WO1998028004A1 - Particule de l'hepatite delta contenant un immunogene de proteine de fusion - Google Patents

Particule de l'hepatite delta contenant un immunogene de proteine de fusion Download PDF

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Publication number
WO1998028004A1
WO1998028004A1 PCT/AU1997/000884 AU9700884W WO9828004A1 WO 1998028004 A1 WO1998028004 A1 WO 1998028004A1 AU 9700884 W AU9700884 W AU 9700884W WO 9828004 A1 WO9828004 A1 WO 9828004A1
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Prior art keywords
virus
hdag
protein
immunogenic polypeptide
hcv
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PCT/AU1997/000884
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English (en)
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Eric James Gowans
Thomas Bernard Macnaughton
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The Crown In The Right Of The Queensland Department Of Health (Sir Albert Sakzewski Virus Research Centre)
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Application filed by The Crown In The Right Of The Queensland Department Of Health (Sir Albert Sakzewski Virus Research Centre) filed Critical The Crown In The Right Of The Queensland Department Of Health (Sir Albert Sakzewski Virus Research Centre)
Priority to AU78716/98A priority Critical patent/AU7871698A/en
Publication of WO1998028004A1 publication Critical patent/WO1998028004A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to an improved therapeutic delivery system and in particular to virus-like particles which may be used to ameliorate or prevent infections.
  • the invention relates to virus-like particles which may be used to ameliorate or protect against infections caused by hepatitis B virus and/or at least another hepatitis virus.
  • the immune response to infection with a micro-organism is divided into a specific and a non-specific response.
  • the non-specific response becomes effective soon after infection and serves to inhibit spread of the invading organism during the time it takes the host to mount the specific response.
  • the specific immune response is also divided into 2 components viz. the humoral (antibody) and the cellular immune responses.
  • humoral (antibody) and the cellular immune responses are effected by different cells of the immune system and although the system involves complex multimolecular interactions, B lymphocytes produce antibodies whereas T lymphocytes are a major component of the cellular response. However, T lymphocytes are also important for the antibody response to infection by providing T cell help.
  • the specific antigen is recognised by soluble antibody or by immunoglobulin (receptors) on the B cell; the immunogenic activity of the antigen is most often but not exclusively dependent on the conformation of the protein that is recognised in solution.
  • T lymphocyte help is necessary for specific B cell expansion; antigen is taken up by antigen presenting cells (APCs), viz. macrophages and dendritic cells, or by B lymphocytes, presented in context with MHC (major histocompatibility complex) Class II to T helper lymphocytes (CD4+) which then stimulate B cell division.
  • APCs antigen presenting cells
  • MHC major histocompatibility complex
  • CD4+ T helper lymphocytes
  • MHC antigens are cell surface glycoproteins which control the recognition of cell and foreign proteins in a complex system of intracellular signalling. The immune response is dependent on the expression of the MHC, sometimes called the human leukocyte antigen (HLA) system.
  • HLA human leukocyte antigen
  • the antigen recognised by the antibody is displayed on the surface of for example a virus particle, then one effect of antibody binding to specific antigen is to neutralise the virus and this in turn results in protection of the host.
  • a viral or bacterial antigen displayed on the surface of a cell can be recognised by antibody and this can result in elimination of the infected cell by a process of antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • the elimination of virus infected cells is most commonly and readily accomplished by CD8+ cytotoxic T cell (CTL).
  • CTL cytotoxic T cell
  • CTL recognise short (8-11 residues long) antigenic sequential peptides which are MHC Class 1 restricted and which are generally derived from endogenous expression.
  • peptides are processed by the cell proteosome machinery then transported to the lumen of the endopiasmic reticulum (ER) by a family of transporter proteins which are encoded in the HLA locus. There, the peptides are examined for the presence of HLA allele-specific binding motifs by MHC Class I molecules. Peptides containing the appropriate motifs are then bound by the MHC Class I protein which then associates with B2-microglobulin and the complex is then transported to the cell surface to be displayed as an integral membrane protein. This complex is then recognised by a CD8+ cell with the appropriate specific T cell receptor (TCR).
  • TCR T cell receptor
  • the peptide antigen which interacts with the Class I MHC molecule is derived from a virus or bacterial protein, then this peptide is seen as foreign and the CTL proceeds to eliminate the target cell.
  • the target peptide/MHC Class I interaction with the specific TCR is stabilised by several accessory interactions. Elimination of the target cell may be the result of the direct transfer of cytotoxic molecules from the effector cell or by the indirect action of cytokines thought to be TNF-a and IFN-g secreted by the cell.
  • CTL epitopes like antibody epitopes, often require T cell help for activity and it is not always possible to recognise T helper epitopes in the amino acid sequence of a protein.
  • 3 types of vaccines are commonly used to prevent infections, namely: i) live attenuated vaccines; ii) killed particle vaccines; and iii) subunit vaccines.
  • the choice can depend on several factors including a knowledge of the specific microorganisms pathogenesis.
