virion-like distribution particles for self-replicating RNA molecules

专利摘要:
VIRION-LIKE DISTRIBUTION PARTICLE FOR SELF-REPLICATING RNA MOLECULES, PHARMACEUTICAL COMPOSITION INCLUDING THE SAME AND ITS USE.Nucleic acid immunization is achieved by delivering a self-replicating RNA encapsulated in a small particle. The RNA encodes an immunogen of interest, and the particle can deliver that RNA by mimicking the delivery function of a natural RNA virus. Thus, the invention provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material that encapsulates a self-replicating RNA molecule that encodes an immunogen. These particles are useful as components in pharmaceutical compositions for immunizing individuals against various diseases.
公开号:BR112013000392A2
申请号:R112013000392-8
申请日:2011-07-06
公开日:2021-05-25
发明作者:Andrew Geall；Christian Mandl；Derek O'Hagan；Manmohan Singh
申请人:Novartis Ag；
IPC主号:

专利说明:

[001] [001] This application claims the benefit of provisional application US 61/361,828 (filed July 6, 2010), the contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD
[002] [002] The invention is in the field of non-viral delivery of self-replicating RNAs for immunization. FUNDAMENTALS OF THE TECHNIQUE
[003] [003] The delivery of nucleic acids for the immunization of animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, viral or non-viral delivery vehicles (or no delivery vehicle at all, in a "naked" vaccine), either replicating or non-replicating vectors, or viral or non-viral vectors. not viral.
[004] There remains a need for advanced and improved nucleic acid vaccines and, in particular, for better modes of delivery of nucleic acid vaccines. DISCLOSURE OF THE INVENTION
[005] [005] According to the invention, nucleic acid immunization is obtained by delivering a self-replicating RNA encapsulated and/or adsorbed to a small particle. The RNA encodes an immunogen of interest, and the particle can deliver that RNA by mimicking the delivery function of a natural virus.
[006] [006] Thus, the invention provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material that encapsulates a self-replicating RNA molecule that encodes an immunogen. The invention also provides a non-virion particle for in vivo delivery of RNA to a vertebrate cell, wherein the particle comprises a delivery material to which a self-replicating RNA molecule encoding an immunogen is adsorbed. These particles are useful as components in pharmaceutical compositions for immunizing individuals against various diseases. The combination of using a non-virion particle to deliver a self-replicating RNA provides a way to elicit a specific strong immune response against the immunogen, while delivering only a low dose of RNA. Furthermore, these particles can easily be manufactured on a commercial scale. the particle
[007] [007] Particles of the invention are non-virion particles, that is, they are not a virion. Therefore, the particle does not comprise a protein capsid. By avoiding the need to create a capsid particle, the invention does not require a packaging cell line, thus allowing for easier extrapolation to commercial production and minimizing the risk that dangerous infectious viruses are inadvertently produced.
[008] [008] Instead of encapsulating the RNA in a virion, the particles of the invention are formed from a delivery material. Several materials are suitable for forming particles that can deliver RNA to a vertebrate cell in vivo. Two delivery materials of particular interest are (i) amphiphilic lipids which can form liposomes and (ii) non-toxic and biodegradable polymers which can form microparticles. If delivery is via liposomes, the RNA must be encapsulated; if the distribution is by polymeric microparticles, the RNA can be encapsulated or adsorbed. A third distribution material of interest is the particulate reaction product of a polymer, a crosslinker, an RNA and a charged monomer.
[009] [009] Thus, one embodiment of a particle of the invention comprises a liposome that encapsulates a self-replicating RNA molecule that encodes an immunogen, while another embodiment comprises a polymeric microparticle that encapsulates a self-replicating RNA molecule that encodes an immunogen, and another embodiment comprises a polymeric microparticle onto which a self-replicating RNA molecule encoding an immunogen is adsorbed. In all three cases, the particles are preferably substantially spherical. In a fourth embodiment, a particle of the invention comprises the particulate reaction product of a polymer, a cross-linker, self-replicating RNA molecule encoding an immunogen and a charged monomer. These particles are formed in molds and therefore can be created in any shape including, but not limited to, spheres.
[0010] [0010] RNA can be encapsulated in particles (particularly if the particle is a liposome). This means that the RNA inside the particles (as in a natural virus) is separated from any external medium by the delivery material, and encapsulation has been shown to protect the RNA from digestion by RNase. Encapsulation can take many forms. For example, in some embodiments (such as in a unilamellar liposome) the delivery material forms an outer layer around an aqueous core containing RNA, while in other embodiments (eg in molded particles) the delivery material forms a matrix in the which RNA is embedded. Particles can include some external RNA (for example, on the surface of particles), but at least half of the RNA (and ideally all of it) is encapsulated. Encapsulation in liposomes is different, for example, from the lipid/RNA complexes disclosed in reference 1.
[0011] [0011] RNA can be adsorbed to particles (particularly if the particle is a polymeric microparticle). This means that the RNA is not separated from any external means by the delivery material, unlike the RNA genome of a natural virus. Particles can include some encapsulated RNA (eg, in the nucleus of a particle), but at least half of the RNA (and ideally all of it) is adsorbed. liposomes
[0012] [0012] Various amphiphilic lipids can form bilayers in aqueous media to encapsulate an aqueous RNA-containing core as a liposome. These lipids can have a zwitterionic, cationic or anionic hydrophilic headgroup. Formation of liposomes from anionic phospholipids dates back to the 1960s and lipids that form cationic liposomes have been studied since the 1990s.
[0013] [0013] Liposomal particles of the invention can be formed from a single lipid or from a mixture of lipids. A mixture may comprise (i) a mixture of anionic lipids, (ii) a mixture of cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) a mixture of anionic lipids and cationic lipids, (v) a mixture of lipids anionic and zwitterionic lipids (vi) a mixture of zwitterionic lipids and cationic lipids, or (vii) a mixture of anionic lipids, cationic lipids and zwitterionic lipids. Likewise, a mixture can comprise both saturated and unsaturated lipids. For example, a mixture can comprise DSPC (zwitterionic saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated). If a lipid blend is used, not all lipid components of the blend need to be amphiphilic, eg one or more amphiphilic lipids may be blended with cholesterol.
[0014] [0014] The hydrophilic part of a lipid can be PEGylated (ie modified by covalently linking a polyethylene glycol). This modification can increase stability and prevent non-specific liposome adsorption. For example, lipids can be conjugated to PEG using techniques such as those disclosed in references 2 and 3. Various lengths of PEG can be used, for example, between 0.5-8kDa.
[0015] [0015] A mixture of DSPC, DlinDMA, PEG-DMG and cholesterol is used in the examples.
[0016] [0016] Liposomal particles are generally divided into three groups: multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and large unilamellar vesicles (LUV). MLVs have multiple bilayers in each vesicle, forming several separate aqueous compartments. SUVs and LUVs have a single bilayer that encapsulates an aqueous core; SUVs typically have a diameter of ≤50nm and LUVs have a diameter >50nm. Liposomal particles of the invention are ideally LUVs with a diameter in the range of 50-220nm. For a composition comprising a population of LUVs with different diameters: (i) at least 80% per number must have diameters in the range 20-220nm, (ii) the mean diameter (Zav, by intensity) of the population is ideally in the range 40-200nm, and/or (iii) the diameters must have a polydispersity index <0.2. The liposome/RNA complexes of reference 1 are expected to have a diameter in the range of 600-800nm and to have a high polydispersity.
[0017] [0017] Techniques for preparing suitable liposomes are well known in the art, for example see references 4 to 6. A useful method is described in reference 7 and involves mixing (i) an ethanolic lipid solution, from (ii) an aqueous solution of nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. Preferred liposomes of the invention can be obtained by this mixing process. polymeric microparticles
[0018] [0018] Various polymers can form microparticles to encapsulate or adsorb RNA according to the invention. The use of a substantially non-toxic polymer means that a recipient can safely receive the particles and the use of a biodegradable polymer means that the particles can be metabolized after distribution to avoid long-term persistence. Useful polymers are also sterilizable to aid in the preparation of pharmaceutical grade formulations.
[0019] Suitable non-toxic and biodegradable polymers include, but are not limited to, poly(α-hydroxy acids), polyhydroxy butyric acids, polylactones (including polycaprolactones), polydioxanones,
[0020] [0020] In some embodiments, microparticles are formed from poly(α-hydroxy acids), such as poly(lactides) ("PLA"), copolymers of lactide and glycolide such as a poly(D,L-lactide -co-glycolide) ("PLG") and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having a lactide/glycolide molar ratio ranging, for example, from 20:80 to 80:20, e.g., 25:75, 40:60, 45:55, 50:50, 55: 45, 60:40, 75:25. Useful PLG polymers include those with molecular weight between, for example, 5,000-200,000 Da, for example, between 10,000-100,000, 20,000-70,000, 30,000-
[0021] [0021] The microparticles ideally have a diameter in the range of 0.02 µm to 8 µm. For a composition comprising a population of microparticles with different diameters, at least 80% should have diameters in the range of 0.03-7 µm.
[0022] [0022] Techniques for preparing suitable microparticles are well known in the art, for example see references 6, 8 (especially chapter 7) and 9. To facilitate RNA adsorption, a microparticle may include a lipid and/or surfactant cationic, for example, as disclosed in references 10 & 11.
[0023] [0023] The microparticles of the invention can have a zeta potential between 40-100 mV.
[0024] [0024] An advantage of microparticles over liposomes is that they are easily lyophilized for stable storage. molded particles
[0025] [0025] A third delivery material of interest is the particulate reaction product of a polymer, a crosslinker, a self-replicating RNA encoding an immunogen and a charged monomer. These four components can be mixed together as a liquid, placed in a mold (eg comprising a perfluoropolyether) and then cured to form particles according to the shape and dimensions of the mold. Details of a suitable method of production are disclosed in ref. 12. These methods provide a biodegradable crosslinked oligomeric polymer nanoparticle.
[0026] [0026] Ideally, the particles have the largest cross-sectional dimension of ≤5 µm. They can have an overall positive charge.
[0027] [0027] Suitable polymers include, but are not limited to: a poly(acrylic acid); a poly(styrene) sulfonate; a carboxymethylcellulose (CMC); a poly(vinyl alcohol); a poly(ethylene oxide); a poly(vinyl pyrrolidone); a dextran; a poly(vinylpyrrolidone-co-vinyl acetate-co-vinyl) alcohol. A preferred polymer is a poly(vinyl pyrrolidinone). The amount of polymer to form the particles may be between 2-75% by weight, for example 10-60% by weight, 20-60% by weight.
[0028] Suitable crosslinkers may include a disulfide and/or ketal. For example, crosslinker may comprise poly(epsilon-caprolactone)-b-dimethacrylate
[0029] [0029] Charged monomers can be anionic or cationic. These include, but are not limited to: [2-(acryloyloxy)ethyl]trimethyl ammonium chloride (AETMAC) and 2-aminoethyl methacrylate hydrochloride (AEM-HCl). The amount of monomer charged to form the particles can be between 2-75% by weight.
[0030] [0030] The amount of RNA to form the particles can be between 0.25-20% by weight.
[0031] [0031] A precure mixture within a mold may include an initiator. For example, the mold may include ≤1% by weight of initiator, ≤0.5% by weight of initiator, or ≤0.1% by weight of initiator. Between 0.1-0.5% of initiator is useful. Photoinitiators such as DEAP and DPT are useful, for example, for use with ultraviolet curing.
[0032] The invention may use some of the materials disclosed in table 1 or in examples 1-15 of reference 12, unless that siRNA components are replaced therewith by self-replicating RNAs as herein. the RNA
[0033] [0033] Particles of the invention include a self-replicating RNA molecule that (unlike siRNA) encodes an immunogen. After in vivo administration of the particles, RNA is released from the particles and translated into a cell to provide the immunogen in situ.
