Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/099003
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Lipid Nanoparticles for Delivering mRNA Vaccines
RELATED APPLICATIONS
[001] This application claims the benefit of priority of U.S.
Provisional Application No.
63/110,965, filed November 6, 2020, U.S. Provisional Application No.
63/212,523, filed June 18,
2021, and EP Priority Application No. 21315198.8, filed October 13, 2021, the
content of each
incorporated by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[002] Messenger RNA (mRNA)-based vaccines provide a promising alternative to
traditional
subunit vaccines, which contain antigenic proteins derived from a pathogen.
Antigenic proteins
are usually recombinantly made and require bacterial fermentation and/or cell
culture, as well as
complex purification. Vaccines based on mRNA allow de novo expression of
complex antigens in
the vaccinated subject, which in turn allows proper post-translational
modification and
presentation of the antigen in its natural conformation. Unlike traditional
technologies, the
manufacture of mRNA vaccines does not require complex and costly bacterial
fermentation, tissue
culture, and purification processes. Moreover, once established, the
manufacturing process for
mRNA vaccines can be used for a variety of antigens, enabling rapid
development and deployment
of mRNA vaccines. Further, mRNA vaccines are inherently safe delivery vectors
as they express
the antigens only transiently and do not integrate into the host genome.
Because antigens encoded
by mRNAs are produced in vivo in the vaccinated individual, mRNA vaccines are
especially
effective in eliciting both humoral and T cell mediated immunity.
[003] RNA, however, is unstable and subject to rapid degradation. There
also are no natural
cell surface receptors that facilitate cellular uptake of RNA. Indeed,
development of mRNA
vaccines has been hampered by inefficient in vivo delivery of mRNA. Thus,
there remains a need
to develop vaccine formulations that can improve mRNA delivery in vivo.
SUMMARY OF THE INVENTION
[004] The present disclosure provides a pharmaceutical composition
comprising nucleic acid
molecules (e.g., mRNA molecules) encapsulated in lipid nanopartieles (LNPs),
wherein each LNP
comprises a cationic lipid at a molar ratio between 35% and 45%, a
polyethylene glycol (PEG)
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conjugated (PEGylated) lipid at a molar ratio between 0.25% and 2.75%, a
cholesterol-based lipid
at a molar ratio between 20% and 35%, and a helper lipid at a molar ratio of
between 25% and
35%, wherein all the molar ratios are relative to the total lipid content of
the LNP. The composition
may be used as a vaccine to elicit immune protection in subjects (e.g., human
subjects) in need
thereof.
[005] In some embodiments, the cationic lipid is OF-02, cKK-E10, GL-HEPES-
E3-E10-DS-3-
E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14.
[006] In some embodiments, the LNP comprises a cationic lipid at a molar
ratio of 40%, a
PEGylated lipid at a molar ratio of 1.5%, a cholesterol-based lipid at a molar
ratio of 28.5%, and
a helper lipid at a molar ratio of 30%.
[007] In some embodiments, the cationic lipid is OF-02, cKK-E10, GL-HEPES-
E3-E10-DS-3-
E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14, the PEGylated
lipid is
dimyristoyl-PEG2000 (DMG-PEG2000), the cholesterol-based lipid is cholesterol,
and/or the
helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).
In particular
embodiments, the LNP comprises OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-
HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%,
DMG-
PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and
DOPE at a molar
ratio of 30%.
[008] In some embodiments, the LNP comprises 1-20, optionally 5-10 or 6-8,
nucleic acid
molecules. In some embodiments, the LNP comprises one or more mRNA molecules
encoding an
antigen (e.g., a viral antigen such as an influenza viral antigen, or a
bacterial antigen).
[009] In some embodiments, the LNP comprises two or more mRNA molecules,
wherein each
mRNA molecule encodes a different antigen, optionally wherein the different
antigens are from
the same pathogen or from different pathogens. In some embodiments, the
composition comprises
two or more LNPs, wherein each LNP comprises an mRNA encoding a different
antigen,
optionally wherein the different antigens are from the same pathogen or from
different pathogens.
[0010] For example, the composition may comprise two, three, four, five, six,
seven, eight, nine,
or more mRNA molecules encoding (i) different hemagglutinin (HA) antigens,
(ii) different
neuraminidase (NA) antigens, or (iii) at least one HA antigen and at least one
NA antigen.
[0011] In some embodiments, mRNA molecule comprises an open reading frame
(ORF)
encoding a respiratory syncytial virus (RSV) F protein antigen.
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[0012] In some embodiments, the RSV F protein antigen comprises an amino acid
sequence with
at least 98% identity to SEQ ID NO: 16 or consists of an amino acid sequence
of SEQ ID NO: 16.
[0013] In some embodiments, the RSV F protein antigen is a pre-fusion protein.
[0014] In some embodiments, the ORF is codon optimized.
[0015] In some embodiments, the mRNA molecule comprises at least one 5'
untranslated region
(5' UTR), at least one 3' untranslated region (3' UTR), and at least one
polyadenylation (poly(A))
sequence.
[0016] In some embodiments, the mRNA comprises at least one chemical
modification.
[0017] In some embodiments, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the mRNA arc chemically modified.
[0018] In some embodiments, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the ORF are chemically modified.
[0019] In some embodiments, the chemical modification is selected from the
group consisting
of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-
methylcytosine, 2-
thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-
aza-uridine, 2-thio-
dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-
thio-
pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-
pseudouridine,
5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-
methoxyuridine, and
2 ' -0-m ethyl uri din e.
[0020] In some embodiments, the chemical modification is selected from the
group consisting
of pseudouridine, Nl-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine,
and a
combination thereof
[0021] In some embodiments, the chemical modification is N1-
methylpseudouridine.
[0022] In some embodiments, the mRNA comprises a nucleic acid sequence with at
least 80%
identity to a nucleic acid sequence set forth in SEQ ID NO: 17.
[0023] In some embodiments, the mRNA comprises a nucleic acid sequence with at
least 80%
identity to a nucleic acid sequence set forth in SEQ ID NO: 21.
[0024] In some embodiments, the mRNA comprises of the following structural
elements:
(i) a 5' cap with the following structure:
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0
H OH OHH <I/1 ITA NH
..)õ
D N NH2
,t731._ " I. I.
0
N 0- 0- 0-
HN I 0 0
N+ - CH3
0-P=0
\CH,
0
(ii) a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ
ID NO: 19;
(iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:
17;
(iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ
ID NO: 20; and
(v) a poly(A) tail.
[0025] In some embodiments, the LNP has an average diameter of 30-200 nm
(e.g., 80-150 nm).
In some embodiments, the composition comprises 1-10, optionally 1, mg/mL of
the LNP. The
composition may be formulated for intramuscular or intradermal injection and
may comprise a
phosphate-buffer saline. In some embodiments, the composition comprising
trehalose, optionally
at 10% (w/v) of the composition.
[0026] In another aspect, the present disclosure provides a method of
preparing the LNP
composition herein, comprising providing an aqueous buffered solution
comprising the nucleic
acid molecule, providing an amphiphilic solution comprising the cationic
lipid, the PEGylated
lipid, the cholesterol-based lipid, and the helper lipid, and mixing the
aqueous buffered solution
and the amphiphilic solution at a ratio of 5:1 to 3:1, optionally 4:1. The
aqueous buffered solution
may be, for example an acidic buffered solution (e.g., comprising 1 mM citrate
and 150 mM
sodium chloride with a pH of about 4.5). The amphiphilic solution may be,
e.g., an ethanol
solution.
[0027] In another aspect, the present disclosure provides a method of
eliciting an immune
response in a subject in need thereof, comprising administering to the
subject, optionally
intramuscularly, intranasally, intravenously, subcutaneously, or
intradermally, a prophylactically
effective amount of the present LNP composition. In some embodiments, the
subject is treated
with one or more (e.g., two) doses of the composition, each dose comprising 1-
250, optionally
2.5., 5, 15, 45, or 135, lag of mRNA. The doses may be given at an interval of
2-24, optionally 4,
8, 12, 16, or 20 weeks, or one, two, three, four, five, or six months.
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[0028] Also provided herein are use of the present composition for the
manufacture of a
medicament for use in treating a subject in need thereof, as well as the
composition for use for use
in treating a subject in need thereof].
[0029] The present disclosure also provides a kit comprising a container
comprising a single-use
or multi-use dosage of the present, optionally wherein the container is a vial
or a pre-filled syringe
or injector.
[0030] In another aspect, the disclosure provides a pharmaceutical composition
comprising a
mRNA molecule encapsulated in a lipid nanoparticle (LNP), wherein the LNP
comprises:
a cationic lipid at a molar ratio between 35% and 45%,
a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio
between 0.25%
and 2.75%,
a cholesterol-based lipid at a molar ratio between 20% and 35%, and
a helper lipid at a molar ratio of between 25% and 35%,
wherein all the molar ratios are relative to the total lipid content of the
LNP;
wherein the mRNA molecule comprises an open reading frame (ORF) encoding an
antigen
derived from influenza virus.
[0031] In another aspect, the disclosure provides a pharmaceutical composition
comprising a
mRNA molecule encapsulated in a lipid nanoparticle (LNP), wherein the LNP
comprises:
a cationic lipid at a molar ratio between 35% and 45%,
a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio
between 0.25%
and 2.75%,
a cholesterol-based lipid at a molar ratio between 20% and 35%, and
a helper lipid at a molar ratio of between 25% and 35%,
wherein all the molar ratios are relative to the total lipid content of the
LNP;
wherein the mRNA molecule comprises an open reading frame (ORF) encoding a
respiratory syncytial virus (RSV) F protein antigen.
[0032] Other features, objects, and advantages of the invention are apparent
in the detailed
description that follows. It should be understood, however, that the detailed
description, while
indicating embodiments and aspects of the invention, is given by way of
illustration only, not
limitation. Various changes and modification within the scope of the invention
will become
apparent to those skilled in the art from the detailed description
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BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1A is a pair of graphs showing the expression of human
erythropoietin (hEPO) in
mice treated with various LNP formulations of hEPO mRNA. Panel a): LNP
formulations "Lipid
A" and "Lipid B" compared to MC3. Bars represent means and standard
deviations. Panel b):
Formulation made with cationic lipid OF-02. PEG: DMG-PEG2000. Cholest:
cholesterol. "Lipid
A": LNP composition containing OF-02, DMG-PEG2000, cholesterol, and DOPE, in
this order,
at a molar ratio of 40:1.5:2K5:30, unless otherwise indicated. "Lipid B": LNP
composition
containing cKK-E10, DMG-PEG2000, cholesterol, and DOPE, in this order, at a
molar ratio of
40:1.5:28.5:30.
[0034] FIG. 1B is a pair of graphs showing expression of hEPO in mice and non-
human primates
(NHPs) using LNP formulations Lipid A and Lipid B.
[0035] FIG. 2A and 2B are a pair of graphs showing that Lipid A and Lipid B
LNP formulations
with mRNA encoding hemagglutinin (HA) of strain A/California/7/2009 (H1N1)
(CA09) induced
robust functional antibodies (FIG. 2A) and protected mice against death or
severe weight loss
(more than 20%) when challenged with a pandemic strain of influenza virus
(FIG. 2B).
Hemagglutinin inhibition (HAT) titers are reported as log10 for serum samples
taken at study days
0, 14, 28, 42, 56, 92, and 107. Bars are geometric means and geometric
standard deviations. Daily
weights were measured after intranasal challenge (day 93) with 4LD50 of
A/Belgium/2009 (H1N1)
(Belgium09). Weights are presented as the percentage of weight lost from the
day of challenge.
Euthanasia occurred for mice losing more than 20% of their starting body
weight and for all mice
14 days post-infection (day 107). rHA: recombinant hemagglutinin. AF03: an oil-
in-water
emulsion adjuvant. Diluent = PBS. LLOQ = lower limit of quantitation. 1/40 =
1/40 minimum
target, which refers to HAT antibody titers associated with 50% reduction in
the risk of influenza
infection or disease in healthy adults (Coudeville et al., BMC Med Res
Methodal. (2010) 10:18).
Dashed line in FIG. 2B = 20% weight loss cut off with respect to weight on the
day of challenge.
[0036] FIG. 3A and 3B are a pair of graphs showing that A/Michigan/45/2015
(Mich15)
neuraminidase (NA) mRNA formulated with Lipid A LNP induced robust functional
antibodies
(FIG. 3A) and protected mice against weight loss and death when challenged
with a pandemic
strain of influenza virus (FIG. 3B). Neuraminidase inhibition (NAT) titers are
reported as log10
for serum samples taken at study days 14, 28, 42, 56, 88, and 114. Daily
weights were observed
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after intranasal challenge (day 89 for the one-dose groups or day 117 for the
two-dose groups)
with 4LD50 of Belgium09. Weights are presented as the percentage of weight
lost from the day of
challenge. Euthanasia occurred for mice losing more than 20% of their starting
body weight and
for all mice 14 days post-infection (day 103 for the 1 dose groups or day 131
for the 2 dose groups).
Bars are means and standard deviations. Upper dashed line in FIG. 3A = upper
limit of
quantitation. Lower dashed line in FIG. 3A = lower limit of quantitation.
Dashed line in FIG.
3B = 20% weight loss cut off with respect to weight on the day of challenge.
mRNA dosed: 0.4
or 0.016 p,g mRNA encoding Mich15 NA. Control: 0.6 p,g mRNA encoding 1-1EPO or
diluent
(PBS).
[0037] FIG. 4 is a graph showing that Lipid A and Lipid B LNP formulations
with CA09 HA
mRNA (10 p,g) induced robust functional antibodies in cynomolgus macaque
monkeys. HAI titers
are reported as 1og2 for serum samples taken at study days 0, 14, 28, 42, and
56.
[0038] FIGs. 5A-C show the MRT1400 mRNA encoding for influenza virus
A/Singapore/
INFIMH160019/2016 (Sing16; H3N2) HA hemagglutinin. FIG. 5A: an alignment of
the wildtype
(WT) gene and a codon-optimized gene (MRT10279) for the HA antigen. FIG. 5B:
the structure
of the mRNA. FIG. SC: the sequence of the mRNA.
[0039] FIG. 6 is a pair of graphs showing that Lipid A and Lipid B LNP
formulations with
MRT1400 or NA mRNA induced robust functional antibodies in mice. First
injection was given
at study day 0 and second injection was given at study day 28. Left Panel: HAI
titers are reported
as log10 for serum samples taken at study days 14, 28, 42, and 56. Right
Panel: NAT titers are
reported as log10 for serum samples taken at study days 14, 28, 42, and 56.
Bars are geometric
means and geometric standard deviations. Dashed line = lower limit of
quantitation.
[0040] FIG. 7A is a graph showing that Lipid A and Lipid B LNP formulations
with MRT 1400
induced robust functional antibodies in NHPs= HAT titers are reported as 1og2
for serum samples
taken at study days 0, 14, 28, 42, and 56. First injection was given at study
day 0 and second
injection was given at study day 28. Bars are means and standard deviation.
Upper dashed line =
1/40 minimum target. Lower dashed line = lower limit of detection.
[0041] FIG. 7B and 7C are a pair of graphs showing that a Lipid A LNP
formulation (MRT5400)
containing MRT1400 mRNA induced functional antibodies (FIG. 7B) and robust
ELIS A titers
(FIG. 7C) in cynomolgus macaque monkeys at four dose levels: 15, 45, 135 and
250 jag of mRNA.
HAT and ELISA titers are reported as 1og2 for serum samples taken at study
days 0, 14, 28, 42,
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and 56. First injection was given at study day 0 and second injection given at
study day 28. Bars
are means and standard deviations. Dash line = 1/40 minimum target.
[0042] FIGs. 8A and 8B are panels of graphs showing the T cell cytokine
response of
cynomolgus macaques after a second vaccination with Lipid A LNP formulation
MRT5400 in
three dose level groups (250 lag, 135 lag, and 45 lag of mRNA). IFN-7 and IL-
13 induced by re-
stimulation with either the recombinant HA (rHA) protein (left panel) or the
pooled peptides (right
panel) were assessed in peripheral blood mononuclear cells (PMBC) on day 42 by
ELISPOT
assays. The frequencies of PBMC secreting IFN-y (FIG. 8A) or IL-13 (FIG. 8B)
were calculated
as spots forming cells (SFC) per million PBMC. Each symbol represents an
individual sample,
and the bar represents the standard deviation.
[0043] FIG. 9A is a pair of graphs showing that Lipid A LNP formulations
containing modified
and unmodified CA09 HA mRNA were comparable as indicated by HAT titers in
vaccinated mice.
HAT titers are reported as 1og2 for serum samples taken at study days 14, 28,
42, and 56. First
injection was given at study day 0 and second injection was given at study day
28. Bars are means
and standard deviation. Upper dashed line = 1/40 minimum target. Lower dashed
line = lower
limit of quantitation.
[0044] FIG. 9B is a pair of graphs showing that Lipid A LNP formulations
containing modified
and unmodified CA09 HA mRNA were comparable as indicated by ELISA titers in
mice. Total
IgG ELISA titers are reported as log10 for serum samples taken at study days
14, 28, 42, and 56.
First injection was given at study day 0 and second injection was given at
study day 28. Dashed
line = lower limit of quantitation.
[0045] FIGs. 10A and 10B are a pair of graphs showing that bivalent Lipid A
LNP formulations
with CA09 HA mRNA and Sing16 HA mRNA induced robust functional antibodies as
assessed
by HAT titers (CA09 (FIG. 10A) and Sing16 (FIG. 10B)) in Balb/c mice at a dose
of 0.4 Jag of
total mRNA. 0.4 jig mRNA was dosed as a co-encapsulated mRNA-LNP formulation,
or each
HA mRNA was separately administered with 0.2 pig going into each leg. Each HA
mRNA was
also co-encapsulated into a formulation with non-coding mRNA to control for
total mRNA
packing into the LNP. The diluent group received mRNA-LNP diluent buffer. HAT
titers are
reported for serum samples taken at study days -2 (baseline), 14, 28, and 42.
FIG. 10B only shows
study days -2 (baseline from pooled sera) and 42. First injection was given at
study day 0 and
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second injection given at study day 28. Bars are geometric means and geometric
standard
deviations. Dashed line = lower limit of quantitation.
[0046] FIG. 11 shows the functional verification of mRNA-LNP Formulations.
Panel (a) is a
graph showing the expression of firefly (FF) luciferase in BALB/c mice: a
single dose of
Luciferase FF mRNA-LNP (5, 1, 0.1, 0.05 jig) was injected in mice (n=4) by IM
route. Luciferin
(3 mg) was injected at the time of whole animal imaging, using IVIS Spectrum,
Perkin Elmer
recording bioluminescence intensity. Images of whole animal average radiance
at 6, 24, 48 and
7211 after injection were taken. Radiance recorded for 1, 0.5, 0.1 and 0.05
p,g dose administrations
of Luc mRNA-LNP are shown in the graph. Panel (b) shows whole animal images
indicating
total flux of luminescence, at 6 to 72 hours. Total flux of luminescence in
groups of mice (n=4)
receiving 0.1 p,g dose of FF-LNP arc shown. Panel (c) shows the expression of
hEPO in BALB/c
mice. A single dose of hEPO mRNA-LNP (0.1 p,g) was injected in BALB/c mice by
IM route.
hEPO expression was quantified in serum at 6 hours and 24 hours after
administration using
ELISA. Bars represent means and standard deviations. Panel (d) shows the
expression of hEPO
in NHP. A single dose of hEPO mRNA-LNP (10 jig) was injected in Cynomolgus
macaques by
IM route. hEPO expression was quantified in serum at 6, 24, 48, 72, and 96
hours after
administration, using ELISA. Bars represent means and standard deviations.
[0047] FIG. 12 shows the serological evaluation of HA mRNA-LNP vaccine in
mice. BALB/c
mice (n=8 per group) were immunized twice IM, 4 weeks apart with 2, 0.4, 0.08,
and 0.016 lag of
either Ca109 HA mRNA-LNP or Sing16 HA mRNA-LNP. ELISA titers recorded for sera
collected at days 14, 28, 42, 56 against CA09 (Ca109) MINI influenza virus
recombinant HA (left
panel) and Singl 6 H3N2 influenza virus recombinant HA (right panel) are
shown.
[0048] FIG. 13 shows the serological evaluation of HA mRNA-LNP vaccine in
mice. BALB/c
mice (n=8 per group) were immunized twice IM, 4 weeks apart with 2, 0.4, 0.08
and 0.016 p,g of
either CA09 HA mRNA-LNP or Sing16 HA mRNA-LNP. Logic) HAI titers recorded
against
CA09 H1N1 influenza virus (left panel) and Sing16 H3N2 influenza virus (right
panel) are shown.
[0049] FIG. 14 shows the serological evaluation of NA mRNA-LNP vaccine in
mice. BALB/c
mice (n=8 per group) were immunized twice IM 4 weeks apart with 2, 0.4, 0.08,
and 0.016 p,g of
either Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Total IgG titers recorded for
sera
collected at days 0, 14, 28, 42, 56 against Mich15 Ni influenza virus
recombinant NA (left panel)
and Sing16 N2 virus recombinant NA (right panel) are shown
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[0050] FIG. 15 shows the serological evaluation of NA mRNA-LNP vaccine in
mice. BALB/c
mice (n=8 per group) were immunized twice TM 4 weeks apart with 2, 0.4, 0.08
and 0.016 1.,ig of
either Mich15 NA mRNA-LNP or Sing16 NA mRNA-LNP. Logio NAT (ELLA) titers
recorded
for sera against Mich2015 (Ni): A/Mallard/Sweden/2002 (H6) chimeric influenza
virus (left
panel) and Singl 6 (N2): A/Mallard/Sweden/2002 (H6) chimeric virus (right
panel) are shown.