  • CID 50 (1 CID 50 is the dose which infects 50% of chimpanzees in a given experiment). Moreover, the duration of protection was very limited. No data are available on the results of challenge of the animals with heterologous virus.
  • the present invention consists of a virus-like particle for use in the treatment or prevention of at least a microorganism infection wherein said particle comprises: at least a antigenic and/or immunogenic polypeptide or part thereof from the microorganism, fused to at least the last 19 ami ⁇ o acid of the COOH terminal sequence of the large protein from Hepatitis D virus (L-HDAg), wherein the fusion protein is at least partially enveloped by Hepatitis B surface antigen (HBsAg).
  • L-HDAg Hepatitis D virus
  • HBsAg Hepatitis B surface antigen
  • the antigenic and or immunogenic polypeptide or part thereof used in the invention should be at least capable of eliciting a humoral and/or a T cell response.
  • the T-cell response may be either a T helper cell response or a cytotoxic T-cell (CTL) response.
  • CTL cytotoxic T-cell
  • the polypeptide or part thereof displays a plurality of epitopes.
  • An epitopic region on a polypeptide is generally relatively small - typically 8 to 10 amino acids or less in length. Fragments of as few as 5 amino acids may characterize an epitopic region.
  • some of the epitopes on the polypeptide should be capable of eliciting a humoral response and/or some should be capable of eliciting a T cell response.
  • the polypeptide should incorporate at least a range of epitopes which contribute to T-cell activity.
  • the polypeptide includes epitopes capable of eliciting a CTL response. Most preferably these epitopes are also substantially conserved between members of the species from which the polypeptide or part thereof was initially selected.
  • the polypeptide or part thereof used in the invention may correspond to part of a natural protein produced by a microorganism or it may be a recombinant protein which contains at least a antigenic and/or immunogenic peptide.
  • the polypeptide or part thereof consists of a plurality of antigenic and/or immunogenic peptides linked together.
  • the polypeptide is selected from regions in a protein or is composed of peptides which have a variety of antigenic and/or immunogenic epitopes and which are substantially homologous between members of the species of microorganism from which the regions or peptides were selected.
  • polypeptide or part thereof used in the invention may be selected from any protein from any microorganism (including but not limited to bacteria, protozoa and viruses), provided that the polypeptide or part thereof displays antigenic and or immunogenic properties.
  • the polypeptide or part thereof displays antigenic and or immunogenic properties.
  • different virus-like particles may be produced to treat different microorganism infections without departing from the substance of the invention.
  • the polypeptide or part thereof is derived from a virus, such as a Hepatitis causing virus.
  • virus-like particles may be generated against Hepatitis C Virus (HCV). Since peptides with lipid tails (lipopeptides) are well known to stimulate cellular immune responses, it is expected that the lipid component of the HBsAg will have a similar effect, perhaps by enhancing the intracellular delivery of the sub-viral particle. On the other hand, individuals who are already anti-HBs positive may respond most favourably to the sub-viral particle vaccine because the particles will be targeted by the antibody to antigen presenting cells. Furthermore, because L-HBsAg is known to be more immunogenic than S-HBsAg, incorporation of L-HBsAg into the particles will mimic the second generation of HBV vaccines and lead to improved rates of response to the HBV component of the vaccine.
  • HCV Hepatitis C Virus
  • the polypeptide or part thereof is preferably derived from either the HCV core protein or the NS3 protein. These proteins, in contrast to other HCV proteins, are highly conserved amongst HCV isolates and are known to contain both CTL and T-helper epitopes that are recognised by a range of HLA types. Some examples of CTL epitopes are described in table 1 , below.
  • CTL epitopes are generally HLA Class I restricted but HLA Class ll-restricted CTL have been described (see Kaneko et al, 1996).
  • the HCV core protein is selected from any peptide or polypeptide that may be produced from amino acids 1 to 191 of the HCV core protein (wherein the amino acid numbering starts at the first amino acid in the core protein).
  • the peptide or polypeptide must, however, be capable of inducing a T cell response against at least one major subtype of HCV.
  • the core protein is between about 120 and about 160 amino acids in length. Most preferably the sequence is about 140 amino acids in length.
  • the length of the polypeptide or part thereof that may be used in the invention is dictated by (i) the length of the amino acid sequence used from L-HDAg and (ii) the overall length of fusion protein which can be efficiently enveloped by HBsAg.
  • the polypeptide or the length of the amino acid sequence used from L-HDAg may be varied depending on the purpose for which the virus-like particles are being used and the method of construction used.
  • the amino acid sequence used from L-HDAg is the last 19 amino acids at the COOH terminus of that protein.
  • the number of amino acids in the polypeptide or part thereof should at least be greater than about 5 amino acids and more particularly about 5 to 500 amino acids in length.
  • the polypeptide sequence or part thereof is about 50 to 200 amino acids long. Most preferably the sequence is about 100 to about 160 amino acid long.
  • the fusion protein which is produced preferably consists of entire core of HCV together with the complete L-HDAg. More preferably the fusion protein contains amino acids 1 to 140 from HCV core.