[0034] [0034] Unlike reference 13, the particulate RNA of the invention is self-replicating. A self-replicating RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy it generates from itself ). A self-replicating RNA molecule is thus typically a strand-+ molecule that can be translated directly upon delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from it. of the distributed RNA. Thus, the distributed RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can themselves be translated to provide in situ expression of a encoded immunogen, or they can be transcribed to provide more transcripts with the same sense as the distributed RNA that are translated to provide in situ expression of the immunogen. The overall result of the sequence of transcripts is a huge amplification of the number of replicon RNAs introduced and thus the encoded immunogen becomes a major polypeptide product of the cells.
[0035] [0035] A suitable system to achieve self-replication in this way is to use an alphavirus-based replicon. These replicons are strand-+ RNAs that lead to the translation of a replicase (or replicase-transcriptase) after delivery to a cell. Replicase is translated as a polyprotein that self-cleaves to provide a replication complex that creates genomic strand copies of distributed strand-+ RNA. These strand-- transcripts can themselves be transcribed to give additional copies of the parental strand-+ RNA and also give a subgenomic transcript that encodes the immunogen. Translation of the subgenomic transcript thus leads to in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons may use a replicase of a Sindbis virus, a Semliki Forest virus, an Eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used, for example the attenuated TC83 mutant of VEEV was used in replicons [14].
[0036] A preferred self-replicating RNA molecule thus encodes (i) an RNA-RNA polymerase
[0037] Considering that natural alphavirus genomes encode virion structural proteins in addition to the non-structural polyprotein replicase, it is preferable that the self-replicating RNA molecules of the invention do not encode alphavirus structural proteins. Thus, a preferred self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in the infectious form. The alphavirus structural proteins that are necessary for perpetuation in wild viruses are absent from self-replicating RNAs of the invention and their place is taken by genes encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural proteins of the virion of alphavirus.
[0038] [0038] Thus, a self-replicating RNA molecule useful with the invention may have two open reading frames. The first open reading frame (5') encodes a replicase; the second open reading frame (3') encodes an immunogen. In some embodiments, the RNA may have additional open reading frame (eg, downstream), e.g., to encode more immunogens (see below) or to encode accessory polypeptides.
[0039] A preferred self-replicating RNA molecule has a 5' cap (eg a 7-methylguanosine). This cap can enhance the in vivo translation of RNA. In some embodiments, the 5' sequence of the self-replicating RNA molecule must be selected to ensure compatibility with the encoded replicase.
[0040] [0040] A self-replicating RNA molecule can have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (eg, AAUAAA) near its 3' end.
[0041] [0041] Self-replicating RNA molecules can be of various lengths, but are typically 5000-25000 nucleotides in length, for example, 8000-15000 nucleotides, or 9000-12000 nucleotides. Thus, the RNA is larger than seen in siRNA distribution.
[0042] [0042] Self-replicating RNA molecules will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR. RNA distributed in double-stranded form (dsRNA) can bind to TLR3 and this receptor can also be triggered by dsRNA that is formed during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA.
[0043] Self-replicating RNA can conveniently be prepared by in vitro transcription (IVT). IVT can use a template (cDNA) created and propagated as a plasmid in bacteria, or created synthetically (eg, by polymerase chain reaction (PCR) and/or gene synthesis engineering methods). For example, a DNA-dependent RNA polymerase (such as bacteriophage RNA polymerases T7, T3 or SP6) can be used to transcribe self-replicating RNA from a DNA template. Appropriate capping reactions and addition of poly-A can be used as needed (although the replicon poly-A is usually encoded by the DNA template). These RNA polymerases may have stringent requirements for the 5' transcribed nucleotides, and in some embodiments, these requirements must be combined with encoded replicase requirements to ensure that the IVT transcribed RNA can efficiently function as a substrate for its self-encoded replicase .
[0044] [0044] As discussed in reference 15, self-replicating RNA can include (in addition to any 5' cap structure) one or more nucleotides that have a modified nucleobase. Thus, the RNA may comprise m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine) ; m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxy-isopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxy-isopentenyl) adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); hn6A (N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); M1I (1-
[0045] [0045] An RNA used with the invention ideally includes only phosphodiester bonds between nucleosides, but in some embodiments may contain phosphoramidate, phosphorothioate and/or methylphosphonate bonds.
[0046] [0046] The amount of RNA per particle may vary, and the number of individual self-replicating RNA molecules per particle may depend on the characteristics of the particle to be used. In general, a particle can include 1-500 RNA molecules. For a liposome, the number of RNA molecules is typically ≤50 per liposome, for example < 20, < 10, < 5 or 1-4. For a polymeric microparticle, the number of RNA molecules will depend on the particle diameter, but can be ≤ 50 per particle (eg, < 20, < 10, < 5, or 1-4) or 50-200 per particle. Ideally, a particle includes less than 10 different species of RNA, for example 5, 4, 3 or 2 different species; more preferably, a particle includes a single species of RNA, that is, all RNA molecules in the particle are of the same length and sequence. the immunogen
[0047] The self-replicating RNA molecules used with the invention encode a polypeptide immunogen. After administration of the particles, the immunogen is translated in vivo and can elicit an immune response from the recipient. The immunogen can trigger an immune response against a bacterium, virus, fungus, or parasite (or, in some modalities, against an allergen; and in other modalities, against a tumor antigen). The immune response can comprise an antibody response (generally including IgG) and/or a cell-mediated immune response. The polypeptide immunogen will typically elicit an immune response that recognizes the corresponding bacterial, viral, fungal, or parasitic (or allergen or tumor) polypeptide, but in some embodiments, the polypeptide can act as a mimotope to elicit an immune response that recognizes a bacterial saccharide , viral, fungal or parasitic. The immunogen will typically be a surface polypeptide, e.g., an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc.
[0048] [0048] Self-replicating RNA molecules can encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens can be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. If the immunogens are expressed as separate polypeptides, then one or more of these can be provided with an upstream IRES or an additional viral promoter element. Alternatively, various immunogens can be expressed from a polyprotein encoding individual immunogens fused to a short autocatalytic protease (eg, foot-and-mouth disease virus protein 2A), or as inteins.
[0049] [0049] Unlike references 1 and 16, RNA encodes an immunogen. For the avoidance of doubt, the invention does not encompass RNA that encodes a firefly luciferase or that encodes an E. coli β-galactosidase fusion protein or that encodes a green fluorescent protein (GFP). Furthermore, the RNA is not mouse thymus total RNA.
[0050] [0050] In some modalities, the immunogen provokes an immune response against one of these bacteria:
[0051] [0051] Neisseria meningitidis: Useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins and factor H-binding protein. A combination of three useful polypeptides is disclosed in the reference 17.
[0052] Streptococcus pneumoniae: Useful polypeptide immunogens are disclosed in reference 18. These include, but are not limited to, the pilus subunit of RrgB, the precursor of beta-N-acetyl-hexosaminidase (spr0057), spr0096, stress proteins general GSP-781 (spr2021, SP2216), serine/threonine kinase StkP (SP1732) and pneumococcal surface adhesin PsaA.
[0053] [0053] Streptococcus pyogenes: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 19 and 20.
[0054] [0054] Moraxella catarrhalis.
[0055] [0055] Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous hemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.
[0056] Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 21, such as a hemolysin, esxA, esxB, ferrichrome binding protein (sta006) and/or lipoprotein sta011.
[0057] [0057] Clostridium tetani: the typical immunogen is tetanus toxoid.
[0058] [0058] Cornynebacterium diphtheriae: the typical immunogen is diphtheria toxoid.
[0059] Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 22 and 23.
[0060] [0060] Pseudomonas aeruginosa
[0061] [0061] Streptococcus agalactiae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 19.
[0062] [0062] Chlamydia trachomatis: Useful immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (eg as disclosed in reference 24. LcrE
[25] [25] and HtrA [26] are two preferred immunogens.
[0063] [0063] Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in reference 27.
[0064] [0064] Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP and/or urease [28].
[0065] [0065] Escherichia coli: Useful immunogens include, but are not limited to, immunogens, derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffuse adhesion E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and meningitis/sepsis-associated E. coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in references 29 and 30. Useful MNEC immunogens are disclosed in reference 31. A useful immunogen for various types of E. coli is AcfD [32].
[0066] [0066] Bacillus anthracis
[0067] [0067] Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in references 33 and 34.
[0068] [0068] Staphylococcus epidermis
[0069] [0069] Staphylococcus epidermis
[0070] [0070] Clostridium perfringens or Clostridium botulinums
[0071] [0071] Legionella pneumophila
[0072] [0072] Coxiella burnetii
[0073] Brucella, such as B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae.
[0074] [0074] Francisella, as F. novicida, F. philomiragia, F. tularensis.
[0075] [0075] Neisseria gonorrhoeae
[0076] [0076] Treponema pallidum
[0077] [0077] Haemophilus ducreyi
[0078] [0078] Enterococcus faecalis or Enterococcus faecium
[0079] [0079] Staphylococcus saprophyticus
[0080] [0080] Yersinia enterocolitica
[0081] [0081] Mycobacterium tuberculosis
[0082] [0082] Rickettsia
[0083] [0083] Listeria monocytogenes
[0084] [0084] Vibrio cholerae
[0085] [0085] Salmonella typhi
[0086] [0086] Borrelia burgdorferi
[0087] [0087] Porphyromonas gingivalis
[0088] [0088] Klebsiella
[0089] [0089] In some modalities, the immunogen provokes an immune response against one of these viruses:
[0090] [0090] Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus, such as hemagglutinin, neuraminidase or M2 matrix proteins. If the immunogen is an influenza A virus hemagglutinin, it can be of any subtype, for example, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.
[0091] [0091] Paramyxoviridae Viruses: Viral immunogens include, but are not limited to, those derived from pneumoviruses (eg respiratory syncytial virus, RSV), rubulaviruses (eg mumps virus), paramyxoviruses
[0092] [0092] Poxviridae: Viral immunogens include, but are not limited to, orthopoxvirus derivatives such as Variola vera, but not limited to, Variola major and Variola minor.
[0093] Picornaviruses: Viral immunogens include, but are not limited to, those derived from Picornaviruses such as Enteroviruses, Rhinoviruses, Heparnaviruses, Cardioviruses, and Aphthoviruses. In one embodiment, the enterovirus is a poliovirus, for example, a type 1, type 2, and/or type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a coxsackie A or B virus.
[0094] Buniaviruses: Viral immunogens include, but are not limited to, those derived from an ortobuniavirus such as California encephalitis virus, a phlebovirus such as Rift valley fever virus, or a nairovirus such as California hemorrhagic fever virus Crimea-Congo.
[0095] [0095] Heparnavirus: Viral immunogens include, but are not limited to, those derived from a heparnavirus such as the hepatitis A virus (HAV).
[0096] [0096] Filoviruses: Viral immunogens include, but are not limited to, those derived from a filovirus, such as an Ebola virus (including an Ebolavirus from Zaire, Cote d'Ivoire, Reston, or Sudan) or a Marburg virus.
[0097] [0097] Togavirus: Viral immunogens include, but are not limited to, those derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Aterivirus. This includes the rubella virus.
[0098] [0098] Flavivirus: Viral immunogens include, but are not limited to, those derived from a Flavivirus such as Tick-Borne encephalitis virus (TBE), Dengue virus (types 1, 2, 3 or 4), virus Yellow fever, Japanese encephalitis virus, Kyasanur forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian Spring-Summer encephalitis virus, Powassan encephalitis virus .
[0099] [0099] Pestivirus: Viral immunogens include, but are not limited to, those derived from a Pestivirus such as bovine viral diarrhea (BVDV), classical swine fever (CSFV) or border disease (BDV).