[0051] FIGs. 16A and 16B show the protective efficacy of CA09 HA mRNA-LNP
vaccine in
mice after lethal A/Belgium/2009 H1N1 virus challenge. Mice (n=8) received two
TM doses of
CA09 HA mRNA-LNP (0.4 p,g each) on day 0 and day 28. Control animals received
two TM doses
of diluent on day 0 and day 28. FIG. 16A shows the HAT titers reported as
Logio for serum
samples taken at study days 0, 14, 28, 42, 56, 92, and 107. FIG. 16B shows
daily weights after
intranasal challenge on day 93 with 4LD50 of A/Belgium/2009 H1N1 strain.
Weights arc presented
as the percentage of weight lost from the day of challenge. Individual lines
represent each animal.
[0052] FIGs. 17A-B show the protective efficacy of a single dose of unmodified
Mich15 NA
mRNA-LNP in mice after lethal A/Belgium/2009 H1N1 virus challenge. Mice (n=16)
were
injected by the TM route with 0.4 p,g or 0.016 p,g of Mich15 NA mRNA-LNP. Half
of the mice
only received one injection (1 dose) on study day 0, while the other half (2
doses) received two
injections given at study day 0 and day 28. Control animals received two TM
doses of hEPO
mRNA-LNP (0.6 p,g) on day 0 and day 28. FIG. 17A shows the NAT titers are
reported as Logio
for serum samples taken at study days 0, 14, 28, 42, 56, 88, and 114. FIG. 17B
shows the daily
weight change after intranasal challenge on day 89 for single dose group and
day 117 (89 days
after second dose) for two dose group with 4LD50 of Belgium09 MINI. Weights
are presented
as the percentage of weight lost from the day of challenge. individual lines
represent each animal.
[0053] FIG. 18 shows the serological evaluation of HA Sing16 HA mRNA-LNP
vaccine in
NHP. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks apart by
IM route,
with 15,45 or 135 p,g of Sing16 HA mRNA-LNP. Serum samples were collected at
days -6, 14,
28, 42, and 56. Logio IgG titers against recombinant HA protein of Sing16
virus are shown.
[0054] FIGs. 19A and 19B show the serological evaluation of HA Sing16 HA mRNA-
LNP
vaccine in NHP. Cynomolgus macaques (n=6 per group) were injected twice, 4
weeks apart by
TM route, with 15, 45 or 135 p,g of Singl 6 HA mRNA-LNP. Serum samples were
collected at
days 0, 14, 28, 42, and 56. Logio HAI titers (FIG. 19A) and Logio micro-
neutralization (MN)
titers (FIG. 19B) against 5ing2016 virus are shown.
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[0055] FIGs. 20A and 20B show T cell responses in NHP vaccinated with Sing16
HA mRNA-
LNP vaccine. Cynomolgus macaques (n=6 per group) were injected twice, 4 weeks
apart by IM
route, with 45, 135, or 250 lag of Sing16 HA mRNA-LNP. T cells were determined
by ELISPOT
on day 42 in PBMC stimulated in vitro with peptide pools to represent the
entire HA open reading
frame. The responses of PBMC secreting IFN-7 (FIG. 20A) or IL-13 (FIG. 20B)
calculated as
spots forming cells (SFC) per million PBMC are shown. Each symbol represents
an individual
sample, and the bar represent the geometric mean for the group.
[0056] FIG. 21 shows the secretion of Sing16 H3-specific IgG by memory B cells
on day 180
in NHP vaccinated with Sing16 HA mRNA-LNP vaccine. Cynomolgus macaques (n=6
per group)
were injected twice, 4 weeks apart by IM route, with 15 or 45 pg of Sing16 HA
mRNA-LNP. The
Human IgG single-color memory B cell EL1SPOT kit (CAT# NC1911372, CTL) was
uscd to
measure Sing16/H3-specific and total IgG+ antibody-secreting cells (ASCs).
Differentiation of
MBCs into ASCs was performed in PBMC collected at day 180 by using a
stimulation cocktail
provided by the kit. The number of IgG and number of Sing16/H3-specific ASCs
was calculated
per million of PBMCs for each animal and the frequency of antigen-specific
ASCs is shown.
[0057] FIG. 22 shows the delivery of bivalent combinations of influenza
vaccine in mice.
BALB/c mice (n=8 per group) were immunized twice TM, 4 weeks apart with a
total 0.4 p,g of
bivalent combinations co-encapsulated mRNA transcripts (1:1 wt/wt, half volume
per each leg) or
0.2 us each monovalent which was separately formulated and immunized different
legs. H1H3
combo constituting CA09 HA mRNA-LNP, Sing16 HA rnRNA-LNP; H3N2 combo of Sing16
HA
mRNA-LNP and Sing16 NA mRNA-LNP and N1N2 combo of Mich15 NA mRNA-LNP and
Perth09 NA mRNA-LNP were tested in sera collected a day 0, 14, 28, 42, against
corresponding
virus. Panel (a) shows HAT titers recorded against CA09 H1N1 influenza virus
and Sing2016
H3N2. Panel (b) shows the HAT and NAT titers recorded against Sing2016 H3N2
and
A/Mallard/Sweden/2002 (H6) chimeric influenza virus and H6N2 A/Perth/09 virus
F191 9D (N2)
virus, respectfully. Panel (c) shows NAT titers recorded against Mich15
(Ni):
A/Mallard/Sweden/2002 (H6) chimeric influenza virus and H6N2 A/Perth/09 virus
F1919D (N2)
virus.
[0058] FIG. 23 shows the delivery of quadrivalent combinations of influenza
vaccines in NHP.
Cynomolgus macaques (n=6 per group) were immunized twice IM, 4 weeks apart
with a total 10
i.tg of quadrivalent combinations of co-encapsulated mRNA transcripts (1:1:1:1
wt/wt).
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H2H3N1N2 combo consisting of CA09 HA mRNA, Sing16 HA mRNA, Mich15 NA mRNA, and
Perth09 NA mRNA. H1H3 combo constituting CA09 HA mRNA, 5ing16 HA mRNA and 2x
non-coding mRNA (ncmRNA); H3N2 combo of Sing16 HA mRNA and Perth09 NA mRNA and
2x non-coding mRNA. N1N2 combo of Mich15 NA mRNA, Perth09 NA mRNA-LNP, and 2x
non-coding mRNA. H1 consisting of CA09 HA mRNA and 3x non-coding mRNA. H3
consisting
of Sing16 HA mRNA and 3x non-coding mRNA. Ni consisting of Mich15 NA mRNA and
3x
non-coding mRNA. N2 consisting of Perth09 NA mRNA and 3x non-coding mRNA.
Inhibitory
titers were tested in sera collected a day 0, 14, 28, 42, against
corresponding virus. Panel (a)
shows the HAT titers recorded against CA09 H1N1 influenza virus and Sing16
H3N2. Panel (b)
shows the NAT titers recorded against Mich15 (Ni): A/Mallard/Sweden/2002 (116)
chimeric
influenza virus and H6N2 Perth/09 virus F1919D (N2) virus.
[0059] FIG. 24 depicts a graph showing the expression of human erythropoietin
(hEPO) in mice
treated with various LNP formulations of hEPO mRNA. LNP formulations "Lipid
A," "Lipid B,"
"Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent means and
standard deviations.
The LNP compositions contain the cationic lipid, DMG-PEG2000, cholesterol, and
DOPE, in this
order, at a molar ratio of 40:1.5:28.5:30.
[0060] FIG. 25 depicts a graph showing the expression of hEPO in non-human
primates (NHPs)
treated with various LNP formulations of hEPO mRNA. LNP formulations "Lipid
A," "Lipid B,"
"Lipid C," "Lipid D," and "Lipid E" are shown. Bars represent means and
standard deviations.
The LNP compositions contain the cationic lipid, DMG-PEG2000, cholesterol, and
DOPE, in this
order, at a molar ratio of 40:1.5:28.5:30.
[0061] FIG. 26 depicts a graph showing HAT titers at day 28 and day 42 post
injection with
various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid B,"
"Lipid C,"
"Lipid D," and "Lipid E" are shown. Bars represent means and standard
deviations. The LNP
compositions contain the cationic lipid, DMG-PEG2000, cholesterol, and DOPE,
in this order, at
a molar ratio of 40:1.5:28.5:30.
[0062] FIG. 27 depicts a graph showing Ca109 H1 HAT titers at day 28 and day
42 post injection
with various LNP formulations of HA mRNA. LNP formulations "Lipid A," "Lipid
B," "Lipid
C," "Lipid D," and "Lipid E" are shown. Bars represent means and standard
deviations. The LNP
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compositions contain the cationic lipid, DMG-PEG2000, cholesterol, and DOPE,
in this order, at
a molar ratio of 40:1.5:28.5:30.
[0063] FIG. 28 depicts a graph showing Sing16 H3 HAT titers at day 28 and day
42 post injection
with various LNP formulations of HA mRNA. LNP formulations "Lipid A,- "Lipid
"Lipid
C," "Lipid D," and "Lipid E" are shown. Bars represent means and standard
deviations. The LNP
compositions contain the cationic lipid, DMG-PEG2000, cholesterol, and DOPE,
in this order, at
a molar ratio of 40:1.5:28.5:30.
[0064] FIG. 29 depicts RSV F protein antibody titers in NHPs immunized with
the FD3 F protein
expressing mRNA. The mRNA was delivered with lipid nanoparticles (LNPs)
containing one of
several cationic lipids. The antibody titers were measured at day 0, 21, and
35 for each antigenic
composition.
[0065] FIG. 30 depicts RSV neutralization titers in NHPs immunized with the
FD3 F protein
expressing mRNA. The mRNA was delivered with lipid nanoparticles (LNPs)
containing one of
several cationic lipids. The antibody titers were measured at day 0, 21, and
35 for each antigenic
composition.
[0066] FIG. 31 depicts HAT titers for quadrivalent and octavalent mRNA-LNP
vaccines
administered to mice for 4 different influenza strains.
[0067] FIG. 32 depicts HINT values for quadrivalent and octavalent mRNA-LNP
vaccines,
administered to ferrets for 4 different influenza strains.
[0068] FIG. 33 depicts NAT titers for quadrivalent and octavalent mRNA-LNP
vaccines,
administered to mice for 4 different influenza strains.
[0069] FIG. 34 depicts NAT titers for quadrivalent and octavalent mRNA-LNP
vaccines,
administered to ferrets for 4 different influenza strains. Samples were
obtained on day 20 (D20)
after the second dose of vaccine.
[0070] FIG. 35 depicts NAI titers for quadrivalent and octavalent mRNA-LNP
vaccines,
administered to ferrets for 4 different influenza strains. Samples were
obtained on day 42 (D42)
after the second dose of vaccine.
[0071] FIG. 36 depicts microneutralization titers for Sing16HA-encoding mRNA
in a Lipid A
LNP formulation, administered to NHPs at 15 pg and 45 pg doses. Samples were
obtained on day
6 (D6) and day42 (D42) after the second dose of vaccine.
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[0072] FIG. 37 depicts microneutralization titers for Sing16HA-encoding mRNA
in a Lipid B
LNP formulation, administered to NHPs at 15 lag and 45 lag doses. Samples were
obtained on day
6 (D6) and day42 (D42) after the second dose of vaccine.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present disclosure provides novel lipid nanoparticle (LNP)
formulations for
delivering mRNA vaccines in vivo and methods of making the vaccines. The LNPs
are made of a
mixture of four lipids: a cationic lipid, a polyethylene glycol (PEG)-
conjugated lipid, a cholesterol-
based lipid, and a helper lipid. The LNPs encapsulate mRNA molecules. The
encapsulated mRNA
molecules can be comprised of naturally-occurring ribonucleotides, chemically
modified
nucleotides, or a combination thereof, and can each or collectively code for
one or more proteins.
[0074] The inventors have discovered the present formulations through
screening combinatorial
libraries of lipid components. The present LNPs encapsulate and protect the
mRNA payload from
degradation and facilitate cellular uptake of the encapsulated mRNA. The LNPs
described herein
have enhanced tran sfecti on efficiency, promote en do s om al escape of the
mRNA, and consequently
have improved potency as demonstrated by enhanced expression in vivo and in
vitro when
compared to industrial formulations described in literature. For example, the
LNPs disclosed
herein have superior stability and/or potency profiles compared to known LNPs,
e.g.,
heptatriaconta-6,9,28,31-tetraen-19-y1 4-(dimethyl amino)butano ate (aka DLin-
MC3 -DMA or
MC3; Semple et al., Nat Biotechnol. (2010) 28:172-6) or di((Z)-non-2-en- 1 -
y1) 9-((4-
(dimethylamino)butanoyl)oxy) heptadecanedioate (aka L319; Maier et al., Mol
Ther. (2013)
21(8):1570-8). As further described below, the present formulations
encapsulating an mRNA
encoding hEPO, when delivered in vivo, led to high levels of erythropoietin
circulating in blood at
6 hours and 24 hours, with an up to 12-fold increase, relative to the
industrial standard, the MC3
LNP formulation. Similarly, high potency has been found with other mRNAs, such
as those
encoding influenza antigens, in both murine and non-human primate models.
[0075] The mRNA vaccines as formulated herein can be used to induce a balanced
immune
response comprising both cellular and immoral immunity. Because the advantages
of the present
LNP formulations are not sequence-specific, these formulations can be used to
deliver mRNAs
that encode a variety of antigens, allowing rapid deployment in epidemic or
pandemic situations.
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Further, the present LNP-formulated mRNA vaccines are highly immunogenic and
therefore
provide significant dose sparing possibility.
I. Compositions of the Present Lipid Nanoparticles
[0076] The present LNPs comprise four categories of lipids: (i) an ionizable
lipid; (ii) a
PEGylated lipid; (iii) a cholesterol-based lipid, and (iv) a helper lipid.
A. Ionizable Lipids
[0077] An ionizable lipid facilitates mRNA encapsulation and may be a cationic
lipid. A cationic
lipid affords a positively charged environment at low pH to facilitate
efficient encapsulation of the
negatively charged mRNA drug substance.
[0078] In some embodiments, the cationic lipid is OF-02:
HO 0
HO
OH
0 OH
OF-02
Formula (1)
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OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains
degradable
ester linkages to attach the diketopiperazine core and the doubly-unsaturated
tails, whereas OF-02
contains non-degradable 1,2-amino-alcohol linkages to attach the same
diketopiperazine core and
the doubly-unsaturated tails (Fenton et al., Adv Mater. (2016) 28:2939; U.S.
Pat. 10,201,618). An
exemplary LNP formulation herein, Lipid A, contains OF-2.
[0079] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS
(2014)
111(10:3955-60; U.S. Pat. 9,512,073):
OH
0
.17
HN
y NH
0
OH
cKK-E10
Formula (II)
An exemplary LNP formulation herein, Lipid B, contains cKK-E10.
[0080] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1
(2-(4-(2-((3-
(B is((Z)-2-hydroxyo ctad cc-9-cn-1 -yl)amino)propyl)distil fancyl)cthyl)p ip
crazin-l-yl)cthyl 4-
(bis(2-11ydroxydecyl)amino)butanoate), which is a HEPES-based disulfide
cationic lipid with a
piperazine core, having the Formula III:
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Cy H
OH
0
OH
Ho
Formula (Ill)
An exemplary LNP formulation herein, Lipid C, contains GL-HEPES-E3-E 1 0-DS-3-
E18-1. Lipid
C has the same composition as Lipid A or Lipid B but for the difference in the
cationic lipid.
[0081] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-
(4-(2-((3-
(bis(2-hydroxydecyl)amino)butyl)disulfaneypethyl)piperazin-l-y1)ethyl
4-(bis(2-
hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic
lipid with a
piperazine core, having the Formula IV:
0
71¨\>
HO
N
HO N
OH
SS /
/ __________________________________________________________ / OH _______
/ /
Formula (IV)
An exemplary LNP formulation herein, Lipid D, contains GL-HEPES-E3-E12-DS-4-
E10. Lipid
D has the same composition as Lipid A or Lipid B but for the difference in the
cationic lipid.
[0082] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14
(244424(3-
(Bis (2-hydroxytetrad ec yl)amino)prop yl)di s ul faneyl)ethyl)piperazin-l-y1)
ethyl 4-(bis(2-
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hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic
lipid with a
piperazine core, having the Formula V:
OH
OH (0)7X\
0
171Th\--N HO
µS
Formula (V)
An exemplary LNP formulation herein, Lipid E, contains GL-HEPES-E3-E12-DS-3-
E14. Lipid
E has the same composition as Lipid A or Lipid B but for the difference in the
cationic lipid.
[0083] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1 (III), GL-HEPES-E3-E12-
DS-4-
E10 (IV), and GL-HEPES-E3-E12-DS-3-E14 (V) can be synthesized according to the
general
procedure set out in Scheme 1:
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Scheme 1: General Synthetic Scheme for Lipids of Formulas (III), (IV), and (V)
NSH
,S N
N S
S
(VI)
R'
TBSO 0
R'
TBSO)'R' TBSO -Th 0NSS N
XNLO'r\L (VII)
TBSO R'
R'
HO NSA
N)
HOAR (VIII)
R'
HO R' IR'yl OH
N,RSH
a HO 0
HO R"
crOH
HOAR (IX)
R'
[0084] Other cationic lipids that can be used include those described in Dong,
supra; and U.S.
Pat. 10,201,618.
B. PEGylated Lipids
100851 The PEGylated lipid component provides control over particle size and
stability of the
nanoparticle. The addition of such components may prevent complex aggregation
and provide
a means for increasing circulation lifetime and increasing the delivery of the
lipid-nucleic acid
pharmaceutical composition to target tissues (Klibanov et al., FEBS Letters
(1990) 268
(1):235-7). These components may be selected to rapidly exchange out of the
pharmaceutical
composition in vivo (see, e.g., U.S. Pat. 5,885,613).
[0086] Contemplated PEGylated lipids include, but are not limited to, a
polyethylene glycol
(PEG) chain of up to 5 kDa in length covalently attached to a lipid with alkyl
chain(s) of
C6 -C20 (e.g., C8 ,C10,C12,C14,C16, or C18) lenght, such as a derivatized
ceramide (e.g., N-
octanoyl-sphingosine-l-tsuccinyhmethoxypolyethylene glycol)] (C8 PEG
ceramide)). In some
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embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-
methoxypolyethylene glycol
(DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol
(DSPE-
PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-
PEG); or
1,2-distearoyl-rae-glycero-polyethelene glycol (D SG-PEG).
[0087] In particularly exemplary embodiments, the PEG has a high molecular
weight, e.g., 2000-
2400 gimol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In particular
embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-
PEG2000,
DSG-PEG2000, or C8 PEG2000.
C. Cholesterol-Based Lipids
[0088] The cholesterol component provides stability to the lipid bilayer
structure within the
nanoparticle. In some embodiments, the LNPs comprise one or more cholesterol-
based lipids.
Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-
N-
ethylcarboxamidocholesterol), 1,4-bis(3-N-ol eyl amino-propyl)piperazine (Gao
et al., Biochem
Bioph_ys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139;
U.S. Pat.
5,744,335), imidazole cholesterol ester ("ICE"; WO 2011/068810), P-sitosterol,
fucosterol,
stigmasterol, and other modified forms of cholesterol. In some embodiments,
the cholesterol-
based lipid used in the LNPs is cholesterol.
D. Helper Lipids
[0089] A helper lipid enhances the structural stability of the LNP and helps
the LNP in endosome
escape. It improves uptake and release of the mRNA drug payload. In some
embodiments, the
helper lipid is a zwitterionic lipid, which has fusogenic properties for
enhancing uptake and release
of the drug payload.
Examples of helper lipids are 1,2-dioleoyl-SN-glycero-3-
phosphoethanolamine (DOPE); 1 ,2-distearoyl-sn-glye ero-3 -phosphocholine (D
SP C); 1 ,2-
diole oyl- sn-glyc ero-3 -phospho-L-s erine (DOP S);
1 ,2-di elai doyl- sn-glycero-3 -
ph osph oeth an olamin e (DEP E); and 1,2-di ol e oyl -sn -gl ycero-3 -ph o
sph och olin e (DPOC),
dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-
phosphocholine (DLPC),
1,2-Distearoylphosphatidylethanolamine (DSPE), and
1 ,2-dilauroyl- sn- glycero-3 -
phosphoethanolamine (DLPE).
[0090] Other exemplary helper lipids are di ol eoylph osph dylch olin e
(DOPC),
dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine
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(POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-
carboxylate
(DOPE-ma!), dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine
(DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-0-
monomethyl PE,
16-0-dimethyl PE, 18-1-trans PE, 1-stearoy1-2-oleoyl-phosphatidyethanolamine
(SOPE), or a
combination thereof.
[0091] In particular embodiments, the helper lipid is DOPE. In further
embodiments, the present
LNPs comprise (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-
E10-DS-3-
E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-
PEG2000;
(iii) cholesterol; and (iv) DOPE.
