WO2008012062A1 - Protéoliposomes antigéniques et procédé de fabrication - Google Patents

Protéoliposomes antigéniques et procédé de fabrication Download PDF

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WO2008012062A1
WO2008012062A1 PCT/EP2007/006561 EP2007006561W WO2008012062A1 WO 2008012062 A1 WO2008012062 A1 WO 2008012062A1 EP 2007006561 W EP2007006561 W EP 2007006561W WO 2008012062 A1 WO2008012062 A1 WO 2008012062A1
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lipid
protein
proteins
membrane
virus
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PCT/EP2007/006561
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English (en)
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Hermann Katinger
Gabriela Stiegler
Andreas Wagner
Heribert Quendler
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Polymun Scientific Immunbiologische Forschung Gmbh
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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is in the field of virology and vaccine development and relates to a process for the preparation of virus-like vesicles composed of liposomes comprising antigenic peptides or proteins, particularly of viral or bacterial origin, preferably HIV proteins, either recombinantly produced or from virus lysates, wherein said peptides or proteins are incorporated in the membrane of the liposomes and have essentially retained their native conformation.
  • the invention further relates to the vesicles obtainable by this process and the use of said vesicles for vaccine preparation.
  • Liposomes have long been used as models for lipid membranes. They have been useful in studying membrane receptors, channel proteins, and membrane-bound enzymes. There is a growing interest in the preparation of lipid vesicles able to encapsulate labile biological substances like proteins, peptides or nucleic acids for biological, pharmaceutical or cosmetical purposes. Recent studies have examined the use of liposomes as potential immunoadjuvants. There are several advantages of liposomes: they are biodegradable and non-toxic and can be prepared quite easily with varying compositions to obtain the most efficacious antigen-presenting liposome formulation. In addition, liposomes have the ability to elicit both a cellular mediated immune response and a humoral immune response. Studies have shown liposomes to be effective immunopotentiators in hepatitis A and influenza vaccines. In addition, liposomes have adjuvant activity in vaccines against protozoan and bacterial organisms.
  • liposomes can be prepared by different methods. After disperging suitable membrane lipids in an aqueous phase and spontaneous formation of multilamellar large vesicles (MLV), mechanical procedures such as ultrasonication, homogenization by a French press or by other high pressure devices or extrusion through polycarbonate membranes with defined pore sizes lead to a reduction in size and number of lamellae of the vesicles.
  • MLV multilamellar large vesicles
  • a second group of preparation procedures uses suitable detergents, e.g. bile salts or alkylglycosides.
  • suitable detergents e.g. bile salts or alkylglycosides.
  • cmc critical micellar concentration
  • they dissolve membrane lipids in an aqueous phase to form mixed micelles, in which the detergents are in equilibrium with free detergent monomers.
  • mixed micelles Upon reducing the monomer concentration, mixed micelles lose their bound detergent molecules and are forced to fuse to larger disc-like aggregates and finally vesiculate and form liposomes when the free detergent concentration drops to about the cmc.
  • Common procedures of detergent removal from the mixed micelles are dilution, gel chromatography, dialysis through hollow fibres or through thin membranes.
  • the major advantage of detergent removal techniques is the possibility to tailor the size of the liposomes and to yield almost exclusively large unilamellar vesicles (LUV).
  • LUV unilamellar vesicles
  • this is the most common method to prepare membrane protein associated liposomes. With this technique, lipids, detergent and membrane proteins are mixed together, forming mixed micelles and by controlled dialysis, vesicles are formed, entrapping the membrane protein.
  • this high quality of the product is only obtained when low lipid concentrations are used. Above ⁇ 2% lipid (w/w), the size distribution of the liposomes becomes broader and the number of lamellae increases.
  • a third group of procedures starts with dissolving the lipids in an organic solvent and mixing it with an aqueous phase.
  • concentration of the organic solvent is then reduced by suitable procedures.
  • Ether can be evaporated from an excess of warm water, ethanol can be diluted or filtrated or the organic solvent is evaporated until the coherent outer organic phase is removed and a phase reversion occurs (reverse phase evaporation).
  • the most frequently applied method for membrane protein reconstitution involves the co-solubilization of membrane proteins and phospholipids. After solubilization the detergent is removed, leading to spontaneous formation of liposomes with bilayer membranes in which protein is incorporated.
  • Detergents can be removed by several ways, like dialysis, dilution or gel filtration. Additionally, detergents can also be removed by adsorption to hydrophobic resins or cyclodextrins. Such methods are disclosed, for instance, in WO 92/13525, WO 97/41834 or WO 01 /26628.
  • the size distribution of the proteoliposomes formed during the process of liposome preparation depends on the fusogenic properties of the detergent and the ratio of detergent to phospholipid, but even more on the method and rate of detergent removal. If detergent removal proceeds very slowly and hence takes a very long time, like in dialysis or detergent absorption processes, large proteoliposomes will form based on fusion events.
  • Lamellarity is also influenced by the rate of detergent removal. Slow removal results in oligo- and multilamellar structures being formed.
