Liposomes containing lipids with pKa values ​​favorable for RNA delivery

Abstract

To provide a composition containing liposomes and RNA molecules encoding an immunogen.SOLUTION: RNA encoding an immunogen is delivered in a liposome for the purposes of immunisation. The liposome includes lipids which have a pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine. These liposomes can have essentially neutral surface charge at physiological pH and are effective for immunisation. In one embodiment, a liposome having a lipid bilayer encapsulating an aqueous core is provided, wherein: (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6; and (ii) the aqueous core includes a RNA which encodes an immunogen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Applications No. 61 / 361,830 (filed July 6, 2010) and No. 61 / 378,837 (filed August 31, 2010), the entirety of both of the aforementioned U.S. Provisional Applications being incorporated herein by reference for all purposes.

[0002] (Technical field) This invention relates to the field of nonviral delivery of RNA for immune purposes. [Background technology]

[0003] (Background technology) The delivery of nucleic acids to immunize animals has been a goal for several years. Various approaches have been tested, including the use of DNA or RNA, the use of viral or nonviral delivery vehicles (or even without a delivery vehicle in "naked" vaccines), the use of replicating or non-replicating vectors, or the use of viral or nonviral vectors.

[0004] There is a need for further improved nucleic acid vaccines. [Overview of the project] [Means for solving the problem]

[0005] (Disclosure of the invention) According to the present invention, RNA encoding an immunogen is delivered in liposomes for the purpose of immunization. The liposomes contain lipids having a pKa in the range of 5.0 to 7.6. Ideally, lipids having a pKa in this range have a tertiary amine; such lipids behave differently from lipids having a quaternary amine group (e.g., DOTAP or DC-Chol). At physiological pH, amines having a pKa in the range of 5.0 to 7.6 have a neutral or reduced surface charge, whereas lipids like DOTAP are strongly cationic. The inventors have found that liposomes formed from quaternary amine lipids (e.g., DOTAP) are less suitable for delivering RNA encoding an immunogen than liposomes formed from tertiary amine lipids (e.g., DLinDMA).

[0006] Accordingly, the present invention provides a liposome having a lipid bilayer encapsulating an aqueous core, wherein (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6, and preferably a lipid having a tertiary amine; and (ii) the aqueous core comprises a liposome containing RNA encoding an immunogen. Since these liposomes are suitable for in vivo delivery of the RNA to vertebrate cells, they are useful as components in pharmaceutical compositions for immunizing subjects against various diseases.

[0007] The present invention also provides a process for preparing RNA-containing liposomes, comprising: (a) mixing RNA and a lipid at a pH less than the pKa of the lipid but greater than 4.5 to form liposomes containing the RNA; and (b) raising the pH of the resulting liposome-containing mixture to a level greater than the pKa of the lipid.

[0008] (Liposomes) The present invention utilizes liposomes in which RNA encoding an immunogen is encapsulated. Thus, the RNA is separated from any external medium by the lipid bilayer of the liposome (as in natural viruses), and this encapsulation has been found to protect the RNA from RNase digestion. The liposomes may contain some external RNA (e.g., on their surface), but at least half (and ideally all) of the RNA is encapsulated within the core of the liposome. The encapsulation within the liposome differs from the lipid / RNA complex disclosed, for example, in Reference 1.

[0009] Various amphiphilic lipids can form a bilayer in an aqueous environment for encapsulating an RNA-containing aqueous core as a liposome. These lipids may have anionic, cationic, or zwitterionic hydrophilic head groups. The liposomes of the present invention include lipids having a pKa in the range of 5.0 to 7.6, and preferred lipids having a pKa in this range have tertiary amines. For example, they may include 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA; pKa 5.8) and / or 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Another suitable lipid having a tertiary amine is 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA). See Figure 3 and Reference 2. Some of the amino acid lipids in Reference 3 may also be used, as may certain of the amino lipids in Reference 4. Further useful lipids having tertiary amines in their head groups are disclosed in Reference 5 (the complete contents thereof are incorporated herein by reference).

[0010] The liposomes of the present invention may be formed from a single lipid or a mixture of lipids, provided that at least one of the lipids has a pKa in the range of 5.0 to 7.6 (and preferably a tertiary amine). Within this pKa range, preferred lipids have a pKa of 5.5 to 6.7 (e.g., between 5.6 to 6.8, between 5.6 to 6.3, between 5.6 to 6.0, between 5.5 to 6.2, or between 5.7 to 5.9). The above pKa is the pH at which 50% of the lipid is charged, which is midway between the point where the lipid is fully charged and the point where the lipid is not fully charged. The above pKa is measured using the method disclosed below in the section titled "pKa Measurement". The above pKa should typically be measured for the lipid alone rather than for the lipid in the context of a mixture that also includes other lipids (e.g., as seen in the pKa of SNALP rather than the pKa of individual lipids, as is done in Reference 6).

[0011] When the liposomes of the present invention are formed from a mixture of lipids, it is preferable that the proportion of those lipids having a pKa within a desired range be between 20 and 80% of the total amount of lipids (e.g., between 30 and 70%, or between 40 and 60%). For example, useful liposomes are shown below, where 40% or 60% of the total lipids are lipids having a pKa within a desired range. The remainder can be made from, for example, cholesterol (e.g., 35-50% cholesterol) and / or DMG (PEGylated as needed) and / or DSPC. Such mixtures are used below. These percentage values ​​are molar percentages.

[0012] Liposomes may contain amphiphilic lipids whose hydrophilic portions are PEGylated (i.e., modified by covalent bonding of polyethylene glycol). This modification can increase the stability of the liposomes and prevent nonspecific adsorption. For example, lipids can be conjugated to PEG using techniques such as those disclosed in References 6 and 7. PEG provides a coating that can confer favorable pharmacokinetic properties to the liposomes. The combination of efficient encapsulation of RNA (particularly self-replicating RNA), cationic lipids with pKas in the range of 5.0–7.6, and a PEGylated surface enables efficient delivery to multiple cell types (including both immune and non-immune cells), thereby inducing a more potent and favorable immune response than when using quaternary amines without PEGylation. PEGs of various lengths can be used, for example, between 0.5 and 8 kDa.

[0013] The lipids used in this invention may be saturated or unsaturated. It is preferable to use at least one unsaturated lipid to prepare liposomes. Figure 3 shows three useful unsaturated lipids. If an unsaturated lipid has two tails, both tails may be unsaturated, or it may have one saturated tail and the other unsaturated tail.

[0014] A mixture of DSPC, DLinDMA, PEG-DMG, and cholesterol is used in the examples. An independent aspect of the present invention is a liposome comprising DSPC, DLinDMA, PEG-DMG, and cholesterol. This liposome preferably encapsulates RNA (e.g., self-replicating RNA, e.g., encoding an immunogen).

[0015] Liposome particles are typically divided into three groups: multilayer vesicles (MLVs); small monolayer vesicles (SUVs); and large monolayer vesicles (LUVs). MLVs have multiple bilayers in each vesicle, forming several distinct aqueous compartments. SUVs and LUVs have a single bilayer encapsulating an aqueous core; SUVs typically have a diameter ≤ 50 nm, and LUVs have a diameter > 50 nm. The liposome particles of the present invention are ideally LUVs having a diameter in the range of 50–220 nm. With regard to compositions comprising a population of LUVs having various diameters: (i) at least 80% (in number) should have a diameter in the range of 20–220 nm, (ii) the average diameter (Zav (in intensity)) of the above population should ideally be in the range of 40–200 nm, and / or (iii) the above diameters should have a polydispersity index < 0.2. The liposome / RNA complex described in Reference 1 is predicted to have a diameter in the range of 600–800 nm and exhibit high polydispersity. The liposome described above may be substantially spherical.

[0016] Techniques for preparing suitable liposomes are well known in the art (see, for example, references 8-10). One useful method is described in reference 11 and comprises the steps of (i) mixing an ethanol solution of lipids, (ii) an aqueous solution of nucleic acids, and (iii) a buffer, followed by mixing, equilibration, dilution, and purification. Preferred liposomes of the present invention can be obtained by this mixing process.

[0017] (Mixing process) As described above, the present invention provides a process for preparing RNA-containing liposomes, comprising the steps of (a) mixing RNA and a lipid at a pH less than the pKa of the lipid but greater than 4.5; and (b) raising the pH to greater than the pKa of the lipid.

[0018] Thus, the cationic lipid is positively charged during liposome formation in step (a), but the subsequent pH change means that most (or all) of the positively charged groups become neutral. This process is advantageous for preparing the liposomes of the present invention, and by avoiding a pH of less than 4.5 during step (a), the stability of the encapsulated RNA is improved.

[0019] The pH in step (a) is greater than 4.5, and ideally greater than 4.8. Using a pH in the range of 5.0 - 6.0 or in the range of 5.0 - 5.5 can provide suitable liposomes.

[0020] The increased pH in step (b) is above the pKa of the lipid. The pH ideally rises to less than 9, and preferably less than 8. Depending on the pKa of the lipid, the pH in step (b) can thus rise to be within the range of 6 - 8 (e.g., to pH 6.5 ± 0.3). The pH increase in step (b) can be achieved by transferring the liposomes to a suitable buffer (e.g., phosphate buffered saline). The pH increase in step (b) is ideally carried out after liposome formation has occurred.

[0021] The RNA used in step (a) can be present in an aqueous solution while being mixed with an organic solution of the lipid (e.g., an ethanolic solution as in reference 11). The mixture can then be diluted to form liposomes, and thereafter, the pH can be raised in step (b).

[0022] (RNA) The present invention is useful for in vivo delivery of RNA encoding an immunogen. The RNA is translated by non - immune cells at the site of delivery, resulting in the expression of the immunogen, and also, it causes immune cells to secrete type I interferon and / or pro - inflammatory cytokines, which provide a local adjuvant effect. The non - immune cells can also secrete type I interferon and / or pro - inflammatory cytokines in response to the RNA.

[0023] Since the above RNA is a plus strand, it can be translated by non-immune cells without any intervening replication step (e.g., reverse transcription). It can also bind to the TLR7 receptor expressed by immune cells, thereby initiating an adjuvant effect.

[0024] Preferred plus strand RNAs are self-replicating. Self-replicating RNA molecules (replicons) can result in the generation of multiple daughter RNAs by transcription from the self-replicating RNA molecule itself (via an antisense copy generated from the self-replicating RNA molecule itself), even when delivered to vertebrate cells without any protein. Self-replicating RNA molecules are thus typically plus strand molecules that can be directly translated after delivery to cells, and this translation provides an RNA-dependent RNA polymerase, which then generates both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the generation of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded immunogen or transcribed to provide additional transcripts of the same sense as the delivered RNA (translated to provide in situ expression of the immunogen). The overall result of this series of transcriptions is a very large amplification in the number of the introduced replicon RNAs, and thus the encoded immunogen becomes the major polypeptide product of the cells.

[0025] As shown below, self-replicating activity is not required for the RNA to provide an adjuvant effect, but it can enhance the post-transfection secretion of cytokines. The self-replicating activity is particularly useful for achieving high-level expression of immunogens by non-immune cells. It can also enhance the apoptosis of the non-immune cells.

