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WO2016039620A2 - Virosomes du virus respiratoire syncytial - Google Patents

Virosomes du virus respiratoire syncytial Download PDF

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Publication number
WO2016039620A2
WO2016039620A2 PCT/NL2014/050628 NL2014050628W WO2016039620A2 WO 2016039620 A2 WO2016039620 A2 WO 2016039620A2 NL 2014050628 W NL2014050628 W NL 2014050628W WO 2016039620 A2 WO2016039620 A2 WO 2016039620A2
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rsv
synthetic
virosomes
virosome
viral
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WO2016039620A3 (fr
Inventor
Antonius Johannes Henrikus STEGMANN
Joan Claudia Maureen SOEI-KEN TJON
Farien Irasia BHOELAN
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Bestewil Holding BV
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Bestewil Holding BV
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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
    • A61K39/12—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
    • A61K39/39—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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18523—Virus like particles [VLP]
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of immunology and vaccinology .
  • the invention relates to Respiratory Syncytial Virus (RSV) virosomes, and virosomal vaccines comprising them.
  • RSV Respiratory Syncytial Virus
  • Vaccines against membrane-containing (enveloped) viruses mostly consist of killed or live attenuated viruses, or a preparation of their proteins (e.g. split virus vaccines or subunit preparations). Vaccination with killed viruses and protein preparations is safer than vaccination with replicating live attenuated or recombinant viruses, because the latter may mutate or revert back to wild-type virus.
  • Subunit vaccines result in fewer local and systemic side effects and also have the clear advantage that they can be prepared from recombinant viral proteins expressed by cells rather than from virus, making production safer and eliminating the risk of
  • live viruses generally induces strong cellular and antibody immune responses, protecting against future infections by the virus, non- replicating vaccines such as protein preparations, particularly membrane protein preparations, may fail to do so, inducing predominantly an antibody response.
  • Infected cells can present material from the infecting pathogen on MHC-1 molecules on their surface, initiating cellular immune responses, such as a cytotoxic T-cell response. Many protein preparations that are not produced within the cell, will not be presented to the immune system in this manner. Live or killed viruses will also be taken up preferentially by specialized phagocytic cells of the immune system, such as dendritic cells, and be presented to other cells of the immune system, triggering an immune responses.
  • phagocytic cells patrol the body, ingesting particles of the size of viruses, but they do not efficiently take up purified proteins such as those from split virus or subunit vaccines.
  • a particular problem with membrane proteins is that these are not soluble in water. Therefore, for successful presentation to antigen-presenting cells these proteins need some form of solubilization, allowing their use in a vaccine.
  • Numerous attempts to reinforce the immune response to subunit or protein preparations by physical or chemical means have been undertaken. The most important principle that emerges from these experiments is that multiple copies of the viral proteins need to be combined in particles that will be taken up efficiently by phagocytic cells. These particles can be virosome-like-p articles, virosomes, Immune-Stimulating Complexes
  • ISCOMs immunological complex lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipids, and the like.
  • adjuvants chemical substances that are meant to stimulate the immune system
  • Virosomes are the reconstituted membranes of enveloped viruses. Virosomes are a particularly useful kind of vaccine composition. Virosomes are generally produced by extraction of membrane proteins and lipids from enveloped viruses with a detergent or short-chain phospholipid, followed by removal of this detergent or short chain phospholipid from the extracted lipids and viral membrane proteins, in fact reconstituting or reforming the characteristic lipid bilayers (envelopes) that surround the viral core or nucleocapsids (W02004/071492, Stegmann T. et al, 1987, EMBO J. 6, 2651- 2659).
  • virosomes can be assembled basically from any integral membrane protein or peripheral membrane proteins, or proteins conjugated to lipid anchors.
  • virosomes are particles of the size that is efficiently taken up by phagocytic cells of the immune system, and they closely mimic the composition, surface architecture and functional activities, particularly the membrane fusion activity, of the native viral envelope. Lipids and integral membrane proteins from the virus will therefore be present in a virosomal membrane.
  • Influenza virus and RSV are two classical examples of enveloped viruses. Enveloped viruses in general carry specific integral membrane proteins (the “spikes") which are required for binding to and entry of cells. These proteins are present on the surface of mature virions in a metastable conformation, known as the "pre-fusion form". After binding of the virus to the cells, the first step in infection of cells by influenza and RSV virus is uptake of intact viral particles by receptor-mediated endocytosis. For these viruses, endocytosis is a prerequisite for fusion between the viral and the endosomal membrane, which leads to infection of the cell.
  • influenza membrane fusion is triggered as the endosomal compartment then becomes mildly acidic due to the activity of an ATP-dependent proton pump present in the membrane of the endosome.
  • the influenza hemagglutinin spike proteins undergo a conformational change (from the "pre-fusion” to the "post-fusion” conformation) which drives fusion of the viral membrane with the membrane of the endosome.
  • the viral nucleocapsid and genetic material DNA or RNA
  • influenza HA and RSV-F show strong similarities; both proteins are type I membrane fusion proteins, produced as a single precursor protein; the protein is then cleaved once by a protease to produce a disulfide-linked dimer of HAl and HA2 or Fl and F2 respectively; three such dimers form the HA or F spike.