  • Lenz et al. J Biol Chem 280(6), 2005; 4095-4101 disclose a method based on the crossflow injection technique described in full detail in WO 02/36257, wherein the detergent is not removed by dialysis or absorption but instead is rapidly diluted after liposome formation. Using this method, detergent- saturated liposomes do not have sufficient time to fuse and will thus primarily yield small unilamellar liposome structures.
  • the present invention which is substantially based on said crossflow injection technique, relates to an efficient and reproducible, optionally semi- continuous or continuous method for protein encapsulation into a liposomal membrane in a closed and typically though not exclusively sterile containment. It is in principle a further development of the method of Lenz et al. and allows for a cheap and easily upscalable way to produce stable viro- somes of a desired, pre-defined size and composition.
  • the detergent dilution technique of the present invention can be used for the preparation of virus- like particles, or more specifically virus-like vesicles hereinafter called virosomes, composed of liposomal bilayer membranes and viral proteins incorporated in the membranes, wherein the proteins maintain their native conformation, and wherein said virus-like vesicles are formed immediately after injection of an ethanolic lipid solution into an aqueous, typically micellar, protein solution.
  • virus- like particles or more specifically virus-like vesicles hereinafter called virosomes, composed of liposomal bilayer membranes and viral proteins incorporated in the membranes, wherein the proteins maintain their native conformation, and wherein said virus-like vesicles are formed immediately after injection of an ethanolic lipid solution into an aqueous, typically micellar, protein solution.
  • the viral proteins can be either native proteins derived from lysates of primary virus strains, preferably of primary HIV strains, or can be syntheti- cally, e.g. recombinantly produced, peptides or proteins, such as recombinant trimeric gp41 and variants thereof.
  • the invention also relates to vesicles obtainable or obtained by this process and to the use of said vesicles for medical, therapeutical and/or diagnostic purposes.
  • the invention relates to the manufacture of vaccines for active and/or passive immunization against viral or non-viral infections, and more particularly to the preparation of a vaccine for prophylactic or therapeutic treatment of infectious diseases, particularly of viral infections, including but not limited to HIV-1 infection.
  • proteoliposome shall refer to any liposome containing attached to or inserted into its lipid bilayer membrane a peptide or protein of viral origin.
  • proteoliposome shall refer to a liposome containing attached to or inserted into its lipid bilayer membrane a peptide or protein of any origin, e.g. of bacterial or viral origin. Proteoliposomes thus encompass virosomes.
  • the proteoliposomes manufactured according to the present invention are typically unilamellar, bilayer lipid vesicles that contain peptides and/or proteins in their bilayer membranes.
  • the present invention in a first aspect relates to a lipid vesicle comprising a lipid bilayer membrane and inserted into said membrane at least one antigenic peptide or protein, wherein said at least one peptide or protein is anchored in the membrane without involvement of a crosslinker in a site- oriented manner such that at least an antigenic part of said peptide or protein extends from the vesicle surface membrane to the exterior and is presented essentially in its native conformation, which has in effect that upon administration to a mammalian immune system said antigen presenting vesicle causes a detectable, typically a strong cellular and/or humoral immune response directed against said antigen or antigens.
  • the immune response may comprise a virus abolishing or even neutralizing activity.
  • the cellular and/or humoral immune response triggered upon parenteral administration of a pharmaceutical composition comprising such a vesicle or, more specifically, virosome will exert at least some protective, i.e. prophylactic or therapeutic effect, that is similar or even comparable to the effect caused during infection or vaccination with the naturally occurring counterparts of said antigens or antigen bearing microorganisms, respectively.
  • the invention relates to such a vesicle, wherein its vesicle membrane further comprises at least one antioxidant, said antioxidant particularly of lipophilic nature, in order to allow for insertion into the lipid bilayer membrane, the antioxidant preferably being vitamin E.
  • said peptide or protein is of viral origin obtained by recombinant technology or from a virus lysate. Also, a combination of both, i.e. reconstituted proteins from virus lysate plus recombinant viral proteins, has proven very successful.
  • the invention shall further encompass any other viral and non-viral antigenic, or more specifically pathogenic, peptides and proteins, such as, for instance peptides and proteins obtained from influenza, herpes, FSME, or polio virus or from non-viral species such as Plasmodium species (malaria) or various pathogenic bacteria including E.coli, cocci species such as streptococcus, staphylococcus, pneumococcus, and others.
  • viruses such as, for instance peptides and proteins obtained from influenza, herpes, FSME, or polio virus or from non-viral species such as Plasmodium species (malaria) or various pathogenic bacteria including E.coli, cocci species such as streptococcus, staphylococcus, pneumococcus, and others.
  • Peptides or proteins containing a substantial lipophilic part in their molecular structure apart from the antigenic domain or domains are particularly preferred as they will get anchored and stabilized by the membrane lipids of the virosomal bilayer membrane.