[0026] One suitable system for achieving self-replication is to use alphavirus-based RNA replication. These positive-strand replicons are translated after delivery to cells to give a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that produces a genomic negative-strand copy of the delivered positive-strand RNA. These negative-strand transcripts can be transcribed to give themselves a further copy of the positive-strand parental RNA and also a subgenomic transcript encoding an immunogen. Thus, translation of the subgenomic transcript results in in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons may use replicases derived from Sindbis virus, Semliki forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Sequences of mutant or wild-type viruses may be used (e.g., the attenuated TC83 variant of VEEV has been used in replicons

[12] ).

[0027] A preferred self-replicating RNA molecule is, therefore, (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an immunogen. The polymerase may be an alphaviral replicase (e.g., comprising one or more of the alphaviral proteins nsP1, nsP2, nsP3, and nsP4).

[0028] While the natural alphavirus genome encodes structural virion proteins in addition to the non-structural replicase polyproteins mentioned above, the self-replicating RNA molecule of the present invention preferably does not encode alphavirus structural proteins. Therefore, the preferred self-replicating RNA can result in the generation of its own genomic RNA copies in cells, but not in the generation of RNA-containing virions. The inability to generate these virions means that, unlike wild-type alphavirus, the self-replicating RNA molecule cannot persist itself in an infectious form. The alphavirus structural proteins necessary for persistence in wild-type viruses are not present in the self-replicating RNA of the present invention, and their positions are occupied by genes encoding the immunogen of the purpose. As a result, the subgenome transcript encodes the immunogen, not the structural alphavirus virion proteins.

[0029] Accordingly, a self-replicating RNA molecule useful in the present invention may have two open reading frames. The first (5' side) open reading frame encodes a replicase; the second (3' side) open reading frame encodes an immunogen. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames to encode, for example, a further immunogen (see below) or an auxiliary polypeptide.

[0030] The self-replicating RNA molecule may have a 5' sequence that is compatible with the replicase encoded above.

[0031] Self-replicating RNA molecules can have various lengths, but they are typically 5,000 to 25,000 nucleotides long (for example, 8,000 to 15,000 nucleotides, or 9,000 to 12,000 nucleotides). Therefore, the above RNA is longer than that observed in siRNA delivery.

[0032] RNA molecules useful in this invention may have a 5' cap (e.g., 7-methylguanosine). This cap can enhance the in vivo translation of the RNA.

[0033] The 5' nucleotide of the RNA molecule useful in this invention may have a 5' triphot group. In capped RNA, this can be linked to 7-methylguanosine via a 5'-to-5' crosslink. The 5' triphot group can enhance RIG-I binding and thus promote the adjuvant effect.

[0034] RNA molecules may have a 3' poly(A) tail. They may also contain a poly(A) polymerase recognition sequence (e.g., AAUAAA) near their 3' end.

[0035] The RNA molecules useful in this invention are typically single-stranded. Single-stranded RNA can generally initiate adjuvant effects by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, which receptor can also be triggered by dsRNA formed either during single-stranded RNA replication or within the secondary structure of single-stranded RNA.

[0036] RNA molecules useful in the present invention may, for convenience, be prepared by in vitro transcription (IVT). IVT may use (cDNA) templates that have been produced and amplified in plasmid form in bacteria or synthesized (e.g., by gene synthesis and / or polymerase chain reaction (PCR) procedures). For example, DNA-dependent RNA polymerases (e.g., bacteriophage T7, T3, or SP6 RNA polymerases) may be used to transcribe RNA from the DNA template. Appropriate capping and poly-A addition reactions may be used as needed (however, the poly-A of the replican is usually encoded within the DNA template). These RNA polymerases may have stringent requirements for the 5' nucleotide to be transcribed, and in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT transcription RNA can function efficiently as a substrate for the replicase it encodes.

[0037] As discussed in Reference 13, the above self-replicating RNA may contain one or more nucleotides having modified nucleic acid bases (in addition to any 5' cap structure). Therefore, the above RNA may include: m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (l-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io 6A(N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine);g6A(N6-glycinylcarbamoyladenosine);t6A(N6-threonylcarbamoyladenosine);ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine);m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6-hydroxynorvalylcarbamoyladenosine);ms 2hn6A(2-methylthio-N6-hydroxynorvalylcarbamoyladenosine);Ar(p)(2'-O-ribosyladenosine (phosphate));I(inosine);m11(1-methylinosine);m'Im(1,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-O-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-formyl(fonnyl)cytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm (N4-acetyl-2-TO-methylcytidine); k2C (lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine);OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queosine); oQ (epoxyqueosine); galQ (galactosyl-queosine); manQ (mannosyl-queosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine); m 5Um(5,2'-O-dimethyluridine);s4U(4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);ho5U(5-hydroxyuridine);mo5U(5-methoxyuridine);cmo5U(uridine 5-oxyacetic acid);mcmo5U(uridine 5-oxyacetic acid methyl ester);chm5U(5-(carboxyhydroxymethyl)uridine));mchm5U(5-(carboxyhydroxymethyl) (5-Methoxycarbonylmethyluridine); mcm5U(5-Methoxycarbonylmethyluridine); mcm5Um(S-Methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U(5-Methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-Aminomethyl-2-thiouridine); mnm5U(5-Methylaminomethyluridine); mnm5s2U(5-Methylaminomethyl-2-thiouridine); mnm5se2U(5-Methylaminomethyl-2-selenouridine); ncm5U(5-Carbamoylmethyluridine); nc m5Um(5-carbamoylmethyl-2'-O-methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cnmm5Um(5-carboxymethylaminomethyl-2-LOmethyluridine);cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine);m62A(N6,N6-dimethyladenosine);Tm(2'-O-methylinosine);m4C(N4-methylcytidine);m4Cm(N4,2-O-dimethylcytidine);hm5C(5-hydroxymethylcytidine);m3U(3-methyluridine);cm5U(5-carboxymethyluridine); m6Am(N6,TO-dimethyladenosine); rn62Am(N6,N6,O-2-trimethyladenosine); m2'7G(N2,7-dimethylguanosine); m2'2'7G(N2,N2,7-trimethylguanosine); m3Um(3,2T-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm(5-formyl-2'-O-methylcytidine); m1Gm(1,2'-O-dimethylguanosine); m'Am((1,2-O-dimethyladenosine)irinomethyluridine); tm5s2U(S-taurinomethyl-2-thiouridine); imG-14(4-demethylguanosine); imG2(isoguanosine);Alternatively, ac6A(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine Syn, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, or a baseless nucleotide. For example, self-replicating RNA may contain one or more modified pyrimidine nucleic acid bases (e.g., pseudouridine and / or 5-methylcytosine residues). However, in some embodiments, the RNA may not contain modified nucleic acid bases and may not contain modified nucleotides (i.e., all nucleotides in the RNA are standard A, C, G, and U ribonucleotides (except for any 5' cap structure, which may include 7'-methylguanosine)). In other embodiments, the RNA may contain a 5' cap containing 7'-methylguanosine, and the first one, two, or three 5' ribonucleotides may be methylated at the 2' position of ribose.

[0038] The RNA used in the present invention ideally contains only phosphodiester bonds between nucleotides, but in some embodiments it may contain phosphoramidate bonds, phosphorothioate bonds, and / or methylphosphonate bonds.

[0039] Ideally, the liposomes contain fewer than 10 different RNA species (e.g., 5, 4, 3, or 2 different species); most preferably, the liposomes contain a single RNA species (i.e., all RNA molecules in the liposomes have the same sequence and length).

[0040] The amount of RNA per liposome can vary. Typically, the number of individual self-replicating RNAs per liposome is ≤50 (e.g., <20, <10, <5, or 1-4).

[0041] (immunogen) The RNA molecules used in this invention encode polypeptide immunogens. After administration of the liposomes, the RNA is translated in vivo, and the immunogen can induce an immune response in the recipient. The immunogen can induce an immune response against bacteria, viruses, fungi, or parasites (or, in some embodiments, against allergens; and in other embodiments, against tumor antigens). The immune response may include an antibody response (usually including IgG) and / or a cell-mediated immune response. The polypeptide immunogen typically induces an immune response that recognizes the corresponding bacterial, viral, fungal, or parasite (or allergen or tumor) polypeptide, but in some embodiments, the polypeptide may act as a mimotop to induce an immune response that recognizes a saccharide of a bacterium, virus, fungus, or parasite. The immunogen is typically a surface polypeptide (e.g., adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc.).

[0042] Self-replicating RNA molecules may encode a single polypeptide immunogen or multiple polypeptides. Multiple immunogens may be presented as a single polypeptide immunogen (fusion polypeptide) or as separate polypeptides. When immunogens are expressed as separate polypeptides, one or more of these may be provided with an upstream IRES or further viral promoter element. Alternatively, multiple immunogens may be expressed from polyproteins encoding individual immunogens fused to a short autocatalytic protease (e.g., foot-and-mouth disease virus 2A protein), or as an intein.

[0043] Unlike references 1 and 14, the above RNA encodes an immunogen. To avoid uncertainty, the present invention does not include RNA encoding firefly luciferase, RNA encoding E. coli β-galactosidase fusion protein, or RNA encoding green fluorescent protein (GFP). Furthermore, the above RNA is not whole mouse thymus RNA.

[0044] In some embodiments, the immunogen induces an immune response against one of these bacteria: Neisseria meningitidis: Useful immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron-acquiring proteins, and H-factor-binding proteins. Three useful polypeptide combinations are disclosed in reference 15.

[0045] Useful polypeptide immunogens for Streptococcus pneumoniae are disclosed in reference 16. These include, but are not limited to, the RrgB ciliary subunit, β-N-acetyl-hexosaminidase precursors (spr0057), spr0096, general stress proteins GSP-781 (spr2021, SP2216), serine / threonine kinase StkP (SP1732), and pneumococcus surface adhesin PsaA.

[0046] Streptococcus pyogenes: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 17 and 18.

[0047] Moraxella catarrhalis Bordetella pertussis: Useful pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), fibrous hemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.

[0048] Staphylococcus aureus: Useful immunogens include, but are not limited to, the polypeptides disclosed in Reference 19 (e.g., hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and / or sta011 lipoprotein).

[0049] Clostridium tetani: Its most representative immunogen is tetanus toxoid.

[0050] Cornynebacterium diphtheriae: Its representative immunogen is diphtheria toxoid.

[0051] Haemophilus influenzae: Useful immunogens include, but are not limited to, the polypeptides disclosed in references 20 and 21.

[0052] Pseudomonas aeruginosa Streptococcus agalactiae: Useful immunogens include, but are not limited to, the polypeptides disclosed in Reference 17.

[0053] 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 (as disclosed, for example, in reference 22). LcrE

[23] and HtrA

[24] are two preferred immunogens.

[0054] Chlamydia pneumoniae: Useful immunogens include, but are not limited to, the polypeptides disclosed in Reference 25.

[0055] Helicobacter pylori: Useful immunogens include, but are not limited to, CagA, VacA, NAP, and / or ureases

[26] .

[0056] Escherichia coli: Useful immunogens include, but are not limited to, immunogens derived from enterotoxin-producing E. coli (ETEC), enteric agglutinating E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraenteropathogenic E. coli (ExPEC), and / or enterohemorrhagic E. coli (EHEC). ExPEC strains include urinary tract pathogenic E. coli (UPEC) and meningitis / sepsis-associated E. coli (MNEC). Useful UPEC polypeptide immunogens are disclosed in references 27 and 28. Useful MNEC immunogens are disclosed in reference 29. AcfD is a useful immunogen for several E. coli types

[30] .