[00100] [00100] Hepadnavirus: Viral immunogens include, but are not limited to, those derived from a Hepadnavirus, such as the hepatitis B virus. A composition may include the hepatitis B virus surface antigen (HBsAg).
[00101] [00101] Other hepatitis viruses: A composition may include an immunogen from a hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus.
[00102] [00102] Rhabdovirus: Viral immunogens include, but are not limited to, those derived from a Rhabdovirus, such as a Lyssavirus (eg, a rabies virus) and Vesiculovirus (VSV).
[00103] [00103] Caliciviridae: Viral immunogens include, but are not limited to, those derived from Calciviridae such as Norwalk virus (Norovirus), and Norwalk-like viruses such as Hawaii virus and snow mountain virus.
[00104] [00104] Coronavirus: Viral immunogens include, but are not limited to, those derived from a SARS coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine transmissible gastroenteritis virus (TGEV). The coronavirus immunogen may be a spike polypeptide.
[00105] [00105] Retroviruses: Viral immunogens include, but are not limited to, those derived from an Oncovirus, a Lentivirus (eg, HIV-1 or HIV-2) or a Foamvirus.
[00106] [00106] Reovirus: Viral immunogens include, but are not limited to, those derived from an orthoreovirus, a rotavirus, an orbivirus, or a coltivirus.
[00107] [00107] Parvovirus: Viral immunogens include, but are not limited to, those derived from Parvovirus B19.
[00108] [00108] Herpesvirus: Viral immunogens include, but are not limited to, those derived from a human herpesvirus, such as, by way of example, Herpes Simplex virus (HSV) (eg, HSV types 1 and 2), chickenpox virus -zoster (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8).
[00109] [00109] Papovaviruses: Viral immunogens include, but are not limited to, those derived from papillomaviruses and polyomaviruses. The papillomavirus (human) may be serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65, for example, from one or more of serotypes 6, 11, 16 and/or 18.
[00110] [00110] Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36).
[00111] [00111] In some modalities, the immunogen elicits an immune response against a virus that infects fish, such as: infectious salmon anemia virus (ISAV), pancreatic salmon disease virus (SPDV), infectious pancreatic necrosis virus (IPNV) , channel catfish virus (CCV), lymphocytic fish disease virus (FLDV), infectious hematopoietic necrosis virus (IHN), herpes koi virus, salmon picorna-like virus (also known as Atlantic salmon picorna-like virus ), landlocked salmon virus (LSV), Atlantic salmon rotavirus (ASR), strawberry trout disease virus (TSD), silver salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).
[00112] [00112] Fungal immunogens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton gallphytone, Trichotonninum, Trichotonnium Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoids, var. ochraceum, Trichophyton violaceum and/or Trichophyton faviform; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatelidei tropicalis, Candida glalase, Candidal. Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Encephaltointestinus e. less common are Brachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovapiora spp. schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizocorpia spp. Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp and Cladosporium spp.
[00113] [00113] In some modalities, the immunogen provokes an immune response against a parasite of the Plasmodium genus, such as P. falciparum, P. vivax, P. malariae or P. ovale. Thus, the invention can be used for immunization against malaria. In some modalities, the immunogen elicits an immune response against a parasite of the Caligidae family, particularly those of the genera Lepeophtheirus and Caligus, eg, sea lice like Lepeophtheirus salmonis or Caligus rogercresseyi.
[00114] [00114] In some modalities, the immunogen elicits an immune response against: pollen allergens (tree, grass, weed, and grass pollen allergens); insect or arachnid allergens (respiratory, saliva and venom allergens, eg mite allergens, cockroach and mosquito allergens, hymenoptera venom allergens); animal hair and dandruff allergens (eg, from dog, cat, horse, rat, mouse, etc.); and food allergens (eg, gliadin). Important allergens to pollen from grasses, grasses and trees originate from the taxonomic orders Fagales, Oleales, Pinales and Platanaceae, including, among others, birch (Betula), alder (Alnus), hazel (Corylus), white poplar ( Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), flat tree (Platanus), the order Poales including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum, the orders Asterales and Urticales including herbs from the genera Ambrosia, Artemisia and Parietaria.
[00115] [00115] In some embodiments, the immunogen is a tumor antigen selected from: (a) testis-cancer antigens such as NY-ESO-1, SSX2, SCP1 as well as the RAGE, BAGE, GAGE and MAGE polypeptide families, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 and MAGE-12 (which can be used, for example, to target melanoma, tumors lung, head and neck, NSCLC, breast, gastrointestinal and bladder; (b) mutated antigens, eg p53 (associated with various solid tumors eg colorectal, lung, head and neck cancer), p21/ Ras (associated with eg melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with eg melanoma), MUMl (associated with eg melanoma), caspase-8 (associated with eg melanoma head and neck cancer), CIA 0205 (associated with eg bladder cancer), HLA-A2-R1701, beta-catenin (associated with eg melanoma), TCR (associated with eg non-lymphoma -Hodgkin's T cells), BCR-abl (associated with, for example, chronic myeloid leukemia), triosephosphate isomerase, KIA 0205, CDC-27 and LDLR-FUT; (c) overexpressed antigens, eg galectin 4 (associated with eg colorectal cancer), galectin 9 (associated with eg Hodgkin's disease), proteinase 3 (associated with eg chronic myeloid leukemia), WT-1 (associated with eg various leukemias), carbonic anhydrase (associated with eg kidney cancer), aldolase A (associated with eg lung cancer), PRAME (associated with eg melanoma ), HER-2/neu (associated with eg breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with eg hepatoma), KSA (associated with eg colorectal cancer ), gastrin (associated with, for example, gastric and pancreatic cancer), telomerase catalytic protein, MUC-1 (associated with, for example, breast and ovarian cancer), G-250 (associated with, for example, breast cancer renal cells), p53 (associated with eg breast, colon cancer) and carcinoembryonic antigen (associated with eg breast cancer, cancer of lung and gastrointestinal tract cancers such as colorectal cancer); (d) shared antigens, eg melanoma-melanocyte differentiation antigens such as MART-1/Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1/TRPI and tyrosinase-related protein -2/TRP2 (associated with, for example, melanoma); (e) prostate-associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with, for example, prostate cancer; (f) immunoglobulin idiotypes (associated with myeloma and B-cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, pi 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens , EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn- 23Hl, TAG-72-4, CA 19-9, CA 72-4, CAM
[00116] The particles of the invention are useful as components in pharmaceutical compositions for immunizing individuals against various diseases. These compositions will typically include a pharmaceutically acceptable carrier in addition to the particles. A detailed discussion of pharmaceutically acceptable carriers is available in reference 35.
[00117] [00117] A pharmaceutical composition of the invention may include one or more small immunopotentiating molecules. For example, the composition may include a TLR2 agonist (for example Pam3CSK4), a TLR4 agonist (for example an aminoalkyl glucosaminide phosphate such as E6020), a TLR7 agonist (for example imiquimod), a TLR8 agonist (eg resiquimod) and/or a TLR9 agonist (eg IC31). Any such agonist ideally has a molecular weight of <2000Da. In case an RNA is encapsulated, in some modalities such agonists are also encapsulated with the RNA, but in other modalities they are unencapsulated. In case an RNA is adsorbed to a particle, in some modalities such agonists are also adsorbed with the RNA, but in other modalities they are desorbed.
[00118] [00118] Pharmaceutical compositions of the invention may include particles in pure water (eg, wfi), or in a buffer, e.g. a phosphate buffer, Tris buffer, a borate buffer, succinate buffer, a histidine buffer , or a citrate buffer. Buffer salts will typically be included in the 5-20mM range.
[00119] [00119] The pharmaceutical compositions of the invention may have a pH between 5.0 and 9.5, for example, between 6.0 and 8.0.
[00120] Compositions of the invention may include sodium salts (eg sodium chloride) to give tonicity. A concentration of 10+2 mg/ml of NaCl is typical, for example around 9 mg/ml.
[00121] [00121] The compositions of the invention may include metal ion chelators. These can prolong RNA stability by removing ions that can accelerate phosphodiester hydrolysis. Thus, a composition may include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc... Such chelators are typically present in between 10-500µM, eg 0.1mM. A citrate salt, such as sodium citrate, can also act as a chelator, while advantageously also providing buffering activity.
[00122] The pharmaceutical compositions of the invention may have an osmolality between 200 mOsm/kg and 400 mOsm/kg, for example between 240-360 mOsm/kg or between 290-310 mOsm/kg.
[00123] [00123] Pharmaceutical compositions of the invention may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Compositions without mercury are preferred and vaccines without preservatives can be prepared.
[00124] [00124] The pharmaceutical compositions of the invention are preferably sterile.
[00125] [00125] The pharmaceutical compositions of the invention are preferably non-pyrogenic, e.g. containing < 1 EU (endotoxin unit, a standard measure) per dose and preferably < 0.1 EU per dose.
[00126] [00126] The pharmaceutical compositions of the invention are preferably gluten free.
[00127] [00127] The pharmaceutical compositions of the invention can be prepared in unit dose form. In some embodiments, a unit dose may have a volume between 0.1-1.0 ml, for example, about 0.5 ml.
[00128] [00128] The compositions can be prepared as injectables, as solutions or suspensions. The composition can be prepared for pulmonary administration, for example, by an inhaler, using a fine spray. The composition may be prepared for nasal, aural or ocular administration, for example, as a spray or drops. Injectables for intramuscular administration are typical.
[00129] The compositions comprise an immunologically effective amount of particles, as well as any other components, as needed. By 'immunologically effective amount' it is meant that administration of that amount to a subject, either as a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, the age, the taxonomic group of the individual to be treated (eg, non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the attending physician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall into a relatively wide range that can be determined through routine trials. The particle and RNA content of compositions of the invention will generally be expressed in terms of the amount of RNA per dose. A preferred dose is ≤100µg of RNA (eg 10-100 µg, such as about 10 µg, 25 µg, 50 µg, 75 µg or 100 µg), but expression can be seen at much lower levels, for example, ≤1 µg/dose, ≤100ng/dose, ≤10ng/dose, ≤1ng/dose, etc.
[00130] The invention also provides a dispensing device (e.g. syringe, nebulizer, spray, inhaler, epidermal patch, etc.) containing a pharmaceutical composition of the invention. This device can be used to administer the composition to a vertebrate individual.
[00131] [00131] The particles of the invention do not include ribosomes. Treatment methods and medical uses
[00132] [00132] In contrast to the particles disclosed in reference 16, the particles and pharmaceutical compositions of the invention are for use in vivo to elicit an immune response against an immunogen of interest.
[00133] The invention provides a method for inducing an immune response in a vertebrate, comprising the step of administering an effective amount of a particle or pharmaceutical composition of the invention. The immune response is preferably protective and preferably involves antibodies and/or cell-mediated immunity. The method can generate a reinforcement response.
[00134] The invention also provides a particle or pharmaceutical composition of the invention for use in a method of inducing an immune response in a vertebrate.
[00135] [00135] The invention also provides the use of a particle of the invention in the manufacture of a drug to induce an immune response in a vertebrate.
[00136] [00136] By inducing an immune response in the vertebrate by these uses and methods, the vertebrate can be protected against various diseases and/or infections, for example, against bacterial and/or viral diseases, as discussed above. The particles and compositions are immunogenic and most preferably are vaccine compositions. Vaccines in accordance with the invention may be prophylactic (i.e. to prevent infection) or therapeutic (i.e., to treat infection), but will typically be prophylactic.
[00137] The vertebrate is preferably a mammal, such as a human or a large veterinary mammal (eg horses, cattle, deer, goats, pigs). If the vaccine is for prophylactic use, the human is preferably a child (eg, a child or infant) or an adolescent; if the vaccine is for therapeutic use, the human is preferably an adolescent or an adult. A vaccine intended for children can also be given to adults, for example, to assess safety, dosage, immunogenicity, etc.