  • the present injection method for liposomal or virosomal vesicle formation at least a share of these proteins or peptides will automatically get fixed to the virosome membrane in the desired site-oriented manner, i.e. extending with a more hydrophilic part from the vesicle surface of the virosome, thus presenting at least one antigenic domain to the surrounding environment in a physiologically functional, typically fully physiological functional, particularly native conformation.
  • Suitable peptides and proteins will thus be selected primarily from the groups of glycoproteins and lipoproteins. However, any antigenic proteinaceous structures artificially combined with one or more lipophilic portions by recombinant or conventional chemical technology are also encompassed herein.
  • the invention relates to such a vesicle, which is a reconstituted virus from a virus lysate and therefore comprises viral proteins and lipids from the original virus or virus lysate, respectively, optionally supplemented with additional lipids.
  • the invention relates to such a vesicle, which is unilamellar and has an average size of 100 - 300, preferably of 120 - 200, most preferably of 130 - 160 nm. That is, the present virosomes may be produced very cost-effectively, for example applying a single step procedure as disclosed herein, in a way such that they very closely resemble their naturally occurring wildtype virus counterparts both in lipid and protein composition but also in size.
  • the invention relates to such a vesicle, which is void of any replicable DNA or RNA. This is very important to make the vesicles safe for medical use.
  • the invention relates to such a vesicle, wherein the peptide or protein comprises at least one HIV-derived peptide or protein, preferably selected from the group consisting of gp41 , gp41 -CTM, gp41 CHRTM5, gp120, gp140, gp1 60, nef, tat, and p24.
  • the invention relates to such a vesicle, wherein the lipid vesicle is a reconstituted virus from a virus lysate and comprises, in addition, recombinant viral protein or antigenic parts thereof such as, for example, gp41 -CTM and/or recombinant gp41 CHRTM5 of HIV-1 .
  • a virus lysate comprises, in addition, recombinant viral protein or antigenic parts thereof such as, for example, gp41 -CTM and/or recombinant gp41 CHRTM5 of HIV-1 .
  • the invention relates to such a vesicle, which further comprises vitamin E in its bilayer membrane, which not only improves the vesicle shelf life but also contributes to the overall performance of the vesicles when utilized as active ingredients in vaccine formulations.
  • the invention relates to such a vesicle, wherein the membrane lipids are selected from the group consisting of di-palmitoyl- phosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidyl-ethanol- amine (PE), phosphatidylglycerol (PG), egg-phosphatidylcholine (E-PC), egg- phosphatidyl-ethanolamine (E-PE), egg phosphatidylglycerol (E-PG), the lipid mixture E80S, and cholesterol. It is within the scope of the present invention that other lipids may be selected for specific purposes, too.
  • DPPC di-palmitoyl- phosphatidylcholine
  • PC phosphatidylcholine
  • PE phosphatidyl-ethanol- amine
  • PG phosphatidylglycerol
  • E-PC egg-phosphatidylcholine
  • E-PE egg-phosphatidyl-
  • the invention relates to a method of making such lipid vesicles, e.g. proteoliposomes or special virosomes, which comprise a lipid bilayer membrane and inserted into said membrane at least one antigenic peptide or protein, the method comprising: a) providing an aqueous phase comprising antigenic peptides or proteins solubilized therein by means of a detergent; b) injecting an ethanolic lipid solution into said aqueous phase under essentially shear-free conditions at a temperature selected from 20 - 40 0C, preferably selected from 28 - 35 0 C, most preferably selected from 29 - 31 0 C and diluting the resulting lipid-enriched aqueous phase using a dilution buffer in order to reduce the detergent concentration below the critical micelle forming concentration, thereby causing spontaneous formation and stabilization of lipid vesicles having incorporated in their lipid bilayer membranes said antigenic peptides or proteins; and c) harvesting the
  • said lipid vesicles contain said peptides or proteins inserted into the membrane in a site-oriented way such that they extend at least in part from the vesicle surface or membrane to the exterior and wherein such extending parts comprise at least one antigenic or pathogenic structure or domain having physiological functionality, optionally full physiological functionality, as compared to the naturally occurring counterpart upon infection of a mammalian immune system.
  • the invention relates to such a method of manufacture, wherein said peptides or proteins are of viral origin and are provided in the form of a virus lysate or as recombinant proteins or as a combination of virus lysate and recombinant proteins, the peptides or proteins preferably comprising at least one HIV protein selected from the group consisting of gp41 , gp41 -CTM, gp41 CHRTM5, gp120, gp140, gp160, nef, tat, and p24.
  • the invention relates to such a method of manufacture, wherein the step of providing said aqueous phase comprises solubilizing said peptides or proteins in a suitable buffer, optionally PBS, in the presence of a suitable detergent at a detergent concentration beyond the critical micelle forming concentration (cmc), thus allowing for micelle formation, the detergent optionally selected from bile salts and alkylglycosides, and preferably selected from the group consisting of octyl-b-D-glucopyranoside (b-OG), 3- [N-(3-cholanamidopropyl)-dimethylammonio]-1 -propanesulfonate (CHAPS), and octaethyleneglycol monododecylether (C 12 E 8 ).