[0057] Bacillus anthracis Yersinia pestis: Useful immunogens include, but are not limited to, those disclosed in references 31 and 32.

[0058] [ka]

[0059] [ka] In some embodiments, the immunogen induces an immune response against one of these viruses: Orthomyxoviruses: Useful immunogens may be derived from influenza A, B, or C viruses (e.g., hemagglutinin, neuraminidase, or matrix M2 protein). If the immunogen is influenza A virus hemagglutinin, it may be derived from any subtype (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16).

[0060] Viruses of the Paramyxoviridae family: Viral immunogens include, but are not limited to, those derived from pneumonia viruses (e.g., respiratory syncytial virus, RSV), rubra viruses (e.g., mumps virus), paramyxoviruses (e.g., parainfluenza virus), metapneumoviruses, and morbilliviruses (e.g., measles virus).

[0061] Poxviridae: Viral immunogens include, but are not limited to, those derived from orthopoxviruses (e.g., *Psathyrrhizon* (including, but not limited to, *Psathyrrhizon* and *Psathyrrhizon*)).

[0062] Picornaviruses: Viral immunogens may include, but are not limited to, those derived from picornaviruses (e.g., enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aftoviruses). In one embodiment, the enterovirus is poliovirus (e.g., type 1, type 2, and / or type 3 poliovirus). In another embodiment, the enterovirus is EV71 enterovirus. In yet another embodiment, the enterovirus is coxsackie A or B virus.

[0063] Bunyaviruses: Viral immunogens include, but are not limited to, those derived from orthobunyaviruses (e.g., California encephalitis virus), phleboviruses (e.g., Rift Valley fever virus), or nairoviruses (e.g., Crimean-Congo hemorrhagic fever virus).

[0064] Heparnavirus: Viral immunogens include, but are not limited to, those derived from heparnavirus (e.g., hepatitis A virus (HAV)).

[0065] Filoviruses: Viral immunogens include, but are not limited to, those derived from filoviruses (e.g., Ebola virus (including Zaire Ebola virus, Ivory Coast Ebola virus, Reston Ebola virus, or Sudan Ebola virus)) or Marburg virus.

[0066] Togaviruses: Viral immunogens include, but are not limited to, those derived from togaviruses (e.g., rubivirus, alphavirus, or arterivirus). This includes rubella virus.

[0067] Flaviviruses: Viral immunogens include, but are not limited to, those derived from flaviviruses (e.g., tick-borne encephalitis (TBE) virus, dengue (types 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kyasanur forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Poissant encephalitis virus).

[0068] Pestivirus: Viral immunogens include, but are not limited to, those derived from pestiviruses (e.g., bovine viral diarrhea (BVDV), classical swine fever (CSFV), or border disease (BDV)).

[0069] Hepadnavirus: Viral immunogens include, but are not limited to, those derived from hepadnavirus (e.g., hepatitis B virus). The composition may contain hepatitis B virus surface antigen (HBsAg).

[0070] Other hepatitis viruses: The composition may contain immunogens derived from hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus.

[0071] Rhabdoviruses: Viral immunogens include, but are not limited to, those derived from rhabdoviruses (e.g., lyssavirus (e.g., rabies virus) and becyclovirus (VSV)).

[0072] Caliciviridae: Viral immunogens include, but are not limited to, those derived from the Caliciviridae family (e.g., Norwalk virus (norovirus), and Norwalk-like viruses (e.g., Hawaiian virus and Snow Mountain virus)).

[0073] Coronaviruses: Viral immunogens include, but are not limited to, those derived from SARS coronavirus, avian infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV). The above coronavirus immunogens may be spike polypeptides.

[0074] Retroviruses: Viral immunogens include, but are not limited to, those derived from oncoviruses, lentiviruses (e.g., HIV-1 or HIV-2), or spumaviruses.

[0075] Reoviruses: Viral immunogens include, but are not limited to, those derived from orthoreovirus, rotavirus, or orbivirus, or cortivirus.

[0076] Parvovirus: Viral immunogens include, but are not limited to, those derived from parvovirus B19.

[0077] Herpesviruses: Viral immunogens include, but are not limited to, those derived from human herpesviruses (for example, herpes simplex virus (HSV) (e.g., HSV types 1 and 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8)).

[0078] Papovavirus: Viral immunogens include, but are not limited to, those derived from papillomaviruses and polyomaviruses. The (human) papillomaviruses mentioned above may be serotypes 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, derived from one or more of serotypes 6, 11, 16, and / or 18).

[0079] Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36).

[0080] In some embodiments, the immunogens induce an immune response to viruses that infect fish (e.g., infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), American catfish virus (CCV), fish lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), carp herpesvirus, salmon picorna-like virus (also known as Atlantic salmon picorna-like virus), cherry salmon virus (LSV), Atlantic salmon rotavirus (ASR), trout disease virus (TSD), silver salmon tumor virus (CSTV), or viral hemorrhagic septic virus (VHSV)).

[0081] Fungal immunogens include dermatophytes (such as the following):

[0082] [ka]

[0083] [ka] It may originate from this.

[0084] In some embodiments, the immunogens induce an immune response against parasites derived from the genus Plasmodium (e.g., P. falciparum, P. vivax, P. malariae, or P. ovale). Therefore, the present invention can be used to immunize against malaria. In some embodiments, the immunogens induce an immune response against parasites derived from the family Caligidae, particularly parasites derived from the genera Lepeophtheirus and Caligus (e.g., sea slaters such as Lepeophtheirus salmonis or Caligus rogercresseyi).

[0085] In some embodiments, the immunogens induce an immune response to: pollen allergens (tree pollen, herbaceous pollen, weed pollen, and grass pollen allergens); insect or arachnid allergens (inhalation, saliva, and venom allergens, e.g., dust mite allergens, cockroach allergens, and worm allergens, Hymenopthera venom allergen); animal hair and dander allergens (e.g., derived from dogs, cats, horses, rats, mice, etc.); and food allergens (e.g., gliadin). Important pollen allergens originating from trees, grasses, and herbs are those derived from the taxonomic orders Fagales, Oleales, and Pinales, and the family Platanaceae (including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), olive (Olea), cedar (Cryptomeria and Juniperus), and plane tree (Platanus)), the order Poales (including herbs of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum), and the orders Asterales and Urticales (including herbs of the genera Ambrosia, Artemisia, and Palietaria). Other important inhalation allergens include those derived from dust mites and storage mites of the genera Dermatophagoides and Euroglyphus (e.g., Lepidoglyphys, Glycyphagus, and Tyrophagus), cockroaches, gnats, and fleas (e.g., Blatella, Periplaneta, Chironomus, and Ctenocepphalides), as well as those derived from mammals (e.g., cats, dogs, and horses), and venomous allergens (such as those derived from stinging or biting insects, e.g., the taxonomic orders Hymenoptera (including bees (Apidae), wasps (Vespidea), and ants (Formicoidae))).

[0086] In some embodiments, the immunogen is a tumor antigen selected from: (a) cancer-testis antigens, e.g., NY-ESO-1, SSX2, SCP1, and polypeptides of the RAGE, BAGE, GAGE, and MAGE families (e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12), which can be used to address tumors of, for example, melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder; (b) mutated antigens, e.g., p53 (associated with various solid tumors (e.g., colorectal cancer, lung cancer, head and neck cancer)), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA (c) Overexpressed antigens, e.g., 0205 (e.g., associated with bladder cancer), HLA-A2-R1701, β-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triose phosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) Overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT 1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast cancer, colon cancer, lung cancer, and ovarian cancer), mammoglobin, alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g., associated with colorectal cancer), gastrin (e.g., associated with pancreatic cancer and gastric cancer), telomerase catalytic protein, MUC-1 (e.g., associated with breast cancer and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast cancer and colon cancer), and carcinoembryonic antigen (e.g., associated with breast cancer, lung cancer, and gastrointestinal cancers (e.g., colorectal cancer));(d) Shared antigens, e.g., melanoma-melanocyte differentiation antigens, e.g., MART-1 / Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) Prostate-related antigens (e.g., PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2 (e.g., associated with prostate cancer)); (f) Immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma). In certain embodiments, tumor immunogens include, but are not limited to, p15, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens (including E6 and E7), hepatitis B virus and hepatitis C virus antigens, human T lymphotropic virus antigens;

[0087] [ka] (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, TPS, etc.

[0088] (Pharmaceutical composition) The liposomes of the present invention are useful as components in pharmaceutical compositions for immunizing subjects against various diseases. These compositions typically include, in addition to the liposomes, a pharmaceutically acceptable carrier. A detailed discussion of pharmaceutically acceptable carriers is available in Reference 33.

[0089] The pharmaceutical composition of the present invention may contain one or more small-molecule immune-enhancing factors. For example, the composition may contain a TLR2 agonist (e.g., Pam3CSK4), a TLR4 agonist (e.g., aminoalkylglucosaminide phosphate (e.g., E6020)), a TLR7 agonist (e.g., imiquimod), a TLR8 agonist (e.g., reximod), and / or a TLR9 agonist (e.g., IC31). Any such agonist ideally has a molecular weight <2000 Da. When RNA is encapsulated, in some embodiments such agonists are also encapsulated together with the RNA, but in other embodiments they are not. When RNA is adsorbed onto particles, in some embodiments such agonists are also adsorbed together with the RNA, but in other embodiments they are not.

[0090] The pharmaceutical composition of the present invention may contain the above-mentioned liposomes in plain water (e.g., WFI) or a buffer (e.g., phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer). The buffer salt is typically included in a range of 5 to 20 mM.

[0091] The pharmaceutical composition of the present invention may have a pH of 5.0 to 9.5 (for example, 6.0 to 8.0).

[0092] The composition of the present invention may contain a sodium salt (e.g., sodium chloride) to provide tonicity. A typical concentration of NaCl is 10 ± 2 mg / ml (e.g., about 9 mg / ml).

[0093] The compositions of the present invention may contain metal ion chelating agents. These can extend RNA stability by removing ions that can accelerate phosphodiester hydrolysis. Accordingly, the compositions may contain one or more of the following: EDTA, EGTA, BAPTA, pentetate, etc. Such chelating agents are typically present at concentrations of 10 to 500 μM (e.g., 0.1 mM). Citrates (e.g., sodium citrate) can also act as chelating agents and, advantageously, provide buffering activity.

[0094] The pharmaceutical compositions of the present invention may have a gravimetric osmolality of 200 mOsm / kg to 400 mOsm / kg (for example, 240 to 360 mOsm / kg, or 290 to 310 mOsm / kg).

[0095] The pharmaceutical composition of the present invention may contain one or more preservatives (for example, thiomersal or 2-phenoxyethanol). A mercury-free composition is preferred, and a preservative-free vaccine can be prepared.

[0096] The pharmaceutical composition of the present invention is preferably sterile.

[0097] The pharmaceutical compositions of the present invention are preferably nonpyrogenic (e.g., containing <1 EU (endotoxin unit, standard scale) per dose, and preferably <0.1 EU per dose).

[0098] The pharmaceutical composition of the present invention is preferably gluten-free.

[0099] The pharmaceutical compositions of the present invention can be prepared in unit dose form. In some embodiments, the unit dose may have a volume of 0.1 to 1.0 ml (for example, about 0.5 ml).

