IMMUNISATION OF LARGE MAMMALS WITH LOW DOSES OF RNA

Abstract

RNA encoding an immunogen is delivered to a large mammal at a dose of between 2 μg and 100 μg. Thus the invention provides a method of raising an immune response in a large mammal, comprising administering to the mammal a dose of between 2 μg and 100 μg of immunogen-encoding RNA. Similarly, RNA encoding an immunogen can be delivered to a large mammal at a dose of 3 ng/kg to 150 ng/kg. The delivered RNA can elicit an immune response in the large mammal.

Claims

1.-12. (canceled)

13. A method of eliciting in a large mammal an antibody response against an immunogen, a cell-mediated immune response against the immunogen, or both, the method comprising administering to the large mammal at least two unit doses, each unit dose comprising a composition comprising lipid particles and messenger ribonucleic acid (mRNA) molecules; the at least two unit doses being sequential and administered at least 1 week apart; the administering comprising contacting the composition with skeletal muscle; the mRNA molecules comprising a sequence that encodes the immunogen; the immunogen comprising a respiratory syncytial virus (RSV) surface fusion glycoprotein (F protein) immunogen or an influenza A virus immunogen; each unit dose comprising between 2 μg and 100 μg of the mRNA molecules; the lipid particles comprising: i) a polyethylene glycol-ylated (PEGylated) lipid, ii) cholesterol, iii) an anionic phospholipid or a zwitterionic phospholipid, and iv) a cationic lipid; the cationic lipid comprising a tertiary amine; the lipid particles encapsulating at least half of the mRNA molecules; and the large mammal being a human or a cow.

14. A method of eliciting in a large mammal an antibody response against an immunogen, a cell-mediated immune response against the immunogen, or both, the method comprising administering to the large mammal at least two unit doses, each unit dose comprising a composition comprising lipid particles and mRNA molecules; the at least two unit doses being sequential and administered at least 1 week apart; the administering comprising contacting the composition with skeletal muscle; the mRNA molecules comprising a sequence that encodes the immunogen; the immunogen comprising a RSV F protein immunogen or an influenza A virus immunogen; each unit dose comprising between 0.5 μg and 1.5 μg of the mRNA molecules per kg of the body mass of the large mammal; the lipid particles comprising: i) a PEGylated lipid, ii) cholesterol, ii) an anionic phospholipid or a zwitterionic phospholipid, and iv) a cationic lipid; the cationic lipid comprising a tertiary amine; the lipid particles encapsulating at least half of the mRNA molecules; and the large mammal being a human or a cow.

15. The method of claim 13, the mRNA molecules comprising a modified nucleotide.

16. The method of claim 14, the mRNA molecules comprising a modified nucleotide.

17. The method of claim 15, the modified nucleotide including a modified pyrimidine nucleotide.

18. The method of claim 16, the modified nucleotide including a modified pyrimidine nucleotide.

19. The method of claim 17, the modified nucleotide further including a 5′ cap.

20. The method of claim 18, the modified nucleotide further including a 5′ cap.

21. The method of claim 13, at least 80% of the lipid particles having diameters from 20 nm to 220 nm.

22. The method of claim 14, at least 80% of the lipid particles having diameters from 20 nm to 220 nm.

23. The method of claim 13, the zwitterionic lipid being 1,2-diastearoyl-sn-glycero-3-phosphocholine (DSPC); the large mammal being human.

24. The method of claim 14, the zwitterionic lipid being DSPC; the large mammal being human.

25. The method of claim 15, the zwitterionic lipid being 1 DSPC; the large mammal being human.

26. The method of claim 16, the zwitterionic lipid being DSPC; the large mammal being a human.

27. The method of claim 17, the zwitterionic lipid being DSPC; the large mammal being a human.

28. The method of claim 18, the zwitterionic lipid being DSPC; the large mammal being a human.

29. The method of claim 19, the zwitterionic lipid being DSPC; the large mammal being a human.

30. The method of claim 20, the zwitterionic lipid being DSPC; the large mammal being a human.

31. The method of claim 21, the zwitterionic lipid being 1 DSPC; the large mammal being a human.

32. The method of claim 22, the zwitterionic lipid being DSPC; the large mammal being a human.

33. The method of claim 23 comprising administering to the human at least three unit doses.

34. The method of claim 24 comprising administering to the human at least three unit doses.

35. The method of claim 25 comprising administering to the human at least three unit doses.

36. The method of claim 26 comprising administering to the human at least three unit doses.

37. The method of claim 27 comprising administering to the human at least three unit doses.

38. The method of claim 28 comprising administering to the human at least three unit doses.

39. The method of claim 29 comprising administering to the human at least three unit doses.

40. The method of claim 30 comprising administering to the human at least three unit doses.

41. The method of claim 31 comprising administering to the human at least three unit doses.

42. The method of claim 32 comprising administering to the human at least three unit doses.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0204] 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.

