U.S. patent application number 17/591421 was filed with the patent office on 2022-08-04 for lyssavirus antigen constructs.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS SA. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS SA. Invention is credited to KATHRYN HASHEY, RASHMI JALAH, PADMA MALYALA, MARCELO SAMSA, OLGA SLACK, ALAN STOKES, DONG YU.
Application Number | 20220241398 17/591421 |
Document ID | / |
Family ID | 1000006269233 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220241398 |
Kind Code |
A1 |
HASHEY; KATHRYN ; et
al. |
August 4, 2022 |
LYSSAVIRUS ANTIGEN CONSTRUCTS
Abstract
Nucleic acid based vaccine constructs encoding Lyssaviral
antigens are useful in preventing and treating diseases.
Self-amplifying RNA molecules encoding Lyssaviral antigens provide
potent and long-lasting immunity.
Inventors: |
HASHEY; KATHRYN;
(Washington, DC) ; MALYALA; PADMA; (Cambridge,
MA) ; SAMSA; MARCELO; (Rockville, MD) ; SLACK;
OLGA; (Cambridge, MA) ; YU; DONG; (Rockville,
MD) ; STOKES; ALAN; (Rockville, MD) ; JALAH;
RASHMI; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS SA |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
SA
RIXENSART
BE
|
Family ID: |
1000006269233 |
Appl. No.: |
17/591421 |
Filed: |
February 2, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16630951 |
Jan 14, 2020 |
11278613 |
|
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PCT/IB2018/055258 |
Jul 16, 2018 |
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17591421 |
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62533312 |
Jul 17, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61K 9/127 20130101; A61K 39/205 20130101 |
International
Class: |
A61K 39/205 20060101
A61K039/205; A61K 9/127 20060101 A61K009/127 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with United States government
support under Agreement No. HR0011-12-3-0001 awarded by DARPA. The
government has certain rights in the invention.
Claims
1. A composition comprising an mRNA encoding a polypeptide
comprising an antigen selected from one or more of a full-length
Lyssavirus glycoprotein (G), RNA polymerase (L), matrix protein
(M), nucleoprotein (N) or phosphoprotein (P), an immunogenic
derivative or an immunogenic fragment thereof and a lipid
nanoparticle delivery system comprising at least one of RV28, RV31,
RV33, RV37, RV39, RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86,
RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99, and
RV101.
2. The composition of claim 1, wherein the mRNA is codon
optimized.
3. The composition of claim 1, wherein the mRNA is codon
pairoptimized.
4. The composition of claim 1, wherein the mRNA comprises a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 2;
SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
5. The composition of claim 1, wherein the mRNA encodes an amino
acid sequence selected from the group consisting of SEQ ID NO: 3;
SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.
6. The composition according to claim 1 comprising an RNA-based
vaccine.
7. The composition according to claim 1, wherein the composition
further comprises a nucleic acid sequence which encodes an
additional antigen.
8. The composition according to claim 1, wherein the composition is
pharmaceutically acceptable for administration to a subject in
combination with a further composition which comprises a nucleic
acid comprising a sequence which encodes an additional antigen.
9. The composition according to claim 1, wherein the composition
comprises one or more adjuvants.
10. A method of inducing an immune response against a disease
caused by a Lyssa virus in a subject in need thereof, which
comprises administering to the subject an immunologically effective
amount of the composition according to claim 8.
11. The method according to claim 8 wherein the subject is human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 16/630,951 filed Jan. 14, 2020, which is a National Phase
Application of International Patent Application Serial No.
PCT/IB2018/055258 filed Jul. 16, 2018, which claims priority to
U.S. Provisional Application No. 62/533,312 filed Jul. 17, 2017.
The entire contents of each of the foregoing applications are
hereby incorporated by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"2801-0329PUS2_ST25.txt" created on Apr. 14, 2022 and is 38,150
bytes in size. The sequence listing contained in this .txt file is
part of the specification and is hereby incorporated by reference
herein in its entirety.
FIELD OF THE INVENTION
[0004] This invention is in the field of treating and preventing
viral diseases. In particular, the present invention relates to
self-amplifying RNA molecules encoding a Lyssavirus antigen. It
includes the use of Lyssavirus antigens for treating and preventing
rabies.
BACKGROUND TO THE INVENTION
[0005] Lyssavirus is an enveloped, single-stranded RNA virus in the
Rhabdoviridae family. Members of the Lyssavirus genus cause rabies
and have the highest fatality rate of all known human viral
pathogens. Rabies is transmitted via the saliva of infected
mammals. A neurotropic virus, it enters the nervous system of its
host, causing an encephalomyelitis that is almost invariably fatal.
Currently there are about 60,000 rabies deaths worldwide yearly,
mostly caused by dog bites in developing countries in Asia and
Africa and by wildlife and bats in North America.
[0006] Rabies presents either in a furious or a paralytic form. The
incubation period varies between about five days and several years
but is typically between about 20 and 90 days. Clinical illness
most often starts with prodromal complaints of malaise, anorexia,
fatigue, headache and fever followed by pain or parathesia at the
site of exposure. Anxiety, agitation or irritability may be
prominent during this period, followed by hyperactivity,
disorientation, seizures, hydrophobia, hypersalivation and,
eventually, paralysis, coma and death.
[0007] Experimentally, RNA vaccines have been derived from
sub-genomic replicons that lack viral structural proteins and
express a heterologous antigen in place of the viral structural
proteins. They can be produced in packaging cell lines that permit
the expression of single-round of infectious particles carrying
RNAs encoding the vaccine antigen. RNA amplification in the
cytoplasm then produces multiple copies of antigen-encoding mRNAs
and creates double stranded RNA intermediates, which are known to
be potent stimulators of innate immunity. Thus, replicon RNA
vaccines can achieve transient high levels of antigen production
without the use of a live virus (Brito et al. (2015) Advances in
Genetics 89:179-233).
[0008] While inserting RNA formulated merely with a buffer, i.e.,
naked RNA, into a cell can induce both gene expression and an
immune response, the in vivo instability of naked RNA limits its
potency as a vaccine. Furthermore, the hydrophilicity and strong
negative charge of RNA impedes its uptake into cells. However,
transfer into the cell cytoplasm can be facilitated. Synthetic
delivery systems, such as lipid nanoparticles and cationic
nanoemulsions have been demonstrated to effectively transfer
nucleic acids, including self-amplifying RNA, into the cell
cytoplasm, where it can amplify and express encoded antigens.
[0009] There remains a need for novel methods of immunizing against
diseases, including diseases caused by Lyssaviruses, which are
highly efficacious, safe, convenient, cost-effective, long-lasting
and induce a broad spectrum of immune responses. Accordingly, there
is a demand for vectors that can effectively deliver vaccine
antigens, specifically, Lyssavirus antigens. While Lyssavirus
prophylaxis is currently available, high numbers of doses are
required both pre- and post-exposure, and compliance is low, which
diminishes the medical benefit. There is a need for an improved
Lyssavirus vaccine with a simplified administration schedule,
increased safety and an enhanced manufacturing profile.
SUMMARY OF THE INVENTION
[0010] The present invention provides constructs useful as
components of immunogenic compositions for the induction of an
immune response in a subject against Lyssavirus diseases, methods
for their use in treatment, and processes for their
manufacture.
[0011] A first aspect of the invention provides a nucleic
acid-based vaccine construct comprising or consisting of a
nucleotide sequence encoding one or more polypeptide comprising or
consisting of a full-length Lyssavirus protein, or an immunogenic
fragment thereof. Alternatively or additionally, the Lyssavirus
protein is selected from the group consisting of glycoprotein (G),
RNA polymerase (L), matrix protein (M), nucleoprotein (N) and
phosphoprotein (P).
[0012] Alternatively or additionally, the polypeptide comprises a
full length Lyssavirus glycoprotein or immunogenic fragment
thereof. Alternatively or additionally, the Lyssavirus glycoprotein
is the Flury high egg passage ("HEP") rabies G protein designated
AGN9427.1 in GenBank. In another preferred embodiment, the
Lyssavirus glycoprotein is the Flury low egg passage ("LEP") rabies
G protein designated GU565703.1 in GenBank. Alternatively or
additionally, the Lyssavirus glycoprotein is a codon optimized
version of Flury LEP rabies G protein. Alternatively or
additionally, the Lyssavirus glycoprotein is a codon pair optimized
version of Flury LEP rabies G protein.
[0013] A second aspect of the invention provides a vector
comprising or consisting of the nucleic acid-based vaccine
construct.
[0014] A third aspect of the invention provides a self-amplifying
RNA molecule comprising or consisting of the nucleic acid-based
vaccine construct. The self-amplifying RNA molecules of the
invention are not encompassed in a virion and the constructs of the
invention do not comprise a protein capsid. By avoiding the need to
create a capsid, the invention does not require a packaging cell
line, thus permitting easier up-scaling for commercial production
and minimising the risk that dangerous infectious viruses will
inadvertently be produced.
[0015] A fourth aspect of the invention provides a DNA molecule
encoding the self-amplifying RNA molecule.
[0016] A fifth aspect of the invention provides a composition
comprising or consisting of one or more of the constructs, vectors,
or self-amplifying RNA molecules as described herein. Alternatively
or additionally, the composition comprises or consists of an
immunologically effective amount of one or more of the constructs,
vectors, or self-amplifying RNA molecules.
[0017] Alternatively or additionally, the composition comprises or
consists of an RNA-based vaccine.
[0018] A sixth aspect of the invention provides a method is
provided for inducing an immune response against a Lyssavirual
disease in a subject in need thereof, which comprises administering
to the subject an immunologically effective amount of a composition
comprising one or more of the constructs, vectors, or
self-amplifying RNA molecules described herein.
[0019] A seventh embodiment of the invention provides a process for
producing an RNA-based vaccine comprising a step of transcribing
the vector or DNA molecule encoding a self-amplifying RNA molecule
described herein to produce an RNA comprising a coding region for a
Lyssavirus antigen.
[0020] An eighth aspect of the invention provides a composition
produced by the process described herein.
[0021] A ninth aspect of the invention provides the use of the
construct, vector, self-amplifying RNA molecule, or composition
described herein for inducing an immune response against a disease
caused by Lyssavirus in a subject is provided.
[0022] A tenth aspect of the invention provides the construct,
vector, self-amplifying RNA molecule, or composition described
herein for use in medicine.
[0023] An eleventh aspect of the invention provides the construct,
vector, self-amplifying RNA molecule, or composition described
herein for use in treating or preventing a disease caused by
Lyssavirus in a subject (e.g., by inducing a protective immune
response).
[0024] A twelfth aspect of the invention provides the use of the
construct, vector, self-amplifying RNA molecule, or composition
described herein in the manufacture of a medicament inducing an
immune response against a Lyssaviral disease in a subject is
provided.
[0025] A thirteenth aspect of the invention provides a human dose
of the construct, vector, self-amplifying RNA molecule, or
composition described herein in the amount of two micrograms or
less that is immunogenic in humans.
DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1A. Schematic representation of SAM Rabies constructs.
"NSP" denotes viral RNA encoding non-structural proteins. The
rabies G antigen is encoded 3' to the NSP. Construct 1: Flury HEP
Rabies G; Construct 2: Flury LEP Rabies G; Construct 3: codon
optimized Flury LEP Rabies G; Construct 4: codon pair optimized
Flury LEP Rabies G.
[0027] FIG. 1B. Alignment of the DNA sequences of Constructs
1-4(SEQ ID NOS. 2, 4, 6 and 8, respectively) This figure was
originally in color and subsequently revised for black and white,
where the specification refers to color related to this figure, the
following key applies to this figure: Yellow=plain text;
Blue=Italics; Green=underline; White=Bold.
[0028] FIG. 2A. Western blot expression of rabies glycoprotein (G)
antigen by Constructs 1-4 in BHK cells.
[0029] FIG. 2B. Western blot expression of rabies glycoprotein (G)
antigen by Constructs 1 and 3 before and after treatment with
peptide N-glycosidase A (PNGase A).
[0030] FIG. 3A. Neutralizing antibody (nAb) titers induced in
Balb/c mice by SAM constructs 1-4 formulated with CNE or LNP on day
14 compared to RABAVERT at day 35 determined by Rapid Fluorescent
Focus Inhibition Test (RFFIT) and expressed as Geometric Mean Titer
(GMT).
[0031] FIG. 3B. Neutralizing antibody (nAb) titers induced in
Balb/c mice by SAM constructs 1-4 formulated with CNE or LNP on day
35 compared to RABAVERT at day 35 determined by Rapid Fluorescent
Focus Inhibition Test (RFFIT) and expressed as Geometric Mean Titer
(GMT).
[0032] FIG. 4. Neutralizing antibody (nAb) titers determined by
RFFIT in Balb/c mice of Construct 4 formulated with CNE or LNP for
six months post immunization. RABAVERT (circles); 1.5 ug Construct
4 in CNE (squares); 0.15 ug Construct 4 in LNP (triangles); 1.5 ug
Construct 4 in LNP (inverted triangles).
[0033] FIG. 5A. Day 14. Neutralizing antibody (nAb) titers
determined by RFFIT in Balb/c mice of Construct 4 formulated with
CNE or LNP, showing the dose response relationship with
immunogenicity. The upper dashed line indicates a benchmark of 100
IU/ml, which is the peak nAb titer observed to be elicited by
RABAVERT at high doses. The lower dashed line indicates the
immunogenicity threshold of 0.5 IU/ml.
[0034] FIG. 5B. Day 35. Neutralizing antibody (nAb) titers
determined by RFFIT in Balb/c mice of Construct 4 formulated with
CNE or LNP, showing the dose response relationship with
immunogenicity. The upper dashed line indicates a benchmark of 100
IU/ml, which is the peak nAb titer observed to be elicited by
RABAVERT at high doses. The lower dashed line indicates the
immunogenicity threshold of 0.5 IU/ml.
[0035] FIG. 6. Neutralizing antibody (nAb) titers determined by
RFFIT in non-human primates of Construct 4 immunizations at weeks
0, 8 and 24 compared to a full human dose of RABAVERT immunizations
at weeks 0, 1 and 3. The top panel shows the neutralizing
anti-rabies antibody titers of four doses of Construct 4 formulated
in CNE. 150 ug (squares); 75 ug (triangles); 15 ug (inverted
triangles); 3 ug (solid circles); RABAVERT (open circles). The
upper dashed line indicates the protective threshold of
immunogenicity and the lower dotted line at log 0.1 indicates the
lower limits of quantitation (LLOQ).
[0036] The bottom panel shows the neutralizing anti-rabies antibody
titers of four doses of Construct 4 formulated in LNP. 75 ug
(triangles); 15 ug (inverted triangles); 3 ug (closed circles);
RABAVERT (open circles). The upper dashed line indicates the
protective threshold of immunogenicity and the lower dotted line at
log 0.1 indicates the lower limits of quantitation (LLOQ).
[0037] FIG. 7. IgG titers determined by ELISA in non-human primates
of Construct 4 immunizations at weeks 0, 8 and 24 compared to a
full human dose of RABAVERT immunizations at weeks 0, 1 and 3. The
top panel shows the anti-rabies IgG titers of four doses of
Construct 4 formulated in CNE. 150 ug (squares); 75 ug (triangles);
15 ug (inverted triangles); 3 ug (closed circles); RABAVERT (open
circles). The upper dashed line indicates the protective threshold
of immunogenicity and the lower dotted line at log 0.1 indicates
the lower limits of quantitation (LLOQ).
[0038] The bottom panel shows the anti-rabies IgG titers of four
doses of Construct 4 formulated in the RV39 LNP. 75 ug (triangles);
15 ug (inverted triangles); 3 ug (closed circles); RABAVERT (open
circles). The upper dashed line indicates the protective threshold
of immunogenicity and the lower dotted line at log 0.1 indicates
the lower limits of quantitation (LLOQ).
[0039] FIG. 8. Dose-response of neutralizing antibody titers
determined by RFFIT in mice of Construct 4 in LNP at doses ranging
from 15 pg to 1.5 ug or CNE 15 ug. The top panel shows the
neutralizing antibody titers for six months following a single dose
at day 1. The bottom panel shows the neutralizing antibody titers
for six months following two doses at days 1 and 22. RABAVERT (open
circles); LNP 1.5 ug (open squares); LNP 0.15 ug (open triangles);
LNP 0.015 ug (open inverted triangles); LNP 0.0015 ug (closed
squares); LNP 0.00015 ug (closed triangles); LNP 0.000015 ug
(closed inverted triangles); CNE 15 ug (closed circles). The upper
dashed line indicates the protective threshold of immunogenicity
and the lower dotted line below log 0.1 indicates the lower limits
of quantitation (LLOQ). The following observations were noted.
Similar antibody levels as previous rabies SAM studies were induced
with a dose response at lower RNA doses. A single immunization with
a dose as low as 15 picograms induced substantial and stable levels
of rabies neutralizing antibodies that were maintained over the six
months' time tested in mice. The levels were boosted by the second
immunization and remained significantly greater than three RABAVERT
immunizations.
