Pegylated liposomes for delivery of immunogen-encoding RNA

Abstract

Nucleic acid immunisation is achieved by delivering RNA encapsulated within a PEGylated liposome. The RNA encodes an immunogen of interest. The PEG has an average molecular mass of between 1 kDa and 3 kDa. Thus the invention provides a liposome having a lipid bilayer encapsulating an aqueous core, wherein: (i) the lipid bilayer comprises at least one lipid which includes a polyethylene glycol moiety, such that polyethylene glycol is present on the liposome's exterior, wherein the average molecular mass of the polyethylene glycol is between 1 kDa and 3 kDa; and (ii) the aqueous core includes a RNA which encodes an immunogen. These liposomes are suitable for in vivo delivery of the RNA to a vertebrate cell and so they are useful as components in pharmaceutical compositions for immunising subjects against various diseases.

Claims

1. A method for raising a protective immune response in a vertebrate, the method comprising administering to the vertebrate an effective amount of a liposome within which at least one ribonucleic acid (RNA) that encodes an immunogen of interest is encapsulated, wherein the immunogen of interest elicits in the vertebrate a protective immune response against a bacterium, a virus, a fungus, a parasite, or an allergen, wherein the liposome comprises at least one lipid that includes a polyethylene glycol (PEG) moiety, wherein the PEG moiety is present on at least the exterior of the liposome, wherein the average molecular mass of the PEG moiety is between 1 kDa and 3 kDa, wherein the at least one lipid that includes the PEG moiety is not a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)].

2. The method of claim 1, wherein the liposome has a diameter in the range of 80-160 nm.

3. The method of claim 1, wherein the liposome further comprises a cationic lipid.

4. The method of claim 1, wherein the liposome further comprises a zwitterionic lipid.

5. The method of claim 1, wherein the at least one RNA that encodes the immunogen of the interest is a self-replicating RNA, which further encodes a RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA.

6. The method of claim 5, wherein the self-replicating RNA has two open reading frames, wherein the first open reading frame encodes an alphavirus replicase and the second open reading frame encodes the immunogen of interest, and wherein the alphavirus replicase comprises the RNA-dependent RNA polymerase.

7. The method of claim 1, wherein the immunogen of interest elicits a protective immune response in the vertebrate against the bacterium, the virus, the fungus, or the parasite.

8. A method for raising a protective immune response in a vertebrate, the method comprising administering to the vertebrate an effective amount of a liposome within which at least one RNA that encodes an immunogen of interest is encapsulated, wherein the immunogen of interest is expressed and elicits in the vertebrate a protective immune response against a bacterium, a virus, a fungus, a parasite, or an allergen, wherein the liposome comprises at least one lipid that includes a PEG moiety, wherein the PEG moiety is present on at least the exterior of the liposome, wherein the PEG moiety has a number-averaged degree of polymerization of ethylene oxide between 22 and 67, wherein the at least one lipid that includes the PEG moiety is not a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)].

9. A method for raising a protective immune response in a vertebrate, the method comprising administering to the vertebrate an effective amount of a liposome within which at least one RNA that encodes an immunogen of interest is encapsulated, wherein the immunogen of interest is expressed and elicits in the vertebrate a protective immune response against a virus, wherein the liposome comprises at least one lipid that includes a PEG moiety, wherein the PEG moiety is present on at least the exterior of the liposome, wherein the PEG moiety has a number-averaged degree of polymerization of ethylene oxide between 22 and 67, wherein the at least one lipid that includes the PEG moiety is not a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)].

10. The method of claim 8, wherein the liposome has a diameter in the range of 80-160 nm.

11. The method of claim 9, wherein the liposome has a diameter in the range of 80-160 nm.

12. The method of claim 8, wherein the liposome further comprises a cationic lipid.

13. The method of claim 9, wherein the liposome further comprises a cationic lipid.

14. The method of claim 8, wherein the liposome further comprises a zwitterionic lipid.

15. The method of claim 9, wherein the liposome further comprises a zwitterionic lipid.

16. The method of claim 8, wherein the at least one RNA that encodes the immunogen of the interest is a self-replicating RNA, which further encodes a RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA.

17. The method of claim 9, wherein the at least one RNA that encodes the immunogen of the interest is a self-replicating RNA, which further encodes a RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA.

18. The method of claim 16, wherein the self-replicating RNA has two open reading frames, wherein the first open reading frame encodes an alphavirus replicase and the second open reading frame encodes the immunogen of interest, and wherein the alphavirus replicase comprises the RNA-dependent RNA polymerase.

19. The method of claim 17, wherein the self-replicating RNA has two open reading frames, wherein the first open reading frame encodes an alphavirus replicase and the second open reading frame encodes the immunogen of interest, and wherein the alphavirus replicase comprises the RNA-dependent RNA polymerase.

20. The method of claim 4, wherein the zwitterionic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

21. The method of claim 14, wherein the zwitterionic lipid is DSPC.

22. The method of claim 15, wherein the zwitterionic lipid is DSPC.

23. The method of claim 1, wherein the liposome further comprises cholesterol.

24. The method of claim 8, wherein the liposome further comprises cholesterol.

25. The method of claim 9, wherein the liposome further comprises cholesterol.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon after RNase treatment (4) replicon encapsulated in liposome (5) liposome after RNase treatment (6) liposome treated with RNase then subjected to phenol/chloroform extraction.

(2) FIG. 2 is an electron micrograph of liposomes.

(3) FIG. 3 shows protein expression (as relative light units, RLU) at days 1, 3 and 6 after delivery of RNA in liposomes with PEGs of different lengths: 1 kDa (triangles); 2 kDa (circles); 3 kDa (squares).

(4) FIG. 4 shows a gel with stained RNA. Lanes show (1) markers (2) naked replicon (3) replicon encapsulated in liposome (4) liposome treated with RNase then subjected to phenol/chloroform extraction.

(5) FIG. 5 shows protein expression at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), as naked RNA (diamonds), or in liposomes (+=0.1 μg, x=1 μg).

(6) FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA.

(7) FIG. 7 shows anti-F IgG titers in animals receiving virion-packaged replicon (VRP or VSRP), 1 μg naked RNA, and 1 μg liposome-encapsulated RNA.

(8) FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1 μg naked RNA, and 0.1 g or 1 μg liposome-encapsulated RNA.

(9) FIG. 9 shows neutralising antibody titers in animals receiving VRP or either 0.1 g or 1 μg liposome-encapsulated RNA.

(10) FIG. 10 shows expression levels after delivery of a replicon as naked RNA (circles), liposome-encapsulated RNA (triangle & square), or as a lipoplex (inverted triangle).

(11) FIG. 11 shows F-specific IgG titers (2 weeks after second dose) after delivery of a replicon as naked RNA (0.01-1 μg), liposome-encapsulated RNA (0.01-10 μg), or packaged as a virion (VRP, 10.sup.6 infectious units or IU).

(12) FIG. 12 shows F-specific IgG titers (circles) and PRNT titers (squares) after delivery of a replicon as naked RNA (1 μg), liposome-encapsulated RNA (0.1 or 1 μg), or packaged as a virion (VRP, 10.sup.6 IU). Titers in naïve mice are also shown. Solid lines show geometric means.