[0100] The above compositions may be prepared as injections (either as solutions or suspensions). The compositions may be prepared for pulmonary administration (e.g., by inhaler) using a fine spray. The compositions may be prepared for administration to the nose, ears, or eyes (e.g., as sprays or drops). Injections for intramuscular administration are typical.

[0101] The composition comprises an immunologically effective amount of liposomes, as well as any other components (if necessary). “Immunologically effective amount” means that the dose administered to an individual, 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 of the individual, the taxonomic group (e.g., non-human primates, primates, etc.), the individual’s ability of the immune system to synthesize antibodies, the desired level of protection, the vaccine prescription, the assessment of the medical condition of the treating physician, and other relevant factors. The amount is expected to fall within a relatively wide range that can be determined through conventional clinical trials. The liposome and RNA content of the composition of the present invention is generally expressed in terms of the amount of RNA per dose. A preferred dose is ≤100 μg RNA (e.g., 10–100 μg (e.g., approximately 10 μg, 25 μg, 50 μg, 75 μg, or 100 μg)), but expression may occur at much lower levels, e.g., ≤1 μg / dose, ≤100 ng / dose, ≤10 ng / dose, ≤1 ng / dose, etc.

[0102] The present invention also provides a delivery device (e.g., a syringe, nebulizer, sprayer, inhaler, skin patch, etc.) containing the pharmaceutical composition of the present invention. This device can be used to administer the composition to a vertebrate subject.

[0103] The liposomes of the present invention do not contain ribosomes.

[0104] (Treatment methods and medical use) In contrast to the particles disclosed in Reference 14, the liposomes and pharmaceutical compositions of the present invention are intended for in vivo use to induce an immune response to an immunogen of interest.

[0105] The present invention provides a method for inducing an immune response in vertebrates, comprising the step of administering an effective amount of the liposomes or pharmaceutical composition of the present invention. The immune response is preferably defensive and preferably comprises antibodies and / or cell-mediated immunity. The method may induce a booster response.

[0106] The present invention also provides liposomes or pharmaceutical compositions for use in methods for inducing an immune response in vertebrates.

[0107] The present invention also provides the use of the liposomes of the present invention in the manufacture of pharmaceuticals for inducing an immune response in vertebrates.

[0108] By using and using these methods to induce an immune response in the vertebrates, the vertebrates can be protected from various diseases and / or infections (e.g., bacterial and / or viral diseases) as discussed above. The liposomes and compositions are immunogenic and, more preferably, vaccine compositions. Vaccines according to the present invention may be either prophylactic (i.e., prevent infection) or therapeutic (i.e., treat infection), but are typically prophylactic.

[0109] The vertebrates mentioned above are preferably mammals, such as humans or large veterinary mammals (e.g., horses, cattle, deer, goats, pigs). If the vaccine is for prophylactic use, the humans are preferably children (e.g., infants or toddlers) or teenagers; if the vaccine is for therapeutic use, the humans are preferably teenagers or adults. Vaccines intended for children may also be administered to adults, for example, to evaluate safety, dosage, immunogenicity, etc.

[0110] Vaccines prepared according to the present invention may be used to treat both children and adults. Therefore, human patients may be under 1 year of age, under 5 years of age, 1–5 years of age, 5–15 years of age, 15–55 years of age, or at least 55 years of age. Patients who are preferable to receive the above vaccine include the elderly (e.g., ≥50 years, ≥60 years, and preferably ≥65 years), young people (e.g., ≤5 years), hospitalized patients, healthcare workers, military personnel and officers, pregnant women, patients with chronic diseases, or immunocompromised individuals. The above vaccine is not only suitable for these groups, but may be used more generally in populations.

[0111] The compositions of the present invention are generally administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or into the interstitial space of tissues; unlike Reference 1, intralingual injection is not typically used in the present invention). Alternative delivery routes include rectal, oral (e.g., tablets, spray), oral, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, ear, lung, or other mucosal administration. Intradermal and intramuscular administration are the two preferred routes. Injections may be administered via a needle (e.g., a subcutaneous needle), but needleless injections may be used instead. A typical intramuscular dose is 0.5 ml.

[0112] The present invention may be used to induce systemic immunity and / or mucosal immunity, preferably to induce enhanced systemic immunity and / or mucosal immunity.

[0113] Dosage may be administered via a single-dose schedule or a multi-dose schedule. Multi-dose immunization may be used in a primary immunization schedule and / or a booster immunization schedule. In a multi-dose schedule, various doses may be administered via the same or different routes (e.g., parenteral primary and mucosal boost, mucosal primary and parenteral boost). Multi-dose immunization may typically be administered at intervals of at least one week (e.g., approximately 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, etc.). In one embodiment, multi-dose immunization may be administered at approximately 6 weeks, 10 weeks, and 14 weeks of age (e.g., at 6 weeks, 10 weeks, and 14 weeks of age, as is often used in the World Health Organization's Expanded Program on Immunisation ("EPI"). In an alternative embodiment, two primary doses are administered approximately two months apart (for example, approximately seven, eight, or nine weeks apart), followed by one or more booster doses approximately six months to one year after the second primary dose (for example, approximately six, eight, ten, or twelve months after the second primary dose). In a further embodiment, three primary doses are administered approximately two months apart (for example, approximately seven, eight, or nine weeks apart), followed by one or more booster doses approximately six months to one year after the third primary dose (for example, approximately six, eight, ten, or twelve months after the third primary dose).

[0114] (General) The particles of the present invention, unless otherwise indicated, are produced using conventional methods within the arts of chemistry, biochemistry, molecular biology, immunology, and pharmacology. Such techniques are well described in the literature; see, for example, references 34-40.

[0115] The term "comprising" encompasses both "including" and "consisting." For example, a composition "comprising" X may consist solely of X or it may also contain something else (e.g., X + Y).

[0116] The term "approximately" in relation to the numerical value x is selective and can mean, for example, x ± 10%.

[0117] The phrase "substantially" does not exclude "completely"; for example, a composition that "substantially does not contain" Y may also completely contain Y. Where necessary, the phrase "substantially" may be omitted from the definition of this invention.

[0118] References to electric charge, cations, anions, and amphoteric ions are addressed at pH 7.

[0119] TLR3 is Toll-like receptor 3. It is a single-pass transmembrane receptor that plays an important role in the innate immune system. Known TLR3 agonists include poly(I:C). "TLR3" is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:11849. The RefSeq sequence of the human TLR3 gene is GI:2459625.

[0120] TLR7 is Toll-like receptor 7. It is a single-pass transmembrane receptor that plays an important 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 of the human TLR7 gene is GI:67944638.

[0121] TLR8 is Toll-like receptor 8. It is a single-pass transmembrane receptor that plays an important role in the innate immune system. Known TLR8 agonists include, for example, reximod. "TLR8" is the approved HGNC name for the gene encoding this receptor, and its unique HGNC ID is HGNC:15632. The RefSeq sequence of the human TLR8 gene is GI:20302165.

[0122] The RIG-I-like receptor ("RLR") family comprises various RNA helicases that play important roles in the innate immune system

[41] . RLR-1 (also known as RIG-I or retinoic acid-inducible gene I) has two caspase-recruiting domains near its N-terminus. The approved HGNC name for the gene encoding the RLR-1 helicase is "DDX58" (for DEAD (Asp-Glu-Ala-Asp) box polypeptide 58), and its 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 melanoma differentiation-associated gene 5) also has two caspase-recruiting domains near its N-terminus. The approved HGNC name for the gene encoding RLR-2 helicase is "IFIH1" (for interferon induced with helicase C domain 1), and its 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 Laboratory of Genetics and Physiology 2) does not have a caspase recruiting domain. The approved HGNC name for the gene encoding RLR-3 helicase is "DHX58" (for DEXH(Asp-Glu-X-His) box polypeptide 58), and its unique HGNC ID is HGNC:29517. The RefSeq sequence for the human RLR-3 gene is GI:149408121.

[0123] PKR is a double-stranded RNA-dependent protein kinase. It plays a crucial 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 of the human PKR gene is GI:208431825. [Brief explanation of the drawing]

[0124] [Figure 1] Figure 1 shows a gel containing stained RNA. The lanes represent (1) marker, (2) bare replicon, (3) replicon after RNase treatment, (4) replicon encapsulated in liposomes, (5) liposomes after RNase treatment, and (6) liposomes treated with RNase and then subjected to phenol / chloroform extraction. [Figure 2] Figure 2 is an electron microscope image of a liposome. [Figure 3] Figure 3 shows the structures of DLinDMA, DLenDMA, and DODMA. [Figure 4] Figure 4 shows a gel containing stained RNA. The lanes represent (1) a marker, (2) a bare replicon, (3) a replicon encapsulated in liposomes, and (4) liposomes treated with RNase and then subjected to phenol / chloroform extraction. [Figure 5] Figure 5 shows protein expression on days 1, 3, and 6 after RNA delivery as virion-packaged replicons (squares), as naked RNA (diamonds), or in liposomes (+=0.1 μg, ×=1 μg). [Figure 6] Figure 6 shows protein expression on days 1, 3, and 6 after delivery of four different doses of liposome-encapsulated RNA. [Figure 7]Figure 7 shows the anti-F IgG titers in animals given virion-packaged replicons (VRP or VSRP), 1 μg of bare RNA, and 1 μg of liposome-encapsulated RNA. [Figure 8] Figure 8 shows the anti-F IgG titers in animals given VRP, 1 μg of bare RNA, and 0.1 g or 1 μg of liposome-encapsulated RNA. [Figure 9] Figure 9 shows the neutralizing antibody titers in animals given either VRP or 0.1 g or 1 μg of liposome-encapsulated RNA. [Figure 10] Figure 10 shows the expression levels after delivery of the replicon as naked RNA (circles), liposome-encapsulated RNA (triangles and squares), or lipoplex (inverted triangle). [Figure 11] Figure 11 shows the F-specific IgG titer (2 weeks after the second dose) after delivery of the replicon as naked RNA (0.01–1 μg), as liposome-encapsulated RNA (0.01–10 μg), or as virions packaged (VRP, 10⁶ infection units, or IU). [Figure 12] Figure 12 shows the F-specific IgG titers (circles) and PRNT titers (squares) after delivery of the replicon as naked RNA (1 μg), as liposome-encapsulated RNA (0.1 or 1 μg), or as packaged as a virion (VRP, 10⁶ IU). Titers in naive mice are also shown. The solid line represents the geometric mean. [Figure 13] Figure 13 shows intracellular cytokine production after restimulation with a synthetic peptide representing the major epitope in the F protein, 4 weeks after the second dose. The y-axis shows the % cytokine+ for CD8+CD4-. [Figure 14A] Figure 14 shows the F-specific IgG titers (mean log10 titer ± standard deviation) of calves 63 days (Figure 14A) and 210 days (Figure 14B) after immunization. The three lines are easily distinguishable at day 63, from bottom to top: PBS negative control; liposomal delivery RNA; and "Triangle 4" product. [Figure 14B] Figure 14 shows the F-specific IgG titers (mean log10 titer ± standard deviation) of calves 63 days (Figure 14A) and 210 days (Figure 14B) after immunization. The three lines are easily distinguishable at day 63, from bottom to top: PBS negative control; liposomal delivery RNA; and "Triangle 4" product. [Figure 15] Figure 15 shows the SEAP expression (relative intensity) on day 6 against the pKa of the lipids used in the liposomes described above. Circles indicate the levels for liposomes with DSPCs, and squares indicate the levels for liposomes without DSPCs; sometimes the squares and circles overlap, so that only the squares are visible for a given pKa. [Figure 16] Figure 16 shows the expression of anti-F titer (RV01, 100%) two weeks after the first administration of the replicon encoding the F protein. The titers are plotted against pKa in the same manner as in Figure 15. Asterisks indicate RV02 using cationic lipids with higher pKa than other lipids. Triangles indicate data for liposomes lacking DSPC; circles indicate data for liposomes containing DSPC. [Figure 17] Figure 17 shows, from left to right, the total IgG titers after replicon delivery in liposomes using RV01, RV16, RV17, RV18, or RV19. The bars represent the mean values. In each case, the upper bar represents 2wp2 (i.e., 2 weeks after the second dose), while the lower bar represents 2wp1. [Figure 18] Figure 18 shows IgG titers in 13 mouse groups. Each circle represents an individual mouse, and the solid line shows the geometric mean. The horizontal dashed line is the detection limit of the assay. The 13 groups are labeled A through M from left to right, as shown below. [Figure 19]Figure 19 shows (A) IL-6 and (B) IFNα (pg / ml) released by pDCs. There are four 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 represents wild-type mice, and the gray bar represents rsq1 mutants. [Modes for carrying out the invention]

[0125] (RNA replicon) Various replicons are used as follows. Generally, these are based on a hybrid alphaviral genome containing a non-structural protein derived from Venezuelan encephalitis virus (VEEV), a packaging signal derived from Sindbis virus, and a 3'UTR derived from Sindbis virus or a VEEV variant. The above replicons are approximately 10 kb long and have a poly-A tail.

