U.S. patent application number 13/808080 was filed with the patent office on 2013-07-04 for liposomes with lipids having an advantageous pka-value for rna delivery.
This patent application is currently assigned to NOVARTIS AG. The applicant listed for this patent is Andrew Geall. Invention is credited to Andrew Geall.
Application Number | 20130171241 13/808080 |
Document ID | / |
Family ID | 44534956 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171241 |
Kind Code |
A1 |
Geall; Andrew |
July 4, 2013 |
LIPOSOMES WITH LIPIDS HAVING AN ADVANTAGEOUS PKA-VALUE FOR RNA
DELIVERY
Abstract
RNA encoding an immunogen is delivered in a liposome for the
purposes of immunisation. The liposome includes lipids which have a
pKa in the range of 5.0 to 7.6 and, preferably, a tertiary amine.
These liposomes can have essentially neutral surface charge at
physiological pH and are effective for immunisation.
Inventors: |
Geall; Andrew; (Littleton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Geall; Andrew |
Littleton |
MA |
US |
|
|
Assignee: |
NOVARTIS AG
BASEL
CH
|
Family ID: |
44534956 |
Appl. No.: |
13/808080 |
Filed: |
July 6, 2011 |
PCT Filed: |
July 6, 2011 |
PCT NO: |
PCT/US2011/043105 |
371 Date: |
March 14, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61361830 |
Jul 6, 2010 |
|
|
|
61378837 |
Aug 31, 2010 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/178.1; 514/44R |
Current CPC
Class: |
A61P 31/12 20180101;
A61P 31/14 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61K
39/39 20130101; A61K 39/00 20130101; A61K 2039/53 20130101; A61K
9/1272 20130101; A61K 39/12 20130101; A61K 2039/55555 20130101;
C12N 2760/18534 20130101; A61P 33/00 20180101; C12N 2770/36134
20130101; A61K 9/127 20130101; A61P 31/10 20180101; C12N 15/86
20130101; A61P 31/04 20180101; A61K 31/7088 20130101; C12N
2710/16134 20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/450 ;
514/44.R; 424/178.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/00 20060101 A61K039/00; A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A liposome having a lipid bilayer encapsulating an aqueous core,
wherein: (i) the lipid bilayer comprises a lipid having a pKa in
the range of 5.0 to 7.6; and (ii) the aqueous core includes a RNA
which encodes an immunogen.
2. The liposome of claim 1, wherein the lipid having a pKa in the
range of 5.0 to 7.6 has a tertiary amine.
3. The liposome of claim 1, wherein pKa in the range of 5.0 to 7.6
is between 5.7-5.9.
4. The liposome of claim 1, wherein the lipid having a pKa in the
range of 5.0 to 7.6 has the formula shown herein for RV01, RV02,
RV03, RV04, RV05, RV06, RV07, RV08, RV09, RV11, RV12, RV16 or
RV17.
5. The liposome of claim 1, having a diameter in the range of
20-220 nm.
6. The liposome of claim 1, wherein the RNA molecule encodes (i) a
RNA-dependent RNA polymerase which can transcribe RNA from the RNA
molecule and (ii) an immunogen.
7. The liposome of claim 5, wherein the RNA molecule has two open
reading frames, the first of which encodes an alphavirus replicase
and the second of which encodes the immunogen.
8. The liposome of claim 1, wherein the RNA molecule is 9000-12000
nucleotides long.
9. The liposome of claim 1, wherein the immunogen can elicit an
immune response in vivo against a bacterium, a virus, a fungus or a
parasite.
10. The liposome of claim 1, wherein the immunogen can elicit an
immune response in vivo against respiratory syncytial virus
glycoprotein F.
11. A pharmaceutical composition comprising the liposome of claim
1.
12. A method for raising a protective immune response in a
vertebrate, comprising the step of administering to the vertebrate
an effective amount of the liposome of claim 1.
13. A process for preparing a RNA-containing liposome, comprising
steps during liposome formation of: (a) mixing RNA with a lipid at
a pH which is below the lipid's pKa but is above 4.5; then (b)
increasing the pH to be above the lipid's pKa.
14. The process of claim 13, wherein: RNA used in step (a) is in
aqueous solution, for mixing with an organic solution of the lipid
to give a mixture which is then diluted to form liposomes; and the
pH is increased in step (b) after liposome formation.
Description
[0001] This application claims the benefit of U.S. provisional
applications 61/361,830 (filed Jul. 6, 2010) and 61/378,837 (filed
Aug. 31, 2010), the complete contents of both of which are hereby
incorporated herein by reference for all purposes.
TECHNICAL FIELD
[0002] This invention is in the field of non-viral delivery of RNA
for immunisation.
BACKGROUND ART
[0003] The delivery of nucleic acids for immunising animals has
been a goal for several years. Various approaches have been tested,
including the use of DNA or RNA, of viral or non-viral delivery
vehicles (or even no delivery vehicle, in a "naked" vaccine), of
replicating or non-replicating vectors, or of viral or non-viral
vectors.
[0004] There remains a need for further and improved nucleic acid
vaccines.
DISCLOSURE OF THE INVENTION
[0005] According to the invention, RNA encoding an immunogen is
delivered in a liposome for the purposes of immunisation. The
liposome includes lipids which have a pKa in the range of 5.0 to
7.6. Ideally the lipid with a pKa in this range has a tertiary
amine; such lipids behave differently from lipids such as DOTAP or
DC-Chol, which have a quaternary amine group. At physiological pH
amines with a pKa in the range of 5.0 to 7.6 have neutral or
reduced surface charge, whereas a lipid such as DOTAP is strongly
cationic. The inventors have found that liposomes formed from
quaternary amine lipids (e.g. DOTAP) are less suitable for delivery
of immunogen-encoding RNA than liposomes formed from tertiary amine
lipids (e.g. DLinDMA).
[0006] Thus the invention provides a liposome having a lipid
bilayer encapsulating an aqueous core, wherein: (i) the lipid
bilayer comprises a lipid having a pKa in the range of 5.0 to 7.6,
and preferably having a tertiary amine; and (ii) the aqueous core
includes a RNA which encodes an immunogen. These liposomes are
suitable for in vivo delivery of the RNA to a vertebrate cell and
so they are useful as components in pharmaceutical compositions for
immunising subjects against various diseases.
[0007] The invention also provides a process for preparing a
RNA-containing liposome, comprising steps of: (a) mixing RNA with a
lipid at a pH which is below the lipid's pKa but is above 4.5, to
form a liposome in which the RNA is encapsulated; and (b)
increasing the pH of the resulting liposome-containing mixture to
be above the lipid's pKa.
The Liposome
[0008] The invention utilises liposomes in which immunogen-encoding
RNA is encapsulated. Thus the RNA is (as in a natural virus)
separated from any external medium by the liposome's lipid bilayer,
and encapsulation in this way has been found to protect RNA from
RNase digestion. The liposomes can include some external RNA (e.g.
on their surface), but at least half of the RNA (and ideally all of
it) is encapsulated in the liposome's core. Encapsulation within
liposomes is distinct from, for instance, the lipid/RNA complexes
disclosed in reference 1.
[0009] Various amphiphilic lipids can form bilayers in an aqueous
environment to encapsulate a RNA-containing aqueous core as a
liposome. These lipids can have an anionic, cationic or
zwitterionic hydrophilic head group. Liposomes of the invention
comprise a lipid having a pKa in the range of 5.0 to 7.6, and
preferred lipids with a pKa in this range have a tertiary amine.
For example, they may comprise
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA; pKa 5.8)
and/or 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
Another suitable lipid having a tertiary amine is
1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA). See FIG. 3 &
ref. 2. Some of the amino acid lipids of reference 3 may also be
used, as can certain of the amino lipids of reference 4. Further
useful lipids with tertiary amines in their headgroups are
disclosed in reference 5, the complete contents of which are
incorporated herein by reference.
[0010] Liposomes of the invention can be formed from a single lipid
or from a mixture of lipids, provided that at least one of the
lipids has a pKa in the range of 5.0 to 7.6 (and, preferably, a
tertiary amine). Within this pKa range, preferred lipids have a pKa
of 5.5 to 6.7 e.g. between 5.6 and 6.8, between 5.6 and 6.3,
between 5.6 and 6.0, between 5.5 and 6.2, or between 5.7 and 5.9.
The pKa is the pH at which 50% of the lipids are charged, lying
halfway between the point where the lipids are completely charged
and the point where the lipids are completely uncharged. It can be
measured in various ways, but is preferably measured using the
method disclosed below in the section entitled "pKa measurement".