  • the HA2 and the Fl subunits provide the C-terminal membrane anchor.
  • the HA2 subunit has an N-terminal "fusion peptide", which is buried in the protein at neutral pH and exposed at low pH to enter the endosomal membrane for fusion.
  • the fusion peptide on Fl is 27 amino acids away from the N- terminus; intra-endosomal cleavage removes these 27 amino acids as a single peptide (Kryzaniak et. al. PloS Pathog 9(4): e 10033309; doi: 10.1371).
  • the F and HA spikes undergo extensive
  • the spike proteins After fusion, viral the spike proteins are in a highly stable post-fusion form. Before fusion, the proteins in their pre-fusion conformation, for as found on example on purified virus preparations, are metastable and can be easily triggered to undergo the conformational change, for example by low pH for influenza HA, at low or high pH, low osmolarity or high salt concentrations for RSV F, and at elevated
  • RSV-F protein in the pre-fusion form.
  • Monoclonal antibodies against an epitope unique to RSV-F in the prefusion form, such as 5C4, AM22 and D25 have been found and proven instrumental in the determination of the pre- fusion conformation (McLellan, J.S. et al. Science 2013, may 31; 340
  • Virosomes that are particularly active in inducing an immune response were found to have maintained the proper functions of the envelope proteins of the native virus, such as membrane fusion, receptor- binding and other activities. Preservation of receptor-binding and
  • virosomes with membrane fusion activity are useful as vaccines, having the safety profile of killed vaccines, while providing the immune systems with the stimulus representative of live vaccines.
  • the lipid bilayer of the virosomes must be in the liquid crystalline phase at the temperature of fusion.
  • the phase transition temperature depends mostly on the acyl chain length of the lipids and their degree of unsaturation, and to a lesser extent on the lipid headgroup. In a mixture of lipid species, the phase transition is broadened. However, if the differences in acyl chain length are considerable,
  • hydrophobic mismatch may occur, leading to the formation of separated patches of lipid, and affecting membrane stability.
  • the plane of the membrane should have near zero curvature, which is best achieved with lipids having a cylindrical shape lipid; an example of such a lipid is phosphatidylcholine (PC); the acyl chains diameter of PC matches that of the headgroup.
  • the headgroup of phosphatidylethanolamine (PE) is much smaller, giving the molecule a conical shape; in the membrane, the tip of the cone will face the membrane/water interface, and the molecule will have a tendency to form a curved, hexagonal phase in water Lee, A.G. Biochim. Biophys. Acta 1666 (2004) 62-87).
  • Membrane fusion is promoted by the presence of such hexagonal phase lipids in the membrane.
  • lipids will not by themselves form bilayers; they need to mixed with lipids producing zero curvature membranes, preferably in the liquid crystalline phase at the temperature of fusion.
  • amphiphilic adjuvants have been incorporated in the membrane of virosomes (WO2004/110486).
  • virus is solubilized with a detergent or short- chain phospholipid, the viral nucleocapsids is removed, and then the adjuvant, dissolved in the same detergent or short-chain phospholipid, is added to the solubilized viral membranes.
  • virosomes that include at least the viral membrane proteins and lipids and the adjuvants.
  • Some amphiphilic adjuvants, such as monophosphoryl lipid A (MPLA) incorporated into the virosomal membrane in this fashion have been shown to be stably integrated in the membrane of RSV virosomes (Stegmann, T et al. Vaccine 2010; 28(34): 5543-50; WO2004/110486) and enhance or alter the immune response following vaccination with RSV virosomes in preclinical trials (Kamphuis, T. et al. Plos One 2012; 7 (5):e36812).
  • MPLA is derived from lipopolysaccharide (LPS), present in the membrane of Gram -negative bacteria, which is well known to be a strong, but toxic, stimulator of the immune system.
  • LPS lipopolysaccharide
  • MPLA produced from the LPS from the membrane of Salmonella Minnesota Re595 by removal of the core carbohydrate and a phosphate has reduced toxicity, and still is a strong immunopotentiator. Removal of the acyl chain at the 3 position from the disaccharide backbone of MPLA produces 3-O-desacyl MPLA, with still further reduced toxicity.
  • 3-O-desacyl MPLA is present in two marketed vaccines for human use. The 3-O-desacyl MPLA present in these vaccines is a mixture of at least 21 molecules with different numbers and lengths of acyl chains.
  • Phosphorylated Hexa Acyl Disaccharide also called GLA
  • 3- D-PHAD which lacks the acyl chain on the 3 position compared to PHAD and is therefore an analogue of 3-O-desacyl MPLA.
  • 3-O-desacyl monophosphoryl lipid A 3-O-D-MPLA
  • 3-O-desacyl monophosphoryl lipid A 3-O-D-MPLA
  • most lipids presents in viruses are derived from mammalian membranes
  • 3-O-D- MPLA has 12-14 carbon atom acyl chains.