  • part or all of the HCV core protein may be inserted into an internal site within L-HDAg. For example amino acids 1 to 40 may be inserted into the nuclear localisation site for L-HDAg or into the proline/glycine rich domain.
  • the fusion protein is selected from SEQ ID NO 1 to SEQ ID NO 3.
  • the fusion protein selected for use in the invention need not, however, be identical to those described.
  • the fusion protein should, however, be substantially homologous to SEQ ID NO 1 to SEQ ID NO 3, while still maintaining substantially all of the biological activity of the fusion proteins described herein.
  • biological activity is meant at least the ability of the fusion protein to be released inside or from a host cell and the respective polypeptides or parts thereof ability to bind to an appropriate MHC molecule and induce a CTL response against at least one major subtype of HCV.
  • CTL response is meant a CD8+ T Lymphocyte response specific for an HCV antigen of interest, wherein CD8+, MHC class l-restricted T Lymphocytes are activated.
  • modifications can be effected at non-critical amino acid positions within a polypeptide without substantially disturbing its biological activity. Such modifications include but are not limited to, substitutions (either conservative or non-conservative), deletions and additions.
  • polypeptides or parts thereof used in the invention may be modified to enhance substantially their CTL inducing activity.
  • HDAg Hepatitis D virus
  • the present invention requires at least the last 19 amino acids of the L- HDAg to be present for the packaging by HBsAg, it will be appreciated that larger forms of the L-HDAg may be present in the fusion protein.
  • any length of amino acids from the L-HDAg may be used in the invention.
  • the entire L-HDAg is used in the fusion protein.
  • FIG. 1 The general principle behind the the development of the therapeutic of the present invention is illustrated in Figure 1. Having regard to figure 1 it can be seen that a fusion protein consisting of a polypeptide which exhibits antigenic and or immunogenic properties is fused to at least the last 19 amino acid tail of the L-HDAg. The fusion protein is then packaged into virus-like particles through the interaction of the 19 amino acid moiety with HBsAg. This process occurs when the 19 amino acid moiety from the L-HDAg and HBsAg are co- expressed in the same cell.
  • virus-like particles using the method of the present invention provides a means of stimulating a hosts immune system against HBV and the polypeptide that is fused to the 19 amino acid tail of the L-HDAg. Thus a dual immunological effect is observed from using the method of the invention.
  • a method for producing virus-like particles containing an antigenic/immunogenic polypeptide or part thereof comprising: incubating host cells transformed with an expression vector containing a sequence encoding a fusion polypeptide containing the antigenic/immunogenic polypeptide or part thereof and at least the last 19 amino acid of the COOH terminal sequence of the large protein from Hepatitis D virus (L-HDAg); in the presence of HBsAg and under conditions which allow expression and packaging of said fusion polypeptide.
  • the HBsAg is expressed in the same host cells as the fusion polypeptide. This may be acheived by co-tranfection of both expression vectors into the host cells.
  • the coding sequence for the antigenic/immunogenic polypeptide or part thereof used in the invention may be derived from any source which expresses the polypeptide or part thereof or a protein containing the polypeptide or part thereof.
  • the coding sequence for the polypeptide or part thereof or polypetide may be selected from the coding region for coat or envelope antigens, from core antigens or from non-structural proteins.
  • Fragments encoding the desired polypeptides may be derived from cDNA clones or genomic clones using conventional restriction digestion or any other method known in the art. Alternatively the fragments may be obtained by synthetic methods.
  • Virus-like particles produced according to the present invention may be expressed in a variety of different expression systems. The selection of the expression system which a researcher wishes to use will to a large extent be based on personal preference. Systems in which the virus-like particles may be expressed include Chinese hamster ovary cells (CHO cells), COS cells, HeLa and MRC-5 cells, all of which have been used in the past to produce vaccines or therapeutic products for use in humans, or any other suitable continuous cell line.
  • CHO cells Chinese hamster ovary cells
  • COS cells COS cells
  • HeLa and MRC-5 cells all of which have been used in the past to produce vaccines or therapeutic products for use in humans, or any other suitable continuous cell line.
  • the particles may also be synthesised in Escherichia coli, in yeast cells or in insect cells infected with recombinant baculovirus. Further, these cells may be used with alternative systems to transient transfection viz. stable transfected cell lines, constitutive or inducible expression, expression from a live recombinant virus. In a highly preferred example of the invention the particles are expressed from transient transfection of DNA into COS 7 cells.
  • the particles may be purified by any protein purification method known in the field. Purification may be achieved by techniques such as, for example, salt fractionation, chromatography on ion exchange resins, affinity chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins. Preferably they are purified by a combination of sucrose and caesium chloride gradient centrifugation using methods which are well described in the literature.
  • a number of methods to administer the virus-like particles to uninfected individuals or to infected patients are available.
  • the method of choice to produce the most effective response will however need to be determined empirically and the methods described below are given as examples and do not limit the method of delivery.
  • therapeutics which contain an immunogenic or antigenic polypeptide or part thereof as the active agent are known to those of ordinary skill in the field.