(13) FIG. 13 shows intracellular cytokine production after restimulation with synthetic peptides representing the major epitopes in the F protein, 4 weeks after a second dose. The y-axis shows the % cytokine+ of CD8+CD4−.

(14) FIG. 14 shows the structure of lipid “RV05”.

(15) FIG. 15 shows F-specific IgG titers (mean log.sub.10 titers±std dev) over 210 days after immunisation of calves. The three lines are easily distinguished at day 63 and are, from bottom to top: PBS negative control; liposome-delivered RNA; and the “Triangle 4” product.

(16) FIG. 16 shows structures of three PEG-conjugated DMG lipids (1-3 kDa).

(17) FIGS. 17A to 17E show structures of various PEG-conjugated lipids, where R is PEG of a desired length.

(18) FIG. 18 shows the structure of a useful “split” PEG-conjugated lipid. The box shows the total MW of PEG in the lipid (which, in the specific example below, was 2000).

MODES FOR CARRYING OUT THE INVENTION

(19) RNA Replicons

(20) Various replicons are used below. In general these are based on a hybrid alphavirus genome with non-structural proteins from venezuelan equine encephalitis virus (VEEV), a packaging signal from VEEV, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10 kb long and has a poly-A tail.

(21) Plasmid DNA encoding alphavirus replicons (named: pT7-mVEEV-FL.RSVF or A317; pT7-mVEEV-SEAP or A306; pSP6-VCR-GFP or A50) served as a template for synthesis of RNA in vitro. The replicons contain the alphavirus genetic elements required for RNA replication but lack those encoding gene products necessary for particle assembly; the structural proteins are instead replaced by a protein of interest (either a reporter, such as SEAP or GFP, or an immunogen, such as full-length RSV F protein) and so the replicons are incapable of inducing the generation of infectious particles. A bacteriophage (T7 or SP6) promoter upstream of the alphavirus cDNA facilitates the synthesis of the replicon RNA in vitro and a hepatitis delta virus (HDV) ribozyme immediately downstream of the poly(A)-tail generates the correct 3′-end through its self-cleaving activity.

(22) Following linearization of the plasmid DNA downstream of the HDV ribozyme with a suitable restriction endonuclease, run-off transcripts were synthesized in vitro using T7 or SP6 bacteriophage derived DNA-dependent RNA polymerase. Transcriptions were performed for 2 hours at 37° C. in the presence of 7.5 mM (T7 RNA polymerase) or 5 mM (SP6 RNA polymerase) of each of the nucleoside triphosphates (ATP, CTP, GTP and UTP) following the instructions provided by the manufacturer (Ambion). Following transcription the template DNA was digested with TURBO DNase (Ambion).

(23) 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; replicons capped in this way are given the “v” prefix e.g. vA317 is the A317 replicon capped by VCE. Post-transcriptionally capped RNA was precipitated with LiCl and reconstituted in nuclease-free water. The concentration of the RNA samples was determined by measuring OD.sub.260nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis.

(24) Liposomal Encapsulation

(25) RNA was encapsulated in liposomes made essentially by the method of references 7 and 41. The liposomes were made of 10% DSPC (zwitterionic), 40% DlinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG. These proportions refer to the % moles in the total liposome.

(26) DlinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of reference 2. DSPC (1,2-Distearoyl-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 from Avanti Polar Lipids.

(27) Briefly, lipids were dissolved in ethanol (2 ml), a RNA replicon was dissolved in buffer (2 ml, 100 mM sodium citrate, pH 6) and these were mixed with 2 ml of buffer followed by 1 hour of equilibration. The mixture was diluted with 6 ml buffer then filtered. The resulting product contained liposomes, with ˜95% encapsulation efficiency. FIG. 2 shows an example electron micrograph of liposomes prepared by these methods. These liposomes contain encapsulated RNA encoding full-length RSV F antigen. Dynamic light scattering of one batch showed an average diameter of 141 nm (by intensity) or 78 nm (by number).

(28) In one particular encapsulation method, fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG-conjugated DMG were weighed and dissolved in 7.55 mL of ethanol. Five different conjugated PEGs were used: PEG-500, PEG-750, PEG-1000, PEG-2000 or PEG-3000. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mL ethanol to make a working lipid stock solution of 2 mL. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution and washed with plenty of MilliQ water before use to decontaminate the vials of RNAses. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL of citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used had a 2 mm internal diameter and a 3 mm outer diameter; obtained from Idex Health Science). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 hour. Then the mixture was loaded in a 5 cc syringe, which was fitted to a piece of FEP tubing and in another 5 cc syringe with equal length of FEP tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using a syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using a Tangential Flow Filtration (TFF) system before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs and were used according to the manufacturer's guidelines. Hollow fiber filtration membranes with a 100 kD pore size cutoff and 20 cm.sup.2 surface area were used. For in vitro and in vivo experiments, formulations were diluted to the required RNA concentration with 1×PBS.

(29) The percentage of encapsulated RNA and RNA concentration were determined by Quant-iT RiboGreen RNA reagent kit (Invitrogen), following manufacturer's instructions. The ribosomal RNA standard provided in the kit was used to generate a standard curve. Liposomes were diluted 10× or 100× in 1×TE buffer (from kit) before addition of the dye. Separately, liposomes were diluted 10× or 100× in 1×TE buffer containing 0.5% Triton X before addition of the dye (to disrupt the liposomes and thus to assay total RNA). Thereafter an equal amount of dye was added to each solution and then ˜180 μL of each solution after dye addition was loaded in duplicate into a 96 well tissue culture plate. The fluorescence (Ex 485 nm, Em 528 nm) was read on a microplate reader. All liposome formulations were dosed in vivo based on the encapsulated amount of RNA.

(30) To obtain smaller liposomes the syringe/tube method was replaced by a method in which the lipid and RNA solutions are mixed in channels on a microfluidic chip. Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 226.7 μL of the stock was added to 1.773 mL ethanol to make a working lipid stock solution of 2 mL. A 4 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Four 20 mL glass vials (with stir bars) were rinsed with RNase Away solution and washed with plenty of MilliQ water before use to decontaminate the vials of RNAses. Two of the vials were used for the RNA working solution (2 mL in each vial) and the others for collecting the lipid and RNA mixes. The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. Syringes containing RNA and the lipids were connected to a Mitos Droplet junction Chip (a glass microfluidic device obtained from Syrris, Part no. 3000158) using PTFE tubing 0.03 inches ID× 1/16 inch OD, (Syrris) using a 4-way edge connector. Two RNA streams and one lipid stream were driven by syringe pumps and the mixing of the ethanol and aqueous phase was done at the X junction (100 μm×105 μm) of the chip. The flow rate of all three streams was kept at 1.5 mL/min, hence the ratio of total aqueous to ethanolic flow rate was 2:1. The tube outlet was positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 hour. Then the mixture was loaded in a 5 cc syringe which was fitted to a piece of PTFE tubing 0.03 inches ID× 1/16 inches OD and in another 5 cc syringe with equal length of PTFE tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 3 mL/min flow rate using a syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using the TFF system before recovering the final product. Hollow fiber filtration membranes with a 100 kDa pore size cutoff and 20 cm.sup.2 surface area were used. For in vitro and in vivo experiments, formulations were diluted to the required RNA concentration with 1×PBS. Whereas liposomes prepared using the syringe/tube method with 75 μg RNA had a Z-average diameter (Zav) of 148 nm and a polydispersity index (pdI) of 0.122, the chip mixing gave liposomes with a Zav of 97 nm and a pdI of 0.086. The proportion of encapsulated RNA decreased slightly from 90% to 87%.