[00138] The vaccines prepared according to the invention can be used to treat both children and adults. Thus, a human patient may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients to receive vaccines are the elderly (eg ≥50 years old, ≥60 years old and preferably ≥65 years old), young people (eg ≤5 years old), hospitalized patients, health care professionals. health, military and armed service personnel, pregnant women, the chronically ill, or immunodeficient patients. However, vaccines are not only suitable for these groups, and can be used more generally in a population.
[00139] [00139] The compositions of the invention will generally be administered directly to a patient. Direct delivery can be accomplished by parenteral injection (eg, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or into the interstitial space of a tissue; unlike reference 1, intraglossal injection is not typically used with this invention). Alternative routes of delivery include rectal, oral (eg, tablet, spray), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, aural, pulmonary or other mucosal administration. Intradermal and intramuscular administration are two preferred routes. Injection can be through a needle (eg a hypodermic needle), but needleless injection can be used as an alternative. A typical intramuscular dose is 0.5 ml.
[00140] [00140] The invention can be used to provoke systemic and/or mucosal immunity, preferably to provoke an enhanced mucosal and/or systemic immunity.
[00141] The dosage can be a single dose schedule or a multiple dose schedule.
[00142] [00142] In some embodiments of the invention, RNA does not include modified nucleotides (see above). In other embodiments, the RNA may optionally include at least one modified nucleotide, provided that one or more of the following features (already disclosed above) are also required:
[00143] [00143] If the RNA is delivered with a liposome, the liposome will comprise DSDMA, DODMA, DLinDMA and/or DLenDMA.
[00144] [00144] If RNA is encapsulated in a liposome, the hydrophilic part of a lipid in the liposome will be pegylated.
[00145] [00145] If RNA is encapsulated in a liposome, at least 80% of the liposomes will have diameters in the range 20-220nm.
[00146] [00146] If the RNA is delivered with a microparticle, the microparticle will be a non-toxic, biodegradable polymer microparticle.
[00147] [00147] If the RNA is delivered with a microparticle, the microparticles will have a diameter in the range of 0.02µm to 8µm.
[00148] [00148] If the RNA is delivered with a microparticle, at least 80% of the microparticles will have a diameter in the range of 0.03-7µm.
[00149] [00149] If the RNA is delivered with a microparticle, the composition will be lyophilized.
[00150] [00150] The RNA has a 3' poly-A tail and the immunogen can trigger an in vivo immune response against a bacterium, a virus, a fungus or a parasite.
[00151] [00151] RNA is delivered in combination with a metal ion chelator with a delivery system selected from (i) liposomes, (ii) biodegradable and non-toxic polymer microparticles. General
[00152] [00152] The practice of the present invention will employ, unless otherwise indicated, the conventional methods of Chemistry, Biochemistry, Molecular Biology, Immunology and Pharmacology, within the knowledge of the art. Such techniques are fully explained in the literature. See, for example, references 36-42, etc.
[00153] [00153] The term "comprising" encompasses "which includes" as well as "consisting of", for example, a composition "comprising" X may consist exclusively of X or may include something additional, for example, X + Y.
[00154] [00154] The term "about" in relation to a numerical value x is optional and means, for example, x±10%.
[00155] [00155] The word "considerably" does not exclude "completely", eg a composition that is "considerably free" of Y may be completely free of Y. If necessary, the word "considerably" can be omitted from the definition of the invention.
[00156] [00156] References to charge, cations, anions, zwitterions, etc. are taken at pH 7.
[00157] [00157] TLR3 is the Toll-like receptor 3. It is a unique transmembrane receptor that plays a key role in the innate immune system. Known TLR3 agonists include poly(LC). "TLR3" is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC: 11849. The RefSeq sequence for the human TLR3 gene is GI:2459625.
[00158] [00158] TLR7 is the Toll-like receptor 7. It is a unique transmembrane receptor that plays a key role in the innate immune system. Known TLR7 agonists include, for example, imiquimod. "TLR7" is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC: 15631. The RefSeq sequence for the human TLR7 gene is GI: 67944638.
[00159] [00159] TLR8 is the Toll-like 8 receptor. It is a unique transmembrane receptor that plays a key role in the innate immune system. Known TLR8 agonists include, for example, resiquimod. "TLR8" is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC: 15632. The RefSeq sequence for the human TLR8 gene is GI:20302165.
[00160] [00160] The family of RIG-1-like receptors ("RLR") includes several RNA helicases that play key roles in the innate immune system [43]. RLR-1 (also known as RIG-1 or retinoic acid-inducible gene 1) has two caspase recruitment domains near its N-terminal end. The approved HGNC name for the gene encoding the RLR-1 helicase is "DDX58" (for the DEAD box (Asp-Glu-Ala-Asp) polypeptide 58) and the unique HGNC ID is HGNC: 19102. The RefSeq sequence for the human RLR-1 gene is GI:77732514. RLR-2 (also known as MDA5 or gene 5 associated with melanoma differentiation) also has two caspase recruitment domains near its N-terminal end. The approved HGNC name for the gene encoding the RLR-2 helicase is "IFIH1" (for interferon induced by domain 1 of helicase C) and the unique HGNC ID is HGNC: 18873. The RefSeq sequence for the human RLR-2 gene is GI: 27886567. RLR-3 (also known as LGP2 or <genetics and physiology laboratory 2>) does not have caspase recruitment domains. The approved HGNC name for the gene encoding the RLR-3 helicase is "DHX58" (for the DEXH (Asp-Glu-X-His) polypeptide 58 box) and the unique HGNC ID is HGNC: 29517. The RefSeq sequence for the human RLR-3 gene is GI: 149408121.
[00161] [00161] PKR is a double-stranded RNA-dependent protein kinase. It plays a key role in the innate immune system. "EIF2AK2" (for eukaryotic translation initiation factor 2 alpha kinase 2) is the approved HGNC name for the gene encoding this enzyme, and its unique HGNC ID is HGNC: 9437. The RefSeq sequence for the human PKR gene is GI:208431825. BRIEF DESCRIPTION OF THE DRAWINGS
[00162] [00162] Figure 1 shows a gel with stained RNA. The streaks show (1) markers (2) pure (naked) replicon (3) replicon after RNase treatment (4) replicon encapsulated in liposome (5) liposome after RNase treatment (6) RNase treated liposome then subjected to extraction with phenol/chloroform.
[00163] [00163] Figure 2 is an electron micrograph of liposomes.
[00164] [00164] Figure 3 shows protein expression (as relative light units, RLU) on days 1, 3 and 6 after RNA delivery as a virion-packed replicon (squares), pure RNA (naked) (triangles), or as microparticles (circles).
[00165] [00165] Figure 4 shows a gel with stained RNA. The streaks show (1) markers (2) pure replicon (naked) (3) replicon encapsulated in liposome (4) liposome treated with RNase then subjected to extraction with phenol/chloroform.
[00166] [00166] Figure 5 shows protein expression on days 1, 3 and 6 after delivery of RNA as a replicon packaged by virion (squares), as pure RNA (naked) (lozangos), or in liposomes (+ = 0, 1µg, x = 1µg).
[00167] [00167] Figure 6 shows protein expression on days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA.
[00168] [00168] Figure 7 shows anti-F IgG titers in animals receiving virion-packed replicon (VRP or VSRP), 1µg pure RNA, and 1µg liposome-encapsulated RNA.
[00169] Figure 8 shows anti-F IgG titers in animals receiving VRP, 1µg of pure RNA, and 0.1g or 1µg of liposome-encapsulated RNA.
[00170] [00170] Figure 9 shows neutralizing antibody titers in animals receiving VRP, 0.1g or 1µg of liposome-encapsulated RNA.
[00171] [00171] Figure 10 shows expression levels after delivery of a replicon as pure RNA (circles), liposome-encapsulated RNA (triangle & square), or as a lipoplex (inverted triangle).
[00172] [00172] Figure 11 shows F-specific IgG titers (2 weeks after the second dose) after delivery of a replicon as pure RNA (0.01-1µg), liposome-encapsulated RNA (0.01-10µg), or packaged as a virion (VRP, 106 infectious units or IU).
[00173] [00173] Figure 12 shows F-specific IgG titers (circles) and PRNT titers (squares) after delivery of a replicon as pure RNA (1µg), liposome-encapsulated RNA (0.1 or 1µg), or packaged as a virion (VRP, 106 IU). Titles in naïve mice are also shown. Solid lines show geometric means.
[00174] [00174] Figure 13 shows intracellular cytokine production after restimulation with synthetic peptides representing the major epitopes on the F protein 4 weeks after a second dose. The y-axis shows % CD8+CD4- cytokine+.
[00175] Figure 14 shows the F-specific IgG titers (mean log10 titers ± pad std) for 63 days (Fig. 14A) and 210 days (Fig. 14B) after immunization of calves. The three lines are easily distinguished at day 63 and are, from bottom to top: PBS negative control; RNA delivered in liposomes; and the product "Triangle 4".
[00176] Figure 15 shows IgG titers of anti-HIV serum in response to pure RNA ("RNA") or liposome-encapsulated ("LNP"), or to DNA delivered by electroporation into muscle.
[00177] [00177] Figure 16 shows IgG titers in 13 groups of mice. Each circle is an individual mouse, and the solid lines show geometric means. The dotted horizontal line is the detection limit for the assay. The 13 groups are, from left to right, A to M, as described below.
[00178] Figure 17 shows (A) IL-6 and (B) IFNα (pg/ml), released by pDC. There are 4 pairs of bars, from left to right: control; immunized with RNA + DOTAP; immunized with RNA + lipofectamine; and immunized with RNA in liposomes. In each pair, the black bar is wild mice, gray is the rsq1 mutant.
[00179] [00179] Various replicons are used below. In general, these are based on a hybrid alphavirus genome with non-structural proteins from the Venezuelan equine encephalitis virus (VEEV), a syndbis virus packaging signal, and a 3' UTR from either a sindbis virus or a mutant VEEV. The replicon is about 10kb long and has a poly-A tail.
[00180] [00180] Plasmid DNA encoding alphavirus replicons (termed: pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for RNA synthesis in vitro. Replicons contain the alphavirus genetic elements necessary for RNA replication, but lack those that encode the gene products needed for particle assembly; structural proteins are instead replaced by a protein of interest (a reporter such as a SEAP or GFP, or an immunogen such as the complete RSV F protein) and therefore the replicons are unable to induce the generation of infectious particles. A bacteriophage promoter (T7 or SP6) upstream of the alphavirus cDNA facilitates replicon RNA synthesis in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A) tail
[00181] [00181] After linearization of plasmid DNA downstream of the HDV ribozyme with an appropriate restriction endonuclease, run-off transcripts were synthesized in vitro using DNA-dependent RNA polymerase derived from bacteriophage T7 or SP6. Transcriptions were performed for 2 hours at 37oC in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). After transcription, the DNA template was digested with DNase TURBO (Ambion). Replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. RNA without cap was submitted to cap insertion post-transcriptionally with Vaccinia capping enzyme (VCE) using the ScriptCap m7G capping system (Epicentre Biotechnologies), as described in the user manual; replicons with cap inserted in this way are prefixed with "v", eg vA317 is the A317 replicon with cap inserted by VCE. RNA with post-transcriptionally inserted cap was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring the OD260nm. The integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis. PLG Adsorption
[00182] [00182] Microparticles were made using 500mg of PLG RG503 (lactide/glycolide molar ratio 50:50, MW ~30kDa) and 20mg of DOTAP using an Omni homogenizer
[00183] [00183] To assess RNA adsorption, 100 µL of the suspension of particles were centrifuged at 10,000 rpm for 5 minutes and the supernatant was collected. PLG/RNA was reconstituted using 1mL of nuclease-free water. To 100 µL of the particle suspension (1 µg of RNA) was added 1 mg of heparin sulfate. The mixture was vortexed and allowed to stand at room temperature for 30 min for RNA desorption. The suspension of particles was centrifuged and the supernatant was collected.