The pKa typically should be measured for the lipid alone rather
than for the lipid in the context of a mixture which also includes
other lipids (e.g. not as performed in reference 6, which looks at
the pKa of a SNALP rather than of the individual lipids).
[0011] Where a liposome of the invention is formed from a mixture
of lipids, it is preferred that the proportion of those lipids
which have a pKa within the desired range should be between 20-80%
of the total amount of lipids e.g. between 30-70%, or between
40-60%. For instance, useful liposomes are shown below in which 40%
or 60% of the total lipid is a lipid with a pKa in the desired
range. The remainder can be made of e.g. cholesterol (e.g. 35-50%
cholesterol) and/or DMG (optionally PEGylated) and/or DSPC. Such
mixtures are used below. These % values are mole percentages.
[0012] A liposome may include an amphiphilic lipid whose
hydrophilic portion is PEGylated (i.e. modified by covalent
attachment of a polyethylene glycol). This modification can
increase stability and prevent non-specific adsorption of the
liposomes. For instance, lipids can be conjugated to PEG using
techniques such as those disclosed in reference 6 and 7. PEG
provides the liposomes with a coat which can confer favourable
pharmacokinetic characteristics. The combination of efficient
encapsulation of a RNA (particularly a self-replicating RNA), a
cationic lipid having a pKa in the range 5.0-7.6, and a PEGylated
surface, allows for efficient delivery to multiple cell types
(including both immune and non-immune cells), thereby eliciting a
stronger and better immune response than when using quaternary
amines without PEGylation. Various lengths of PEG can be used e.g.
between 0.5-8 kDa.
[0013] Lipids used with the invention can be saturated or
unsaturated. The use of at least one unsaturated lipid for
preparing liposomes is preferred. FIG. 3 shows three useful
unsaturated lipids. If an unsaturated lipid has two tails, both
tails can be unsaturated, or it can have one saturated tail and one
unsaturated tail.
[0014] A mixture of DSPC, DLinDMA, PEG-DMG and cholesterol is used
in the examples. An independent aspect of the invention is a
liposome comprising DSPC, DLinDMA, PEG-DMG & cholesterol. This
liposome preferably encapsulates RNA, such as a self-replicating
RNA e.g. encoding an immunogen.
[0015] Liposomal particles are usually divided into three groups:
multilamellar vesicles (MLV); small unilamellar vesicles (SUV); and
large unilamellar vesicles (LUV). MLVs have multiple bilayers in
each vesicle, forming several separate aqueous compartments. SUVs
and LUVs have a single bilayer encapsulating an aqueous core; SUVs
typically have a diameter.ltoreq.50 nm, and LUVs have a diameter
>50 nm. Liposomal particles of the invention are ideally LUVs
with a diameter in the range of 50-220 nm. For a composition
comprising a population of LUVs with different diameters: (i) at
least 80% by number should have diameters in the range of 20-220
nm, (ii) the average diameter (Zav, by intensity) of the population
is ideally in the range of 40-200 nm, and/or (iii) the diameters
should have a polydispersity index<0.2. The liposome/RNA
complexes of reference 1 are expected to have a diameter in the
range of 600-800 nm and to have a high polydispersity. The liposome
can be substantially spherical.
[0016] Techniques for preparing suitable liposomes are well known
in the art e.g. see references 8 to 10. One useful method is
described in reference 11 and involves mixing (i) an ethanolic
solution of the lipids (ii) an aqueous solution of the nucleic acid
and (iii) buffer, followed by mixing, equilibration, dilution and
purification. Preferred liposomes of the invention are obtainable
by this mixing process.
Mixing Process
[0017] As mentioned above, the invention provides a process for
preparing a RNA-containing liposome, comprising steps of: (a)
mixing RNA with a lipid at a pH which is below the lipid's pKa but
is above 4.5; then (b) increasing the pH to be above the lipid's
pKa.
[0018] Thus a cationic lipid is positively charged during liposome
formation in step (a), but the pH change thereafter means that the
majority (or all) of the positively charged groups become neutral.
This process is advantageous for preparing liposomes of the
invention, and by avoiding a pH below 4.5 during step (a) the
stability of the encapsulated RNA is improved.
[0019] The pH in step (a) is above 4.5, and is ideally above 4.8.
Using a pH in the range of 5.0 to 6.0, or in the range of 5.0 to
5.5, can provide suitable liposomes.
[0020] The increased pH in step (b) is above the lipid's pKa. The
pH is ideally increased to a pH less than 9, and preferably less
than 8. Depending on the lipid's pKa, the pH in step (b) may thus
be increased to be within the range of 6 to 8 e.g. to pH
6.5.+-.0.3. The pH increase of step (b) can be achieved by
transferring the liposomes into a suitable buffer e.g. into
phosphate-buffered saline. The pH increase of step (b) is ideally
performed after liposome formation has taken place.
[0021] RNA used in step (a) can be in aqueous solution, for mixing
with an organic solution of the lipid (e.g. an ethanolic solution,
as in ref. 11). The mixture can then be diluted to form liposomes,
after which the pH can be increased in step (b).
The RNA
[0022] The invention is useful for in vivo delivery of RNA which
encodes an immunogen. The RNA is translated by non-immune cells at
the delivery site, leading to expression of the immunogen, and it
also causes immune cells to secrete type I interferons and/or
pro-inflammatory cytokines which provide a local adjuvant effect.
The non-immune cells may also secrete type I interferons and/or
pro-inflammatory cytokines in response to the RNA.
[0023] The RNA is +-stranded, and so it can be translated by the
non-immune cells without needing any intervening replication steps
such as reverse transcription. It can also bind to TLR7 receptors
expressed by immune cells, thereby initiating an adjuvant
effect.
[0024] Preferred +-stranded RNAs are self-replicating. A
self-replicating RNA molecule (replicon) can, when delivered to a
vertebrate cell even without any proteins, lead to the production
of multiple daughter RNAs by transcription from itself (via an
antisense copy which it generates from itself). A self-replicating
RNA molecule is thus typically a +-strand molecule which can be
directly translated after delivery to a cell, and this translation
provides a RNA-dependent RNA polymerase which then produces both
antisense and sense transcripts from the delivered RNA. Thus the
delivered RNA leads to the production of multiple daughter RNAs.
These daughter RNAs, as well as collinear subgenomic transcripts,
may be translated themselves to provide in situ expression of an
encoded immunogen, or may be transcribed to provide further
transcripts with the same sense as the delivered RNA which are
translated to provide in situ expression of the immunogen. The
overall results of this sequence of transcriptions is a huge
amplification in the number of the introduced replicon RNAs and so
the encoded immunogen becomes a major polypeptide product of the
cells.
[0025] As shown below, a self-replicating activity is not required
for a RNA to provide an adjuvant effect, although it can enhance
post-transfection secretion of cytokines. The self-replicating
activity is particularly useful for achieving high level expression
of the immunogen by non-immune cells. It can also enhance apoptosis
of the non-immune cells.
[0026] One suitable system for achieving self-replication is to use
an alphavirus-based RNA replicon. These +-stranded replicons are
translated after delivery to a cell to give of a replicase (or
replicase-transcriptase). The replicase is translated as a
polyprotein which auto-cleaves to provide a replication complex
which creates genomic --strand copies of the +-strand delivered
RNA. These --strand transcripts can themselves be transcribed to
give further copies of the +-stranded parent RNA and also to give a
subgenomic transcript which encodes the immunogen. Translation of
the subgenomic transcript thus leads to in situ expression of the
immunogen by the infected cell. Suitable alphavirus replicons can
use a replicase from a sindbis virus, a semliki forest virus, an
eastern equine encephalitis virus, a venezuelan equine encephalitis
virus, etc. Mutant or wild-type virus sequences can be used e.g.
the attenuated TC83 mutant of VEEV has been used in replicons
[12].
[0027] A preferred self-replicating RNA molecule thus encodes (i) a
RNA-dependent RNA polymerase which can transcribe RNA from the
self-replicating RNA molecule and (ii) an immunogen. The polymerase
can be an alphavirus replicase e.g. comprising one or more of
alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
[0028] Whereas natural alphavirus genomes encode structural virion
proteins in addition to the non-structural replicase polyprotein,
it is preferred that a self-replicating RNA molecule of the
invention does not encode alphavirus structural proteins. Thus a
preferred self-replicating RNA can lead to the production of
genomic RNA copies of itself in a cell, but not to the production
of RNA-containing virions. The inability to produce these virions
means that, unlike a wild-type alphavirus, the self-replicating RNA
molecule cannot perpetuate itself in infectious form. The
alphavirus structural proteins which are necessary for perpetuation
in wild-type viruses are absent from self-replicating RNAs of the
invention and their place is taken by gene(s) encoding the
immunogen of interest, such that the subgenomic transcript encodes
the immunogen rather than the structural alphavirus virion
proteins.