  • a suitable buffer optionally PBS
  • a suitable detergent at a detergent concentration beyond the critical micelle forming concentration (cmc)
  • the detergent optionally selected from bile salts and alkylglycosides, and
  • the invention relates to such a method of manufacture, wherein the peptides or proteins are provided in the form of a virus lysate that has been subjected to a treatment of nucleic acid digestion prior to the aforementioned solubilization step, preferably to an enzymatic DNA and/or RNA digestion, optionally using Benzonase® as a digesting enzyme.
  • the invention relates to such a method of manufacture, wherein the virus lysate has been subjected to a reverse transcriptase inactivating treatment, in order to render the virosomes replication-inactive, for a safe use as vaccine ingredients.
  • the invention relates to such a method of manufacture, wherein the detergent concentration for solubilizing the peptides or proteins is selected from within a range of from 0.01 - 2% (w/v).
  • the invention relates to such a method of manufacture, wherein after injection of the ethanolic lipid solution in step (b) the resulting aqueous phase is subjected to a dilution by at least a factor of 3, preferably by at least a factor of 5, and most preferably by a factor of between 5 and 10.
  • the dilution factor may well exceed 10 and may, for example, be in a range of from 10-100.
  • the invention relates to such a method of manufacture, wherein said dilution is typically carried out within 2 - 120 seconds, preferably 5 - 60 seconds, after injection of the ethanolic lipid solution.
  • the invention relates to such a method of manufacture, wherein said method is operated in a semi-continuous or continuous mode, which is one of the big advantages of the cross-flow injection technique. It is possible to carry out the entire process under aseptic, GMP-compliant conditions in a discontinuous, semi-continuous or continuous mode.
  • the invention relates to such a vesicle for use as a medicament for prophylactic or therapeutic treatment of viral and non-viral infections, particularly for use as a vaccine for active or passive immuni- zation.
  • the vesicle may be part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.
  • the invention relates to such a use in the manufacture of a pharmaceutical composition, typically a vaccine, for active or passive immunization suitable in the prophylactic or therapeutic treatment of viral or non-viral infections, particularly of HIV-1 or influenza virus infection.
  • the invention relates to such a use, wherein said pharmaceutical composition is adapted for intramuscular, subcutaneous, or mucosal, e.g. intranasal or vaginal, administration.
  • the pharmaceutical composition typically a vaccine preparation, may be in the form of a liquid virosome suspension for injection, or in the form of a sprayable liquid preparation, e.g. for intranasal delivery, or in the form of a gel, ointment, or cream for other topical, particularly mucosal applications.
  • Further galenic forms of preparation are within reach of the ordinarily skilled artisan and may be useful as well.
  • Fig.1 exhibits the ELISA results upon immunization of rabbits using the virosomal vaccine preparations according to the present invention.
  • Fig.2 exhibits part of the results of Fig.1 at an enlarged view.
  • Fig.3 exhibits results of a PBMC neutralization assay using sera from rabbits immunized with the virosomal vaccine preparations according to the present invention.
  • A1 , B1 , C1 , D1 , E1 , F1 relate to serum samples pre-incubated with 125 ⁇ l IL-2 medium;
  • A2, B2, C2, D2, E2, F2 relate to serum samples pre-incubated with H9/PF cells;
  • F1 , F2 positive controls using neutralizing anti-HIV antibody 2F5;
  • E1 , E2 negative controls (pre-immunization sera);
  • A1 through D2 experimental samples.
  • proteoliposomes In order to gain a better control in the formation of proteoliposomes, the inventors developed a technique, where they combine the crossflow injection technique with detergent dilution in one operational step.
  • the hardware for manufacturing the present lipid vesicles comprises the crossflow injection module, vessels for the polar phase (PBS buffer solution and protein/detergent solution), an ethanol/lipid solution vessel and a nitro- gen pressure device.
  • the crossflow injection modules are made of two stainless steel tubes welded together in a substantially right angle thus forming either a cross or a T. At the connecting point the tubes are in liquid connection with each other through a common orifice, i.e. the injection hole.
  • a lipid mixture is dissolved in 95% ethanol at physiological temperature, preferably between 25 and 40 0 C and most preferably at a temperature at or near 30 0 C, i.e. at 30 ⁇ 1 0 C.
  • the protein/detergent solution is also tempered at physiological temperature, preferably between 25 and 40 0 C and most preferably at a temperature at or near 30 0 C, i.e. at 30 ⁇ 1 0 C, thereby preventing thermal damage of the biological material, e.g. of viral proteins, to generate proteoliposomes containing proteins having maintained their native conformation.
  • this lower temperature range instead of the higher temperature range operated by Lenz et al. results in proteoliposomes, e.g.
  • the observed significant difference in proteo- liposome performance may be due to the complex phase transition reactions right after injection of the lipid phase into the polar aqueous proteinaceous phase, i.e. when the micelle forming peptide or protein molecules solubilized by the detergent change their solution behavior upon reduction of the stabilizing detergent concentration.