[00184] [00184] For RNAse stability, 100 µL of the particle suspension were incubated with 6.4mAU of RNase A at room temperature for 30 min. RNAse was inactivated with 0.126mAU of Proteinase K at 55 C for 10 min. 1mg of heparin sulfate was added for RNA desorption followed by centrifugation. The RNA supernatant samples were mixed with dye in formaldehyde sample buffer, heated to 65°C for 10 min and analyzed using a 1% denaturing gel (460ng RNA loaded per lane).
[00185] [00185] To assess expression, Balb/c mice were immunized with 1µg of RNA in 100 µL of intramuscular injection volume (50µL/leg) on day 0. Sera were collected on days 1, 3 and 6. Protein expression was determined using a chemiluminescence assay. As shown in Figure 3, expression was higher when RNA was distributed by PLG (triangles) than without any distribution particle (circles). liposomal encapsulation
[00186] [00186] The RNA was encapsulated in liposomes made by the method of references 7 and 44. The liposomes were made of 10% DSPC (zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2% DMG conjugated to PEG (2kDa PEG). These proportions refer to %> moles in the total liposome.
[00187] DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 2. DSPC (1,2-diastearoyl-sn-glycero-3-phosphocholine) was purchased from Genzyme . Cholesterol was obtained from Sigma-Aldrich. PEG-conjugated DMG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol), ammonium salt), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane, salt chloride) and DC-col (3-[N-(N',N'-dimethylaminoethane)-carbamoyl]) cholesterol hydrochloride were from Avanti Polar Lipids.
[00188] [00188] Briefly, lipids were dissolved in ethanol (2 ml), an RNA replicon was dissolved in buffer (2 ml, 100mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 balance time. The mixture was diluted in 6ml of buffer then filtered. The resulting product contained liposomes, with ~95% encapsulation efficiency.
[00189] [00189] For example, in a given method, fresh lipid stock solutions were prepared in ethanol. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 ml of ethanol. The freshly prepared lipid stock solution was gently oscillated at 37oC for about 15 min to form a homogeneous mixture. Then, 755 µL of the stock was added to 1.245 mL of ethanol to make a 2 mL working lipid stock solution. This amount of lipid was used to form liposomes with 250 µg of RNA. A 2 ml working RNA solution was also prepared from a ~1µg/µL stock solution in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with magnets) were rinsed with RNase Away solution (Molecular BioProducts) and rinsed with plenty of MilliQ water prior to use to decontaminate the RNase vials. One of the vials was used for the RNA working solution and the other to collect the lipid and RNA mixtures (as described below). Working RNA and lipid solutions were heated at 37oC for 10 min before loading into 3cc luer-lok syringes. 2 ml of citrate buffer (pH 6) was loaded into another 3 cc syringe. Syringes containing RNA and lipids were connected to a T-mixer (PEEKTM 500 µm ID junction, Idex Health Science) using FEP (fluorinated ethylene-propylene) tubes; all FEP tubes used had an inner diameter of 2 mm and an outer diameter of 3mm; obtained from Idex Health Science). The output of the T-mixer was also with FEP tubes.
[00190] [00190] Figure 2 shows an example of an electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA that encodes complete RSV F antigen. Dynamic light scattering from a batch showed an average diameter of 141nm (by intensity) or 78nm (by number).
[00191] The percentage of encapsulated RNA and RNA concentration was determined by the Quant-iT RiboGreen RNA reagent kit (Invitrogen) following the manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10x or 100x in 1X TE buffer (from kit) prior to addition of dye. Separately, liposomes were diluted 10x or 100x in 1X TE buffer containing 0.5% Triton X prior to addition of dye (to disrupt liposomes and thus assess total RNA). Then, an equal amount of dye was added to each solution and then ~180 µl of each solution after dye addition was loaded in duplicate into a 96-well tissue culture plate. Fluorescence (Ex 485 nm, Em 528 nm) was read on a microplate reader. All liposome formulations were dosed in vivo based on the amount of encapsulated RNA.
[00192] [00192] Encapsulation in liposomes has been shown to protect RNA from RNase digestion.
[00193] [00193] To assess in vivo RNA expression, a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1X Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50µL per leg with a RNA dose of 0.1µg or 1µg. The same vector was also administered without the liposomes (in PBS 1X without RNase) at 1µg. Virion-packed replicons were also tested. Virion-packed replicons used herein (referred to as "VRPs") were obtained with the methods of reference 45, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the VEEV genome engineered to contain the 3' UTR of the sindbis virus and a syndbis virus packaging signal (PS) packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the sindbis virus glycoprotein and capsid genes.
[00194] [00194] As shown in Figure 5, encapsulation increased SEAP levels by about ½ log at the ^g dose, and on day 6 expression from the encapsulated dose of O.^g corresponded to levels seen with no dose. encapsulated from g. On day 3, expression levels exceeded those achieved with VRPs (squares). Thus, expressed increased when RNA was formulated in liposomes compared to pure RNA control, even at a lower dose of 10x. Expression was also higher compared to the VRP control, but the expression kinetics were very different (see Figure 5). RNA delivery with electroporation resulted in increased expression compared to pure RNA control, but these levels were lower than those of liposomes.
[00195] [00195] Experiments with additional SEAP have shown a clear dose-response in vivo, with expression seen after delivery of as little as 1ng of RNA (Figure 6). Additional experiments comparing the expression of encapsulated and pure replicons indicated that 0.01µg of encapsulated RNA was equivalent to 1µg of pure RNA. With a dose of 0.5µg of RNA, the encapsulated material gave a 12-fold greater expression on day 6; with 0.1µg of dose levels were 24 times higher on day 6.
[00196] [00196] Rather than evaluating mean levels in the group, individual animals were also studied. Although many animals did not respond to pure replicons, encapsulation eliminated non-responders.
[00197] [00197] Additional experiments replaced DlinDMA with DOTAP. Although DOTAP liposomes gave better expression than pure replicon, they were inferior to DlinDMA liposomes (difference 2-3 times on day 1).
[00198] [00198] To assess immunogenicity in vivo, a replicon was constructed to express the complete F protein of respiratory syncytial virus (RSV). This was distributed neat (1µg), encapsulated in liposomes (0.1 or 1µg), or packaged in virions (106 IU; "VRP") on days 0 and 21. Figure 7 shows anti-F IgG titers 2 weeks later the second dose, and liposomes clearly enhance immunogenicity. Figure 8 shows titers 2 weeks later, at which point there was no statistical difference between RNA encapsulated at 0.1µg, RNA encapsulated at 1µg, and the VRP group. Neutralizing titers (measured as 60% plaque reduction, "PRNT60") were not significantly different in these three groups 2 weeks after the second dose (Figure 9). Figure 12 shows PRNT and IgG titres 4 weeks after the second dose.
[00199] [00199] Figure 13 confirms that RNA elicits a robust CD8 T cell response.
[00200] Additional experiments compared F-specific IgG titers in mice receiving VRP, 0.1µg liposome-encapsulated RNA, or 1µg liposome-encapsulated RNA. Titer ratios (VRP:liposome) at various time points after the second dose were as follows: 2 weeks 4 weeks 8 weeks 0.1µg 2.9 1.0 1.1 1µg 2.3 0.9 0.9
[00201] [00201] Thus, liposome-encapsulated RNA elicits essentially the same magnitude of immune response as seen with virion delivery.
[00202] [00202] Additional experiments showed superior F-specific IgG responses at a dose of 10µg, equivalent responses at doses of 1µg and 0.1µg, and a lower response at a dose of 0.01µg. Figure 11 shows IgG titres in mice that receive replicon in pure form at 3 different doses, in liposomes at 4 different doses, or as VRP (106 IU). The response seen with 1µg liposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the greater response seen with 10µg liposome-encapsulated RNA was statistically significant (p < 0.05) compared to both of these groups.
[00203] [00203] An additional study confirmed that the 0.1µg of liposome-encapsulated RNA gave much greater anti-F IgG responses (15 days after the second dose) than 0.1µg of delivered DNA, and was still more immunogenic than 20µg of plasmid DNA encoding the F antigen, delivered by electroporation (ElgenTM DNA Delivery System, Inovio).
[00204] The mice showed few visual signs of distress (weight loss, etc.) after receiving liposome-encapsulated RNA replicon, although a transient weight loss of 3-4% was seen after a second dose of 10µg RNA . In contrast, delivery of 10µg of liposome-encapsulated DNA led to 8-10% weight loss. Mechanism of action
[00205] Bone marrow-derived dendritic cells (pDC) were obtained from wild-type mice or from the mutant strain "Resq" (rsq1). The mutant strain has a point mutation at the amino-terminal end of its TLR7 receptor that abolishes TLR7 signaling without affecting ligand binding [46]. Cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000 or inside a liposome. As shown in Figure 17, IL-6 and INFa were induced in WT cells, but this response was almost completely abrogated in mutant mice. These results show that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines.
[00206] In general, liposome-delivered RNA replicons have been shown to induce several serum cytokines up to 24 hours after intramuscular injection (IFN-α, IP-10 (CXCL-10), IL-6, KC, IL-5, IL -13, MCP-1 and MIP-a), although only MIP-1 was induced by pure RNA and the liposome alone induced only IL-6.
[00207] IFN-α has been shown to contribute to the immune response to the replicon encoding liposome-encapsulated RSV-F, because an anti-IFNa receptor antibody (IFNAR1) reduced F-specific serum IgG by a 10-fold reduction after 2 vaccinations.
[00208] [00208] Liposome-delivered RNA replicons in general have been seen to trigger a balanced IgG1:IgG2 subtype profile in mice, sometimes with a greater IgG2a/IgG1 ratio than seen with electroporated DNA or with protein/ MF59 (ie, a Th1 type immune response). Liposome Fabrication Methods
[00209] In general, eight different methods have been used for the preparation of liposomes according to the invention. These are referred to in the text as methods (A) to (H) and differ mainly with regard to filtering and TFF steps. Details are as follows:
[00210] [00210] Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG DMG 2000 were weighed and dissolved in 7.55 ml of ethanol.
[00211] [00211] As method (A), except that after oscillation, 226.7 µL of the stock was added to 1.773 mL of ethanol to make a 2 mL working lipid stock solution, thus modifying the lipid ratio: RNA.
[00212] [00212] As method (B) except that Mustang filtration was omitted, then the liposomes were from the 20 mL glass vial for TFF dialysis.
[00213] [00213] Like method C, except TFF used polyethersulfone (PES) hollow fiber membranes (part number 1 (PC 1 - 100E- 100-0 IN) with a pore size cut-off limit of 100 kD and surface area of 20 cm2.
[00214] [00214] Like method (D) except that Mustang membrane was used, as in method (A).
[00215] Like method (A) except that Mustang filtration was omitted, then the liposomes were from the 20 ml glass vial for TFF dialysis.
[00216] [00216] As method (D), except that a 4 mL working RNA solution was prepared from a ~1µg/µL stock solution in 100 mM citrate buffer (pH 6). Then four 20 ml glass vials were prepared in the same way. Two of them were used for the RNA working solution (2 ml in each vial) and the others to collect the lipid and RNA mixtures, as in (C). Instead of using a T-mixer, syringes containing RNA and lipids were connected to a Droplet Mitos Junction Chip (a glass microfluidic device obtained from Syrris, Part No. 3000158) using PTFE tubing (0.03 inches in diameter) internal x 1/16 inch outside diameter) which uses a 4-way edge connector (Syrris).