E. Molar Ratios of the Lipid Components
[0092] The inventors have discovered that specific molar ratios of the above
components arc
important for the LNPs' effectiveness in delivering mRNA. The molar ratio of
the cationic lipid,
the PEGylated lipid, the cholesterol-based lipid, and the helper lipid is A:
B: C: D, where A + B +
C + D = 100%. In some embodiments, the molar ratio of the cationic lipid in
the LNPs relative to
the total lipids (i.e., A) is 35-45% (e.g., 38-42% such as 40%). In some
embodiments, the molar
ratio of the PEGylated lipid component relative to the total lipids (i.e., B)
is 0.25-2.75% (e.g., 1-
2% such as 1.5%). In some embodiments, the molar ratio of the cholesterol-
based lipid relative to
the total lipids (i.e., C) is 20-35% (e.g., 27-30% such as 28.5%). In some
embodiments, the molar
ratio of the helper lipid relative to the total lipids (i.e., D) is 25-35%
(e.g., 28-32% such as 30%).
In some embodiments, the (PEGylated lipid + cholesterol) components have the
same molar
amount as the helper lipid. In some embodiments, the LNPs contain a molar
ratio of the cationic
lipid to the helper lipid that is more than 1.
[0093] In particular embodiments, the LNPs contain a cationic lipid, a
PEGylated lipid, a
cholesterol-based lipid, and a helper lipid at a molar ratio of 40: 1.5: 28.5:
30. In further specific
embodiments, the LNPs contain (i) OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1,
GL-
HEPES-E3-E 1 2-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii)
cholesterol; and (iv) DOPE at 40: 1.5: 28.5: 30.
[0094] To calculate the actual amount of each lipid to be put into an LNP
formulation, the molar
amount of the cationic lipid is first determined based on a desired N/P ratio,
where N is the number
of nitrogen atoms in the cationic lipid and P is the number of phosphate
groups in the mRNA to
be transported by the LNP. Next, the molar amount of each of the other lipids
is calculated based
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on the molar amount of the cationic lipid and the molar ratio selected. These
molar amounts are
then converted to weights using the molecular weight of each lipid.
F. Active Ingredients of the LNPs
[0095] The active ingredient of the present LNP vaccine composition is an mRNA
that encodes
an antigen of interest. The antigen may be a polypeptide derived from a virus,
for example,
influenza virus, coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, or MERS-related
virus), Ebola
virus, Dengue virus, human immunodeficiency virus (HIV), hepatitis A virus
(HAY), hepatitis B
virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), respiratory
syncytial virus
(RSV), rhinovirus, cytomegalovirus (CMV), zika virus, human papillomavirus
(HPV), human
metapneumovirus (hMPV), human parainfluenza virus type 3 (PIV3), Epstein-Barr
virus (EBV),
chikungunya virus, or respiratory syncytial virus (RSV).
[0096] The antigen also may be derived from a bacterium, for example,
Staphylococcus aureus,
Moraxella (e.g., Moraxella catarrhalis; causing otitis, respiratory
infections, and/or sinusitis),
Chlamydia trachomatis (causing chlamydia), borrelia (e.g., Borrelia
burgdorferi causing Lyme
Disease), Bacillus anthracis (causing anthrax), Salmonella typhi (causing
typhoid fever),
Mycobacterium tuberculosis (causing tuberculosis), Propionibacterium acnes
(causing acne), or
non-typeable Haemophilus influenzae.
[0097] Where desired, the LNP or the LNP formulation may be multi-valent. In
some
embodiments, the LNP may carry mRNAs that encode more than one antigen, such
as two, three,
four, five, six, seven, eight, nine, ten, or more antigens, from the same or
different pathogens. For
example, the LNP may carry multiple mRNA molecules, each encoding a different
antigen; or
carry a polycistronic mRNA that can be translated into more than one antigen
(e.g., each antigen-
coding sequence is separated by a nucleotide linker encoding a self-cleaving
peptide such as a 2A
peptide). An LNP carrying different mRNA molecules typically comprises
(encapsulate) multiple
copies of each mRNA molecule. For example, an LNP carrying or encapsulating
two different
mRNA molecules typically carries multiple copies of each of the two different
mRNA molecules.
[0098] In some embodiments, a single LNP formulation may comprise multiple
kinds (e.g., two,
three, four, five, six, seven, eight, nine, ten, or more) of LNPs, each kind
carrying a different
mRNA
[0099] Examples of multi-valent LNP vaccines are those containing mRNAs
encoding two or
n-lore antigens from the above-listed pathogens, such as LNP vaccines
comprising mRNAs
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encoding polypeptides derived from influenza virus. In some embodiments, the
multi-valent LNP
vaccines contain mRNA molecules encoding polypeptides derived from two or more
(e.g., three,
four, five, six, seven, eight, nine, or ten) influenza viral proteins selected
from hemagglutinin (e.g.,
hemagglutinin 1 (HA 1) and hemagglutinin 2 (HA2)), neuraminidase (NA),
nucleoprotein (NP),
matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein I (NS1),
and non-structural
protein 2 (NS2). In further embodiments, the multi-valent LNP vaccines
containing two or more
(e.g., three, four five, six, seven, eight, or more) mRNA molecules encoding
antigenic polypeptides
derived from an HA protein, from an NA protein, and from both HA and NA
proteins. In some
embodiments, the mRNA molecules encoding antigenic polypeptides are derived
from different
influenza strains.
[00100] In certain embodiments, the composition may comprise one or more mRNA
molecules
encoding antigens of influenza A, B and C viruses. In one embodiment, the
composition may
comprise one or more mRNA molecules encoding HA and/or NA antigens of
influenza A and
influenza B viruses. In one embodiment, the HA antigens of influenza A viruses
are selected from
subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15,
H16, H17, and
H18. In one embodiment, the NA antigens of influenza A viruses are selected
from subtypes Ni,
N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. In one embodiment, the HA and NA
antigens
of Influenza B viruses are from the Influenza B/Yamagata lineage. In one
embodiment, the HA
and NA antigens of Influenza B viruses are from the Influenza B/Victoria
lineage. In some
embodiments, the one or more HA and NA antigens are from influenza virus
strains recommended
by the World Health Organization (WHO) in their annual recommendation for
influenza vaccine
formulations.
[00101] In certain embodiments, at least one of the one or more influenza
virus proteins comprises
an influenza virus HA protein and/or an influenza virus NA protein having a
molecular sequence
identified or designed from a machine learning model, and in certain
embodiments, at least one of
the one or more ribonucleic acid molecules encode one or more influenza virus
proteins having a
molecular sequence identified or designed from a machine learning model.
[00102] In certain embodiments, the composition comprises two, three, four,
five, six, seven,
eight, nine, or more mRNA molecules encoding (i) one or more HA antigens, (ii)
one or more NA
antigens, or (iii) a combination of one or more HA antigens and NA antigens.
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[00103] In one embodiment, the composition comprises two, three, four, five,
six, seven, eight,
nine, or more mRNA molecules encoding (i) one or more HA antigens, (ii) one or
more NA
antigens, or (iii) a combination of one or more HA antigens and NA antigens,
selected from H1N1,
H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H1ON7 subtypes
and/or B/Yamagata and B/Victoria lineages.
[00104] In one embodiment, the composition comprises one mRNA molecule
encoding an H3
HA antigen, one mRNA molecule encoding an H1 HA antigen, one mRNA molecule
encoding an
HA antigen from the Influenza B/Yamagata lineage, and one mRNA molecule
encoding an HA
antigen from the Influenza B/Victoria lineage.
[00105] In one embodiment, the composition comprises one mRNA molecule
encoding an 113
HA antigen, one mRNA molecule encoding an N2 NA antigen, one mRNA molecule
encoding an
HI HA antigen, one mRNA molecule encoding an NI NA antigen, one mRNA molecule
encoding
an HA antigen from the Influenza B/Yamagata lineage, one mRNA molecule
encoding an NA
antigen from the Influenza B/Yamagata lineage, one mRNA molecule encoding an
HA antigen
from the Influenza B/Victoria lineage, and one mRNA molecule encoding an NA
antigen from the
Influenza B/Victoria lineage.
[00106] In an embodiment, the composition comprises further comprise one or
more mRNA
molecules encoding a machine learning influenza virus HA having a molecular
sequence identified
or designed from a machine learning model, wherein the one or more machine
learning influenza
virus HA may be selected from an Hi HA, an H3 HA, an HA from a B/Victoria
lineage, an HA
from a B/Yamagata lineage, or a combination thereof.
[00107] When selecting one or more machine learning influenza virus HAs, any
machine learning
algorithm may be used. For example, envisioned herein are any of the machine
learning algorithms
and methods disclosed in PCT Application Nos. WO 2021/080990 Al, entitled
Systems and
Methods for Designing Vaccines, and WO 2021/080999 Al, entitled Systems and
Methods for
Predicting Biological Responses, both of which are incorporated by reference
in their entireties
herein.
[00108] The mRNA molecule may be unmodified (i.e., containing only natural
ribonucleotides
A, U, C, and/or G linked by phosphodiester bonds), or chemically modified
(e.g., including
nucleotide analogs such as pseudouridines (e.g., N-1-methyl pseudouridine), 2'-
fluoro
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ribonucleotides, and 2'-methoxy ribonucleotides, and/or phosphorothioate
bonds). The mRNA
molecule may comprise a 5' cap and a polyA tail.
[00109] RSV F Protein:
[00110] Respiratory syncytial virus (RSV) is a negative-sense, single-stranded
RNA virus
belonging to the Pneumoviridae family. RSV can cause infection of the
respiratory tract. RSV is
an enveloped virus with a glycoprotein (G protein), small hydrophobic protein
(SH protein), and
a fusion protein (F protein) on the surface.
[00111] The RSV F protein is responsible for fusion of viral and host cell
membranes and takes
on at least three conformations (pre-fusion, intermediate, and post-fusion
conformations). In the
pre-fusion conformation (pre-fusion, Pre-F), the F protein exists in a
trimeric form with the major
antigenic site 0 exposed. Sitc 0 serves as a primary target of neutralizing
antibodies produccd by
RSV-infected subjects (see, Coultas et al., Thorax. 74: 986-993. 2019;
McLellan et al., Science.
340(6136): 1113-7. 2013). After binding to its target on the host cell
surface, Pre-F undergoes a
conformational change during which site 0 is no longer exposed. Pre-F
transitions into a transient
intermediate conformation, enabling the F protein to insert into the host cell
membrane, leading to
fusion of the viral and host cell membranes. A final conformational shift
results in a more stable
and elongated form of the protein (post-fusion, Post-F). Site II and Site IV
of the F protein are
specific to Post-F, while Site I is present in both the Pre-F and Post-F
conformations (McLellan et
al., J. Virol. 85(15): 7788-7796. 2011).
[00112] As used herein, the term "F protein" or "RSV F protein" refers to the
protein of RSV
responsible for driving fusion of the viral envelope with host cell membrane
during viral entry.
[00113] As used herein, the term "RSV F polypeptide" or "F polypeptide" refers
to a polypeptide
comprising at least one epitope of F protein.
[00114] As used herein, the term "post-fusion" with respect to RSV F refers to
a stable
conformation of RSV F that occurs after merging of the virus and cell
membranes.
[00115] As used herein, the term "pre-fusion" with respect to RSV F refers to
a conformation of
RSV F that is adopted before virus-cell interaction.
[00116] Provided herein are mRNA molecules that encode for antigenic RSV F
polypeptides.
[00117] In some embodiments, the mRNA molecule comprises an open reading frame
(ORF)
encoding a respiratory syncytial virus (RSV) F protein antigen.
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[00118] In some embodiments, the RSV F protein antigen comprises a sequence
having at least
85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to an amino acid sequence set
forth in SEQ
ID NO: 16.
[00119] In some embodiments, the RSV F protein antigen comprises an amino acid
sequence with
at least 98% identity to SEQ ID NO: 16 or consists of an amino acid sequence
of SEQ ID NO: 16.
[00120] In some embodiments, the mRNA comprises a nucleic acid sequence with
at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the
nucleic acid sequence
set forth in SEQ ID NO: 17.
[00121] In some embodiments, the mRNA comprises a nucleic acid sequence with
at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the
nucleic acid sequence
set forth in SEQ ID NO: 21.
[00122] In some embodiments, the RSV F protein antigen is a pre-fusion
protein.
[00123] In some embodiments, wherein the ORF is codon optimized.
[00124] In some embodiments, wherein the mRNA molecule comprises at least one
5'
untranslated region (5' UTR), at least one 3' untranslated region (3' UTR),
and at least one
polyadenylation (poly(A)) sequence.
[00125] In some embodiments, the mRNA comprises at least one chemical
modification.
[00126] In some embodiments, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the mRNA are chemically modified.
[00127] In some embodiments, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the ORF are chemically modified.
[00128] In some embodiments, the chemical modification is selected from the
group consisting
of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-
methylcytosine, 2-
thio-l-methy1-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-
aza-uridine, 2-thio-
dthydropseudouridine, 2-th i o-dihydrouri din e,
2-th i o -pseudouri din e, 4-m ethox y-2-th o-
pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-
pseudouridine,
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5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-
methoxyuridine, and
2'-0-methyl uridine.
[00129] In some embodiments, the chemical modification is selected from the
group consisting
of pseudouridine, Nl-methylpseudouridine, 5-methyleytosine, 5-methoxyuridine,
and a
combination thereof. In some embodiments, the chemical modification is Ni -
methylps eudouri dine.
[00130] In some embodiments, the mRNA comprises of the following structural
elements:
(i) a 5' cap with the following structure:
0
OH OH <op II' NH
0 0 0 NX kr NH2
0
H2N N N 0- 0 0-
0 0
N+ -0¨P=0 Chia
0 CH3 0 =
(ii) a 5' untranslated region (5' UTR) having the nucleic acid sequence of SEQ
ID NO:
19;
(iii) a protein coding region having the nucleic acid sequence of SEQ ID NO:
17;
(iv) a 3' untranslated region (3' UTR) having the nucleic acid sequence of SEQ
ID NO:
20; and
(v) a poly(A) tail.
G. Buffer and Other Components
[00131] To stabilize the nucleic acid and/or LNPs (e.g., to prolong the shelf-
life of the vaccine
product), to facilitate administration of the LNP pharmaceutical composition,
and/or to enhance in
vivo expression of the nucleic acid, the nucleic acid and/or LNP can be
fommlated in combination
with one or more carriers, targeting ligands, stabilizing reagents (e.g.,
preservatives and
antioxidants), and/or other pharmaceutically acceptable excipients. Examples
of such excipients
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are parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride,
chelators (e.g., EDTA)
and the like.
[00132] The LNP compositions of the present disclosure can be provided as a
frozen liquid form
or a lyophilized form. A variety of cryoprotectants may be used, including,
without limitations,
sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The
cryoprotectant may
constitute 5-30% (w/v) of the LNP composition. In some embodiments, the LNP
composition
comprises trehalose, e.g., at 5-30% (e.g., 10%) (w/v). Once formulated with
the cryoprotectant,
the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20 C
to -80 C.
[00133] The LNP compositions may be provided to a patient in an aqueous
buffered solution ¨
thawed if previously frozen, or if previously lyophilized, reconstituted in an
aqueous buffered
solution at bedside. In particularly exemplary embodiments, the buffered
solutionis isotonic and
suitable for e.g., intramuscular or intradermal injection. In some
embodiments, the buffered
solution is a phosphate-buffered saline (PBS).
II. RNA
[00134] The present LNP vaccine compositions of the disclosure may comprise an
RNA molecule
(e.g., mRNA) that encodes an antigen of interest. The RNA molecule of the
present disclosure
may comprise at least one ribonucleic acid (RNA) comprising an ORF encoding an
antigen of
interest. In certain embodiments, the RNA is a messenger RNA (mRNA) comprising
an ORF
encoding an antigen of interest. In certain embodiments, the RNA (e.g., mRNA)
further comprises
at least one 5' UTR, 3' UTR, a poly(A) tail, and/or a 5' cap.
II. A. 5' Cap
[00135] An mRNA 5' cap can provide resistance to nucleases found in most
eukaryotic cells and
promote translation efficiency. Several types of 5' caps are known. A 7-
methylguanosine cap
(also referred to as "m7G" or "Cap-0"), comprises a guanosine that is linked
through a 5' ¨ 5' -
triphosphate bond to the first transcribed nucleotide.
[00136] A 5' cap is typically added as follows: first, an RNA terminal
phosphatase removes one
of the terminal phosphate groups from the 5' nucleotide, leaving two terminal
phosphates;
guanosine triphosphate (GTP) is then added to the terminal phosphates via a
guanylyl transferase,
producing a 5 '5 '5 triphosphate linkage; and the 7-nitrogen of guanine is
then methylated by a
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methyltransferase. Examples of cap structures include, but are not limited to,
m7G(5')ppp,
(5'(A,G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are
described in U.S.
Publication No. US 2016/0032356 and U.S. Publication No. US 2018/0125989,
which are
incorporated herein by reference.
[00137] 5'-capping of polynucleotides may be completed concomitantly during
the in vitro-
transcription reaction using the following chemical RNA cap analogs to
generate the 5'-guanosine
cap structure according to manufacturer protocols: 3'-0-Me-m7G(5')ppp(5')G
(the ARCA cap);
G(5 ')ppp(5 ')A; G(5 ')ppp(5')G; m7G(5 ')ppp(5 ')A;
m7G(5')ppp(5 ')G;
m7G(5')ppp(5')(2'0MeA)pG; m7G(5')ppp(5')(2'0MeA)pU; m7G(5')ppp(5')(2'0MeG)pG
(New
England BioLabs, Ipswich, MA; TriLink Biotechnologies). 5 '-capping of
modified RNA may be
completed post-transcriptionally using a vaccinia virus capping enzyme to
generate thc Cap 0
structure: m7G(5')ppp(5')G. Cap 1 structure may be generated using both
vaccinia virus capping
enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-0-methyl.
Cap 2
structure may be generated from the Cap 1 structure followed by the 2'-0-
methylation of the 5'-
antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap 3 structure
may be generated
from the Cap 2 structure followed by the 2 '-0-methylation of the 5'-
preantepenultimate nucleotide
using a 2'-0 methyl-transferase.
[00138] In certain embodiments, the mRNA of the disclosure comprises a 5' cap
selected from
the
group consisting of 3'-0-Me-m7G(5')ppp(5')G (the ARCA cap), G(5
')ppp(5')A,
G(5 ')ppp(5 ')G, m7G(5')ppp(5 ')A,
m7G(5')ppp(5 ')G, m7G(5')ppp(5)(2P0MeA)pG,
m7G(5')ppp(5')(2'0MeA)pU, and m7G(51)ppp(51)(2`0MeG)pG.
[00139] In certain embodiments, the mRNA of the disclosure comprises a 5' cap
of:
OH OH
0 0 0 N 1\r- NH,
II II II
I I I
0
a a a
Hi14
HA:1TX 0 0
I ==
-0-P=0 CH3
0 CH, 0
II. B. Untranslated Region (UTR)
[00140] In some embodiments, the mRNA of the disclosure includes a 5' and/or
3' untranslated
region (UTR). In mRNA, the 5' UTR starts at the transcription start site and
continues to the start
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codon but does not include the start codon. The 3' UTR starts immediately
following the stop
codon and continues until the transcriptional termination signal.
[00141] In some embodiments, the mRNA disclosed herein may comprise a 5' UTR
that includes
one or more elements that affect an mRNA's stability or translation. In some
embodiments, a 5'
UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5'
UTR may be
about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR is at
least about 10
nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in
length, about 40
nucleotides in length, about 50 nucleotides in length, about 100 nucleotides
in length, about 150
nucleotides in length, about 200 nucleotides in length, about 250 nucleotides
in length, about 300
nucleotides in length, about 350 nucleotides in length, about 400 nucleotides
in length, about 450
nucleotides in length, about 500 nucleotides in length, about 550 nucleotides
in length, about 600
nucleotides in length, about 650 nucleotides in length, about 700 nucleotides
in length, about 750
nucleotides in length, about 800 nucleotides in length, about 850 nucleotides
in length, about 900
nucleotides in length, about 950 nucleotides in length, about 1,000
nucleotides in length, about
1,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500
nucleotides in length,
about 3,000 nucleotides in length, about 3,500 nucleotides in length, about
4,000 nucleotides in
length, about 4,500 nucleotides in length or about 5,000 nucleotides in
length.
[00142] In some embodiments, the mRNA disclosed herein may comprise a 3' UTR
comprising
one or more of a polyadenylation signal, a binding site for proteins that
affect an mRNA's stability
of location in a cell, or one or more binding sites for miRNAs. In some
embodiments, a 3' UTR
may be 50 to 5,000 nucleotides in length or longer. In some embodiments, a 3'
UTR may be 50
to 1,000 nucleotides in length or longer. In some embodiments, the 3' UTR is
at least about 50
nucleotides in length, about 100 nucleotides in length, about 150 nucleotides
in length, about 200
nucleotides in length, about 250 nucleotides in length, about 300 nucleotides
in length, about 350
nucleotides in length, about 400 nucleotides in length, about 450 nucleotides
in length, about 500
nucleotides in length, about 550 nucleotides in length, about 600 nucleotides
in length, about 650
nucleotides in length, about 700 nucleotides in length, about 750 nucleotides
in length, about 800
nucleotides in length, about 850 nucleotides in length, about 900 nucleotides
in length, about 950
nucleotides in length, about 1,000 nucleotides in length, about 1,500
nucleotides in length, about
2,000 nucleotides in length, about 2,500 nucleotides in length, about 3,000
nucleotides in length,
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about 3,500 nucleotides in length, about 4,000 nucleotides in length, about
4,500 nucleotides in
length, or about 5,000 nucleotides in length.