[0040] FIG. 9: Specific polyfunctional CD8+ T cell responses in
mice by a single 15 ug dose of Construct 4 formulated in CNE. The
Th1 cytokines IL-2, TNF alpha, interferon gamma and CD107a were
stimulated by rabies antigenic peptides. Their expression in CD4+
and CD8+ T cells are shown in the top and bottom panels
respectively. The vaccinated group (8 mice) was compared to a
saline control group (5 mice). Cells from each group were exposed
to either rabies antigenic peptides or a media control.
[0041] FIG. 10. Dose-response of neutralizing antibody titers
determined by RFFIT in mice of Construct 4 at doses ranging from 15
pg to 0.15 ug in the six months following two doses (days 1 and
22). Construct 4 was formulated either with LNP RV39 or LNP RV94.
RABAVERT (open circles); 0.15 ug RV39 (open squares); 0.15 ug RV94
(dashed squares); 0.0015 ug RV39 open triangles); 0.0015 ug RV94
(dashed triangles); 0.000015 ug RV39 (open inverted triangles);
0.000015 ug RV94 (dashed inverted triangles). It was observed that
both RV39 and RV94 LNP formulations gave similar rabies nAb titers
but with greater longevity observed for RV39.
[0042] FIG. 11. Dose-response of IgG determined by ELISA in mice of
Construct 4 at doses ranging from 15 pg to 0.15 ug for six months
following two doses (days 1 and 22). Construct 4 was formulated
either with LNP RV39 or LNP RV94. RABAVERT (open circles); 0.15 ug
RV39 (open squares); 0.15 ug RV94 (dashed squares); 0.0015 ug RV39
(open triangles); 0.0015 ug RV94 (dashed triangles); 0.000015 ug
RV39 (open inverted triangles); 0.000015 ug RV94 (dashed inverted
triangles).
DESCRIPTION OF THE SEQUENCES
[0043] SEQ ID NO: 1--Vector Backbone--VEE TC83 (empty vector)
[0044] SEQ ID NO: 2--Construct 1 Coding Sequence
[0045] SEQ ID NO: 3--Construct 1 Amino Acid Sequence
[0046] SEQ ID NO: 4--Construct 2 Coding Sequence
[0047] SEQ ID NO: 5--Construct 2 Amino Acid Sequence
[0048] SEQ ID NO: 6--Construct 3 Coding Sequence
[0049] SEQ ID NO: 7--Construct 3 Amino Acid Sequence
[0050] SEQ ID NO: 8--Construct 4 Coding Sequence
[0051] SEQ ID NO: 9--Construct 4 Amino Acid Sequence
DETAILED DESCRIPTION OF THE INVENTION
[0052] Lyssavirus Vaccines
[0053] Lyssavirus, a genus in the Rhabdoviridae family, is an
enveloped virus with a single-stranded antisense RNA genome. It is
a neurotropic virus that spreads through the central nervous system
causing severe inflammation of the brain and spinal cord. The RNA
encodes five genes, a glycoprotein (G), a viral RNA polymerase (L),
a matrix protein (M), a nucleoprotein (N) and a phosphoprotein (P).
The G protein is a major target of protective neutralizing
antibodies.
[0054] The Lyssavirus genus comprises seven genotypes, the
following six of which have been associated with cases of human
rabies: rabies virus (RABV, genotype 1), Mokola virus (genotype 3),
Duvenhage virus (genotype 4), European bat Lyssavirus (genotype 5),
European bat Lyssavirus 2 (genotype 6), and Australian bat
Lyssavirus (genotype 7). Once symptoms develop, rabies is nearly
one hundred percent fatal.
[0055] Vaccination is one of the most effective methods for
preventing infectious diseases. However, a single administration of
an antigen is often not sufficient to confer full immunity and/or a
long-lasting response. Rabies currently requires multi-dose
vaccination. Approaches to establishing strong and lasting immunity
to specific pathogens include addition of adjuvants to vaccines
and/or repeated vaccination, i.e. boosting an immune response by
administration of one or more further doses of antigen. Such
further administrations may be performed with the same vaccine
(homologous boosting) or with a different vaccine (heterologous
boosting). The most common approach for homologous boosting is not
only to administer the same vaccine, but also to administer it in
the same dose as the earlier administration.
[0056] Rabies vaccines are currently used primarily for
post-exposure prophylaxis, only a small percentage of rabies
vaccine doses are used for pre-exposure prophylaxis. The
intervention schedule is defined by the World Health Organization
based on the seriousness and the type of the wound via which the
virus gains entry and may include additional treatment with
anti-rabies immunoglobulin. Pre-exposure prophylaxis typically
involves two to three visits for two to three intramuscular doses
with boosters timed according to the exposure risk. Post-exposure
prophylaxis typically involves three to five visits for four to
five intramuscular doses or four visits for four intradermal doses.
In some less-developed countries, immunization is still performed
by propagating rabies virus in the brains of an infected animal,
inactivating the virus and providing 14-21 daily injections given
subcutaneously into the abdominal wall.
[0057] Several rabies vaccines are currently available for human
use in both pre-exposure and post-exposure prophylaxis and are
approved by regulatory agencies when administered in certain dosage
regimens. IMOVAX (Sanofi Pasteur) is provided as freeze-dried
rabies virus prepared from strain PM-1503-3M obtained from the
Wistar Institute. It is harvested from infected human diploid cells
then inactivated. Both pre- and post-exposure prophylaxis consists
of three doses administered intramuscularly on days 0, 7 and 21 or
28. VERORAB (Sanofi Pasteur) is provided as freeze-dried rabies
virus prepared from strain PM/WI 38 1503-3M obtained from the
Wistar Institute. It is harvested from Vero cells then inactivated.
Pre-exposure prophylaxis consists of three doses administered
intramuscularly on days 0, 7 and 21 or 28. Post-exposure
prophylaxis consists of five doses administered intramuscularly on
days 0, 3, 7, 14 and 28. VAXIRAB/LYSSAVAC (Zydus Cadila/Novavax is
provided as freeze-dried rabies virus prepared from the Pitman
Moore strain of the rabies virus. It is produced in duck embryo
cells then inactivated. Pre-exposure prophylaxis consists of three
doses administered intramuscularly on days 0, 7 and 21 or 28.
Post-exposure prophylaxis consists of five doses administered
intramuscularly on days 0, 3, 7, 14 and 28. Post-exposure
prophylaxis can also be administered intradermally, injected at
each of two sites on days 0, 3, 7 and 28. RABAVERT (GSK) is
provided as a freeze-dried rabies virus prepared from the Flury LEP
(low egg passage) strain. It is grown in primary cultures of
chicken fibroblasts then inactivated. Pre-exposure prophylaxis
consists of three doses administered intramuscularly on days 0, 7
and 21 or 28. Post-exposure prophylaxis consists of five doses
administered intramuscularly on days 0, 3, 7, 14 and 28.
[0058] The rabies viral strain Flury LEP present in RABAVERT was
obtained from the American Type Culture Collection as the 59.sup.th
egg passage. The growth medium for the propagation of the virus is
a synthetic cell culture medium with the addition of human albumin,
processed bovine gelatin (polygeline) and antibiotics. The virus is
inactivated with beta-propiolactone and further processed by zonal
centrifugation in a sucrose density gradient. The vaccine is
lyophilized after addition of a stabilizer solution of buffered
polygeline and potassium glutamate. The potency of one dose (1.0
ml) RABAVERT is approximately 2.5 IU rabies antigen.
[0059] Nucleic acid-based rabies vaccines have been attempted in
the past but have proven inferior to RABAVERT. A rabies DNA vaccine
encoding the glycoprotein gene and a rabies self-amplifying RNA
vaccine encoding the glycoprotein gene were directly compared to
RABAVERT. Mice vaccinated with RABAVERT demonstrated a more robust
T cell proliferative response, increased cytokine production and
higher antibody titers than those vaccinated with either a Sindbis
virus RNA replicon expressing rabies G protein or a DNA vaccine
expressing rabies G protein. The Sindbis viral replicon vaccine was
also inferior to RABAVERT in protecting against a rabies challenge
(Saxena et al. (2009) Veterinary Microbiol. 136:36).
[0060] Antigens, Variants, Fragments and Constructs
[0061] The present invention provides constructs useful as
components of immunogenic compositions for the induction of an
immune response in a subject against diseases caused by
Lyssaviruses. These constructs are useful for the expression of
antigens, methods for their use in treatment, and processes for
their manufacture. A "construct" is a genetically engineered
molecule. A "nucleic acid construct" refers to a genetically
engineered nucleic acid and may comprise DNA, RNA, or non-naturally
occurring nucleic acid monomers.
[0062] In some embodiments, the constructs disclosed herein encode
wild-type polypeptide sequences of a Lyssavirus, a variant, or a
fragment thereof. The constructs may further encode a polypeptide
sequence heterologous to the polypeptide sequences of a Lyssavirus.
In some embodiments, the constructs encode wild-type, variants
and/or fragments of polypeptide sequences of a Lyssavirus protein
of the CVS11, CVS-N2C, Evelyn Rokitniki Abelseth (ERA), Flury,
Pitman Moore or Wistar strains. Such antigens may be derived from
the rabies viral glycoprotein (G), RNA polymerase (L), matrix
protein (M), nucleoprotein (N) and phosphoprotein (P).
[0063] A "variant" of a polypeptide sequence includes amino acid
sequences having one or more amino acid additions, substitutions
and/or deletions when compared to the reference sequence. The
variant may comprise an amino acid sequence which is at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
identical to a full-length wild-type polypeptide, for example, to a
polypeptide according to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7
or SEQ ID NO: 9. Alternatively, or in addition to, a fragment of a
polypeptide may comprise an immunogenic fragment (i.e. an
epitope-containing fragment) of the full-length polypeptide which
may comprise or consist of a contiguous amino acid sequence of at
least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 20, or more amino acids which is identical to a
contiguous amino acid sequence of the full-length polypeptide.
[0064] Where a Lyssavirus G antigen is a variant of a wild-type
Lyssavirus glycoprotein, the variant may comprise or consist of an
amino acid sequence which is at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% identical to a full-length
wild-type polypeptide, for example, to a polypeptide according to
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
Alternatively, or in addition, a fragment of a polypeptide may
comprise an immunogenic fragment (i.e. an epitope-containing
fragment) of the full-length polypeptide which may comprise or
consist of a contiguous amino acid sequence of at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17, at least 18, at least 20, or
more amino acids which is identical to a contiguous amino acid
sequence of the full-length polypeptide.
[0065] As used herein, the term "antigen" refers to a molecule
containing one or more epitopes (e.g., linear, conformational or
both) that will stimulate a host's immune system to make a humoral
and/or cellular antigen-specific immunological response (i.e. an
immune response which specifically recognizes a naturally occurring
polypeptide). An "epitope" is that portion of an antigen that
determines its immunological specificity.
[0066] T- and B-cell epitopes can be identified empirically (e.g.
using PEPSCAN or similar methods). They can also be predicted by
known methods (e.g. using the Jameson-Wolf antigenic index,
matrix-based approaches, TEPITOPE, neural networks, OptiMer &
EpiMer, ADEPT, Tsites, hydrophilicity or antigenic index.
[0067] Alternatively or additionally, the constructs herein encode
a Lyssavirus G antigen. By "Lyssavirus G antigen" is intended the
amino acid sequence, or a nucleotide sequence encoding the amino
acid sequence, of a wild-type Lyssavirus glycoprotein, variant, or
fragment thereof. FIG. 1 identifies the nucleic acid coding
sequences of several full-length Lyssavirus G protein variants.
[0068] Alternatively or additionally, the cross-protective breadth
of a vaccine construct can be increased by comprising a medoid
sequence of an antigen. By "medoid" is meant a sequence with a
minimal dissimilarity to other sequences. Alternatively or
additionally, a vector of the invention comprises a medoid sequence
of the rabies G glycoprotein or immunogenic fragment thereof.
Alternatively or additionally, a self-amplifying RNA construct of
the invention comprises a medoid sequence of the rabies G
glycoprotein. Alternatively or additionally, the medoid sequence is
derived from a natural viral strain with the highest average
percent of amino acid identity among all rabies G protein sequences
annotated in the NCBI database. Alternatively or additionally, the
medoid sequence of the rabies G glycoprotein is NCBI strain
AGN94271.
[0069] As a result of the redundancy in the genetic code, a
polypeptide can be encoded by a variety of different nucleic acid
sequences. Coding is biased to use some synonymous codons, codons
that encode the same amino acid, more than others. By "codon
optimized" it is intended that modifications in the codon
composition of a recombinant nucleic acid are made without altering
the amino acid sequence. Codon optimization has been used to
improve mRNA expression in different organisms by using
organism-specific codon-usage frequencies.
[0070] In addition to, and independently from, codon bias, some
synonymous codon pairs are used more frequently than others. This
codon pair bias means that some codon pairs are overrepresented and
others are underrepresented. Codon pair deoptimization has been
used to reduce viral virulence. For example, it has been reported
that polioviruses modified to contain underrepresented codon pairs
demonstrated a decreased translation efficiency and were attenuated
compared to wild type poliovirus (WO 2008/121992; Coleman et al.
(2008) Science 320:1784). Coleman et al. demonstrated that
engineering a synthetic attenuated virus by codon pair
deoptimization can produce viruses that encode the same amino acid
sequences as wild type but use different pairwise arrangements of
synonymous codons. Viruses attenuated by codon pair deoptimization
generated up to 1000-fold fewer plaques compared to wild type,
produced fewer viral particles and required about 100 times as many
viral particles to form a plaque.
[0071] In contrast, polioviruses modified to contain codon pairs
that are overrepresented in the human genome acted in a manner
similar to wild type RNA and generated plaques identical in size to
wild type RNA (Coleman et al. (2008) Science 320:1784). This
occurred despite the fact that the virus with overrepresented codon
pairs contained a similar number of mutations as the virus with
underrepresented codon pairs and demonstrated enhanced translation
compared to wild type. This observation suggests that codon pair
optimized constructs would be expected to act in a manner similar
to their non-codon pair optimized counterparts and would not be
expected to provide a functional advantage.
[0072] Alternatively or additionally, a construct of the invention
comprises a codon optimized nucleic acid sequence. Alternatively or
additionally, a self-amplifying RNA construct of the invention
comprises a codon optimized sequence of the rabies glycoprotein or
an immunogenic derivative or fragment thereof. Alternatively or
additionally, a self-amplifying RNA construct of the invention
comprises a codon optimized sequence of the Flury LEP wild type
rabies glycoprotein or an immunogenic derivative or fragment
thereof.
[0073] Alternatively or additionally, a construct of the invention
comprises a codon pair optimized nucleic acid sequence.
Alternatively or additionally, a self-amplifying RNA construct of
the invention comprises or consists of a codon pair optimized
sequence of the rabies glycoprotein or an immunogenic derivative or
fragment thereof. Alternatively or additionally, a self-amplifying
RNA construct of the invention comprises or consists of a codon
pair optimized sequence of the Flury LEP wild type rabies
glycoprotein or an immunogenic derivative or fragment thereof.
[0074] Alternatively or additionally, the constructs herein encode
a Lyssavirus L antigen. By "Lyssavirus L antigen" is intended the
amino acid sequence, or a nucleotide sequence encoding the amino
acid sequence, of a known wild-type Lyssavirus RNA polymerase,
variant, or fragment thereof. Thus, where a Lyssavirus L antigen is
a variant of a wild-type Lyssavirus RNA polymerase, the variant may
comprise an amino acid sequence which is at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
a full-length wild-type polypeptide. Alternatively, or in addition,
a fragment of a polypeptide may comprise an immunogenic fragment
(i.e. an epitope-containing fragment) of the full-length
polypeptide which may comprise a contiguous amino acid sequence of
at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 20, or more amino acids which is identical to a
contiguous amino acid sequence of the full-length polypeptide.
[0075] Alternatively or additionally, the constructs herein encode
a Lyssavirus M antigen. By "Lyssavirus M antigen" is intended the
amino acid sequence, or a nucleotide sequence encoding the amino
acid sequence, of a known wild-type Lyssavirus matrix protein,
variant, or fragment thereof. Thus, where a Lyssavirus M antigen is
a variant of a wild-type Lyssavirus matrix protein, the variant may
comprise an amino acid sequence which is at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
a full-length wild-type polypeptide. Alternatively, or in addition,
a fragment of a polypeptide may comprise an immunogenic fragment
(i.e. an epitope-containing fragment) of the full-length
polypeptide which may comprise a contiguous amino acid sequence of
at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 20, or more amino acids which is identical to a
contiguous amino acid sequence of the full-length polypeptide.
[0076] Alternatively or additionally, the constructs herein encode
a Lyssavirus N antigen. By "Lyssavirus N antigen" is intended the
amino acid sequence, or a nucleotide sequence encoding the amino
acid sequence, of a wild-type Lyssavirus nucleoprotein, variant, or
fragment thereof. Thus, where a Lyssavirus N antigen is a variant
of a wild-type Lyssavirus nucleoprotein, the variant may comprise
an amino acid sequence which is at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% identical to a
full-length wild-type polypeptide. Alternatively, or in addition, a
fragment of a polypeptide may comprise an immunogenic fragment
(i.e. an epitope-containing fragment) of the full-length
polypeptide which may comprise a contiguous amino acid sequence of
at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 20, or more amino acids which is identical to a
contiguous amino acid sequence of the full-length polypeptide.