[0126] Plasmid DNA encoding alphavirus replicons (names: pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) was used as a template for in vivo RNA synthesis. These replicons contain the alphavirus genetic elements necessary for RNA replication but lack the elements encoding the gene products required for particle assembly; the structural proteins are replaced instead by the target protein (either a reporter (e.g., SEAP or GFP) or an immunogen (e.g., full-length RSV F protein)), thus preventing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA promotes the synthesis of the replicon RNA in vitro, and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A) tail generates the precise 3' end via its autocleavage activity.

[0127] After linearizing the plasmid DNA downstream of the HDV ribozyme using an appropriate restriction endonuclease, a run-off transcript was synthesized in vitro using T7 or SP6 bacteriophage-derived DNA-dependent RNA polymerase. Transcription was performed at 37°C for 2 hours in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each nucleoside triphoside phosphate (ATP, CTP, GTP, and UTP), according to instructions provided by the manufacturer (Ambion). After transcription, the template DNA was digested with TURBO DNase (Ambion). The replicon RNA was precipitated with LiCl and reconstituted in nuclease-free water. Uncapped RNA was capped post-transcriptionally with vaccinia capping enzyme (VCE) using the ScriptCap m7G capping system (Epicentre Biotechnologies) as outlined in the user manual; the prefix "v" was added to the thus capped replicons (e.g., vA317 is the A317 replicon capped by VCE). The post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the above RNA samples was measured in OD. 260nm This was determined by measuring [the relevant parameter]. The integrity of the above in vitro transcript was confirmed by denatured agarose gel electrophoresis.

[0128] (Liposome encapsulation) RNA was encapsulated in liposomes prepared according to the methods described in references 11 and 42. These liposomes were prepared from 10% DSPC (amphoteric), 40% DLinDMA (cationic), 48% cholesterol, and 2% PEG-conjugated DMG (2kDa PEG). These percentages refer to the % moles of the total liposomes.

[0129] DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure in Reference 6. 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, chloride salt), and DC-chol (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were obtained from Avanti Polar Lipids.

[0130] In short, the lipid was dissolved in ethanol (2 ml), and the RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6). These were mixed with 2 ml of buffer and then equilibrated for 1 hour. The mixture was diluted with 6 ml of buffer and then filtered. The resulting product contained liposomes and had an encapsulation efficiency of approximately 95%.

[0131] For example, in one specific method, a fresh lipid stock solution was 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 shaken at 37°C for approximately 15 minutes to form a homogeneous mixture. Then, 755 μL of the stock was added to 1.245 mL of ethanol to prepare 2 mL of working lipid stock solution. This amount of lipid was used to form liposomes containing 250 μg of RNA. Another 2 mL working solution of RNA was also prepared from a stock solution of approximately 1 μg / μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stirring bars) were rinsed with RNase Away solution (Molecular BioProducts) and washed with a large amount of MilliQ water before use to remove RNase contamination from the vials. One of the vials described above was used for the RNA working solution, and the other was used to collect the lipid and RNA mixture (as described later). The working lipid solution and RNA solution were heated at 37°C for 10 minutes and then placed in a 3cc Luerlock syringe. 2mL of citrate buffer (pH 6) was placed in another 3cc syringe. The syringes containing RNA and lipids were mixed using an FEP tube (ethylene fluoride-propylene; all FEP tubes used had an inner diameter of 2mm and an outer diameter of 3mm) in a T-mixer (PEEK). TMA 500 μm ID junction (Idex Health Science) was connected. The outlet from the T mixer was also an FEP tube. A third syringe containing the citrate buffer was connected to a separate FEP tube. All syringes were then operated using a syringe pump at a flow rate of 7 mL / min. The tube outlets were positioned to collect the mixture into a 20 mL glass vial (while stirring). The stirring bar was removed, and the ethanol / aqueous solution was allowed to equilibrate to room temperature for 1 hour. 4 ml of the mixture was placed in a 5 cc syringe and connected to one of the FEP tubes, and another 5 cc syringe was connected to an FEP tube of equal length, and an equal volume of 100 mM citrate buffer (pH 6) was added. The two syringes were operated using the syringe pump at a flow rate of 7 mL / min, and the final mixture was collected into a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) was passed through a Mustang Q membrane (an anion exchange support obtained from Pall Corporation that binds and removes anionic molecules). Prior to using this membrane for the liposomes, 4 mL of 1 M NaOH, 4 mL of 1 M NaCl, and 10 mL of 100 mM citrate buffer (pH 6) were successively passed through the membrane. The liposomes were heated at 37°C for 10 minutes and then passed through the membrane. Next, the liposomes were concentrated to 2 mL by tangential flow filtration, dialyzed against 10-15 volumes of 1×PBS, and the final product was collected. The TFF system and hollow fiber filtration membrane were purchased from Spectrum Labs (Rancho Dominguez) and used according to the manufacturer's guidelines. 100 kD pore size cutoff and 8 cm 2 A polysulfone hollow fiber filtration membrane with a large surface area was used. For in vitro and in vivo experiments, the formulation was diluted with 1×PBS to the required RNA concentration.

[0132] Figure 2 shows exemplary electron micrographs of liposomes prepared by these methods. These liposomes contain encapsulated, full-length RSV F antigen-encoding RNA. Dynamic light scattering of one batch showed an average diameter of 141 nm (in intensity) or 78 nm (in number).

[0133] The percentage of encapsulated RNA and the RNA concentration were determined using the Quant-iT RiboGreen RNA reagent kit (Invitrogen) according to the manufacturer's instructions. A standard curve was created using the ribosomal RNA standard provided in the kit. Liposomes were diluted to 10× or 100× in 1× TE buffer (from the kit), and then the dye was added. Separately, liposomes were diluted to 10× or 100× in 1× TE buffer containing 0.5% Triton X, and then the dye was added (to disrupt the liposomes and thus to assay the total RNA). Equal volumes of dye were then added to each solution, and approximately 180 μL of each solution after dye addition was placed in a double-decker 96-well tissue culture plate. Fluorescence (excitation 485 nm, emission 528 nm) was read using a microplate reader. All liposomal formulations were administered in vivo based on the amount of encapsulated RNA.

[0134] Liposomal encapsulation was shown to protect RNA from RNase digestion. In the experiment, 3.8 mAU of RNase A / μg RNA was used and incubated at room temperature for 30 minutes. RNase was inactivated with proteinase K at 55°C for 10 minutes. The RNA was then extracted from the lipids into the aqueous phase by adding a 1:1 v / v mixture of phenol:chloroform:isoamyl alcohol to the sample. The sample was mixed by vortexing for several seconds and then centrifuged at 12k RPM for 15 minutes. The aqueous phase (containing the RNA) was removed and used for RNA analysis. Before loading (400 ng RNA / well), all samples were incubated with formaldehyde hydroding dye, denatured at 65°C for 10 minutes, and cooled to room temperature. The molecular weight of the RNA constructs was estimated using Ambion Millennium markers. The gels were electrophoresed at 90V. The gel described above was stained with 0.1% SYBR gold in water, according to the manufacturer's guidelines, by shaking it at room temperature for 1 hour. Figure 1 shows that RNase completely digests RNA in the absence of inclusion (lane 3). The RNA was undetectable after inclusion (lane 4), and no changes were observed even when these liposomes were treated with RNase (lane 4). Undigested RNA was observed after RNase-treated liposomes were subjected to phenol extraction (lane 6). Even after 1 week at 4°C, the above RNA could be observed without any fragmentation (Figure 4, arrow). In vivo protein expression remained unchanged after 6 weeks and one freeze-thaw cycle at 4°C. Therefore, liposome-encapsulated RNA is stable.

[0135] To evaluate the in vivo expression of the above RNA, a reporter enzyme (SEAP; secreted alkaline phosphatase), rather than an immunogen, was encoded in the replicon. Expression levels were measured in serum diluted 1:4 in 1× Phospha-Light dilution buffer using a chemiluminescent alkaline phosphatase substrate. 8-10 week old BALB / c mice (5 mice / group) were intramuscularly injected at a dose of 0.1 μg or 1 μg RNA (50 μl / leg) on ​​day 0. The same vector was also administered at 1 μg without the above liposome (in RNase-free 1× PBS). Virion-packaged replicons were also tested. The virion-packaged replicons used herein (referred to as "VRP") were obtained by the method described in Reference 43. Here, the alphavirus replicons were derived from mutant VEEV or were chimeric from the genome of VEEV engineered to contain the 3'UTR and SINDOBIS virus packaging signal (PS) of SINDOBIS virus, and were packaged into BHK cells by co-electroporating them with deficient helper RNA encoding the SINDOBIS virus capsid and glycoprotein genes.

[0136] As shown in Figure 5, inclusion increased SEAP levels by approximately 1 / 2 logarithm at a 1 μg dose, and on day 6, expression from a 0.1 μg inclusion dose was comparable to the level observed with a 1 μg uninclusion dose. By day 3, expression levels exceeded those achieved with VRP (square). Thus, expression was increased when the RNA was administered in liposomes, even at 10 × lower doses compared to the naked RNA control. Expression was also higher compared to the VRP control, but the expression dynamics were very different (see Figure 5). Delivery of the RNA using electroporation resulted in increased expression compared to the naked RNA control, but these levels were lower than those in liposomes.

[0137] To evaluate whether the effects observed in the above liposome group were solely due to the components of the liposomes or related to the inclusion process, the replicons were administered in inclusion form (0.1 μg RNA using two different purification protocols), mixed with the liposomes after their formation (uninclusiond "lipoplex", 0.1 μg RNA), or as naked RNA (1 μg). Figure 10 shows that the lipoplex gave the lowest level of expression, indicating that inclusion is essential for strong expression.