(31) Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8 mAU of RNase A per microgram of RNA, incubated for 30 minutes at room temperature. RNase was inactivated with Proteinase K at 55° C. for 10 minutes. A 1:1 v/v mixture of sample to 25:24:1 v/v/v, phenol:chloroform:isoamyl alcohol was then added to extract the RNA from the lipids into the aqueous phase. Samples were mixed by vortexing for a few seconds and then placed on a centrifuge for 15 minutes at 12k RPM. The aqueous phase (containing the RNA) was removed and used to analyze the RNA. Prior to loading (400 ng RNA per well) all the samples were incubated with formaldehyde loading dye, denatured for 10 minutes at 65° C. and cooled to room temperature. Ambion Millennium markers were used to approximate the molecular weight of the RNA construct. The gel was run at 90 V. The gel was stained using 0.1% SYBR gold according to the manufacturer's guidelines in water by rocking at room temperature for 1 hour. FIG. 1 shows that RNase completely digests RNA in the absence of encapsulation (lane 3). RNA is undetectable after encapsulation (lane 4), and no change is seen if these liposomes are treated with RNase (lane 4). After RNase-treated liposomes are subjected to phenol extraction, undigested RNA is seen (lane 6). Even after 1 week at 4° C. the RNA could be seen without any fragmentation (FIG. 4, arrow). Protein expression in vivo was unchanged after 6 weeks at 4° C. and one freeze-thaw cycle. Thus liposome-encapsulated RNA is stable.

(32) To assess in vivo expression of the RNA a reporter enzyme (SEAP; secreted alkaline phosphatase) was encoded in the replicon, rather than an immunogen. Expression levels were measured in sera diluted 1:4 in 1×Phospha-Light dilution buffer using a chemiluminescent alkaline phosphate substrate. 8-10 week old BALB/c mice (5/group) were injected intramuscularly on day 0, 50 μl per leg with 0.1 μg or 1 μg RNA dose. The same vector was also administered without the liposomes (in RNase free 1×PBS) at 1 μg. Virion-packaged replicons were also tested. Virion-packaged replicons used herein (referred to as “VRPs”) were obtained by the methods of reference 42, where the alphavirus replicon is derived from the mutant VEEV or a chimera derived from the genome of VEEV engineered to contain the 3′ UTR of Sindbis virus and a Sindbis virus packaging signal (PS), packaged by co-electroporating them into BHK cells with defective helper RNAs encoding the Sindbis virus capsid and glycoprotein genes.

(33) As shown in FIG. 5, encapsulation increased SEAP levels by about % log at the 1 μg dose, and at day 6 expression from a 0.1 μg encapsulated dose matched levels seen with 1 μg unencapsulated dose. By day 3 expression levels exceeded those achieved with VRPs (squares). Thus expressed increased when the RNA was formulated in the liposomes relative to the naked RNA control, even at a 10× lower dose. Expression was also higher relative to the VRP control, but the kinetics of expression were very different (see FIG. 5). Delivery of the RNA with electroporation resulted in increased expression relative to the naked RNA control, but these levels were lower than with liposomes.

(34) To assess whether the effect seen in the liposome groups was due merely to the liposome components, or was linked to the encapsulation, the replicon was administered in encapsulated form (with two different purification protocols, 0.1 μg RNA), or mixed with the liposomes after their formation (a non-encapsulated “lipoplex”, 0.1 μg RNA), or as naked RNA (1 μg). FIG. 10 shows that the lipoplex gave the lowest levels of expression, showing that shows encapsulation is essential for potent expression.

(35) Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1 ng RNA (FIG. 6). Further experiments comparing expression from encapsulated and naked replicons indicated that 0.01 μg encapsulated RNA was equivalent to 1 μg of naked RNA. At a 0.5 μg dose of RNA the encapsulated material gave a 12-fold higher expression at day 6; at a 0.1 μg dose levels were 24-fold higher at day 6.

(36) Rather than looking at average levels in the group, individual animals were also studied. Whereas several animals were non-responders to naked replicons, encapsulation eliminated non-responders.

(37) Further experiments replaced DlinDMA with DOTAP. Although the DOTAP liposomes gave better expression than naked replicon, they were inferior to the DlinDMA liposomes (2- to 3-fold difference at day 1).

(38) To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1 μg), encapsulated in liposomes (0.1 or 1 μg), or packaged in virions (10.sup.6 IU; “VRP”) at days 0 and 21. FIG. 7 shows anti-F IgG titers 2 weeks after the second dose, and the liposomes clearly enhance immunogenicity. FIG. 8 shows titers 2 weeks later, by which point there was no statistical difference between the encapsulated RNA at 0.1 μg, the encapsulated RNA at 1 μg, or the VRP group. Neutralisation titers (measured as 60% plaque reduction, “PRNT60”) were not significantly different in these three groups 2 weeks after the second dose (FIG. 9). FIG. 12 shows both IgG and PRNT titers 4 weeks after the second dose.

(39) FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

(40) Further experiments compared F-specific IgG titers in mice receiving VRP, 0.1 μg liposome-encapsulated RNA, or 1 μg liposome-encapsulated RNA. Titer ratios (VRP: liposome) at various times after the second dose were as follows:

(41) TABLE-US-00001 2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1 1 μg 2.3 0.9 0.9

(42) Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery.

(43) Further experiments showed superior F-specific IgG responses with a 10 μg dose, equivalent responses for 1 μg and 0.1 μg doses, and a lower response with a 0.01 μg dose. FIG. 11 shows IgG titers in mice receiving the replicon in naked form at 3 different doses, in liposomes at 4 different doses, or as VRP (10.sup.6 IU). The response seen with 1 μg liposome-encapsulated RNA was statistically insignificant (ANOVA) when compared to VRP, but the higher response seen with 10 μg liposome-encapsulated RNA was statistically significant (p<0.05) when compared to both of these groups.

(44) A further study confirmed that the 0.1 μg of liposome-encapsulated RNA gave much higher anti-F IgG responses (15 days post-second dose) than 0.1 μg of delivered DNA, and even was more immunogenic than 20 μg plasmid DNA encoding the F antigen, delivered by electroporation (Elgen™ DNA Delivery System, Inovio).