[0077] Alternatively or additionally, the constructs herein encode
a Lyssavirus P antigen. By "Lyssavirus P antigen" is intended the
amino acid sequence, or a nucleotide sequence encoding the amino
acid sequence, of a known wild-type Lyssavirus phosphoprotein,
variant, or fragment thereof. Thus, where a Lyssavirus P antigen is
a variant of a wild-type Lyssavirus phosphoprotein, the variant may
comprise an amino acid sequence which is at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
a full-length wild-type polypeptide. Alternatively, or in addition,
a fragment of a polypeptide may comprise an immunogenic fragment
(i.e. an epitope-containing fragment) of the full-length
polypeptide which may comprise a contiguous amino acid sequence of
at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 20, or more amino acids which is identical to a
contiguous amino acid sequence of the full-length polypeptide.
[0078] Alternatively or additionally, a construct encodes more than
one component of a polypeptide. The components are juxtaposed
immediately next to the adjacent component, i.e., without any
intervening amino acids. Alternatively or additionally, a linker
group of 1, 2, 3, 4, or 5 amino acids is present between one or
more of the polypeptide components.
[0079] Alternatively or additionally, the construct comprises an
RNA nucleic acid sequence comprising one or more antigens. Multiple
antigens can be co-delivered by the constructs of the invention.
Constructs of the invention comprise or consist of recombinant
polycistronic nucleic acid molecules that contain a first sequence
encoding a first Lyssavirus antigen and, optionally, a second
antigen, which may or may not be a Lyssavirus antigen. If desired,
one or more additional sequences, encoding additional antigens, for
example a third antigen, a fourth antigen, a fifth antigen, etc.
can be present in the recombinant RNA. Alternatively or
additionally, constructs of the invention can be polycistronic.
[0080] Polypeptides
[0081] By "polypeptide" is meant a plurality of covalently linked
amino acid residues defining a sequence and linked by amide bonds.
The term is used interchangeably with peptide. The term peptide
also embraces post-translational modifications introduced by
chemical or enzyme-catalyzed reactions, as are known in the art.
The term can refer to a variant or fragment of a polypeptide.
[0082] Alternatively or additionally, a polypeptide herein is in a
non-naturally occurring form (e.g. a recombinant or modified form).
Polypeptides of the invention may have covalent modifications at
the C-terminus and/or N-terminus. They can also take various forms
(e.g. native, fusions, glycosylated, non-glycosylated, lipidated,
non-lipidated, phosphorylated, non-phosphorylated, myristoylated,
non-myristoylated, monomeric, multimeric, particulate, denatured,
etc.). The polypeptides can be naturally or non-naturally
glycosylated (i.e. the polypeptide may have a glycosylation pattern
that differs from the glycosylation pattern found in the
corresponding naturally occurring polypeptide).
[0083] Non-naturally occurring forms of polypeptides herein may
comprise one or more heterologous amino acid sequences (e.g.
another antigen sequence, another signal sequence, a detectable
tag, or the like) in addition to Lyssavirus antigen sequence. For
example, a polypeptide herein may be a fusion protein.
Alternatively, or in addition, the amino acid sequence or chemical
structure of the polypeptide may be modified (e.g. with one or more
non-natural amino acids, by covalent modification, and/or or by
having a different glycosylation pattern, for example, by the
removal or addition of one or more glycosyl groups) compared to a
naturally-occurring polypeptide sequence.
[0084] Alternatively or additionally, the construct encodes a
polypeptide having a sequence selected from the group consisting of
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.
Alternatively or additionally, the construct encodes a polypeptide
which is at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identical to a sequence selected from the group
consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID
NO: 9. Alternatively or additionally, the construct encodes a
polypeptide which comprises a fragment of a full-length sequence
selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7 and SEQ ID NO: 9, wherein the fragment comprises a
contiguous stretch of the amino acid sequence of the full-length
sequence up to 1, 10, 25, 50, 100, 200, 400, 450 or 475 amino acids
shorter than full-length sequence.
[0085] Nucleic Acids
[0086] The term "nucleic acid" means a polymeric form of
nucleotides of any length, which contain deoxyribonucleotides,
ribonucleotides, and/or their analogs. It includes DNA, RNA and
DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those
containing modified backbones (e.g. peptide nucleic acids (PNAs) or
phosphorothioates) or modified bases. Thus the nucleic acid of the
disclosure includes mRNA, DNA, cDNA, recombinant nucleic acids,
branched nucleic acids, plasmids, vectors, etc. Where the nucleic
acid takes the form of RNA, it may or may not have a 5' cap.
[0087] The present inventors disclose herein nucleic acids
comprising one or more nucleic acid sequence which encodes a
Lyssavirus antigen. A nucleic acid, as disclosed herein, can take
various forms (e.g. single-stranded, double-stranded, vector,
etc.). Nucleic acids may be circular or branched, but will
typically be linear.
[0088] The nucleic acids used herein are preferably provided in
purified or substantially purified form i.e., substantially free
from other nucleic acids (e.g. free from naturally-occurring
nucleic acids), particularly from other Lyssavirus or host cell
nucleic acids, typically being at least about 50% pure (by weight),
and usually at least about 90% pure.
[0089] Nucleic acids may be prepared in many ways e.g., by chemical
synthesis (e.g., phosphoramidite synthesis of DNA) in whole or in
part, by digesting longer nucleic acids using nucleases (e.g.,
restriction enzymes), by joining shorter nucleic acids or
nucleotides (e.g., using ligases or polymerases), from genomic or
cDNA libraries.
[0090] The nucleic acids herein comprise a sequence which encodes
at least one Lyssavirus antigen. Typically, the nucleic acids of
the invention will be in recombinant form, i.e., a form which does
not occur in nature. For example, the nucleic acid may comprise one
or more heterologous nucleic acid sequences (e.g., a sequence
encoding another antigen and/or a control sequence such as a
promoter or an internal ribosome entry site) in addition to the
sequence encoding the Lyssavirus antigen. The nucleic acid may be
part of a vector, i.e., part of a nucleic acid construct designed
for transduction/transfection of one or more cell types. Vectors
may be, for example, expression vectors which are designed to
express a nucleotide sequence in a host cell, or viral vectors
which are designed to result in the production of a recombinant
virus or virus-like particle.
[0091] Alternatively, or in addition, the sequence or chemical
structure of the nucleic acid may be modified compared to a
naturally-occurring sequence which encodes a Lyssavirus antigen.
The sequence of the nucleic acid molecule may be modified, e.g. to
increase the efficacy of expression or replication of the nucleic
acid, or to provide additional stability or resistance to
degradation. Alternatively or additionally, a vaccine construct of
the invention is resistant to RNAse digestion in an in vitro
assay.
[0092] The nucleic acid encoding the polypeptides described above
may be modified to increase translation efficacy and/or half-life.
For example, the nucleic acid may be codon optimized or codon-pair
optimized. A poly A tail (e.g., of about 30, about 40 or about 50
adenosine residues or more) may be attached to the 3' end of the
RNA to increase its half-life. The 5' end of the RNA may be capped
with a modified ribonucleotide with the structure m7G (5')ppp(5')N
(cap 0 structure) or a derivative thereof, which can be
incorporated during RNA synthesis or can be enzymatically
engineered after RNA transcription (e.g., by using Vaccinia Virus
Capping Enzyme (VCE) consisting of mRNA triphosphatase,
guanylyl-transferase and guanine-7-methytransferase, which
catalyzes the construction of N7-monomethylated cap 0 structures).
Cap 0 structure plays an important role in maintaining the
stability and translational efficacy of the RNA molecule. The 5'
cap of the RNA molecule may be further modified by a 2
'-O-Methyltransferase which results in the generation of a cap 1
structure (m7Gppp [m2'-O] N), which may further increase
translation efficacy.
[0093] The nucleic acids may comprise one or more nucleotide
analogs or modified nucleotides. As used herein, "nucleotide
analog" or "modified nucleotide" refers to a nucleotide that
contains one or more chemical modifications (e.g., substitutions)
in or on the nitrogenous base of the nucleoside (e.g., cytosine
(C), thymine (T), uracil (U), adenine (A) or guanine (G)). A
nucleotide analog can contain further chemical modifications in or
on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose,
modified ribose, modified deoxyribose, six-membered sugar analog,
or open-chain sugar analog), or in or on the phosphate moiety. Many
modified nucleosides and modified nucleotides are commercially
available.
[0094] Modified nucleobases which can be incorporated into modified
nucleosides and nucleotides and be present in the RNA molecules
include: m5C (5-methylcytidine); m5U (5-methyluridine); m6A
(N6-methyladenosine); s2U (2-thiouridine); Um (2'-0-methyluridine);
mIA (1-methyladenosine); m2A (2-methyladenosine); Am
(2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine);
i6A (N6-isopentenyladenosine); ms2i6A
(2-methylthio-N6isopentenyladenosine); 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'-0-ribosyladenosine (phosphate)); I (inosine); mil
(1-methylinosine); m'Im (I,2'-0-dimethylinosine); m3C
(3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine);
ac4C (N4-acetylcytidine); f5C (5-fonnylcytidine); m5Cm
(5,2-O-dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C
(lysidine); mIG (1-methylguanosine); m2G (N2-methylguanosine); m7G
(7-methylguanosine); Gm (2'-0-methylguanosine); m22G
(N2,N2-dimethylguanosine); m2Gm (N2,2'-0-dimethylguanosine); m22Gm
(N2,N2,2'-0-trimethylguanosine); Gr(p) (2'-0-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'-0-dimethyluridine); s4U
(4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um
(2-thio-2'-0-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'-0-methyluridine); cmnm5U
(5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1
aminomethyl-2-L-Omethyl uridine); cmnm5s2U
(5-carboxymethylaminomethyl-2-thiouridine); m62A
(N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C
(N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C
(5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U
(5-carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am
(N6,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine);
m2'2'7G (N2,N2,7-trimethylguanosine); m3Um
(3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); .English
Pound.5Cm (5-formyl-2'-0-methylcytidine); mIGm
(I,2'-0-dimethylguanosine); m'Am (1,2-0-dimethyl adenosine)
irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine));
iniG-14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A
(N6-acetyladenosine); hypoxanthine; inosine; 8-oxo-adenine;
7-substituted derivatives thereof, dihydrouracil; pseudouracil;
2-thiouracil; 4-thiouracil; 5-aminouracil; 5-(Ci-Ce)-alkyluracil;
5-methyluracil; 5-(C2-C6)-alkenyluracil; 5-(C2-Ce)-alkynyluracil;
5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouracil;
5-bromouracil; 5-hydroxycytosine; 5-(Ci-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; hydrogen (abasic residue); m5C; m5U; m6A; s2U; W; or
2'-0-methyl-U. Many of these modified nucleobases and their
corresponding ribonucleosides are available from commercial
suppliers.
[0095] Alternatively or additionally, the construct comprises a DNA
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. Alternatively
or additionally, the construct comprises a nucleic acid sequence
which is at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identical to a sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID
NO: 8. Alternatively or additionally, the construct comprises a
nucleic acid sequence which comprises a fragment of a full-length
sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, wherein the fragment
comprises a contiguous stretch of the nucleic acid sequence of the
full-length sequence up to 1, 10, 25, 50, 100, 200, 300, 400, 450
or 475 nucleic acids shorter than the full-length sequence.
[0096] Nucleic Acid Based Vaccines
[0097] The present invention discloses compositions comprising a
nucleic acid sequence which encodes a polypeptide comprising a
Lyssavirus antigen, variant or fragment thereof. Such compositions
may be a nucleic acid-based vaccine. A further composition
comprising a nucleic acid sequence which encodes one or more
additional Lyssavirus antigens may also be provided as a nucleic
acid-based vaccine. Alternatively or additionally, a composition
comprises a nucleic acid sequence encoding a Lyssavirus antigen
from a first Lyssavirus strain and an additional nucleic acid
sequence encoding an additional Lyssavirus antigen from one or more
other strains of Lyssavirus. Alternatively or additionally, a
composition comprises a nucleic acid sequence encoding a Lyssavirus
antigen and an additional Lyssavirus antigen. Alternatively, an
additional non-Lyssavirus antigen may be encoded.
[0098] The nucleic acid may, for example, be RNA (i.e., an
RNA-based vaccine) or DNA (i.e., a DNA-based vaccine, such as a
plasmid DNA vaccine). Alternatively or additionally, the nucleic
acid-based vaccine is an RNA-based vaccine. Alternatively or
additionally, the RNA-based vaccine comprises a self-amplifying RNA
molecule. The self-amplifying RNA molecule may be an
alphavirus-derived RNA replicon.
[0099] As used herein, the term "alphavirus" has its conventional
meaning in the art and includes various species such as Venezuelan
equine encephalitis virus (VEE e.g., Trinidad donkey, TC83CR,
etc.), Semliki Forest virus (SFV), Sindbis virus, Ross River virus,
Western equine encephalitis virus, Eastern equine encephalitis
virus, Chikungunya virus, S.A. AR86 virus, Everglades virus,
Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus,
O' nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus,
Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki
virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus,
Ndumu virus, and Buggy Creek virus. The term alphavirus may also
include chimeric alphaviruses that contain genome sequences from
more than one alphavirus.
[0100] An "alphavirus replicon particle" (VRP) or "replicon
particle" is an alphavirus replicon packaged with alphavirus
structural proteins.
[0101] An "alphavirus replicon" (or "replicon") is an RNA molecule
which can direct its own amplification in vivo in a target cell.
The replicon encodes the polymerase(s) which catalyzes RNA
amplification and contains cis RNA sequences required for
replication which are recognized and utilized by the encoded
polymerase(s). An alphavirus replicon typically contains the
following ordered elements: 5' viral sequences required in cis for
replication, sequences which encode biologically active alphavirus
nonstructural proteins (nsP1, nsP2, nsP3, nsP4), 3' viral sequences
required in cis for replication, and a polyadenylate tract. An
alphavirus replicon also may contain one or more viral subgenomic
"junction region" promoters directing the expression of
heterologous nucleotide sequences, which may be modified in order
to increase or reduce viral transcription of the subgenomic
fragment and heterologous sequence(s) to be expressed.
[0102] "Self-amplifying RNA," and "RNA replicon" are used
interchangeably to mean RNA with the ability to replicate itself.
The self-amplifying RNA molecules of the invention comprise mRNA
encoding one or more antigens. This mRNA can replace nucleic acid
sequences encoding structural proteins required for the production
of infectious virus. The RNA can be produced in vitro by enzymatic
transcription, thereby avoiding manufacturing issues associated
with cell culture production of vaccines. After immunization with a
self-amplifying RNA molecule of the invention, replication and
amplification of the RNA molecule occur in the cytoplasm of the
transfected cell and the nucleic acid is not integrated into the
genome. As the RNA does not integrate into the genome and transform
the target cell, self-amplifying RNA vaccines do not pose the
safety hurdles faced by recombinant DNA vaccines.
[0103] Self-amplifying RNA molecules are known in the art and can
be produced by using replication elements derived from, e.g.,
alphaviruses, and substituting structural viral proteins with a
nucleotide sequence encoding a protein of interest. A
self-amplifying RNA molecule is typically a plus-strand molecule
which can be directly translated after delivery to a cell. This
translation provides an 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 antigen (e.g., a Lyssavirus antigen), or
may be transcribed to provide further transcripts with the same
sense as the delivered RNA, which are then translated to provide in
situ expression of the antigen. The overall result of this sequence
of transcriptions is a huge amplification in the number of the
introduced replicon RNAs and so the encoded antigen becomes a major
polypeptide product of the cells.
[0104] One suitable system for achieving self-replication in this
manner is to use an alphavirus-based replicon. These replicons are
plus-stranded RNAs which lead to the translation of a replicase (or
replicase-transcriptase) following their delivery to a cell. The
replicase is translated as a polyprotein which auto-cleaves to
provide a replication complex which creates genomic-strand copies
of the plus-strand delivered RNA. These minus-strand transcripts
can themselves be transcribed to give further copies of the
plus-stranded parent RNA and also to give a subgenomic transcript
which encodes the antigen. Translation of the subgenomic transcript
leads to in situ expression of the antigen 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.
[0105] Self-amplifying RNAs contain the basic elements of mRNA,
i.e., a cap, 5'UTR, 3'UTR and a poly(A) tail. They additionally
comprise a large open reading frame (ORF) that encodes
non-structural viral genes and one or more subgenomic promoter. The
nonstructural genes, which include a polymerase, form intracellular
RNA replication factories and transcribe the subgenomic RNA at high
levels. This mRNA encoding the vaccine antigen(s) is amplified in
the cell, resulting in high levels of mRNA and antigen
expression.