  • Viral membrane proteins have transmembrane domains spanning the thicker mammahan membranes and consequently cannot readily be incorporated in membrane bilayers with 12- 14 carbon acyl chains; their incorporation in thinner membranes leads to protein aggregation and induces acyl chain ordering (Killian, J.A. Biochim. Biophys. Acta 1376 (1998) 401-416), which affects membrane fusion.
  • Membrane fusion proteins from mammalian viruses will not induce fusion efficiently if embedded in short-chain lipids, and 12- 14 carbon acyl lipids form membranes that are in the gel phase, not the liquid crystalline phase, at 37°C. Therefore, the longer chain mammalian lipids are preferred as constituents of virosomal membranes for optimal insertion of viral membrane proteins, but adjuvants are most readily accommodated in shorter chain phospholipids.
  • a mismatch between the membrane lipid and membrane-incorporated adjuvant can profoundly affect membrane formation and stability; mismatched membrane material will tend to aggregate in lumps rather than form virosomes.
  • One solution for this problem is to use a broad mixture of natural lipids containing acyl chains with different lengths, thus more readily capable of accommodating some shorter acyl chains on adjuvants and/or to use a broad mixture of adjuvant chain lengths.
  • mixtures are ideal, but for the production of vaccines, such mixtures of natural adjuvants are problematic, since they are difficult to characterize and standardize.
  • synthetic or essentially pure adjuvants are preferred. Synthetic essentially pure adjuvants or can be accommodated in membranes produced from mixed-acyl chains lipids, but, likewise, these lipids will be difficult to characterize and standardize. Therefore, synthetic or essentially pure lipids are preferred in vaccines.
  • the modal diameter of the virosome populations produced from a mixture of viral membrane extract, added lipids and adjuvants should be significantly smaller than 220 nm.
  • a defined phospholipid mixture combined with specific adjuvant at a defined relative phospholipid/adjuvant yielded sufficiently homogeneous virosomes capable of eliciting immune responses to the incorporated RSV protein.
  • This specific choice for synthetic amphiphilic adjuvants and synthetic lipids in RSV virosomal membranes improves the quality of the formulation for use as a human vaccine and enhances the stability and immunogenicity of virosome-based vaccines.
  • the improved RSV virosomes can be readily produced e.g. by solubilizing the membranes of enveloped viruses with a short-chain phospholipid, followed by removal of the viral nucleic acids by centrifugation.
  • lipids and the adjuvants of the invention are added, solubilized in the same short-chain phospholipid, and the membrane is then reformed by removal of the short - chain phospholipid, resulting in the formation of virosomes. Surprisingly, it was found that the pre-fusion conformation of RSV F is preserved in these virosomes.
  • the present invention provides compositions for the formation of RSV virosomes from a mixture of solubilized viral membranes, synthetic lipids, and synthetic adjuvants, capable of inducing an immune response against RSV, and with long term physicochemical stability.
  • the invention provides a RSV virosome comprising: (i) Lipids and proteins extracted from the membrane of Respiratory Syncytial Virus; (ii) synthetic adjuvant chosen from the group of PHAD and 3-D- PHAD; (iii) at least one synthetic or essentially pure phosphatidylcholine (PC) species and at least one synthetic or essentially pure
  • phosphatidylethanolamine at a molar ratio of 3: 1 to 1:3, characterized in that the acyl chains have between 14 and 18 carbon atoms, the total number of unsaturated bonds in the acyl chains being four , and that the molar ratio of total synthetic phospholipid to adjuvant is between 1.5 and 10;
  • sterol or sterol derivative like cholesterol, at a ratio of 0-30 mol% of total added phospholipid; and (v) optionally, one or more further antigens.
  • the expression "synthetic or essentially pure” refers to an exogenously added, non-viral phospholipid species of defined quality, purity and chemical structure. It does not refer to purified or semi-purified phospholipids of mixed fatty acid composition that are extracted from natural sources, such as tissue-derived, plant-derived or egg-derived PC and PE.
  • the synthetic or essentially pure PC and PE species are manufactured according to the guidelines of Good Manufacturing Practice (cGMP). Synthetic or essentially pure PC and PE species are commercially available, for example from Avanti Polar Lipids, Alabaster, AL.
  • said at least one synthetic or essentially pure PC species and said at least one synthetic or essentially pure PE species are the only non-viral phospholipids in said virosome.
  • RSV virosomes with desirable properties were obtained when the acyl chains of synthetic PE and PC have between 14 and 18 carbon atoms and the total number of unsaturated bonds in the acyl chains of is four.
  • both PC and PE contain acyl chains with unsaturated bonds.
  • the acyl chains can be mono- or di-unsaturated. In view of their reduced susceptibility for oxidation, mono-unsaturated acyl chains are preferred.
  • a RSV virosome wherein each of the acyl chains of PC and PE all contain one unsaturated bond.
  • the PC and/or the PE species is a symmetric phospholipid, i.e. comprising identical acyl chains at the sn-1 and sn-2 position of the glycerol backbone.
  • an RSV virosome according to the invention typically comprises one synthetic or essentially pure PC species and one synthetic or essentially pure PE species.
  • the acyl chains in synthetic PC and/or PE have 16 or 18 carbon atoms, preferably 18 carbon atoms.