[0138] These findings were confirmed by in vivo studies using liposome delivery. Mice were given various combinations of (i) self-replicating RNA replicons encoding the full-length RSV F protein, (ii) self-replicating GFP-coding RNA replicons, (iii) GFP-coding RNA replicons with nsP4 knockout to eliminate self-replication, and (iv) the full-length RSV F protein. A total of 13 groups were given:

[0139] [Table 1] The results in Figure 18 indicate that the F-specific IgG response required inclusion in the liposomes described above, rather than mere co-delivery (compare groups C and D). Comparisons of groups K, L, and M show that the RNA provided an adjuvant effect on the co-delivered protein, and this effect was observed in both replicated and non-replicated RNAs.

[0140] Further SEAP experiments demonstrated a clear dose-response in vivo. Expression was observed even after delivery of approximately 1 ng of RNA (Figure 6). Further experiments comparing expression from inclusion replicons with expression from bare replicons showed that 0.01 μg of inclusion RNA was equivalent to 1 μg of bare RNA. At a dose of 0.5 μg of RNA, the inclusion material resulted in 12-fold higher expression at day 6; at a dose level of 0.1 μg, it was 24-fold higher at day 6.

[0141] In addition to examining the average level within the above groups, individual animals were also studied. Some animals were non-responders to naked replicons, whereas mounting eliminated non-responders.

[0142] In further experiments, DLinDMA was substituted with DOTAP. DOTAP liposomes showed better expression than the naked replicons, but they were inferior to DLinDMA liposomes (a 2-3 fold difference on day 1). DOTAP has a quaternary amine and therefore a positive charge in terms of delivery, while DLinDMA has a tertiary amine.

[0143] To evaluate immunogenicity in vivo, replicons were constructed and expressed full-length F protein derived from RSV (respiratory syncytial virus). These were then packaged in virions (1 μg), encapsulated in liposomes (0.1 or 1 μg), or in virions (10 μg). 6 IU ("VRP") was delivered on days 0 and 21. Figure 7 shows the IgG titer of anti-F two weeks after the second dose. The liposomes clearly enhance immunogenicity. Figure 8 shows the titer after two weeks. Up to this point, there were no statistically significant differences between the 0.1 μg inclusion RNA, the 1 μg inclusion RNA, and the VRP group. Neutralization titer (measured as a 60% plaque reduction, "PRNT60") did not differ significantly among these three groups two weeks after the second dose (Figure 9). Figure 12 shows both IgG and PRNT titers four weeks after the second dose.

[0144] Figure 13 confirms that the above RNA induces a robust CD8 T cell response.

[0145] In further experiments, F-specific IgG titers were compared in mice given VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. The titer ratios (VRP:liposome) at various time points after the second dose were as follows:

[0146] [Table 2] Therefore, the liposome-encapsulated RNA described above induces an immune response essentially equivalent to that observed with virion delivery.

[0147] Further experiments showed a superior F-specific IgG response at a 10 μg dose, which was equal to the responses at 1 μg and 0.1 μg doses, while a lower response was observed at 0.01 μg dose. Figure 11 shows the naked form at three different doses, in liposomes at four different doses, or in VRP(10 6 The IgG titer in animals given the above replicons is expressed as IU. The response observed with 1 μg liposome-encapsulated RNA was not statistically significant when compared to VRP (ANOVA), but the higher response observed with 10 μg liposome-encapsulated RNA was statistically significant when compared to both of these groups (p<0.05).

[0148] Further studies have shown that the 0.1 μg liposome-encapsulated RNA evokes a significantly higher anti-F IgG response than 0.1 μg delivered DNA (15 days after the second dose), and that electroporation (Elgen) is also effective. TM Immunogenicity was confirmed from 20 μg plasmid DNA encoding the F antigen, delivered by the DNA Delivery System (Inovio).

[0149] Further studies were conducted in cotton rats (Sigmodon hispidis) instead of mice. At a dose of 1 μg, liposome encapsulation increased F-specific IgG titer 8.3-fold and PRNT titer 9.5-fold compared to naked RNA. The magnitude of the antibody response was 5 × 10⁻⁶. 6 It was equivalent to that induced by IU VRP. Both naked RNA and liposome-encapsulated RNA showed RSV challenge (1 × 10⁶). 5 The above cotton rats were protected from plaque-forming units and reduced lung viral load by at least 3.5 log. Inclusion increased the reduction by approximately twofold.

[0150] Large animal studies were conducted in cattle. Cattle were immunized on day 0 and day 21 with a 66 μg replicon encoding the full-length RSV F protein, formulated within liposomes. PBS alone was used as a negative control, and an approved vaccine (Fort Dodge's "Triangle 4," containing dead viruses) was used as a positive control. Figure 14 shows F-specific IgG titers over a 63-day period starting from the first immunization. The RNA replicon was immunogenic in cattle but yielded lower titers than the approved vaccine. All vaccinated cattle showed F-specific antibodies after a second dose, and titers were very stable for a period of 2–6 weeks after the second dose (and particularly stable with respect to the RNA vaccine).

[0151] (Mechanism of action) Bone marrow-derived dendritic cells (pDCs) were obtained from wild-type mice or the “Resq” (rsq1) mutant line. The mutant line had a point mutation at the amino terminus of its TLR7 receptor, which disrupted TLR7 signaling without affecting ligand binding

[44] . These cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000, or liposome-encapsulated replicon RNA. As shown in Figure 19, IL-6 and INFα were induced in WT cells, but this response was almost completely eliminated in mutant mice. These results indicate that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can induce immune cells to secrete both high levels of interferon and pro-inflammatory cytokines.

[0152] (pKa measurement) The pKa of lipids is measured in water at standard temperature and pressure using the following technique: • A 2 mM solution of lipids in ethanol is prepared by weighing the lipids and dissolving them in ethanol. A 0.3 mM solution of the fluorescent probe toluenenitrosulfonic acid (TNS) in an ethanol:methanol 9:1 mixture is prepared by first preparing a 3 mM solution of TNS in methanol, and then diluting it to 0.3 mM with ethanol. Prepare aqueous buffer solutions containing sodium phosphate, sodium citrate, sodium acetate, and sodium chloride at concentrations of 20 mM, 25 mM, 20 mM, and 150 mM, respectively. Divide the buffer solutions into eight parts and adjust their pH with either 12N HCl or 6N NaOH to 4.44-4.52, 5.27, 6.15-6.21, 6.57, 7.10-7.20, 7.72-7.80, 8.27-8.33, and 10.47-11.12. Mix 400 μL of 2 mM lipid solution and 800 μL of 0.3 mM TNS solution. Add 7.5 μL of the probe / lipid mixture to 242.5 μL of buffer in a 1 mL 96-well plate. Do this for all eight buffers. After mixing, transfer 100 μL of each probe / lipid / buffer mixture to a clear-bottom 250 μL black 96-well plate (e.g., Model COSTAR 3904, Corning). A convenient method for performing this mixing is to use a Tecan Genesis RSP150 high-throughput liquid handler and Gemini Software. • Measure the fluorescence of each probe / lipid / buffer mixture using 322 nm excitation and 431 nm emission (auto-cutoff 420 nm) (e.g., with a SpectraMax M5 spectrophotometer and SoftMax pro 5.2 software). After measurement, subtract the background fluorescence values ​​of the empty wells in the 96-well plate from each probe / lipid / buffer mixture. Then, normalize the fluorescence intensity values ​​to the values ​​at the lowest pH. Next, plot the normalized fluorescence intensity against pH to provide the optimal line. • Find the optimal point on the line where the normalized fluorescence intensity equals 0.5. Find the pH corresponding to the normalized fluorescence intensity equal to 0.5 and consider it to be the pKa of the lipid.

[0153] This method yields a pKa of 5.8 for DLinDMA. The pKa values ​​measured by this method for cationic lipids in Reference 5 are as follows:

[0154] (Encapsulation in liposomes using alternative cationic lipids) Instead of using DlinDMA, cationic lipids from Reference 5 are used. These lipids can be synthesized as disclosed in Reference 5.

[0155] The liposomes formed above using DlinDMA will hereafter be referred to as the "RV01" series. In the "RV02" to "RV12" series, the DlinDMA was replaced with various cationic lipids as described below. Two different types of liposomes were formed using either 2% PEG2000-DMG and either (01) 40% of the cationic lipid, 10% DSPC, and 48% cholesterol, or (02) 60% of the cationic lipid and 38% cholesterol. Thus, a comparison between (01) liposomes and (02) liposomes demonstrates the effect of neutral amphoteric lipids.

[0156] RV02 liposomes were prepared using the following cationic lipids (pKa>9, without tertiary amines):

[0157] [ka] RV03 liposomes were constructed using the following cationic lipid (pKa 6.4):

[0158] [ka] RV04 liposomes were constructed using the following cationic lipid (pKa 6.62):

[0159] [ka] RV05 liposomes were prepared using the following cationic lipid (pKa 5.85):

[0160] [ka] RV06 liposomes were constructed using the following cationic lipid (pKa 7.27):

[0161] [ka] RV07 liposomes were prepared using the following cationic lipid (pKa 6.8):

[0162] [ka] RV08 liposomes were constructed using the following cationic lipid (pKa 5.72):

[0163] [ka] RV09 liposomes were constructed using the following cationic lipid (pKa 6.07):

[0164] [ka] RV10 liposomes were prepared for comparison using the following cationic lipid (pKa 7.86):

[0165] [ka] RV11 liposomes were constructed using the following cationic lipid (pKa 6.41):

[0166] [ka] RV12 liposomes were constructed using the following cationic lipids (pKa 7):

[0167] [ka] RV16 liposomes were constructed using the following cationic lipids (pKa 6.1)

[45] :

[0168] [ka] RV17 liposomes were constructed using the following cationic lipids (pKa 6.1)

[45] :

[0169] [ka] RV18 liposomes were prepared using DODMA. RV19 liposomes were prepared using DOTMA, and RV13 liposomes were prepared using DOTAP. All of these have a quaternary amine head group.

[0170] These liposomes were characterized and tested with the SEAP reporter described above. The table below shows the size (Z-mean and polydispersity index) of the above liposomes, the percentage of RNA inclusion in each liposome, and the SEAP activity detected on days 1 and 6 after injection. SEAP activity is associated with the "RV01(02)" liposomes made from DLinDMA, cholesterol, and PEG-DMG:

[0171] [Table 3] Figure 15 plots the SEAP level on day 6 against the pKa of the cationic lipids described above. The best results are observed when the lipids have a pKa between 5.6 and 6.8, and ideally between 5.6 and 6.3.

[0172] These liposomes were also used to deliver replicons encoding the full-length RSV F protein. The total IgG titer for the F protein two weeks after the first dose (2wp1) is plotted against pKa in Figure 16. The best results are observed when the above pKa is between 5.7 and 5.9, but the pKa alone is not sufficient to guarantee a high titer (e.g., the lipid still needs to support liposome formation). (RSV immunogenicity) Further studies were conducted using a self-replicating replicon (vA317) encoding the RSV F protein. BALB / c mice (4 or 8 animals per group) were intramuscularly inoculated (50 μL / leg) on ​​both days 0 and 21 with either the replicon alone (1 μg), or with the RV01 or RV05 lipids (see above; pKa 5.8 or 5.85), or with the replicon formulated as liposomes with RV13. The RV01 liposomes contained 40% DLinDMA, 10% DSPC, 48% cholesterol, and 2% PEG-DMG, but with varying amounts of RNA. The RV05(01) liposomes contained 40% cationic lipids, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; the RV05(02) liposomes contained 60% cationic lipids, 38% cholesterol, and 2% PEG-DMG. The RV13 liposomes contained 40% DOTAP, 10% DPE, 48% cholesterol, and 2% PEG-DMG. For comparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigen was delivered either by electroporation or via RV01(10) liposomes (0.1 μg DNA). Four mice were used as a naive control group.