  • the same preparations can be used with the virus-like particles of the present invention.
  • therapeutics are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the particles encapsulated in liposomes.
  • the virus-like particles may be formulated into therapeutics with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients which may be used in such a formulation include, water, saline, ethanol, dextrose glycerol, or the like and combinations thereof.
  • the virus-like particle formulation may also contain minor amounts of auxiliary substances such as adjuvants, wetting, pH buffering agents, or emulsifying agents which enhance the effectiveness of the vaccine.
  • Suitable adjuvants which may be include in such formulations for example, aluminium hydroxide, N-acetyl-muramy1-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramy1 -L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L- alanyl-D-isoglutaminyl-L-ala ⁇ ine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)methylamine (MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS).
  • thr-MDP N-acetyl-muramy1-L-threonyl-D-isoglutamine
  • nor-MDP N-acety
  • Virus-like particles may also be formulated into therapeutics as neutral or salt forms.
  • Pharmaceutically acceptable salts include, for example, the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from in- organic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamins, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • Virus-like particle formulations may be administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity of virus-like particles to be administered will generally be in the range of 5 micrograms to 250 micrograms of particles per dose. However this will depend on the subject to be treated, the capacity of the subject's immune system to respond, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • Formulations may be administered by the intradermal, subcutaneous or intramuscular routes, or by other routes including oral, aerosol, parenteral, intravenous, i ⁇ traperitoneal, rectal or vaginal administration.
  • the virus-like particles may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. All the above formulations are commonly used in the pharmaceutical industry and are known to suitably qualified practitioners.
  • the virus-like particles should be delivered with diluents (water, saline etc) and/or delivery vehicles (tablets, capsules) which do not interfere with the activity of the particles.
  • Oral formulations may include such normally employed excipients as, for example, pharmaceutical grades of ma ⁇ nitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders
  • Rectal or vaginal administration also requires specific formulation into acceptable forms that contain lubricants and or emulsifying agents.
  • such formulations usually include, traditional binders and carriers such as, polyalkylene glycols or triglycerides.
  • the therapeutic may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of delivery may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at I-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • the therapeutic containing the virus-like particles may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins.
  • Figure 1 illustrates the method by which virus-like particles, consistent with the present invention, are made.
  • Figure 2 illustrates the sequence of the HCV cDNA insert of pA2.
  • Figure 3 illustrates the sequence of the HCV cDNA insert of pA3.
  • Figure 4 illustrates the sequence of the HCV cDNA insert of pA10.
  • Figure 5 illustrates the PCR cloning strategy for clones pA2, pA3 and pA10.
  • Figure 6 illustrates the DNA and Amino-Acid Sequence of pTBM-HBsAg (ayw3).
  • Figure 7 illustrates the sequence of the L-HDAg gene.
  • Figure 8 illustrates the DNA sequence of L-HDAg 19aa tail and alignment with other group 1 isolates of HDV.
  • Figure 9 illustrates the steps in the construction of plasmid pECE-C/d.
  • Figure 10 illustrates the general cloning strategy for the construction of the partial core protein expression vectors.
  • Figure 11 illustrates the sequence of the chimeric insert of plasmid pC ⁇ 27core120a.
  • Figure 12 illustrates the HCV-HDV sequence in plasmid pC ⁇ 27core120.
  • Figure 13 illustrates the HCV-HDV sequence in plasmid pC ⁇ 27core140.
  • Figure 14 illustrates the HCV-HDV sequence in plasmid pC ⁇ 27core161.
  • Figure 15 illustrates that a product with a size consistent with the expected size of the core-delta fusion protein was detectable by SDS- PAGE.
  • Figure 16 provides a western blot of the product of in vitro translated RNA from pC ⁇ 27 corel 20a.
  • Figure 17 provides a western blot of secreted core/HDAg fusion protein.
  • the figure shows that doublet bands that were identical in size to those detected in cell lysates could be detected in cell culture fluids (CCF) from COS7 cells transfected with pC ⁇ 27core140.
  • CCF cell culture fluids
  • No HCV core antigen was detected in the CCF from COS7 cells transfected with pC ⁇ 27core120 or pC ⁇ 27core161.
  • HCV core antigen in the CCF was dependent on co- expression of HBsAg.
  • Figure 18 is a schematic representation of three HCV L-HDAg fusion constructs according to the invention.
  • Figure 19 is an imi ⁇ unoblot of secreted particles from Cos 7 (lanes 1 and
  • Huh 7 (lane 2) cells transfected with genes for HCV core 140 full length HDAg fusion protein and HBsAg.
  • Human anti-HDAg was used in lanes 1 and 2 and human anti-HCV was used in lane 3.
  • the arrow indicates the position of the full length chimeric protein.
  • Figure 20 is an immunoblot, with human anti-HDAg, of secreted particles from Huh 7 cells transfected with genes for full length HCV core full length HDAg fusion protein and HBsAg.
  • the arrow indicates the position of the full length chimeric protein.