  • the liposome suspensions were usually produced at an injection pressure of 5 bar and an injection buffer flow rate of 250 ml/min.
  • an aqueous micellar protein/detergent solution is pumped from a vessel A to a vessel B. While pumping the micellar protein/detergent solution through the crossflow injection module the ethanol/lipid solution is injected, substantially at a right angle, into the aqueous phase of micellar protein/detergent solution, followed by immediate dilution with PBS in vessel B (one way mode).
  • the lipid/ethanol solution used for the preparation of liposomes of the present invention typically contained E8OS, which is a mixture of egg- phosphatidylcholine (E-PC), egg-phosphatidyl-ethanolamine (E-PE), egg phosphatidylglycerol (E-PG), egg phosphatidyl-ethanolamine (E-PE) (all from Lipoid GmbH (Germany)), and cholesterol (from Solvay Pharmaceuticals (Netherlands)), preferably in a molar ratio of 6.7 : 0.4 : 1 .7 : 1 .1 .
  • Lipid concentration per ml aqueous phase ranged from 10 to 25 ⁇ mol/ml aqueous phase.
  • other lipid composition may also be suitable.
  • Vitamin E lipid-soluble or hydrophobic antioxidant
  • Vitamin E was added - based on total lipids - in an amount of 15 - 20 %, preferably in an amount of 17 - 18 %, but may also be useful at lower concentrations, e.g. between 0 and 15%, as well as at higher concentrations of up to 30%, based on total lipids.
  • the proteins used in the examples for incorporation in the liposomal membrane were selected from native proteins derived from lysates of primary virus strains, preferably of primary HIV strains, and proteins that were recombinantly produced, preferably recombinant trimeric gp41 and variants thereof. It is pointed out, however, that any other antigenic protein or peptide comprising a substantial lipophilic portion may be successfully employed according to the present invention, for instance lipoproteins or glycoproteins, and antigenic parts thereof comprising such a lipophilic portion.
  • the virus strains HIV-1 92UG001 and HIV-1 92UG029 were preferably used as primary virus strains.
  • the non-ionic detergent octaethylene glycol monododecylether (Ci 2 E 8 ) was preferred as a detergent due to its high molecular weight of 538.8 Da and its low cmc of 0.1 1 mM (0.006%). It was used at a concentration well above the cmc of 0.006%, preferably at a concentration between 0.01 % and 0.02% and most preferably at a concentration of about 0.0156% (w/v).
  • virus production media used herein were typically protein free and did not contain any ingredients of animal sources, in order to avoid contamination with adventitious pathogens.
  • the main steps to ensure that no infectious viruses are present in final proteliposomal preparations comprise the above-mentioned detergent treatment and, additionally, a Benzonase ® degradation step. It could be shown by co-culture experiments that by treatment of virus supernatants with detergent C 12 E 8 and Benzonase ® complete abrogation of infectivity was obtained.
  • octyl-b-D-gluco- pyranoside (b-OG) or CHAPS octyl-b-D-gluco- pyranoside
  • b-OG octyl-b-D-gluco- pyranoside
  • CHAPS octyl-b-D-gluco- pyranoside
  • Recombinant gp41 and variants thereof were contained in the injection buffer at a concentration of 1 - 2 mg/ml.
  • the detergent concentration is reduced by a factor of 5 to 10, i.e. to a concentration of 0.15% - 0.3% for b-OG and CHAPS, or to 0,003% for C12E8 , resulting in a stable liposome solution.
  • proteoliposomes according to the present invention may therefore be produced to be of the size of virions and to comprise a lipid composition closely similar to the one of a virus membrane.
  • Example 1 Preparation and Characterization of HIV Virosomes a) Virosome preparation
  • Virosomes containing either recombinant trimeric HIV-1 gp41 or native HIV- 1 proteins from lysates derived from primary HIV-1 strains are produced.
  • gp41 antigens that expose epitopes of the well known broadly neutralizing monoclonal antibodies (mAbs) 2F5 and/or 4E10 in a fully functional way were generated.
  • mAbs broadly neutralizing monoclonal antibodies
  • the following gp41 constructs were produced in high quantities and with high purity and provided for incorporation into liposomes:
  • gp41 -CTM which contains the authentic trimeric transmembrane region and the membrane proximal epitopes of mAbs 2F5 and 4E10.
  • gp41 -int which represents the pre-hairpin intermediate structure of gp41 , containing the trimeric transmembrane region and the C-terminal extracellular part of gp41 linked to a trimeric coiled coil region at the N- terminus.
  • gp41 -CHRTM5 which contains the gp41 HR2 region, the epitopes for mAbs 2F5 and 4E10, the Env transmembrane region and a few residues of the cytoplasmic domain of gp41 .
  • the gp41 antigens were produced in large quantities from E. coli expression systems (2 to 5 mg from 1 liter E. coli culture) and purified.