[0106] Alternatively or additionally, the self-amplifying RNA
molecule described herein encodes (i) an RNA-dependent RNA
polymerase which can transcribe RNA from the self-amplifying RNA
molecule and (ii) a Lyssavirus antigen. The polymerase can be an
alphavirus replicase e.g., comprising one or more of the
non-structural alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
[0107] Whereas natural alphavirus genomes encode structural virion
proteins in addition to the non-structural replicase polyprotein,
alternatively or additionally, the self-amplifying RNA molecules do
not encode alphavirus structural proteins. Thus, the
self-amplifying 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-amplifying 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-amplifying RNAs of the
present disclosure 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.
[0108] Thus a self-amplifying RNA molecule useful with the
invention may have two open reading frames. The first open reading
frame encodes a replicase; the second open reading frame encodes an
antigen. Alternatively or additionally, the RNA may have one or
more additional (e.g. downstream) open reading frames, e.g. to
encode further antigen(s) or to encode accessory polypeptides.
[0109] Alternatively or additionally, the self-amplifying RNA
molecule disclosed herein has a 5' cap (e.g. a 7-methylguanosine).
This cap can enhance in vivo translation of the RNA. Alternatively
or additionally, the 5' sequence of the self-amplifying RNA
molecule must be selected to ensure compatibility with the encoded
replicase.
[0110] A self-amplifying 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.
[0111] Self-amplifying RNA molecules can have various lengths but
they are typically 5000-25000 nucleotides long. Self-amplifying RNA
molecules 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.
[0112] The self-amplifying RNA 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 example, a DNA-dependent RNA
polymerase, such as the bacteriophage T7, T3 or SP6 RNA
polymerases, can be used to transcribe the self-amplifying 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.
[0113] The self-amplifying RNA can include, in addition to any 5'
cap structure, one or more nucleotides having a modified
nucleobase. An 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.
[0114] The self-amplifying RNA molecule may encode a single
heterologous polypeptide antigen (i.e., a Lyssavirus antigen) or,
optionally, two or more heterologous polypeptide antigens linked
together in a way that each of the sequences retains its identity
(e.g., linked in series) when expressed as an amino acid sequence.
The heterologous polypeptides generated from the self-amplifying
RNA may then be produced as a fusion polypeptide or engineered in
such a manner as to result in separate polypeptide or peptide
sequences.
[0115] The self-amplifying RNA molecules described herein may be
engineered to express multiple nucleotide sequences, from two or
more open reading frames, thereby allowing co-expression of
proteins, such as one, two or more Lyssavirus antigens (e.g. one,
two or Lyssavirus antigens) together with cytokines or other
immunomodulators, which can enhance the generation of an immune
response. Such a self-amplifying RNA molecule might be particularly
useful, for example, in the production of various gene products
(e.g., proteins) at the same time, for example, as a bivalent or
multivalent vaccine.
[0116] If desired, the self-amplifying RNA molecules can be
screened or analyzed to confirm their therapeutic and prophylactic
properties using various in vitro or in vivo testing methods that
are known to those of skill in the art. For example, the Rapid
Fluorescent Focus Inhibition Test (RFFIT) can measure the level of
rabies virus neutralizing activity. Vaccines comprising a
self-amplifying RNA molecule can be tested for their effect on the
induction of proliferation or on the effector function of a
particular lymphocyte type of interest, e.g., B cells, T cells, T
cell lines or T cell clones. For example, spleen cells from
immunized mice can be isolated and the capacity of cytotoxic T
lymphocytes to lyse autologous target cells that contain a
self-amplifying RNA molecule encoding a Lyssavirus antigen. In
addition, T helper cell differentiation can be analyzed by
measuring proliferation or production of TH1 (IL-2 and IFN-.gamma.)
and/or TH2 (IL-4 and IL-5) cytokines by ELISA or directly in CD4+ T
cells by cytoplasmic cytokine staining and flow cytometry.
[0117] Self-amplifying RNA molecules that encode a Lyssavirus
antigen can also be tested for ability to induce humoral immune
responses, as evidenced, for example, by induction of B cell
production of antibodies specific for a Lyssavirus antigen of
interest. These assays can be conducted using, for example,
peripheral B lymphocytes from immunized individuals. Such assay
methods are known to those of skill in the art. Other assays that
can be used to characterize the self-amplifying RNA molecules can
involve detecting expression of the encoded Lyssavirus antigen by
the target cells. For example, fluorescent activated cell sorting
(FACS) can be used to detect antigen expression on the cell surface
or intracellularly. Another advantage of FACS selection is that one
can sort for different levels of expression, as sometimes a lower
expression may be desired. Other suitable methods for identifying
cells which express a particular antigen involve panning using
monoclonal antibodies on a plate or capture using magnetic beads
coated with monoclonal antibodies.
[0118] Alternatively or additionally, a DNA sequence encoding a
self-amplifying RNA molecule is provided, and can be selected, from
the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6
and SEQ ID NO: 8. Alternatively or additionally, DNA sequence
encoding a self-amplifying RNA molecule comprises a sequence which
is at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% identical to a sequence selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID
NO: 8. Alternatively or additionally, the DNA sequence encoding a
self-amplifying RNA molecule comprises or consists of a fragment of
a full-length sequence selected from the group consisting of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 wherein the
fragment comprises or consists of a contiguous stretch of the
nucleic acid sequence of the full-length sequence up to 1, 10, 25,
50, 100, 200, 300, 400, 450 or 475 nucleic acids shorter than
full-length sequence.
[0119] Lipid-Based Delivery Systems
[0120] The nucleic acid-based vaccines of the invention may
comprise a non-viral delivery system, e.g., a lipid-based delivery
system. These systems can efficiently deliver a self-amplifying RNA
vaccine to the interior of a cell, where it can then replicate and
express the encoded antigen(s).
[0121] The delivery system may have adjuvant effects which enhance
the immunogenicity of the encoded Lyssavirus antigen. For example,
the nucleic acid molecule may be encapsulated in liposomes or
non-toxic biodegradable polymeric microparticles. Alternatively or
additionally, the nucleic acid-based vaccine comprises a lipid
nanoparticle (LNP) delivery system. Alternatively or additionally,
the nucleic molecule may be delivered as a cationic nanoemulsion
(CNE). Alternatively or additionally, the nucleic acid-based
vaccine may comprise a naked nucleic acid, such as naked RNA (e.g.
mRNA), but lipid-based delivery systems are preferred.
[0122] "Lipid nanoparticles (LNPs)" are non-virion liposome
particles in which a nucleic acid molecule (e.g. RNA) can be
encapsulated. LNP delivery systems and non-toxic biodegradable
polymeric microparticles, and methods for their preparation are
known in the art. The particles can include some external RNA (e.g.
on the surface of the particles), but at least half of the RNA (and
preferably all of it) is encapsulated. Liposomal particles can, for
example, be formed of a mixture of zwitterionic, cationic and
anionic lipids which can be saturated or unsaturated, for example
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (zwitterionic,
saturated), 1,2-dilinoleyoxy-3-dimethylaminopropane (DIinDMA)
(cationic, unsaturated), and/or 1,2-dimyristoyl-rac-glycerol (DMG)
(anionic, saturated). Preferred LNPs for use with the invention
include a zwitterionic lipid which can form liposomes, optionally
in combination with at least one cationic lipid (such as
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium
methyl-sulfate (DOTAPBis(2-methacryloyl)oxyethyl disulfide (DSDMA),
2,3-Dioleyloxy-1-(dimethylamino)propane (DODMA),
1,2-dilinoleyoxy-3-dimethylaminopropane (DLinDMA),
N,N-dimethyl-3-aminopropane (DLenDMA), etc.). A mixture of DSPC,
DIinDMA, PEG-DMG and cholesterol is particularly effective.
Alternatively or additionally, the LNPs are RV01 liposomes.
##STR00001##
[0123] Alternatively or additionally, the LNP comprises neutral
lipids, cationic lipids, cholesterol and polyethylene glycol (PEG)
and forms nanoparticles that encompass the self-amplifying RNA. In
some embodiments, the cationic lipids herein comprise the structure
of Formula I:
##STR00002##
[0124] wherein n=an integer from 1 to 3 and
[0125] (i) R.sub.1 is CH.sub.3, R.sub.2 and R.sub.3 are both H, and
Y is C; or
[0126] (ii) R.sub.1 and R.sub.2 are collectively CH.sub.2--CH.sub.2
and together with the nitrogen form a five-, six-, or
seven-membered heterocycloalkyl, R.sub.3 is CH.sub.3, and Y is C;
or
[0127] (iii) R.sub.1 is CH.sub.3, R.sub.2 and R.sub.3 are both
absent, and Y is O;
[0128] wherein o is 0 or 1;
[0129] wherein X is:
##STR00003##
wherein R.sub.4 and R.sub.5 are independently a C.sub.10-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; or
[0130] (ii) --CH(--R.sub.6)--R.sub.7, wherein [0131] (1) R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8 or --C.sub.p--R.sub.8; [0132]
(2) R.sub.7 is --(CH.sub.2).sub.p--O--C(O)--R.sub.8' or
--C.sub.p'--R.sub.8', [0133] (3) p and p' are independently 0, 1,
2, 3 or 4; and [0134] (4) R.sub.8 and R.sub.8', are independently a
[0135] (A) --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions;
[0136] (B) --C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; [0137] (C) --C.sub.6-16 saturated
hydrocarbon chain; [0138] (D) --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; [0139] (E)
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and [0140] (F) --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0141] In an embodiment, R.sub.1 is CH.sub.3, R.sub.2 and R.sub.3
are both H, and Y is C. In some embodiments, R.sub.1 and R.sub.2
are collectively CH.sub.2--CH.sub.2 and together with the nitrogen
form a five-, six-, or seven-membered heterocycloalkyl, R.sub.3 is
CH.sub.3, and Y is C. In some embodiments, R.sub.1 is CH.sub.3,
R.sub.2 and R3 are both absent, and Y is O.
[0142] In an embodiment, X is
##STR00004##
wherein R.sub.4 and R.sub.5 are independently a C.sub.10-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0143] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0144] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0145] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0146] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0147] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0148] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0149] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0150] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0151] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0152] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0153] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0154] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0155] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having one
or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0156] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0157] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0158] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0159] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0160] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0161] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.8-20 hydrocarbon chain having one or two cis alkene groups
at either or both of the omega 6 and 9 positions.
[0162] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0163] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated hydrocarbon chain.
[0164] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0165] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0166] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated or unsaturated hydrocarbon chain.
[0167] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0168] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0169] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0170] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0171] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0172] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0173] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0174] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0175] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0176] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0177] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0178] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0179] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions.
[0180] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12
saturated or unsaturated hydrocarbon chain.
[0181] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C.sub.6-16 saturated hydrocarbon chain.
[0182] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0183] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12
saturated or unsaturated hydrocarbon chain.
[0184] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.8-20 hydrocarbon chain having one or two cis
alkene groups at either or both of the omega 6 and 9 positions; and
R.sub.8' is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0185] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0186] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7--C.sub.p'--R.sub.8',
p and p' are independently 0, 1, 2, 3 or 4; R.sub.8 is
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0187] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16,
saturated hydrocarbon chain.
[0188] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0189] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0190] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated
or unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16,
saturated or unsaturated hydrocarbon chain.
[0191] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C.sub.8-20 hydrocarbon chain having one or two cis alkene
groups at either or both of the omega 6 and 9 positions.
[0192] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0193] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C.sub.6-16 saturated hydrocarbon chain.
[0194] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0195] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12
saturated or unsaturated hydrocarbon chain.
[0196] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
R.sub.8 is a --C.sub.6-16 saturated hydrocarbon chain; and R.sub.8'
is a --C.sub.6-16 saturated or unsaturated hydrocarbon chain.
[0197] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0198] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.3-12 saturated or
unsaturated hydrocarbon chain.
[0199] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0200] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0201] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0202] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0203] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0204] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0205] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0206] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0207] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0208] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0209] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having one
or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0210] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4; and
R.sub.7 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0211] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0212] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0213] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0214] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8, R.sub.7 is
--C.sub.p'--R.sub.8', p and p' are independently 0, 1, 2, 3 or 4;
and R.sub.8 is a --C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0215] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0216] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0217] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0218] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0219] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a
--C[--C--O--OC(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0220] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20 hydrocarbon chain having
one or two cis alkene groups at either or both of the omega 6 and 9
positions; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0221] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0222] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0223] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0224] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0225] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0226] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0227] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having one
or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0228] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0229] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0230] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0231] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0232] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0233] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.8-20 hydrocarbon chain having one or two cis alkene groups
at either or both of the omega 6 and 9 positions.
[0234] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.3-12 saturated or
unsaturated hydrocarbon chain.
[0235] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated hydrocarbon chain.
[0236] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0237] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0238] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated or unsaturated hydrocarbon chain.
[0239] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0240] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0241] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0242] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0243] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0244] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0245] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p'--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0246] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0247] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0248] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0249] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0250] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is
--(CH.sub.2).sub.p--O--C(O)--R.sub.8', p and p' are independently
0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0251] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0252] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0253] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0254] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0255] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0256] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0257] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0258] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0259] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0260] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0261] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0262] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is
a-C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0263] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain
having one or two cis alkene groups at either or both of the omega
6 and 9 positions.
[0264] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0265] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a --C.sub.6-16 saturated
hydrocarbon chain.
[0266] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0267] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0268] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; R.sub.8 is a --C.sub.6-16 saturated
hydrocarbon chain; and R.sub.8' is a --C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0269] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.8-20 hydrocarbon chain having one
or two cis alkene groups at either or both of the omega 6 and 9
positions.
[0270] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0271] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated hydrocarbon
chain.
[0272] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C(--C.sub.6-16)--C.sub.6-16 saturated or
unsaturated hydrocarbon chain.
[0273] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0274] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain; and R.sub.8' is a --C.sub.6-16 saturated or unsaturated
hydrocarbon chain.
[0275] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.8-20
hydrocarbon chain having one or two cis alkene groups at either or
both of the omega 6 and 9 positions.
[0276] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0277] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated hydrocarbon chain.
[0278] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0279] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0280] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--C(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain; and R.sub.8' is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain.
[0281] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.8-20 hydrocarbon chain having one or two cis alkene groups
at either or both of the omega 6 and 9 positions.
[0282] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.1-3--C(--O--C.sub.6-12)--O--C.sub.6-12 saturated or
unsaturated hydrocarbon chain.
[0283] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.8 is
--C.sub.p--R.sub.8, R.sub.7--C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.7 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated hydrocarbon chain.
[0284] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C(--C.sub.6-16)--C.sub.6-16 saturated or unsaturated hydrocarbon
chain.
[0285] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8'', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C[--C--O--C(O)--C.sub.4-12]--C--O--OC(O)--C.sub.4-12 saturated or
unsaturated hydrocarbon chain.
[0286] In an embodiment, X is --CH(--R.sub.6)--R.sub.7, R.sub.6 is
--C.sub.p--R.sub.8, R.sub.7 is --C.sub.p--R.sub.8', p and p' are
independently 0, 1, 2, 3 or 4; and R.sub.8 is a --C.sub.6-16
saturated or unsaturated hydrocarbon chain; and R.sub.8' is a
--C.sub.6-16 saturated or unsaturated hydrocarbon chain.
[0287] In an embodiment, an exemplary cationic lipid is RV28 having
the following structure:
##STR00005##
[0288] In an embodiment, an exemplary cationic lipid is RV31 having
the following structure:
##STR00006##
[0289] In an embodiment, an exemplary cationic lipid is RV33 having
the following structure:
##STR00007##
[0290] In an embodiment, an exemplary cationic lipid is RV37 having
the following structure:
##STR00008##
[0291] In an embodiment, the LNP comprises the cationic lipid RV39,
i.e. 2,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl
4-(dimethylamino)butanoate):
##STR00009##
[0292] In an embodiment, an exemplary cationic lipid is RV42 having
the following structure:
##STR00010##
[0293] In an embodiment, an exemplary cationic lipid is RV44 having
the following structure:
##STR00011##
[0294] In an embodiment, an exemplary cationic lipid is RV73 having
the following structure:
##STR00012##
[0295] In an embodiment, an exemplary cationic lipid is RV75 having
the following structure:
##STR00013##
[0296] In an embodiment, an exemplary cationic lipid is RV81 having
the following structure:
##STR00014##
[0297] In an embodiment, an exemplary cationic lipid is RV84 having
the following structure:
##STR00015##
[0298] In an embodiment, an exemplary cationic lipid is RV85 having
the following structure:
##STR00016##
[0299] In an embodiment, an exemplary cationic lipid is RV86 having
the following structure:
##STR00017##
[0300] In an embodiment, an exemplary cationic lipid is RV88 having
the following structure:
##STR00018##
[0301] In an embodiment, an exemplary cationic lipid is RV91 having
the following structure:
##STR00019##
[0302] In an embodiment, an exemplary cationic lipid is RV92 having
the following structure:
##STR00020##
[0303] In an embodiment, an exemplary cationic lipid is RV93 having
the following structure:
##STR00021##
[0304] In an embodiment, an exemplary cationic lipid is
2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)hexadecyl)oxy)-5-oxopent-
yl)propane-1,3-diyl dioctanoate (RV94), having the following
structure:
##STR00022##
[0305] In an embodiment, an exemplary cationic lipid is RV95 having
the following structure:
##STR00023##
[0306] In an embodiment, an exemplary cationic lipid is RV96 having
the following structure:
##STR00024##
[0307] In an embodiment, an exemplary cationic lipid is RV97 having
the following structure:
##STR00025##
[0308] In an embodiment, an exemplary cationic lipid is RV99 having
the following structure:
##STR00026##
[0309] In an embodiment, an exemplary cationic lipid is RV101
having the following structure:
##STR00027##
[0310] In an embodiment, the cationic lipid is selected from the
group consisting of: RV39, RV88, and RV94.