[00217] [00217] As in method (A), except that the 2mL of working lipid stock solution was made by mixing 120.9 µL of the lipid stock with 1.879 mL of ethanol. In addition, after mixing in the T-mixer, the liposomes from the 20 ml vial were loaded into Pierce's Slide-A-Lyzer dialysis cassette (Thermo Scientific, extra strength, 0.5-3 ml capacity) and dialyzed against 400 -500 mL of 1X PBS overnight at 4oC in an autoclaved plastic container before recovering the final product. Expression in BHK
[00218] [00218] Liposomes with different lipids were incubated with BHK cells overnight and evaluated for protein expression potency. From a baseline with RV05, lipid expression could be increased 18x by adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposome, 10x by adding 10% 18:2 (cis) phosphatidylcholine and 900x using RV01 instead.
[00219] [00219] In general, in vivo studies have shown that unsaturated lipid tails tend to increase titers of IgG produced against encoded antigens. RSV immunogenicity
[00220] [00220] The vA317 self-replicating replicon encoding the F protein of RSV was administered to BALB/c mice, 4 or 8 animals per group, by bilateral intramuscular vaccinations (50 µL per leg) on days 0 and 21 with the replicon (1 µg) alone or formulated as liposomes with DlinDMA ("RV01") or DOTAP ("RV13"). RV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2% PEG-DMG, but with different amounts of RNA. RV13 liposomes had 40% DOTAP, 10% DPE, 48% cholesterol and 2% PEG-DMG. For comparison, pure plasmid DNA (20 µg) expressing the same RSV-F antigen was delivered using electroporation or with RV01(10) liposomes (0.1 µg DNA). Four mice were used as a naive control group.
[00221] [00221] Liposomes were prepared by method (D) or method (B). For some liposomes made by method (D), double or half the amount of RNA was used. The mean Z particle diameter, polydispersity index and liposome encapsulation efficiency were as follows: RV Zav (nm) pdI % of encapsulation Preparation RV01 (10) 158.6 0.088 90.7 (A) RV01 (08) 156, 8 0.144 88.6 (A) RV01 (05) 136.5 0.136 99 (B) RV01 (09) 153.2 0.067 76.7 (A) RV01 (10) 134.7 0.147 87.8* (A) RV01 (02) 128.3 0.179 97 (A) * For this RV01(10) formulation, the nucleic acid was DNA not RNA
[00222] Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were collected from mice on day 49 for T cell analysis.
[00223] [00223] F-specific serum IgG titers (GMT) were as follows: RV Day 14 Day 36 Plasmid DNA 439 6712 pure RNA A317 pure 78 2291 RV01 (10) 3020 26170 RV01 (08) 2326 9720 RV01 (05) 5352 54907 RV01 (09) 4428 51316 DNA RV01 (10) 5 13 RV13 (02) 644 3616
[00224] [00224] The proportion of T cells that are cytokine-positive and specific for the F51-66 peptide of RSV is as follows, showing only figures that are statistically significantly above zero: RV CD4+CD8-CD4-CD8+ IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Plasmid 0.04 0.07 0.10 0.57 0.29 0.66 Pure DNA Pure A317 RNA 0.04 0.05 0.08 0.57 0.23 0.67 RV01 (10) 0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.04 0.06 0.46 0.30 0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09) 0.06 0.08 0.09 1.62 0.67 1.71 DNA RV01 (10) 0.03 0.08 RV13 ( 02) 0.03 0.04 0.06 1.15 0.41 1.18
[00225] Thus, liposome formulations significantly enhanced immunogenicity relative to pure RNA controls, as determined by increased F-specific IgG titers and T cell frequencies. Plasmid DNA formulated with liposomes, or delivered neat using electroporation , was significantly less immunogenic than liposome-formulated self-replicating RNA.
[00226] RV01 RNA vaccines were more immunogenic than RV13 vaccine. RV01 has a tertiary amine in the headgroup with a pKa of about 5.8 and also includes unsaturated alkyl tails. RV13 has unsaturated alkyl tails, but its group head has a quaternary amine and is very strongly cationic. Liposomes - requirement for encapsulation
[00227] [00227] To assess whether the effect seen in the liposome groups was due merely to the liposome components,
[00228] [00228] Additional experiments used three different RNAs: (i) 'vA317' replicon which expresses RSV-F, ie the surface fusion glycoprotein of VRS; (ii) 'vA17' replicon which expresses GFP; and (iii) 'vA336' which is defective in replication and encodes GFP.
[00229] [00229] The RNAs were distributed pure or with liposomes made by method (D). Empty liposomes were made by method (D) but without any RNA. Four liposome formulations had these characteristics: RNA Polydispersity Size Encapsulation RNA Particle Zav (nm) vA317 155.7 0.113 86.6% vA17 148.4 0.139 92% vA336 145.1 0.143 92.9% Void 147.9 0.147 -
[00230] [00230] BALB/c mice, 5 animals per group, received bilateral intramuscular vaccines (50 per leg) on days 0 and 21 with:
[00231] [00231] Group 1 pure suto-replicating RSV-F RNA (vA317, 0.1µg)
[00232] [00232] Group 2 self-replicating RSV-F RNA (vA317, 0.1 µg) encapsulated in liposomes
[00233] [00233] Group 3 self-replicating RSV-F RNA (vA317, 0.1 µg) added to empty liposomes
[00234] [00234] Group 4 F subunit protein (5 µg)
[00235] [00235] Serum was collected for antibody analysis on days 14, 35 and 51. F-specific serum IgG titers (GMT) were measured; if an individual animal had a titer of <25 (limit of detection), it was assigned a titer of 5. In addition, spleens were collected from mice on day 51 for T-cell analysis to determine which cells were cytokine- positive and specific for the peptide F51-66 of RSV (CD4+) or for the F peptides of RSV F85-93 and F249-258 (CD8+).
[00236] [00236] IgG titers were as follows, in the groups and in unimmunized control mice: Day 1 2 3 4 - 14 22 1819 5 5 5 290 32533 9 19877 5 51 463 30511 18 20853 5
[00237] [00237] Neutralizing titers of RSV serum on day 51 were as follows: Day 1 2 3 4 51 35 50 24 38
[00238] [00238] Animals showing RSV F-specific CD4+ splenic T cells on day 51 were as follows, where a number (% positive cells) is given only if the stimulated response is statistically significantly above zero: Cytokine 1 2 3 4
[00239] [00239] Animals showing RSV F-specific CD8+ splenic T cells on day 51 were as follows, where a number is given only if the stimulated response is statistically significantly above zero: Cytokine 1 2 3 4 IFN-γ 0.37 0.87 IL2 0.11 0.40 0.04 IL5 TNFα 0.29 0.79 0.06
[00240] [00240] Thus, RNA encapsulation in liposomes is required for high immunogenicity, since a simple mixture of RNA and liposomes (group 3) was not immunogenic (in fact, less immunogenic than pure RNA).
[00241] [00241] In other studies, mice received various combinations of (i) self-replicating RNA replicon encoding complete F protein of RSV (ii) RNA replicon encoding self-replicating GFP (iii) RNA replicon encoding GFP with a knockout in nsP4 that eliminates the self-replication (iv) complete F protein of RSV. 13 groups received in total: A - - B 0.1µg of (i), pure - C 0.1µg of (i), encapsulated in - liposome D 0.1µg of (i), with liposomes -
[00242] [00242] The results in Figure 16 showed that F-specific IgG responses required liposome encapsulation rather than mere co-distribution (compare C & D groups). A comparison of groups K, L and M shows that RNA provided an adjuvant effect against the co-distributed protein, and this effect was seen with both replicating and non-replicating RNA. The immunogenicity of RSV in different mouse strains
[00243] The replicon "vA142" encodes the complete wild-type surface fusion glycoprotein (F) of RSV, but with the fusion peptide deleted, and the 3' end is formed by ribozyme-mediated cleavage. It was tested on three different mouse strains.
[00244] [00244] BALB/c mice received bilateral intramuscular vaccines (50 µL per leg) on days 0 and
[00245] Group 1 received pure replicon (1 µg).
[00246] [00246] Group 2 received 1µg of replicon distributed in liposomes "RV01(37)" with 40% DlinDMA, 10% DSPC, 48% Choi, 2% DMG conjugated to PEG.
[00247] [00247] Group 3 received the same as group 2, but in 0.1µg of RNA.
[00248] [00248] Group 4 received 1µg of replicon in liposomes "RV17(10)" (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG).
[00249] [00249] Group 5 was 1µg replicon in liposomes "RV05(11)" (40% RV07 lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG).
[00250] [00250] Group 6 received 0.1µg of replicon in liposomes "RV 17(10)".
[00251] Group 7 received 5 µg of RSV-F subunit protein with aluminum hydroxide adjuvant.
[00252] [00252] Group 8 was a naive control (2 animals)
[00253] [00253] Sera were collected for antibody analysis on days 14, 35 and 49. F-specific serum IgG GMTs were: Day 1 2 3 4 5 6 7 8 14 82 2463 1789 2496 1171 1295 1293 5 1538 34181 25605 23579 13718 8887 73809 5
[00254] [00254] On day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7
[00255] RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers of combinations from 2-5 mice, 1 combination per group): Day 1 2 3 4 5 6 7 8 35 <20 143 20 101 32 30 111 <20 49 <20 139 <20 83 41 32 1009 <20
[00256] [00256] Spleens were collected on day 49 for T cell analysis. The mean net frequencies of F-specific cytokine-positive T cells (CD4+ or CD8+) were as follows, showing only figures that were statistically significantly above zero ( specific for RSV peptides F51-66, F 164-178, F309-323 for CD4+, or for F85-93 and F249-258 for CD8+ peptides): CD4+CD8- CD4-CD8+ IFNγ IL2 IL5 TNFα IFNγ IL2 group IL5 TNFα 1 0.03 0.06 0.08 0.47 0.29 0.48 2 0.05 0.10 0.08 1.35 0.52 1.11 3 0.03 0.07 0.06 0.64 0.31 0.61 4 0.05 0.09 0.07 1.17 0.65 1.09 0.03 0.08 0.07 0.65 0.28 0.58 6 0.05 0.07 0.07 0.74 0.36 0.66 7 0.02 0.04 0.04 8
[00257] [00257] C57BL/6 mice were immunized in the same way, but a 9th group received VRPs (1x106 IU)
[00258] [00258] Sera were collected for antibody analysis on days 14, 35 & 49. F-specific IgG titers (GMT) were: Day 1 2 3 4 5 6 7 8 9 14 1140 2133 1026 2792 3045 1330 2975 5 1101 1721 5532 3184 3882 9525 2409 39251 5 12139
[00259] [00259] On day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7 8 IgG1 66 247 14 328 468 92 56258 79 IgG2a 2170 7685 5055 6161 1573 2944 35 14229
[00260] RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers of combinations of 2-5 mice, 1 combination per group): Day 1 2 3 4 5 6 7 8 9 35 <20 27 29 22 36 <20 28 <20 <20 49 <20 44 30 23 36 <20 33 <20 37
[00261] [00261] Spleens were collected on day 49 for T cell analysis. The mean net frequencies of F-specific cytokine-positive T cells (CD8+) were as follows, showing only figures that were statistically significantly above zero (specific for the RSV peptides F85-93 and F249-258): CD4-CD8+ group IFNγ IL2 IL5 TNFα 1 0.42 0.13 0.37
[00262] [00262] Nine groups of C3H/HeN mice were similarly immunized. F-specific IgG titres (GMT) were: Day 1 2 3 4 5 6 7 8 9 14 5 2049 1666 1102 298 984 3519 5 806 152 27754 19008 17693 3424 6100 62297 5 17249
[00263] On day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows: IgG 1 2 3 4 5 6 7 8 IgG1 5 1323 170 211 136 34 83114 189 IgG2a 302 136941 78424 67385 15667 27085 3800 72727
[00264] RSV serum neutralizing antibody titers on days 35 and 49 were as follows: Day 1 2 3 4 5 6 7 8 9 35 <20 539 260 65 101 95 443 <20 595 49 <20 456 296 35 82 125 1148 <20 387
[00265] [00265] Thus, three different lipids (RVOl, RV05, RV17; pKa 5.8, 5.85, 6.1) were tested in three different inbred mouse strains. For all 3 strains, RVOl was more effective than RV17; for the BALB/c and C3H strains, RV05 was less effective than RVOl or RV17, but it was more effective in the B6 strain. In all cases, however, liposomes were more effective than two cationic nanoemulsions that were tested in parallel. The immunogenicity of CMV
[00266] [00266] RV01 liposomes with DLinDMA as the cationic lipid were used to deliver RNA replicons encoding cytomegalovirus (CMV) glycoproteins. The "vA160" replicon encodes complete H and L glycoproteins (gH/gL), while the "vA322" replicon encodes a soluble form (gHsol/gL). The two proteins are under the control of subgenomic promoters separated into a single replicon; co-administration of two separate vectors, one encoding gH and one encoding gL, did not yield good results.