(45) Liposome Manufacturing Methods

(46) In general, eight different methods have been used for preparing liposomes according to the invention. These are referred to in the text as methods (A) to (H) and they differ mainly in relation to filtration and TFF steps. Details are as follows: (A) Fresh lipid stock solutions in ethanol were prepared. 37 mg of DlinDMA, 11.8 mg of DSPC, 27.8 mg of Cholesterol and 8.07 mg of PEG DMG 2000 were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently rocked at 37° C. for about 15 min to form a homogenous mixture. Then, 755 μL of the stock was added to 1.245 mL ethanol to make a working lipid stock solution of 2 mL. This amount of lipids was used to form liposomes with 250 μg RNA. A 2 mL working solution of RNA was also prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Three 20 mL glass vials (with stir bars) were rinsed with RNase Away solution (Molecular BioProducts, San Diego, CA) and washed with plenty of MilliQ water before use to decontaminate the vials of RNases. One of the vials was used for the RNA working solution and the others for collecting the lipid and RNA mixes (as described later). The working lipid and RNA solutions were heated at 37° C. for 10 min before being loaded into 3 cc luer-lok syringes. 2 mL of citrate buffer (pH 6) was loaded in another 3 cc syringe. Syringes containing RNA and the lipids were connected to a T mixer (PEEK™ 500 μm ID junction, Idex Health Science, Oak Harbor, WA) using FEP tubing (fluorinated ethylene-propylene; al FEP tubing has a 2 mm internal diameter×3 mm outer diameter, supplied by Idex Health Science). The outlet from the T mixer was also FEP tubing. The third syringe containing the citrate buffer was connected to a separate piece of FEP tubing. All syringes were then driven at a flow rate of 7 mL/min using a syringe pump. The tube outlets were positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 hour. 4 ml of the mixture was loaded into a 5 cc syringe, which was connected to a piece of FEP tubing and in another 5 cc syringe connected to an equal length of FEP tubing, an equal amount of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 7 mL/min flow rate using the syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, the mixture collected from the second mixing step (liposomes) were passed through a Mustang Q membrane (an anion-exchange support that binds and removes anionic molecules, obtained from Pall Corporation, AnnArbor, MI, USA). Before passing 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 Mustang membrane. Liposomes were warmed for 10 min at 37° C. before passing through the membrane. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using TFF before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes (part number P/N: X1AB-100-20P) with a 100 kD pore size cutoff and 8 cm.sup.2 surface area were used. For in vitro and in vivo experiments, formulations were diluted to the required RNA concentration with 1×PBS. (B) As method (A) except that, after rocking, 226.7 μL of the stock was added to 1.773 mL ethanol to make a working lipid stock solution of 2 mL, thus modifying the lipid:RNA ratio. (C) As method (B) except that the Mustang filtration was omitted, so liposomes went from the 20 mL glass vial into the TFF dialysis. (D) As method (C) except that the TFF used polyethersulfone (PES) hollow fiber membranes (part number P-C.sub.1-100E-100-01N) with a 100 kD pore size cutoff and 20 cm.sup.2 surface area. (E) As method (D) except that a Mustang membrane was used, as in method (A). (F) As method (A) except that the Mustang filtration was omitted, so liposomes went from the 20 mL glass vial into the TFF dialysis. (G) As method (D) except that a 4 mL working solution of RNA was prepared from a stock solution of ˜1 μg/μL in 100 mM citrate buffer (pH 6). Then four 20 mL glass vials were prepared in the same way. Two of them were used for the RNA working solution (2 mL in each vial) and the others for collecting the lipid and RNA mixes, as in (C). Rather than use T mixer, syringes containing RNA and the lipids were connected to a Mitos Droplet junction Chip (a glass microfluidic device obtained from Syrris, Part no. 3000158) using PTFE tubing (0.03 inches internal diameter× 1/16 inch outer diameter) using a 4-way edge connector (Syrris). Two RNA streams and one lipid stream were driven by syringe pumps and the mixing of the ethanol and aqueous phase was done at the X junction (100 μm×105 μm) of the chip. The flow rate of all three streams was kept at 1.5 mL/min, hence the ratio of total aqueous to ethanolic flow rate was 2:1. The tube outlet was positioned to collect the mixtures in a 20 mL glass vial (while stirring). The stir bar was taken out and the ethanol/aqueous solution was allowed to equilibrate to room temperature for 1 h. Then the mixture was loaded in a 5 cc syringe, which was fitted to another piece of the PTFE tubing; in another 5 cc syringe with equal length of PTFE tubing, an equal volume of 100 mM citrate buffer (pH 6) was loaded. The two syringes were driven at 3 mL/min flow rate using a syringe pump and the final mixture collected in a 20 mL glass vial (while stirring). Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1×PBS using TFF, as in (D). (H) As method (A) except that the 2 mL working lipid stock solution was made by mixing 120.9 μL of the lipid stock with 1.879 mL ethanol. Also, after mixing in the T mixer the liposomes from the 20 mL vial were loaded into Pierce Slide-A-Lyzer Dialysis Cassette (Thermo Scientific, extra strength, 0.5-3 mL capacity) and dialyzed against 400-500 mL of 1×PBS overnight at 4° C. in an autoclaved plastic container before recovering the final product.

(47) RSV Immunogenicity

(48) The vA317 self-replicating replicon encoding RSV F protein was administered to BALB/c mice, 4 or 8 animals per group, by bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon (1 μg) alone or formulated as liposomes with DlinDMA (“RV01”) or DOTAP (“RV13”) or the lipid shown in FIG. 14 (“RV05”). The RV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2% PEG-DMG, but with differing amounts of RNA. The RV05 liposomes had either 40% RV05, 10% DSPC, 48% cholesterol and 2% PEG-DMG or 60% RV05, 38% cholesterol and 2% PEG-DMG. The RV13 liposomes had 40% DOTAP, 10% DOPE, 48% cholesterol and 2% PEG-DMG. In all cases the PEG was PEG-2000 (i.e. 2 kDa PEG). For comparison, naked plasmid DNA (20 μg) expressing the same RSV-F antigen was delivered either using electroporation or with RV01(10) liposomes (0.1 μg DNA). Four mice were used as a naïve control group.

(49) Liposomes were prepared by method (A) or method (B). For some liposomes made by method (A) a double or half amount of RNA was used. Z average particle diameter and polydispersity index were:

(50) TABLE-US-00002 RV Zav (nm) pdI Preparation RV01 (10) 158.6 0.088 (A) RV01 (08) 156.8 0.144 (A) RV01 (05) 136.5 0.136 (B) RV01 (09) 153.2 0.067 (A) RV01 (10) 134.7 0.147 (A) RV05 (01) 148 0.127 (A) RV05 (02) 177.2 0.136 (A) RV13 (02) 128.3 0.179 (A)

(51) Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were harvested from mice at day 49 for T cell analysis.