[0029] Thus a self-replicating RNA molecule useful with the
invention may have two open reading frames. The first (5') open
reading frame encodes a replicase; the second (3') open reading
frame encodes an immunogen. In some embodiments the RNA may have
additional (e.g. downstream) open reading frames e.g. to encode
further immunogens (see below) or to encode accessory
polypeptides.
[0030] A self-replicating RNA molecule can have a 5' sequence which
is compatible with the encoded replicase.
[0031] Self-replicating RNA molecules can have various lengths but
they are typically 5000-25000 nucleotides long e.g. 8000-15000
nucleotides, or 9000-12000 nucleotides. Thus the RNA is longer than
seen in siRNA delivery.
[0032] A RNA molecule useful with the invention may have a 5' cap
(e.g. a 7-methylguanosine). This cap can enhance in vivo
translation of the RNA.
[0033] The 5' nucleotide of a RNA molecule useful with the
invention may have a 5' triphosphate group. In a capped RNA this
may be linked to a 7-methylguanosine via a 5'-to-5' bridge. A 5'
triphosphate can enhance RIG-I binding and thus promote adjuvant
effects.
[0034] A RNA molecule may have a 3' poly-A tail. It may also
include a poly-A polymerase recognition sequence (e.g. AAUAAA) near
its 3' end.
[0035] A RNA molecule useful with the invention will typically be
single-stranded. Single-stranded RNAs can generally initiate an
adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKR.
RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and
this receptor can also be triggered by dsRNA which is formed either
during replication of a single-stranded RNA or within the secondary
structure of a single-stranded RNA.
[0036] A RNA molecule useful with the invention can conveniently be
prepared by in vitro transcription (IVT). IVT can use a (cDNA)
template created and propagated in plasmid form in bacteria, or
created synthetically (for example by gene synthesis and/or
polymerase chain-reaction (PCR) engineering methods). For instance,
a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or
SP6 RNA polymerases) can be used to transcribe the RNA from a DNA
template. Appropriate capping and poly-A addition reactions can be
used as required (although the replicon's poly-A is usually encoded
within the DNA template). These RNA polymerases can have stringent
requirements for the transcribed 5' nucleotide(s) and in some
embodiments these requirements must be matched with the
requirements of the encoded replicase, to ensure that the
IVT-transcribed RNA can function efficiently as a substrate for its
self-encoded replicase.
[0037] As discussed in reference 13, the self-replicating RNA can
include (in addition to any 5' cap structure) one or more
nucleotides having a modified nucleobase. Thus the RNA can comprise
m5C (5-methylcytidine), m5U (5-methyluridine), m6A
(N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine),
m1A (1-methyladenosine); m2A (2-methyladenosine); Am
(2'-O-methyladenosine); ms2 m6A (2-methylthio-N6-methyladenosine);
i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6
isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine);
ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A
(N6-glycinylcarbamoyladenosine); t6A (N6-threonyl
carbamoyladenosine); ms2t6A (2-methylthio-N6-threonyl
carbamoyladenosine); m6t6A
(N6-methyl-N6-threonylcarbamoyladenosine); hn6A
(N6.-hydroxynorvalylcarbamoyl adenosine); ms2hn6A
(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)
(2'-O-ribosyladenosine (phosphate)); I (inosine); m11
(1-methylinosine); m'Im (1,2'-O-dimethylinosine); m3C
(3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine);
ac4C(N4-acetylcytidine); f5C (5-formylcytidine); m5 Cm
(5,2-O-dimethylcytidine); ac4Cm (N4-acetyl2TOmethylcytidine); k2C
(lysidine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G
(7-methylguanosine); Gm (2'-O-methylguanosine); m22G
(N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm
(N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine
(phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW
(hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG
(wyosine); mimG (methylguanosine); Q (queuosine); oQ
(epoxyqueuosine); galQ (galtactosyl-queuosine); manQ
(mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi
(7-aminomethyl-7-deazaguanosine); G (archaeosine); D
(dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U
(4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um
(2-thio-2'-O-methyluridine); acp3U
(3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U
(5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U
(uridine 5-oxyacetic acid methyl ester); chm5U
(5-(carboxyhydroxymethyl)uridine)); mchm5U
(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U
(5-methoxycarbonyl methyluridine); mcm5Um
(S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U
(5-methoxycarbonylmethyl-2-thiouridine); nm5s2U
(5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine);
mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U
(5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl
uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U
(5-carboxymethylaminomethyluridine); cnmm5Um
(5-carboxymethylaminomethyl-2-L-Omethyluridine); cmnm5s2U
(5-carboxymethylaminomethyl-2-thiouridine); m62A
(N6,N6-dimethyladenosine); Tm (2'-O-methylinosine);
m4C(N4-methylcytidine); m4 Cm (N4,2-O-dimethylcytidine); hm5C
(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U
(5-carboxymethyluridine); m6 Am (N6,T-O-dimethyladenosine); rn62 Am
(N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine);
m2'2'7G (N2,N2,7-trimethylguanosine); m3Um
(3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5 Cm
(5-formyl-2'-.beta.-methylcytidine); m1Gm
(1,2'-O-dimethylguanosine); m'Am (1,2-O-dimethyl adenosine)
irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine));
imG-14 (4-demethyl guanosine); imG2 (isoguanosine); or ac6A
(N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine,
7-substituted derivatives thereof, dihydrouracil, pseudouracil,
2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil,
5-methyluracil, 5-(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil,
5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil,
5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine,
5-methylcytosine, 5-(C2-C6)-alkenylcytosine,
5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,
5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine,
7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine,
7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine,
8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine,
2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted
7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted
purine, or an abasic nucleotide. For instance, a self-replicating
RNA can include one or more modified pyrimidine nucleobases, such
as pseudouridine and/or 5-methylcytosine residues. In some
embodiments, however, the RNA includes no modified nucleobases, and
may include no modified nucleotides i.e. all of the nucleotides in
the RNA are standard A, C, G and U ribonucleotides (except for any
5' cap structure, which may include a 7'-methylguanosine). In other
embodiments, the RNA may include a 5' cap comprising a
7'-methylguanosine, and the first 1, 2 or 3 5' ribonucleotides may
be methylated at the 2' position of the ribose.
[0038] A RNA used with the invention ideally includes only
phosphodiester linkages between nucleosides, but in some
embodiments it can contain phosphoramidate, phosphorothioate,
and/or methylphosphonate linkages.
[0039] Ideally, a liposome includes fewer than 10 different species
of RNA e.g. 5, 4, 3, or 2 different species; most preferably, a
liposome includes a single RNA species i.e. all RNA molecules in
the liposome have the same sequence and same length.
[0040] The amount of RNA per liposome can vary. The number of
individual self-replicating RNA molecules per liposome is typically
.ltoreq.50 e.g. <20, <10, <5, or 1-4 per liposome.
The Immunogen
[0041] RNA molecules used with the invention encode a polypeptide
immunogen. After administration of the liposomes the RNA is
translated in vivo and the immunogen can elicit an immune response
in the recipient. The immunogen may elicit an immune response
against a bacterium, a virus, a fungus or a parasite (or, in some
embodiments, against an allergen; and in other embodiments, against
a tumor antigen). The immune response may comprise an antibody
response (usually including IgG) and/or a cell-mediated immune
response. The polypeptide immunogen will typically elicit an immune
response which recognises the corresponding bacterial, viral,
fungal or parasite (or allergen or tumour) polypeptide, but in some
embodiments the polypeptide may act as a mimotope to elicit an
immune response which recognises a bacterial, viral, fungal or
parasite saccharide. The immunogen will typically be a surface
polypeptide e.g. an adhesin, a hemagglutinin, an envelope
glycoprotein, a spike glycoprotein, etc.
[0042] Self-replicating RNA molecules can encode a single
polypeptide immunogen or multiple polypeptides. Multiple immunogens
can be presented as a single polypeptide immunogen (fusion
polypeptide) or as separate polypeptides. If immunogens are
expressed as separate polypeptides then one or more of these may be
provided with an upstream IRES or an additional viral promoter
element. Alternatively, multiple immunogens may be expressed from a
polyprotein that encodes individual immunogens fused to a short
autocatalytic protease (e.g. foot-and-mouth disease virus 2A
protein), or as inteins.
