once with PBS, detached with 0.25 mM EDTA (in PBS) for 10 min at 37° C., and resuspended in binding buffer. After washing, cell-bound HCV-LP were analyzed by flow cytometry.
SYTO 12-labeled HCV-LP were pre-incubated with 20 μg/ml of anti-E2 (ALP98), anti-E1 (A4), or anti-C mAbs for 2 h at 4° C. and were then incubated with Ad-NKNT-3 cells for 1 h (
NKNT-3 cells were transduced with recombinant AdCANCre. HCV-LP binding was performed at 3 days post-transduction using 2×105 cells incubated with 1.5 or 2.5 μg of SYTO 12-labeled HCV-LP (
NKNT-3 cells were used as is or transduced with recombinant AdCANCre. (
This application claims the benefit of U.S. Provisional Patent Application No. 404,183 filed Aug. 16, 2002, which is incorporated herein by reference.
This invention is supported, at least in part, by funding from the National Institutes of Health, USA. The U.S. government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/25674 | 8/18/2003 | WO | 5/18/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60404183 | Aug 2002 | US |