(52) F-specific serum IgG titers (GMT) were as follows:

(53) TABLE-US-00003 RV Day 14 Day 36 Naked DNA plasmid 439 6712 Naked A317 RNA 78 2291 RV01 (10) 3020 26170 RV01 (08) 2326 9720 RV01 (05) 5352 54907 RV01 (09) 4428 51316 RV05 (01) 1356 5346 RV05 (02) 961 6915 RV01 (10) DNA 5 13 RV13 (02) 644 3616

(54) The proportion of T cells which are cytokine-positive and specific for RSV F51-66 peptide are as follows, showing only figures which are statistically significantly above zero:

(55) TABLE-US-00004 CD4+ CD8− CD4− CD8+ RV IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Naked DNA plasmid 0.04 0.07 0.10 0.57 0.29 0.66 Naked A317 RNA 0.04 0.05 0.08 0.57 0.23 0.67 RV01 (10) 0.07 0.10 0.13 1.30 0.59 1.32 RV01 (08) 0.02 0.04 0.06 0.46 0.30 0.51 RV01 (05) 0.08 0.12 0.15 1.90 0.68 1.94 RV01 (09) 0.06 0.08 0.09 1.62 0.67 1.71 RV01 (10) DNA 0.03 0.08 RV13 (02) 0.03 0.04 0.06 1.15 0.41 1.18

(56) Thus the liposome formulations significantly enhanced immunogenicity relative to the naked RNA controls, as determined by increased F-specific IgG titers and T cell frequencies. Plasmid DNA formulated with liposomes, or delivered naked using electroporation, was significantly less immunogenic than liposome-formulated self-replicating RNA.

(57) Further RV01 liposomes were prepared by method (H), again using 2 kDa PEG conjugated to DMG, and either encapsulating 150 μg RNA (vA375 replicon encoding surface fusion glycoprotein of RSV) or encapsulating only buffer. Thus these liposomes had 4000 DlinDMA, 10% DSPC, 48% Chol, and 2% PEG-DMG. Sizes and encapsulation were as follows:

(58) TABLE-US-00005 RV Zav (nm) pdI RNA Encapsulat.sup.n RV01 (36) 152.1 0.053 + 92.5% RV01 (36) 144 0.13 − −

(59) The liposomes were administered to BALB/c mice (10 per group) by bilateral intramuscular injection (50 μl per leg) on days 0 & 21. Doses were 0.01, 0.03, 0.1, 0.3 or 1 μg. F-specific serum IgG and PRNT60 titers (GMT) were as follows, 2 weeks after the first or second injection:

(60) TABLE-US-00006 RV RNA (μg) 2wp1 2wp2 PRNT60 (2wp2) Buffer control 0 − − 10 RV01 (36) 0 − − 10 RV01 (36) 0.01 3399 50691 37 RV01 (36) 0.03 3446 53463 83 RV01 (36) 0.1 8262 76808 238 RV01 (36) 0.3 5913 82599 512 RV01 (36) 1 8213 85138 441

(61) Cytomegalovirus Immunogenicity

(62) RV01 liposomes with DLinDMA as the cationic lipid and 2 kDa PEG were used to deliver RNA replicons encoding CMV glycoproteins. The “vA160” replicon encodes full-length glycoproteins H and L (gH/gL), whereas the “vA322” replicon encodes a soluble form (gHsol/gL). The two proteins are under the control of separate subgenomic promoters in a single replicon; co-administration of two separate vectors, one encoding gH and one encoding gL, did not give good results.

(63) BALB/c mice, 10 per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0, 21 and 42 with VRPs expressing gH/gL (1×10.sup.6 IU), VRPs expressing gHsol/gL (1×10.sup.6 IU) and PBS as the controls. Two test groups received 1 μg of the vA160 or vA322 replicon formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; made using method (D) but with 150 μg RNA batch size).

(64) The vA160 liposomes had a Zav diameter of 168.8 nm, a pdI of 0.144, and 87.4% encapsulation. The vA322 liposomes had a Zav diameter of 162 nm, a pdI of 0.131, and 90% encapsulation.

(65) The replicons were able to express two proteins from a single vector.

(66) Sera were collected for immunological analysis on day 63 (3wp3). CMV neutralization titers (the reciprocal of the serum dilution producing a 50% reduction in number of positive virus foci per well, relative to controls) were as follows:

(67) TABLE-US-00007 gH/gL VRP gHsol/gL VRP gH/gL liposome gHsol/gL liposome 4576 2393 4240 10062

(68) RNA expressing either a full-length or a soluble form of the CMV gH/gL complex thus elicited high titers of neutralizing antibodies, as assayed on epithelial cells. The average titers elicited by the liposome-encapsulated RNAs were at least as high as for the corresponding VRPs.

(69) Repeat experiments confirmed that the replicon was able to express two proteins from a single vector. The RNA replicon gave a 3wp3 titer of 11457, compared to 5516 with VRPs.

(70) Expression Kinetics

(71) A self-replicating RNA replicon (“vA311”) that expresses a luciferase reporter gene (luc) was used for studying the kinetics of protein expression after injection. BALB/c mice, 5 animals per group, received bilateral intramuscular vaccinations (50 μL per leg) on day 0 with: Group 1 DNA expressing luciferase, delivered using electroporation (10 μg) Group 2 self-replicating RNA (1 μg) formulated in liposomes (40% DlinDMA, 10% DSPC, 48% cholesterol, 2% PEG-2000 conjugated to DMG Group 3 self-replicating RNA (1 μg) formulated with a cationic nanoemulsion (CNE17) Group 4 self-replicating RNA (1 μg) formulated with a different cationic nanoemulsion Group 5 VRP (1×10.sup.6 IU) expressing luciferase

(72) Prior to vaccination mice were depilated. Mice were anesthetized (2% isoflurane in oxygen), hair was first removed with an electric razor and then chemical Nair. Bioluminescence data was then acquired using a Xenogen IVIS 200 imaging system (Caliper Life Sciences) on days 3, 7, 14, 21, 28, 35, 42, 49, 63 and 70. Five minutes prior to imaging mice were injected intraperitoneally with 8 mg/kg of luciferin solution. Animals were then anesthetized and transferred to the imaging system. Image acquisition times were kept constant as bioluminescence signal was measured with a cooled CCD camera.

(73) In visual terms, luciferase-expressing cells were seen to remain primarily at the site of RNA injection, and animals imaged after removal of quads showed no signal.

(74) In quantitative terms, luciferase expression was measured as average radiance over a period of 70 days (p/s/cm.sup.2/sr), and results were as follows for the 5 groups:

(75) TABLE-US-00008 Days 1 2 3 4 5  3 8.69E+07 3.33E+06 2.11E+06 9.71E+06 1.46E+07  7 1.04E+08 8.14E+06 1.83E+07 5.94E+07 1.64E+07 14 8.16E+07 2.91E+06 9.22E+06 3.48E+07 8.49E+05 21 1.27E+07 3.13E+05 6.79E+04 5.07E+05 6.79E+05 28 1.42E+07 6.37E+05 2.36E+04 4.06E+03 2.00E+03 35 1.21E+07 6.12E+05 2.08E+03 42 1.49E+07 8.70E+05 49 1.17E+07 2.04E+05 63 9.69E+06 1.72E+03 70 9.29E+06

(76) The self-replicating RNA formulated with cationic nanoemulsions showed measurable bioluminescence at day 3, which peaked at day 7 and then reduced to background levels by days 28 to 35. When formulated in liposomes the RNA showed measurable bioluminescence at day 3, which peaked at day 7 and reduced to background levels by day 63. RNA delivered using VRPs showed enhanced bioluminescence at day 21 when compared to the formulated RNA, but expression had reduced to background levels by day 28. Electroporated DNA showed the highest level of bioluminescence at all time points measured and levels of bioluminescence did not reduce to background levels within the 70 days of the experiment.

