VACCINE FOR ELICITING IMMUNE RESPONSE COMPRISING LIPID FORMULATIONS AND RNA ENCODING MULTIPLE IMMUNOGENS

Abstract

Provided are vaccines for eliciting an immune response. The vaccines for eliciting an immune response comprise RNA encoding an immunogen, which 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.

Claims

1. A formulation comprising: a first species of ribonucleic acid (RNA) molecules comprising a sequence that encodes a first immunogen; a second species of RNA molecules comprising a sequence that encodes a second immunogen; and lipids comprising first lipids, second lipids, polyethylene glycol-conjugated (PEG-conjugated) lipids, and a cholesterol, wherein: the lipids encapsulate at least half of the first species of RNA molecules and at least half of the second species of RNA molecules; the second lipids comprise anionic lipids or neutral zwitterionic lipids; the first lipids comprise a tertiary amine and have a pKa from 5.0 to 7.6; at least half of the first lipids are neutrally charged when the first lipids are at a pH that is above the pKa; and at least half of the first lipids are positively charged when the first lipids are at a pH that is below the pKa; and whereby the pKa is defined by the following: (1) admixing the first lipids with ethanol and fluorescent probe 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS), thereby obtaining a lipid/TNS mixture; (2) separately admixing each of a plurality of a sodium salt buffer with a portion of the lipid/TNS mixture, wherein the sodium salt buffer comprises 20 mM sodium phosphate, 25 mM sodium citrate, 20 mM sodium acetate, and 150 mM sodium chloride, wherein each of the plurality of the sodium salt buffer has a different pH and the plurality of the sodium salt buffer has a range of pH from 4.4 to 11.12, thereby obtaining a plurality of pH-varied lipid/TNS mixtures: (3) measuring the absolute fluorescence at a wavelength of 431 nm with an excitation wavelength of 322 nm and a cut-off below a wavelength of 420 nm of each of the plurality of the pH-varied lipid/TNS mixtures, thereby obtaining an absolute fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures: (4) measuring the absolute fluorescence at a wavelength of 431 nm with an excitation wavelength of 322 nm and a cut-off below a wavelength of 420 nm of an empty vessel used in the measuring of (3), thereby obtaining a blank fluorescence: (5) subtracting the blank fluorescence from each of the absolute fluorescences of the plurality of the pH-varied lipid/TNS mixtures, thereby obtaining a blank-subtracted fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures: (6) normalizing each of the blank-subtracted fluorescences of the plurality of the pH-varied lipid/TNS mixtures to the blank-subtracted fluorescence of the pH-varied lipid/TNS mixture that was obtained from the admixing in (2) with the sodium salt buffer that had the lowest pH of the first sodium salt buffers, thereby obtaining a relative fluorescence for each of the plurality of the pH-varied lipid/TNS mixtures, the relative fluorescence being 1 for the pH-varied lipid/TNS mixture that was obtained from the admixing in (2) with the sodium salt buffer that had the lowest pH of the first sodium salt buffers: (7) obtaining a line of best fit of the pHs of the sodium salt buffers versus the respective relative fluorescences of the plurality of pH-varied lipid/TNS mixtures; and (8) defining the pKa as the pH on the line of best fit at which a relative fluorescence of 0.5 is obtained.

2. The formulation of claim 1, wherein the first immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.

3. The formulation of claim 2, wherein the second immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.

4. The formulation of claim 3, wherein the first immunogen and second immunogen comprise viral immunogens.

5. The formulation of claim 1, wherein the formulation is immunogenic in vivo against the first immunogen and the second immunogen.

6. The formulation of claim 5, wherein the formulation elicits at least an antibody response.

7. The formulation of claim 5, wherein the formulation elicits at least a cell-mediated immune response.

8. The formulation of claim 1, further comprising a third species of RNA molecules comprising a sequence that encodes a third immunogen.

9. The formulation of claim 8, wherein the lipids encapsulate at least half of the third species of RNA molecules.

10. The formulation of claim 8, wherein the third immunogen comprises a viral immunogen, a bacterial immunogen, a fungal immunogen, or a parasitic immunogen.