[0173] Liposomes were prepared by method (A) or method (B). In method (A), a fresh lipid stock solution in ethanol was prepared. 37 mg of cationic lipid, 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 shaken at 37 °C for about 15 minutes to form a homogeneous mixture. Then, 226.7 μL of the above stock was added to 1.773 mL of ethanol to prepare 2 mL of a working lipid stock solution. This amount of lipid was used to form liposomes with 75 μg RNA, giving an 8:1 nitrogen to phosphate ratio (except that in RV01(08) and RV01(09) this ratio was modified to 4:1 or 16:1). A 2 mL working solution of RNA (or DNA for RV01(10)) was also prepared from an approximately 1 μg / μL stock solution in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stirrers) were rinsed with RNase Away solution (Molecular BioProducts) and washed with a large amount of MilliQ water before use to remove RNase contamination in the above vials. One of the above vials was used for the above RNA working solution, and the others were used to collect the above lipid and RNA mixture (as described later). The above working lipid and RNA solutions were heated at 37 °C for 10 minutes and then filled into a 3 cc syringe. 2 mL of citrate buffer (pH 6) was filled into another 3 cc syringe. The syringe containing RNA and the above lipid was connected using an FEP tube to a T mixer (PEEK TMIt was connected to a 500 μm ID junction. The outlet from the above T mixer was also an FEP tube. A third syringe containing the above citrate buffer was connected to a separate section of the FEP tube. Next, all syringes were operated at a flow rate of 7 mL / min using a syringe pump. The outlets of the tubes were positioned to collect the above mixture into a 20 mL glass vial (while stirring). The stirring bar was removed and the ethanol / aqueous solution was equilibrated to room temperature for 1 hour. The above mixture was then packed into a 5 cc syringe attached to the section of the FEP tube, and another 5 cc syringe with an equal volume of 100 mM citrate buffer (pH 6) was packed into a separate 5 cc syringe with an FEP tube of equal length. The above two syringes were operated at a flow rate of 7 mL / min using a syringe pump, and the final mixture was collected into a 20 mL glass vial (while stirring). Next, the liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using a TFF, after which the final product was collected. The TFF system and hollow fiber filtration membrane were purchased from Spectrum Labs and used according to the manufacturer's guidelines. 100 kD pore size cutoff and 20 cm 2 A polyethersulfone (PES) hollow fiber filtration membrane with surface area (part number P-C1-100E-100-01N) was used. For in vitro and in vivo experiments, the formulation was diluted to the required RNA concentration in 1×PBS.

[0174] Preparation method (B) differs from method (A) in two ways. First, after collection in the 20 mL glass vial (but before TFF concentration), the mixture was passed through a Mustang Q membrane (an anion exchange support that binds to and removes anionic molecules, obtained from Pall Corporation, Ann Arbor, MI, USA). This membrane was washed first with 4 mL of 1 M NaOH, then with 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6), and the liposomes were heated at 37°C for 10 minutes and then filtered. Second, the hollow fiber filtration membrane was polysulfone (part number P / N: X1AB-100-20P).

[0175] The Z-mean particle diameter, polydispersity index, and encapsulation efficiency of the above liposomes were as follows:

[0176] [Table 5] Serum was collected on days 14, 36, and 49 for antibody analysis. Spleen was collected from mice on day 49 for T-cell analysis.

[0177] The F-specific serum IgG titer (GMT) was as follows:

[0178] [Table 6] The percentage of T cells that are cytokine-positive and specific to RSV F51-66 peptides is as follows, and only numbers statistically significant above zero are shown:

[0179] [Table 7] Therefore, the liposomal formulations significantly increased immunogenicity compared to the naked RNA control, as determined by increased F-specific IgG titer and T cell frequency. Plasmid DNA, formulated with liposomes or delivered naked using electroporation, was significantly less immunogenic than liposomally formulated self-replicating RNA.

[0180] The RV01 RNA vaccine and RV05 RNA vaccine described above were more immunogenic than the RV13 (DOTAP) vaccine described above. These formulations had comparable physical characteristics and were formulated with the same self-replicating RNA, but they contained different cationic lipids. Both RV01 and RV05 have a tertiary amine with a pKa of approximately 5.8 in their head group and also contain an unsaturated alkyl tail. RV13 has an unsaturated alkyl tail, but its head group has a quaternary amine and is very cationic. These results suggest that lipids with tertiary amines having a pKa in the range of 5.0 to 7.6 are superior to lipids like DOTAP (which are highly cationic when used in liposome delivery systems for RNA).

[0181] (Further alternatives to DLinDMA) The cationic lipid (DLinDMA) in the RV01 liposome was replaced with RV16, RV17, RV18, or RV19. The total IgG titer is shown in Figure 17. The lowest result was observed with RV19 (i.e., DOTMA quaternary amine).

[0182] (BHK expression) Liposomes containing different lipids were incubated overnight with BHK cells, and their protein expression efficacy was evaluated. From baseline with RV05 lipid expression, expression could be increased by 18× by adding 10% 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposomes, by 10× by adding 10% 18:2(cis) phosphatidylcholine, and by 900× by using RV01 instead.

[0183] (RSV immunogenicity in various mouse strains) The replicon "vA142" encodes the full-length wild-type surface fusion (F) glycoprotein of RSV, but its fusion peptide is deleted, and its 3' end is formed by ribozyme-mediated cleavage. This was tested in three different mouse strains.

[0184] BALB / c mice were administered bilateral intramuscular vaccine (50 μL / leg) on ​​days 0 and 22. The animals were divided into eight test groups (5 animals per group) and a naive control group (2 animals): Group 1 was given a bare replicon (1 μg). Group 2 was given a 1 μg replicon delivered in liposome "RV01(37)" containing 40% DlinDMA, 10% DSPC, 48% Chol, and 2% PEG-conjugated DMG. Group 3 was given the same sample as Group 2, but the amount of RNA was 0.1 μg. Group 4 was given 1 μg of replicon in "RV17(10)" liposomes (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG). Group 5 was given 1 μg of replicon in "RV05(11)" liposomes (40% RV07 lipid, 30% 18:2 PE (DLoPE), 28% cholesterol, 2% PEG-DMG). Group 6 was given 0.1 μg of replicon in "RV17(10)" liposomes. Group 7 was given 5 μg of RSV-F subunit protein with aluminum hydroxide adjuvant added. Group 8 was a naive control (two animals).

[0185] Serum was collected on days 14, 35, and 49 for antibody analysis. The F-specific serum IgG GMT was as follows:

[0186] [Table 8] On day 35, the F-specific IgG1 titers and IgG2a titers (GMT) were as follows:

[0187] [Table 9] The RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers from a pool of 2-5 mice (1 pool per group):

[0188] [Table 10] On day 49, the spleen was collected for T cell analysis. The mean net frequency of F-specific cytokine-positive T cells (CD4+ or CD8+) was as follows (only the numbers are shown, which were statistically significant above zero (specific to the RSV peptides F51-66, F164-178, F309-323 for CD4+, or peptides F85-93 and F249-258 for CD8+):

[0189] [Table 11] C57BL / 6 mice were immunized in the same way, but the ninth group had VRP (1 × 10⁻¹⁰) expressing the full-length wild-type surface fusion glycoprotein (fusion peptide deletion) of RSV. 6 IU) Give.

[0190] Serum was collected on days 14, 35, and 49 for antibody analysis. The F-specific IgG titers (GMT) were as follows:

[0191] [Table 12] On day 35, the F-specific IgG1 titers and IgG2a titers (GMT) were as follows:

[0192] [Table 13] The RSV serum neutralizing antibody titers on days 35 and 49 were as follows (data are 60% plaque reduction neutralizing titers from a pool of 2-5 mice (1 pool per group):

[0193] [Table 14] On day 49, the spleen was collected for T cell analysis. The mean net F-specific cytokine-positive T cell frequency (CD8+) was as follows (this shows only the numbers that were statistically significant above zero (specific to RSV peptides F85-93 and F249-258):

[0194] [Table 15] Nine groups of C3H / HeN mice were immunized in the same manner. The F-specific IgG titers (GMT) were as follows:

[0195] [Table 16] On day 35, the F-specific IgG1 titers and IgG2a titers (GMT) were as follows:

[0196] [Table 17] The RSV serum neutralizing antibody titers on days 35 and 49 were as follows:

[0197] [Table 18] Therefore, three different lipids (RV01, RV05, and RV17; pKa values ​​of 5.8, 5.85, and 6.1) were tested in three different inbred mouse strains. In all three strains, RV01 was more effective than RV17; in the BALB / c and C3H strains, RV05 was less effective than RV01 and RV17, but was more effective in the B6 strain. However, in all cases, the above liposomes were more effective than two cationic nanoemulsions tested in parallel.

[0198] (CMV immunogenicity) RNA replicons encoding cytomegalovirus (CMV) glycoproteins were delivered using RV01 liposomes containing DLinDMA as a cationic lipid. The "vA160" replicon encodes full-length glycoproteins H and L (gH / gL), while the "vA322" replicon encodes the soluble form (gHsol / gL). Both proteins are under the control of separate subgenome promoters within a single replicon; co-administration of two separate vectors (one encoding gH and the other gL) did not yield favorable results.

[0199] BALB / c mice (10 mice / group) were given VRP (1 × 10) expressing gH / gL on day 0, day 21, and day 42. 6 IU), VRP (1×10) expressing gHsol / gL 6 IU) and PBS as a control were administered bilaterally via intramuscular vaccination (50 μL / leg). The two test groups were given 1 μg of either the vA160 replicon or the vA322 replicon, formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; prepared using the method (A) discussed above, but with a 150 μg RNA batch size).

[0200] The vA160 liposomes described above had a Zav diameter of 168 nm, a pdI of 0.144, and 87.4% encapsulation. The vA322 liposomes described above had a Zav diameter of 162 nm, a pdI of 0.131, and 90% encapsulation.

[0201] The above replicon was able to express two types of proteins from a single vector.

[0202] On day 63 (3wp3), serum was collected for immunological analysis. The CMV neutralizing titer (the reciprocal of the serum dilution that results in a 50% reduction in the number of positive virus focuses / well compared to the control) was as follows:

[0203] [Table 19] RNA expressing either the full-length or soluble form of the CMV gH / gL complex induced high titer neutralizing antibodies when assayed in epithelial cells. The average titer induced by the liposome-encapsulated RNA was at least as high as that for its corresponding VRP.