[00143] In some embodiments, the mRNA disclosed herein may comprise a 5' or 3'
UTR that is
derived from a gene distinct from the one encoded by the mRNA transcript
(i.e., the UTR is a
heterologous UTR).
[00144] In certain embodiments, the 5' and/or 3' UTR sequences can be derived
from mRNA
which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid
cycle enzymes) to
increase the stability of the mRNA. For example, a 5' UTR sequence may include
a partial
sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to
improve the nuclease
resistance and/or improve the half-life of the mRNA. Also contemplated is the
inclusion of a
sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3'
end or
untranslated region of the mRNA. Generally, these modifications improve the
stability and/or
pharmacokinetic properties (e.g., half-life) of the mRNA relative to their
unmodified counterparts,
and include, for example, modifications made to improve such mRNA resistance
to in vivo
nuclease digestion.
[00145] Exemplary 5' UTRs include a sequence derived from a CMV immediate-
early 1 (IE1)
gene (U.S. Publication Nos. 2014/0206753 and 2015/0157565, each of which is
incorporated
herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 22) (U.S.
Publication No.
2016/0151409, incorporated herein by reference).
[00146] In various embodiments, the 5' UTR may be derived from the 5' UTR of a
TOP gene.
TOP genes are typically characterized by the presence of a 5'-terminal
oligopyrimidine (TOP)
tract. Furthermore, most TOP genes are characterized by growth-associated
translational
regulation. However, TOP genes with a tissue specific translational regulation
are also known. In
certain embodiments, the 5' UTR derived from the 5' UTR of a TOP gene lacks
the 5' TOP motif
(the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017/0029847,
2016/0304883,
2016/0235864, and 2016/0166710, each of which is incorporated herein by
reference).
[00147] In certain embodiments, the 5' UTR is derived from a ribosomal protein
Large 32 (L32)
gene (U.S. Publication No. 2017/0029847, supra).
[00148] In certain embodiments, the 5' UTR is derived from the 5' UTR of an
hydroxysteroid
(17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016/0166710,
supra).
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[00149] In certain embodiments, the 5' UTR is derived from the 5' UTR of an
ATP5A1 gene
(U.S. Publication No. 2016/0166710, supra).
[00150] In some embodiments, an internal ribosome entry site (IRES) is used
instead of a 5' UTR.
[00151] In some embodiments, the 5'UTR comprises a nucleic acid sequence set
forth in SEQ ID
NO: 19. In some embodiments, the 3'UTR comprises a nucleic acid sequence set
forth in SEQ ID
NO: 20. The 5' UTR and 3'UTR are described in further detail in W02012/075040,
incorporated
herein by reference.
II. C. Polyadenylated Tail
[00152] As used herein, the terms "poly(A) sequence," "poly(A) tail," and
"poly(A) region" refer
to a sequence of adenosine nucleotides at the 3' end of the mRNA molecule. The
poly(A) tail may
confer stability to the mRNA and protect it from exonuclease degradation. The
poly(A) tail may
enhance translation. In some embodiments, the poly(A) tail is essentially
homopolymeric. For
example, a poly(A) tail of 100 adenosine nucleotides may have essentially a
length of 100
nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at
least one nucleotide
different from an adenosine nucleotide (e.g., a nucleotide that is not an
adenosine nucleotide). For
example, a poly(A) tail of 100 adenosine nucleotides may have a length of more
than 100
nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide,
or a stretch of
nucleotides, that are different from an adenosine nucleotide). In certain
embodiments, the poly(A)
tail comprises the
sequence
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AA (SEQ ID NO: 23).
[00153] The "poly(A) tail," as used herein, typically relates to RNA. However,
in the context of
the disclosure, the term likewise relates to corresponding sequences in a DNA
molecule (e.g., a
"poly(T) sequence").
[00154] The poly(A) tail may comprise about 10 to about 500 adenosine
nucleotides, about 10 to
about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides,
or about 40 to about
150 adenosine nucleotides. The length of the poly(A) tail may be at least
about 10, 50, 75, 100,
150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides.
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[00155] In some embodiments where the nucleic acid is an RNA, the poly(A) tail
of the nucleic
acid is obtained from a DNA template during RNA in vitro transcription. In
certain embodiments,
the poly(A) tail is obtained in vitro by common methods of chemical synthesis
without being
transcribed from a DNA template. In various embodiments, poly(A) tails are
generated by
enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using
commercially
available polyadenylation kits and corresponding protocols, or alternatively,
by using immobilized
poly(A)polymerases, e.g., using methods and means as described in
W02016/174271.
[00156] The nucleic acid may comprise a poly(A) tail obtained by enzymatic
polyadenylation,
wherein the majority of nucleic acid molecules comprise about 100 (+/-20) to
about 500 (+1-50)
or about 250 (+/-20) adenosine nucleotides.
[00157] In some embodiments, the nucleic acid may comprise a poly(A) tail
derived from a
template DNA and may additionally comprise at least one additional poly(A)
tail generated by
enzymatic polyadenylation, e.g., as described in W02016/091391, incorporated
herein by
reference.
[00158] In certain embodiments, the nucleic acid comprises at least one
polyadenylation signal.
[00159] In various embodiments, the nucleic acid may comprise at least one
poly(C) sequence.
[00160] The term "poly(C) sequence," as used herein, is intended to be a
sequence of cytosine
nucleotides of up to about 200 cytosine nucleotides. In some embodiments, the
poly(C) sequence
comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100
cytosine nucleotides,
about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine
nucleotides, or about 10
to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence
comprises about
cytosine nucleotides.
II. D. Chemical Modification
25 [00161] The mRNA disclosed herein may be modified or unmodified. In some
embodiments, the
mRNA may comprise at least one chemical modification. In some embodiments, the
mRNA
disclosed herein may contain one or more modifications that typically enhance
RNA stability.
Exemplary modifications can include backbone modifications, sugar
modifications, or base
modifications. In some embodiments, the disclosed mRNA may be synthesized from
naturally
30 occurring nucleotides and/or nucleotide analogues (modified nucleotides)
including, but not
limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T),
cytosine (C), and
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uracil (U)). In certain embodiments, the disclosed mRNA may be synthesized
from modified
nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g.,
1-methyl-adenine, 2-
methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-
isopentenyl-
adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-
cytosine, 2,6-
diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-
methyl-guanine,
inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-
uracil, 4-thio-uracil, 5-
earboxymethylaminomethy1-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-
fluoro-uraci1, 5-
brom o-uracil, S -carboxymetliyl am in om yl -uracil, 5-m eth yl -2-th o-
uracil, S -methyl -uraci I, N-
uracil-5-oxy acetic acid methyl ester, 5-methylaminomethyl-uracil, 5-
methoxyaminomethy1-2-
thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-
oxyacetic acid methyl
ester, uracil-5-oxyacctic acid (v), 1-mcthyl-pscudouracil, qucosinc, 13-D-
mannosyl-qucosinc,
phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates,
7-
deazaguanosine, 5-methylcytosine, and inosine.
[00162] In some embodiments, the disclosed mRNA may comprise at least one
chemical
modification including, but not limited to, pseudouridine, Ni-
methylpseudouridine, 2-thiouridine,
4' -thiouridine, 5-methyl cyto sine, 2-thio-l-methy1-1 -de aza-pseudouri dine,
2-thio-l-methyl-
pseudouri dine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-
dihydrouridine, 2-thio-
pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-
l-methyl-
pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-
methyluridine, 5-
methyluridine, 5-methoxyuridine, and 2'-0-methyl uridine.
[00163] In some embodiments, the chemical modification is selected from the
group consisting
of ps eudouri din e, N1-methylpseudouri dine, 5-methyl cyto sin e, 5-
m eth ox yuri dine, and a
combination thereof.
[00164] In some embodiments, the chemical modification comprises N1-
methylpseudouridine.
[00165] In some embodiments, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the mRNA are chemically modified.
[00166] In some embodiments, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%,
at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%
of the uracil nucleotides
in the ORF are chemically modified.
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[00167] The preparation of such analogues is described, e.g., in U.S. Pat. No.
4,373,071, U.S. Pat.
No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No.
4,500,707, U.S.
Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S.
Pat. No. 5,132,418,
U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642.
II. E. mRNA Synthesis
[00168] The mRNAs disclosed herein may be synthesized according to any of a
variety of
methods. For example, mRNAs according to the present disclosure may be
synthesized via in
vitro transcription (IVT). Some methods for in vitro transcription are
described, e.g., in Geall et
al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods
Enzymol. 530:101-
14. Briefly, 1VT is typically performed with a linear or circular DNA template
containing a
promoter, a pool of ribonucleotide triphosphates, a buffer system that may
include DTT and
magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or 5P6 RNA
polymerase), DNase
I, pyrophosphatase, and/or RNase inhibitor. The exact conditions may vary
according to the
specific application. The presence of these reagents is generally undesirable
in a final mRNA
product and these reagents can be considered impurities or contaminants which
can be purified or
removed to provide a clean and/or homogeneous mRNA that is suitable for
therapeutic use. While
mRNA provided from in vitro transcription reactions may be desirable in some
embodiments,
other sources of mRNA can be used according to the instant disclosure
including wild-type mRNA
produced from bacteria, fungi, plants, and/or animals.
III. Processes for Making the Present LNP Vaccines
[00169] The present LNPs can be prepared by various techniques presently known
in the art. For
example, multilamellar vesicles (MLV) may be prepared according to
conventional techniques,
such as by depositing a selected lipid on the inside wall of a suitable
container or vessel by
dissolving the lipid in an appropriate solvent, and then evaporating the
solvent to leave a thin film
on the inside of the vessel or by spray drying. An aqueous phase may then be
added to the vessel
with a vortexing motion that results in the formation of MLVs. Unilamellar
vesicles (ULV) can
then be formed by homogenization, sonication or extrusion of the multilamellar
vesicles. In
addition, unilamellar vesicles can be formed by detergent removal techniques.
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[00170] Various methods are described in US 2011/0244026, US 2016/0038432, US
2018/0153822, US 2018/0125989, and PCT/U52020/043223 (filed July 23, 2020) and
can be used
to practice the present invention. One exemplary process entails encapsulating
mRNA by mixing
it with a mixture of lipids, without first pre-forming the lipids into lipid
nanoparticles, as described
in US 2016/0038432. Another exemplary process entails encapsulating mRNA by
mixing pre-
formed LNPs with mRNA, as described in US 2018/0153822.
[00171] In some embodiments, the process of preparing mRNA-loaded LNPs
includes a step of
heating one or more of the solutions to a temperature greater than ambient
temperature, the one or
more solutions being the solution comprising the pre-formed lipid
nanoparticles, the solution
comprising the mRNA and the mixed solution comprising the LNP-encapsulated
mRNA. In some
embodiments, the process includes the stcp of heating one or both of the rnRNA
solution and the
pre-formed LNP solution, prior to the mixing step. In some embodiments, the
process includes
heating one or more of the solutions comprising the pre-formed LNPs, the
solution comprising the
mRNA and the solution comprising the LNP-encapsulated mRNA, during the mixing
step. In
some embodiments, the process includes the step of heating the LNP-
encapsulated mRNA, after
the mixing step. In some embodiments, the temperature to which one or more of
the solutions is
heated is or is greater than about 30 C, 37 C, 40 C, 45 C, 50 C, 55 C, 60 C,
65 C, or 70 C. In
some embodiments, the temperature to which one or more of the solutions is
heated ranges from
about 25-70 C, about 30-70 C, about 35-70 C, about 40-70 C, about 45-70 C,
about 50-70 C, or
about 60-70 C. In some embodiments, the temperature is about 65 C.
[00172] Various methods may be used to prepare an mRNA solution suitable for
the present
invention. In some embodiments, mRNA may be directly dissolved in a buffer
solution described
herein. In some embodiments, an mRNA solution may be generated by mixing an
mRNA stock
solution with a buffer solution prior to mixing with a lipid solution for
encapsulation. In some
embodiments, an mRNA solution may be generated by mixing an mRNA stock
solution with a
buffer solution immediately before mixing with a lipid solution for
encapsulation. In some
embodiments, a suitable mRNA stock solution may contain mRNA in water or a
buffer at a
concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6
mg/ml, 0.8 mg/ml, 1.0
mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml,
3.0 mg/ml, 3.5
mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.
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[00173] In some embodiments, an mRNA stock solution is mixed with a buffer
solution using a
pump. Exemplary pumps include but are not limited to gear pumps, peristaltic
pumps and
centrifugal pumps. Typically, the buffer solution is mixed at a rate greater
than that of the mRNA
stock solution. For example, the buffer solution may be mixed at a rate at
least lx, 2x, 3x, 4x, 5x,
6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock
solution. in some
embodiments, a buffer solution is mixed at a flow rate ranging between about
100-6000 ml/minute
(e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-
2400 ml/minute,
2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420
ml/minute). In
some embodiments, a buffer solution is mixed at a flow rate of, or greater
than, about 60 ml/minute,
100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300
ml/minute,
340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600
ml/minute,
1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000
ml/minute.
[00174] In some embodiments, an mRNA stock solution is mixed at a flow rate
ranging between
about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute,
about 30-60
ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360
ml/minute, about
360-480 ml/minute, or about 480-600 ml/minute). In some embodiments, an mRNA
stock solution
is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15
ml/minute, 20
ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45
ml/minute, 50 ml/minute,
60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400
ml/minute, 500
ml/minute, or 600 ml/minute.
[00175] The process of incorporation of a desired mRNA into a lipid
nanopartiele is referred to
as "loading." Exemplary methods are described in Lasic et al., FEBS Lett.
(1992) 312:255-8. The
LNP-incorporated nucleic acids may be completely or partially located in the
interior space of the
lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or
associated with the
exterior surface of the lipid nanoparticle membrane. The incorporation of an
mRNA into lipid
nanoparticles is also referred to herein as "encapsulation" wherein the
nucleic acid is entirely or
substantially contained within the interior space of the lipid nanoparticle.
[00176] Suitable LNPs may be made in various sizes. In some embodiments,
decreased size of
lipid nanoparticles is associated with more efficient delivery of an mRNA.
Selection of an
appropriate LNP size may take into consideration the site of the target cell
or tissue and to some
extent the application for which the lipid nanoparticle is being made.
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[00177] A variety of methods known in the art are available for sizing of a
population of lipid
nanoparticles. Particularly exemplary methods herein utilize Zetasizer Nano ZS
(Malvern
Panalytical) to measure LNP particle size. In one protocol, 10 !al of an LNP
sample are mixed
with 990 p,1 of 10% trehalose. This solution is loaded into a cuvette and then
put into the Zetasizer
machine. The z-average diameter (nm), or cumulants mean, is regarded as the
average size for the
LNPs in the sample. The Zetasizer machine can also be used to measure the
polydispersity index
(PDI) by using dynamic light scattering (DLS) and cumulant analysis of the
autocorrelation
function. Average LNP diameter may be reduced by sonication of formed LNP.
Intermittent
sonication cycles may be alternated with quasi-elastic light scattering (QELS)
assessment to guide
efficient lipid nanoparticle synthesis.
[00178] In some embodiments, the majority of purified LNPs, i.c., greater than
about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs,
have a size
of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130
nm, about 125
nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm,
about 95 nm, about
90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all
(e.g., greater than
80 or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm
(e.g., about 145 nm,
about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about
115 nm, about
110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or
about 80 nm).
[00179] In some embodiments, the LNPs in the present composition have an
average size of less
than 150 nm, less than 120 nrn, less than 100 nm, less than 90 nm, less than
80 nm, less than 70
nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.
[00180] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%,
98%, 99% of the LNPs in the present composition have a size ranging from about
40-90 nm (e.g.,
about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70
nm (e.g., 55-
65 nm) arc particular suitable for pulmonary delivery via nebulization.
[00181] In some embodiments, the dispersity, or measure of heterogeneity in
size of molecules
(PDI), of LNPs in a pharmaceutical composition provided by the present
invention is less than
about 0.5. In some embodiments, an LNP has a PDI of less than about 0.5, less
than about 0.4,
less than about 0.3, less than about 0.28, less than about 0.25, less than
about 0.23, less than about
0.20, less than about 0.18, less than about 0.16, less than about 0.14, less
than about 0.12, less than
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about 0.10, or less than about 0.08. The PDI may be measured by a Zetasizer
machine as described
above.
[00182] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%,
or 99% of the purified LNPs in a pharmaceutical composition provided herein
encapsulate an
mRNA within each individual particle. In some embodiments, substantially all
(e.g., greater than
80% or 90%) of the purified lipid nanoparticles in a pharmaceutical
composition encapsulate an
mRNA within each individual particle. In some embodiments, a lipid
nanoparticle has an
encapsulation efficiency of between 50% and 99%; or greater than about 60, 65,
70, 75, 80, 85,
90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an
encapsulation
efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).
[00183] In some embodiments, an LNP has a N/P ratio of between 1 and 10. In
some
embodiments, a lipid nanoparticle has a N/P ratio above 1, about 1, about 2,
about 3, about 4, about
5, about 6, about 7, or about 8. In further embodiments, a typical LNP herein
has an N/P ratio of
4.
[00184] In some embodiments, a pharmaceutical composition according to the
present invention
contains at least about 0.5 ug, 1 ug, 5 ug, 10 ug, 100 ug, 500 ug, or 1000 ug
of encapsulated
mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 ug
to 1000 jig,
at least about 0.5 ug, at least about 0.8 ug, at least about 1 ug, at least
about 5 1.ig, at least about 8
ug, at least about 10 us, at least about 50 mg, at least about 100 pig, at
least about 500 us, or at least
about 1000 lug of encapsulated mRNA.
[00185] In some embodiments, mRNA can be made by chemical synthesis or by in
vitro
transcription (IVT) of a DNA template. An exemplary process for making and
purifying mRNA
is described in Example 1. In this process, in an IVT process, a cDNA template
is used to produce
an mRNA transcript and the DNA template is degraded by a DNase. The transcript
is purified by
depth filtration and tangential fl ow filtration (TFF). The purified
transcript is further modified by
adding a cap and a tail, and the modified RNA is purified again by depth
filtration and TFF.
[00186] The mRNA is then prepared in an aqueous buffer and mixed with an
amphiphilic solution
containing the lipid components of the LNPs. An amphiphilic solution for
dissolving the four lipid
components of the LNPs may be an alcohol solution. In some embodiments, the
alcohol is ethanol.
The aqueous buffer may be, for example, a citrate, phosphate, acetate, or
succinate buffer and may
have a pH of about 3.0-7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0,
about 5.5, about 6.0, or
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about 6.5. The buffer may contain other components such as a salt (e.g.,
sodium, potassium, and/or
calcium salts). In particular embodiments, the aqueous buffer has 1 mM
citrate, 150 mM NaCl,
pH 4.5.
[00187] An exemplary, nonlimiting process for making an mRNA-LNP composition
is described
in Example 1. The process involves mixing of a buffered mRNA solution with a
solution of lipids
in ethanol in a controlled homogeneous manner, where the ratio of lipids:mRNA
is maintained
throughout the mixing process. In this illustrative example, the mRNA is
presented in an aqueous
buffer containing citric acid m on oh ydrate, tri -sodium citrate dill ydrate,
and sodium chloride. The
mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaC1, pH
4.5). The lipid
mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a
cholesterol-based lipid, and a
helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the
ethanol lipid solution
are mixed at a volume ratio of 4:1 in a "T" mixer with a near "pulseless" pump
system. The
resultant mixture is then subjected for downstream purification and buffer
exchange. The buffer
exchange may be achieved using dialysis cassettes or a TFF system. TFF may be
used to
concentrate and buffer-exchange the resulting nascent LNP immediately after
formation via the T-
mix process. The diafiltration process is a continuous operation, keeping the
volume constant by
adding appropriate buffer at the same rate as the permeate flow.
IV. Packaging and Use of the mRNA-LNP Vaccines
[00188] The mRNA-LNP vaccines can be packaged for parenteral (e.g.,
intramuscular,
intradermal or subcutaneous) administration or nasopharyngeal (e.g.,
intranasal) administration.
The vaccine compositions may be in the form of an extemporaneous formulation,
where the LNP
composition is lyophilized and reconstituted with a physiological buffer
(e.g., PBS) just before
use. The vaccine compositions also may be shipped and provided in the form of
an aqueous
solution or a frozen aqueous solution and can be directly administered to
subjects without
reconstitution (after thawing, if previously frozen).
[00189] Accordingly, the present disclosure provides an article of
manufacture, such as a kit, that
provides the mRNA-LNP vaccine in a single container, or provides the mRNA-LNP
vaccine in
one container and a physiological buffer for reconstitution in another
container. The contain er(s)
may contain a single-use dosage or multi-use dosage. The containers may be pre-
treated glass
vials or ampules. The article of manufacture may include instructions for use
as well.
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[00190] In particular embodiments, the mRNA-LNP vaccine is provided for use in
intramuscular
(IM) injection. The vaccine can be injected to a subject at, e.g., his/her
deltoid muscle in the upper
arm. In some embodiments, the vaccine is provided in a pre-filled syringe or
injector (e.g., single-
chambered or multi-chambered). In some embodiments, the vaccine is provided
for use in
inhalation and is provided in a pre-filled pump, aerosolizer, or inhaler.
[00191] The mRNA-LNP vaccines are administered to subjects in need thereof in
a
prophylactically effective amount, i.e., an amount that provides sufficient
immune protection
against a target pathogen for a sufficient amount of time (e.g., one year, two
years, five years, ten
years, or life-time). Sufficient immune protection may be, for example,
prevention or alleviation
of symptoms associated with infections by the pathogen. In some embodiments,
multiple doses
(e.g., two doses) of the vaccine arc injected to subjects in need thereof to
achieve the desired
prophylactic effects. The doses (e.g., prime and booster doses) may be
separated by an interval of
e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months,
four months, five
months, six months, one year, two years, five years, or ten years.