(77) Delivery Volume

(78) Hydrodynamic delivery employs the force generated by the rapid injection of a large volume of solution to overcome the physical barriers of cell membranes which prevent large and membrane-impermeable compounds from entering cells. This phenomenon has previously been shown to be useful for the intracellular delivery of DNA vaccines.

(79) A typical mouse delivery volume for intramuscular injection is 50 μl into the hind leg, which is a relatively high volume for a mouse leg muscle. In contrast, a human intramuscular dose of ˜0.5 ml is relatively small. If immunogenicity in mice would be volume-dependent then the replicon vaccines' efficacy might be due, at least in part, on hydrodynamic forces, which would not be encouraging for use of the same vaccines in humans and larger animals.

(80) The vA317 replicon was delivered to BALB/c mice, 10 per group, by bilateral intramuscular vaccinations (5 or 50 per leg) on day 0 and 21: Group 1 received naked replicon, 0.2 μg in 50 μL per leg Group 2 received naked replicon, 0.2 μg in 5 μL per leg Group 3 received emulsion-formulated replicon (0.2 μg, 50 μL per leg) Group 4 received emulsion-formulated replicon (0.2 μg, 5 μL per leg) Group 5 received liposome-formulated replicon (0.2 μg, 50 μL per leg) Group 6 received liposome-formulated replicon (0.2 μg, 5 μL per leg)

(81) The liposomes for groups 5 & 6 were 40% DlinDMA, 10% DSPC, 48% cholesterol, and 2% PEG-2000 conjugated to DMG.

(82) Serum was collected for antibody analysis on days 14 and 35. F-specific serum IgG GMTs were:

(83) TABLE-US-00009 Day 1 2 3 4 5 6 14 42 21 783 760 2669 2610 35 241 154 2316 2951 17655 18516

(84) Thus immunogenicity of the formulated replicon did not vary according to the delivered volume, thus indicating that these RNA vaccines do not rely on hydrodynamic delivery for their efficacy.

(85) Cotton Rats

(86) A study was performed in cotton rats (Sigmodon hispidis) instead of mice. At a 1 μg dose liposome encapsulation increased F-specific IgG titers by 8.3-fold compared to naked RNA and increased PRNT titers by 9.5-fold. The magnitude of the antibody response was equivalent to that induced by 5×10.sup.6 IU VRP. Both naked and liposome-encapsulated RNA were able to protect the cotton rats from RSV challenge (1×10.sup.5 plaque forming units), reducing lung viral load by at least 3.5 logs. Encapsulation increased the reduction by about 2-fold.

(87) Further work in cotton rats used four different replicons: vA317 expresses full-length RSV-F; vA318 expresses truncated (transmembrane and cytoplasmic tail removed) RSV-F; vA142 expresses RSV-F with its fusion peptide deleted; vA140 expresses the truncated RSV-F also without its peptide. Cotton rats, 4 to 8 animals per group, were given intramuscular vaccinations (100 μL in one leg) on days 0 and 21 with the four different replicons at two doses (1.0 and 0.1 μg) formulated in liposomes made using 2 kDa PEG-conjugated DMG by method (D), but with a 150 μg RNA batch size. Control groups received a RSV-F subunit protein vaccine (5 μg) adjuvanted with alum (8 animals/group), VRPs expressing full-length RSV-F (1×10.sup.6 IU, 8 animals/group), or naïve control (4 animals/group). Serum was collected for antibody analysis on days 0, 21 and 34.

(88) F-specific serum IgG titers and RSV serum neutralizing antibody titers on day 21 and 34 were:

(89) TABLE-US-00010 Group IgG, day 21 IgG, day 34 NT, day 21 NT, day 34   1 μg vA317 915 2249 115 459 0.1 μg vA317 343 734 87 95   1 μg vA318 335 1861 50 277 0.1 μg vA318 129 926 66 239   1 μg vA142 778 4819 92 211 0.1 μg vA142 554 2549 78 141   1 μg vA140 182 919 96 194 0.1 μg vA140 61 332 29 72   5 μg F trimer 13765 86506 930 4744 subunit/alum   1 × 10.sup.6 IU VRP-F 1877 19179 104 4528 full Naïve 5 5 10 15

(90) All four replicons evaluated in this study (vA317, vA318, vA 142, vA 140) were immunogenic in cotton rats when delivered by liposome, although serum neutralization titers were at least ten-fold lower than those induced by adjuvanted protein vaccines or by VRPs. The liposome/RNA vaccines elicited serum F-specific IgG and RSV neutralizing antibodies after the first vaccination, and a second vaccination boosted the response effectively. F-specific IgG titers after the second vaccination with 1 μg replicon were 2- to 3-fold higher than after the second vaccination with 0.1 μg replicon. The four replicons elicited comparable antibody titers, suggesting that full length and truncated RSV-F, each with or without the fusion peptide, are similarly immunogenic in cotton rats.

(91) Further work in cotton rats again used the vA317, vA318 and vA 142 replicons. Cotton rats, 2-8 animals per group, were given intramuscular vaccinations (100 μL in one leg) on days 0 and 21 with the replicons (0.1 or 1 μg) encapsulated in RV01 liposomes (with PEG-2000) made by method (D) but with a 150 μg RNA batch size. Control groups received the RSV-F subunit protein vaccine (5 μg) adjuvanted with alum or VRPs expressing full-length RSV-F (1×10.sup.6 IU, 8 animals/group). All these animals received a third vaccination (day 56) with RSV-F subunit protein vaccine (5 μg) adjuvanted with alum. In addition there was a naïve control (4 animals/group). In addition, an extra group was given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 56 with 1 μg vA317 RNA in liposomes but did not receive a third vaccination with the subunit protein vaccine.

(92) Serum was collected for antibody analysis on days 0, 21, 35, 56, 70, plus days 14, 28 & 42 for the extra group. F-specific serum IgG titers (GMT) were as follows:

(93) TABLE-US-00011 Day 21 Day 35 Day 56 Day 70   1 μg vA318 260 1027 332 14263 0.1 μg vA318 95 274 144 2017   1 μg vA142 483 1847 1124 11168 0.1 μg vA142 314 871 418 11023   1 μg vA317 841 4032 1452 13852   1 × 10.sup.6 VRP (F-full) 2075 3938 1596 14574   5 μg F trimer subunit/alum 12685 54526 25846 48864 Naïve 5 5 5 5

(94) Serum neutralisation titers were as follows (60% RSV neutralization titers for 2 pools of 3-4 animals per group, GMT of these 2 pools per group):

(95) TABLE-US-00012 Day 21 Day 35 Day 56 Day 70   1 μg vA318 58 134 111 6344 0.1 μg vA318 41 102 63 6647   1 μg vA142 77 340 202 5427 0.1 μg vA142 35 65 56 2223   1 μg vA317 19 290 200 4189   1 × 10.sup.6 VRP (F-full) 104 1539 558 2876   5 μg F trimer subunit/alum 448 4457 1630 3631 Naïve 10 10 10

(96) Serum titers and neutralising titers for the extra group were as follows:

(97) TABLE-US-00013 Day 14 21 28 35 42 56 70 IgG 397 561 535 501 405 295 3589 NT 52 82 90 106 80 101 1348

(98) Thus the replicons are confirmed as immunogenic in cotton rats, eliciting serum F-specific IgG and RSV neutralizing antibodies after the first vaccination. A second vaccination boosted the responses effectively. F-specific IgG titers after the second vaccination with 1.0 μg replicon were 1.5 to 4-fold higher than after the second vaccination with 0.1 μg replicon.