[0204] It is understood that this invention has been described only by example, and that modifications may be made while remaining within the scope and spirit of the invention. The present invention also relates to the following embodiments. [1] A liposome having a lipid bilayer encapsulating an aqueous core, wherein: (i) the lipid bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6; and (ii) the aqueous core comprises RNA encoding an immunogen. [2] The liposome according to [1] above, wherein the lipid having a pKa in the range of 5.0 to 7.6 has a tertiary amine. [3] Liposomes as described in any of [1] or [2] above, wherein the pKa in the range of 5.0 to 7.6 is between 5.7 and 5.9. [4] The liposomes described in [1] above, wherein the lipid having a pKa in the range of 5.0 to 7.6 has the formula shown herein for RV01, RV02, RV03, RV04, RV05, RV06, RV07, RV08, RV09, RV11, RV12, RV16, or RV17. [5] A liposome according to any of [1] to [4] above, having a diameter in the range of 20 to 220 nm. [6] The RNA molecule is (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the RNA molecule, and (ii) a liposome according to any one of [1] to [5] above. [7] The liposome described in [5] above, wherein the RNA molecule has two open reading frames, the first of which encodes an alphavirus replicase and the second of which encodes the immunogen. [8] The RNA molecule is a liposome according to any of [1] to [7] above, having a length of 9,000 to 12,000 nucleotides. [9] The immunogen is a liposome according to any one of [1] to [8] above, which can induce an immune response in vivo against bacteria, viruses, fungi, or parasites.

[10] The immunogen is a liposome according to any one of [1] to [9] above, which can induce an immune response in vivo against respiratory syncytial virus glycoprotein F.

[11] A pharmaceutical composition comprising the liposome described in any of [1] to

[10] above.

[12] A method for inducing a protective immune response in a vertebrate, the method comprising administering to the vertebrate an effective amount of the liposomes described in [1] to

[10] above, or the pharmaceutical composition described in

[11] above.

[13] A process for preparing RNA-containing liposomes, the process comprising, in the course of liposome formation, (a) mixing RNA and a lipid at a pH less than the pKa of the lipid but greater than 4.5; and then (b) raising the pH to greater than the pKa of the lipid.

[14] The process described in

[13] above, wherein the RNA used in step (a) is present in an aqueous solution to be mixed with the organic solution of the lipid to give a mixture, the mixture is then diluted to form liposomes; and the pH is raised in step (b) after liposome formation.

[0205]

number

[0206]

number

Claims

1. A composition comprising liposomes and RNA molecules encoding immunogens, The immunogen includes respiratory syncytial virus (RSV) immunogen, coronavirus spike polypeptide immunogen, or influenza A virus immunogen. The RNA molecule encoding the immunogen includes a polyA tail, The liposome has a lipid bilayer, and the lipid bilayer encapsulates at least half of the RNA molecule encoding the immunogen within an aqueous core. The aforementioned lipid bilayer contains lipids including tertiary amines, The lipid containing the tertiary amine has a pKa in the range of 5.8 to 7.

0. The pKa of the lipid containing the tertiary amine in the range of 5.8 to 7.0 is 1) Mix 400 μL of 2 mM of the lipid in 100% by volume ethanol with 800 μL of 0.3 mM fluorescent probe toluenenitrosulfonic acid (TNS) in 90% by volume ethanol and 10% by volume methanol to obtain a lipid / TNS mixture; 2) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a first buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid / buffer mixture; dispense 100 μL of the first lipid / buffer mixture into the first well of a 96-well plate having a clear bottom; 3) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a second buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 5.27, thereby obtaining a second lipid / buffer mixture; dispense 100 μL of the second lipid / buffer mixture into the second well of the 96-well plate; 4) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a third buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid / buffer mixture, and dispense 100 μL of the third lipid / buffer mixture into the third well of the 96-well plate; 5) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fourth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid / buffer mixture; dispense 100 μL of the fourth lipid / buffer mixture into the fourth well of the 96-well plate; 6) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fifth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid / buffer mixture, and dispense 100 μL of the fifth lipid / buffer mixture into the fifth well of the 96-well plate; 7) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a sixth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid / buffer mixture, and dispense 100 μL of the sixth lipid / buffer mixture into the sixth well of the 96-well plate; 8) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a seventh buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid / buffer mixture, and dispense 100 μL of the seventh lipid / buffer mixture into the seventh well of the 96-well plate; 9) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of an eighth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid / buffer mixture, and dispense 100 μL of the eighth lipid / buffer mixture into the eighth well of the 96-well plate; 10) Using an excitation wavelength of 322 nm and cutting off wavelengths below 420 nm, the absolute fluorescence of each of the first to eighth wells and the empty well of the 96-well plate at a wavelength of 431 nm was measured. 11) Subtract the absolute fluorescence of the empty well from the absolute fluorescence of each of the first to eighth wells, thereby obtaining the blank removal fluorescence for each of the first to eighth lipid / buffer mixtures; 12) The blank removal fluorescence of each of the first to eighth lipid / buffer mixtures is normalized relative to the blank removal fluorescence of the first lipid / buffer mixture, thereby obtaining a relative fluorescence for each of the first to eighth lipid / buffer mixtures, with the relative fluorescence of the first lipid / buffer mixture set to 1; 13) Plot the relative fluorescence of the first to eighth lipid / buffer mixtures against the pH of the first to eighth lipid / buffer mixtures; 14) Find the pH corresponding to the relative fluorescence of 0.5 from the graph obtained in 13), and determine the pH corresponding to the relative fluorescence of 0.5 as the pKa of the lipid. Determined by, composition.

2. A composition comprising liposomes and self-replicating RNA molecules encoding immunogens, The immunogen includes respiratory syncytial virus (RSV) immunogen, coronavirus spike polypeptide immunogen, or influenza A virus immunogen. The self-replicating RNA molecule encoding the immunogen includes a poly-A tail, The liposome has a lipid bilayer, and the lipid bilayer encapsulates at least half of the self-replicating RNA molecules encoding the immunogen within an aqueous core. The aforementioned lipid bilayer contains lipids including tertiary amines, The lipid containing the tertiary amine has a pKa in the range of 5.0 to 7.

6. The pKa of the lipid containing the tertiary amine in the range of 5.0 to 7.6 is 1) Mix 400 μL of 2 mM of the lipid in 100% by volume ethanol with 800 μL of 0.3 mM fluorescent probe toluenenitrosulfonic acid (TNS) in 90% by volume ethanol and 10% by volume methanol to obtain a lipid / TNS mixture; 2) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a first buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 4.44 to 4.52, thereby obtaining a first lipid / buffer mixture; dispense 100 μL of the first lipid / buffer mixture into the first well of a 96-well plate having a clear bottom; 3) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a second buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 5.27, thereby obtaining a second lipid / buffer mixture; dispense 100 μL of the second lipid / buffer mixture into the second well of the 96-well plate; 4) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a third buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.15 to 6.21, thereby obtaining a third lipid / buffer mixture, and dispense 100 μL of the third lipid / buffer mixture into the third well of the 96-well plate; 5) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fourth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 6.57, thereby obtaining a fourth lipid / buffer mixture; dispense 100 μL of the fourth lipid / buffer mixture into the fourth well of the 96-well plate; 6) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a fifth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.10 to 7.20, thereby obtaining a fifth lipid / buffer mixture, and dispense 100 μL of the fifth lipid / buffer mixture into the fifth well of the 96-well plate; 7) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a sixth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 7.72 to 7.80, thereby obtaining a sixth lipid / buffer mixture, and dispense 100 μL of the sixth lipid / buffer mixture into the sixth well of the 96-well plate; 8) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of a seventh buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 8.27 to 8.33, thereby obtaining a seventh lipid / buffer mixture, and dispense 100 μL of the seventh lipid / buffer mixture into the seventh well of the 96-well plate; 9) Mix 7.5 μL of the lipid / TNS mixture obtained in 1) with 242.5 μL of an eighth buffer, which is a sodium salt buffer containing 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, and is adjusted to have a pH of 10.47 to 11.12, thereby obtaining an eighth lipid / buffer mixture, and dispense 100 μL of the eighth lipid / buffer mixture into the eighth well of the 96-well plate; 10) Using an excitation wavelength of 322 nm and cutting off wavelengths below 420 nm, the absolute fluorescence of each of the first to eighth wells and the empty well of the 96-well plate at a wavelength of 431 nm was measured. 11) Subtract the absolute fluorescence of the empty well from the absolute fluorescence of each of the first to eighth wells, thereby obtaining the blank removal fluorescence for each of the first to eighth lipid / buffer mixtures; 12) The blank removal fluorescence of each of the first to eighth lipid / buffer mixtures is normalized relative to the blank removal fluorescence of the first lipid / buffer mixture, thereby obtaining a relative fluorescence for each of the first to eighth lipid / buffer mixtures, with the relative fluorescence of the first lipid / buffer mixture set to 1; 13) Plot the relative fluorescence of the first to eighth lipid / buffer mixtures against the pH of the first to eighth lipid / buffer mixtures; 14) Find the pH corresponding to the relative fluorescence of 0.5 from the graph obtained in 13), and determine the pH corresponding to the relative fluorescence of 0.5 as the pKa of the lipid. Determined by, composition.

3. The composition according to claim 2, wherein the lipid containing the tertiary amine has a pKa in the range of 5.8 to 7.

0.

4. The composition according to claim 1 or 2, wherein the lipid containing the tertiary amine has a pKa in the range of 6.07 to 7.

0.

5. The composition according to any one of claims 1 to 4, wherein the liposomes have a diameter in the range of 20 to 220 nm.

6. The composition according to claim 2, wherein the self-replicating RNA molecule encoding the immunogen has two open reading frames, the first of which encodes alphavirus replicase, and the second of which encodes the immunogen.

7. The composition according to any one of claims 1 to 6, wherein the lipid bilayer further comprises polyethylene glycol-conjugated (PEG-conjugated) lipids and / or cholesterol.

8. The composition according to any one of claims 1 to 7, wherein the PEG-conjugated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)].

9. The composition according to any one of claims 1 to 8, wherein the lipid bilayer contains cholesterol in a proportion of 35 mol% to 50 mol% of the total lipids forming the lipid bilayer.

10. The composition according to any one of claims 1 to 9, wherein the immunogen comprises two or more immunogens.

11. The composition according to any one of claims 1 to 10, wherein the lipid bilayer contains lipids comprising the tertiary amine in a proportion of 40 mol% to 60 mol% of the total lipids forming the lipid bilayer.

12. The composition according to any one of claims 1 to 11, wherein the lipid bilayer further comprises 1,2-diastearoyl-sn-glycero-3-phosphocholine.

13. The composition according to any one of claims 1 to 12, wherein the lipid bilayer encapsulates 71% to 97.6% of the RNA molecule encoding the immunogen within the aqueous core.

14. The composition according to any one of claims 1 to 12, wherein the lipid bilayer encapsulates 85.3% to 97.6% of the RNA molecule encoding the immunogen within the aqueous core.

15. A pharmaceutical composition comprising the composition described in any one of claims 1 to 14.

16. The pharmaceutical composition according to claim 15 for inducing a protective immune response in vertebrates.

17. A pharmaceutical composition according to claim 15 or 16 for inducing a cell-mediated immune response to the immunogen and / or an antibody response to the immunogen in vivo.

18. A process for preparing the composition according to claim 1 or 2, the process comprising, during liposome formation, (i) mixing an RNA molecule encoding the immunogen with a lipid at a pH below the pKa of the lipid but above 4.5; and then (ii) raising the pH above the pKa of the lipid.

19. A process according to claim 18, wherein the RNA molecule encoding the immunogen used in step (i) is present in an aqueous solution to be mixed with the organic solution of the lipid to yield a mixture, the mixture is then diluted to form liposomes; and the pH is increased in step (ii) after liposome formation.

20. Use of the composition according to any one of claims 1 to 14 for producing a pharmaceutical product for inducing a protective immune response in vertebrates.

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