[00192] In some embodiments, a single dose of the mRNA-LNP vaccine contains 1-
50 pg of
mRNA (e.g., monovalent or multivalent). For example, a single dose may contain
about 2.5 pg,
about 5 p,g, about 7.5 lag, about 10 jig, about 12.5 lag, or about 15 lag of
the mRNA for intramuscular
(IM) injection. In further embodiments, a multi-valent single dose of an LNP
vaccine contains
multiple (e.g., 2, 3, or 4) kinds of LNPs, each for a different antigen, and
each kind of LNP has an
mRNA amount of, e.g., 2.5 jig, about 5 jig, about 7.5 jug, about 10 jig, about
12.5 jug, or about 15
[00193] In another aspect, the present invention provides methods of
immunizing a subject against
one or more influenza viruses. The present invention further provides methods
of eliciting an
immune response against one or more influenza viruses in a subject. In some
embodiments, the
present methods comprise administering to the subject an effective amount of a
composition
described herein to a subject.
[00194] In various embodiments, the methods of immunizing provided herein
elicit a broadly
protective immune response against multiple epitopes within one or more
influenza viruses. In
various embodiments, the methods of immunizing provided herein elicit a
broadly neutralizing
immune response against one or more influenza viruses. In some embodiments,
the immune
response comprises an antibody response. Accordingly, in various embodiments,
the composition
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described herein can offer broad cross-protection against different types of
influenza viruses. In
some embodiments, the composition offers cross-protectionagainst avian, swine,
seasonal, and/or
pandemic influenza viruses. In some embodiments, the composition offers cross-
protection
against one or more influenza A, B, or C subtypes. In some embodiments, the
composition offers
cross-protection against multiple strains of influenza A Hl-subtype viruses
(e.g., H1N1), influenza
A H3-subtype viruses (e.g., H3N2), influenza A H5-subtype viruses (e.g.,
H5N1), and/or influenza
B viruses (e.g., Yamagata lineage, Victoria lineage).
[00195] In some embodiments, the methods of the invention are capable of
eliciting an improved
immune response against one or more seasonal influenza strains. Exemplary
seasonal strains
include, without limitation, A/Puerto Rico/8/1934, A/Fort Monmouth/1/1947,
A/Chile/1/1983,
A/Texas/36/1991, A/Singapore/6/1986, A/Beijing/32/1992, A/New
Caledonia/20/1999,
A/Solomon Islands/03/2006, A/Brisbane/59/2007, A(H3N2) virus antigenically
like the cell-
propagated prototype virus ANictori a/361/201 1 , A/Beijing/262/95 (H1N1)-like
virus,
A/Brisbane/02/2018 (H1N1)pdm09-like virus, A/Brisbane/10/2007 (H3N2)-like
virus,
A/California/7/2004 (H3N2)-like virus, A/California/7/2009 (H1N1)-like virus,
A/California/7/2009 (H1N1)pdm09-like virus, A/Cambodia/e0826360/2020 (H3N2)-
like virus,
A/Fujian/411/2002 (H3N2) - like virus, A/Fujian/411/2002 (H3N2)-like virus,
A/Guangdong-
Maonan/S WL1536/2019 (H1N1)pdm09-like virus-like virus, A/Hawaii/70/2019
(H1N1)p dm09-
like virus-like virus, A/Hong Kong/2671/2019 (H3N2)-like virus, A/Hong
Kong/45/2019 (H3N2)-
like virus, A/Hong Kong/4801/2014 (H3N2)-like virus, A/Kansas/14/2017 (H3N2)-
like virus,
A/Michigan/45/2015 (H1N1)pdm09-like virus, A/Moscow/10/99 (H3N2)-like virus,
A/New
Caledonia/20/99 (H1N1)-like virus, A/Perth/16/2009 (H3N2)-like virus,
A/Singapore/I4FIMH-
16-0019/2016 (H3N2)-like virus, A/Solomon Islands/3/2006 (H1N1)-like virus,
A/South
Australia/34/2019 (H3N2)-like virus, A/Switzerland/8060/2017 (H3N2)-like
virus,
A/Switzerland/9715293/2013 (H3 N2)-like virus,
A/Sydney/5/97 (H 3 N2)-like virus,
A/Texas/50/2012 (H3N2)-like virus, A/Victoria/2570/2019 (H1N1)pdm09-like
virus,
ANictoria/2570/2019 (H1N1)pdm09-like virus -like virus, A/Victoria/361/2011
(H3N2) -like
virus, A/Wellington/1/2004 (H3N2)-like virus, A/Wisconsin/588/2019 (H1N1)pdm09-
like virus,
A/Wisconsin/588/2019 (H1N1)pdm09-like virus-like virus, A/Wisconsin/67/2005
(H3N2)-like
virus, B/Beijing/184/93-like virus, B/Brisbane/60/2008-like virus,
B/Colorado/06/2017-like virus
(B/Victoria/2/87 lineage), B/Florida/4/2006-like virus, B/Hong Kong/330/2001-
like virus,
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B/Malaysia/2506/2004-like virus, B/Massachusetts/2/2012-like virus,
B/Phuket/3073/2013
(B/Yamagata lineage)-like virus, B/Phuket/3073/2013 -like virus,
B/Phuket/3073/2013-like virus
(B/Yamagata/16/88 lineage), B/Shangdong/7/97-like virus, B/Shanghai/361/2002-
like virus,
B/Sichuan/379/99-like virus, B/Washington/02/2019 (B/Victoria lineage)-like
virus,
B/Washington/02/2019-like (B/Victoria lineage) virus, and B/Wisconsin/1/2010-
like virus. In
some embodiments, the methods of the invention are capable of eliciting an
improved immune
response against one or more pandemic influenza strains. Exemplary pandemic
strains include,
without limitation, A/C al i forni a/07/2009, A/Cali forni a/04/2009,
A/Belgium/145/2009, A/South
Carolina/01/1918, and A/New Jersey/1976. Pandemic subtypes include, in
particular, the H1N1,
H5N1, 112N2, 113N2, 119N2, 117N7, 117N3, 117N9 and Hi 0N7 subtypes. In some
embodiments,
the methods of the invention arc capable of eliciting an improved immune
response against one or
more swine influenza strains. Exemplary swine strains include, without
limitation, A/New
Jersey/1976 isolates and A/California/07/2009. In some embodiments, the
methods of the
invention are capable of eliciting an improved immune response against one or
more avian
influenza strains. Exemplary avian strains include, without limitation, H5N1,
H7N3, H7N7,
H7N9, and H9N2. Additional influenza pandemic, seasonal, avian and/or swine
strains are known
in the art.
[00196] In some embodiments, the present invention provides methods of
preventing or treating
influenza infections by administering the composition of the invention to a
subject in need thereof
In some embodiments, the subject is suffering from or susceptible to an
influenza infection. In
some embodiments, a subject is considered to be suffering from an influenza
infection if the subject
is displaying one or more symptoms commonly associated with influenza
infection. in some
embodiments, the subject is known or believed to have been exposed to the
influenza virus. In
some embodiments, a subject is considered to be susceptible to an influenza
infection if the subject
is known or believed to have been exposed to the influenza virus. In some
embodiments, a subject
is known or believed to have been exposed to the influenza virus if the
subject has been in contact
with other individuals known or suspected to have been infected with the
influenza virus and/or if
the subject is or has been present in a location in which influenza infection
is known or thought to
be prevalent.
[00197] In various embodiments, the composition as described herein may be
administered prior
to or after development of one or more symptoms of influenza infection. In
some embodiments,
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the composition is administered as a prophylactic. In such embodiments, the
methods of the
invention are effective in preventing or protecting a subject from influenza
virus infection. In
some embodiments, the composition of the present invention is used as a
component of a seasonal
and/or pandemic influenza vaccine or as part of an influenza vaccination
regimen intended to
confer long-lasting (multi-season) protection. In some embodiments, the
composition of the
presenting invention is used to treat the symptoms of influenza infection.
[00198] In some embodiments, the subject is a non-human mammal. In some
embodiments, the
subject is a farm animal or a pet (e.g., a dog, a cat, a sheep, cattle, and/or
a pig). In some
embodiments, the subject is a non-human primate. In some embodiments, the
subject is an avian
(e.g., a chicken, a duck, a goose and/or a turkey).
[00199] In some embodiments, the subject is a human. In certain embodiments,
thc subject is an
adult, an adolescent, or an infant. In some embodiments, the human subject is
younger than 6
months of age. In some embodiments, the human subject is 6 months of age or
older, is 6 months
through 35 months of age, is 36 months through 8 years of age, or is 9 years
of age or older. In
some embodiments, the human subject is an elderly aged 55 years or older, such
as 60 years of age
or older, or 65 years of age or older. Also contemplated by the present
invention are the
administration of the composition and/or performance of the methods of
treatment in utero.
[00200] Unless otherwise defined herein, scientific and technical terms used
in connection with
the present invention shall have the meanings that are commonly understood by
those of ordinary
skill in the art. Exemplary methods and materials are described below,
although methods and
materials similar or equivalent to those described herein can also be used in
the practice or testing
of the present invention. In case of conflict, the present specification,
including definitions, will
control. Generally, nomenclature used in connection with, and techniques of,
cell and tissue
culture, molecular biology, virology, immunology, microbiology, genetics,
analytical chemistry,
synthetic organic chemistry, medicinal and pharmaceutical chemistry, and
protein and nucleic acid
chemistry and hybridization described herein are those well-known and commonly
used in the art.
Enzymatic reactions and purification techniques are performed according to
manufacturer's
specifications, as commonly accomplished in the art or as described herein.
Further, unless
otherwise required by context, singular terms shall include pluralities and
plural terms shall include
the singular. Throughout this specification and embodiments, the words "have"
and "comprise,"
or variations such as "has," "having," "comprises," or "comprising," will be
understood to imply
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the inclusion of a stated integer or group of integers but not the exclusion
of any other integer or
group of integers. All publications and other references mentioned herein are
incorporated by
reference in their entirety. Although a number of documents are cited herein,
this citation does
not constitute an admission that any of these documents forms part of the
common general
knowledge in the art. As used herein, the term "approximately" or "about" as
applied to one or
more values of interest refers to a value that is similar to a stated
reference value. In certain
embodiments, the term refers to a range of values that fall within 10%, 9%,
8%, 7%, 6%, 5%, 4%,
3%, 2%, 10
/0 or less in either direction (greater than or less than) of the stated
reference value
unless otherwise stated or otherwise evident from the context.
[00201] In order that this invention may be better understood, the following
examples are set forth.
These examples arc for purposes of illustration only and arc not to be
construed as limiting the
scope of the invention in any manner.
EXAMPLES
Example 1: Optimization of LNP Formulations
[00202] This Example describes a study in which a series of LNP formulations
for mRNA
vaccines was prepared from combinatorial libraries of various components.
Rationally designed
novel cationic lipids were synthesized. Altogether, more than 150 lipids and
more than 430
formulations were tested. Human erythropoietin (hEPO) mRNA was used as a test
mRNA. In the
lead formulations described below, the mRNA was formulated into LNP using
combinations of
the cation ic lipids and the three other lipids ¨helper lipids; cholesterol-
based lipids; and PEGylated
lipids ¨ in various permutations of combinations.
[00203] The LNP formulations consisted of four lipid components ¨ ionizable
lipid, helper lipid
DOPE, cholesterol, and PEGylated lipid DMG-PEG-2K. The PEGylated lipid molar
fraction was
held constant at 1.5%, while the ionizable lipid and the different helper
lipids and their molar ratios
were evaluated to identify the optimized ratios based on the hEPO screening
studies.
[00204] Citrate buffer (1 mM citrate, 150 mM NaCl, pH 4.5) was used in the
preparation of LNP
formulation. mRNA solution added to the citrate buffer was mixed with the
lipids in ethanol
solution during the formulation process. The pH and the concentration of the
buffer were selected
to achieve the high rate of mRNA encapsulation in the LNP formulation.
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[00205] The LNP formulation process included mixing the lipid ethanol solution
and the mRNA
citrate solution in a 'T' mixer using a pump system. The resultant solution
was then subjected to
buffer exchange using TFF/ dialysis tubes. The concentration of the final
formulation in 10%
(w/v) trehalose was adjusted based on dosing needs.
[00206] Mouse in vivo expression of hEPO protein was used as a surrogate to
measure the potency
of the LNPs to delivery mRNA in vivo. In this study, a single dose of hEPO
mRNA (0.1 lug)
formulated in LNPs derived from various combinations of the components was
injected into mice
intramuscularly (TM). Serum collected at 6 hours and 24 hours after
administration was tested for
hEPO levels using ELISA. MC3 formulation, an industry benchmark, was used a
reference for
the calculation of fold-increase in hEPO expression (Angew, Chem Int Ed.
(2012) 51:8529-33).
[00207] The level of hEPO expression seen for each LNP formulation indicated
the formulation's
ability to deliver mRNA into cells. The initial formulations included 2-
dioleoyl-sn-glyeero-3-
phosphoethanolarnine (DOPE; helper lipid), DMG-PEG2000, and cholesterol at the
molar ratio of
cationic lipid: DMG-PEG2000: cholesterol: DOPE at 40:1.5:28.5:30. These
formulations were
found to have robust potency when compared to MC3 formulations.
[00208] Further formulations were tested. Optimized formulations Lipid A LNP
and Lipid B LNP
are shown in Table 1. The mRNA in these formulations can be modified or
unmodified and may
encode an antigen derived from a virus such as influenza or SARS-CoV-2.
Table 1. Composition of Exemplary LNP Formulations
Components Function Description
mRNA Active substance mRNA Construct
Cationic Lipid OF-02 Ionizable lipid,
facilitates mRNA
(A) or cKK-E10 (B) encapsulation
lipid DOPE Zwitterionic lipid,
enhances uptake and
nanoparticle release of drug payload
(LNP)
Del ivery
Cholesterol Provides stability to
lipid bilayer
DMG-PEG-2K Provides control and
stability to the
lipid bilayer
Trehalose Excipient Cryoprotectant
Water for Injection (WFI) Diluent N/A
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[00209] In Table 1, the final dosing for a human vaccine would be dilution of
the above final bulk
product in phosphate-buffered saline (PBS) based on the intended single human
dose. The WFI
amount is calculated based upon nominal of final drug product. Trehalose
content in the
formulation corresponds to 10% (100 mg/mL) trehalose dihydrate, converted to
an anhydrous basis
using the ratio of the molecular weight values of anhydrous trehalose and
trehalose dihydrate.
E00210] The molar ratios of lipid components in two optimized formulations ¨
Lipid A and Lipid
B LNP formulations ¨ are shown in Table 2 (CL: cationic lipid).
Table 2. Molar Ratios of Lipid Components in Exemplary LNPs
CL LNP Code Molar Ratios of CL: DMG-PEG2000: Cholesterol:
DOPE
OF-02 Lipid A 40: 1.5: 28.5: 30
cKK-E10 Lipid B 40: 1.5: 28.5: 30
[00211] As shown in Table 3 and FIG. 1A, the fold increase of hEPO expression
for Lipid A and
Lipid B compared to MC3 indicates the superiority of these LNPs over MC3 for
the delivery of
mRNA. In the table below, "P2" means PEG2000; "Times MC3" means the fold of
increase over
MC3; and "Std Dev" means standard deviation.
Table 3. In vivo Delivery of hEPO mRNA in Mice
Times
Std
Study # Cationic lipid Formulation Composition
MCI Dev
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
1 1.74
0.97
(P2 low DOPE) 40:3:27:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DSPC
0.18 0.17
(P2 w/ DSPC) 50:1.5:38.5:10
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
2 OF-02 5.04
1.79
40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
3 7.35
3.90
(high DOPE) 40:1.5:13.5:45
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
4 OF-02
16.19 7.86
40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
5 OF
12.13 6.56
40:1.5:28.5:30
6 cKK-E10
Cationic lipid: DMG-PEG2000: cholesterol: DOPE 5.41
3.46
40:1.5:28.5:30
cKK-E10 Cationic lipid: DMG-PEG2000: cholesterol:
DEPE
7 5.77
2.09
(DEPE) 40:1.5:28.5:30
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Times
Std
Study # Cationic lipid Formulation Composition
MC3 Dev
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
6.59 2.50
(177 nm) 40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
4.94 1.75
(161 nm) 40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
7.40 3.54
(153 nm) 40:1.5:28.5:30
8
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
7.15 3.86
(133 nm) 40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
5.91 2.79
(115 nm) 40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
10.54 4.38
(118 nm) 40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DSPC
OF-02 (DSPC) 0.00
0.00
40:5:25:30
Cationic lipid: DMG-PEG2000: cholesterol: DSPC
OF-02 (DSPC) 0.00
0.00
40:3.5:26.5:30
9
Cationic lipid: DMG-PEG2000: cholesterol: DSPC
OF-02 (DSPC) 0.00
0.00
40:2:28:30
Cationic lipid: DMG-PEG2000: cholesterol: DSPC
OF-02 (DSPC) 0.99
0.70
40:2:53:5
Cationic lipid: DMG-PEG2000: cholesterol: DOPS
OF-02 (DOPS) 3.26
1.97
40:1.5:28.5:30
OF-02 (DEPE) Cationic lipid: DMG-PEG2000: cholesterol:
DEPE
11.83 6.89
40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DOPC
OF-02 (DOPC) 3.32
1.20
40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
OF-02 7.14
3.37
40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
cKK-E10 5.58
2.01
40:1.5:28.5:30
OF-02 Cationic lipid: DMG-PEG2000: cholesterol:
DOPE
11 8.81
3.22
(PD lot) 40:1.5:28.5:30
Cationic lipid: DMG-PEG2000: cholesterol: DOPE
cKK-E10 5.16
3.25
40:1.5:28.5:30
[00212] FIG. 1B shows hEPO expression in mice and non-human primates (NHPs)
using LNPs
Lipid A and Lipid B. A single dose of hEPO mRNA (0.1 lig for mice and 10 lig
for NHPs)
formulated with Lipid A or Lipid B was injected intramuscularly. Serum hEPO
levels were
5 quantified at 6, 24, 48, and 72 hours after administration using
ELISA. The data show prolonged
hEPO protein expression in vivo even beyond 4 days in mice and NHPs.
[00213] One of the key process parameters identified during optimization was
the flow rate during
initial mixing step. Formulations with different final LNP sizes (ranging from
108-177 nm) were
prepared by changing these flow rates during mixing, allowing additional
control on process and
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product attributes. The higher the flow rate, the smaller the particle size.
When the flow rate
reached 375 ml/min, producing an average LNP size of 108 nM, there was a
markedly increased
potency. The impact of size on potency of LNP was noted as a measure of fold
increase in hEPO
expression over MC3 as Table 4.
Table 4. LNP Size Optimization
Formulation Total Flow rate Size PD! Encapsulation Cationic
Times
Lot# (ml/min) (nm) (%) Lipid
MC3
1 250 108 0.077 99 MC3
1.00
2 62.5 177 0.086 94 OF-02
6.59
3 75 161 0.075 95 OF-02
4.94
2-88 87.5 152 0.116 97 OF-02
7.40
2-89 125 133 0.089 97 OF-02
7.15
2-90 250 115 0.076 98 OF-02
5.91
2-91 375 108 0.042 98 OF-02
10.54
*PDI: polydispersity index.
[00214] The above screening data show that helper lipid DOPE was effective in
promoting protein
expression. The data also led to determination of the promising molar
composition of the four
lipids (0E-02 or cKK-E10: DMG-PEG-2K: cholesterol: DOPE = 40:1.5:28.5:30). LNP
formulations in 10% trehalose were characterized for all parameters including
particle size, PDI,
mRNA encapsulation, and mRNA integrity. All the tested batches showed the
desired
characteristics and stability in freeze/thaw cycling. The long-term stability
of the formulation at -
80 C in 10% (w/v) trehalose was assessed. Lipid A and Lipid B formulations
were shown to be
highly stable.
Example 2: Influenza H1N1 LNP Vaccine Formulations
[00215] Influenza pandemics can occur when a novel influenza virus emerges in
the human
population. Such pandemics remain a major threat to public health, requiring
vigilant attention
and preparedness with countermeasures to be used in the event of sustained
human-to-human
spread of the virus. In the experiments described in this Example,
hemagglutinin (HA) from a
highly pathogenic H1N1 strain A/California/7/2009 (CA09), the cause of the
2009 flu pandemic,
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was used as a prototype antigen to evaluate the potency of mRNA vaccines
prepared with LNP
formulations of Lipid A and Lipid B.
[00216] The HA mRNA was prepared as described above. Citrate buffer (1 mM
citrate, 150 mM
NaCl, pH 4.5) was used in the preparation of the LNP compositions. A citrate
buffer containing
the mRNA was mixed with the lipids in ethanol solution during the formulation
process. The pH
and the concentration of the buffer were selected to achieve the high
encapsulation rate of mRNA
in the LNP formulations. The two solutions (mRNA in citrate buffer and lipids
in ethanol solution)
were mixed in a "T" mixer using a pump system, resulting in a homogeneous
pulseless flow,
wherein the lipids and the mRNA were mixed at a constant ratio throughout the
process. This was
critical to achieve a homogeneous formulation with the desired size and a low
PDI, an indicator of
a more homogeneous size distribution. This process resulted in high mRNA
encapsulation, which
is critical for achieving high potency. The resultant solution was then
subjected to buffer exchange
using TFF/di al ysi s tubes.