  • the total number of carbon atoms in the acyl chains of PC and PE is at least 70. Very good results are obtained if the total number of carbon atoms is 72.
  • RSV virosomes comprising one or more selected from the group consisting of synthetic l,2-dioleoyl-sn.-glycero-3- phosphocholine (DOPC), l,2-choleoyl-S7 -glycero-3-phosphoetanolamine (DOPE), l,2-dipalmitoleoyl-S7 -glycero-3-phosphoethanolamine (PPPE) and l-palmitoyl-2-hnoleoyl-S7 -glycero-3-phosphoethanolamine (PLPE).
  • DOPC synthetic l,2-dioleoyl-sn.-glycero-3- phosphocholine
  • DOPE l,2-choleoyl-S7 -glycero-3-phosphoetanolamine
  • PPPE l,2-dipalmitoleoyl-S7 -glycero-3-phosphoethanolamine
  • PLPE l-palmitoyl-2
  • the RSV virosome comprises synthetic PC consisting of synthetic DOPC and synthetic PE consisting of DOPE.
  • An improved RSV virosome of the invention is furthermore characterized in that the molar ratio between the synthetic or essentially pure PC species and synthetic or essentially pure PE species is between 3: 1 to 1:3.
  • said ratio is between 2: 1 to 1:2.
  • synthetic PC is used in an amount equal to or exceeding that of synthetic PE. Accordingly, provided is an RSV virosome comprising synthetic PC species and synthetic PE species at a molar ratio of between 3: 1 to 1: 1, preferably 2: 1 to 1: 1. In a specific aspect, PC is present in excess of PE, for instance at a molar ratio of between 3: 1 up to 1: 1, preferably 2: 1 up to 1: 1. In another, preferred embodiment, synthetic PC is used in an amount equal to or less than that of synthetic PE. Accordingly, provided is an RSV virosome comprising synthetic PC species and synthetic PE species at a molar ratio of between 1: 1 to 1:3, preferably 1: 1 to 1:2. In a specifically preferred aspect, PE is present in excess of PC, for instance at a molar ratio of between 3: 1 up to 1: 1, preferably 2: 1 up to 1: 1.
  • the addition of further non-viral lipids may enhance one or more desirable properties of the virosomes.
  • a sterol or sterol derivative can be added to increase the storage stability of the virosomes.
  • examples of sterol derivatives that can be incorporated into virosomes of the invention include cholesterol hemisuccinate, phytosterols such as lanosterol, ergosterol, and vitamin D and vitamin D related compounds
  • the RSV virosome of the invention comprises sterol (derivative) at a ratio of 5-30 mol% of total added phospholipid, preferably 10-25 mol%, more preferably about 20 mol%.
  • the virosome comprises DOPC, DOPE and cholesterol, preferably wherein DOPE is present in excess e.g. at least 1.5-fold, of DOPC.
  • a molar ratio of total synthetic phospholipid to synthetic adjuvant between 1.5 and 10 is of relevance for the properties of an RSV virosome. Good results were obtained when the ratio is between 3 and 6, preferably between 3.5 and 5. In a specific aspect, the ration is between 3.7 and 4.5, like 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5.
  • An RSV virosome of the invention is characterized by the presence of a synthetic adjuvant selected from PHAD (phosphorylated hexaacyl disaccharide) and the 3-O-desacyl derivate thereof, 3-D-PHAD. Both are known in the art as synthetic TLR-4 agonists.
  • PHAD is also referred to in the art as Glycopyranoside Lipid A, or GLA. See Lousada-Dietrich et al., , Vaccine. 2011 Apr 12;29(17):3284-92.
  • the RSV virosome contains PHAD, which has the following structure (designations 14 indicate the total number of carbon atoms in each acyl chain):
  • the synthetic adjuvant may be used in an RSV virosome of the invention at a ratio of 0.01-2.5 mg per mg of viral protein, preferably at a ratio of 0.1-2 mg per mg of viral protein, hke 0.5-1.5 mg per mg of viral protein, more preferably at about 1 mg per mg of viral protein, optionally in combination with a molar ratio of total synthetic phospholipid to adjuvant is between 3 and 6, preferably between 3.5 and 5.
  • PHAD or 3-D-PHAD is present at a ratio of 0.01-0.1 or 0.05-0.3 or 0.1-0.5 or 0.1- 1 or 0.2- 1.5 or 0.3-2 or 0.6-2 or 0.2-0.8 or 1 to 2 or 1.4-2.2 per mg of viral protein
  • the RSV virosome comprises about 400-450 nmol DOPC, about 800-900 nmol DOPE, about 250-350 nmol 3-D-PHAD and about 200-300 nmol cholesterol per mg of viral membrane protein
  • a virosome according to the invention comprises proteins extracted from the membrane of Respiratory Syncytial Virus, such as the RSV proteins F, G, SH and/or M. Preferably, it comprises at least RSV F protein.
  • the virosomes comprise the F protein of RSV in the pre-fusion conformation, which is the target of most RSV- neutralizing activity in human sera. See McLellan et al., (Science 2013: Vol. 340 no. 6136 pp. 1113- 1117) describing the structure of RSV-F trimer bound to a prefusion-specific neutralizing antibody.