  • Figure 21 is an immunoblot of secreted particles from Cos 7 cells co- transfected with genes expressing HBsAg and full length L-HDAg containing an internal insertion of a portion of the HCV core protein.
  • the insertions were amino acids 1-40 of the HCV core and were made into the Apa1 (nt222) or Sma1 (nt490) sites of L-HDAg with (+) or without (-) wild type L-HDAg.
  • Human anti-HDAg was used and the large and small arrows indicate secreted fusion protein and L-HDAg respectively.
  • Figure 22 provides a graph of BALB/c mice vaccinated with HBsAg-HCV core particle (HBsAg boost, Bs28 treated P815 in vitro restimulation).
  • primers recognise a sequence in pBluescript KS on either side of the multiple cloning site.
  • the reaction was performed according to the manufacturer's instructions and the extension products ethanol precipitated.
  • the DNA was dried and analysed with the Applied Biosystems 373 sequencer.
  • RNA from the sample was prepared by the addition of guanidine isothiocyanate, sodium acetate and phenol-chloroform, as described (Chomczynski and Sacchi, Anal Biochem 162; 156-159, 1987), incubated on ice for 5-10min and the RNA precipitated by the addition of an equal volume of isopropanol. The sample was then centrifuged and the RNA dried then re-dissolved in distilled water. An aliquot of the RNA sample was mixed with random hexamer primers
  • the second strand synthesis was performed by the addition of DNA polymerase 1 from Esch coli (Boehringer, Mannheim), RNaseH
  • the dsDNA sample was then subjected to the sequence-independent single- strand amplification (SISPA) procedure (Reyes and Kim. Mol Cell Probes 5; 473-1991 ). Briefly, the dsDNA sample was ligated to a dsDNA molecule composed of 2 complementary synthetic oligonucleotides which, prior to re- annealing, were previously phosphorylated by the action of polynucleotide kinase at 37°C for 1 h, using T4 DNA ligase (Boehringer, Mannheim) at 15°C overnight then 65°C for 15min.
  • the nucleotide sequence of the complementary primers is:
  • the ligation reaction may result in the formation of a dimer of the ds oligonucleotide resulting in reconstitution of an EcoRV site; thus, prior to SISPA amplification with primer RT-A, the product was digested with the restriction enzyme EcoRV. This step ensured that only ds DNA molecules generated by the reverse transcriptase that were ligated to the ds oligonucleotide were amplified.
  • the sequence of the primers is: sense primer (#156) 5' - GAGGTCTCGTAGACCGTGCA - 3' (-22 to -3 of 5' UTR) anti-sense primer (#155) 5' - CCGGTGCTCCCTGTTGCATAGTTCACG - 3' (residues 1-7 represent sequences designed to facilitate cloning while the remainder represent nt 501 - 482 of the HCV genome) NB.
  • HCV nucleotide numbering is based on nucleotide number 1 representing the start of the long open reading frame.
  • Clone A3 was generated in a similar manner, using the SISPA product and primers #157 and #402.
  • the sequence of these primers is: primer (#157) 5' - CCGGTGCTCGGTCGTCCCCACCACAAC - 3' (nt 1540 - 1559, excluding 7 nt at the 5' end to facilitate cloning) primer (#402) 5' - TGGCATGGGATATGATGATG - 3'
  • Primer #157 was designed from an HCV RNA published sequence (Okamoto et al., (1990) Jap J Exp Med 60; 167-177), and primer #402 from a sequence published by Choo et al (Proc NatI Acad Sci, USA 88; 2451-2455, 1991 ).
  • the product of this reaction, (614 bp) was blunt-end cloned into pBluescript KS linearised by Sma1 to generate clone pA3.
  • the sequence of clone pA3 is shown in Figure 3.
  • primers 1A and 2A respectively were designed to amplify a region corresponding to nt 358-1030.
  • the sequence of these primers is;
  • primer (#2A) 5'-TTTCTTGTGGGATCCGGAGT - 3' (1011-1030) primer (#1A) - 5'-GGTAAGGTCATCGATACCCT - 3' (358-367).
  • a serum sample from a HBsAg-positive patient represented the source of virus.
  • the virus DNA was purified by protease digestion followed by phenol extraction and ethanol precipitation. Briefly, the virus was pelleted through a 20% sucrose cushion at 39000 rpm for 5h in a Beckman SW41 rotor and the pellet was then mixed with a solution of proteinase K and SDS, and incubated at 37°C for 4h. The solution was then extracted with an equal volume of phenol:chloroform:isopropanol (25:24:1 ) and the upper aqueous layer containing the virus DNA removed. The DNA was precipitated by the addition of sodium acetate to 0.4M and 2.5vol of absolute ethanol. The DNA was pelleted, dried and re-dissolved in distilled water. The gene for HBsAg was amplified by PCR using primers designed from a published sequence of HBV DNA (Galibert et al., J Virol 41 ; 51-65, 1982). The sequence of the primers is:
  • This reaction produced a 681 bp product that was cloned into pBluescript KS to create pTBM-LHBsAg and sequenced.