11. The formulation of claim 10, wherein the first species of RNA molecules further comprise a sequence that encodes a replicase, and wherein the first species of RNA molecules are self-replicating.

12. The formulation of claim 11, wherein the second species of RNA molecules further comprise a sequence that encodes the replicase, and wherein the second species of RNA molecules are self-replicating.

13. The formulation of claim 12, wherein the third species of RNA molecules further comprise a sequence that encodes the replicase, and wherein the third species of RNA molecules are self-replicating.

14. The formulation of claim 1, wherein the neutral zwitterionic lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

15. The formulation of claim 1, wherein the PEG-conjugated lipids comprise a PEG that has a molecular weight of 2000 Daltons.

16. The formulation of claim 1, wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

17. The formulation of claim 1, wherein the first immunogen comprises a cytomegalovirus (CMV) immunogen, a herpes simplex virus (HSV) immunogen, a varicella-zoster virus (VZV) immunogen, an Epstein-Barr virus (EBV) immunogen, a papillomavirus immunogen, a polyomavirus immunogen, a coronavirus immunogen, an oncovirus immunogen, a lentivirus immunogen, a flavivirus immunogen, an orthomyxovirus immunogen, a paramyxovirus immunogen, or a picornavirus immunogen.

18. The formulation of claim 17, wherein the second immunogen comprises the CMV immunogen, the HSV immunogen, the VZV immunogen, the EBV immunogen, the papillomavirus immunogen, the polyomavirus immunogen, the coronavirus immunogen, the oncovirus immunogen, the lentivirus immunogen, the flavivirus immunogen, the orthomyxovirus immunogen, the paramyxovirus immunogen, or the picornavirus immunogen.

19. The formulation of claim 18, wherein the first immunogen comprises the coronavirus immunogen and the second immunogen comprises the influenza virus immunogen.

20. The formulation of claim 1, wherein the first lipids are from 30 mol % to 60 mol % of the lipids.

21. The formulation of claim 1, wherein the first immunogen comprises a tumor polypeptide.

22. The formulation of claim 21, wherein the formulation is immunogenic against the tumor polypeptide.

23. The formulation of claim 1, wherein the pKa is from 5.6 to 6.8.

24. The formulation of claim 17, wherein the first immunogen and the second immunogen comprise CMV immunogens.

25. The formulation of claim 1, wherein the neutral zwitterionic lipids are at most 30 mol % of the lipids, and wherein the neutral zwitterionic lipids comprise DSPC or dimyristoyl phosphatidylethanolamine (DMPE).

26. The formulation of claim 1, wherein the cholesterol is from 30 mol % to 50 mol % of the lipids.

27. The formulation of claim 1, wherein the PEG-conjugated lipids are from 1 mol % to 6 mol % of the lipids.

28. The formulation of claim 27, and wherein the PEG-conjugated lipids comprise 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

29. The formulation of claim 1, wherein: the neutral zwitterionic lipids are at most 30 mol % of the lipids, the cholesterol is from 30 mol % to 50 mol % of the lipids, the PEG-conjugated lipids are from 1 mol % to 6 mol % of the lipids, the neutral zwitterionic lipids comprise DSPC, and the PEG-conjugated lipids comprise 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N—[methoxy(polyethylene glycol)] or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

30. The formulation of claim 1, wherein the first species and the second species of RNA molecules further comprise a 7′-methylguanosine, a triphosphate bridge, and a 5′ first ribonucleoside; wherein the 7′-methylguanosine is linked 5′-to-5′ to the 5′ first ribonucleoside by the triphosphate bridge; and wherein the first ribonucleoside comprises a 2′-methylated ribose.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0143] 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.

[0144] FIG. 2 is an electron micrograph of liposomes.

[0145] FIG. 3 shows the structures of DLinDMA, DLenDMA and DODMA.

[0146] 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.

[0147] 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).

[0148] FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA.

[0149] 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.

[0150] 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.

[0151] FIG. 9 shows neutralising antibody titers in animals receiving VRP or either 0.1 g or 1 μg liposome-encapsulated RNA.

[0152] 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).

[0153] 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).

[0154] 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.

[0155] 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−.