[0205] FIG. 2 is an electron micrograph of liposomes.

[0206] FIG. 3 shows protein expression (as relative light units, RLU) at days 1, 3 and 6 after delivery of RNA as a virion-packaged replicon (squares), naked RNA (triangles), or as microparticles (circles).

[0207] 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.

[0208] 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).

[0209] FIG. 6 shows protein expression at days 1, 3 and 6 after delivery of four different doses of liposome-encapsulated RNA.

[0210] 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.

[0211] 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.

[0212] FIG. 9 shows neutralising antibody titers in animals receiving VRP or either 0.1 g or 1 μg liposome-encapsulated RNA.

[0213] 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).

[0214] 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).

[0215] 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.

[0216] 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−.

[0217] FIGS. 14A & 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 four lines are easily distinguished at day 63 and are, from bottom to top: PBS negative control; liposome-delivered RNA; emulsion-delivered RNA; and the “Triangle 4” product.

MODES FOR CARRYING OUT THE INVENTION

[0218] RNA Replicons

[0219] 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 10 kb long and has a poly-A tail.

[0220] 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.

[0221] 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.260 nm. Integrity of the in vitro transcripts was confirmed by denaturing agarose gel electrophoresis.

[0222] PLG Adsorption

[0223] Microparticles were made using 500 mg of PLG RG503 (50:50 lactide/glycolide molar ratio, MW ˜30 kDa) and 20 mg DOTAP using an Omni Macro Homogenizer. The particle suspension was shaken at 150 rpm overnight and then filtered through a 40 μm sterile filter for storage at 2-8° C. Self-replicating RNA was adsorbed to the particles. To prepare 1 mL of PLG/RNA suspension the required volume of PLG particle suspension was added to a vial and nuclease-free water was added to bring the volume to 900 μL. 100 μL RNA (10 μg/mL) was added dropwise to the PLG suspension, with constant shaking. PLG/RNA was incubated at room temperature for 30 min. For 1 mL of reconstituted suspension, 45 mg mannitol, 15 mg sucrose and 250-500 μg of PVA were added. The vials were frozen at −80° C. and lyophilized.

[0224] To evaluate RNA adsorption, 100 μL particle suspension was centrifuged at 10,000 rpm for 5 min and supernatant was collected. PLG/RNA was reconstituted using 1 mL nuclease-free water. To 100 μL particle suspension (1 μg RNA), 1 mg heparin sulfate was added. The mixture was vortexed and allowed to sit at room temperature for 30 min for RNA desorption. Particle suspension was centrifuged and supernatant was collected.

[0225] For RNAse stability, 100 μL particle suspension was incubated with 6.4 mAU of RNase A at room temperature for 30 min. RNAse was inactivated with 0.126 mAU of Proteinase K at 55° C. for 10 min. 1 mg of heparin sulfate was added to desorb the RNA followed by centrifugation. The supernatant samples containing RNA were mixed with formaldehyde load dye, heated at 65° C. for 10 min and analyzed using a 1% denaturing gel (460 ng RNA loaded per lane).

[0226] To assess expression, Balb/c mice were immunized with 1 μg RNA in 100 μL intramuscular injection volume (50 μL/leg) on day 0. Sera were collected on days 1, 3 and 6. Protein expression was determined using a chemiluminescence assay. As shown in FIG. 3, expression was higher when RNA was delivered by PLG (triangles) than without any delivery particle (circles).

[0227] Cationic Nanoemulsion

[0228] An oil-in-water emulsion was prepared by microfluidising squalene, span 85, polysorbate 80, and varying amounts of DOTAP. Briefly, oil soluble components (squalene, span 85, cationic lipids, lipid surfactants) were combined in a beaker, lipid components were dissolved in organic solvent. The resulting lipid solution was added directly to the oil phase. The solvent was allowed to evaporate at room temperature for 2 hours in a fume hood prior to combining the aqueous phase and homogenizing the sample to provide a homogeneous feedstock. The primary emulsions were passed three to five times through a Microfluidizer with an ice bath cooling coil. The batch samples were removed from the unit and stored at 4° C.