  • the sequence of the HBsAg gene is shown in Figure 6.
  • Plasmid pSV-HBsAg was transfected into COS7 and HuH7 cells using the DOTAP procedure. Five days later, the cell monolayers were examined for expression of HBsAg by immunofluorescence and the cell culture fluid for secreted HBsAg by ELISA. The immunofluorescence pattern of the HBsAg expressed in the cells was typically cytoplasmic and the ELISA was positive for HBsAg. These results proved that the HBsAg was not only expressed from pSV-HBsAg but was also secreted.
  • a serum sample from a HDV RNA-positive patient represented the source of the virus; the RNA was purified by the GIT extraction method described above.
  • the L-HDAg gene was amplified by RT-PCR.
  • the RT step was performed using random hexamer primers (Pharmacia) and the cDNA amplified by PCR using the following primers which include BamHI restriction enzyme sites to facilitate cloning;
  • the product of this reaction was a 465bp amplicon. This was digested with BamHI and cloned into the BamHI site of pCDNA3 to create pC-LHDAgl The sequence of the L-HDAg gene is shown in figure 7. Expression of L-HDAg.
  • the gene for the C-terminal 19aa region of HDAg was assembled from overlapping primers which were designed from examination of the consensus nucleotide sequences of the genomes of group 1 isolates of HDV.
  • the sequence of the primers including restriction enzyme sites to facilitate cloning is:
  • the primers were heated to 85°C for 5 min then allowed to re-anneal as the mixture was cooled to room temperature.
  • the product of this reaction was end filled with T4 DNA polymerase (Boehringer, Mannheim) to ensure that the hybrid was completely double stranded.
  • the DNA was double digested with BamHI and Xho1 and directionally cloned into the corresponding sites in pcDNA3 which was previously digested with these enzymes and this resulted in the construction of pC ⁇ 27basic.
  • the full length core gene was amplified using primers #156C and #WYH-5 from pA2-A10; the sequence of the primers including Bam H1 and EcoR1 restriction enzyme sites to facilitate cloning is:
  • the 600bp product of this reaction was directionally cloned into pECE (Leland et al, (1986), Cell 45; 721-732) which was previously digested with Bgl II and EcoR1 to create pECE-HCVcore; this plasmid was then digested with EcoR1 , made blunt ended by the action of the Klenow component of DNA polymerase 1 (Boehringer, Mannheim) and then digested with Xba1.
  • HCV corel 20a-HDAg The region corresponding to aa1-120 of the HCV core protein was fused to the 19aa COOH terminus of the L-HDAg to produce a chimeric fusion protein.
  • the expression plasmid was constructed in the following manner.
  • the region coding for core amino acids 1 - 120 was amplified by PCR from pA2- A10 using primers TM1 and TM2.
  • the sequence of these primers including the underlined BamHI sites to facilitate cloning is: primer #TM1
  • HCV core 120-HDAg The core gene region 1-120 was also amplified from pA2-A10 using primers TM2 and TM3 then ligated to the gene for the 19aa tail of L-HDAg.
  • Primer TM3 is a modified version of primer TM1 that was designed to eliminate a potential loop in the 5' end of the mRNA transcribed from the plasmid, as it was considered that this loop may reduce the efficiency of protein translation from the mRNA.
  • the sequence of primer TM3 including a BamHI site to facilitate cloning is: primer TM3-5'-AAAGGATCCAAAATGAGTACTAACCCTAAACCCCAA-3'.
  • the expression plasmid was constructed in the following manner:
  • the region coding for core amino acids 1-140 was amplified by PCR from pA2- A10 using primers TM3 and TM4 which include a BamHI site to facilitate cloning.
  • the sequence of the primers is: primer #TM3-as above primer #TM4-5'-AAAGGATCCGACAAGCGGGATGTACCCCAT-3'.
  • the product of this reaction was digested with BamHI and ligated into the BamHI site of pC ⁇ 27basic to yield pC ⁇ 27core140.
  • the sequence of the chimeric HCV-HDAg gene in this plasmid is shown in Figure 13.
  • HCV corel 61 -HDAg The core protein aa1 -161 was fused to the 19aa L-HDAg COOH terminus using a similar strategy.
  • the expression plasmid was constructed by amplifying the core gene coding for 1-161 from pA2-A10 using primers TM3 and TM5 which also contain a BamHI site for ease of cloning. The sequence of these primers is: primer #TM3-as above primer #TM5-5'-AAAGGATCCGCCGTCCTCCAGAACCCGGAC-3'
  • DOTAP transfection of COS7 and THT1 cells 10 ⁇ g of DNA (5ug of each plasmid) in 100 ⁇ l of HBS was added to 50 ⁇ l DOTAP (Boehringer Mannheim) and 50 ⁇ l HBS, then incubated at room temperature for 10min. The cell culture medium in a 25cm 2 flask was replaced with DMEM+1 % FCS and the tranfection mixture then added. The cells were incubated at 37°C overnight then re-fed with DMEM+5% FCS and incubated for the desired period.