  • a virosome of the invention comprises RSV-F protein which is recognized by a monoclonal antibody against an epitope unique to RSV-F in the pre-fusion conformation, such as 5C4, AM22 or D25. It was surprisingly found that the pre-fusion form of the RSV-F protein was completely preserved in a virosome having the defined lipid composition of the invention.
  • the capability of the virosomes of the invention to fuse with a host cell is thus dependent on the presence of an appropriate viral fusion protein.
  • lipid composition of the bilayer of the reconstituted viral membrane is further dependent of the lipid composition of the bilayer of the reconstituted viral membrane, as virosomes composed of certain synthetic lipids and viral fusion proteins have been described in the art that are incapable of fusion.
  • the lipid composition of the virosomes is thus preferably chosen such that the membranes are capable of fusion with appropriate host cells at an appropriate pH.
  • One preferred lipid composition that provides the virosomes with fusion activity is a lipid composition that comprises natural lipids of a virus.
  • natural lipids of a virus is herein understood to mean those lipids that are present in the membrane of a virus grown on cells, preferably mammalian, insect or plant cells, or grown on embryonated eggs.
  • the natural lipids of a virus are thus preferably obtained or isolated from virus particles thus grown, as opposed to synthetic lipids.
  • functionally reconstituted viral membranes of the invention comprise purified lipids from other sources, i.e. synthetic PE and PC, in addition to the natural lipids. Typically, the total amount of added
  • phospholipids is in the range of about 500-3000 nmol per mg of viral protein, preferably about 700- 2800 nmol, more preferably about 800-2600 nmol, per mg of viral protein like about 800, 900, 1000, 1200, 1400, 1700 or 2500 nmol per mg of viral protein. Depending on the efficiency of viral lipid extraction, this corresponds to about a 2 to 12-fold excess of synthetic lipid over viral lipids.
  • the invention also provides a method for providing an RSV virosome according to the invention.
  • This preferably comprises the functional reconstitution of RSV by solubilizing the membranes of enveloped viruses with a detergent or short-chain phospholipid, mostly followed by removal of the viral nucleic acids.
  • Other molecules such as lipids, adjuvants or proteins can be added to the solubilized membrane material.
  • the membrane is then reformed by removal of the detergent or short-chain phospholipid, forming the virosomal membrane as a result.
  • the added molecules will be included within the virosomes or integrated in the virosomal membrane.
  • the method contacting the enveloped virus with a solution containing a short-chain phospholipid or a detergent allowing solubilisation of the viral envelope of said virus, further comprising removing short-chain phospholipid or detergent from said solution allowing formation of a functionally reconstituted viral envelope.
  • the phospholipid or detergent is removed by dialysis, filtration, or absorption onto hydrophobic beads.
  • Preferred short-chain phospholipid have a critical micelle concentration (cmc) of larger than 0.1 mM, preferably larger than 0.3 mM, more preferably larger than 1 mM. Very good results were obtained with 1,2-diheptanoyl-sn- phosphatidylcholine or 1,2-dicaproyl-sn-phosphatidylcholine.
  • cmc critical micelle concentration
  • the present invention provides for a pharmaceutical preparation comprising as active ingredient RSV virosomes according to the invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable stabilizing agents, osmotic agents, buffering agents, dispersing agents, and the like may also be incorporated into the pharmaceutical compositions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the virosomes to the patient.
  • Pharmaceutically acceptable carriers for intranasal delivery are exemplified by powdered containing lyophihzed virosomes, water, buffered saline solutions, glycerin, polysorbate 20, cremophor EL, and an aqueous mixture of caprylic/capric glyceride, and may be buffered to provide a neutral pH environment.
  • Pharmaceutically acceptable carriers for intranasal delivery are exemplified by powdered containing lyophihzed virosomes, water, buffered saline solutions, glycerin, polysorbate 20, cremophor EL, and an aqueous mixture of caprylic/capric glyceride, and may be buffered to provide a neutral pH environment.
  • Pharmaceutically acceptable carriers for intranasal delivery are exemplified by powdered containing lyophihzed virosomes, water, buffered saline solutions, glycerin, poly
  • parenteral delivery are exemplified by sterile buffered 0.9% (w/v) NaCl or 5% (w/v) glucose optionally supplemented with a 20% albumin.
  • Preparations for parental administration must be sterile.
  • the parental route for administration of the virosome is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intraarterial or intralesional routes.
  • a typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 0.1 - 1 ml of phosphate buffered saline and 1 to 100 ⁇ £, preferably 15-50 ⁇ £ (of antigen protein) of the virosomes of the present invention.
  • the active ingredient can be administered in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • Liquid dosage forms for oral administration can contain excipients like coloring and flavoring to increase patient acceptance.
  • parenterally, orally or intranasally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack
  • the invention provides an immunogenic
  • composition or vaccine comprising an adjuvanted RSV virosome according to the invention.
  • immunogenic refers to the capacity of eliciting an immune response in a host animal, including producing an antibody response and/or a cell mediated immune response (for example, involving cytotoxic T lymphocytes (CTL)).