[0156] FIGS. 14A and 14B show F-specific IgG titers (mean log.sub.10 titers±std dev) over 63 days (FIG. 14A) and 210 days (FIG. 14B) 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.

[0157] FIG. 15 shows SEAP expression (relative intensity) at day 6 against pKa of lipids used in the liposomes. Circles show levels for liposomes with DSPC, and squares for liposomes without DSPC; sometimes a square and circle overlap, leaving only the square visible for a given pKa.

[0158] FIG. 16 shows anti-F titers expression (relative to RV01, 100%) two weeks after a first dose of replicon encoding F protein. The titers are plotted against pKa in the same way as in FIG. 15. The star shows RV02, which used a cationic lipid having a higher pKa than the other lipids. Triangles show data for liposomes lacking DSPC; circles are for liposomes which included DSPC.

[0159] FIG. 17 shows total IgG titers after replicon delivery in liposomes using, from left to right, RV01, RV16, RV17, RV18 or RV19. Bars show means. The upper bar in each case is 2wp2 (i.e. 2 weeks after second dose), whereas the lower bar is 2wp1.

[0160] FIG. 18 shows IgG titers in 13 groups of mice. Each circle is an individual mouse, and solid lines show geometric means. The dotted horizontal line is the assay's detection limit. The 13 groups are, from left to right, A to M as described below.

[0161] FIGS. 19A and 19B show IL-6 (FIG. 19A) and IFNα (FIG. 19B) (pg/ml) released by pDC. There are 4 pairs of bars, from left to right: control; immunised with RNA+DOTAP; immunised with RNA+lipofectamine; and immunised with RNA in liposomes. In each pair the black bar is wild-type mice, grey is rsq1 mutant.

MODES FOR CARRYING OUT THE INVENTION

RNA Replicons

[0162] 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 sindbis virus, and a 3′ UTR from Sindbis virus or a VEEV mutant. The replicon is about 10kb long and has a poly-A tail.

[0163] 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.

[0164] 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). 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.

Liposomal Encapsulation

[0165] RNA was encapsulated in liposomes made by the method of references 11 and 42. The liposomes were made of 10% DSPC (zwitterionic), 40% DLinDMA (cationic), 48% cholesterol and 2% PEG-conjugated DMG (2 kDa PEG). These proportions refer to the % moles in the total liposome.

[0166] DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane) was synthesized using the procedure of 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 (3p-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids. 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol was obtained from NOF Corporation (catalog #GM-020).

[0167] 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.

[0168] For example, in one particular method, fresh lipid stock solutions were prepared in ethanol. 37 mg of DLinDMA, 11.8 mg of DSPC, 27.8 mg of cholesterol and 8.07 mg of PEG-DMG were weighed and dissolved in 7.55 mL of ethanol. The freshly prepared lipid stock solution was gently 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) 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 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 pm ID junction, Idex Health Science) using FEP tubing (fluorinated ethylene-propylene; all FEP tubing used has a 2 mm internal diameter and a 3 mm outer diameter). 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 h. 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). Before 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 it. 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 by tangential flow filtration before recovering the final product. The TFF system and hollow fiber filtration membranes were purchased from Spectrum Labs (Rancho Dominguez) and were used according to the manufacturer's guidelines. Polysulfone hollow fiber filtration membranes 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.

[0169] 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).

[0170] 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.

[0171] Encapsulation in liposomes was shown to protect RNA from RNase digestion. Experiments used 3.8mAU 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 12 k 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.

[0172] 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 43, 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.

[0173] As shown in FIG. 5, encapsulation increased SEAP levels by about ½ 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.

[0174] 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.