[00217] In a mouse study, efficacy of Lipid A and Lipid B CA09 HA formulations
were assessed
in a head-to-head comparison to MC3 LNP formulation as well as recombinant HA
(rHA). CA09
(H1) HA mRNA (0.4 p,g) formulated with different cationic lipids was injected
intramuscularly
into Balb/C mice (n=8) on day 0 (DO) and day 28 (D28). Immunogenicity of the
vaccines, as
indicated by HA inhibition (HAT) titers, is shown in FIG. 2A. The data show
that two
immunizations of Lipid A or Lipid B on day 0 (DO) and day 28 (D28) elicited
high HAT titers and
allowed complete protection of animals from homologous viral challenge
(Belgium09 H1N1 virus)
(FIG. 2B). During 14 days of post challenge observation, no obvious signs of
morbidity (weight
loss) were observed within the Lipid A and Lipid B treated groups, while a
small number of
animals within the recombinant protein control group demonstrated morbidity
(FIG. 2B).
[00218] Similarly, mRNA encoding neuraminidase (NA) from the Mich15 influenza
strain
(Mich 1 5 Ni) was formulated with Lipid A and evaluated for its potency. Two
doses (0.4 or 0.016
1.1,g) of NA mRNA formulated with Lipid A were injected intramuscularly into
Balb/c mice (n=8).
The control groups (n=8) were injected with 0.6 lag of hEPO mRNA or with
diluent. Half of the
mice received only one injection (1 dose) on study day 0, while the other half
received two
injections (2 doses) given at study day 0 and day 28. The data show that this
N1 Lipid A
formulation elicited robust immune response, as indicated by NA inhibition
(NAT) titers (FIG.
3A). The data further show that the mice treated with either one dose or two
doses of the vaccine
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were protected from lethal viral challenge by Belgium09 H1N1 (FIG. 3B). The
level of protection
correlated with the NAT titers of vaccine treatment groups versus the negative
control groups
(hEPO and diluent).
[00219] The CA09 H1 mRNA formulated with the present LNPs was also tested in
an NHP model.
The mRNA (10 lag) was formulated with Lipid A and Lipid B, and injected
intramuscularly into
eynomolgus macaque monkeys (n=6) on study days 0 and 28. Detectable HAT
priming by day 14
and a significant boost in HAT titer by day 28 for all LNPs were observed
(FIG. 4, right panel).
ELISA data also demonstrated significant priming over baseline by day 14 for
all doses tested with
a robust boost detected two weeks after the boost (FIG. 4, left panel). The
results show that the
present H1 mRNA formulations resulted in robust immune responses as indicated
by HAT and
endpoint EL1SA titers.
Example 3: Influenza 113N2 LNP Vaccine Formulation
[00220] This Example describes experiments in which mRNA-LNP vaccine
formulations for
influenza strain Sing16 (H3N2) were evaluated for potency. One of the mRNAs
used in these
experiments is MRT1400. MRT1400 is a biosynthetic codon-optimized HA-H3
(influenza virus
hemagglutinin, H3 subtype) messenger RNA (CO-HA-H3 mRNA) manufactured by in
vitro
transcription.
[00221] The protein sequence for influenza virus hemagglutinin, H3 subtype, is
shown below:
MKTIIALSYI LCLVFAQKIP GNDNSTATLC LGHHAVPNGT IVKTITNDRI
EVTNATELVQ NSSIGEICDS 2HQILDGENC TLIDALLGDP QCDGFQNKKW
DLFVERSKAY SNCYPYDVPD YASLRSLVAS SGTLEFKNES FNWTGVTQNG
TSSACIRGSS SSFFSRLNWL THLNYTYPAL NVTMPNKEQF DKLYIWGVHH
PGTDKDQIFL YAQSSGRITV STKRSQQAVI PNIGSRPRIR DIPSRISIYW
TIVKPGDILL INSTGNLIAP RGYFKIRSGK SSIMRSDAPI GKCKSECITP
NGSIPNDKPF QNVNRITYGA CPRYVKHSTL KLATGMRNVP EKQTRGIFGA
IAGFIENGWE GMVDGWYGFR HQNSEGRGQA ADLKSTQAAI DQINGKLNRL
IGKMEKDHQ lEKEDSEVEG RVQDLEKYVE DTKIDLWSYN AELLVALENQ
HTIDLTDSEM NKLYEKTKKQ LRENAEDMGN GCK_LYHKCD NACIESIRNE
TYDHNVYRDE ALNNRFQIKG VELKSGYKDW ILWISFAISC FLLCVALLGF
IMWACQKGNI RCNICI* (SEQ ID NO:1)
[00222] The coding sequence for this protein was codon-optimized. The codon-
optimized
sequence encoding the protein is shown in FIG. SA (SEQ ID NO:2), where the
wildtype sequence
is shown as SEQ ID NO:3. The mRNA structure and sequence are shown in FIGs. 5B
and 5C,
respectively. As shown in the figures, the HA-H3 mRNA coding sequence is
flanked by 5' and 3'
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untranslated regions (UTRs) of 140 and 100 nucleotides, respectively. The
biosynthetic HA-H3
mRNA also contains a 5' cap structure consisting of a 7-methyl guanosine (m7G)
residue linked
via an inverted 5'-5' triphosphate bridge to the first nucleoside of the 5'
UTR, which is itself
modified by 2'-0-ribose methylation. The 5' cap is essential for initiation of
translation by the
ribosome. The entire linear structure is terminated at the 3' end by a tract
of approximately 100 to
500 adenosine nucleosides (polyA). The polyA region confers stability to the
mRNA and is also
thought to enhance translation. All of these structural elements are naturally
occurring components
used to promote the efficient translation of the HA -H3 mRNA.
[00223] A DNA plasmid was constructed for producing the codon-optimized mRNA
sequence by
in vitro transcription. In vitro transcription (IVT) reaction was carried out
using RNA polymerase.
The reaction mixes were precipitated. The precipitated RNA samples were loaded
onto individual
depth filtration cassette, washed with 80% ethanol and re-dissolved with
recirculating water. A
second aliquot of water was pumped through in a manner similar to the first
step. This step was
repeated one more time. The pooled eluates were subjected to
ultrafiltration/diafiltration using a
50 kD hollow fiber TFF cassette. Each IVT TFF pool was then diluted in
preparation for cap and
tail reactions. Cap-tail reactions were precipitated and the RNA from the
reaction was purified
and collected as described above. The filtered mRNA was stored at -20 C until
use.
[00224] In these experiments, mRNA encoding Sing16 NA (N2) or Sing16 HA (H3;
MRT1400
mRNA) antigens was formulated with Lipid A or Lipid B LNPs and injected
intramuscularly into
Balb/c mice (n=8) on DO and D28 at 0.4 pg of mRNA per dose. For comparison, 1
lag of
recombinant Singl 6 H3 or Singl 6 N2 protein with an oil-in-water emulsion
adjuvant (AF03) was
injected by the intramuscular route into Balb/c mice (n=8). Immune responses
were measured by
NAT and HAT assays.
[00225] The data show that animals immunized with NA (N2) mRNA demonstrated
detectable
NA I priming by day 14 and a significant boost in NA I titer by day 28 (FIG.
6, right panel). The
data also show that HA Sing16 Lipid A and Lipid B formulations elicited robust
HAT responses
after boosting on day 28 (FIG. 6, left panel).
[00226] Similarly, the Sing16 HA mRNA Lipid A and Lipid B vaccines were
evaluated in non-
human primates (NHPs), cynomolgus macaque monkeys (n=6). The HA Sing 16 mRNA
(50 p,g)
formulated with Lipid A or Lipid B was injected by the intramuscular route
into the monkeys. The
first injection was given at study day 0 and the second injection was given at
study day 28. The
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data show that the vaccines elicited robust immune functional responses
boosted on day 28 (FIG.
7A).
[00227] In addition, four dose levels of HA Sing16 mRNA formulated in Lipid A
(i.e., MRT5400
vaccine) ¨ 15, 45, 135 and 250 ¨ were evaluated in NHPs. The first
immunization was given
at study day 0 second immunization at study day 28. All NHPs demonstrated IgG
binding and
HAT titers for all doses tested with no differences in immune response between
the various doses
tested at two weeks after the second injection at D42 (FIGs. 7B and 7C).
[00228] The Sing16 HA mRNA Lipid A vaccine was also evaluated for a T cell
response in NHPs
after the second vaccination. Peripheral blood mononuclear cells (PBMCs) were
collected at day
42 and incubated overnight with either the Sing16 113 recombinant protein or
the peptide pools
representing the entire HA open reading frame. Cytokines induced by the re-
stimulation were
assessed in ELISPOT assays. The frequencies of PBMC secreting IFN-y, a Thl
cytokine (FIG.
8A), or IL-13, a Th2 cytokine (FIG. 8B) were calculated as spot-forming cells
(SFC) per million
PBMC. The majority of animals in the three dose level groups tested (250 i.tg,
135 itg, and 45 lug)
demonstrated the presence of high frequency of IFN-y secreting cells, with
over 100 SFCs per
million PBMCs (FIG. 8A). A dose-response was not observed, as the animals in
the lower and
higher dose level groups showed comparable frequencies of IFN-y secreting
cells. In contrast, the
presence of IL-13 cytokine secreting cells was not detected in any of the
groups tested and at any
dose level (FIG. 8B). These data presented clear evidence for a Thl-biased
cellular response and
a lack of Th2 response to the HA antigen following vaccination in NHPs.
Example 4: Influenza LNP Vaccine Formulations with Modified mRNA
[00229] This Example describes experiments comparing the potency of vaccines
containing
unmodified (unmodified non-replicating or "UNR") and modified (modified non-
replicating or
"MN R") mRNA. UN R CA09 HA mRNA and MNR CA09 HA mRNA were prepared by in vitro
transcription. In MNR, all uridines were replaced by pseudouridines.
[00230] Five different doses (0.016, 0.08, 0.4, 2, and 10 i(g) of CA09 HA mRNA
(either modified
or unmodified) formulated with Lipid A were injected by the intramuscular
route into Balb/c mice
(n=15). The data show that the LNP formulations increased the stability and
delivery efficiency
of naked mRNA (UNR), for the potency between UNR and MNR mRNA was comparable
as
indicated by HAT titers (FIG. 9A). ELISA data for Balb/c mice also
demonstrated significant
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priming over baseline by day 14 for all doses tested (both UNR and MNR mRNAs),
with a robust
boost detected two weeks after the boost. The data also show that UNR and MNR
mRNAs were
comparable in eliciting ELISA titers (FIG. 9B).
[00231] In conclusion, the present dose titration study demonstrated that
unmodified and modified
CA09 HA mRNA formulated with Lipid A elicited statistically indistinguishable
immune
responses in Balb/c mice, as indicated by either HAT or by endpoint ELISA
assay. Balb/c mice
immunized with the four higher doses of UNR and MNR mRNA demonstrate
detectable HAT
priming by day 14 and a significant boost in HAT titer by day 42 for all
doses. These day-14
priming titers represent both a dose effect and dose sparing potential for
generating detectable
titers over a 125-fold range. The second injection titers at the same dose
range confirms the
robustness of thc immune response to this mRNA-LNP formulation. Similar
results wcrc also
observed in non-human primates.
Example 5: Multi-Valent Influenza Vaccine LNP Formulation
[00232] This Example describes a study using a Lipid A-based LNP vaccine
containing mRNA
encoding CA09 HA (as described in Example 2) and mRNA encoding Sing16 HA (as
described
in Example 3).
[00233] More specifically, CA09 HA mRNA and Sing16 HA mRNA co-encapsulated in
Lipid A
were evaluated in Balb/c mice (n=8). mRNA-LNP was administered as two mRNAs co-
encapsulated or dosed separately as singly encapsulated mRNAs. For both
approaches, a total of
0.4 jig LNP formulation was injected into mice by intramuscular injection. The
first injection was
given at study day 0 and the second injection was given at study day 28. The
data show that the
vaccines elicited robust immune functional responses. There did not appear to
be any difference
between the two administration approaches. These data show that co-
encapsulation did not cause
hindrance or interference between the two mRNAs.
Example 6: Further Studies on Multi-Valent Influenza Vaccine LNP Formulations
[00234] A panel of unmodified mRNAs encoding CA09 HA, Sing16 HA, Sing16 NA,
Mich15
NA, A/Perth/16/2009 influenza virus (Perth09 NA), and reporter antigens of
firefly luciferase (FF)
and hEPO were prepared. LNP formulations for HA and NA mRNA-LNP preparation
were then
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tested for expression in vitro, the immune responses in animals, and for
potency in preclinical
models. For the studies in this Example, all of the LNP formulations were the
Lipid A formulation.
Materials and Methods
mRNA-LNP Preparations
[00235] mRNA transcripts encoding for hEPO, FF, CA09 HA, Sing16 HA, Mich15 NA,
and
Sing16 NA were synthesized by in vitro transcription employing RNA polymerase
with a plasmid
DNA template encoding the desired gene using unmodified nucleotides. The
resulting purified
precursor mRNA was reacted further via enzymatic addition of a 5' cap
structure (Cap 1) and a 3'
poly(A) tail of approximately 200 nucleotides in length as determined by gel
electrophoresis and
purified. All mRNA preparations were analyzed for purity, integrity, and
percentage of Cap 1
before storage at -20 C. Preparation of mRNA/lipid nanopartiele (LNP)
formulations was
described above. Briefly, an ethanolic solution of a mixture of lipids
(ionizable lipid,
phosphatidylethanolamine, cholesterol and polyethylene glycol-lipid) at a
fixed lipid and mRNA
ratio were combined with an aqueous buffered solution of target mRNA at an
acidic pH under
controlled conditions to yield a suspension of uniform LNPs. Upon
ultrafiltration and diafiltration
into a suitable diluent system, the resulting nanoparticle suspensions were
diluted to final
concentration, filtered, and stored frozen at -80 C until use. The mRNA-LNP
formulations were
characterized for size by dynamic light scattering, percentage encapsulation
and were stored at -
80 C at lmg/mL until further use by dilution with suitable buffer. hEPO-LNPs
and FF-LNPs were
utilized to check level of expression of target protein in vivo.
Visualization of S-Proteins Expressed in IfeLa cells
[00236] Immunocytochemistry-immunofluorescence analysis of influenza NA and HA-
proteins
was performed in HeLa cells transfected with bivalent H3N2 (Sing16 HA and
Perth09 NA)
mRNAs LNPs) using method described previously (Kalnin et al., npj Vaccines
(2021) 6:61). Cells
were fixed in 4% paraformaldehyde and subjected antibody staining for HA
(GeneTex
G1X40258), NA, and ER marker Calnexin (Abeam ab22595) was performed. Images
were
captured on confocal microscope followed by image analysis for quantification
of HA and NA
colocalization to the ER, mean signal intensity, and percent of cell area.
Flow Cytometry
[00237] Human skeletal muscle cells (HskMCs, Lonza) were cultured in M199
(Life
Technologies) supplemented with GlutaMAX (Life Technologies), streptomycin,
penicillin
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(Gibco), and 20% heat inactivated FBS (VWR) at 37 C with 5% CO,. The cells
were harvested
by trypsinization, washed with PBS, and electroporated using human primary
muscle cell
transfection kit on Nucleofector 2b (Lonza) with 12 mg of rnRNA per 106 cells
following
manufacturer's electroporation program D-033. Post 24 hour harvested cells
were fixed,
permeabilized with CytofixTm/Perm (BD) and stained with CA09 HA (Immune Tech),
Sing16 HA
(30-2F11-F7-A5, GeneTex), Mich15 NA (6G6, Immune Tech) and Sing16 NA (40017-
RP01, Sino
Biologicals) specific Ab followed by PE conjugated goat anti-mouse IgG
secondary Ab (Southern
Biotech) or AF647 conjugated goat anti-rabbit IgG (Life Technologies). Then
the antibody-
labeled cells were acquired by Fortessa (BD) and the expression of each
protein was analyzed by
FlowJoTM (Tre e Star).
Cryogenic Transmission Electron Microscopy
[00238] A PELCO easiGlowTM device was used to plasma-clean the grids prior to
LNP sample
application, and a Vitrobot Mark IV System (TherrnoFisher) with the chamber
held at 100%
humidity and 18 C was used for plunge freezing. A 3.0 p1 droplet of LNP sample
was dispensed
onto 300 mesh R2/1 QUANTIFOILED grids with carbon film and gold bars. Grids
were blotted
for 4 seconds, held in place for 10 seconds, and then immediately plunge
frozen in liquid ethane
for storage and transfer to a Krios microscope. Exposures were collected using
a Titan Krios
transmission electron microscope (ThermoFisher) equipped with a BioQuantum
energy filter and
K3 direct electron detector (Gatan) operating in counting mode. Calibrated
physical pixel size at
the detector was 1.38 A, corresponding to 64,000x magnification. A total of
3,141 69-frame movie
exposures were collected at a dose per frame of 1.045 e/A2 with defocus
between -U.S to -1.7 p.m.
For each movie exposure, patch-based motion correction, binning of super-
resolution pixels, and
frame dose-weighting was performed using RELION-3.1.34. From corrected images,
over 700
candidate particle coordinates were extracted. Subsequent data analysis was
done with MATLAB
R2019a with image processing toolbox.
Immunization of Mice and NHPs for Expression Studies
[00239] Groups of four cynomolgus macaques (NHPs) (male and female) and four
to eight male
BALB/c mice were administered intramuscularly either dose of 10 lag (NHP) or
1, 0.5, 0.1, and
0.05 lig (mice) with hEPO-LNP prepared in the same ratio as the one intended
to be used for
HA/NA mRNA-LNP formulations. Blood samples were taken pre-administration, and
at 6h, 24h,
48h, 72h, and 96h post administration to monitor for serum hEPO expression via
an ELISA using
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R and D Systems, Quantikinek IVD ELISA, Human Erythropoietin Immunoassay kit
as per
manufacturers protocol, and reported as final values of mIU/m1 and ng/ml.
Briefly, microplate
wells, precoated with a mouse monoclonal antibody specific for EPO were
incubated with
specimen or standard. After removing excess specimen or standard, wells were
incubated with a
rabbit anti-EPO polyclonal antibody conjugated to horseradish peroxidase.
During the second
incubation, the antibody-enzyme conjugate bound to the immobilized EPO. Excess
conjugate was
removed by washing. A chromogen was added to the wells and was oxidized by the
enzyme
reaction to form a blue colored complex. The reaction was stopped by the
addition of acid, which
turned the blue to yellow. The amount of color generated was directly
proportional to the amount
of conjugate bound to the EPO antibody complex, which, in turn, was directly
proportional to the
amount of EPO in the specimen or standard. The absorbance of this complex was
measured, and
a standard curve was generated by plotting absorbance versus the concentration
of the EPO
standards. The EPO concentration of the unknown specimen was determined by
comparing the
optical density of the specimen to the standard curve. The standards used in
this assay were
recombinant hEPO calibrated against the Second International Reference
Preparation (67/343), a
urine-derived form of human erythropoietin.
Immunization of Mice and NHPs for Immunogenieity Studies
[00240] Groups of Balb/c mice (Mus muscu/us) as per the treatment group were
immunized under
isoflurane anesthesia with a dose of 0.05 mL of designated vaccine preparation
or diluent via the
IM route in the quadriceps, on day 0 in one hind leg and day 28 in the
contralateral leg. Mice that
lost more than 20% of their initial body weight and displayed severe clinical
signs were eutlianized
after the veterinarian's assessment of the animal's health prior to the study
termination.
[00241] Naive male and female Mauritius origin Cynomolgus macaques (Macaca
fascicularis)
were selected for the study. Animals weighed > 2kg and were >2 years of age at
the start of the
study. Animals selected for the study underwent comprehensive physical
examinations prior to
assignment to the study. The pre-assignment assessment of health status
included a hands-on
veterinarian examination and blood sample collections for CBC analysis as
applicable per NIRC
SOPs. Animals were generally housed in pairs and acclimated for at least 3
days prior to the start
of the study. Groups consisted of up to 6 animals per treatment group. All
animals were
immunized under ketamine HC1 (10 mg/kg, IM) or telazol (4-8 mg/kg, IM)
sedation with a dose
of 0.5 ml of their respected vaccine preparation or diluent via the IM route
in one forelimb of each
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animal, targeting the deltoid, on Study Day 0. Twenty-eight days after the
first immunization took
place, a second immunization was given to the animals in the contralateral
limb.
Immunization of Mice and NHPs for Challenge Studies
[00242] Mice were inoculated with the challenge strain approximately 9-12
weeks after the last
immunization. Vials of stock virus were thawed and diluted to the appropriate
concentration in
ice-cold sterile PBS. All mice were challenged with a total volume of 50 tl
containing 105.54
TCID50 of Belgium09 virus in PBS which equated to 4LD50. Virus challenge was
performed inside
the biosafety cabinet in an enhanced ABSL2 laboratory. Mice were first
anesthetized with an IP
injection of a Ketamine/Xylazine solution (50 mg/kg Ketamine and 5 mg/kg
Xylazine), and then
challenged IN (dropwise into both nostrils; 25 pi per nostril) with a total
volume of 50 p,1 of
influenza virus using a micropipette. Following the challenge procedure, mice
were placed in
dorsal recumbency and observed until recovery from anesthesia. Daily body
weights were taken
following HI NI challenge. Any individual animal with a single observation >
20% body weight
loss was euthanized. The weight measurements were either recorded daily post
challenge until
euthanasia in the online database, Pristimag (Version 7.5.0 Build 8), or
written on study specific
working sheets.