[0043] Unlike references 1 and 14, the RNA encodes an immunogen.
For the avoidance of doubt, the invention does not encompass RNA
which encodes a firefly luciferase or which encodes a fusion
protein of E. coli .beta.-galactosidase or which encodes a green
fluorescent protein (GFP). Also, the RNA is not total mouse thymus
RNA.
[0044] In some embodiments the immunogen elicits an immune response
against one of these bacteria: [0045] Neisseria meningitidis:
useful immunogens include, but are not limited to, membrane
proteins such as adhesins, autotransporters, toxins, iron
acquisition proteins, and factor H binding protein. A combination
of three useful polypeptides is disclosed in reference 15. [0046]
Streptococcus pneumoniae: useful polypeptide immunogens are
disclosed in reference 16. These include, but are not limited to,
the RrgB pilus subunit, the beta-N-acetyl-hexosaminidase precursor
(spr0057), spr0096, General stress protein GSP-781 (spr2021,
SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal
surface adhesin PsaA. [0047] Streptococcus pyogenes: useful
immunogens include, but are not limited to, the polypeptides
disclosed in references 17 and 18. [0048] Moraxella catarrhalis.
[0049] Bordetella pertussis: Useful pertussis immunogens include,
but are not limited to, pertussis toxin or toxoid (PT), filamentous
haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3. [0050]
Staphylococcus aureus: Useful immunogens include, but are not
limited to, the polypeptides disclosed in reference 19, such as a
hemolysin, esxA, esxB, ferrichrome-binding protein (sta006) and/or
the sta011 lipoprotein. [0051] Clostridium tetani: the typical
immunogen is tetanus toxoid. [0052] Cornynebacterium diphtheriae:
the typical immunogen is diphtheria toxoid. [0053] Haemophilus
influenzae: Useful immunogens include, but are not limited to, the
polypeptides disclosed in references 20 and 21. [0054] Pseudomonas
aeruginosa [0055] Streptococcus agalactiae: useful immunogens
include, but are not limited to, the polypeptides disclosed in
reference 17. [0056] Chlamydia trachomatis: Useful immunogens
include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398,
OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as
disclosed in reference 22. LcrE [23] and HtrA [24] are two
preferred immunogens. [0057] Chlamydia pneumoniae: Useful
immunogens include, but are not limited to, the polypeptides
disclosed in reference 25. [0058] Helicobacter pylori: Useful
immunogens include, but are not limited to, CagA, VacA, NAP, and/or
urease [26]. [0059] Escherichia coli: Useful immunogens include,
but are not limited to, immunogens derived from enterotoxigenic E.
coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering
E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal
pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC).
ExPEC strains include uropathogenic E. coli (UPEC) and
meningitis/sepsis-associated E. coli (MNEC). Useful UPEC
polypeptide immunogens are disclosed in references 27 and 28.
Useful MNEC immunogens are disclosed in reference 29. A useful
immunogen for several E. coli types is AcfD [30]. [0060] Bacillus
anthracia [0061] Yersinia pestis: Useful immunogens include, but
are not limited to, those disclosed in references 31 and 32. [0062]
Staphylococcus epidermis [0063] Clostridium perfringens or
Clostridium botulinums [0064] Legionella pneumophila [0065]
Coxiella burnetii [0066] Brucella, such as B. abortus, B. canis, B.
melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae. [0067]
Francisella, such as F. novicida, F. philomiragia, F. tularensis.
[0068] Neisseria gonorrhoeae [0069] Treponema pallidum [0070]
Haemophilus ducreyi [0071] Enterococcus faecalis or Enterococcus
faecium [0072] Staphylococcus saprophyticus [0073] Yersinia
enterocolitica [0074] Mycobacterium tuberculosis [0075] Rickettsia
[0076] Listeria monocytogenes [0077] Vibrio cholerae [0078]
Salmonella typhil [0079] Borrelia burgdorferi [0080] Porphyromonas
gingivalis [0081] Klebsiella
[0082] In some embodiments the immunogen elicits an immune response
against one of these viruses: [0083] Orthomyxovirus: Useful
immunogens can be from an influenza A, B or C virus, such as the
hemagglutinin, neuraminidase or matrix M2 proteins. Where the
immunogen is an influenza A virus hemagglutinin it may be from any
subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,
H13, H14, H15 or H16. [0084] Paramyxoviridae viruses: Viral
immunogens include, but are not limited to, those derived from
Pneumoviruses (e.g. respiratory syncytial virus, RSV),
Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g.
parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g.
measles). [0085] Poxyiridae: Viral immunogens include, but are not
limited to, those derived from Orthopoxvirus such as Variola vera,
including but not limited to, Variola major and Variola minor.
[0086] Picornavirus: Viral immunogens include, but are not limited
to, those derived from Picornaviruses, such as Enteroviruses,
Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In one
embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2
and/or type 3 poliovirus. In another embodiment, the enterovirus is
an EV71 enterovirus. In another embodiment, the enterovirus is a
coxsackie A or B virus. [0087] Bunyavirus: Viral immunogens
include, but are not limited to, those derived from an
Orthobunyavirus, such as California encephalitis virus, a
Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such
as Crimean-Congo hemorrhagic fever virus. [0088] Heparnavirus:
Viral immunogens include, but are not limited to, those derived
from a Heparnavirus, such as hepatitis A virus (HAV). [0089]
Filovirus: Viral immunogens include, but are not limited to, those
derived from a Filovirus, such as an Ebola virus (including a
Zaire, Ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus.
[0090] Togavirus: Viral immunogens include, but are not limited to,
those derived from a Togavirus, such as a Rubivirus, an Alphavirus,
or an Arterivirus. This includes rubella virus. [0091] Flavivirus:
Viral immunogens include, but are not limited to, those derived
from a Flavivirus, such as Tick-borne encephalitis (TBE) virus,
Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese
encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis
virus, St. Louis encephalitis virus, Russian spring-summer
encephalitis virus, Powassan encephalitis virus. [0092] Pestivirus:
Viral immunogens include, but are not limited to, those derived
from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical
swine fever (CSFV) or Border disease (BDV). [0093] Hepadnavirus:
Viral immunogens include, but are not limited to, those derived
from a Hepadnavirus, such as Hepatitis B virus. A composition can
include hepatitis B virus surface antigen (HBsAg). [0094] Other
hepatitis viruses: A composition can include an immunogen from a
hepatitis C virus, delta hepatitis virus, hepatitis E virus, or
hepatitis G virus. [0095] Rhabdovirus: Viral immunogens include,
but are not limited to, those derived from a Rhabdovirus, such as a
Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV). [0096]
Caliciviridae: Viral immunogens include, but are not limited to,
those derived from Calciviridae, such as Norwalk virus (Norovirus),
and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain
Virus. [0097] Coronavirus: Viral immunogens include, but are not
limited to, those derived from a SARS coronavirus, avian infectious
bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine
transmissible gastroenteritis virus (TGEV). The coronavirus
immunogen may be a spike polypeptide. [0098] Retrovirus: Viral
immunogens include, but are not limited to, those derived from an
Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus.
[0099] Reovirus: Viral immunogens include, but are not limited to,
those derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or
a Coltivirus. [0100] Parvovirus: Viral immunogens include, but are
not limited to, those derived from Parvovirus B19. [0101]
Herpesvirus: Viral immunogens include, but are not limited to,
those derived from a human herpesvirus, such as, by way of example
only, Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2),
Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),
Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human
Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8). [0102]
Papovaviruses: Viral immunogens include, but are not limited to,
those derived from Papillomaviruses and Polyomaviruses. The (human)
papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18,
31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one or
more of serotypes 6, 11, 16 and/or 18. [0103] Adenovirus: Viral
immunogens include those derived from adenovirus serotype 36
(Ad-36).
[0104] In some embodiments, the immunogen elicits an immune
response against a virus which infects fish, such as: infectious
salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV),
infectious pancreatic necrosis virus (IPNV), channel catfish virus
(CCV), fish lymphocystis disease virus (FLDV), infectious
hematopoietic necrosis virus (1HNV), koi herpesvirus, salmon
picorna-like virus (also known as picorna-like virus of atlantic
salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus
(ASR), trout strawberry disease virus (TSD), coho salmon tumor
virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).