[0175] In vivo studies using liposomal delivery confirmed these findings. Mice received various combinations of (i) self-replicating RNA replicon encoding full-length RSV F protein (ii) self-replicating GFP-encoding RNA replicon (iii) GFP-encoding RNA replicon with a knockout in nsP4 which eliminates self-replication (iv) full-length RSV F-protein. 13 groups in total received:

TABLE-US-00001 A — — B 0.1 μg of (i), naked — C 0.1 μg of (i), encapsulated in — liposome D 0.1 μg of (i), with separate — liposomes E 0.1 μg of (i), naked 10 μg of (ii), naked F 0.1 μg of (i), naked 10 μg of (iii), naked G 0.1 μg of (i), encapsulated in 10 μg of (ii), naked liposome H 0.1 μg of (i), encapsulated in 10 μg of (iii), naked liposome I 0.1 μg of (i), encapsulated in 1 μg of (ii), encapsulated in liposome liposome J 0.1 μg of (i), encapsulated in 1 μg of (iii), encapsulated in liposome liposome K 5 μg F protein — L 5 μg F protein 1 μg of (ii), encapsulated in liposome M 5 μg F protein 1 μg of (iii), encapsulated in liposome

[0176] Results in FIG. 18 show that F-specific IgG responses required encapsulation in the liposome rather than mere co-delivery (compare groups C & D). A comparison of groups K, L and M shows that the RNA provided an adjuvant effect against co-delivered protein, and this effect was seen with both replicating and non-replicating RNA.

[0177] Further SEAP experiments showed a clear dose response in vivo, with expression seen after delivery of as little as 1ng 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.

[0178] 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.

[0179] 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). Whereas DOTAP has a quaternary amine, and so have a positive charge at the point of delivery, DLinDMA has a tertiary amine.

[0180] To assess in vivo immunogenicity a replicon was constructed to express full-length F protein from respiratory syncytial virus (RSV). This was delivered naked (1Ig), 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 0.1Ig, the encapsulated RNA at 1Ig, 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.

[0181] FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

[0182] 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:

TABLE-US-00002 2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1   1 μg 2.3 0.9 0.9

[0183] Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery.

[0184] 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.

[0185] 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).

[0186] A further 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.

[0187] A large-animal study was performed in cattle. Cows were immunised with 66 μg of replicon encoding full-length RSV F protein at days 0 and 21, formulated inside liposomes. 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). FIGS. 14 show F-specific IgG titers over 63 day and 210 day periods, respectively, starting from the first immunisation. The RNA replicon was immunogenic in the cows, 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 vaccine).

Mechanism of Action

[0188] Bone marrow derived dendritic cells (pDC) were obtained from wild-type mice or the “Resq” (rsq1) mutant strain. The mutant strain has a point mutation at the amino terminus of its TLR7 receptor which abolishes TLR7 signalling without affecting ligand binding as disclosed in reference 44. The cells were stimulated with replicon RNA formulated with DOTAP, lipofectamine 2000 or inside a liposome. As shown in FIGS. 19A and 19B, IL-6 and INFα, respectively, were induced in WT cells but this response was almost completely abrogated in mutant mice. These results shows that TLR7 is required for RNA recognition in immune cells, and that liposome-encapsulated replicons can cause immune cells to secrete high levels of both interferons and pro-inflammatory cytokines.

pKa Measurement

[0189] The pKa of a lipid is measured in water at standard temperature and pressure using the following technique: [0190] 2 mM solution of lipid in ethanol is prepared by weighing the lipid and dissolving in ethanol. 0.3 mM solution of fluorescent probe 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) in ethanol:methanol 9:1 is prepared by first making 3 mM solution of TNS in methanol and then diluting to 0.3 mM with ethanol. [0191] An aqueous buffer containing sodium phosphate, sodium citrate sodium acetate and sodium chloride, at the concentrations 20 mM, 25 mM, 20 mM and 150 mM, respectively, is prepared. The buffer is split into eight parts and the pH adjusted either with 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. 400 μL of 2 mM lipid solution and 800 μL of 0.3 mM TNS solution are mixed. [0192] 7.5 μL of probe/lipid mix are added to 242.5 μL of buffer in a 1 mL 96 well plate. This is done with all eight buffers. After mixing, 100 μL of each probe/lipid/buffer mixture is transferred to a 250 μL black with clear bottom 96 well plate (e.g. model COSTAR 3904, Corning). A convenient way of performing this mixing is to use the Tecan Genesis RSP150 high throughput liquid handler and Gemini Software. [0193] Fluorescence of each probe/lipid/buffer mixture is measured (e.g. with a SpectraMax M5 spectrophotometer and SoftMax pro 5.2 software) with 322 nm excitation, 431 nm emission (auto cutoff at 420 nm). [0194] After the measurement, the background fluorescence value of an empty well on the 96 well plate is subtracted from each probe/lipid/buffer mixture. The fluorescence intensity values are then normalized to the value at lowest pH. The normalized fluorescence intensity is then plotted against pH and a line of best fit is provided. [0195] The point on the line of best fit at which the normalized fluorescence intensity is equal to 0.5 is found. The pH corresponding to normalized fluorescence intensity equal to 0.5 is found and is considered the pKa of the lipid.