  • CTL cytotoxic T lymphocytes
  • the virosome composition according to the invention has a narrow size distribution.
  • a virosome may have a diameter (particle size) generally in the range of 40 to 200 nm, and in particular a diameter from 50 nm to 150 nm, and in particular from 70 nm to 130 nm.
  • the RSV virosomes have a homogeneous size distribution with less than 15%, preferably less than 10% of the virosomes having a particle size above 150 nm, and less than 15%, preferably less than 10% below 50 nm.
  • the modal diameter is preferably below 90 nm, preferably below 85 mn.
  • the modal diameter is in the range of 55-90 nm, preferably 58-82 nm, like 59-80 nm, 65-80 nm, 65-77 nm, 68-75 nm, 68-78 nm, 69-74 nm.
  • an RSV virosome according to the invention for use as medicament, for instance for raising an immune responses against respiratory syncytial virus.
  • the invention provides an RSV virosome for use in a method of prophylaxis or treatment of an RSV infection.
  • a method for reducing infection and/or replication of RSV in a subject comprising administering to the subject a virosome composition according to the invention.
  • the invention relates to a method of vaccinating a mammal against a viral infection comprising administering the RSV virosome composition according to the invention to a human subject.
  • the invention provides a kit for immunizing a human subject against a viral infection comprising a RSV virosome according to the invention.
  • the human subject may be of any age, for example from about 1 month to 100 years old, e.g., from about 2 months to about 80 years old, e.g., from about 1 month to about 3 years old, from about 3 years to about 50 years old, from about 50 years to about 75 years old.
  • Figure 1 Size distribution of virosomal samples measured by single particle tracking. As dilutions of the virosomes were made such as to obtain the best optical signal, absolute particle numbers cannot be compared. Notice the narrow distribution of particles without added adjuvant (panel A), with natural adjuvant (panel B), the broadening for virosomes containing natural lipids and synthetic rather than natural adjuvant (panels C and D), and the more extensive broadening when using both synthetic lipids and synthetic adjuvant (panel E).
  • FIG. 4 Anti-RSV serum IgG of mice as measured by ELISA 14 days after the first (panel A) or the second injection (panel B) of virosomes.
  • Figure 5 Equilibrium density gradient analysis of virosomes containing DOPC/DOPE and 3-D-PHAD.
  • Panel A 300 nmol 3-D-PHAD added per mg.
  • Panel B 600 nmol 3-D-PHAD per mg of viral protein.
  • Example 2 Figure 6. Mice were vaccinated on day 0 and 14 with 5 ⁇ g of a virosome with DOPC:DOPE (2:1) per mg of viral protein, and 0 , 200, 300 and 600 nmol of 3-D-PHAD. The spleen cells were stimulated with an RSV-F derived H2-K(d)peptide, KYKNAVTEL. The ELIspot assay data show the number of RSV-F peptide-specific spot-forming cells.
  • Figure 8 RSV neutralizing antibodies elicited by virosomes made from 425 nmol DOPC and 850 nmol DOPE plus 300 nmol 3-D-PHAD per mg of viral protein ("1:2 300"), 850 nmol DOPC and 1700 nmol DOPE plus 651 nmol 3- D-PHAD per mg of viral protein ("1:2 651”), or 1700 nmol of DOPC, 850 nmol of DOPE, 510 nmol of cholesterol plus 651 nmol of 3-D-PHAD per mg of viral protein ("1:2 651 + chol”), vaccination as in Figure 7.
  • the invention is exemplified by the following non-limiting examples.
  • Example 1 RSV Virosomes containing natural or synthetic lipids, and natural or synthetic adjuvant.
  • This Example compares the effect of natural and synthetic lipids, and natural and synthetic adjuvants on RSV virosome size and size distribution. All lipids and adjuvants used in these examples were acquired from Avanti Polar Lipids. Egg phosphatidylcholine (eggPC), consisting of a blend of PC molecules with different acyl chain lengths representing the acyl chains of egg yolk was produced from chicken eggs. Phosphatidylethanolamine (eggPE) was made from the egg PC by transphosphatidylation. Natural MPLA was isolated from the lipopolysaccharide (LPS) of Salmonella minnesota strain Re595.
  • LPS lipopolysaccharide
  • This preparation contains a variety of molecules differing in the number and size of the acyl chains; the mono- phosphorylated disaccharide backbone is common to all species.
  • the 3-O-D- MPLA was prepared from the natural MPLA by alkaline hydrolysis (US 4,912,094).
  • 3-D-PHAD is a synthetic version of 3-O-D-MPLA; all acyl chains have 14 carbon atoms.
  • Virosomes were produced as known in the art. Briefly, to a 2.5 ml solution of purified RSV virus, strain A2, at 6.2 mg/ml of protein, 278 ⁇ of 500 mM dicaproyl phosphatidylcholine (DCPC) was added; after 30 min on ice the sample was spun for 30 min at 40 krpm in a table-top S100 AT 4 rotor (Sorvall discovery M120-SE table top ultracentrifuge); the supernatant was harvested, filtered through an 0.1 ⁇ filter, and found to contain 3.58 mg of protein in 2.45 ml; the supernatant was divided into 5 aliquots.