[0229] This emulsion is thus similar to the commercial MF59 adjuvant, but supplemented by a cationic DOTAP to provide a cationic nanoemulsion (“CNE”). The final composition of emulsion “CNE17” was squalene (4.3% by weight), span 85 (0.5% by weight), polysorbate 80 (0.5% by weight), DOTAP (1.4 mg/ml), in 10 mM citrate buffer, pH 6.5.

[0230] RNA adsorbs to the surface of the oil droplets in these cationic emulsions. To adsorb RNA a RNA solution is diluted to the appropriate concentration in RNase free water and then added directly into an equal volume of emulsion while vortexing lightly. The solution is allowed to sit at room temperature for approximately 2 hours to allow adsorption. The resulting solution is diluted to the required RNA concentration prior to administration.

[0231] Liposomal Encapsulation

[0232] RNA was encapsulated in liposomes made by the method of references 11 and 50. 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.

[0233] 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 (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) were from Avanti Polar Lipids.

[0234] 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.

[0235] 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 gL 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/gL 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 μm ID junction, Idex Health Science) 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 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.

[0236] 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).

[0237] 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.

[0238] 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 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.

[0239] 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 51, 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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).

[0245] 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.

[0246] FIG. 13 confirms that the RNA elicits a robust CD8 T cell response.

[0247] 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-00001 2 weeks 4 weeks 8 weeks 0.1 μg 2.9 1.0 1.1   1 μg 2.3 0.9 0.9

[0248] Thus the liposome-encapsulated RNA induces essentially the same magnitude of immune response as seen with virion delivery.

[0249] 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.

[0250] 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).

[0251] 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.

[0252] Host Defence Responses at Higher RNA Doses

[0253] Mice were used to see if host defence responses (innate or adaptive immunity) might limit the immune response to encoded antigens at higher RNA doses.

[0254] Three different RNAs were used for this study: (i) ‘vA317’ replicon that expresses RSV-F i.e. the surface fusion glycoprotein of RSV; (ii) ‘vA17’ replicon that expresses GFP; and (iii) ‘vA336’ that is replication-defective and encodes GFP.

[0255] RNAs were delivered either naked or with liposomes made with 40% DlinDMA, 10% DSPC, 48% Chol, and 2% PEG-DMG (proportions are % moles of total liposome). These liposomes were prepared in 75 μg batches. 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 gL 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. 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 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. 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 h. 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.

[0256] The four liposome formulations had the following characteristics:

TABLE-US-00002 RNA Particle Size Zav (nm) Polydispersity RNA Encapsulation vA317 155.7 0.113 86.6% vA17 148.4 0.139 .sup. 92% vA336 145.1 0.143 92.9% Empty 147.9 0.147 —

[0257] BALB/c mice, 5 animals per group, were given bilateral intramuscular vaccinations (50 μL per leg) on days 0 and 21 with: [0258] Group 1 naked self-replicating RSV-F RNA (vA317, 0.1 μg) [0259] Group 2 self-replicating RSV-F RNA (vA317, 0.1 μg) encapsulated in liposomes [0260] Group 3 self-replicating RSV-F RNA (vA317, 0.1 μg) added to empty liposomes [0261] Group 4 a mixture of self-replicating RSV-F RNA (vA317, 0.1 μg) and self-replicating GFP RNA (vA17, 10 μg) [0262] Group 5 a mixture of self-replicating RSV-F RNA (vA317, 0.1 μg) and replication-defective GFP RNA (vA336, 10 μg) [0263] Group 6 a mixture of self-replicating RSV-F RNA formulated in liposomes (vA317, 0.1 μg) and self-replicating GFP RNA (vA17, 10 μg) [0264] Group 7 a mixture of self-replicating RSV-F RNA formulated in liposomes (vA317, 0.1 μg) and replication-defective GFP RNA (vA336, 10 μg) [0265] Group 8 a mixture of self-replicating RSV-F RNA formulated in liposomes (vA317, 0.1 μg) and self-replicating GFP RNA formulated in liposomes (vA17, 1 μg) [0266] Group 9 a mixture of self-replicating RSV-F RNA formulated in liposomes (vA317, 0.1 μg) and replication-defective GFP RNA formulated in liposomes (vA336, 1 μg) [0267] Group 10 F subunit protein (5 μg)