Blood Collection
[00243] For mice, blood was collected via submandibular or orbital sinus
bleeds (in-life bleed,
pre-study and on study days 14, 28, and 42 approximately 200 1,11) and cardiac
puncture (terminal
bleed, day 56) from all animals under sedation. Mice were bled on pre-study to
obtain a base-line
pre-immune serum sample and for pre-screening purposes. Processing of the
serum, blood
samples were collected into SST tubes and allowed to clot for 30 minutes to 1
hour at room
temperature. The samples were then centrifuged 1000 ¨ 1300 g for 5-10 minutes
with brakes off.
Serum was collected using a P200 pipettor, divided into two 0.5 ml cryovials,
and stored at -20 C.
All bleeds were documented on specimen collection and processing logs,
indicating the time of
sample collection and the technician responsible for performing the procedure.
A portion of the
serum samples were evaluated in the HAT or ELLA and ELISA assays for antibody
titers.
[00244] NHPs were bled for serum isolation while under anesthesia administered
intramuscularly
using10 mg/kg ketamine/1 mg/kg acepromazine (days -4, 2, 7, 14, 28, 30, 35,
42, 56, 90, and 180).
The volume of blood withdrawn did not exceed established guidelines with
respect to percentage
of body weight and animal's physical condition. Blood was withdrawn from
anesthetized NHPs
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using femoral venipuncture using a Vacutainer 21 ga x 1" blood collection
needle or Abbott
Butterfly 23 ga x 3/4" tubing attached to BD Vacutainer SSTI'm gel tubes.
Serum was isolated by
spinning the tubes at room temperature at a speed of 1200 x g for 10 minutes.
Serum was then
aliquoted into labeled cryovials (1 ml/vial) and stored at < -20 C. A portion
of the serum samples
were evaluated in the HAT or ELLA and ELISA assays for antibody titers. For
PBMCs, NHPs
were pre-bled before vaccination and again approximately 42-63 days after the
first injection. For
this purpose, blood was collected into BD Vacutainer tubes containing heparin
anticoagulant.
Briefly, anticoagulated blood samples were diluted in PBS and subjected to
gradient density
centrifugation for 30 minutes at 400 x g using Histopaque separation solution
(Sigma). The
opaque interface containing mononuclear cells was then collected, washed three
times in PBS
using a low speed (250 x g) centrifugation for the last centrifugation to
reduce the number of
platelets. The live vs. dead PBMC were enumerated using a Nexcelom Cellometer
K2. The
PBMC were cryopreserved in FBS with 10% DMSO using Mr. Frosty freezing boxes.
The boxes
were placed immediately into a -80 C freezer for 24 hours and then transferred
for storage in a
liquid nitrogen tank.
ELISA
[00245] The antibody ELIS As were performed using recombinantly produced
Sing16 NA protein,
5ing16 HA protein, or CA09 HA protein. The proteins were captured on 96 well
high binding
polystyrene plates at a concentration of 2p,g/m1 in carbonate-bicarbonate
buffer. The plates were
covered and incubated overnight (16 4 hours) at 2-8 C. After overnight
incubation, the antigen
coated plates were washed 5 times with a washing buffer (PBS, 0.5% Tween20)
and blocked with
a blocking solution (10% BSA in PBS) for 60 30 minutes at room temperature.
Test samples,
naïve control, and the reference sample were diluted in a sample diluent (PBS
10% BSA 0.5%
Tween 20) and added to wells in duplicates followed by incubation at room
temperature for 90
minutes. Plates were washed 5 times with the washing buffer, and goat anti-
mouse R P for mouse
sera or goat anti-monkey HRP for NHP sera was added at a dilution of 1:10,000.
The plates were
then incubated 30 minutes at room temperature and the excess HRP-IgG was
washed with the
washing buffer. Sure-Blue TMB substrate was added to each plate and the
reaction was stopped
after about 10 minutes with TMB stop solution. The plates were then read at
450 nm with a
Thermo Labsystems MultiskanTM spectrophotometer. The anti-antigen (HA or NA)
specific
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antibody titers were expressed as a reciprocal of the highest serum dilution
with an absorbance
value >0.3.
HAI Assay
[00246] HAT assays were performed using the Sing16 H3N2 and the CA09 H1N1
virus stocks
(BIOQUAL, Inc.). Sera were treated with receptor-destroying enzyme (RDE) by
diluting one-part
serum with three parts enzyme and incubated overnight in a 37 C water bath.
Enzyme was
inactivated by a 30-minute incubation period at 56 C followed by addition of
six parts PBS for a
final dilution of 1/10. HAT assays were performed in V-bottom 96-well plates
using four
hemagglutinating units (HAU) of virus and 0.5% turkey RBC. The reference serum
for each strain
was included as a positive control on every assay plate. Each plate also
included a back-titration
to confirm the antigen dose (4 HAU/25p,1) as well as a negative control sample
(PBS or naïve
control serum). The HAT titer was determined as the highest dilution of serum
resulting in
complete inhibition of hemagglutination. Results were only valid for plates
with the appropriate
back-titration result (verifying 4 HAU/25 pi added) and a reference serum
titer within 2-fold of
the expected titer.
NAI Assay
[00247] The method for the enzyme-linked lectin assay (ELLA) assay was used to
determine
neuraminidase-inhibiting (NAT) antibody titers. The source of antigen (virus
NA) was titrated,
and a standard amount was selected for incubation with serial dilutions of
serum. Titration of sera
was performed with serial dilutions of sera (heat inactivated at 56 C for 1
hour) and a standard
amount of virus was added to duplicate wells of a fetuin-coated plate. This
mixture was then
incubated overnight (16-18 hours); the next day, HRP-conjugated peanut
agglutinin PNA (diluted
to 2.5 pg/ml) was added to the washed plate and incubated for 2 hours at room
temperature.
Substrate (ODP in sodium citrate) was added and incubated for 10 minutes to
develop the color.
And then stop buffer (1N sulfuric acid) was added to stop the reaction. Plates
were scanned for
absorbance at OD 490 nm. The reduction or absence of color relative to a viral
control indicated
inhibition of NA activity due to the presence of NA-specific antibodies. NAT
titers (ICso values)
were calculated from the OD readings and the results were graphed in GraphPad
Prism. If ELLA
titration curves did not allow a good fit to determine a reliable IC50 value,
the samples were
retested using a different dilution scheme to reach the 50% endpoint.
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T cell ELISPOT Assay
[00248] Complete medium (DMEM1640 + 10% heat-inactivated FCS) was prewarmed in
a 37 C
water bath. PBMCs were quickly thawed in a 37 C water bath and transferred
dropwise to conical
tubes with the prewarmed medium. The tubes were centrifuged at 1,500 rpm for 5
mins and the
cells were resuspended and counted using a Guava cell counter. Monkey IFN-y
ELISPOT kit
(Mabtech 3421M-4APW) and IL-13 ELISPOT kit (Mabtech 3470M-4APW) were used.
Precoated plates provided by the kits were washed four times with sterile PBS
and blocked with
200 pl of complete medium in 37 C incubator for at least 30 minutes. Sing16 H3
peptides pool
(Genscript Custom Order) (at 1 lag/m1 of each peptide) were used as recall
antigens in the assay.
Two lag/m1 of ConA (Sigma CAT#C5275) was used as a positive control. Fifty al
of recall
antigens and 300,000 of PBMCs in 50 glwere added to each well for stimulation.
The plates were
placed in a 37 C, 5% CO2 humidified incubator for 48 hours.
[00249] After the incubation, cells were removed, plates were washed 5 times
with PBS, and
100 1 of 1 lag/m1 biotinylated anti-IFN-y or anti-IL-13 detection antibodies
were added to each
well in the plates. After a 2 hour incubation, the plates were washed 5 times
with PBS and
incubated with 100 jal of a 1:1000 dilution of streptavidin in each well for
one hour at room
temperature. Plates were developed with 100 1 of BCIP/NBT substrate solution
until the spots
emerged. Plates were rinsed by tap water, air-dried and scanned and counted
using CTL
ImmunoSpotO Reader (Cellular Technology Ltd.). The data was reported as spots
forming cells
(SFC) per million PBMCs.
Memory B cell (MBC) ELISPOT Assay
[00250] Human IgG Single-Color memory B cell ELISPOT kit (CAT# NC1911372, CTL)
was
used per manufacturer's instruction to measure Sing16 H3-specific and total
IgG antibody-
secreting cells (ASCs). Differentiation of MBCs into ASCs was performed in
PBMC using a
stimulation cocktail provided by the kit. Briefly, frozen PBMCs were quickly
thawed in a 37 C
water bath, mixed with DNase I (CAT# 90083, Fisher Scientific) and transferred
into the tube
containing pre-warmed complete culture medium (CM) (RPMI 1640, (CAT# 22400-
089, Gibco)
containing 10% FCS (CAT # SH30073.03, HyCloneTm), and 1%
penicillin/streptomycin (CAT#
P4333, Sigma) and centrifuged at 1,500 rpm for 5 minutes. Cell pellet was re-
suspended in 5 ml
of complete medium at 2x106 cells per ml and transferred to a T25 flask for 1
hour in 5% CO2
incubator at 37 C. The volume of cell suspension was then adjusted to 6 ml and
B-Poly-S was
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added at 1:1000 dilution. Cells were left in the CO2 incubator for stimulation
for 4 days. PVDF
microplates supplied by the kit were pre-wetted with 70% ethanol, rinsed and
coated overnight
with 80 pl/well of either anti-human IgG capture Ab provided by the kit or
Sing16/H3 recombinant
protein at 4 jig/ml.
[00251] Cells were harvested after 4 days of stimulation, washed, and counted
and adjusted to the
designated concentration in the CM. Coated microplates were washed with PBS,
blocked for 1
hour with the CM and emptied out. Cell suspension at 100 p1/well was added to
the plates and
incubated in CO? incubator at 37C for 18firs. After washing, 80 p1/well of
1:400 diluted anti-
human IgG biotin detection antibody was added to the plate and incubated at
room temperature
for 2 hours. Following washing, Streptavidin-AP at 1:1000 dilution was added
to the plate at 80
p1/well for 1 hour. Freshly prepared Substrate solution was added and
incubated at RI for 18 min.
Plates were rinsed by tap water, air-dried and scanned and counted using CTL
ImmunoSpotO
Reader (Cellular Technology Ltd). For each individual animal the number of IgG
and number of
Sing16/H3-specific ASCs was calculated per million of PBMCs. The frequency of
antigen-
specific ASCs was calculated as % of antigen-specific ASCs to the total IgG +
ASCs. To assess
assay background the negative control wells on every plate were coated with
PBS (no background
was detected).
Statistical Analysis
[00252] For estimating the Tmax of Radiance, a non-parametric method was used
to estimate the
Tmax of individual subject based on observed data. For estimating the half-
life of Radiance,
assuming exponential decay model for radiance after reaching the maximum
value, a linear model
was -fitted to log transformed data per subject during the time course from
the maximum radiance
to decay to baseline (we estimate the baseline using the average of radiance
in saline group). The
half-life was estimated as the time point when the log radiance had reached
the middle point
between maximum and baseline values. For analysis of different readouts with
results summarized
as geometric mean, SE model based geometric means and SEs were estimated from
a mixed effect
model for repeated measures where the response was the log transformed
readouts, vaccination
was fixed effect and time was repeated measure; log-based means and SE
estimates from the model
were then back transformed to get geometric means and SEs. For weight change,
over descriptive
statistical analysis was used. Medians and ranges of each group of the maximum
% body weight
loss from baseline (Day 0) over time were reported to evaluate the worse
scenarios; medians and
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ranges of each group of the % body weight change from baseline at the last
observation were
reported to evaluate the body weight recovery.
Antigen Sequences
[00253] The sequence of the Perth09 N2 antigen used here is:
MNPNQKI ITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTI IE
RNITEIVYLINTTIEKEICPKLAEYRNWSKPQCDITGFAPFSKDNSIRLSAGGDIWVIR
EPYVSCDP DKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHL GTKQVCIA
WSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKE I LRTQESECVCINGT
CTVVMTDGSASGKADTKILFIEEGKIVHTSTLSGSAQHVEECSCYPRYPGVRCVCRDNW
KGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAF
DDGNDVWMGRT I SEKSRLGYETFKVIEGWSNPKSKLQINRQVIVDRGNRSGYSGI FSVE
GKSCINRCEYVEL IRGRKEE TEVLWT SNS IVVFCGTSGTYGTGSWPDGADINLMP I *
(SEQ ID NO:4)
[00254] The sequence of the Mich15 NI antigen used here is:
MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENN
TWVNQTYVNI SNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIR
EPFI SCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCP I GEVPSPYNSRFESV
AWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQE SECACVNG
SCFTIMTDGPSDGQASYKI FRIEKGKI IKSVEMKAPNYHYEECSCYPDSSE ITCVCRDN
WHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYG
NGVWIGRIKS I SSRKGFEMIWDPNGWTGIDNKFS IKQDIVGINEWSGYSGS FVQHPELT
GLDCIRPCFWVEL IRGRPEENT IWTSGSS SFCGVNSDTVGWSWP DGAELP FT I DK*
(SEQ ID NO:5)
[00255] The sequence of the Sing16 H3 antigen used here is:
MKTI IALSYILCLVFAQKI PGNDNSTATLCLGHHAVPNGT IVKT INDRIEVINATELV
QNSS IGE I CDSPHQILDGENCTL I DALLGDPQCDGFQNKKWDLFVERSKAY SNCYPYDV
PDYASLRSLVASSGTLEFKNESFNWTGVTQNGT SSACIRGSSSSFFSRLNWLTHLNYTY
PALNVIMPNKEQFDKLYIWGVHHPGT DKDQI FLYAQSSGRITVSTKRSQQAVI PNIGSR
PRIRDIPSRISIYWTIVKPGDILLINSIGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKS
ECITPNGS I PNDKP FQNVNRITYGACPRYVKHS TLKLATGMRNVPEKQTRGI FGAIAGF
IENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEK
EFSEVEGRVQDLEKYVEDTKI DLWSYNAELLVALENQHT I DLTDSEMNKLFEKTKKQLR
ENAE DMGNGCFKIYHKCDNACIES IRNE TYDHNVYRDEALNNRFQIKGVELKS GYKDW I