[0311] Compositions and methods for the synthesis of compounds
having Formula I and RV28, RV31, RV33, RV37, RV39, RV42, RV44,
RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94,
RV95, RV96, RV97, RV99, and RV101 can be found in PCT/US2014/070882
(publication number WO/2015/095340) and PCT/US2014/070891
(publication number WO/2015/095346), filed 17 Dec. 2014; as well as
PCT/US2015/048535 (publication number WO/2016/037053), filed 4 Sep.
2015.
[0312] The ratio of RNA to lipid can be varied. The ratio of
nucleotide (N) to phospholipid (P) can be in the range of, e.g.,
1N:1P, 2N:1P, 3N:1P, 4N:1P, 5N:1P, 6N:1P, 7N:1P, 8N:1P, 9N:1P, or
10N:1P. The ratio of nucleotide (N) to phospholipid (P) can be in
the range of, e.g., 1N:1P to 10N:1P, 2N:1P to 8N:1P, 2N:1P to 6N:1P
or 3N:1P to 5N:1P. Alternatively or additionally, the ratio of
nucleotide (N) to phospholipid (P) is 4N:1P.
[0313] Alternatively or additionally, the nucleic acid-based
vaccine comprises a cationic nanoemulsion (CNE) delivery system.
Cationic oil-in water emulsions can be used to deliver negatively
charged molecules, such as RNA molecules, to the interior of a
cell. The emulsion particles comprise a hydrophobic oil core and a
cationic lipid, the latter of which can interact with the RNA,
thereby anchoring it to the emulsion particle. In a CNE delivery
system, the nucleic acid molecule (e.g., RNA) which encodes the
antigen is complexed with a particle of a cationic oil-in-water
emulsion.
[0314] Thus, in a nucleic acid-based vaccine of the invention, an
RNA molecule encoding a Lyssavirus antigen may be complexed with a
particle of a cationic oil-in-water emulsion. The particles
typically comprise an oil core (e.g. a plant oil or squalene) that
is in liquid phase at 25.degree. C., a cationic lipid (e.g.
phospholipid) and, optionally, a surfactant (e.g. sorbitan
trioleate, polysorbate 80); polyethylene glycol can also be
included. Alternatively or additionally, the CNE comprises squalene
and a cationic lipid, such as
1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP). In an
embodiment, the CNE is an oil in water emulsion of DOTAP and
squalene stabilized with polysorbate
[0315] The LNP and CNE delivery systems of the invention can be
particularly effective in eliciting both humoral and cellular
immune responses. Advantages of these delivery systems also include
the absence of a limiting anti-vector immune response.
[0316] Pharmaceutical Compositions, Immunogenic Compositions
[0317] The disclosure provides compositions comprising a nucleic
acid comprising a sequence which encodes a Lyssavirus polypeptide,
for example a Lyssavirus antigen. The composition may be a
pharmaceutical composition, e.g., an immunogenic composition or a
vaccine composition. Accordingly, the composition may also comprise
a pharmaceutically acceptable carrier. In some embodiments, the
Lyssavirus is a rabies virus.
[0318] A "pharmaceutically acceptable carrier" includes any carrier
that does not itself induce the production of antibodies harmful to
the individual receiving the composition. The compositions of the
invention may also contain a pharmaceutically acceptable diluent,
such as water, sterile pyrogen-free water, saline,
phosphate-buffered physiologic saline, glycerol, etc. Additionally,
auxiliary substances, such as wetting or emulsifying agents, pH
buffering substances, and the like, may be present.
[0319] Pharmaceutical compositions may include the constructs,
nucleic acid sequences, and/or polypeptide sequences described
elsewhere herein in plain water (e.g. water for injection "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. Pharmaceutical compositions may have a pH between 5.0 and
9.5, e.g. between 6.0 and 8.0. Compositions may include sodium
salts, e.g. sodium chloride, to give tonicity.
[0320] A concentration of 10.+-.2 mg/ml NaCl is typical, e.g. about
9 mg/ml. Compositions 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 10-500 .mu.M, e.g., 0.1 mM. A citrate
salt, such as sodium citrate, can also act as a chelator, while
advantageously also providing buffering activity.
[0321] Pharmaceutical compositions 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. Pharmaceutical compositions may include
one or more preservatives, such as thiomersal or 2-phenoxyethanol.
Mercury-free compositions are preferred, and preservative-free
vaccines can be prepared. Pharmaceutical compositions may be
aseptic or sterile. Pharmaceutical compositions may be
non-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard
measure) per dose, and preferably <0.1 EU per dose.
Pharmaceutical compositions may be gluten free. Pharmaceutical
compositions may be prepared in unit dose form. Alternatively or
additionally, a unit dose may have a volume of between 0.1-2.0 ml,
e.g. about 1.0 or 0.5 ml.
[0322] A composition of the invention may be administered with or
without an adjuvant. Alternatively or additionally, the composition
may comprise, or be administered in conjunction with, one or more
adjuvants (e.g. vaccine adjuvants), in particular where the
composition comprises an immunologically effective amount of a
nucleic acid encoding a Lyssavirus antigen.
[0323] By "adjuvant" is meant an agent that augments, stimulates,
activates, potentiates or modulates the immune response to an
active ingredient of the composition. The adjuvant effect may occur
at the cellular or humoral level or both. Adjuvants stimulate the
response of the immune system to the actual antigen but have no
immunological effect themselves. Alternatively or additionally,
adjuvented compositions of the invention may comprise one or more
immunostimulants. By "immunostimulant" it is meant an agent that
induces a general, temporary increase in a subject's immune
response, whether administered with the antigen or separately.
[0324] Methods of Use/Uses
Methods are provided for inducing an immune response against a
disease caused by a Lyssavirus in a subject in need thereof
comprising a step of administering an immunologically effective
amount of a construct or composition as disclosed herein. In some
embodiments are provided the use of the constructs or compositions
disclosed herein for inducing an immune response to a Lyssavirus
antigen in a subject in need thereof. In some embodiments are
provided use of the construct or composition as disclosed herein in
the manufacture of a medicament inducing an immune response to a
Lyssavirus in a subject.
[0325] By "subject" is intended a vertebrate, such as a mammal e.g.
a human or a veterinary mammal. In some embodiments the subject is
human.
[0326] Routes of Administration
[0327] Compositions disclosed herein will generally be administered
directly to a subject. Direct delivery may be accomplished by
parenteral injection, e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, intradermally, or to the
interstitial space of a tissue. Self-amplifying RNA encoding
Lyssavirus antigens can be given either prophylactically or
therapeutically to individuals of all ages. When given
prophylactically, e.g., administered to residents of or travelers
to areas endemic for rabies, the dosing schedule may consist of
three doses, two doses or one dose. Alternatively or additionally,
one dose is administered prophylactically. When given
therapeutically, e.g., administered after a rabies exposure, the
dosing schedule may consist of five doses, four doses, three doses,
two doses or one dose. In a preferred embodiment, one or two doses
are administered therapeutically.
[0328] As used herein, administration of a composition "followed
by" administration of a composition indicates that a time interval
has elapsed between administration of a first composition and
administration of a second composition, regardless of whether the
first and second compositions are the same or different.
[0329] Processes of Manufacturing and Formulations
[0330] Alternatively or additionally, the process of manufacturing
a self-amplifying RNA comprises a step of in vitro transcription
(IVT). In some embodiments, the process of manufacturing a
self-amplifying RNA comprises a step of IVT to produce an RNA,
followed by a capping 5' dinucleotide m7G(5')ppp(5')G reaction and
further comprises a step of combining the RNA with a non-viral
delivery system. Alternatively or additionally, the process of
manufacturing a self-amplifying RNA comprises a step of IVT to
produce an RNA, and further comprises a step of combining the RNA
with a lipid based delivery system.
[0331] Sequence Identity
[0332] Identity with respect to a sequence is defined herein as the
percentage of amino acid residues in the candidate sequence that
are identical with the reference amino acid sequence after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
[0333] Sequence identity can be determined by standard methods that
are commonly used to compare the similarity in position of the
amino acids of two polypeptides. Using a computer program such as
BLAST or FASTA, two polypeptides are aligned for optimal matching
of their respective amino acids (either along the full length of
one or both sequences or along a pre-determined portion of one or
both sequences). The programs provide a default opening penalty and
a default gap penalty, and a scoring matrix such as PAM 250 or
swgapdnamt can be used in conjunction with the computer program. In
an embodiment, the gap opening penalty is 15, the gap extension
penalty is 6.66, the gap separation penalty range is eight and the
percent identity for alignment delay is 40. By way of example, the
percent identity can be calculated as the total number of identical
matches multiplied by 100 and then divided by the sum of the length
of the longer sequence within the matched span and the number of
gaps introduced into the shorter sequences in order to align the
two sequences.
[0334] Where the present disclosure refers to a sequence by
reference to a UniProt or GenBank accession code, the sequence
referred to is the current version as of the filing date of the
present application.
[0335] The skilled person will recognise that individual
substitutions, deletions or additions to a protein which alters,
adds or deletes a single amino acid or a small percentage of amino
acids is an "immunogenic derivative" where the alteration(s)
results in the substitution of an amino acid with a functionally
similar amino acid or the substitution/deletion/addition of
residues which do not impact the immunogenic function.
[0336] Conservative substitution tables providing functionally
similar amino acids are well known in the art. In general, such
conservative substitutions will fall within one of the amino-acid
groupings specified below, though in some circumstances other
substitutions may be possible without substantially affecting the
immunogenic properties of the antigen. The following eight groups
each contain amino acids that are typically conservative
substitutions for one another: [0337] 1) Alanine (A), Glycine (G);
[0338] 2) Aspartic acid (D), Glutamic acid (E); [0339] 3)
Asparagine (N), Glutamine (Q); [0340] 4) Arginine (R), Lysine (K);
[0341] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
[0342] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); [0343]
7) Serine (S), Threonine (T); and [0344] 8) Cysteine (C),
Methionine (M)
[0345] Suitably such substitutions do not occur in the region of an
epitope, and do not therefore have a significant impact on the
immunogenic properties of the antigen.
[0346] Immunogenic derivatives may also include those wherein
additional amino acids are inserted compared to the reference
sequence. Suitably such insertions do not occur in the region of an
epitope, and do not therefore have a significant impact on the
immunogenic properties of the antigen. One example of insertions
includes a short stretch of histidine residues (e.g. 2-6 residues)
to aid expression and/or purification of the antigen in
question.
[0347] Immunogenic derivatives include those wherein amino acids
have been deleted compared to the reference sequence. Suitably such
deletions do not occur in the region of an epitope, and do not
therefore have a significant impact on the immunogenic properties
of the antigen.
[0348] The skilled person will recognise that a particular
immunogenic derivative may comprise substitutions, deletions and
additions (or any combination thereof).
[0349] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. The term "plurality" refers to two or more.
Additionally, numerical limitations given with respect to
concentrations or levels of a substance, such as solution component
concentrations or ratios thereof, and reaction conditions such as
temperatures, pressures and cycle times are intended to be
approximate. The term "about" used herein is intended to mean the
amount .+-.10%.
[0350] The present invention will now be further described by means
of the following non-limiting examples. The results shown in the
Examples below demonstrate that self-amplifying RNA encoding
Lyssaviral antigens convey potent and long-lasting immunity when
formulated in various lipid compositions. Their ease of
manufacture, effectiveness at low doses and superiority to existing
vaccines provides a significant breakthrough in rabies prevention
and post-exposure treatment.
EXAMPLES
Example 1: Construct Design of SAM with Lyssavirus Antigens
[0351] The present inventors initiated work on a Lyssavirus vaccine
using synthetic, self-amplifying mRNA ("SAM") derived from
alphavirus replicons. The SAM vector derived from VEE TC-83 (SEQ ID
NO: 1) was chosen as the backbone for cloning. Lyssaviral antigens
of interest were expressed by this vector and evaluated for robust
antigen production, immunogenicity, and efficacy using in vitro and
in vivo models.
TABLE-US-00001 TABLE 1 SAM-Rabies G Protein Constructs Rabies G
Protein SEQ ID NO Construct 1 Medoid SEQ ID NO: 2 Flury HEP SEQ ID
NO: 3 Construct 2 Flury LEP SEQ ID NO: 4 RABAVERT SEQ ID NO: 5
Construct 3 Codon SEQ ID NO: 6 optimized SEQ ID NO: 7 Construct 4
Codon pair SEQ ID NO: 8 optimized SEQ ID NO: 9
[0352] SAM Rabies constructs of the invention are exemplified by
the constructs described in Table 1 and FIG. 1A. These constructs
all express rabies full length glycoprotein ("G"). Construct 1
encodes a medoid G protein sequence and is closely related to the
Flury-HEP and ERA strains of rabies virus. Construct 2 encodes the
wild-type sequence of the Flury-LEP strain, which is the strain of
the licensed RABAVERT vaccine (GenBank GU565703.1). Constructs 3
and 4 are derived from the wild-type sequence of the Flury-LEP
strain. Construct 3 was codon optimized using a proprietary
bioinformatics platform provided by GENEWIZ. Construct 4 was
codon-pair optimized based on the table used for codon pair
deoptimization described by Coleman et al. (2008) Science 320:1784.
Codon pair optimization was performed manually without the use of
bioinformatics tools or computational algorithms, using the high
scores identified by Coleman et al. An aligned comparison of the
DNA sequences of Constructs 1-4 is shown in FIG. 1B.
Example 2: In Vitro Expression of SAM Lyssaviral Antigens
[0353] Western blot analysis was performed to determine whether the
transgenes were expressed from the SAM rabies constructs. BHK cells
(1.times.10.sup.6) were transfected with 2.5 ug RNA from Constructs
1-4. After 20 hours, cell extracts were harvested and the
production of rabies G protein was analysed by SDS gel
electrophoresis followed by western blot analysis with the mouse
anti-rabies glycoprotein antibody MAB8727 (Millipore Sigma,
Billerica Mass., US). After incubation with primary antibody, the
membrane was washed and then incubated with the
peroxidase-conjugated anti-mouse antibody 115-035-003 (Jackson
ImmunoResearch Laboratories, Inc., West Grove Pa., US). Finally the
assay was developed by electrochemiluminescence (ECL) using
standard techniques (GE Healthcare RPN2106, Little Chalfont,
UK).
[0354] FIG. 2A demonstrates that BHK cells transfected with
Constructs 1-4 all expressed rabies G protein. Molecular weight
standards showing the location of 39 kDa, 51 kDa and 64 kDa
proteins are indicated to the left of the blots. The lower band of
39 kDa is actin protein and was used as a standard for the total
amount of protein in each lane. Each of the four constructs
expressed rabies G protein, as shown in the first four lanes. The
molecular weight of the protein expressed by Construct 1 is lower
than that of the protein expressed by Constructs 2-4. This
difference was attributed to a potentially different glycosylation
pattern. Bioinformatic analysis predicts that Construct 1 contains
two N-glycosylation sites and two O-glycosylation sites and that
Constructs 2, 3 and 4 contain three or four N-glycosylation sites
and five O-glycosylation sites.
[0355] FIG. 2B demonstrates that treating these samples with
peptide N-glycosidase A (PNGase A) to remove the N-linked
carbohydrate chains markedly reduced the difference in molecular
weight, indicating that a difference in N-glycosylation accounts
for at least some of the observed difference in molecular
weight.
Example 3: In Vivo Immunogenicity
[0356] The immunogenicity of the SAM rabies constructs was
evaluated in parallel with RABAVERT in BALB/c mice (FIG. 3A and
FIG. 3B). Each of the four constructs shown in FIG. 1A and Table 1
was formulated with either a cationic nanoemulsion (CNE) or a lipid
nanoparticle (LNP). The animals were immunized on days 0 and 21
with the SAM vaccine constructs and on days 0, 7 and 21 with
RABAVERT, according to the licensed RABAVERT dosing schedule. Each
group consisted of 10 mice. Sera were collected on days 14 and 35
to evaluate the animals' immunogenicity against rabies. Serology
was performed by the micro rapid Fluorescent Focus Inhibition Test
for rabies (RFFIT) to determine the titer of anti-rabies
neutralizing antibodies (nAb) (Smith et al. Bull. World Health
Organ. 48:535 (1973)). The World Health Organization (WHO)
guidelines recommend using the RFFIT to determine nAb titers and
considers an Ab titer of 0.5 IU/ml to be an adequate response to a
rabies vaccine (WHO Position Paper (2010) Vaccine 28:7140).