[0105] Fungal immunogens may be derived from Dermatophytres,
including: Epidermophyton floccusum, Microsporum audouini,
Microsporum canis, Microsporum distortum, Microsporum equinum,
Microsporum gypsum, Microsporum nanum, Trichophyton concentricum,
Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum,
Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton
quinckeanum, Trichophyton rubrum, Trichophyton schoenleini,
Trichophyton tonsurans, Trichophyton verrucosum, T verrucosum var.
album, var. discoides, var. ochraceum, Trichophyton violaceum,
and/or Trichophyton faviforme; or from Aspergillus fumigatus,
Aspergillus flavus, Aspergillus niger, Aspergillus nidulans,
Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus,
Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans,
Candida enolase, Candida tropicalis, Candida glabrata, Candida
krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei,
Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,
Candida guilliermondi, Cladosporium carrionii, Coccidioides
immitis, Blastomyces dermatidis, Cryptococcus neoformans,
Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae,
Microsporidia, Encephalitozoon spp., Septata intestinalis and
Enterocytozoon bieneusi; the less common are Brachiola spp,
Microsporidium spp., Nosema spp., Pleistophora spp.,
Trachipleistophora spp., Vittaforma spp Paracoccidioides
brasiliensis, Pneumocystis carinii, Pythiumn insidiosum,
Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces
boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix
schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium
marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp.,
Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus
spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp,
Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium
spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp,
Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium
spp.
[0106] In some embodiments the immunogen elicits an immune response
against a parasite from the Plasmodium genus, such as P.
falciparum, P. vivax, P. malariae or P. ovale. Thus the invention
may be used for immunising against malaria. In some embodiments the
immunogen elicits an immune response against a parasite from the
Caligidae family, particularly those from the Lepeophtheirus and
Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or
Caligus rogercresseyi.
[0107] In some embodiments the immunogen elicits an immune response
against: pollen allergens (tree-, herb, weed-, and grass pollen
allergens); insect or arachnid allergens (inhalant, saliva and
venom allergens, e.g. mite allergens, cockroach and midges
allergens, hymenopthera venom allergens); animal hair and dandruff
allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food
allergens (e.g. a gliadin). Important pollen allergens from trees,
grasses and herbs are such originating from the taxonomic orders of
Fagales, Oleales, Pinales and platanaceae including, but not
limited to, birch (Betula), alder (Alnus), hazel (Corylus),
hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and
Juniperus), plane tree (Platanus), the order of Poales including
grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis,
Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and
Urticales including herbs of the genera Ambrosia, Artemisia, and
Parietaria. Other important inhalation allergens are those from
house dust mites of the genus Dermatophagoides and Euroglyphus,
storage mite e.g. Lepidoglyphys, Glycyphagus and Tyrophagus, those
from cockroaches, midges and fleas e.g. Blatella, Periplaneta,
Chironomus and Ctenocepphalides, and those from mammals such as
cat, dog and horse, venom allergens including such originating from
stinging or biting insects such as those from the taxonomic order
of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants
(Formicoidae).
[0108] In some embodiments the immunogen is a tumor antigen
selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2,
SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for
example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5,
MAGE-6, and MAGE-12 (which can be used, for example, to address
melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and
bladder tumors; (b) mutated antigens, for example, p53 (associated
with various solid tumors, e.g., colorectal, lung, head and neck
cancer), p21/Ras (associated with, e.g., melanoma, pancreatic
cancer and colorectal cancer), CDK4 (associated with, e.g.,
melanoma), MUM1 (associated with, e.g., melanoma), caspase-8
(associated with, e.g., head and neck cancer), CIA 0205 (associated
with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated
with, e.g., melanoma), TCR (associated with, e.g., T-cell
non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic
myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27,
and LDLR-FUT; (c) over-expressed antigens, for example, Galectin 4
(associated with, e.g., colorectal cancer), Galectin 9 (associated
with, e.g., Hodgkin's disease), proteinase 3 (associated with,
e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g.,
various leukemias), carbonic anhydrase (associated with, e.g.,
renal cancer), aldolase A (associated with, e.g., lung cancer),
PRAME (associated with, e.g., melanoma), HER-2/neu (associated
with, e.g., breast, colon, lung and ovarian cancer), mammaglobin,
alpha-fetoprotein (associated with, e.g., hepatoma), KSA
(associated with, e.g., colorectal cancer), gastrin (associated
with, e.g., pancreatic and gastric cancer), telomerase catalytic
protein, MUC-1 (associated with, e.g., breast and ovarian cancer),
G-250 (associated with, e.g., renal cell carcinoma), p53
(associated with, e.g., breast, colon cancer), and carcinoembryonic
antigen (associated with, e.g., breast cancer, lung cancer, and
cancers of the gastrointestinal tract such as colorectal cancer);
(d) shared antigens, for example, melanoma-melanocyte
differentiation antigens such as MART-1/Melan A, gp100, MC.sup.1R,
melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase
related protein-1/TRP1 and tyrosinase related protein-2/TRP2
(associated with, e.g., melanoma); (e) prostate associated antigens
such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with
e.g., prostate cancer; (f) immunoglobulin idiotypes (associated
with myeloma and B cell lymphomas, for example). In certain
embodiments, tumor immunogens include, but are not limited to, p15,
Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr
virus antigens, EBNA, human papillomavirus (HPV) antigens,
including E6 and E7, hepatitis B and C virus antigens, human T-cell
lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met,
mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16,
TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA
125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43,
CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50,
MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2
binding protein/cyclophilin C-associated protein), TAAL6, TAG72,
TLP, TPS, and the like.
Pharmaceutical Compositions
[0109] Liposomes of the invention are useful as components in
pharmaceutical compositions for immunising subjects against various
diseases. These compositions will typically include a
pharmaceutically acceptable carrier in addition to the liposomes. A
thorough discussion of pharmaceutically acceptable carriers is
available in reference 33.
[0110] A pharmaceutical composition of the invention may include
one or more small molecule immunopotentiators. For example, the
composition may include a TLR2 agonist (e.g. Pam3CSK4), a TLR4
agonist (e.g. an aminoalkyl glucosaminide phosphate, such as
E6020), a TLR7 agonist (e.g. imiquimod), a TLR8 agonist (e.g.
resiquimod) and/or a TLR9 agonist (e.g. IC31). Any such agonist
ideally has a molecular weight of <2000Da. Where a RNA is
encapsulated, in some embodiments such agonist(s) are also
encapsulated with the RNA, but in other embodiments they are
unencapsulated. Where a RNA is adsorbed to a particle, in some
embodiments such agonist(s) are also adsorbed with the RNA, but in
other embodiments they are unadsorbed.
[0111] Pharmaceutical compositions of the invention may include the
liposomes in plain water (e.g. w.f.i.) or in a buffer e.g. a
phosphate buffer, a Tris buffer, a borate buffer, a succinate
buffer, a histidine buffer, or a citrate buffer. Buffer salts will
typically be included in the 5-20 mM range.
[0112] Pharmaceutical compositions of the invention may have a pH
between 5.0 and 9.5 e.g. between 6.0 and 8.0.
[0113] Compositions of the invention may include sodium salts (e.g.
sodium chloride) to give tonicity. A concentration of 10.+-.2 mg/ml
NaCl is typical e.g. about 9 mg/ml.
[0114] Compositions of the invention may include metal ion
chelators. These can prolong RNA stability by removing ions which
can accelerate phosphodiester hydrolysis. Thus a composition may
include one or more of EDTA, EGTA, BAPTA, pentetic acid, etc. Such
chelators are typically present at between 10-50004 e.g. 0.1 mM. A
citrate salt, such as sodium citrate, can also act as a chelator,
while advantageously also providing buffering activity.
[0115] Pharmaceutical compositions of the invention may have an
osmolality of between 200 mOsm/kg and 400 mOsm/kg, e.g. between
240-360 mOsm/kg, or between 290-310 mOsm/kg.
[0116] Pharmaceutical compositions of the invention may include one
or more preservatives, such as thiomersal or 2-phenoxyethanol.
Mercury-free compositions are preferred, and preservative-free
vaccines can be prepared.
[0117] Pharmaceutical compositions of the invention are preferably
sterile.
[0118] Pharmaceutical compositions of the invention are preferably
non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard
measure) per dose, and preferably <0.1 EU per dose.
[0119] Pharmaceutical compositions of the invention are preferably
gluten free.
[0120] Pharmaceutical compositions of the invention may be prepared
in unit dose form. In some embodiments a unit dose may have a
volume of between 0.1-1.0 ml e.g. about 0.5 m1.
[0121] The compositions may be prepared as injectables, either as
solutions or suspensions. The composition may be prepared for
pulmonary administration e.g. by an inhaler, using a fine spray.
The composition may be prepared for nasal, aural or ocular
administration e.g. as spray or drops. Injectables for
intramuscular administration are typical.