[0196] This method gives a pKa of 5.8 for DLinDMA. The pKa values measured by this method for cationic lipids of reference 5 are included below.

Encapsulation in Liposomes Using Alternative Cationic Lipids

[0197] As an alternative to using DlinDMA, the cationic lipids of reference 5 are used. These lipids can be synthesised as disclosed in reference 5.

[0198] The liposomes formed above using DlinDMA are referred to hereafter as the “RV01” series. The DlinDMA was replaced with various cationic lipids in series “RV02” to “RV12” as described below. Two different types of each liposome were formed, using 2% PEG2000-DMG with 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 of the (01) and (02) liposomes shows the effect of the neutral zwitterionic lipid.

[0199] RV02 liposomes were made using the following cationic lipid (pKa >9, without a tertiary amine):

##STR00001##

[0200] RV03 liposomes were made using the following cationic lipid (pKa 6.4):

##STR00002##

[0201] RV04 liposomes were made using the following cationic lipid (pKa 6.62):

##STR00003##

[0202] RV05 liposomes were made using the following cationic lipid (pKa 5.85):

##STR00004##

[0203] RV06 liposomes were made using the following cationic lipid (pKa 7.27):

##STR00005##

[0204] RV07 liposomes were made using the following cationic lipid (pKa 6.8):

##STR00006##

[0205] RV08 liposomes were made using the following cationic lipid (pKa 5.72):

##STR00007##

[0206] RV09 liposomes were made using the following cationic lipid (pKa 6.07):

##STR00008##

[0207] RV10 liposomes were made for comparison using the following cationic lipid (pKa 7.86):

##STR00009##

[0208] RV11 liposomes were made using the following cationic lipid (pKa 6.41):

##STR00010##

[0209] RV12 liposomes were made using the following cationic lipid (pKa 7):

##STR00011##

[0210] RV16 liposomes were made using the following cationic lipid (pKa 6.1) as disclosed in reference 45:

##STR00012##

[0211] RV17 liposomes were made using the following cationic lipid (pKa 6.1) as disclosed in reference 45:

##STR00013##

[0212] RV18 liposomes were made using DODMA. RV19 liposomes were made using DOTMA, and RV13 liposomes were made with DOTAP, both having a quaternary amine headgroup.

[0213] These liposomes were characterised and were tested with the SEAP reporter described above. The following table shows the size of the liposomes (Z average and polydispersity index), the % of RNA encapsulation in each liposome, together with the SEAP activity detected at days 1 and 6 after injection. SEAP activity is relative to “RV01(02)” liposomes made from DlinDMA, cholesterol and PEG-DMG:

TABLE-US-00003 Lipid % SEAP SEAP RV pKa Zav (pdI) encapsulation day 1 day 6 RV01 (01) 5.8 154.6 (0.131) 95.5 80.9 71.1 RV01 (02) 5.8 162.0 (0.134) 85.3 100 100 RV02 (01) >9 133.9 (0.185) 96.5 57 45.7 RV02 (02) >9 134.6 (0.082) 97.6 54.2 4.3 RV03 (01) 6.4 158.3 (0.212) 62.0 65.7 44.9 RV03 (02) 6.4 164.2 (0.145) 86 62.2 39.7 RV04 (01) 6.62 131.0 (0.145) 74.0 91 154.8 RV04 (02) 6.62 134.6 (0.117) 81.5 90.4 142.6 RV05 (01) 5.85 164.0 (0.162) 76.0 76.9 329.8 RV05 (02) 5.85 177.8 (0.117) 72.8 67.1 227.9 RV06 (01) 7.27 116.0 (0.180) 79.8 25.5 12.4 RV06 (02) 7.27 136.3 (0.164) 74.9 24.8 23.1 RV07 (01) 6.8 140.6 (0.184) 77 26.5 163.3 RV07 (02) 6.8 138.6 (0.122) 87 29.7 74.8 RV 08 (01) 5.72 176.7 (0.185) 50 76.5 187 RV08 (02) 5.72 199.5 (0.191) 46.3 82.4 329.8 RV09 (01) 6.07 165.3 (0.169) 72.2 65.1 453.9 RV09 (02) 6.07 179.5 (0.157) 65 68.5 658.2 RV10 (01) 7.86 129.7 (0.184) 78.4 113.4 47.8 RV10 (02) 7.86 147.6 (0.131) 80.9 78.2 10.4 RV11 (01) 6.41 129.2 (0.186) 71 113.6 242.2 RV11 (02) 6.41  139 (0198) 75.2 71.8 187.2 RV12 (01) 7 135.7 (0.161) 78.8 65 10 RV12 (02) 7 158.3 (0.287) 69.4 78.8 8.2

[0214] FIG. 15 plots the SEAP levels at day 6 against the pKa of the cationic lipids. The best results are seen where the lipid has a pKa between 5.6 and 6.8, and ideally between 5.6 and 6.3.

[0215] These liposomes were also used to deliver a replicon encoding full-length RSV F protein. Total IgG titers against F protein two weeks after the first dose (2wpl) are plotted against pKa in FIG. 16. The best results are seen where the pKa is where the cationic lipid has a pKa between 5.7-5.9, but pKa alone is not enough to guarantee a high titer e.g. the lipid must still support liposome formation.

RSV Immunogenicity

[0216] Further work was carried out with a self-replicating replicon (vA317) encoding RSV F protein. BALB/c mice, 4 or 8 animals per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with the replicon (1p g) alone or formulated as liposomes with the RV01 or RV05 lipids (see above; pKa of 5.8 or 5.85) or with RV13. The RV01 liposomes had 40% DlinDMA, 10% DSPC, 48% cholesterol and 2% PEG-DMG, but with differing amounts of RNA. The RV05(01) liposomes had 40% cationic lipid, 48% cholesterol, 10% DSPC, and 2% PEG-DMG; the RV05(02) liposomes had 60% cationic lipid, 38% cholesterol, and 2% PEG-DMG. The RV13 liposomes had 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 using electroporation or with RV01(10) liposomes (0.1 g DNA). Four mice were used as a naïve control group.

[0217] Liposomes were prepared by method (A) or method (B). In method (A) fresh lipid stock solutions in ethanol were 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 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. This amount of lipids was used to form liposomes with 75 μg RNA to give 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, for RV01(10), DNA) 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) 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 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 pm ID junction) using FEP tubing. 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. 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 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. Polyethersulfone (PES) hollow fiber filtration membranes (part number P-C1-100E-100-01N) 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.

[0218] Preparation method (B) differed in two ways from method (A). Firstly, 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 and removes anionic molecules, obtained from Pall Corporation, Ann Arbor, Mich., USA). This membrane was first washed with 4 mL of 1 M NaOH, 4 mL of 1 M NaCl and 10 mL of 100 mM citrate buffer (pH 6) in turn, and liposomes were warmed for 10 min at 37° C. before being filtered. Secondly, the hollow fiber filtration membrane was Polysulfone (part number P/N: X1AB-100-20P).

[0219] The Z average particle diameter, polydispersity index and encapsulation efficiency of the liposomes were as follows:

TABLE-US-00004 RV Zav (nm) pdI % encapsulation Preparation RV01 (10) 158.6 0.088 90.7 (A) RV01 (08) 156.8 0.144 88.6 (A) RV01 (05) 136.5 0.136 99   (B) RV01 (09) 153.2 0.067 76.7 (A) RV05 (01) 148 0.127 80.6 (A) RV05 (02) 177.2 0.136 72.4 (A) RV01 (10) 134.7 0.147   87.8 * (A) RV13 (02) 128.3 0.179 97   (A) * For this RV01(10) formulation the nucleic acid was DNA not RNA

[0220] Serum was collected for antibody analysis on days 14, 36 and 49. Spleens were harvested from mice at day 49 for T cell analysis.