[00267] [00267] BALB/c mice, 10 per group, received bilateral intramuscular vaccines (50 µL per leg) on days 0, 21 and 42 with VRPs expressing gH/gL (1x106 UI), VRPs expressing gHsol/gL (1x106 UI) and PBS as controls. Two test groups received 1µg of the liposome-formulated vA160 or vA322 replicon (40% DlinDMA, 10% DSPC, 48% Choi, 2% PEG-DMG; made using method (D), but with 150 µg of batch size RNA).
[00268] The vA160 liposomes had a Zav diameter of 168nm, a pdl of 0.144 and 87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162nm, a pdl of 0.131 and 90% encapsulation.
[00269] [00269] The replicons were able to express two proteins from a single vector.
[00270] [00270] Sera were collected for immunological analysis on day 63 (3wp3). CMV neutralizing titers (the reciprocal of serum dilution producing a 50% reduction in the number of positive virus foci per well, relative to controls) were as follows: gH/gL of VRP gHsol/gL of gH/gL of gHsol/gL of VRP liposome liposome 4576 2393 4240 10062
[00271] [00271] RNA expressing a complete or soluble form of the CMV gH/gL complex thus elicited high neutralizing antibody titers as analyzed in epithelial cells. The mean titers elicited by RNAs encapsulated in liposomes were at least as high as the corresponding VRPs.
[00272] [00272] Repeated experiments confirmed that the replicon was able to express two proteins from a single vector. The RNA replicon gave a 3wp3 titer of 11457, compared to 5516 with VRPs.
[00273] [00273] Additional experiments used different replicons in addition to vA160. The vA526 replicon expresses the CMV pentameric complex (gH-gL-UL128-UL130-UL-131) under the control of three subgenomic promoters: the first drives the expression of gH; the second drives the expression of gL; the third drives expression of the UL128-2A-UL130-2A-UL131 polyprotein, which contains two 2A cleavage sites between the three UL genes. The vA527 replicon expresses the CMV pentameric complex through three subgenomic promoters and two IRESs: the first subgenomic promoter drives the expression of gH; the second subgenomic promoter drives expression of gL; the third subgenomic promoter drives UL128 expression; UL130 is under the control of an EMCV IRES; UL131 is under the control of an EV71 IRES. These three replicons were distributed by liposome (method (H), with 150µg batch size) or by VRPs.
[00274] [00274] BALB/c mice, 10 groups of 10 animals, received bilateral intramuscular vaccines (50 µL per leg) on days 0, 21 and 42 with:
[00275] [00275] Group 1 VRPs expressing gH FL/gL (1x106 IU)
[00276] [00276] Group 2 pentameric, 2A VRP (1x105 UI)
[00277] [00277] Group 3 pentameric, 2A VRP (1x106 IU)
[00278] [00278] Group 4 pentameric, IRES VRP (1x105 UI)
[00279] [00279] Group 5 vA160 of self-replicating RNA (1µg) formulated in liposomes
[00280] [00280] Group 6 vA256 of self-replicating RNA (1µg) formulated in liposomes
[00281] [00281] Group 7 vA527 of self-replicating RNA (1µg) formulated in liposomes
[00282] [00282] Group 8 vA160 of self-replicating RNA (1µg) formulated in a cationic nanoemulsion
[00283] [00283] Group 9 vA256 of self-replicating RNA (^g) formulated in a cationic nanoemulsion
[00284] [00284] Group 10 vA527 of self-replicating RNA (^g) formulated in a cationic nanoemulsion.
[00285] [00285] Sera were collected for immunological analysis on days 21 (3wpl), 42 (3wp2) and 63 (3wp3).
[00286] [00286] The CMV serum neutralizing titers on days 21, 42 and 63 were: Vaccine group 3wp1 3wp2 3wp3 1 126 6296 26525 2 N/A N/A 6769 3 N/A 3442 7348 4 N/A N/A 2265
[00287] [00287] Therefore, self-replicating RNA can be used to express multiple antigens from a single vector and to elicit a potent and specific immune response. The replicon can express five antigens (CMV pentameric complex (gH-gL-UL128-UL130-UL-131) and elicit a potent immune response. The self-replicating RNA distributed in liposomes was able to elicit high titers of neutralizing antibody, as evaluated in epithelial cells, at all time points evaluated (3wp1, 3wp2 and 3wp3) These responses were superior to the corresponding VRPs and to the cationic nanoemulsions.
[00288] [00288] The hydrodynamic distribution employs the force generated by the rapid injection of a large volume of solution to overcome the physical barriers of cell membranes that prevent the entry of large and membrane-impermeable compounds into cells. This phenomenon has previously been shown to be useful for the intracellular delivery of DNA vaccines.
[00289] [00289] A typical mouse volume of distribution for intramuscular injection is 50 µL for the hind leg, which is a relatively high volume for a mouse leg muscle. In contrast, a human intramuscular dose of ~0.5ml is relatively small. If immunogenicity in mice were volume dependent, then the efficacy of replicon vaccines would be due, at least in part, to hydrodynamic forces, which would not be encouraging to use the same vaccines in humans and larger animals.
[00290] [00290] The vA317 replicon was distributed to BALB/c mice, 10 per group, by bilateral intramuscular vaccines (5 or 50 per leg) on day 0 and 21:
[00291] [00291] Group 1 received pure replicon, 0.2µg in 50 µL per leg
[00292] [00292] Group 2 received pure replicon, 0.2 µg in 5 µL per leg
[00293] [00293] Group 3 received liposome-formulated replicon (0.2 µg, 50 µL per leg)
[00294] [00294] Group 4 received liposome-formulated replicon (0.2 µg, 5 µL per leg)
[00295] [00295] Serum was collected for antibody analysis on days 14 and 35. F-specific serum IgG GMTs were: Day 1 2 3 4 14 42 21 2669 2610 241 154 17655 18516
[00296] Thus, the immunogenicity of the formulated replicon did not vary according to the volume distributed, thus indicating that these RNA vaccines do not depend on hydrodynamic distribution for their efficacy. expression kinetics
[00297] [00297] A self-replicating RNA replicon ("vA31 1") expressing a luciferase (luc) reporter gene was used to study the protein expression kinetics after injection. BALB/c mice, 5 animals per group, received bilateral intramuscular vaccines (50 µL per leg) on day 0, with:
[00298] [00298] Group 1 DNA expressing luciferase, distributed using electroporation (10 µg)
[00299] [00299] Group 2 self-replicating RNA (1 µg) formulated in liposomes
[00300] [00300] Group 3 self-replicating RNA (1µg) formulated with a cationic nanoemulsion
[00301] [00301] Group 4 self-replicating RNA (1 µg) formulated with a cationic nanoemulsion
[00302] [00302] Group 5 VRP (1x106 IU) expressing luciferase
[00303] [00303] Before vaccination, the mice were shaved. The mice were anesthetized (2% isoflurane in oxygen), the hair first removed with an electric slide and then chemical Nair. Bioluminescence data were then acquired using a Xenogen IVIS 200 imaging system (Caliper Life Sciences) on days 3, 7, 14, 21, 28, 35, 42, 49, 63, and 70. Five minutes prior to imaging, mice were injected intraperitoneally with 8 mg/kg of luciferin solution. The animals were then anesthetized and transferred to the imaging system. Image acquisition times were kept constant as the bioluminescence signal was measured with a cooled CCD camera.
[00304] [00304] Visually, cells expressing luciferase were seen to remain mainly at the site of RNA injection and animals visualized after removal of the quadriceps showed no signal.
[00305] [00305] In quantitative terms, luciferase expression was measured as mean luminosity over a period of 70 days (p/s/cm2/sr), and the results were as follows for 5 groups: Day 1 2 3 4 5 3 8.69E+07 3.33E+06 2.11E+06 9.71E+06 1.46E+07 7 1.04E+08 8.14E+06 1.83E+07 5.94E+07 1.64E+ 07 14 8.16E+07 2.91E+06 9.22E+06 3.48E+07 8.49E+05 21 1.27E+07 3.13E+05 6.79E+04 5.07E+05 6, 79E+05 28 1.42E+07 6.37E+05 2.36E+04 4.06E+03 2.00E+03 1.21E+07 6.12E+05 2.08E+03 42 1.49E+07 8.70E+05 49 1.17E+07 2.04E+05 63 9.69E+06 1.72E+03 70 9.29E+06
[00306] [00306] The self-replicating RNA formulated with cationic nanoemulsions showed measurable bioluminescence on day 3, which peaked on day 7 and then was reduced to basal levels on days 28 to 35. When formulated in liposomes, the RNA showed measurable bioluminescence on day 3, which peaked on day 7 and was reduced to basal levels by day 63. RNA delivered using VRPs showed increased bioluminescence on day 21 when compared to formulated RNA, but expression had been reduced to basal levels on day 21. day 28. The electroporated DNA showed the highest level of bioluminescence at all times measured and the bioluminescence levels were not reduced to basal levels in the 70 days of the experiment.
[00307] RNA encapsulated in liposomes encoding HIV gp140 was delivered to mice, intramuscularly, intradermally, or subcutaneously. All three pathways led to elevated serum IgG levels of HIV-specific antibodies (Figure 15), exceeding the titers seen in response to electroporated intramuscular DNA. sigmodon
[00308] [00308] A study was performed on Sigmodon (Sigmodon hispidis) rather than mice. At one dose, liposome encapsulation increased F-specific IgG titers by 8.3 times compared to pure RNA and increased PRNT titers by 9.5 times. The magnitude of the antibody response was equivalent to that induced by 5x106 IU of VRP. Both pure RNA and liposome-encapsulated RNA were able to protect sigmodons from RSV challenge (1x105 plate-forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction about 2-fold.
[00309] [00309] Additional work on sigmodons utilized four different replicons: vA317 expresses complete RSV-F; vA318 expresses truncated RSV-F (transmembrane and cytoplasmic tail removed); vA142 expresses RSV-F with its fusion peptide deleted; vA140 expresses the truncated RSV-F also without its peptide. Sigmodons, 4 to 8 animals per group, received intramuscular vaccines (100 µL in one leg) on days 0 and 21 with the four different replicons in two doses (1.0 and 0.1 µg) formulated in liposomes made by method (D) , but with an RNA batch size of 150 µg. Control groups received an RSV-F subunit protein vaccine (5 µg) with alum adjuvant (8 animals/group), VRPs expressing complete RSV-F (1x106 IU, 8 animals/group), or naive control (4 animals/group) ). Serum was collected for antibody analysis on days 0, 21 and 34.