[0268] Serum was collected for antibody analysis on days 14, 35 and 51. F-specific specific serum IgG titers (GMT) were measured; if an individual animal had a titer of <25 (limit of detection), it was assigned a titer of 5. In addition, spleens were harvested from mice at day 51 for T cell analysis, to determine cells which were cytokine-positive and specific for RSV F51-66 peptide (CD4+) or for RSV F peptides F85-93 and F249-258 (CD8+).

[0269] IgG titers were as follows in the 10 groups and in non-immunised control mice:

TABLE-US-00003 Day 1 2 3 4 5 6 7 8 9 10 — 14 22 1819 5 5 24 174 1130 44 347 5 5 35 290 32533 9 5 746 6887 13171 773 4364 19877 5 51 463 30511 18 10 1076 7201 14426 922 4697 20853 5

[0270] RSV serum neutralization titers at day 51 were as follows:

TABLE-US-00004 Day 1 2 3 4 5 6 7 8 9 10 51 35 50 24 25 31 31 54 34 24 38

[0271] Animals showing RSV F-specific CD4+ splenic T cells on day 51 were as follows, where a number (% positive cells) is given only if the stimulated response was statistically significantly above zero:

TABLE-US-00005 Cytokine 1 2 3 4 5 6 7 8 9 10 IFN-γ 0.04 0.02 0.02 IL2 0.02 0.06 0.02 0.02 0.02 0.02 IL5 0.01 TNFα 0.03 0.05 0.02 0.02

[0272] Animals showing RSV F-specific CD8+ splenic T cells on day 51 were as follows, where a number is given only if the stimulated response was statistically significantly above zero:

TABLE-US-00006 Cytokine 1 2 3 4 5 6 7 8 9 10 IFN-γ 0.37 0.87 0.37 0.40 0.49 0.06 0.54 IL2 0.11 0.40 0.15 0.18 0.20 0.03 0.23 0.04 IL5 TNFα 0.29 0.79 0.35 0.42 0.40 0.53 0.06

[0273] These results show that host defence responses can limit the immune response to the delivered vector. For instance, groups 2 and 6-9 used the same self-replicating antigen-encoding vector, delivered in liposomes, but groups 6-9 also had a 100-fold or 10-fold excess of GFP-encoding vector, delivered either naked or inside liposomes, and either self-replicating or replication-defective. The extra RNA reduced anti-RSV responses, particularly if it was self-replicating and/or encapsulated.

[0274] Further experiments aimed to see if host responses to RNA might limit protein expression. Thus expression was followed for only 6 days, before an adaptive response (antibodies, T cells) would be apparent. The “vA306” replicon encodes SEAP; the “vA17” replicon encodes GFP; the “vA336” replicon encodes GFP but cannot self-replicate; the “vA336*” replicon is the same as vA336 but was prepared with 10% of uridines replaced with 5-methyluridine; the “vA336**” replicon is the same as va336 but 100% of its uridine residues are MSU. BALB/c mice were given bilateral intramuscular vaccinations (50 μL per leg) on day 0. Animals, 35 total, were divided into 7 groups (5 animals per group) and were immunised as follows: [0275] Group 1 Naïve control. [0276] Group 2 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes. [0277] Group 3 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336, 1.0 μg, GFP) formulated in liposomes. [0278] Group 4 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336*, 1.0 μg, GFP) formulated in liposomes. [0279] Group 5 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with non-replicating RNA (vA336**, 1.0 μg, GFP) formulated in liposomes. [0280] Group 6 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with empty liposomes at the same lipid dose as groups 2-5. [0281] Group 7 were given bilateral intramuscular vaccinations (50 μL per leg) on day 0 with RNA (vA306, 0.1 μg, SEAP) formulated in liposomes mixed with self-replicating RNA (vA17, 1.0 μg, GFP) formulated in liposomes.