[0221] F-specific serum IgG titers (GMT) were as follows:

TABLE-US-00005 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

[0222] 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: PGP-39J 2

TABLE-US-00006 CD4+CD8− CD4−CD8+ RV IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα Naked 0.04 0.07 0.10 0.57 0.29 0.66 DNA plasmid Naked 0.04 0.05 0.08 0.57 0.23 0.67 A317 RNA 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 RV05 (01) 0.06 0.04 0.19 RV05 (02) 0.05 0.07 0.11 0.64 0.35 0.69 RV01 (10) 0.03 0.08 DNA RV13 (02) 0.03 0.04 0.06 1.15 0.41 1.18

[0223] 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.

[0224] The RV01 and RV05 RNA vaccines were more immunogenic than the RV13 (DOTAP) vaccine. These formulations had comparable physical characteristics and were formulated with the same self-replicating RNA, but they contain different cationic lipids. RV01 and RV05 both have a tertiary amine in the headgroup with a pKa of about 5.8, and also include unsaturated alkyl tails. RV13 has unsaturated alkyl tails but its headgroup has a quaternary amine and is very strongly cationic. These results suggest that lipids with tertiary amines with pKas in the range 5.0 to 7.6 are superior to lipids such as DOTAP, which are strongly cationic, when used in a liposome delivery system for RNA.

Further Alternatives to DLinDMA

[0225] The cationic lipid in RV01 liposomes (DLinDMA) was replaced by RV16, RV17, RV18 or RV19. Total IgG titers are shown in FIG. 17. The lowest results are seen with RV19 i.e. the DOTMA quaternary amine.

BHK Expression

[0226] Liposomes with different lipids were incubated with BHK cells overnight and assessed for protein expression potency. From a baseline with RV05 lipid expression could be increased 18× by adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) to the liposome, 10× by adding 10% 18:2 (cis) phosphatidylcholine, and 900× by instead using RV01.

RSV Immunogenicity in Different Mouse Strains

[0227] Replicon “vA142” encodes the full-length wild type surface fusion (F) glycoprotein of RSV but with the fusion peptide deleted, and the 3′ end is formed by ribozyme-mediated cleavage. It was tested in three different mouse strains.

[0228] BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 22. Animals were divided into 8 test groups (5 animals per group) and a naïve control (2 animals): [0229] Group 1 were given naked replicon (1 μg). [0230] Group 2 were given 1 g replicon delivered in liposomes “RV01(37)” with 40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-conjugated DMG. [0231] Group 3 were given the same as group 2, but at 0.1 g RNA. [0232] Group 4 were given 1 g replicon in “RV17(10)” liposomes (40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG). [0233] Group 5 were 1 g replicon in “RV05(11)” liposomes (40% RV07 lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG). [0234] Group 6 were given 0.1 g replicon in “RV17(10)” liposomes. [0235] Group 7 were given 5 g RSV-F subunit protein adjuvanted with aluminium hydroxide. [0236] Group 8 were a naïve control (2 animals)

[0237] Sera were collected for antibody analysis on days 14, 35 and 49. F-specific serum IgG GMTs were:

TABLE-US-00007 Day 1 2 3 4 5 6 7 8 14 82 2463 1789 2496 1171 1295 1293 5 35 1538 34181 25605 23579 13718 8887 73809 5

[0238] At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

TABLE-US-00008 IgG 1 2 3 4 5 6 7 IgG1 94 6238 4836 7425 8288 1817 78604 IgG2a 5386 77064 59084 33749 14437 17624 24

[0239] RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group):

TABLE-US-00009 Day 1 2 3 4 5 6 7 8 35 <20 143 20 101 32 30 111 <20 49 <20 139 <20 83 41 32 1009 <20

[0240] Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD4+ or CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F51-66, F164-178, F309-323 for CD4+, or for peptides F85-93 and F249-258 for CD8+):

TABLE-US-00010 CD4+CD8− CD4−CD8+ Group IFNγ IL2 IL5 TNFα IFNγ IL2 IL5 TNFα 1 0.03 0.06 0.08 0.47 0.29 0.48 2 0.05 0.10 0.08 1.35 0.52 1.11 3 0.03 0.07 0.06 0.64 0.31 0.61 4 0.05 0.09 0.07 1.17 0.65 1.09 5 0.03 0.08 0.07 0.65 0.28 0.58 6 0.05 0.07 0.07 0.74 0.36 0.66 7 0.02 0.04 0.04 8

[0241] C57BL/6 mice were immunised in the same way, but a 9th group received VRPs (1×10.sup.6 IU) expressing the full-length wild-type surface fusion glycoprotein of RSV (fusion peptide deletion).