[0357] FIG. 3A shows the results of the serology test to detect
neutralizing anti-rabies antibodies on day 14, i.e., two weeks
after a single dose of the SAM rabies vaccines or 35 days after
three doses of RABAVERT given on days 0, 7 and 21. Fourteen days
post-immunization, one dose of the SAM rabies vaccines comprising
constructs 2, 3 and 4 and formulated with LNP were as effective in
eliciting neutralizing antibodies as three doses of RABAVERT, i.e.
producing nAb titers of approximately 100 IU/ml. At 14 days
post-immunization, the nAb titers of all tested constructs rose
above the titer of 0.5 IU/ml, a surrogate marker of the threshold
of effectiveness for protection against a naturally occurring
rabies infection. This immunogenicity threshold has been documented
to correlate with protection efficacy in humans and newborn pigs
and has also been cited in a recent publication that used a mouse
model (Schnee et al. (2016) PLoS Negl. Trop. Dis. 10:e0004746,
e.g., at p. 15). The starred line (top of graph) indicates that the
0.15 ug dose of Construct 4 in LNP was statistically significantly
more potent than the 0.15 ug dose of Construct 2 in LNP at day
14.
[0358] FIG. 3B shows the results of the serology test to detect
neutralizing antibodies on day 35, i.e., two weeks following two
doses of the SAM rabies vaccine administered on days 0 and 21 or
three doses of RABAVERT administered on days 0, 7 and 21. The
dotted line indicates the nAb titer in response to RABAVERT
(approximately 100 IU/ml). (Note the difference in the scale of the
RFFIT titers between FIG. 3A and FIG. 3B.) The 0.15 ug doses of
Constructs 1, 2, 3 and 4 were all statistically significantly more
potent than RABAVERT at day 35.
[0359] Thirty-five days post-immunization, two doses of the SAM
rabies vaccines comprising Constructs 2, 3 and 4 formulated with
1.5 ug CNE were as effective in eliciting neutralizing antibodies
as three doses of RABAVERT. Two doses of the SAM rabies constructs
2, 3 and 4 formulated with either 0.15 or 1.5 ug LNP RV39 markedly
outperformed RABAVERT, producing approximately a ten-fold higher
titer of neutralizing antibodies.
Example 4: SAM Rabies Vaccines Provide Long-Term Immunogenicity
[0360] The ability of SAM rabies vaccines to confer long term
immunogenicity was examined and the results are shown in FIG. 4.
Construct 4 was formulated in amounts of 1.5 ug with CNE (square),
0.15 ug with LNP (triangle) or 1.5 ug with LNP (inverted triangle).
RABAVERT (circle) was administered in three doses on days 0, 7 and
21 at a dilution factor of 1/25 of the human clinical dose.
Neutralizing antibody titers of mice immunized with each of these
formulations were measured by RFFIT at days 56, 90 and 180 days
post-immunization. The dotted line denotes the threshold of
immunogenicity for a rabies vaccine of 0.5 IU/ml.
[0361] At day 14, 1.5 ug Construct 4 RNA formulated with CNE, 0.15
ug Construct 4 RNA formulated with LNP or 1.5 ug Construct 4 RNA
formulated with LNP elicited neutralizing antibodies to rabies at
levels well above the immunogenicity threshold of effectiveness. By
day 35 and at subsequent time points, the LNP-formulated SAM
vectors elicited titers equal to or greater than that of RABAVERT.
At day 35 and subsequent time points, each of the SAM Construct 4
vectors demonstrated immunogenicity equivalent or superior to
RABAVERT. The LNP formulated vectors demonstrated a dose-dependent
effect, the 1.5 ug dose formulated with RV39 was more potent than
the 0.15 ug dose. By day 56, RABAVERT titers began to decline. In
contrast, the titers of the SAM rabies constructs remained
constant.
[0362] Immunogenicity was further examined in a dose range study
and the results are shown in FIG. 5. In this study, decreasing
doses of Construct 4 were formulated with either LNP or CNE.
Construct 4 was formulated with LNP in decreasing amounts of 4.5
ug, 1.5 ug, 0.5 ug, 0.167 ug, 0.055 ug, 0.0185 ug, 0.006 ug, 0.002
ug or 0.0007 ug RNA or with CNE in amounts of 15 or 1.5 ug RNA.
Balb/c mice were immunized either on days 0 and 21 with Construct 4
at the doses shown or on days 0, 7 and 21 with RABAVERT. Each group
consisted of 10 mice. Sera were collected on days 14 and 35 and
analysed by RFFIT for neutralizing antibodies. The immunogenicity
threshold of effectiveness is indicated by the lower dashed line
and the historically observed peak RABAVERT titer is indicated by
the upper dashed lines in both panels.
[0363] FIG. 5A demonstrates that at 14 days post-immunization, one
dose of SAM rabies in amounts of 0.055 to 4.5 ug RNA, formulated
with LNP was as effective as RABAVERT. Even at the very low doses
of 2.0 ng and 0.7 ng RNA, mice immunized with SAM rabies formulated
with LNP produced neutralizing antibodies well above the
immunogenicity threshold for effectiveness, as did mice immunized
with SAM rabies in amounts of 15 ug or 1.5 ug RNA when formulated
with CNE. In contrast, when diluted one-thousand-fold, the
effectiveness of RABAVERT fell almost to ineffective levels (data
not shown).
[0364] FIG. 5B shows the results of the serology test for detecting
neutralizing antibodies on day 35, i.e., following two doses of the
SAM rabies vaccine administered on days 0 and 21 or three doses of
RABAVERT administered on days 0, 7 and 21. The potency of the SAM
rabies vaccine increased at all doses and with both lipid
formulations compared to the levels observed on day 14. At day 35,
two doses of SAM rabies vaccine formulated in LNP, from the very
low amount of 0.7 ng up to 4.5 ug RNA, significantly outperformed
the three-dose regimen of RABAVERT. SAM rabies vaccine formulated
in CNE at a dose of 15 ug RNA also outperformed RABAVERT.
Example 6: In Vivo Immunogenicity in Non-Human Primates
[0365] Both SAM LNP and SAM CNE formulations were well-tolerated
and induced functional immune responses in non-human primates.
Thirty five female rhesus macaques, between three years and four
and one half years of age (>4.3 kg body weight) were immunized
intramuscularly with either RABAVERT, Construct 4 formulated with
CNE or Construct 4 formulated with LNP in the doses shown in Table
2. The RABAVERT dose was a full human dose and the dosing schedule
was the same as that used in humans, i.e., weeks 0, 1 and 3. Serum
was collected on days 1, 8, 15, 22, 36, 57, 71, 85, 113, 141, 169,
183 and 197 for RFFIT neutralization studies and measurement of
total IgG by ELISA. Peripheral blood mononuclear cells (PBMC) were
obtained from whole blood in days 1, 22, 36, 57, 71, 113, 141, 169,
183 and 197 for T cell intracellular cytokine staining (ICS)
assays.
TABLE-US-00002 TABLE 2 SAM-Rabies G Protein Immunogenicity in
Non-human Primates No. Dosing Group Animals Vaccine Dose
Formulation Regimen 1 4 RABAVERT full N/A Day 1, 8, human dose 15
(weeks 0, 1, 3) 2 4 Construct 4 150 ug RNA CNE56 Day 1, 3 4
Construct 4 75 ug RNA CNE56 57, 169 4 4 Construct 4 15 ug RNA CNE56
(weeks 5 4 Construct 4 3 ug RNA CNE56 0, 8, 24) 6 5 Construct 4 75
ug RNA LNP RV39 7 5 Construct 4 15 ug RNA LNP RV39 8 5 Construct 4
3 ug RNA LNP RV39
[0366] As shown in FIG. 6, both SAM rabies formulated in CNE and
SAM rabies formulated in LNP induced high and long-lasting levels
of rabies neutralizing antibodies as measured by RFFIT.
[0367] The top panel of FIG. 6 shows the neutralizing anti-rabies
antibody titers of four doses of Construct 4 formulated in CNE,
compared to RABAVERT. All four doses induced antibody levels well
above the protective threshold (dashed line). The antibody levels
were boosted by the second and third SAM vaccination and the
boosted titers were superior to those achieved with RABAVERT. A
dose response was observed; the 150 ug (open squares) and 75 ug
(open triangles) doses produced higher antibody titers than the 15
(inverted open triangles) and 3 ug (solid circles) doses. Even at
the very low dose of 3 ug RNA, Construct 4 formulated in CNE
elicited a high and sustained neutralizing anti-rabies antibody
titer at higher levels than RABAVERT (open circles).
[0368] The bottom panel of FIG. 6 shows the neutralizing
anti-rabies antibody titers of four doses of Construct 4 formulated
in LNP, compared to RABAVERT. The SAM LNP titers were higher than
the SAM CNE titers. All three doses induced antibody levels well
above the protective threshold (dashed line). The antibody levels
were boosted by the second and third SAM vaccination and the
boosted titers were superior to those achieved with RABAVERT. A
dose response was observed with the 75 ug (open triangles), 15 ug
(inverted open triangles) and 3 ug (solid circles) doses. Even at
the very low dose of 3 ug RNA, Construct 4 formulated in LNP
elicited a high and sustained neutralizing anti-rabies antibody
titer at higher levels than RABAVERT (open circles).
[0369] As shown in FIG. 7, both SAM-RG-CNE and SAM-RG-LNP induced
high and long-lasting levels of anti-rabies IgG, measured by a
standard ELISA method. The IgG response followed a pattern similar
to that of the neutralizing antibody response.
[0370] The top panel of FIG. 7 shows the rabies IgG binding
antibody levels induced by Construct 4 formulated in CNE compared
to RABAVERT. All four doses induced IgG levels well above the
protective threshold (dashed line). The IgG levels were boosted by
the second and third SAM vaccinations and the boosted titers were
superior to those achieved with RABAVERT. A dose response was
observed; the 150 ug (open squares) and 75 ug (open triangles)
doses produced higher antibody titers than the 15 ug (inverted open
triangles) and 3 ug (solid circles) doses. Even at the very low
dose of 3 ug RNA, SAM-RG-co2 formulated in CNE elicited a high and
sustained anti-rabies IgG titer at higher levels than RABAVERT
(open circles).
[0371] The bottom panel of FIG. 7 shows the anti-rabies IgG titers
of four doses of Construct 4 formulated in LNP, compared to
RABAVERT. The SAM LNP titers were higher than the SAM CNE titers.
All three doses induced antibody levels well above the protective
threshold (dashed line). The antibody levels were boosted by the
second and third SAM vaccinations and the boosted titers were
superior to those achieved with RABAVERT. A dose response was
observed with the 75 ug (open triangles), 15 ug (inverted open
triangles) and 3 ug (solid circles) doses. Even at the very low
dose of 3 ug RNA, Construct 4 formulated in LNP elicited a high and
sustained anti-rabies IgG titer at higher levels than RABAVERT
(open circles).
Example 7: Dose-Response of Construct 4 by RFFIT and ELISA
[0372] Experiment 1
[0373] Female Balb/C mice (6-8 weeks old) were vaccinated by
intramuscular injection either with one-tenth the clinical dose of
RABAVERT on days 1, 8 and 22, with Construct 4 on day 1 or with
Construct 4 on days 1 and 22 at the doses shown in Table 3. Serum
was collected on days 15, 36, 57, 91 and 181 and an RFFIT virus
neutralization assay was performed.
TABLE-US-00003 TABLE 3 Dose Response of LNP Compared to CNE
Injection Group Vaccine Dose Formulation (day) 1 RABAVERT 1/10
clinical LNP 1, 8, 22 2 Construct 4 1.5 ug LNP 1 3 Construct 4 0.15
ug LNP 1 4 Construct 4 0.015 ug LNP 1 5 Construct 4 0.0015 ug LNP 1
6 Construct 4 0.00015 ug LNP 1 7 Construct 4 0.000015 ug LNP 1 8
Construct 4 15 ug CNE 1 9 Construct 4 1.5 ug LNP 1, 22 10 Construct
4 0.15 ug LNP 1, 22 11 Construct 4 0.015 ug LNP 1, 22 12 Construct
4 0.0015 ug LNP 1, 22 13 Construct 4 0.00015 ug LNP 1, 22 14
Construct 4 0.000015 ug LNP 1, 22 15 Construct 4 15 ug CNE 1,
22
[0374] The results of the RFFIT assay are shown in FIG. 8. The
upper dotted line shows the protective threshold of 0.5 IU/ml
neutralizing antibodies and the lower dotted line shows the lower
limit of quantitation (LLOQ) of the assay (below log 0.1). A
dose-response relationship was observed with both the one and two
dose regimens. A single immunization with very low doses of SAM
RNA, at least as low as 15 picograms, induced very high and stable
levels of neutralizing antibodies (top panel). The levels were
boosted by the second immunization and remained significantly
higher than the neutralizing antibody levels induced by three doses
of RABAVERT (bottom panel).
[0375] The results of the ELISA assay are shown in FIG. 8 and are
similar to those observed with the RFFIT. The upper dotted line
shows the protective threshold of 0.5 IU/ml neutralizing antibodies
and the lower dotted line shows the lower limit of quantitation
(LLOQ) of the assay (below log 0.1). A dose-response relationship
was observed with both the one and two dose regimens. A single
immunization with very low doses of SAM RNA, at least as low as 15
picograms, induced very high and stable levels of rabies IgG (top
panel). The levels were boosted by the second immunization and
remained significantly higher than the neutralizing antibody levels
induced by three doses of RABAVERT (bottom panel).
[0376] Spleens were removed from five mice immunized with one 15 ug
dose of Construct 4 formulated in CNE. Splenic T cells were
stimulated with brefeldin A and intracellular cytokines were
detected by flow cytometry. The cell mediated immune response is
shown in FIG. 9. One dose of the SAM rabies CNE vaccine induced
high levels of the Th1 cytokines IL2, TNF alpha, interferon gamma
and CD107a, predominantly from CD8+ T cells (top panel) and also
from CD4+ T cells (bottom panel), thus the T cell response was
polyfunctional. The Th2 response (IL-4, IL-13) and the Th17
(IL-17A, IL-17F) responses were negligible.
[0377] Experiment 2
[0378] Female Balb/C mice (6-8 weeks old) were vaccinated by
intramuscular injection either with one-tenth the clinical dose of
RABAVERT on days 1, 8 and 22, with Construct 4 on day 1 or with
Construct 4 on days 1 and 22 at the doses shown in Table 4 and
formulated with either LNP RV29 or LNP RV94. Serum was collected on
days 15, 36, 57, 91 and 181 and both an RFFIT virus neutralization
assay and an ELISA assay for total IgG were performed.
TABLE-US-00004 TABLE 4 Dose Response of LNP RV29 and LNP RV94
Injection Group Vaccine Dose Formulation (day) 1 RABAVERT 1/10
clinical 1, 8, 22 2 Construct 4 0.15 ug LNP RV29 1, 22 3 Construct
4 0.0015 ug LNP RV29 1, 22 4 Construct 4 0.000015 ug LNP RV29 1, 22
5 Construct 4 0.15 ug LNP RV94 1, 22 6 Construct 4 0.0015 ug LNP
RV94 1, 22 7 Construct 4 0.000015 ug LNP RV94 1, 22
[0379] The results of the RFFIT assay are shown in FIG. 10. The
upper dotted line shows the protective threshold of 0.5 IU/ml
neutralizing antibodies and the lower dotted line shows the lower
limit of quantitation (LLOQ) of the assay (below log 0.1). Similar
to the results in Experiment 1, very low doses of SAM RNA, at least
as low as 15 picograms, induced very high and stable levels of
neutralizing antibodies. The LNP RV39 and the LNP RV94 formulations
induced similarly high neutralizing antibody levels.
[0380] The results of the ELISA assay are shown in FIG. 11 and, as
in Experiment 1, are similar to those observed with the RFFIT. The
upper dotted line shows the protective threshold of 0.5 IU/ml
neutralizing antibodies and the lower dotted line shows the lower
limit of quantitation (LLOQ) of the assay (below log 0.1). Very low
doses of SAM RNA, at least as low as 15 picograms, induced very
high and stable levels of rabies IgG.
[0381] Therapeutics administered by intramuscular injection in
animals should be scaled to humans according to relative body
weight (FDA Guidance for Industry: Estimating the Maximum Safe
Starting Dose in Initial Clinical Trials for Therapeutics in Adult
Healthy Volunteers (2005) US Dept. Health and Human Services; Nair
et al. J Basic Clin. Pharma. 7:27-31 (2016)). An average adult body
weight of 50 kg and an average mouse body weight of 20 grams was
used to convert a low dose (i.e. 15 picograms) of mRNA administered
using the SAM LNP platform to adult humans based on body weight.
Using these average body weights, a scaling factor of 2500 is
obtained (50,000 grams/20 grams=2500). Using this scaling factor to
convert a mouse dose of vaccine into an equivalent dose in humans
based on body weight, the low dose of 15 picograms in the mouse is
equivalent to a human dose of 38 nanograms (3.8.times.10.sup.-8
grams). The calculation is: 15 picograms.times.2500=38
nanograms.