  • H9 cells a subclone of the HuT79 cell line
  • Primary HIV-1 92UG029 (subtype A) was used for preparations and immunization experiments.
  • C 12 E 8 was chosen for further work.
  • the solubilization step led to complete abrogation of infectivity as evidenced by co-culture experiments.
  • the virus lysates were treated with Benzonase ® (Merck KGaA, Darmstadt). This endonuclease approved by the FDA and EMEA for GMP production of biopharmaceuticals and vaccines attacks all forms of DNA and RNA over a great range of operating conditions and reduces them to oligonucleotides down to approximately three to five base pairs in length.
  • RNA/DNA degradation was confirmed by using the RiboGreen RNA Quantitation Kit (Molecular Probes) as well as RT-PCR.
  • the one step detergent dilution technique for the generation of HIV virosomes is based on the crossflow injection technique and is preferably carried out as set out hereinafter:
  • the preparation system consists of the crossflow injection module, vessels for the polar phase (e.g. PBS buffer solution and protein/detergent solution), an ethanol/lipid solution vessel and a nitrogen pressure device.
  • the crossflow injection modules used for these studies are made of two stainless steel tubes welded together forming a cross or a T. At the connecting point the modules are adapted with an injection hole (typically 250 ⁇ m in diameter).
  • the lipid mixture was dissolved in stirred 95% ethanol at 30 0 C.
  • the protein/detergent solution was also tempered at 3O 0 C thereby ensuring a least possible degeneration of proteins.
  • C 12 E 8 was used as a preferred detergent.
  • octyl-b-D-glucopyranoside (b-OG) or CHAPS was preferably used as the detergent and recombinant gp41 was contained in the injection buffer at a concentration of 1 - 2 mg/ml.
  • the lipid/ethanol solution used for the preparation of liposomes usually contained E80S, egg phosphatidylglycerol (E-PG) and egg phosphatidyl-ethanolamine (E-PE) (all from Lipoid GmbH (Germany)) and cholesterol (from Solvay Pharmaceuticals (Netherlands)) in a molar ratio of 6.7 : 0.4 : 1 .7 : 1 .1. Lipid concentration per ml aqueous phase ranged between 10 - 25 ⁇ mol/ml aqueous phase and concentration increased after filtration procedures.
  • E-PG egg phosphatidylglycerol
  • E-PE egg phosphatidyl-ethanolamine
  • the liposome suspensions were produced at 5 bar injection pressure and an injection buffer flow rate of 250 ml/min.
  • the protein/detergent solution was pumped from one vessel to the other.
  • the micellar solution was pumped from vessel A to a vessel B thereby linearly passing through the injection module. While the micellar protein solution is passing through the module the ethanol/lipid solution is being injected from an essentially right angle into said aqueous protein phase passing by said injection hole, followed by immediate dilution of the resulting mixture containing the spontaneously formed proteoliposomes with e.g. PBS in vessel B.
  • the detergent concentration is reduced by a factor of about 5 to 10, i.e. down to a concentration of about 0.15% - 0,3% (b-OG and CHAPS) or 0.003% (C 12 E 8 ) resulting in a stable liposome suspension.
  • Liposome size and size distribution measurement was performed by Dynamic- Laser-Light-Scattering with a Malvern Nano ZS. This system is equipped with a 4 mW Helium/Neon Laser at 633nm wavelength and measures the liposome samples with the non invasive backscatter technology at a detection angle of 171 deg. Liposomes were diluted with PBS-buffer and the experiments were carried out at room temperature. Mean diameters of 1 10 nm - 120 nm having a polydispersity index of 0.18 - 0.21 depending on the preparation parameters, the chosen lipid concentration and detergent could be measured with high accuracy and very narrow size distributions, thus closely resembling the natural virus particle.
  • the average amount of encapsulated and non-entrapped recombinant gp41 was determined by SDS-Page on a Novex system and Western blot analysis.
  • the virosome sample was separated from free protein by diafiltration.
  • the membrane incorporated gp41 was determined in the retentate and the non-entrapped protein was quantified in the filtrate.
  • the filtered virosome sample (retentate) and the unbound protein (filtrate) were spotted onto an electrophoresis gel (e.g. NOVEX Tris/Glycine gel).
  • the gel was electro-blotted onto PVDF lmmobilon P 0.45 ⁇ M membrane (Millipore) for 2 hours and viral membrane antigens were specifically stained with hmAb 2F5 and 4E10. These antibodies can be visualized with anti human IgG conjugated with alkaline phosphatase. It was impressively shown that nearly all admitted gp41 was integrated into the lipid bilayer system and that no degradation of the membrane protein occurred. The protein content from primary virosome preparations was also analyzed by SDS-PAGE and Western Blot analysis: p24, gp41 and gp120 were detected.
  • FACS Vantage Becton Dickinson, San Jose, CA
  • 5W Argon Laser Coherent Innova 305, St. Clara, CA
  • Membrane incorporated proteins can be detected via FACS by an antibody sandwich consisting of hmAb 2F5 and 4E10 plus quantum red marked anti human IgG antibody and FITC.