  • DCPC dicaproyl phosphatidylcholine
  • phosphatidyl ethanolamine (eggPE) 2 1 plus 143 nmol of synthetic 3-
  • all the preparations contained 200 nmol of adjuvant and 850 nmol of added lipid per mg of viral protein.
  • this (2: 1) molar ratio of PC to PE approximately planar membranes can be formed that have sufficient PE for membrane fusion.
  • the solutions were diluted and analyzed by single particle tracking analysis on a
  • Nanosight® instrument the track lengths of individual Brownian
  • Virosomes without added adjuvant and with added natural lipids show the narrowest size distribution; an almost symmetrical peak is observed, and less than 4% of the particles is bigger than 150nm ( Figure 1 and Table 1).
  • Table 1 gives the modal size of the particles and shows the percentage of virosome particles larger than 150 nm.
  • the larger particles that were detected could be aggregates of smaller virosomes, or larger virosomes. Such particles would be difficult to filter across an 0.22 ⁇ filter, and therefore a substantial fraction of virosomes above 150 nm would result in substantial losses.
  • virosomes made from 200 nmol 3-D-PHAD, 566 nmol DOPC and 266 nmol DOPE were passed through a cellulose acetate filter with a nominal pore size of 0.22 ⁇ (the "tortuous path" type of filter that is commonly used in sterile filtration) at 1 mg of protein/ml, 63% of the preparation was retained.
  • the density of the virosomes was analyzed by equilibrium density gradient centrifugation loaded on 10-60 % sucrose gradients, which were spun for 66 hrs in a Sorvall AH 650 rotor at 50 krpm. Samples from the gradient were analyzed for sucrose concentration by refractometry, giving a measure of density, phosphate (from both lipid and adjuvant), and protein. As shown in Figures 2 and 3, all phosphate was contained in a single virosome peak, indicating that lipids and adjuvant are both included in the virosomes; the bulk of the protein was also found in this peak. Western blotting confirmed the presence of the viral membrane proteins, F and G in the peak.
  • Protein present in the dense fractions at the bottom of the peak mainly contained viral N and M protein.
  • Substantial peak broadening was with virosomes containing synthetic adjuvant, or synthetic lipids, and the most substantial peak broadening with the composition that contained both synthetic adjuvant and synthetic lipid.
  • Table 1 Summary of size distribution of virosomes of different compositions Preparations 1-5 are from example 1, 6-8 as in example 2, 9-11 from example 3.
  • mice Five groups of six to eight week old Balb/C mice were vaccinated on day 0 and 14 by intramuscular injection with 5 ⁇ g of each of the virosome preparations 1 to 5 of Example 1 (see also entries 1-5 of Table 1). A sixth group was injected with buffer only (HNE). Blood samples were taken on day 0, 14 and 28. A live virus challenge was administered intranasally on day 28. Five days later the mice were sacrificed and virus titers were measured. All virosome preparations protected equally well against a live virus challenge. The IgG titers 14 days after the first and 14 days after the second injection, measured as presented in Kamphuis, T. et al. Plos One 2012; 7 (5):e36812, are presented in Figures 4A and 4B, respectively.
  • 3-D-PHAD 200 nmol of 3-D-PHAD could be included per mg of viral protein, but that produced inhomogeneous particles.
  • Currently marketed human vaccines typically contain about 1 to 2.5 mg of 3-O-D-MPLA per mg of protein; 1 mg of 3-D-PHAD is 651 nmol. It was found that with 850 nmol of DOPC:DOPE at a 2: 1 ratio, 651 nmol could not be incorporated, such mixtures would form an aggregated lump of material rather than forming virosomes.
  • To produce virosomes containing more 3-D-PHAD one solution is to add more lipid. However, this will dilute out the membrane proteins, potentially affecting the immune response.
  • Table 1 shows the effect of adding more DOPC and DOPE at a 2: 1 molar ratio on the modal size and percentage of particles larger than 150nm. Clearly, narrow size distributions were obtained.
  • Figure 5 shows the sucrose density equilibrium gradient analysis of virosomes 6 (300 nmol 3-D-PHAD added per mg; panel A) and 8 (600 nmol 3 D PHAD per mg of viral protein; panel B). As expected, the density of the virosomes containing more lipid and adjuvant is lower, but for both preparations fairly homogeneous particles were observed.
  • peptides from RSV virus could be exposed on MHC-I molecules on antigen-presenting cells, giving rise to a CD8 T-cell response.
  • five groups of six to eight week old Balb/C mice were vaccinated on day 0 and 14 with 5 ⁇ g of each of the virosome preparations with compositions like those of the virosomes 4, 5 6 and 8 of Table 1 (with increasing amounts of DOPC:DOPE (2: 1) per mg of viral protein, and 0,200, 300 and 600 nmol of 3-D-PHAD).
  • a fifth group was injected with buffer only (HNE).