[0122] Compositions comprise an immunologically effective amount of
liposomes, as well as any other components, as needed. By
`immunologically effective amount`, it is meant that the
administration of that amount to an individual, either in a single
dose or as part of a series, is effective for treatment or
prevention. This amount varies depending upon the health and
physical condition of the individual to be treated, age, the
taxonomic group of individual to be treated (e.g. non-human
primate, primate, etc.), the capacity of the individual's immune
system to synthesise antibodies, the degree of protection desired,
the formulation of the vaccine, the treating doctor's assessment of
the medical situation, and other relevant factors. It is expected
that the amount will fall in a relatively broad range that can be
determined through routine trials. The liposome and RNA content of
compositions of the invention will generally be expressed in terms
of the amount of RNA per dose. A preferred dose has .ltoreq.100
.mu.g RNA (e.g. from 10-100 .mu.g, such as about 10 .mu.g, 25
.mu.g, 50 .mu.g, 75 .mu.g or 100 .mu.g), but expression can be seen
at much lower levels e.g. .ltoreq.1 .mu.g/dose, .ltoreq.100
ng/dose, .ltoreq.10 ng/dose, .ltoreq.1 ng/dose, etc
[0123] The invention also provides a delivery device (e.g. syringe,
nebuliser, sprayer, inhaler, dermal patch, etc.) containing a
pharmaceutical composition of the invention. This device can be
used to administer the composition to a vertebrate subject.
[0124] Liposomes of the invention do not include ribosomes.
Methods of Treatment and Medical Uses
[0125] In contrast to the particles disclosed in reference 14,
liposomes and pharmaceutical compositions of the invention are for
in vivo use for eliciting an immune response against an immunogen
of interest.
[0126] The invention provides a method for raising an immune
response in a vertebrate comprising the step of administering an
effective amount of a liposome or pharmaceutical composition of the
invention. The immune response is preferably protective and
preferably involves antibodies and/or cell-mediated immunity. The
method may raise a booster response.
[0127] The invention also provides a liposome or pharmaceutical
composition of the invention for use in a method for raising an
immune response in a vertebrate.
[0128] The invention also provides the use of a liposome of the
invention in the manufacture of a medicament for raising an immune
response in a vertebrate.
[0129] By raising an immune response in the vertebrate by these
uses and methods, the vertebrate can be protected against various
diseases and/or infections e.g. against bacterial and/or viral
diseases as discussed above. The liposomes and compositions are
immunogenic, and are more preferably vaccine compositions. Vaccines
according to the invention may either be prophylactic (i.e. to
prevent infection) or therapeutic (i.e. to treat infection), but
will typically be prophylactic.
[0130] The vertebrate is preferably a mammal, such as a human or a
large veterinary mammal (e.g. horses, cattle, deer, goats, pigs).
Where the vaccine is for prophylactic use, the human is preferably
a child (e.g. a toddler or infant) or a teenager; where the vaccine
is for therapeutic use, the human is preferably a teenager or an
adult. A vaccine intended for children may also be administered to
adults e.g. to assess safety, dosage, immunogenicity, etc.
[0131] Vaccines prepared according to the invention may be used to
treat both children and adults. Thus a human patient may be less
than 1 year old, less than 5 years old, 1-5 years old, 5-15 years
old, 15-55 years old, or at least 55 years old. Preferred patients
for receiving the vaccines are the elderly (e.g. .gtoreq.50 years
old, .gtoreq.60 years old, and preferably .gtoreq.65 years), the
young (e.g. .ltoreq.5 years old), hospitalized patients, healthcare
workers, armed service and military personnel, pregnant women, the
chronically ill, or immunodeficient patients. The vaccines are not
suitable solely for these groups, however, and may be used more
generally in a population.
[0132] Compositions of the invention will generally be administered
directly to a patient. Direct delivery may be accomplished by
parenteral injection (e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, intradermally, or to the
interstitial space of a tissue; unlike reference 1, intraglossal
injection is not typically used with the present invention).
Alternative delivery routes include rectal, oral (e.g. tablet,
spray), buccal, sublingual, vaginal, topical, transdermal or
transcutaneous, intranasal, ocular, aural, pulmonary or other
mucosal administration. Intradermal and intramuscular
administration are two preferred routes. Injection may be via a
needle (e.g. a hypodermic needle), but needle-free injection may
alternatively be used. A typical intramuscular dose is 0.5 ml.
[0133] The invention may be used to elicit systemic and/or mucosal
immunity, preferably to elicit an enhanced systemic and/or mucosal
immunity.
[0134] Dosage can be by a single dose schedule or a multiple dose
schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc. Multiple doses will
typically be administered at least 1 week apart (e.g. about 2
weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks,
about 10 weeks, about 12 weeks, about 16 weeks, etc.). In one
embodiment, multiple doses may be administered approximately 6
weeks, 10 weeks and 14 weeks after birth, e.g. at an age of 6
weeks, 10 weeks and 14 weeks, as often used in the World Health
Organisation's Expanded Program on Immunisation ("EPI"). In an
alternative embodiment, two primary doses are administered about
two months apart, e.g. about 7, 8 or 9 weeks apart, followed by one
or more booster doses about 6 months to 1 year after the second
primary dose, e.g. about 6, 8, 10 or 12 months after the second
primary dose. In a further embodiment, three primary doses are
administered about two months apart, e.g. about 7, 8 or 9 weeks
apart, followed by one or more booster doses about 6 months to 1
year after the third primary dose, e.g. about 6, 8, 10, or 12
months after the third primary dose.
General
[0135] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., references 34-40, etc.
[0136] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0137] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0138] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0139] References to charge, to cations, to anions, to zwitterions,
etc., are taken at pH 7.
[0140] TLR3 is the Toll-like receptor 3. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR3 agonists include poly(I:C). "TLR3" is the
approved HGNC name for the gene encoding this receptor, and its
unique HGNC ID is HGNC:11849. The RefSeq sequence for the human
TLR3 gene is GI:2459625.
[0141] TLR7 is the Toll-like receptor 7. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR7 agonists include e.g. imiquimod. "TLR7"
is the approved HGNC name for the gene encoding this receptor, and
its unique HGNC ID is HGNC:15631. The RefSeq sequence for the human
TLR7 gene is GI:67944638.
[0142] TLR8 is the Toll-like receptor 8. It is a single
membrane-spanning receptor which plays a key role in the innate
immune system. Known TLR8 agonists include e.g. resiquimod. "TLR8"
is the approved HGNC name for the gene encoding this receptor, and
its unique HGNC ID is HGNC:15632. The RefSeq sequence for the human
TLR8 gene is GI:20302165.
[0143] The RIG-1-like receptor ("RLR") family includes various RNA
helicases which play key roles in the innate immune system [41].
RLR-1 (also known as RIG-I or retinoic acid inducible gene I) has
two caspase recruitment domains near its N-terminus. The approved
HGNC name for the gene encoding the RLR-1 helicase is "DDX58" (for
DEAD (Asp-Glu-Ala-Asp) box polypeptide 58) and the unique HGNC ID
is HGNC:19102. The RefSeq sequence for the human RLR-1 gene is
GI:77732514. RLR-2 (also known as MDA5 or melanoma
differentiation-associated gene 5) also has two caspase recruitment
domains near its N-terminus. The approved HGNC name for the gene
encoding the RLR-2 helicase is "IFIH1" (for interferon induced with
helicase C domain 1) and the unique HGNC ID is HGNC:18873. The
RefSeq sequence for the human RLR-2 gene is GI: 27886567. RLR-3
(also known as LGP2 or laboratory of genetics and physiology 2) has
no caspase recruitment domains. The approved HGNC name for the gene
encoding the RLR-3 helicase is "DHX58" (for DEXH (Asp-Glu-X-His)
box polypeptide 58) and the unique HGNC ID is HGNC:29517. The
RefSeq sequence for the human RLR-3 gene is GI:149408121.
[0144] PKR is a double-stranded RNA-dependent protein kinase. It
plays a key role in the innate immune system. "EIF2AK2" (for
eukaryotic translation initiation factor 2-alpha kinase 2) is the
approved HGNC name for the gene encoding this enzyme, and its
unique HGNC ID is HGNC:9437. The RefSeq sequence for the human PKR
gene is GI:208431825.
BRIEF DESCRIPTION OF DRAWINGS
[0145] 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.
[0146] FIG. 2 is an electron micrograph of liposomes.
[0147] FIG. 3 shows the structures of DLinDMA, DLenDMA and
DODMA.
[0148] 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.