  • CCF from the transfected cells were then examined.
  • the samples were clarified, then centrifuged through a 20% sucrose cushion at 38000 rpm for 5h at 4°C in a Beckman SW-41 rotor.
  • the pellets were dissolved in SDS-PAGE loading buffer then examined by immunoblot to detect secreted HCV core antigen.
  • virus-like particles constructed according to the examples using a fusion HCV/HDV protein and HBsAg were immunogenic with respect to HCV and HBV. That is the virus-like particles are capable of stimulating HLA class I restricted CTL responses against the core protein for HCV and the surface antigen of HBV.
  • the gene encoding aa 1-140 of HCV core was excised by PCR from plasmid pC ⁇ 27c140 (aa 1 -140 fused to C-terminal 19aa of L-HDAg) by BamHI digestion and ligated into the BamHI site of a pCDNA derived plasmid containing full length L-HDAg cloned between BamHI and Xba1 sites.
  • the full length gene for HCV core was amplified by PCR from the Australian HCV isolate (in plasmid pECEcore) and blunt-end cloned into the BamHI site of plasmid pC ⁇ 27/FL (see above).
  • the upstream primer for the PCR was the same as for the C140 insert.
  • the downstream primer was as follows:
  • plasmids contain aa 1-40 of HCV core (amplified by PCR) inserted into the Apa1 or the Sma1 sites respectively of full length L-HDAg in plasmid pSV27 (see above)
  • mice Two groups of 3 mice were vaccinated with the virus-like particles, composed of 140 amino acids of HCV core fused with the 19 amino acid tail of HDAg, and enveloped by HBsAg.
  • the particles were prepared by co-transfection of COS7 cells using DOTAP. The cell culture supernatant 5 days post transfection was then centrifuged over a 20% sucrose cushion and the particles resuspended in PBS 100 ⁇ l of this preparation was injected by the intraperitoneal route, and 2 weeks later the mice were boosted.
  • mice were sacrificed, the spleens removed and the cells stimulated and expanded in vitro with specific peptide.
  • control P815 cells or peptide pulsed P815 cells were incubated with the expanded effector cells in a classical 51 Cr release assay. The results are shown in figure 22; the level of background killing was high (approximately 40%), but there was a clear increase in cell killing in the HBsAg- and the HCV core-peptide pulsed cells.
  • an effectortarget ratio of 100:1 70.5% and 67% of the cells respectively were killed.
  • HCV core peptides amino acids 129- 140 and amino acids 132-140 respectively
  • HBsAg peptide amino acids 28-39.
  • the sequence of the peptides used in these studies were: HCV core amino acids 129 - 140 GFADLMGYIPLV; HCV core amino acids 132 - 140 DLMGYIPLV;
  • GAG TCC TTC TGA AGG CTC GCC AGC GTT GGA GCA CCT TCC GCT GTT GGA Leu Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin Pro
  • ATC CCC AAG GCT CGC CGA CCC GAG GGC AGG GCC TGG GCT CAG CCC GGG TAG GGG TTC CGA GCG GCT GGG CTC CCG TCC CGG ACC CGA GTC GGG CCC lie Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gin Pro Gly 250 260 270 280
  • GAG TCC TTC TGA AGG CTC GCC AGC GTT GGA GCA CCT TCC GCT GTT GGA Leu Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin Pro
  • ATC CCC AAG GCT CGC CGA CCC GAG GGC AGG GCC TGG GCT CAG CCC GGG TAG GGG TTC CGA GCG GCT GGG CTC CCG TCC CGG ACC CGA GTC GGG CCC lie Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gin Pro Gly
  • GAG TCC TTC TGA AGG CTC GCC AGC GTT GGA GCA CCT TCC GCT GTT GGA Leu Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin Pro
  • ATC CCC AAG GCT CGC CGA CCC GAG GGC AGG GCC TGG GCT CAG CCC GGG TAG GGG TTC CGA GCG GCT GGG CTC CCG TCC CGG ACC CGA GTC GGG CCC lie Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gin Pro Gly

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Abstract

Particule de type viral destinée à être utilisée dans le traitement ou la prévention d'au moins une infection due à un micro-organisme. Ladite particule comprend au moins un polypeptide antigène et/ou immunogène ou une partie dudit polypeptide extrait du micro-organisme, fusionné avec au moins les 19 derniers acides aminés de la séquence terminale COOH de la grande protéine du virus de l'hépatite D (L-HDAg), ladite protéine de fusion étant au moins partiellement enveloppée par l'antigène de surface de l'hépatite B (HBsAg).