  • the virosome sample was split into four groups: Group 1 : gP41 - virosomes + FITC; Group 2: gP41 - virosomes + 2F5 + FITC; Group 3: gP41 - virosomes + anti human IgG quantum red + FITC; Group 4: gP41 - virosomes + 2F5 + anti human IgG quantum red + FITC Because of the intensive FITC signal, this configuration allows virosome detection in the FACS. Groups 2 and 3 were done to determine unspecific binding and unspecific signals and virosomes of group 4 should show specific membrane antigen - antibody binding.
  • gp41 ctm PBS, 1 % beta OG
  • gp41 ctm incorporated into liposomes PBS
  • EGS ethyleneglycol bis(-succinimidylsuccinate)
  • mice vaccination studies lmmunogenicity of trimeric gp41 in solution and when incorporated into liposomes was tested.
  • BALB/c inbred female mice 6-8 weeks of age were obtained from the Institute of Experimental Animals Breeding at Himberg, Austria. The animals were housed under standard conditions and were provided with standard diet and water ad libitum. Mice were immunized intraperitoneal ⁇ (i.p.) with gp41 ctm virosomes or with gp41 ctm alone, or as a control, with empty liposomes. The second immunization was performed three weeks later with the same preparations.
  • mice were bled from Sinus orbitalis and sera were stored at -20 0 C before further assaying.
  • the reactivity of the sera against gp41 ctm was tested using a standard ELISA protocol.
  • gp41 CTM protein 2.5 ⁇ g/ml carbonate buffer, pH 9.6 was used as a coating antigen.
  • Serial dilutions of sera in PBS/Tween containing 1 % skim milk were added to the coated plates and the mixtures incubated for 1 .5 h at room temperature. Bound antibodies were detected with goat anti-mouse IgG I - and lgG2a ( ⁇ - chain-specific) conjugated with horse-radish peroxidase (Zymed).
  • mice immunized with gp41 ctm liposomes showed a strong IgG I and lgG2a specific immune response against gp41 ctm up to a reciprocal titer of 2560.
  • 3 mice out of 5 immunized with soluble gp41 ctm showed a weak response with reciprocal titers between 250 and 640.
  • low reciprocal titers for IgA ⁇ 150
  • mice immunized with gp41 ctm liposomes no IgA response was determined in the gp41 ctm group. This indicates that gp41 ctm has poor antigenicity by itself.
  • gp41 ctm shows considerable antigenicity after only two rounds of immunization.
  • Inhibition of HIV-1 replication by serum samples was assessed in a standard syncytium inhibition assay. Experiments were performed with sera from mice immunized with gp41 or gp41 -containing liposomes. Sera from mice, which had received liposomes only, were run in parallel as negative controls. AA-2 cells were used as an indicator cell line with syncytium formation as a read-out.
  • Sera from the control group (liposomes only) showed unspecific inhibition of syncytium formation up to a maximum titer of 1 :28.2.
  • two gp41 ctm liposome derived sera neutralized the HIV-1 RF strain at a slightly higher titer than observed by control sera (1 :33.6 and 1 :40).
  • a third serum derived from gp41 ctm liposome immunization showed a significantly higher titer of 1 :57 while the other two sera tested showed neutralizing titers comparable to the negative control.
  • only one serum from the group immunized with soluble gp41 ctm showed a potential neutralizing titer slightly above the control group.
  • proteoliposomes derived from virus lysates (strain HIV-1 92UG029) and recombinant gp41 was tested.
  • the proteoliposomes were produced according to the method disclosed in Example 1 .
  • Proteoliposomes of group 1 and 2 additionally contained 17.6 % of vitamin E, based on total lipids.
  • the experiment comprised three groups of rabbits each comprising four animals. They were immunized four times intraperitoneally with three weeks between the first three injections and two weeks between the third and the fourth injection. Pre-immunization sera were obtained as control samples. The final blood samples were taken one and a half week after the last boost. All sera were centrifuged (1200 rpm, 4 0 C, 10 min) and supernatants aliquoted before storage at -20 0 C.
  • gp41 CHRTM5, gp41 , gp120, gp160 and several other epitopes like nef, tat, p24 was tested using a standard ELISA protocol.
  • gp41 CHRTM5, gp41 , gp120, gp160 nef, tat and p24 protein (1 - 2 ⁇ g/ml carbonate buffer, pH 9.6) were used as coating antigens.
  • Serial dilutions of sera in PBS/Tween containing 1 % skim milk were added to the coated plates and the mixtures incubated for 1 .5 h at room temperature.
  • Bound antibodies were detected with goat anti-rabbit IgG (whole chain) conjugated with peroxidase (Sigma). Following additional washing steps plates were stained with o-phenyl- enediamine dihydrochloride (OPD) as a substrate. The reaction was stopped with 2.5 M H 2 SO 4 and the plates were measured. Sera were analyzed by ELISA using the blank wells' mean plus three times their standard deviation as cut-off.