[0282] Serum SEAP activity (relative light units) at days 0, 3 and 6 were as follows (GMT):

TABLE-US-00007 Day 1 Day 3 Day 6 1 898 1170 2670 2 1428 4219 28641 3 1702 9250 150472 4 1555 8005 76043 5 1605 8822 91019 6 10005 14640 93909 7 1757 6248 53497

[0283] Replication-competent RNA encoding GFP suppressed the expression of SEAP more than replication-defective GFP RNA, suggesting a strong host defence response against replicating RNA which leads to suppression of SEAP expression. It is possible that interferons induced in response to the GFP RNA suppressed the expression of SEAP. Under the host response/suppression model, blocking host recognition of RNA would be expected to lead to increased SEAP expression, but 5′ methylation of U residues in the GFP RNA was not associated with increased SEAP, suggesting that host recognition of RNA was insensitive to 5′ methylation.

[0284] Delivery Volume

[0285] 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.

[0286] 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.

[0287] 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: [0288] Group 1 received naked replicon, 0.2μg in 50 μL per leg [0289] Group 2 received naked replicon, 0.2 μg in 5 μL per leg [0290] Group 3 received emulsion-formulated replicon (0.2 μg, 50 μL per leg) [0291] Group 4 received emulsion-formulated replicon (0.2 μg, 5 μL per leg) [0292] Group 5 received liposome-formulated replicon (0.2 μg, 50 μL per leg) [0293] Group 6 received liposome-formulated replicon (0.2 μg, 5 μL per leg)

[0294] Serum was collected for antibody analysis on days 14 and 35. F-specific serum IgG GMTs were:

TABLE-US-00008 Day 1 2 3 4 5 6 14 42 21 783 760 2669 2610 35 241 154 2316 2951 17655 18516

[0295] 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.

[0296] Large Mammal Study

[0297] 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 either inside liposomes or with the CNE17 emulsion. 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. One cow was mistakenly vaccinated with the CNE17-based vaccine on day 86 instead of Triangle 4 and so its data were excluded from day 100 onwards.

[0298] 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.

[0299] The liposomes had the same proportion of DlinDMA, DSPC, cholesterol and PEG-DMG as mentioned above. 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 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 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. 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. 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, 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, Mich., 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 filter. Next, liposomes were concentrated to 2 mL and dialyzed against 10-15 volumes of 1× PBS using 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. Polyethersulfone (PES) 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.

[0300] 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.

[0301] 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

[0302] FIG. 14A shows F-specific IgG titers over the first 63 days. The RNA replicon was immunogenic in the cows using both delivery systems, 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). The titers with the liposome delivery system were more tightly clustered than with the emulsion.

[0303] FIG. 14B shows F-specific serum IgG titers (GMT) over 210 days, and measured values up to day 202 were as follows:

TABLE-US-00009 3wpl 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 CNE17 5 5 34 46 56 773 538 70 8297 4843 2073 Triangle 4 5 5 1784 721 514 3406 2786 336 13376 4775 2133

[0304] The emulsion-adjuvanted vaccine induced a neutralising response when assayed without complement, with higher titers than Triangle 4 (although more variable). RSV serum neutralizing antibody titers were as follows:

TABLE-US-00010 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 CNE17 10 10 13 28 44 52 14 64 57 40 Triangle 4 12 15 13 39 38 41 13 24 26 15

[0305] The data from this study provide proof of concept for RNA replicon RSV vaccines in large animals, with two of the five calves in the emulsion-adjuvanted group demonstrating good neutralizing antibody titers after the third vaccination, as measured by the complement-independent HRSV neutralization assay. Although the emulsion-adjuvanted vaccines appear to be more immunogenic than the liposome-adjuvanted vaccines, one complicating factor is that 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 liposome-adjuvanted vaccine would have been more immunogenic if fresh material had been used for all vaccinations.

[0306] 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 regardless of the formulation. Furthermore, both RNA vaccines elicited F-specific serum IgG titers that were detected in a few calves after the second vaccination and in all calves after the third.

[0307] MF59-adjuvanted RSV-F was able to boost the IgG response in all previously vaccinated calves, and to boost complement-independent HRSV neutralization titers of calves previously vaccinated with RNA.

[0308] 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 [52,53] and so it is very encouraging that immune responses were induced with only 66 μg of RNA.

[0309] 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.

TABLE-US-00011 TABLE 1 useful phospholipids DDPC 1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA 1,2-Dierucoyl-sn-Glycero-3-Phosphate DEPC 1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE 1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG 1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLOPC 1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine 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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