[0242] Sera were collected for antibody analysis on days 14, 35 & 49. F-specific IgG titers (GMT) were:

TABLE-US-00011 Day 1 2 3 4 5 6 7 8 9 14 1140 2133 1026 2792 3045 1330 2975 5 1101 35 1721 5532 3184 3882 9525 2409 39251 5 12139

[0243] At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

TABLE-US-00012 IgG 1 2 3 4 5 6 7 8 IgG1 66 247 14 328 468 92 56258 79 IgG2a 2170 7685 5055 6161 1573 2944 35 14229

[0244] RSV serum neutralizing antibody titers at days 35 and 49 were as follows (data are 60% plaque reduction neutralization titers of pools of 2-5 mice, 1 pool per group):

TABLE-US-00013 Day 1 2 3 4 5 6 7 8 9 35 <20 27 29 22 36 <20 28 <20 <20 49 <20 44 30 23 36 <20 33 <20 37

[0245] Spleens were harvested at day 49 for T cell analysis. Average net F-specific cytokine-positive T cell frequencies (CD8+) were as follows, showing only figures which were statistically significantly above zero (specific for RSV peptides F85-93 and F249-258): Group CD4-CD8+

TABLE-US-00014 CD4−CD8+ Group IFNγ IL2 IL5 TNFα 1 0.42 0.13 0.37 2 1.21 0.37 1.02 3 1.01 0.26 0.77 4 1.26 0.23 0.93 5 2.13 0.70 1.77 6 0.59 0.19 0.49 7 0.10 0.05 8 9 2.83 0.72 2.26

[0246] Nine groups of C3H/HeN mice were immunised in the same way. F-specific IgG titers (GMT) were:

TABLE-US-00015 Day 1 2 3 4 5 6 7 8 9 14 5 2049 1666 1102 298 984 3519 5 806 35 152 27754 19008 17693 3424 6100 62297 5 17249

[0247] At day 35 F-specific IgG1 and IgG2a titers (GMT) were as follows:

TABLE-US-00016 IgG 1 2 3 4 5 6 7 8 IgG1 5 1323 170 211 136 34 83114 189 IgG2a 302 136941 78424 67385 15667 27085 3800 72727

[0248] RSV serum neutralizing antibody titers at days 35 and 49 were as follows:

TABLE-US-00017 Day 1 2 3 4 5 6 7 8 9 35 <20 539 260 65 101 95 443 <20 595 49 <20 456 296 35 82 125 1148 <20 387

[0249] Thus three different lipids (RV01, RV05, RV17; pKa 5.8, 5.85, 6.1) were tested in three different inbred mouse strains. For all 3 strains RV01 was more effective than RV17; for BALB/c and C3H strains RV05 was less effective than either RV01 or RV17, but it was more effective in B6 strain. In all cases, however, the liposomes were more effective than two cationic nanoemulsions which were tested in parallel.

CMV Immunogenicity

[0250] RV01 liposomes with DLinDMA as the cationic lipid were used to deliver RNA replicons encoding cytomegalovirus (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.

[0251] 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 g of the vA160 or vA322 replicon formulated in liposomes (40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-DMG; made using method (A) as discussed above, but with 150 μg RNA batch size).

[0252] The vA160 liposomes had a Zav diameter of 168 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.

[0253] The replicons were able to express two proteins from a single vector.

[0254] 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:

TABLE-US-00018 gH/gL VRP gHsol/gL VRP gH/gL liposome gHsol/gL liposome 4576 2393 4240 10062

[0255] 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.

[0256] 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.

REFERENCES

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