[00310] F-specific serum IgG titers and RSV serum neutralizing antibody titers on days 21 and 34 were: Group IgG, IgG, NT, NT, day 21 day 34 day 21 day 34 1 µg vA317 915 2249 115 459 0.1 µg of vA317 343 734 87 95 1 µg of vA318 335 1861 50 277 0.1 µg of vA318 129 926 66 239 1 µg of vA142 778 4819 92 211 0.1 µg of vA142 554 2549 78 141 1 µg vA140 182 919 96 194 0.1 µg vA140 61 332 29 72 µg 13765 subunit 86506 930 4744 F trimer/alum 1x106 IU VRP-F 1877 19179 104 4528 complete Naïve 5 5 10 15
[00311] [00311] All four replicons evaluated in this study (vA317, vA318, vA142, vA140) were immunogenic in sigmodons when distributed by liposome, although serum neutralization titers were at least ten times lower than those induced by adjuvant or protein vaccines by VRPs. Liposome/RNA vaccines elicited serum F-specific IgG and RSV neutralizing antibodies after the first vaccination, and a second vaccination enhanced the response effectively. The F-specific IgG titers after the second vaccination with µg replicon were 2-3 times higher than after the second vaccination with 0.1 µg replicon. The four replicons elicited comparable antibody titers, suggesting that complete and truncated RSV-F, each with or without the fusion peptide, are similarly immunogenic in sigmodons.
[00312] [00312] Additional work on sigmodons again used the vA317, vA318 and vA142 replicons. Sigmodons, 2-8 animals per group, received intramuscular vaccines (100 µL in one leg) on days 0 and 21 with the replicons (0.1 or 1 µg) scapulated in RVOI liposomes made by method (D), but with a size of 150 µg RNA lot. Control groups received RSV-F subunit protein vaccine (5 µg) with alum adjuvant or VRPs expressing complete RSV-F (1x106 IU, 8 animals/group). All these animals received a third vaccination (day 56) with RSV-F subunit protein vaccine (5 µg) with alum adjuvant. In addition, there was a naïve control (4 animals/group). In addition, an extra group received bilateral intramuscular vaccines (50 µL per leg) on days 0 and 56 with 1 µg of vA317 RNA in liposomes, but did not receive a third vaccination with the protein subunit vaccine.
[00313] Serum was collected for antibody analysis on days 0, 21, 35, 56, 70, in addition to days 14, 28 & 42 for the extra group. The F-specific serum IgG (GMT) titers were as follows: day 21 day 35 day 56 day 70
[00314] Serum neutralizing titers were as follows (60% of RSV neutralizing titers for 2 combinations of 3-4 animals per group, GMT of these 2 combinations per group): day 21 day 35 day 56 day 70 1 µg of vA318 58 134 111 6344 0.1 µg of vA318 41 102 63 6647 1 µg of vA142 77 340 202 5427 0.1 µg of vA142 35 65 56 2223 1 µg of vA317 19 290 200 4189 1x106 VRP (Full F) 104 1539 558 2876 5 µg 448 subunit 4457 1630 3631 F trimer/alum Naïve 10 10 10
[00315] [00315] Serum titers and neutralizing titers for the extra group were as follows: Day 14 21 28 35 42 56 70 IgG 397 561 535 501 405 295 3589 NT 52 82 90 106 80 101 1348
[00316] Thus, replicons are confirmed to be immunogenic in sigmodons, eliciting serum F-specific IgG and RSV neutralizing antibodies after the first vaccination. A second vaccination effectively boosted responses. F-specific IgG titers after the second vaccination with 1.0 µg replicon were 1.5 to 4 times higher than after the second vaccination with 0.1 µg replicon.
[00317] The third vaccination (protein on day 56) did not increase titers in sigmodons previously vaccinated with F trimer subunit + alum, but provided a large increase to titers in sigmodons previously vaccinated with replicon. In most cases, serum neutralizing titers to RSV after two replicon vaccines followed by protein boost were greater than or equal to titers induced by two or three sequential protein vaccines.
[00318] This study also evaluated the kinetics of the antibody response to 1.0 µg of vA317. Neutralizing titers of RSV and F-specific serum IgG induced by a single vaccine peaked around day 21 and were maintained until at least day 56 (50-70% drop in F-specific IgG titer, little change in the RSV neutralization titer). A second homologous vaccine was given to these animals on day 56 and increased antibody titers to a level at least equal to that achieved when the second vaccine was administered on day 21.
[00319] [00319] Additional experiments involved a viral challenge. The vA368 replicon encodes the complete wild-type surface fusion glycoprotein of RSV with the deleted fusion peptide, with expression driven by the EV71 IRES. Sigmodons, 7 per group, received intramuscular vaccines (100 µL per leg) on days 0 and 21 with vA368 in liposomes prepared by method (H), RNA batch size of 175 µg, or with VRPs having the same replicon. A control group received 5 µg of protein with alum adjuvant, and a naïve control group was also included.
[00320] All groups received an intranasal (i.n.) challenge with 1x106 PFU of RSV four weeks after the final immunization. Serum was collected for antibody analysis on days 0, 21 and 35. Lung viral titers were measured 5 days after challenge. The results were as follows: Liposome VRP Naïve Protein F-Specific Serum IgG Titers (GMT) Day 21 370 1017 28988 5 Day 35 2636 2002 113843 5 Neutralizing Titers (GMT) Day 21 47 65 336 10 Day 35 308 271 5188 10 Pulmonary viral load (ufp per gram of lung) Day 54 422 225 124 694110
[00321] Thus, the RNA vaccine reduced the lung viral load by more than three logs, from approximately 106 PFU/g in unvaccinated control sigmodons to less than 103 PFU/g in vaccinated sigmodons. Large Mammal Study
[00322] [00322] A large animal study was carried out in cattle. Calves (4-6 weeks old, -60-80 kg, 5 per group) were immunized with 66µg of replicon vA317 encoding the complete RSV F protein on days 0, 21, 86 and 146. Replicons were formulated in liposomes . PBS alone was used as a negative control and a licensed vaccine was used as a positive control ("Triangle 4" from Fort Dodge, which contains inactivated virus). All calves received 15 µg F protein adjuvanted with the MF59 emulsion on day 146. One cow was mistakenly vaccinated with the wrong vaccine on day 86 instead of Triangle 4 and then her data was excluded from day 100 onwards.
[00323] The RNA vaccines encoded F from human RSV, whereas the "Triangle 4" vaccine contains F from bovine RSV, but the F protein from RSV is highly conserved between BRSV and HRSV.
[00324] [00324] Liposomes were made by method (E), except that a batch size of 1.5 mg of RNA was used.
[00325] [00325] The calves received 2 ml of each experimental vaccine, administered intramuscularly as 2 x 1 ml on each side of the neck. In contrast, the "Triangle 4" vaccine was administered as a single 2 ml dose in the neck.
[00326] Serum was collected for antibody analysis on days 0, 14, 21, 35, 42, 56, 63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167, 174, 181 , 188, 195 and 202. If an individual animal had a titer below the detection limit it was assigned a titer of 5
[00327] Figure 14A shows F-specific IgG titers during the first 63 days. The RNA replicon was immunogenic in cows via liposomes, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose and titers were very stable for 2 to 6 weeks after the second dose (and were particularly stable for RNA vaccines).
[00328] [00328] Figure 14B shows F-specific serum IgG (GMT) titers at 210 days, and the values measured up to day 202 were as follows: D0 3wp1 2wp2 5wp2 ~9wp2 2wp3 8wp3 2wp4 5wp4 8wp4 D21 D35 D56 D86 D100 D146 D160 D181 D202 PBS 5 5 5 5 5 5 5 46 98 150 Liposome 5 5 12 11 20 768 74 20774 7022 2353 Triangle 5 5 1784 721 514 3406 336 13376 4775 2133 4
[00329] The RSV serum neutralizing antibody titers were as follows: D0 2wp2 5wp2 2wp3 3wp3 4wp3 8wp3 2wp4 3wp4 4wp4 D35 D56 D100 D107 D107 D146 D160 D167 D174 PBS 12 10 10 14 18 20 14 10 10 10 Liposome 13 10 10 20 13 17 13 47 26 21 Triangle 12 15 13 39 38 41 13 24 26 15 4
[00330] The material used for the second liposome dose was not freshly prepared, and the same batch of RNA showed a decrease in potency in a mouse immunogenicity study. Therefore, it is possible that the vaccine would have been more immunogenic if fresh material had been used for all vaccines.
[00331] [00331] When evaluated with complement, neutralizing antibodies were detected in all vaccinated cows. In this trial, all vaccinated calves had good neutralizing antibody titers after the second RNA vaccine. In addition, the RNA vaccine elicited F-specific serum IgG titers that were detected in some calves after the second vaccine and in all calves after the third.
[00332] RSV-F with MF59 adjuvant was able to increase the IgG response in all previously vaccinated calves, and to increase complement-independent neutralization titers of previously RNA-vaccinated calves.
[00333] [00333] Proof of concept for RNA vaccines in large animals is particularly important in light of the previously observed loss of potency with DNA-based vaccines, when moving from small animal models to larger animals and humans. A typical dose for a cow DNA vaccine would be 0.5-1 mg [47,48] and therefore it is very encouraging that immune responses were induced with just 66 µg of RNA.
[00334] [00334] It will be understood that the invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention. Table 1: Useful Phospholipids DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE 1, 2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol...) DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine
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权利要求:
Claims (16)
[1]
1. Non-virion particle for in vivo delivery of RNA into a vertebrate cell, characterized by the fact that (a) the particle comprises a delivery material (i) that encapsulates a self-replicating RNA molecule that encodes an immunogen, or (ii) wherein a self-replicating RNA molecule encoding an immunogen is adsorbed, and (b) the RNA does not include modified nucleotides.
[2]
2. Particle according to claim 1, characterized in that the particle is a liposome and the RNA is encapsulated in it.
[3]
3. Particle according to claim 1, characterized in that the particle is a non-toxic and biodegradable polymeric microparticle and the RNA is adsorbed to it.
[4]
4. Particle according to claim 1, characterized in that the particle is a biodegradable crosslinked oligomeric polymer nanoparticle formed by the reaction of a polymer, a crosslinker, a charged monomer and RNA.
[5]
5. Particle according to claim 2, characterized in that the liposome comprises a lipid with a cationic headgroup.
[6]
6. Particle according to claim 2 or 5, characterized in that the liposome comprises a lipid with a zwitterionic headgroup.
[7]
7. Particle according to any one of claims 2, 5 and 6, characterized in that the liposome has a diameter in the range of 50 to 220nm.
[8]
8. Particle according to claim 3, characterized in that the particle comprises a poly(D,L-lactide-co-glycolide).
[9]
9. Particle according to claim 3 or 8, characterized in that the particle has a diameter of 30nm to 7 µm.
[10]
10. Particle according to any one of claims 1 to 9, characterized in that the self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen.
[11]
11. Particle according to claim 10, characterized in that the RNA molecule has two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen.
[12]
12. Particle according to any one of claims 1 to 11, characterized by the fact that the RNA molecule has a length of 9000 to 12000 nucleotides.
[13]
13. Particle according to any one of claims 1 to 12, characterized in that the immunogen can provoke an immune response in vivo against a bacterium, a virus, a fungus or a parasite.
[14]
14. Particle according to claim 13, characterized in that the immunogen can provoke an immune response in vivo against the respiratory syncytial virus F glycoprotein.
[15]
15. Pharmaceutical composition, characterized in that it comprises a particle as defined in any one of claims 1 to 14.
[16]
Use of the particle as defined in any one of claims 1 to 14 or the pharmaceutical composition as defined in claim 15, characterized in that it is for the preparation of a medicament for inducing a protective immune response in a vertebrate.

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法律状态:
2021-06-15| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI N? 10196/2001, QUE MODIFICOU A LEI N? 9279/96, A CONCESS?O DA PATENTE EST? CONDICIONADA ? ANU?NCIA PR?VIA DA ANVISA. CONSIDERANDO A APROVA??O DOS TERMOS DO PARECER N? 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL N? 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVID?NCIAS CAB?VEIS. |

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2021-08-17| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-09-14| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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