[0149] 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 .mu.g, x=1 .mu.g).
[0150] FIG. 6 shows protein expression at days 1, 3 and 6 after
delivery of four different doses of liposome-encapsulated RNA.
[0151] FIG. 7 shows anti-F IgG titers in animals receiving
virion-packaged replicon (VRP or VSRP), 1 .mu.g naked RNA, and 1
.mu.g liposome-encapsulated RNA.
[0152] FIG. 8 shows anti-F IgG titers in animals receiving VRP, 1
.mu.g naked RNA, and 0.1 g or 1 .mu.g liposome-encapsulated
RNA.
[0153] FIG. 9 shows neutralising antibody titers in animals
receiving VRP or either 0.1 g or 1 .mu.g liposome-encapsulated
RNA.
[0154] 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).
[0155] FIG. 11 shows F-specific IgG titers (2 weeks after second
dose) after delivery of a replicon as naked RNA (0.01-1 .mu.g),
liposome-encapsulated RNA (0.01-10 .mu.g), or packaged as a virion
(VRP, 10.sup.6 infectious units or IU).
[0156] FIG. 12 shows F-specific IgG titers (circles) and PRNT
titers (squares) after delivery of a replicon as naked RNA (1
.mu.g), liposome-encapsulated RNA (0.1 or 1 .mu.g), or packaged as
a virion (VRP, 10.sup.6 IU). Titers in nauve mice are also shown.
Solid lines show geometric means.
[0157] 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-.
[0158] FIG. 14 shows 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] FIG. 19 shows (A) IL-6 and (B) IFN.alpha. (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
[0164] 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.
[0165] 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.
[0166] 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.degree. 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
[0167] 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.
[0168] 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
(3.beta.-[N--(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
hydrochloride) were from Avanti Polar Lipids.
[0169] 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
.about.95% encapsulation efficiency.
[0170] 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.degree. C. for about 15 min
to form a homogenous mixture. Then, 755 .mu.L 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
.mu.g RNA. A 2 mL working solution of RNA was also prepared from a
stock solution of .about.1 .mu.g/.mu.L 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.degree. 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.TM.
500 .mu.m 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.degree. C.
before passing through the membrane. Next, liposomes were
concentrated to 2 mL and dialyzed against 10-15 volumes of
1.times.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.times.PBS.
[0171] 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).
[0172] 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.times. or 100.times. in 1.times.TE buffer
(from kit) before addition of the dye. Separately, liposomes were
diluted 10.times. or 100.times. in 1.times.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 .about.180 .mu.L 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.
[0173] 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.degree. C. for 10 minutes. A
1:1 v/v mixture of sample to 25:24:1 v/v/v,
phenol:chloroform:isoamyl alcohol was then added to extract the RNA
from the lipids into the aqueous phase. Samples were mixed by
vortexing for a few seconds and then placed on a centrifuge for 15
minutes at 12k RPM. The aqueous phase (containing the RNA) was
removed and used to analyze the RNA. Prior to loading (400 ng RNA
per well) all the samples were incubated with formaldehyde loading
dye, denatured for 10 minutes at 65.degree. 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.degree. C. the RNA could be seen without any
fragmentation (FIG. 4, arrow). Protein expression in vivo was
unchanged after 6 weeks at 4.degree. C. and one freeze-thaw cycle.
Thus liposome-encapsulated RNA is stable.
[0174] 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.times. 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, 50g1 per leg
with 0.1 .mu.g or 1 .mu.g RNA dose. The same vector was also
administered without the liposomes (in RNase free 1.times.PBS) at 1
.mu.g. 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.
[0175] As shown in FIG. 5, encapsulation increased SEAP levels by
about 1/2 log at the 1 .mu.g dose, and at day 6 expression from a
0.1 .mu.g encapsulated dose matched levels seen with 1 .mu.g
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.times. 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.
[0176] 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 .mu.g RNA), or
mixed with the liposomes after their formation (a non-encapsulated
"lipoplex", 0.1 .mu.g RNA), or as naked RNA (1 .mu.g). FIG. 10
shows that the lipoplex gave the lowest levels of expression,
showing that shows encapsulation is essential for potent
expression.
[0177] 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 .mu.g of (i), naked -- C 0.1 .mu.g of
(i), encapsulated in liposome -- D 0.1 .mu.g of (i), with separate
liposomes -- E 0.1 .mu.g of (i), naked 10 .mu.g of (ii), naked F
0.1 .mu.g of (i), naked 10 .mu.g of (iii), naked G 0.1 .mu.g of
(i), encapsulated in liposome 10 .mu.g of (ii), naked H 0.1 .mu.g
of (i), encapsulated in liposome 10 .mu.g of (iii), naked I 0.1
.mu.g of (i), encapsulated in liposome 1 .mu.g of (ii),
encapsulated in liposome J 0.1 .mu.g of (i), encapsulated in
liposome 1 .mu.g of (iii), encapsulated in liposome K 5 .mu.g F
protein -- L 5 .mu.g F protein 1 .mu.g of (ii), encapsulated in
liposome M 5 .mu.g F protein 1 .mu.g of (iii), encapsulated in
liposome
[0178] 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.
[0179] 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 .mu.g
encapsulated RNA was equivalent to 1 .mu.g of naked RNA. At a 0.5
.mu.g dose of RNA the encapsulated material gave a 12-fold higher
expression at day 6; at a 0.1 .mu.g dose levels were 24-fold higher
at day 6.
[0180] 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. 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.
[0181] 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 .mu.g), encapsulated in
liposomes (0.1 or 1 .mu.g), 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 .mu.g, the encapsulated RNA at 1 .mu.g, 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.
[0182] FIG. 13 confirms that the RNA elicits a robust CD8 T cell
response.
[0183] Further experiments compared F-specific IgG titers in mice
receiving VRP, 0.1 .mu.g liposome-encapsulated RNA, or 1 .mu.g
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 .mu.g 2.9 1.0 1.1 1
.mu.g 2.3 0.9 0.9
[0184] Thus the liposome-encapsulated RNA induces essentially the
same magnitude of immune response as seen with virion delivery.
[0185] Further experiments showed superior F-specific IgG responses
with a 10 .mu.g dose, equivalent responses for 1 .mu.g and 0.1
.mu.g doses, and a lower response with a 0.01 .mu.g 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 .mu.g liposome-encapsulated
RNA was statistically insignificant (ANOVA) when compared to VRP,
but the higher response seen with 10 .mu.g liposome-encapsulated
RNA was statistically significant (p<0.05) when compared to both
of these groups.
[0186] A further study confirmed that the 0.1 .mu.g of
liposome-encapsulated RNA gave much higher anti-F IgG responses (15
days post-second dose) than 0.1 .mu.g of delivered DNA, and even
was more immunogenic than 20 .mu.g plasmid DNA encoding the F
antigen, delivered by electroporation (Elgen.TM. DNA Delivery
System, Inovio).
[0187] A further study was performed in cotton rats (Sigmodon
hispidis) instead of mice. At a 1 .mu.g 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.times.10.sup.6 IU VRP. Both naked and liposome-encapsulated RNA
were able to protect the cotton rats from RSV challenge
(1.times.10.sup.5 plaque forming units), reducing lung viral load
by at least 3.5 logs. Encapsulation increased the reduction by
about 2-fold.
[0188] A large-animal study was performed in cattle. Cows were
immunised with 66 .mu.g 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). FIG. 14 shows F-specific IgG titers over a 63 day period
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
[0189] 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 [44]. The cells were stimulated with replicon RNA
formulated with DOTAP, lipofectamine 2000 or inside a liposome. As
shown in FIG. 19, IL-6 and INF.alpha. 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
[0190] The pKa of a lipid is measured in water at standard
temperature and pressure using the following technique: [0191] 2 mM
solution of lipid in ethanol is prepared by weighing the lipid and
dissolving in ethanol. 0.3 mM solution of fluorescent probe toluene
nitrosulphonic 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. [0192] 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 .mu.L of 2 mM
lipid solution and 800 .mu.L of 0.3 mM TNS solution are mixed.
[0193] 7.5 .mu.L of probe/lipid mix are added to 242.5 .mu.L of
buffer in a 1 mL 96 well plate. This is done with all eight
buffers. After mixing, 100 .mu.L of each probe/lipid/buffer mixture
is transferred to a 250 .mu.L 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. [0194] 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). [0195] 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. [0196]
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.
[0197] 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
[0198] As an alternative to using DlinDMA, the cationic lipids of
reference 5 are used. These lipids can be synthesised as disclosed
in reference 5.
[0199] 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.