(99) The third vaccination (protein at day 56) did not boost titers in cotton rats previously vaccinated with F trimer subunit + alum, but it did provide a large boost to titers in cotton rats previously vaccinated with replicon. In most cases the RSV serum neutralization titers after two replicon vaccinations followed by protein boost were equal to or greater than titers induced by two or three sequential protein vaccinations.

(100) This study also evaluated the kinetics of the antibody response to 1.0 μg vA317. F-specific serum IgG and RSV neutralization titers induced by a single vaccination reached their peak around day 21 and were maintained through at least day 56 (50-70% drop in F-specific IgG titer, little change in RSV neutralization titer). A homologous second vaccination was given to these animals on day 56, and boosted antibody titers to a level at least equal to that achieved when the second vaccination was administered on day 21.

(101) Further experiments involved a viral challenge. The vA368 replicon encodes the full-length wild type surface fusion glycoprotein of RSV with the fusion peptide deleted, with expression driven by the EV71 IRES. Cotton rats, 7 per group, were given intramuscular vaccinations (100 μL per leg) on days 0 and 21 with vA368 in liposomes prepared by method (H), 175 μg RNA batch size, or with VRPs having the same replicon. The liposomes included 2 kDa PEG, conjugated to DMG. A control group received 5 μg alum-adjuvanted protein, and a naïve control group was also included.

(102) All groups received an intranasal challenge (i.n.) with 1×10.sup.6 PFU RSV four weeks after the final immunization. Serum was collected for antibody analysis on days 0, 21, 35. Viral lung titers were measured 5 days post challenge. Results were as follows:

(103) TABLE-US-00014 Liposome VRP Protein Naïve F-specific Serum IgG titers (GMT) Day 21 370 1017 28988 5 Day 35 2636 2002 113843 5 Neutralising titers (GMT) Day 21 47 65 336 10 Day 35 308 271 5188 10 Lung viral load (pfu per gram of lung) Day 54 422 225 124 694110

(104) Thus the RNA vaccine reduced the lung viral load by over three logs, from approximately 10.sup.6 PFU/g in unvaccinated control cotton rats to less than 10′ PFU/g in vaccinated cotton rats.

(105) Large Mammal Study

(106) A large-animal study was performed in cattle. Calves (4-6 weeks old, ˜60-80 kg, 5 per group) were immunised with 66 μg of replicon vA317 encoding full-length RSV F protein at days 0, 21, 86 and 146. The replicons were formulated inside liposomes made by method (E) but with a 1.5 mg RNA batch size; they had 40% DlinDMA, 10% DSPC, 48% cholesterol, and 2% PEG-2000 conjugated to DMG. PBS alone was used as a negative control, and a licensed vaccine was used as a positive control (“Triangle 4” from Fort Dodge, containing killed virus). All calves received 15 μg F protein adjuvanted with the MF59 emulsion on day 146.

(107) The RNA vaccines encoded human RSV F whereas the “Triangle 4” vaccine contains bovine RSV F, but the RSV F protein is highly conserved between BRSV and HRSV.

(108) Calves received 2 ml of each experimental vaccine, administered intramuscularly as 2×1 ml on each side of the neck. In contrast, the “Triangle 4” vaccine was given as a single 2 ml dose in the neck.

(109) Serum was collected for antibody analysis on days 0, 14, 21, 35, 42, 56, 63, 86, 100, 107, 114, 121, 128, 135, 146, 160, 167, 174, 181, 188, 195, and 202. If an individual animal had a titer below the limit of detection it was assigned a titer of 5.

(110) FIG. 15 shows F-specific IgG titers over 210 days. Over the first 63 days the RNA replicon was immunogenic in the cows via liposomes, although it gave lower titers than the licensed vaccine. All vaccinated cows showed F-specific antibodies after the second dose, and titers were very stable from the period of 2 to 6 weeks after the second dose (and were particularly stable for the RNA vaccines). Titres up to day 202 were as follows:

(111) TABLE-US-00015 3wp1 2wp2 5wp2 ~9wp2 2wp3 5wp3 8wp3 2wp4 5wp4 8wp4 D0 D21 D35 D56 D86 D100 D121 D146 D160 D181 D202 PBS 5 5 5 5 5 5 5 5 46 98 150 Liposome 5 5 12 11 20 768 428 74 20774 7022 2353 Triangle 4 5 5 1784 721 514 3406 2786 336 13376 4775 2133

(112) RSV serum neutralizing antibody titers were as follows:

(113) TABLE-US-00016 2wp2 5wp2 2wp3 3wp3 4wp3 8wp3 2wp4 3wp4 4wp4 D0 D35 D56 D100 D107 D114 D146 D160 D167 D174 PBS 12 10 10 14 18 20 14 10 10 10 Liposome 13 10 10 20 13 17 13 47 26 21 Triangle 4 12 15 13 39 38 41 13 24 26 15

(114) The material used for the second liposome dose was not freshly prepared, and the same lot of RNA showed a decrease in potency in a mouse immunogenicity study. Therefore it is possible that the vaccine would have been more immunogenic if fresh material had been used for all vaccinations.

(115) When assayed with complement, neutralizing antibodies were detected in all vaccinated cows. In this assay, all vaccinated calves had good neutralizing antibody titers after the second RNA vaccination Furthermore, the RNA vaccine elicited F-specific serum IgG titers that were detected in a few calves after the second vaccination and in all calves after the third.

(116) MF59-adjuvanted RSV-F was able to boost the IgG response in all previously vaccinated calves, and to boost complement-independent neutralization titers of calves previously vaccinated with RNA.

(117) Proof of concept for RNA vaccines in large animals is particularly important in light of the loss in potency observed previously with DNA-based vaccines when moving from small animal models to larger animals and humans. A typical dose for a cow DNA vaccine would be 0.5-1 mg [43, 44] and so it is very encouraging that immune responses were induced with only 66 μg of RNA.

(118) Effect of PEG Length

(119) As mentioned above, liposomes were prepared using DMG to which five different PEGs were conjugated. The average molecular weight of the PEG was 500 Da, 750 Da, 1 kDa, 2 kDa or 3 kDa.

(120) Liposomes formed using the shortest PEGs (500 Da and 750 Da) were unstable or aggregated during TFF purification. PEG-750 gave liposomes with a significantly higher Zaverage diameter (669 nm) and polydispersity index (0.21), with 77% encapsulation. The PEG-500 liposomes visibly aggregated in solution during the TFF process and the experiment was terminated. Thus these short PEG liposomes were unstable, but the longer PEGs formed stable liposomes.