PCT/AU1997/000884 1996-12-24 1997-12-24 Particule de l'hepatite delta contenant un immunogene de proteine de fusion WO1998028004A1 (fr)

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WO2000028009A1 (fr) * 1998-11-11 2000-05-18 Melbourne Health Compositions biologiques, leurs constituants et leurs applications
WO2001002551A2 (fr) * 1999-06-30 2001-01-11 Evotec Oai Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
WO2002010416A1 (fr) * 2000-07-31 2002-02-07 The Crown In The Right Of The Queensland Department Of Health Particules de type virus ameliorees fondees sur une petite proteine d'enveloppe provenant de l'hepatite b (aghbs)
AU2001276182B2 (en) * 2000-07-31 2006-05-04 Monash University Improved virus like particles based on small envelope protein from hepatitis B (HBsAg-S)
EP1219705B1 (fr) * 2000-12-29 2007-11-28 Evotec AG Particules du type virus, leur préparation et leur utilisation en criblage pharmaceutique et en analyse fonctionelle de génomes
US7419802B2 (en) 1999-06-30 2008-09-02 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7476517B2 (en) 1999-06-30 2009-01-13 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
WO2012174220A1 (fr) 2011-06-14 2012-12-20 Globeimmune, Inc. Compositions à base de levure, et procédés de traitement ou de prévention d'une infection par le virus delta de l'hépatite
US9029341B2 (en) 2010-08-17 2015-05-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US10513703B2 (en) 2014-11-10 2019-12-24 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) iRNA compositions and methods of use thereof
US11324820B2 (en) 2017-04-18 2022-05-10 Alnylam Pharmaceuticals, Inc. Methods for the treatment of subjects having a hepatitis b virus (HBV) infection
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US11492623B2 (en) 2018-08-13 2022-11-08 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof

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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028009A1 (fr) * 1998-11-11 2000-05-18 Melbourne Health Compositions biologiques, leurs constituants et leurs applications
WO2001002551A2 (fr) * 1999-06-30 2001-01-11 Evotec Oai Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
WO2001002551A3 (fr) * 1999-06-30 2001-11-08 Evotec Biosystems Ag Particules de type viral, preparation et utilisation de ces dernieres, de preference dans le criblage pharmaceutique et la genomique fonctionnelle
US7419802B2 (en) 1999-06-30 2008-09-02 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
US7476517B2 (en) 1999-06-30 2009-01-13 Evotec Ag Virus like particles, their preparation and their use preferably in pharmaceutical screening and functional genomics
WO2002010416A1 (fr) * 2000-07-31 2002-02-07 The Crown In The Right Of The Queensland Department Of Health Particules de type virus ameliorees fondees sur une petite proteine d'enveloppe provenant de l'hepatite b (aghbs)
AU2001276182B2 (en) * 2000-07-31 2006-05-04 Monash University Improved virus like particles based on small envelope protein from hepatitis B (HBsAg-S)
EP1219705B1 (fr) * 2000-12-29 2007-11-28 Evotec AG Particules du type virus, leur préparation et leur utilisation en criblage pharmaceutique et en analyse fonctionelle de génomes
AU2011292261B2 (en) * 2010-08-17 2015-05-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of Hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US10793860B2 (en) 2010-08-17 2020-10-06 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (SINA)
AU2019264591B2 (en) * 2010-08-17 2022-05-26 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US9029341B2 (en) 2010-08-17 2015-05-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US10407682B2 (en) 2010-08-17 2019-09-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B Virus (HBV) gene expression using short interfering nucleic acid (siNA)
US9464290B2 (en) 2010-08-17 2016-10-11 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US9879262B2 (en) 2010-08-17 2018-01-30 Sirna Therapeutics, Inc. RNA interference mediated inhibition of hepatitis B virus (HBV) gene expression using short interfering nucleic acid (siNA)
US9579377B2 (en) 2011-06-14 2017-02-28 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
US9987352B2 (en) 2011-06-14 2018-06-05 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
EA030381B1 (ru) * 2011-06-14 2018-07-31 Глоубиммьюн, Инк. Иммунотерапевтическая композиция, ее применения и способы лечения или профилактики инфекции, вызванной вирусом гепатита дельта
WO2012174220A1 (fr) 2011-06-14 2012-12-20 Globeimmune, Inc. Compositions à base de levure, et procédés de traitement ou de prévention d'une infection par le virus delta de l'hépatite
EP2720716A1 (fr) * 2011-06-14 2014-04-23 Globeimmune, Inc. Compositions à base de levure, et procédés de traitement ou de prévention d'une infection par le virus delta de l'hépatite
EP2720716A4 (fr) * 2011-06-14 2015-03-11 Globeimmune Inc Compositions à base de levure, et procédés de traitement ou de prévention d'une infection par le virus delta de l'hépatite
US10513703B2 (en) 2014-11-10 2019-12-24 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) iRNA compositions and methods of use thereof
US10640770B2 (en) 2014-11-10 2020-05-05 Alnylam Pharmaceuticals, Inc. Hepatitis D virus (HDV) iRNA compositions and methods of use thereof
US11060091B2 (en) 2014-11-10 2021-07-13 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) iRNA compositions and methods of use thereof
US11324820B2 (en) 2017-04-18 2022-05-10 Alnylam Pharmaceuticals, Inc. Methods for the treatment of subjects having a hepatitis b virus (HBV) infection
US11492623B2 (en) 2018-08-13 2022-11-08 Alnylam Pharmaceuticals, Inc. Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof

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