[0200] RV02 liposomes were made using the following cationic lipid
(pKa>9, without a tertiary amine):
##STR00001##
[0201] RV03 liposomes were made using the following cationic lipid
(pKa 6.4):
##STR00002##
[0202] RV04 liposomes were made using the following cationic lipid
(pKa 6.62):
##STR00003##
[0203] RV05 liposomes were made using the following cationic lipid
(pKa 5.85):
##STR00004##
[0204] RV06 liposomes were made using the following cationic lipid
(pKa 7.27):
##STR00005##
[0205] RV07 liposomes were made using the following cationic lipid
(pKa 6.8):
##STR00006##
[0206] RV08 liposomes were made using the following cationic lipid
(pKa 5.72):
##STR00007##
[0207] RV09 liposomes were made using the following cationic lipid
(pKa 6.07):
##STR00008##
[0208] RV10 liposomes were made for comparison using the following
cationic lipid (pKa 7.86):
##STR00009##
[0209] RV11 liposomes were made using the following cationic lipid
(pKa 6.41):
##STR00010##
[0210] RV12 liposomes were made using the following cationic lipid
(pKa 7):
##STR00011##
[0211] RV16 liposomes were made using the following cationic lipid
(pKa 6.1) [45]:
##STR00012##
[0212] RV17 liposomes were made using the following cationic lipid
(pKa 6.1) [45]:
##STR00013##
[0213] 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.
[0214] 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 Zav % SEAP SEAP RV pKa (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
[0215] 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.
[0216] 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 (2wp1) 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
[0217] 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 .mu.L per leg) on days 0 and 21 with the replicon (1 .mu.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 .mu.g) expressing
the same RSV-F antigen was delivered either using electroporation
or with RV01(10) liposomes (0.1 .mu.g DNA). Four mice were used as
a nauve control group.
[0218] 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.degree. C. for about 15 min to form a homogenous
mixture. Then, 226.7 .mu.L 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 .mu.g 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 .mu.g/.mu.L 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.degree. 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.TM. 500 .mu.m 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.times.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-Cl-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.times.PBS.
[0219] 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.degree. C. before beign
filtered. Secondly, the hollow fiber filtration membrane was
Polysulfone (part number P/N: X1AB-100-20P).
[0220] 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
[0221] Serum was collected for antibody analysis on days 14, 36 and
49. Spleens were harvested from mice at day 49 for T cell
analysis.
[0222] 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
[0223] 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:
TABLE-US-00006 CD4+CD8- CD4-CD8+ RV IFN.gamma. IL2 IL5 TNF.alpha.
IFN.gamma. IL2 IL5 TNF.alpha. 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
[0224] 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.
[0225] 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
[0226] 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
[0227] 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.times. by
adding 10% 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE)
to the liposome, 10.times. by adding 10% 18:2 (cis)
phosphatidylcholine, and 900.times. by instead using RV01.
RSV Immunogenicity in Different Mouse Strains
[0228] 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.
[0229] BALB/c mice were given bilateral intramuscular vaccinations
(50 .mu.L per leg) on days 0 and 22. Animals were divided into 8
test groups (5 animals per group) and a nauve control (2 animals):
[0230] Group 1 were given naked replicon (1 .mu.g). [0231] Group 2
were given 1 .mu.g replicon delivered in liposomes "RV01(37)" with
40% DlinDMA, 10% DSPC, 48% Chol, 2% PEG-conjugated DMG. [0232]
Group 3 were given the same as group 2, but at 0.1 .mu.g RNA.
[0233] Group 4 were given 1 .mu.g replicon in "RV17(10)" liposomes
(40% RV17 (see above), 10% DSPC, 49.5% cholesterol, 0.5% PEG-DMG).
[0234] Group 5 were 1 .mu.g replicon in "RV05(11)" liposomes (40%
RV07 lipid, 30% 18:2 PE (DLoPE, 28% cholesterol, 2% PEG-DMG).
[0235] Group 6 were given 0.1 .mu.g replicon in "RV17(10)"
liposomes. [0236] Group 7 were given 5 .mu.g RSV-F subunit protein
adjuvanted with aluminium hydroxide. [0237] Group 8 were a nauve
control (2 animals)
[0238] 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
[0239] 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
[0240] 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
[0241] 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 IFN.gamma. IL2 IL5
TNF.alpha. IFN.gamma. IL2 IL5 TNF.alpha. 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
[0242] C57BL/6 mice were immunised in the same way, but a 9th group
received VRPs (1.times.10.sup.6 IU) expressing the full-length
wild-type surface fusion glycoprotein of RSV (fusion peptide
deletion).
[0243] 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
[0244] 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
[0245] 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
[0246] 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):
TABLE-US-00014 CD4-CD8+ Group IFN.gamma. IL2 IL5 TNF.alpha. 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
[0247] 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
[0248] 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
[0249] 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
[0250] 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
[0251] 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.
[0252] BALB/c mice, 10 per group, were given bilateral
intramuscular vaccinations (50 .mu.L per leg) on days 0, 21 and 42
with VRPs expressing gH/gL (1.times.10.sup.6 IU), VRPs expressing
gHsol/gL (1.times.10.sup.6 IU) and PBS as the controls. Two test
groups received 1 .mu.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 .mu.g RNA batch
size).
[0253] 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.
[0254] The replicons were able to express two proteins from a
single vector.
[0255] 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
[0256] 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.
[0257] 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
[0258] [1] Johanning et al. (1995) Nucleic Acids Res 23:1495-1501.
[0259] [2] WO2005/121348. [0260] [3] WO2008/137758. [0261] [4]
WO2009/086558. [0262] [5] WO2011/076807. [0263] [6] Heyes et al.
(2005) J Controlled Release 107:276-87. [0264] [7] WO2005/121348.
[0265] [8] Liposomes: Methods and Protocols, Volume 1:
Pharmaceutical Nanocarriers: Methods and Protocols. (ed. Weissig).
Humana Press, 2009. ISBN 160327359X. [0266] [9] Liposome
Technology, volumes I, II & III. (ed. Gregoriadis). Informa
Healthcare, 2006. [0267] [10] Functional Polymer Colloids and
Microparticles volume 4 (Microspheres, microcapsules &
liposomes). (eds. Arshady & Guyot). Citus Books, 2002. [0268]
[11] Jeffs et al. (2005) Pharmaceutical Research 22 (3):362-372.
[0269] [12] WO2005/113782. [0270] [13] WO2011/005799. [0271] [14]
E1 Ouahabi et al. (1996) FEBS Letts 380:108-12. [0272] [15]
Giuliani et al. (2006) Proc Natl Acad Sci USA 103(29): 10834-9.
[0273] [16] WO2009/016515. [0274] [17] WO02/34771. [0275] [18]
WO2005/032582. [0276] [19] WO2010/119343. [0277] [20]
WO2006/110413. [0278] [21] WO2005/111066. [0279] [22]
WO2005/002619. [0280] [23] WO2006/138004. [0281] [24]
WO2009/109860. [0282] [25] WO02/02606. [0283] [26] WO03/018054.
[0284] [27] WO2006/091517. [0285] [28] WO2008/020330. [0286] [29]
WO2006/089264. [0287] [30] WO2009/104092. [0288] [31]
WO2009/031043. [0289] [32] WO2007/049155. [0290] [33] Gennaro
(2000) Remington: The Science and Practice of Pharmacy. 20th
edition, ISBN: 0683306472. [0291] [34] Methods In Enzymology (S.
Colowick and N. Kaplan, eds., Academic Press, Inc.) [0292] [35]
Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C.
C. Blackwell, eds, 1986, Blackwell Scientific Publications) [0293]
[36] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual,
3rd edition (Cold Spring Harbor Laboratory Press). [0294] [37]
Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC
Press, 1997) [0295] [38] Ausubel et al. (eds) (2002) Short
protocols in molecular biology, 5th edition (Current Protocols).
[0296] [39] Molecular Biology Techniques: An Intensive Laboratory
Course, (Ream et al., eds., 1998, Academic Press) [0297] [40] PCR
(Introduction to Biotechniques Series), 2nd ed. (Newton &
Graham eds., 1997, Springer Verlag) [0298] [41] Yoneyama &
Fujita (2007) Cytokine & Growth Factor Reviews 18:545-51.
[0299] [42] Maurer et al. (2001) Biophysical Journal, 80:
2310-2326. [0300] [43] Perri et al. (2003) J Virol 77:10394-10403.
[0301] [44] Iavarone et al. (2011) J Immunol 186; 4213-22. [0302]
[45] WO2011/057020.
* * * * *