[0382] Therefore, based on the in vivo data generated in mice shown
above and this mouse to human conversion, vaccines using the SAM
LNP vaccine platform generate appropriate and effective immune
responses at doses in the nanogram range in adult humans.
Example 8: SAM Rabies Vaccine Protects Against a Lethal Rabies
Challenge
[0383] The capacity of SAM vaccines to protect against a lethal
rabies viral challenge was tested and compared to a saline control.
The lethal dose of the Ps P4 bat isolate live rabies virus was
determined by titration and determined to be a 1:2.5 dilution of a
stock virus at a concentration of 1.times.10.sup.4 tissue culture
infectious dose 50% (TCID.sub.50/ml), delivered intramuscularly.
Clinical signs of rabies were observed from days 7-12 and confirmed
by a direct fluorescent antibody (DFA) test of the brains at
necropsy on day 12.
[0384] Female ICR mice approximately 4-6 weeks old were immunized
in groups of eight with SAM rabies vaccines in the formulations and
dosage regimens shown in Table 5
TABLE-US-00005 TABLE 5 Lethal Rabies Challenge. Dosing Group
Vaccine Dose Formulation Regimen (Days) 1 RABAVERT 1/10.sup.th N/A
1, 8, 22 clinical dose 2 Saline -- -- 1, 22 3 Construct 4 1.5 ug
LNP RV39 1, 22 4 Construct 4 1.5 ug LNP RV94 1, 22 5 Construct 4
1.5 ug CNE56 1, 22 6 Construct 4 1.5 ug LNP RV39 1 7 Construct 4
1.5 ug LNP RV94 1 8 Construct 4 1.5 ug CNE56 1
[0385] Sixty days after the first immunization, the mice were
challenged with a lethal dose of Ps P4 bat isolate live rabies, a
1:2.5 dilution of a stock virus at a concentration of
1.times.10.sup.4 tissue culture infectious dose 50%
(TCID.sub.50/ml), delivered intramuscularly. All mice in the saline
group showed clinical signs of rabies and 100% mortality was
observed by day 8. None of the vaccinated mice showed any clinical
signs of rabies and when they were sacrificed at day 31
post-challenge a DFA test confirmed the absence of rabies virus in
their brains.
[0386] All of the SAM formulations were safe and well tolerated.
Body weight was monitored and no significant aberrant changes in
were observed. Injection site reaction was monitored at 6, 24 and
48 hours after each dose and graded according to the modified
Draize method. All formulations were well tolerated and Draize
scores were zero (no edema or erythema and eschar formation) for
all of the LNP formulations.
[0387] RFFIT and ELISA assays were performed and the results were
similar to those shown above. High levels of neutralizing
antibodies and IgG were induced by a single immunization. Both LNP
RV39 and LNP94 formulations were more potent in inducing
neutralizing antibodies than CNE56. LNP RV39 and LNP RV94 induced
similar levels of neutralizing antibodies. IgG levels correlated
with neutralizing antibody levels.
Sequence CWU 1
1
1017746DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1ataggcggcg catgagagaa gcccagacca
attacctacc caaaatggag aaagttcacg 60ttgacatcga ggaagacagc ccattcctca
gagctttgca gcggagcttc ccgcagtttg 120aggtagaagc caagcaggtc
actgataatg accatgctaa tgccagagcg ttttcgcatc 180tggcttcaaa
actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa
240gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt
ccgatgagat 300gtgcggaaga tccggacaga ttgtataagt atgcaactaa
gctgaagaaa aactgtaagg 360aaataactga taaggaattg gacaagaaaa
tgaaggagct cgccgccgtc atgagcgacc 420ctgacctgga aactgagact
atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480aagtcgctgt
ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag
540ccaataaggg agttagagtc gcctactgga taggctttga caccacccct
tttatgttta 600agaacttggc tggagcatat ccatcatact ctaccaactg
ggccgacgaa accgtgttaa 660cggctcgtaa cataggccta tgcagctctg
acgttatgga gcggtcacgt agagggatgt 720ccattcttag aaagaagtat
ttgaaaccat ccaacaatgt tctattctct gttggctcga 780ccatctacca
cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact
840tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc
gacgggtacg 900tcgttaaaag aatagctatc agtccaggcc tgtatgggaa
gccttcaggc tatgctgcta 960cgatgcaccg cgagggattc ttgtgctgca
aagtgacaga cacattgaac ggggagaggg 1020tctcttttcc cgtgtgcacg
tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080tggcaacaga
tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta
1140tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac
cttttgcccg 1200tagtggccca ggcatttgct aggtgggcaa aggaatataa
ggaagatcaa gaagatgaaa 1260ggccactagg actacgagat agacagttag
tcatggggtg ttgttgggct tttagaaggc 1320acaagataac atctatttat
aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380atttccactc
attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa
1440caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt
accgccgagg 1500acgtacaaga agctaagtgc gcagccgatg aggctaagga
ggtgcgtgaa gccgaggagt 1560tgcgcgcagc tctaccacct ttggcagctg
atgttgagga gcccactctg gaagccgatg 1620tcgacttgat gttacaagag
gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680aggttaccag
ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg
1740ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa
gtcatagtga 1800taacacactc tggccgaaaa gggcgttatg ccgtggaacc
ataccatggt aaagtagtgg 1860tgccagaggg acatgcaata cccgtccagg
actttcaagc tctgagtgaa agtgccacca 1920ttgtgtacaa cgaacgtgag
ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980gagcgctgaa
cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg
2040aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc
actgggctag 2100ggctcacagg cgagctggtg gatcctccct tccatgaatt
cgcctacgag agtctgagaa 2160cacgaccagc cgctccttac caagtaccaa
ccataggggt gtatggcgtg ccaggatcag 2220gcaagtctgg catcattaaa
agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280aagaaaactg
tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg
2340ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag
accctgtata 2400ttgacgaagc ttttgcttgt catgcaggta ctctcagagc
gctcatagcc attataagac 2460ctaaaaaggc agtgctctgc ggggatccca
aacagtgcgg tttttttaac atgatgtgcc 2520tgaaagtgca ttttaaccac
gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580gttgcactaa
atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa
2640cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc
aaacctaagc 2700aggacgatct cattctcact tgtttcagag ggtgggtgaa
gcagttgcaa atagattaca 2760aaggcaacga aataatgacg gcagctgcct
ctcaagggct gacccgtaaa ggtgtgtatg 2820ccgttcggta caaggtgaat
gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880tcctactgac
ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga
2940taaaaacact gactgccaag taccctggga atttcactgc cacgatagag
gagtggcaag 3000cagagcatga tgccatcatg aggcacatct tggagagacc
ggaccctacc gacgtcttcc 3060agaataaggc aaacgtgtgt tgggccaagg
ctttagtgcc ggtgctgaag accgctggca 3120tagacatgac cactgaacaa
tggaacactg tggattattt tgaaacggac aaagctcact 3180cagcagagat
agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg
3240gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg
gataactccc 3300cgtcgcctaa catgtacggg ctgaataaag aagtggtccg
tcagctctct cgcaggtacc 3360cacaactgcc tcgggcagtt gccactggaa
gagtctatga catgaacact ggtacactgc 3420gcaattatga tccgcgcata
aacctagtac ctgtaaacag aagactgcct catgctttag 3480tcctccacca
taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg
3540gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg
gttgactggt 3600tgtcagaccg gcctgaggct accttcagag ctcggctgga
tttaggcatc ccaggtgatg 3660tgcccaaata tgacataata tttgttaatg
tgaggacccc atataaatac catcactatc 3720agcagtgtga agaccatgcc
attaagctta gcatgttgac caagaaagct tgtctgcatc 3780tgaatcccgg
cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa
3840gcatcattgg tgctatagcg cggcagttca agttttcccg ggtatgcaaa
ccgaaatcct 3900cacttgaaga gacggaagtt ctgtttgtat tcattgggta
cgatcgcaag gcccgtacgc 3960acaatcctta caagctttca tcaaccttga
ccaacattta tacaggttcc agactccacg 4020aagccggatg tgcaccctca
tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080gagtgattat
aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc
4140tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga
aaagcgcgac 4200tggtcaaagg tgcagctaaa catatcattc atgccgtagg
accaaacttc aacaaagttt 4260cggaggttga aggtgacaaa cagttggcag
aggcttatga gtccatcgct aagattgtca 4320acgataacaa ttacaagtca
gtagcgattc cactgttgtc caccggcatc ttttccggga 4380acaaagatcg
actaacccaa tcattgaacc atttgctgac agctttagac accactgatg
4440cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag
gaagcagtgg 4500ctaggagaga agcagtggag gagatatgca tatccgacga
ctcttcagtg acagaacctg 4560atgcagagct ggtgagggtg catccgaaga
gttctttggc tggaaggaag ggctacagca 4620caagcgatgg caaaactttc
tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680atatagcaga
aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca
4740tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc
gaagagtcgg 4800aagcctccac accacctagc acgctgcctt gcttgtgcat
ccatgccatg actccagaaa 4860gagtacagcg cctaaaagcc tcacgtccag
aacaaattac tgtgtgctca tcctttccat 4920tgccgaagta tagaatcact
ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980caccgaaagt
gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag
5040acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct
gaacaaccac 5100cacttataac cgaggatgag accaggacta gaacgcctga
gccgatcatc atcgaagagg 5160aagaagagga tagcataagt ttgctgtcag
atggcccgac ccaccaggtg ctgcaagtcg 5220aggcagacat tcacgggccg
ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280ccgactttga
tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca
5340gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag
tttctggcgc 5400gaccggtgcc tgcgcctcga acagtattca ggaaccctcc
acatcccgct ccgcgcacaa 5460gaacaccgtc acttgcaccc agcagggcct
gctcgagaac cagcctagtt tccaccccgc 5520caggcgtgaa tagggtgatc
actagagagg agctcgaggc gcttaccccg tcacgcactc 5580ctagcaggtc
ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga
5640ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt
gatgcgggtg 5700catacatctt ttcctccgac accggtcaag ggcatttaca
acaaaaatca gtaaggcaaa 5760cggtgctatc cgaagtggtg ttggagagga
ccgaattgga gatttcgtat gccccgcgcc 5820tcgaccaaga aaaagaagaa
ttactacgca agaaattaca gttaaatccc acacctgcta 5880acagaagcag
ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta
5940ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc
taccgaaccc 6000tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc
cttttcaagc cccaaggtcg 6060cagtggaagc ctgtaacgcc atgttgaaag
agaactttcc gactgtggct tcttactgta 6120ttattccaga gtacgatgcc
tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180ctgccagttt
ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac
6240ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac
gtcctggcag 6300ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga
attgcccgta ttggattcgg 6360cggcctttaa tgtggaatgc ttcaagaaat
atgcgtgtaa taatgaatat tgggaaacgt 6420ttaaagaaaa ccccatcagg
cttactgaag aaaacgtggt aaattacatt accaaattaa 6480aaggaccaaa
agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca
6540taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact
ccaggaacaa 6600aacatactga agaacggccc aaggtacagg tgatccaggc
tgccgatccg ctagcaacag 6660cgtatctgtg cggaatccac cgagagctgg
ttaggagatt aaatgcggtc ctgcttccga 6720acattcatac actgtttgat
atgtcggctg aagactttga cgctattata gccgagcact 6780tccagcctgg
ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg
6840acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac
gcagagctgt 6900tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat
acatttgccc actaaaacta 6960aatttaaatt cggagccatg atgaaatctg
gaatgttcct cacactgttt gtgaacacag 7020tcattaacat tgtaatcgca
agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080cagcattcat
tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag
7140acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg
gtgggcgaga 7200aagcgcctta tttctgtgga gggtttattt tgtgtgactc
cgtgaccggc acagcgtgcc 7260gtgtggcaga ccccctaaaa aggctgttta
agcttggcaa acctctggca gcagacgatg 7320aacatgatga tgacaggaga
agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380gtattctttc
agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca
7440tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc
tacctgagag 7500gggcccctat aactctctac ggctaacctg aatggactac
gacatagtct agtccgccaa 7560gtgataaggc gcgcccaccc agcggccgca
tacagcagca attggcaagc tgcttacata 7620gaactcgcgg cgattggcat
gccgccttaa aatttttatt ttatttttct tttcttttcc 7680gaatcggatt
ttgtttttaa tatttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 7740aaaaaa
774621572DNARabies lyssavirus 2atggtgcctc aagccctgct gttcgtgccc
ctgctggtct tctccctctg ctttggcaag 60ttccccatct acaccatccc tgacaagctc
ggcccctggt cccccattga catacatcac 120ctcagctgcc ccaacaacct
ggtggtggag gatgagggct gcacaaacct gagcggcttc 180tcctatatgg
aactcaaggt gggctatatc tccgccatca aggtcaatgg attcacatgc
240accggcgtcg tgacagaggc tgaaacatac accaactttg tgggctacgt
caccaccaca 300ttcaagagga agcacttcag gcccacccct gacgcttgca
gggctgccta caattggaag 360atggctggcg accccaggta tgaggagtcc
ctgcacaatc cctaccccga ctaccattgg 420ctcaggacag tcaagaccac
caaggagtcc ctggtcatta tctcccctag cgtggccgac 480ctagacccgt
atgacaaaag cctgcactcc agggtcttcc ctagcggcaa atgctccggc
540attacagtga gctccaccta ctgcagcaca aaccacgact acaccatctg
gatgcctgag 600aatcctaggc tcggcacctc ctgtgacata tttacaaata
gcaggggcaa gagggcttcc 660aaaggcagca aaacctgcgg ctttgtcgac
gaaagaggcc tgtacaagtc cctcaagggc 720gcttgtaaac tcaagctgtg
cggagtgctg ggactcagac tcatggacgg cacatgggtg 780gccatgcaga
ccagcgatga gaccaagtgg tgcccccccg atcagctggt gaatctgcac
840gacttcaggt ccgacgaaat tgagcacctc gtggtcgagg agctggtgaa
gaagagagaa 900gagtgcctgg atgctctgga gtccatcatg accaccaaat
ccgtgtcctt cagaaggctg 960agccacctca ggaagctggt ccccggcttt
ggcaaggcct acacaatttt caataagaca 1020ctgatggagg ccgatgctca
ctacaaatcc gtgaggacct ggaacgagat catcccctcc 1080aaaggctgcc
tgagggtggg aggaagatgc cacccccacg tcaacggcgt cttcttcaac
1140ggcattatcc tcggacccga tggccatgtc ctgatccctg aaatgcaaag
ctccctgctg 1200cagcagcaca tggaactcct ggagagctcc gtcatccccc
tgatgcaccc tctcgctgac 1260cccagcaccg tgtttaaaga cggcgatgag
gccgaggact tcgtggaagt gcatctgcct 1320gatgtgcata agcaagtcag
cggcgtcgat ctgggcctgc ctaattgggg caagtatgtc 1380ctgctctccg
ccggagctct gattgccctg atgctgatca tcttcctgat gacctgctgc
1440agaagagtca acagacctga gagcacccaa agatccctcg gcggaaccgg
aaggaaggtc 1500agcgtgacca gccagtccgg caaagtgatt tcctcctggg
agagctataa aagcggcgga 1560gagaccaggc tg 15723524PRTRabies
lyssavirus 3Met Val Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe
Ser Leu1 5 10 15Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys
Leu Gly Pro 20 25 30Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro
Asn Asn Leu Val 35 40 45Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly
Phe Ser Tyr Met Glu 50 55 60Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys
Val Asn Gly Phe Thr Cys65 70 75 80Thr Gly Val Val Thr Glu Ala Glu
Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95Val Thr Thr Thr Phe Lys Arg
Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110Cys Arg Ala Ala Tyr
Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125Glu Ser Leu
His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140Lys
Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp145 150
155 160Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Ser
Gly 165 170 175Lys Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser
Thr Asn His 180 185 190Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg
Leu Gly Thr Ser Cys 195 200 205Asp Ile Phe Thr Asn Ser Arg Gly Lys
Arg Ala Ser Lys Gly Ser Lys 210 215 220Thr Cys Gly Phe Val Asp Glu
Arg Gly Leu Tyr Lys Ser Leu Lys Gly225 230 235 240Ala Cys Lys Leu
Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255Gly Thr
Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro 260 265
270Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu
275 280 285His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys
Leu Asp 290 295 300Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser
Phe Arg Arg Leu305 310 315 320Ser His Leu Arg Lys Leu Val Pro Gly
Phe Gly Lys Ala Tyr Thr Ile 325 330 335Phe Asn Lys Thr Leu Met Glu
Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350Thr Trp Asn Glu Ile
Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360 365Arg Cys His
Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380Gly
Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu385 390
395 400Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met
His 405 410 415Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp
Glu Ala Glu 420 425 430Asp Phe Val Glu Val His Leu Pro Asp Val His
Lys Gln Val Ser Gly 435 440 445Val Asp Leu Gly Leu Pro Asn Trp Gly
Lys Tyr Val Leu Leu Ser Ala 450 455 460Gly Ala Leu Ile Ala Leu Met
Leu Ile Ile Phe Leu Met Thr Cys Cys465 470 475 480Arg Arg Val Asn
Arg Pro Glu Ser Thr Gln Arg Ser Leu Gly Gly Thr 485 490 495Gly Arg
Lys Val Ser Val Thr Ser Gln Ser Gly Lys Val Ile Ser Ser 500 505
510Trp Glu Ser Tyr Lys Ser Gly Gly Glu Thr Arg Leu 515
52041572DNARabies lyssavirus 4atggttcctc aggttctttt gtttgtaccc
ctcctgggtt tttcattgtg tttcgggaag 60ttccccattt acacgatacc agacaaactt
ggtccctgga gccctattga catacaccat 120ctcagctgtc caaataacct
ggttgtggag gacgaaggat gtaccaacct gtccgagttc 180tcttacatgg
aacttaaagt gggatacatc tcagccataa aagtgaacgg gttcacttgc
240acaggtgttg tgacagaggc agaaacctac accaactttg ttggttatgt
cacaaccaca 300ttcaagagaa agcatttccg ccccacccca gacgcatgta
gagccgcgta taactggaag 360atggccggtg accccagata tgaagagtct
ctacacaatc cgtaccccga ctaccattgg 420cttcgaactg taaaaaccac
caaagagtct ctcgttatca tatccccaag tgtgacagat 480ttggacccat
atgacaaatc ccttcactca agggtcttcc ctggcggaaa ttgctcagga
540ataacggtgt cctcgaccta ctgctcaact aatcatgatt acaccatttg
gatgcctgag 600aatctgagac tagggacatc ttgtgacatt tttaccaata
gcagagggaa gagggcatcc 660aaaggaggca agacttgcgg ctttgtggat
gaaagaggcc tgtataagtc tctaaaggga 720gcatgcaaac tcaagttatg
tggagttctc ggacttagac ttatggatgg aacatgggtc 780gcgatgcaaa
catcagatga gaccaaatgg tgccctccag gtcagttggt gaatttgcac
840gactttcgct cagacgagat tgagcatctc gttgtggaag agttagtcaa
gaaaagagag 900gagtgtctgg atgcactaga gtccatcatg accaccaagt
cagtgagttt cagacgtctc 960agtcacttga gaaaacttgt ccctgggttt
ggaaaagcat ataccatatt caacaaaacc 1020ttgatggagg ctgatgctca
ctacaagtct gtccggacct ggaatgagat catcccctca 1080aaagggtgtt
tgagagttgg ggggaggtgt catccccatg tgaacggggt gtttttcaat
1140ggtataatat tagggtctga cggccatgtt ctaatcccag agatgcagtc
atccctcctc 1200cagcaacata tggagttgtt ggaatcttca gttatccccc
tgatgcaccc cttggcagac 1260ccttctacag ttttcaaaga cggtgatgag
gttgaggatt ttgttgaagt tcacctcccc 1320gatgtgcatg aacaggtctc
aggagttgaa ctgggtctcc cgaactgggg gaagtatgta 1380ttgatgattg
caggggcctt gattgccctg atgttgataa ttttcctgat gacatgttgc
1440agaagagtca atcgaccaga atctacgcaa agcagtcttg gagagacagg
gagaaatgtg 1500tcagtcactt cccaaagcgg aaaagtcata tcttcatggg
agtcatataa gagtggaggc 1560gagaccagac tg 15725524PRTRabies
lyssavirus 5Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe
Ser Leu1 5 10 15Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys
Leu Gly Pro 20 25 30Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro
Asn Asn Leu Val 35 40 45Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu
Phe Ser Tyr Met Glu 50 55 60Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys
Val Asn Gly Phe Thr Cys65 70 75 80Thr Gly Val Val Thr Glu Ala Glu
Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95Val Thr Thr Thr Phe Lys Arg
Lys His Phe Arg Pro Thr Pro Asp Ala
100 105 110Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg
Tyr Glu 115 120 125Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp
Leu Arg Thr Val 130 135 140Lys Thr Thr Lys Glu Ser Leu Val Ile Ile
Ser Pro Ser Val Thr Asp145 150 155 160Leu Asp Pro Tyr Asp Lys Ser
Leu His Ser Arg Val Phe Pro Gly Gly 165 170 175Asn Cys Ser Gly Ile
Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His 180 185 190Asp Tyr Thr
Ile Trp Met Pro Glu Asn Leu Arg Leu Gly Thr Ser Cys 195 200 205Asp
Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Gly Lys 210 215
220Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys
Gly225 230 235 240Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu
Arg Leu Met Asp 245 250 255Gly Thr Trp Val Ala Met Gln Thr Ser Asp
Glu Thr Lys Trp Cys Pro 260 265 270Pro Gly Gln Leu Val Asn Leu His
Asp Phe Arg Ser Asp Glu Ile Glu 275 280 285His Leu Val Val Glu Glu
Leu Val Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300Ala Leu Glu Ser
Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu305 310 315 320Ser
His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330
335Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg
340 345 350Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val
Gly Gly 355 360 365Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn
Gly Ile Ile Leu 370 375 380Gly Ser Asp Gly His Val Leu Ile Pro Glu
Met Gln Ser Ser Leu Leu385 390 395 400Gln Gln His Met Glu Leu Leu
Glu Ser Ser Val Ile Pro Leu Met His 405 410 415Pro Leu Ala Asp Pro
Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu 420 425 430Asp Phe Val
Glu Val His Leu Pro Asp Val His Glu Gln Val Ser Gly 435 440 445Val
Glu Leu Gly Leu Pro Asn Trp Gly Lys Tyr Val Leu Met Ile Ala 450 455
460Gly Ala Leu Ile Ala Leu Met Leu Ile Ile Phe Leu Met Thr Cys
Cys465 470 475 480Arg Arg Val Asn Arg Pro Glu Ser Thr Gln Ser Ser
Leu Gly Glu Thr 485 490 495Gly Arg Asn Val Ser Val Thr Ser Gln Ser
Gly Lys Val Ile Ser Ser 500 505 510Trp Glu Ser Tyr Lys Ser Gly Gly
Glu Thr Arg Leu 515 52061572DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 6atggtccctc aggtgctcct
gttcgtccct ctgctcggct tctccctctg ctttggcaag 60ttccccatct acacaatccc
cgataagctc ggcccttgga gccctattga catccaccac 120ctctcctgtc
ccaacaacct ggtggtggag gacgagggat gcaccaacct gagcgagttc
180tcatacatgg agctgaaggt gggctatatt agcgccatca aggtcaacgg
cttcacatgc 240accggagtcg tgaccgaggc cgagacctac acaaacttcg
tcggctacgt cacaacaacc 300ttcaagagga aacatttcag acccacccct
gacgcttgca gggccgctta caattggaag 360atggctggcg accccagata
cgaggagagc ctccacaacc cctaccctga ctaccactgg 420ctcaggaccg
tgaagaccac caaggagtcc ctcgtgatca tctcccccag cgtcacagac
480ctcgaccctt atgataagag cctccactcc agggtgttcc ctggcggcaa
ctgttccggc 540atcaccgtct cctccaccta ctgcagcacc aaccacgact
acaccatctg gatgcctgag 600aacctgaggc tgggcaccag ctgcgacatc
ttcaccaata gcaggggcaa gagggcctcc 660aagggaggaa agacctgcgg
atttgtggat gagaggggcc tctacaagtc actgaagggc 720gcctgcaagc
tgaaactctg cggcgtgctg ggactgaggc tcatggacgg aacctgggtc
780gctatgcaaa catccgacga gaccaagtgg tgtccccccg gccagctcgt
gaatcttcat 840gacttcagga gcgacgaaat cgagcacctc gtggtggagg
aactggtcaa gaagagggag 900gagtgcctcg acgctctcga gtccatcatg
accaccaaga gcgtgtcatt tagaagactg 960agccacctga ggaagctggt
ccccggcttc ggcaaagcct acaccatctt caacaagacc 1020ctgatggagg
ccgatgctca ctacaagagc gtcaggacct ggaacgagat catccccagc
1080aaaggctgcc tgagagtggg aggaaggtgt cacccccacg tgaacggcgt
cttcttcaac 1140ggcatcattc tcggaagcga cggacacgtc ctgattcccg
agatgcagag ctcactcctg 1200cagcagcata tggagctcct ggaaagctcc
gtcattcctc tgatgcatcc cctcgctgat 1260ccctccacag tcttcaaaga
tggcgacgag gtggaggact ttgtggaagt gcacctcccc 1320gatgttcatg
agcaagtctc cggagtggaa ctgggcctcc ccaactgggg caagtacgtc
1380ctcatgattg ctggcgctct catcgccctg atgctgatca tcttcctgat
gacctgctgc 1440agaagggtca atagacccga gagcactcag tccagcctcg
gcgagaccgg cagaaatgtg 1500agcgtgacct cccaatccgg caaagtcatc
agctcctggg agagctacaa atccggagga 1560gaaacaaggc tg
15727524PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Met Val Pro Gln Val Leu Leu Phe Val Pro Leu
Leu Gly Phe Ser Leu1 5 10 15Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile
Pro Asp Lys Leu Gly Pro 20 25 30Trp Ser Pro Ile Asp Ile His His Leu
Ser Cys Pro Asn Asn Leu Val 35 40 45Val Glu Asp Glu Gly Cys Thr Asn
Leu Ser Glu Phe Ser Tyr Met Glu 50 55 60Leu Lys Val Gly Tyr Ile Ser
Ala Ile Lys Val Asn Gly Phe Thr Cys65 70 75 80Thr Gly Val Val Thr
Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95Val Thr Thr Thr
Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110Cys Arg
Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120
125Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val
130 135 140Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val
Thr Asp145 150 155 160Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg
Val Phe Pro Gly Gly 165 170 175Asn Cys Ser Gly Ile Thr Val Ser Ser
Thr Tyr Cys Ser Thr Asn His 180 185 190Asp Tyr Thr Ile Trp Met Pro
Glu Asn Leu Arg Leu Gly Thr Ser Cys 195 200 205Asp Ile Phe Thr Asn
Ser Arg Gly Lys Arg Ala Ser Lys Gly Gly Lys 210 215 220Thr Cys Gly
Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly225 230 235
240Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp
245 250 255Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp
Cys Pro 260 265 270Pro Gly Gln Leu Val Asn Leu His Asp Phe Arg Ser
Asp Glu Ile Glu 275 280 285His Leu Val Val Glu Glu Leu Val Lys Lys
Arg Glu Glu Cys Leu Asp 290 295 300Ala Leu Glu Ser Ile Met Thr Thr
Lys Ser Val Ser Phe Arg Arg Leu305 310 315 320Ser His Leu Arg Lys
Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335Phe Asn Lys
Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350Thr
Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360
365Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu
370 375 380Gly Ser Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser
Leu Leu385 390 395 400Gln Gln His Met Glu Leu Leu Glu Ser Ser Val
Ile Pro Leu Met His 405 410 415Pro Leu Ala Asp Pro Ser Thr Val Phe
Lys Asp Gly Asp Glu Val Glu 420 425 430Asp Phe Val Glu Val His Leu
Pro Asp Val His Glu Gln Val Ser Gly 435 440 445Val Glu Leu Gly Leu
Pro Asn Trp Gly Lys Tyr Val Leu Met Ile Ala 450 455 460Gly Ala Leu
Ile Ala Leu Met Leu Ile Ile Phe Leu Met Thr Cys Cys465 470 475
480Arg Arg Val Asn Arg Pro Glu Ser Thr Gln Ser Ser Leu Gly Glu Thr
485 490 495Gly Arg Asn Val Ser Val Thr Ser Gln Ser Gly Lys Val Ile
Ser Ser 500 505 510Trp Glu Ser Tyr Lys Ser Gly Gly Glu Thr Arg Leu
515 52081572DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 8atggtgcccc aggtgctgct ctttgtgccc
ctgctgggct tcagcctctg cttcggcaag 60ttccccatct acaccatccc agacaagctg
gggccttgga gccccatcga catccaccac 120ctgagctgcc ccaacaacct
ggtggtggaa gatgaaggct gcaccaacct gagcgagttc 180tcctacatgg
agctgaaggt gggctacatc tctgccatca aggtgaatgg cttcacctgc
240actggagtgg tcacagaggc cgagacctac accaactttg tgggctatgt
gaccaccacc 300ttcaagagga agcacttccg gcccacccca gatgcctgcc
gggccgccta caactggaag 360atggctgggg acccccgcta tgaggagagc
ctgcacaacc cctacccaga ctaccactgg 420ctgaggacag tgaagaccac
caaggagagc ctggtgatca tcagccccag cgtgacagac 480ctggacccct
atgacaagag cctgcacagc cgggtgttcc ctggcggcaa ctgcagcggc
540atcaccgtga gcagcaccta ctgcagcacc aaccacgact acaccatctg
gatgccagaa 600aacctgcggc tgggcaccag ctgtgacatc ttcaccaaca
gccggggcaa gagggccagc 660aagggcggca agacctgtgg ctttgtggat
gagcggggcc tctacaagag cctgaagggg 720gcctgcaagc tgaagctctg
tggggtgctg ggcctgcggc tgatggatgg cacctgggtg 780gccatgcaga
cctcagatga gaccaagtgg tgccccccag gccagctggt gaacctgcat
840gacttccgca gcgacgagat tgagcacctg gtggtggagg agctggtgaa
gaagagggag 900gagtgcctgg atgccctcga gagcatcatg accaccaaga
gcgtgtcctt ccgccgcctg 960agccacctgc ggaagctggt gcctggcttt
ggaaaggcct acaccatctt caacaagacc 1020ctgatggagg cagatgccca
ctacaagagt gtgcggacct ggaatgagat catccccagc 1080aagggctgcc
tgcgggtggg cggccggtgc cacccccacg tgaatggagt gttcttcaat
1140ggcatcatcc tgggcagcga cggccacgtg ctgatccctg agatgcagag
cagcctgctg 1200cagcagcaca tggagctgct ggagagctct gtcatccccc
tgatgcaccc cctcgctgac 1260cccagcaccg tgttcaagga tggagatgaa
gtggaggact tcgtggaggt gcacctgcca 1320gatgtgcacg agcaggtgtc
tggcgtggag ctgggcctgc ccaactgggg caagtacgtg 1380ctgatgattg
ctggcgccct gatcgccctg atgctgatca tcttcctgat gacctgctgc
1440cggcgggtga acagacctga gagcacccag agcagcctgg gagagactgg
aagaaatgtg 1500tctgtcacca gccagagcgg caaggtgatc agcagctggg
agagctacaa gagtggcggc 1560gagaccaggc tg 15729524PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe Ser Leu1 5
10 15Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly
Pro 20 25 30Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn
Leu Val 35 40 45Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser
Tyr Met Glu 50 55 60Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn
Gly Phe Thr Cys65 70 75 80Thr Gly Val Val Thr Glu Ala Glu Thr Tyr
Thr Asn Phe Val Gly Tyr 85 90 95Val Thr Thr Thr Phe Lys Arg Lys His
Phe Arg Pro Thr Pro Asp Ala 100 105 110Cys Arg Ala Ala Tyr Asn Trp
Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125Glu Ser Leu His Asn
Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140Lys Thr Thr
Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Thr Asp145 150 155
160Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Gly Gly
165 170 175Asn Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr
Asn His 180 185 190Asp Tyr Thr Ile Trp Met Pro Glu Asn Leu Arg Leu
Gly Thr Ser Cys 195 200 205Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg
Ala Ser Lys Gly Gly Lys 210 215 220Thr Cys Gly Phe Val Asp Glu Arg
Gly Leu Tyr Lys Ser Leu Lys Gly225 230 235 240Ala Cys Lys Leu Lys
Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255Gly Thr Trp
Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro 260 265 270Pro
Gly Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu 275 280
285His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp
290 295 300Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg
Arg Leu305 310 315 320Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly
Lys Ala Tyr Thr Ile 325 330 335Phe Asn Lys Thr Leu Met Glu Ala Asp
Ala His Tyr Lys Ser Val Arg 340 345 350Thr Trp Asn Glu Ile Ile Pro
Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360 365Arg Cys His Pro His
Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380Gly Ser Asp
Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu385 390 395
400Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His
405 410 415Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu
Val Glu 420 425 430Asp Phe Val Glu Val His Leu Pro Asp Val His Glu
Gln Val Ser Gly 435 440 445Val Glu Leu Gly Leu Pro Asn Trp Gly Lys
Tyr Val Leu Met Ile Ala 450 455 460Gly Ala Leu Ile Ala Leu Met Leu
Ile Ile Phe Leu Met Thr Cys Cys465 470 475 480Arg Arg Val Asn Arg
Pro Glu Ser Thr Gln Ser Ser Leu Gly Glu Thr 485 490 495Gly Arg Asn
Val Ser Val Thr Ser Gln Ser Gly Lys Val Ile Ser Ser 500 505 510Trp
Glu Ser Tyr Lys Ser Gly Gly Glu Thr Arg Leu 515 520106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(6)This sequence may encompass 2-6 residues
10His His His His His His1 5
* * * * *