  • mice Fourteen days after the second immunization (Day 28) five of ten mice per group were sacrificed and spleens were harvested. The spleen cells were stimulated with an RSV-F derived H2-K(d)pe tide, KYKNAVTEL, that has been extensively characterized in Balb/C mice ( Chang et al. J. Immunol (2001); 167;4254) and the numbers of cytokine-producing cells were assayed using an interferon-gamma ELISpot (Kamphuis, T. et al. Plos One 2012; 7 (5):e36812). Statistical analysis was performed using the two- sided Mann Whitney U test.
  • 3D PHAD (median 20; mean 19.60 ⁇ 7.829) induced higher numbers of RSV- F peptide-specific spot-forming cells compared to numbers induced by virosomes with 200 nmol (median 5; mean 04.40 ⁇ 3.507) or 300 nmol 3D PHAD (median 6; mean 6.20 ⁇ 1.924) (pO.01). Control animals showed no RSV-F peptide-specific IFN- ⁇ spot-forming CD8 T cells.
  • Example 4 Optimization of including synthetic adjuvant in
  • Example 5 Effect of various synthetic lipids at virosome properties
  • Table 1 lists a variety of synthetic PC and PE species, other phospholipids and cholesterol that were used to produce virosomes containing 3-D-PHAD, and summarizes the size distribution and storage stability of the virosomes. For some preparations, the virosomes aggregated leading to their
  • Example 6 Immunogenicity of selected virosome compositions
  • Equivalent anti-RSV IgG titers ( Figure 7) and neutralizing antibody titers ( Figure 8) were obtained in a study comparing 3 preparations of virosomes, made from either 425 nmol DOPC and 850 nmol DOPE plus 300 nmol 3-D- PHAD per mg of viral protein; 850 nmol DOPC and 1700 nmol DOPE plus 651 nmol 3-D-PHAD per mg of viral protein; or 1700 nmol of DOPC, 850 nmol of DOPE, 510 nmol of cholesterol plus 651 nmol of 3-D-PHAD per mg of viral protein, respectively.
  • Each of these compositions protected equally well against a live virus challenge.
  • Synagis® (palivizumab) is a humanized mouse monoclonal antibody that recognizes an epitope present on both the pre-fusion and on post-fusion conformation of the RSV-F protein; 5C4 is a mouse monoclonal antibody that specifically recognizes the pre-fusion conformation of RSV-F (McLellan, J.S. et al. Science (2013) 340 (6136): 1113).
  • Synagis® was used to quantify F on intact virus, and the 5C4/ Synagis® ratio to determine the relative concentration of F that is present in the pre-fusion
  • HRP Peroxidase
  • goat-anti human-HRP and goat-anti-mouse-HRP for Synagis® and 5C4 respectively (both antibodies from Bethyl Labs).
  • OPD ortho-phenyl-diamine
  • the reaction was stopped with H2SO4 after 30 min, and the absorbance at 492 nm was read in an ELISA reader. Semi-logarithmic plots of the data points were fitted by linear extrapolation; for all fits, the r 2 was >0.95.
  • Table 2 shows the 5C4/Synagis ratio calculated either by division of the slopes of the curves, or the absorbance ratio calculated for a protein concentration of 50 ⁇ g/ml. It demonstrates that the pre-fusion form of the RSV-F protein is completely preserved in these virosomes. 5C4/Synagis 5C4/Synagis sample (slope) (A 492 at 50 ⁇ )
  • Table 2 Repeated measurements over the course of two months showing the stability of the 5C4 epitope on RSV-F in the context of a virosome of the present invention.

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Abstract

La présente invention concerne le domaine de l'immunologie et de la vaccinologie, en particulier les virosomes du virus respiratoire syncytial (VRS), et des vaccins les comprenant. L'invention porte plus particulièrement sur un virosome du VRS comprenant les éléments suivants : (i) des lipides et des protéines extraites de la membrane du virus respiratoire syncytial ; (ii) un adjuvant synthétique choisi parmi le PHAD (disaccharide hexa-acylé phosphorylé) et son dérivé 3-O-désacyle, 3-D-PHAD ; (iii) au moins une espèce synthétique ou sensiblement pure de phosphatidylcholine et au moins une phosphatidyléthanolamine synthétique ou sensiblement pure dans un rapport molaire de 3/1 à 1/3, caractérisées en ce que les chaînes acyles comportent entre 4 et 18 atomes de carbone, le nombre total de liaisons insaturées dans les chaînes acyle étant de quatre, et le rapport molaire du phospholipide synthétique totale par rapport à l'adjuvant étant compris entre 1,5 et 10 ; (iv) un stérol ou un dérivé de stérol dans un rapport de 0 à 30 % en mole du phospholipide total ajouté ; et v) éventuellement un ou plusieurs antigènes.
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WO2020109485A1 (fr) 2018-11-29 2020-06-04 Catalent U.K. Swindon Zydis Limited Vaccin orodispersible comprenant des virosomes
US11224571B2 (en) 2018-11-29 2022-01-18 Catalent U.K. Swindon Zydis Limited Oral dispersible vaccine comprising virosomes
US11523988B2 (en) 2018-11-29 2022-12-13 Catalent U.K. Swindon Zydis Limited Oral dispersible vaccine comprising virosomes
EP4004036A4 (fr) * 2019-07-30 2023-11-15 Verndari, Inc. Vaccins à particules de type virus

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