(121) The different PEG lengths (FIG. 16) had a small effect on liposome diameter and polydispersity index. The Z-average diameter was 197 nm (0.119 pdI) for the 1 kDa PEG, 142 nm (0.137 pdI) for the 2 kDa PEG, and 147 nm (0.075 pdI) for the 3 kDa PEG. RNA encapsulation increased gradually as the PEG length increased, from 81.7% to 85.9% to 91.5% (although this relationship was not always seen in subsequent experiments).

(122) The liposomes were administered to mice by intramuscular injection on day 0. Serum SEAP levels were measured at days 1, 3 and 6 by chemiluminescent assay. As shown in FIG. 3, the three PEG lengths were all effective, but varying the length of the PEG had some effect on serum SEAP levels, with PEG 2000 giving the highest expression.

(123) Different Lipids and PEG Lengths

(124) The vA317 replicon was administered in liposomes having a variety of different lipids with different PEG lengths. The liposomes all had 40% DlinDMA, 10% DSPC and 48% cholesterol, but the remaining 2% was varied, with different PEGylated lipids (e.g. FIGS. 17A to 17E) and different PEG lengths.

(125) Physical characteristics of the liposomes, made by method (H), were:

(126) TABLE-US-00017 Name PEGylated lipid PEG length Zav (nm) pdI % encapsulat.sup.n A DMG 2000 136.3 0.087 85.35 B DMG 3000 120.9 0.087 72.06 C DMG 1000 175.9 0.111 92.52 D FIG. 17A 2000 157.9 0.094 97.44 E FIG. 17D 2000 122.2 0.122 77.84 F FIG. 17E 2000 129.8 0.125 82.57 G Cholesterol 2000 122.9 0.087 87.1 H FIG. 17C 2000 138 0.137 78.48 I FIG. 17B 2000 113.4 0.091 89.12

(127) BALB/c mice, 8 per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon, either naked (1 μg) or encapsulated in these liposomes (0.1 μg). Serum was collected for antibody analysis on days 14, and 35.

(128) F-specific serum IgG titers (GMT) were as follows, 2 weeks after the two injections (2wp1):

(129) TABLE-US-00018 RV 2wp1 2wp2 Naked RNA 216 1356 A 3271 15659 B 3860 22378 C 1691 7412 D 1025 1767 E 1618 9536 F 2684 11221 G 3514 10566 H 4142 22810 I 952 10410

(130) The results show a trend, indicating that higher molecular weight PEG head groups are more immunogenic. As the length of DMG-conjugated PEG increases from 1000 Da to 3000 Da the 2wp2 F-specific IgG titers increase from 7412 to 15659 to 22378.

(131) Changing the linker region from ester to ether did not impact the titers substantially. Also, at the same molecular weight of the head group (2000) there was a trend that increasing the length of the lipid tails lowers the titers (H with C14 dialkyl vs. I with C18 dialkyl). Replacing a PEG di-alkyl lipid tail with cholesterol had little impact on immunogenicity (A with DMG vs. G with cholesterol).

(132) Similar experiments were performed with different lipids in which the 2 kDa of PEG is split into 2×1 kDa groups (FIG. 18, with total MW in the boxed region being 2000). The vA317 replicon was again used, with BALB/c mice, 8 per group, given bilateral intramuscular vaccinations (50 μL per leg) on days 0 & 21 with 1 μg naked RNA or 0.1 μg liposome-encapsulated RNA. The liposomes all had 40% cationic lipid (DlinDMA), 10% DSPC and 48% cholesterol, but the remaining 2% was varied, with different PEGylated lipids (but all with 2 kDa PEG). They were made by method (H).

(133) Physical characteristics of the liposomes were:

(134) TABLE-US-00019 Name PEGylated lipid Zav (nm) pdI % encapsul.sup.n A DMG 121 0.101 84.84 B Split; R = C14 saturated 141.3 0.049 95.41 C Split; R = C16 saturated 114.6 0.101 96.79 D Split; R = C18 saturated 116.5 0.088 98.63 E Split; R = C18, 1 129.4 0.149 93.37 unsaturated

(135) Further liposomes were made with RV05. The liposomes had 40% cationic lipid (RV05) and 2% PEGylated DMG (2 kDa PEG), while the remaining components varied (but cholesterol was always included). The liposomes were made by method (H) but with pH 5. Physical characteristics were:

(136) TABLE-US-00020 Name Other components Zav (nm) pdI % encapsul.sup.n F 10% DSPC, 48% chol 102.2 0.12 76.81 G 10% DSPC, 46% chol, 2% αGC 103.7 0.107 72.58 H 10% DPyPE, 48% chol 99.6 0.115 78.34 I 10% 18:3 PC, 48% chol 130 0.14 87.92 J 10% 18:2 PC, 48% chol 101.1 0.133 76.64 K 30% 18:2 PC, 28% chol 134.3 0.158 57.76 αGC = α-galactosylceramide

(137) BALB/c mice, 8 per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon, either naked (1 μg) or encapsulated (0.1 μg). Serum was collected for antibody analysis on days 14, and 35. F-specific serum IgG titers (GMT) were as follows, 2 weeks after the two injections (2wp1):

(138) TABLE-US-00021 RV 2wp1 2wp2 Naked RNA 321 915 A 2761 17040 B 866 3657 C 1734 5209 D 426 2079 E 2696 15794 F 551 955 G 342 2531 H 1127 3881 I 364 1741 J 567 5679 K 1251 5303

(139) Splitting the PEG head groups thus lowered in vivo titers. Including a double bond (1 degree of instauration per alkyl tail) in the PEG lipid tails increased IgG titers, 6 fold at day 14 and 7 fold at day 35. For a cationic lipid with an asymmetrical lipid tails (alkyl + cholesterol), changing the neutral lipid from DSPC (saturated C18 lipid tail) to 18:2 or 18:3 PC (with 2 and 3 unsaturated double bonds per tail) increased total IgG titers. Comparable results were observed with replacement of DSPC with DPyPE.

(140) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(141) TABLE-US-00022 TABLE 1 useful phospholipids DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE 1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidy1-rac-(1-glycerol . . . ) DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA 1,2-Dilauroyl-sn-Glycero-3-Phosphate DLPC 1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE 1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . ) DLPS 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine DMG 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine DMPA 1,2-Dimyristoyl-sn-Glycero-3-Phosphate DMPC 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . ) DMPS 1,2-Dimyristoyl-sn-Glycero-3-phosphatidylserine DOPA 1,2-Dioleoyl-sn-Glycero-3-Phosphate DOPC 1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE 1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . ) DOPS 1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine DPPA 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . ) DPPS 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA 1,2-Distearoyl-sn-Glycero-3-Phosphate DSPC 1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE 1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG 1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . . ) DSPS 1,2-Distearoyl-sn-Glycero-3-phosphatidylserine EPC Egg-PC HEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPC Hydrogenated Soy PC LYSOPC MYRISTIC 1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC 1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC 1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin MPPC 1-Myristoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine MSPC 1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC 1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC 1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE 1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG 1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . . ] PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC 1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC 1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC 1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

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