  • Fig. 1 and Fig. 2 disclose the immunization titers obtained in this experiment.
  • Serum derived from group 1 exerted a highly increased reactivity towards tat and p24 and some increased reactivity towards gp41 , gp 140 and nef as compared to the pre-immunization control serum.
  • Serum derived from group 2 also showed a highly increased reactivity towards tat and p24 and some increased reactivity towards gp41 , gp140 and nef as compared to the pre-immunization serum.
  • PBMC neutralization assays were carried out with the sera obtained from the animals at day 35 and at day 66 after the first immunization (also indicated by column headers in tables 2 and 3).
  • the 50% / 90% / 95% inhibiting concentration (IC50, IC90, IC95) was determined by p24-ELISA Experiments were performed with 4 replicates per dilution step. Pre-immunization sera were taken as control samples. The neutralizing mAb 2F5 was used as comparative control. All assays included a virus titration of the inoculum to confirm the infectious titer.
  • Another serum (66/07; Table 3) derived from group 2 showed a strong neutralizing activity, too, up to a dilution of 1 :640. Most of the other sera tested showed neutralizing activities in the range of the negative controls (-1 /02, -1 /05, -1 /06, -1 /07).

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Abstract

La présente invention concerne une vésicule lipidique qui contient une membrane bicouche lipidique et au moins un peptide antigénique ou une protéine antigénique, inséré dans ladite membrane, ledit ou lesdits peptides ou ladite ou lesdites protéines étant ancrés dans la membrane sans qu'un agent de réticulation ne soit impliqué, selon un mode d'orientation par rapport au site tel qu'au moins une partie antigénique dudit peptide ou de ladite protéine s'étend de la membrane superficielle de la vésicule vers l'extérieur et est sensiblement présentée sous une conformation physiologiquement fonctionnelle, particulièrement sous sa conformation native. Lors de son administration à un système immunitaire mammalien, ladite vésicule provoque en outre une forte réponse immune. L'invention concerne également un procédé de fabrication desdites vésicules lipidiques, ainsi que des procédés permettant de les utiliser en tant que médicaments et de fabriquer des vaccins destinés au traitement prophylactique ou thérapeutique d'infections virales ou non virales.
PCT/EP2007/006561 2006-07-24 2007-07-24 Protéoliposomes antigéniques et procédé de fabrication WO2008012062A1 (fr)

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RU2575830C2 (ru) * 2010-06-24 2016-02-20 Дженентек, Инк. Композиции и способы, включающие алкилгликозиды, для стабилизации содержащих белки составов
CN111560352A (zh) * 2020-06-02 2020-08-21 新疆农业大学 一种病毒样囊泡及其制备方法和应用

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US10188735B2 (en) 2010-06-24 2019-01-29 Genentech, Inc. Compositions and methods for stabilizing protein-containing formulations
WO2011163458A3 (fr) * 2010-06-24 2012-06-07 Genentech, Inc. Compositions et méthodes pour stabiliser des préparations contenant des protéines
JP2013533244A (ja) * 2010-06-24 2013-08-22 ジェネンテック， インコーポレイテッド タンパク質含有製剤の安定化のためのアルキルグリコシドを含む組成物及び方法
US9254321B2 (en) 2010-06-24 2016-02-09 Genentech, Inc. Compositions and methods for stabilizing protein-containing formulations
RU2575830C2 (ru) * 2010-06-24 2016-02-20 Дженентек, Инк. Композиции и способы, включающие алкилгликозиды, для стабилизации содержащих белки составов
CN103068367B (zh) * 2010-06-24 2016-09-07 弗·哈夫曼-拉罗切有限公司 用于稳定含有蛋白质的制剂的含有烷基糖苷的组合物和方法
CN103068367A (zh) * 2010-06-24 2013-04-24 弗·哈夫曼-拉罗切有限公司 用于稳定含有蛋白质的制剂的含有烷基糖苷的组合物和方法
KR102058307B1 (ko) 2010-06-24 2019-12-20 제넨테크, 인크. 단백질-함유 제제를 안정화시키기 위한 알킬글리코시드 함유 조성물 및 방법
US11103584B2 (en) 2010-06-24 2021-08-31 Genentech, Inc. Compositions and methods for stabilizing protein-containing formulations
US11938189B2 (en) 2010-06-24 2024-03-26 Genentech, Inc Compositions and methods for stabilizing protein-containing formulations
EP3586826A1 (fr) * 2010-06-24 2020-01-01 F. Hoffmann-La Roche AG Compositions et procédés de stabilisation de formulations contenant des protéines
EP4008313A1 (fr) * 2010-06-24 2022-06-08 F. Hoffmann-La Roche AG Compositions et procédés de stabilisation de formulations contenant des protéines
CN111560352B (zh) * 2020-06-02 2023-03-24 新疆农业大学 一种病毒样囊泡及其制备方法和应用
CN111560352A (zh) * 2020-06-02 2020-08-21 新疆农业大学 一种病毒样囊泡及其制备方法和应用

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