LWI SFAI SCFLLCVALLGFIMWACQKGNIRCNI CI * (SEQ ID NO:6)
[00256] The sequence of the Sing16 N2 antigen used here is:
MNPNQKI I T IGSVSLT I ST I CFFMQIAIL ITTVTLHFKQYEFNSP PNNQVMLCEPTI IE
RNITEIVYLINTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVIR
EPYVSCDP DKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHL GTKQVCIA
WSSSSCHDGKAWLHVCITGDDKNATASFIYNGRL I DSVVSWSKDI LRTQESECVCINGT
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CTVVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNW
KGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLNPNNEEGGHGVKGWAF
DDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVE
GKSCINRC FYVEL IRGRKEE TEVLWT SNS IVVFCGTSGTYGTGSWPDGADLNLMHI *
(SEQ ID NO:7)
[00257] The sequence of the CA09 H1 antigen used here is:
MKAI LVVLLYTFATANADTLC I GYHANNSTDTVDTVLEKNVIVTHSVNLLE DKHNGKLC
KLRGVAPLHLGKCNIAGWILGNPECE SL SIAS SWSYIVE TPS SDNGTCYPG DFI DYEE L
REQLSSVSSFERFE I FPKTS SWPNHDSNKGVTAACPHAGAKS FYKNL IWLVKKGNSYPK
LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPK
VRDREGRMNYYWTLVE PGDK I TFEATGNLVVPRYAFAMERNAGSG IIIS DT PVHDCNTT
CQTPKGAINTSLPFQNIHP I T I GKCPKYVKSTKLRLATGLRNI PS IQSRGL FGAIAGF I
EGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAI DE I TNKVNSVIEKMNTQFTAVGKE
FNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKN
NAKE I GNGCFE FYHKCDNTCME SVKNGTYDYPKYSEEAKLNREE I DGVKLE STRIYQIL
AIYSTVAS SLVLVVSLGAI S FWMCSNGSLQCRI C I * (SEQ ID NO: 24)
[00258] The sequence of the HA strain A/California/7/2009 (H1N1) (CA09)
antigen mRNA
open reading frame (ORF) used here is:
AUGAAAGCUAUCCUGGUCGUCUUGCUGUAUACUUUCGCCACUGCCAACGCCGACA
CCCUGUGUAUCGGUUACCACGCGAACAACUCCACCGACACUGUGGACACCGUGCU
CGAAAAGAACGUGACCGUGACUCAUUCUGUGAAUCUGCUCGAGGACAAGCACAAC
GGAAAGUUGUGCAAGCUGCGCGGAGUGGCACCGCUGCACCU UGGAAAG UGCAAC
AUUGCCGGAUGGAUCCUGGGAAACCCGGAGUGCGAAAGCCUGAGCACCGCGUCCU
CAUGGUCCUACAUCGUGGAAACCCCGUCCUCUGACAACGGCACCUGUUACCCCGG
CGAUUUCAUCGACUACGAAGAACUGCGGGAGCAGCUGUCCUCCGUGUCCUCGUUU
GAACGCUUCGAGAUUUUCCCUAAGACCUCCAGCUGGCCUAAUCACGAUAGCAACA
AGGGCGUGACGGCAGCCUGCCCGCACGCCGGAGCAAAGUCAUUCUACAAGAAUCU
GAUUUGGCUCGUGAAGAAAGGGAACUCAUACCCCAAGCUGUCCAAGUCGUACAUC
AACGACAAGGGAAAGGAAGUGCUCGUGCUCUGGGGGAUCCACCACCCAUCCACCU
CCGCCGACCAGCAGAGCCUGUACCAGAACGCCGAUGCUUACGUGUUUGUGGGUUC
CAGCCGGUACUCCAAGAAGUUCAAGCCUGAAAUCGCGAUCAGGCCUAAAGUCCGG
GACCGCGAGGGCCGCAUGAACUACUACUGGACUCUCGUGGAGCCUGGAGACAAGA
UCACCUUCGAGGCCACCGGAAAUCUCGUGGUGCCACGCUACGCUUUCGCCAUGGA
ACGG A ACGCCGGA A GCGGC AUCAUC AUUA GCGAUACUCCUGUGC AUGACUGUA AC
ACCACGUGCCAGACACCCAAGGGCGCCAUCAACACCAGCCUGCCGUUUCAAAACA
UCCAUCCCAU UACCAU UGGGAAGUGCCCCAAAUACGUCAAGU CCACCAAGCUGAG
GCUGGCGACCGGACUGCGGAACAUUCCGAGCAUCCAGUCGAGAGGCCUGUUCGGU
GCCAUCGCGGGAUUCAUCGAGGGCGGCUGGACUGGAAUGGUGGACGGUUGGUAC
GGGUAUCACCACCAAAACGAACAGGGAUCAGGCUACGCGGCCGAUUUGAAGUCCA
CCCAGAACGCCATJUGAUGAAAUCACCAACAAGGUCAACUCCGUGATJUGAGAAGAU
GAAUACUCAAUUCACCGCCGUGGGCAAAGAAUUCAAUCACCUGGAGAAGAGAAU
AGAGAACCUGAACAAGAAGGUCGACGACGGGUUCCUCGACAUCUGGACCUAUAAC
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GCCGAGUUGCUCGUGCUGCUGGAAAACGAACGGACCCUGGACUAUCACGACUCGA
ACGU GAAGAACC UG UACGAGAAAG UCCGC UCGCAACU GAAGAACAACGCCAAGGA
AAUCGGAAAUGGUUGCUUCGAGUUCUACCAUAAGUGCGACAACACUUGCAUGGA
GUCCGUGAAGAACGGCACUUACGAUUACCCCAAGUACUCCGAAGAGGCUAAACUU
AACCGGGAAGAGAUCGAUGGCGUGAAGCUCGAGUCCACCAGAAUCUACCAGAUUC
UCGCCAUCUACUCGACUGUG G CAUCGAG CCUCGUCCUUGUCGUGUCCCUG G G GGC
CAUUUCAUUCUGG AUGUGCUCCA A CGGGUCCCUGC A GUGCCGG AUUUGCAUCUA A
(SEQ ID NO: 8)
[00259] The sequence of the A/Michigan/45/2015 (Mich15) neuraminidase (NA)
antigen
mRNA open reading frame (ORF) used here is:
AUGAACCCAAACCAGAAAAUCAUCACGAUUGGCUCGAUUUGCAUGACCAUUGGA
AUGGCGAACCUUAUCCUCCAAAUUGGCAACAUUAUCUCGAUCUGGGUCAGCCACU
CGAUCCAGAUCGGCAACCAAUCCCAGAUUGAAACUUGCAACCAGAGCGUGAUUAC
UUACGAAAACAACACGUGGGUGAACCAGACUUACGUCAAUAUUAGCAACACUAA
CUUCGCCGCUGGGCAGAGCGUCGUCAGCGUGAAGCUCGCCGGAAAUUCCUCGCUC
UGCCCCGUGUCCGGCUGGGCGAUCUACAGCAAGGAUAACAGCGUCCGGAUUGGUA
GCAAGGGCGACGUUUUCGUGAUCCGCGAACCCUUCAUAUCAUGCUCCCCGCUCGA
AUGUCGCACGUUCUUCCUGACCCAAGGCGCCCUGCUGAACGACAAGCACUCCAAU
GGCACUAUCAAGGAUCGGAGCCCUUACCGGACCUUGAUGUCCUGCCCUAUUGGAG
AAGUGCCUUCACCAUAUAACUCGCGCUUUGAAAGCGUGGCUUGGUCAGCCUCCGC
CUGCCAUGACGGGAUUAACUGGCUGACCAUUGGCAUAAGCGGCCCCGAUUCCGGC
GCCGUGGCCGUCCUGAAGUACAACGGGAUCAUCACCGACACCAUUAAGUCCUGGC
GCAACAACAUCCUGAGGACCCAGGAGUCCGAGUGCGCGUGCGUGAACGGGUCCUG
CUUUACCAUCAUGACCGACGGACCGUCCGACGGUCAAGCCUCGUACAAGAUCUUC
CGGAUCGAGAAAGGAAAGAUCAUCAAGAGCGUGGAGAUGAAGGCCCCGAACUAC
CAC UACGAG GAA UGUU CA U GC UAUCCCGACUCGUCCGAGAU U AC U UGCGUGUGCC
GCGACAAUUGGCACGGAUCCAACAGGCCGUGGGUCAGCUUCAACCAGAACCUUGA
AUACCAGAUGGGAUACAUUUGCAGCGGAGUGUUCGGGGACAACCCUCGCCCGAAC
GACAAGACCGGAUCGUGUGGGCCCGUGUCCUCCAACGGCGCAAACGGCGUCAAGG
GAUUUUCCUUCA A AUA CGGG A A CGGGGUCUGG AUCGG A CGG A CCA A G A GCAUUU
CAAGCAGAAAGGGAUUCGAGAUGAUUUGGGACCCGAACGGCUGGACUGGUACCG
AUAACAAAUUCAGCAUCAAGCAGGACAUCGUGGGAAUUAACGAGUGGUCCGGUU
ACUCCGGGAGCUUCGUGCAGCAUCCCGAACUCACUGGACUGGACUGCAUUCGGCC
GUGCUUUUGGGUGGAAUUGAUCCGGGGCAGACCUGAGGAGAACACGAUUUGGAC
CUCCGGCUCCUCGAUCUCGUUCUGCGGAGUGAACUCCGACACCGUGGGAUGGUCC
UGGCCCGACGGUGCAGAGCUGCCCUUCACCAUUGAUAAGUAA (SEQ ID NO: 9)
[00260] The sequence of the A/Singapore.INFIMH160019/2016 (Sing] 6; H3N2) HA
hcmagglutinin antigen mRNA open reading frame (ORF) used here is:
AUGAAAACCAUAAUCGCGCUCUCAUACAUACUUUGCCUGGUCUUUGCCCAAAAGA
UCCCUGGCAACGACAACUCAACCGCGACCCUUUGCCUCGGCCAUCACGCCGUGCC
GAACGGCACUAUCGUCAAGACCAUCACAAACGACCGCAUCGAAGUGACCAACGCG
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ACUGAGCUAGUGCAGAACUCCAGCAUUGGAGAGAUUUGCGAUUCUCCACACCAAA
UCCUGGACGGAGAGAAUUGUACCUUGAUCGACGCGCUGCUGGGGGAUCCGCAGU
GCGACGGAUUCCAGAACAAGAAAUGGGACCUUUUCGUGGAACGGAGCAAGGCAU
ACUCGAAUUGCUACCCCUACGAUGUGCCCGACUACGCCUCGCUGCGGUCCUUGGU
CGCUUCCUCCGGGACCCUGGAAUUCAAAAACGAGAGCUUUAAUUGGACCGGAGUG
ACCCAGAAUGGCACCUCGAGCGCCUGCAUUCGGGGCUCCUCCUCGAGCUUCUUCA
GCCGCCUGAACUGGCUCACUCACCUCAACUACACCUACCCGGCACUGAACGUGAC
CAUGCCGAACAAGGAACAAUUCGACAAGCUCUACAUUUGGGGGGUGCAUCACCCG
GGUACCGAUAAGGACCAGAUCUUCCUCUACGCCCAAUCCUCGGGCCGGAUCACCG
UGUCCACUAAGCGCUCGCAGCAGGCCGUGAUCCCGAACAUUGGAAGCAGACCCCG
CAUUCGCGACAUUCCAUCGAGGAUCUCGAUCUACUGGACGAUUGUCAAGCCUGGC
GACAUCCUCCUCAUUAACUCCACCGGGAACCUCAUCGCCCCUCGGGGUUAUUUCA
AGAUCCGCAGCGGGAAGUCCUCCAUCAUGAGAAGCGAUGCCCCCAUUGGAAAGUG
CAAGUCCGAGUGUAUCACACCUAACGGAAGCAUUCCCAAUGACAAGCCAUUCCAG
A ACGUGA AC A GA AUUACCUACGGAGCUUGCCCUCGCUACGUCA A AC AUUCGACCC
UCAAGUUGGCGACUGGAAUGCGCAACGUGCCGGAGAAGCAAACCCGGGGGAUCU
UCGGGGCUAUCGCGGGAUUCAUCGAAAAUGGAUGGGAAGGAAUGGUCGAUGGUU
GGUACGGUUUCAGACACCAGAACUCCGAGGGGCGGGGCCAGGCCGCAGACCUGAA
GUCCACUCAGGCCGCGAUUGACCAGAUCAACGGAAAGCUCAACAGACUCAUUGGA
AAGACCAACGAAAAGUUCCACCAAAUCGAAAAGGAAUUCUCCGAAGUGGAGGGC
CGGGUGCAAGACCUGGAGAAGUACGUGGAGGACACUAAGAUCGACCUUUGGAGC
UAUAACGCAGAACUCCUUGUGGCCCUGGAAAACCAGCACACCAUCGACCUGACCG
AUUCAGAGAUGAACAAGCUCUUUGAGAAAACUAAGAAGCAACUCCCiCiCiAAAACG
CUGAGGACAUGGGAAAUGGAUGCUUUAAGAUCUACCACAAGUGCGACAACGCCU
GCAUUGAGUCCAUACGGAACGA AACUUACGACCAUAACGUCUACCGGGAUGAAGC
CCUGAACAACAGAUUCCAGAUCAAGGGCGUGGAGCUGAAGUCCGGCUACAAAGA
IJUGGAUCCUGUGGAIJUUCCUUCGCGAIJUIJCAUGCUIJCIJUGCUCUGCGUGGCCCUC
CUGGGAUUCAUAAUGUGGGCCUGUCAGAAGGGCAACAUUAGGUGCAACAUAUGC
AUAUAA (SEQ ID NO: 10)
[00261] The sequence of the Perth/16/2009 (H3N2) NA antigen mRNA open reading
frame
(ORF) used here is:
AUGAACCCUAACCAGAAGAUCAUCACAAUUGGAAGCGUGUCCCUGACCAUUUCGA
CGAUUUGCUUCUUCAUGCAAAUCGCGAUCUUGAUUACCACCGUCACCCUGCAUUU
CAAGCAAUACGAAUUCAACUCCCCGCCAAACAACCAAGUCAUGCUCUGCGAGCCC
ACCAUCAUCGAACGCAACAUCACCGAGAUCGUGUACCUL ACCAACACUACCAUCG
AAAAGGAGAUUUGCCCCAAGUUGGCCGAAUACCGGAACUGGAGCAAGCCCCAGUG
UGACAUCACGGGAUUUGCGCCAUUCAGCAAGGAUAACUCGAUCAGACUUUCCGCC
GGGGGCGACAUUUGGGUCACUCGGGAGCCUUACGUGAGCUGCGACCCGGACAAGU
GCUACCAAIJUCGCACUCGGACAGGGIJACCACCCUGAACAACGUCCAUAGCAACAA
CACCGUGCGCGAUAGAACCCCGUACCGCACCCUCCUCAUGAACGAACUGGGAGUG
CCGUUCCACUUGGGA ACCA A ACA AGUCUGCAUUGCAUGGUCCUCCUCCUCCUGCC
ACGACGGCAAAGCCUGGCUUCACGUUUGCAUCACCGGCGACGACAAGAAUGCGAC
GGCCUCCUUCAUAUACAAUGGUAGACUCGUGGAUAGCGUGGUGUCAUGGUCCAA
GGAAAUUCUCAGGACUCAGGAGUCAGAGUGCGUGUGCAUCAACGGGACUUGCAC
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UGUCGUGAUGACCGACGGAUCGGCCUCCGGAAAGGCCGACACUAAGAUCCUCUUC
A U CGAGGAGGGAAAGA U CG U GCACAC U U CUACCCUGAGCGGCUCGGCUCAGCAU G
UCGAAGAGUGCLICGUGCUACCCCCGGUAUCCCGGGGUCCGCUGCGUGUGCCGGGA
CAAUUGGAAAGGCUCAAACCGCCCCAUCGUGGACAUUAACAUCAAGGACCACUCC
AUCGUGAGCUCCUACGUAUGCAGCGGGCUGGUCGGGGAUACCCCGCGGAAGAACG
AUUCCUCGUCCUCCUCCCACUGCCUGGACCCUAACAACGAAGAG G GAG GCCACGG
AGUGAAGGGAUGGGCUUUUGACGAUGGCAACGACGUGUGGAUGGGCAGGACUAU
UUCCGAAAAGUCCCGGCUGGGAUACGAAACCUUCAAGGUCAUCGAGGGCUGGUCC
AACCCGAAGUCAAAGCUCCAGAUCAACCGCCAGGUCAUCGUGGAUAGGGGCAAUA
GAUCCGGCUACUCCGGGAUCUUCAGCGUGGAAGGGAAGUCCUGCAUUAACCGAUG
CUUCUACGUGGAACUCAUUCGGGGUCGGAAGGAGGAAACCGAAGUGCUGUGGAC
UUCGAACUCAAUCGUGGUGUUUUGUGGGACCUCCGGAACUUACGGAACUGGGUC
CUGGCCUGACGGUGCCGACAUCAACCUUAUGCCGAUCUAA (SEQ ID NO: 11)
[00262] The sequence of the A/Wisconsin/588/2019 antigen mRNA open reading
frame (ORF)
used here is:
AUGAAAGCCAUCCUUGUUGUCAUGCUGUACACAUUCACCACCGCAAAUGCGGAUA
CCCUGUGUAUCGGCUACCACGCAAAUAAUUCCACCGACACCGUUGAUACCGUCCU
GGAAAAGAACGUGACAGUGACUCACAGCGUCAAUCUCCUUGAGGAUAAACAUAA
UGGCAAGCUGUGCAAGCUGAGAGGCGUGGCUCCCCUGCAUCUGGGAAAGUGCAAC
AUCGCUGGUUGGAUCCUCGGGAACCCAGAGUGUGAGUCCCUCUCAACCGCACGGU
CUUGGUCAUACAUCGUGGAGACUAGCAAUUCAGACAACGGCACAUGCUACCCCGG
UGACUUCAUUAACUACGAGGAGCUGAGAGAACAGCUGAGUUCCGUGUCAUCCUU
CG AGAG AUUCG A A AUCUUCCCC A A A A CCUCCUCCUGGCCCA AUC AUGACUCCG A C
AAUGGAGUGACAGCCGCUUGUCCCCACGCCGGUGCCAAGAGUUUCUAUAAGAACC
UCAUCUGGCUGGIJGAAAAAGGGCAAGUCCIJAUCCCAAAAIJIJAACCAGACCIJACAU
UAACGAUAAGGGGAAAGAAGUCCUGGUCCUGUGGGGGAUACACCACCCCCCUACC
AUCGCCG A CCA GC A GUCUCUGUAUC A G A A CGCCG A CGCCUACGUGUUCGUGGGUA
CCAGCCGU UAUAGUAAAAAG U UCAAGCCAGAAAU UGCCACCAGACCUAAGGUGCG
CG A CCA GG A GGGCCGC AUG A A CUA CUA CUGG A CCCUGGUGG A A CCUGGCG A CA AG
AUUACAUUCGAGGCCACUGGGAACCUGGUGGCACCCAGAUACGCCUUUACAAUGG
AACGGGAUGCUGGGAGCGGAAUCAUUAUCUCCGAUACCCCUGUCCACGACUGCAA
UACUACCUGUCAGACCCCAGAAGGCGCUAUCAAUACCUCUCUGCCUUUCCAAAAC
GUGCACCCUAUCACUAUCGGGAAAUGUCCCAAGUAUGUGAAAAGCACCAAACUGC
GCCUGGCAACCGGUCUGAGAAAUGUGCCCUCCAUCCAGUCCCGCGGCUUGUUCGG
UGCAAUCGCUGGCUUUAUCGAGGGUGGCUGGACUGGAAUGGUCGAUGGCUGGUA
CGGCUACCAUCACCAGAACGAGCAGGGGUCCGGGUAUGCUGCCGACCUGAAAAGC
ACUCAGAACGCCAUCGAUAAAAUCACUAACAAGGUGAACUCCGUGAUCGAAAAG
AUGAAUACACAGUUCACAGCAGUUGGCAAGGAGUUCAACCACCUGGAAAAACGG
AUAGAGAACCUGAAIJAAGAAAGUCGAUGAUGGCUIJIJCUGGACAIJCUGGACIJIJAC
AAUGCCGAGCUGCUGGUGCUCCUGGAAAACGAGCGGACACUGGAUUAUCACGACU
CAAACGUGAAGAACCUGUAUGAAAAGGUGCGUAACCAGCUGAAAAACAACGCCA
AGGAAAUCGGCAAUGGCUGUUUCGAAUUUUACCACAAGUGUGAUAAUACCUGUA
UGGAGAGCGUUAAGAACGGGACUUACGACUACCCAAAAUACAGCGAGGAGGCCA
AGCUGAACCGGGAGAAGAUCGACGGCGUCAAACUCGACUCCACUAGAAUAUACCA
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GAUUCUCGCCAUCUAUAGCACAGUGGCAUCAAGUCUCGUCCUGGUGGUGUCACUG
GGAGCCAUCAGCUUUUGGAU G U GCAGCAA U GGA U CCC U CCAG U G UAGGA U C U GC
AUCUAA (SEQ ID NO: 12)
[00263] The sequence of the A/Tasmania/503/2020 antigen mRNA open reading
frame (ORF)
used here is:
AUGAAGACCAUCAUCGCUCUGUCCUACAUCCUGUGCCUGGUGUUUGCUCAGAAAA
UCCCCGGGAAUGACAAUUCCACUGCCACUCUCUGCCUGGGCCAUCAUGCCGUGCC
AAAUGGAACCAUUGUCAAGACUAUAACAAAUGACCGCAUCGAAGUGACCAACGC
UACCGA GCUGGUUC A GA AC A GC A GUAUUGGA GA A AUCUGCGAUUCCCCAC ACC A G
AUACUGGAUGGCGGCAACUGCACCCUGAUCGACGCACUGCUGGGUGACCCUCAGU
GCGACGGAUUUCAGAAUAAGGAGUGGGACCUUUUCGUUGAGCGCAGCAGAGCCA
AUAGCAACUGCUACCCGUACGACGUGCCGGAUUACGCCAGUCUUCGAAGCCUGGU
CGCAUCCAGCGGGACACUGGAGUUUAAGAAUGAGUCCUUUAAUUGGACAGGCGU
GAAGCAGAACGGGACUAGCAGCGCAUGCAUUCGGGGCAGUAGCUCAUCCUUCUUU
AGCCGACUGAACUGGCUGACCCACCUCAACUACACAUACCCCGCACUGAA UGUGA
CUAUGCCA A AC A A A GA AC A GUUUG AC A A A CUGUA C AUCUGGGG A GUGC A CCAUCC
UAGCACAGACAAGGACCAGAUCAGCCUGUUUGCCCAGCCCAGCGGCAGGAUUACC
GUGUCCACAAAACGGUCACAGCAAGCCGUGAUCCCUAAUAUUGGAUCCCGCCCCC
GGAUAAGGGACAUCCCUAGUCGCAUCAGUAUCUACUGGACCAUCGUGAAGCCCGG
AGAUAUCUUGCUCAUCAAUAGCACUGGCAACCUCAUUGCCCCCAGGGGCUAUUUU
AAGAUCAGAAGCGGCAAGUCCAGCAUUAUGCGCAGCGACGCACCCAUUGGCAAGU
GCAAGUCCGAGUGCAUCACUCCUAAUGGGUCCAUCCCAAACGACAAGCCAUUCCA
AAAUGUCAACAGAAUCACCUACGGGGCUUGCCCCCGCUACGUGAAGCAGAGUACA
CUGAAACUGGCCACCGGGAUGCGCAACGUGCCCGAGAAGCAAACUAGAGGCAUCU
UUGGAGCUAUCGCUGGCUUCAUUGAGAAUGGCUGGGAGGGUA UGGUGGACGGCU
GGUACGGAU UCCGCCACCAGAAUAGCGAAGGCAGAGGCCAGGCAGCAGACU UGAA
GUCCACCCAGGCCGCCAUUGAUCAGAUCAACGGCAAACUGAAUCGGCUUAUUGGA
AAAACAAACGAGAAGUUCCAUCAGAUUGAGAAGGAGUUUAGCGAGGUGGAGGGC
CGCGUGCAGGAUCUGGAAAAGUACGUUGAAGACACCAAGAUCGACCUGUGGUCA
UA CA AUGC A G A GCUGCUCGUUGCCCUGG A A A AUCA GC A C A CA AUUG A CCUUA C A G
ACUCCGAAAUGAAUAAGCUCUUUGAAAAGACCAAGAAGCAGCUGCGCGAGAACG
CCGAGGAUAUGGGGAACGGUUGUUUUAAGAUCUACCACAAGUGUGACAACGCCU
GCAUUGGGUCCAUCCGAAAUGAAACAUACGACCACAACGUGUAUAGAGAUGAGG
CCCUGAACAACCGAUUCCAGAUUAAGGGAGUCGAGCUGAAGAGUGGCUAUAAGG
ACUGGAUCCUGUGGAUCUCAUUCGCCAUGUCAUGCUUCCUUCUGUGUAUUGCUCU
GCUCGGCUUCAUCAUGUGGGCUUGCCAGAAAGGCAAUAUCCGGUGCAACAUCUGC
AUCUAA (SEQ ID NO: 13)
[00264] The sequence of the B/Washington/02/2019 antigen mRNA open reading
frame (ORF)
used here is:
AUGAAAGCAAUCAUAGUGCUGCUGAUGGUGGUGACUAGCAAUGCCGAUCGGAUC
UGCACCGGCAUCACUUCCAGUAACAGCCCUCAUGUGGUCAAAACCGCCACACAGG
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GCGAGGUGAACGUGACCGGAGUGAUUCCACUGACAACUACACCAACGAAGAGUCA
CU UCGCCAACCUGAAGGGCACCGAAACACGAGGCAAGCUCUGCCCCAAGUGUCUG
AAUUGCACCGACCUGGACGUCGCUUUGGGCCGCCCUAAAUGUACCGGCAAAAUAC
CUUCCGCCAGAGUGUCCAUCCUGCACGAGGUGCGCCCCGUGACCUCCGGGUGUUU
UCCCAUAAUGCACGACCGCACUAAAAUCCGCCAGCUGCCCAAUCUUCUGAGGGGG
UACGAACAUGUCAGGCUGUCCACUCACAACGUGAUCAACGCAGAAGACGCCCCCG
GAAGGCCUUAUGAGAUUGGAACCAGUGGGUCCUGCCCAAACAUUACCAACGGCAA
CGGCUUCUUCGCCACUAUGGCCUGGGCCGUGCCAAAGAACAAGACCGCCACCAAC
CCCCUGACAAUUGAAGUCCCUUACAUCUGCACAGAGGGAGAGGAUCAGAUCACCG
UGUGGGGGUUUCACUCUGAUAACGAAACUCAGAUGGCCAAGCUGUACGGGGAUU
CUAAACCCCAGAAGUUCACCAGUAGCGCUAACGGGGUGACCACCCAUUAUGUGUC
UCAGAUCGGAGGUUUCCCAAAUCAGACCGAGGACGGCGGACUGCCCCAGUCUGGA
AG GAUCGUAGUG GACUAUAUG GUG CAGAAGAGUG G AAAAACCG G CACCAUUACC
UAUCAGCGCGGCAUACUGCUGCCACAGAAGGUGUGGUGUGCUUCCGGCAGGUCCA
AGGUUAUCAAAGGGUCCCUCCCCCUGAUCGGCGAAGCAGAUUGUCUGCACGAGAA
GUACGGCGGACUGAAUAAGAGCAAACCCUACUACACCGGAGAACACGCUAAGGCA
AUUGGGAAUUGUCCGAUCUGGGUGAAGACGCCCCUGAAACUGGCCAAUGGCACA
AAAUACCGGCCCCCCGCUAAGCUGCUGAAGGAACGGGGGUUCUUCGGCGCCAUAG
CCGGCUUUCUGGAGGGAGGCUGGGAGGGCAUGAUAGCCGGGUGGCACGGCUACA
CUUCCCAUGGGGCUCACG GGGUGGCUGUGGCCGCCGACCUGAAGUCUACGCAGGA
AGCUAUCAACAAAAUCACUAAGAACCUGAACAGCCUGUCGGAAUUGGAGGUCAA
GAAUCUGCAGCGGCUGAGCGGCGCCAUGGAUGAGCUGCACAAUGAGAUCCUGGA
GCUUGACGAGAAGGUCGAUGAUCUUCGGGCCGAUACAAUUAGUAGCCAAAUUGA
GUUGGCCGUGCUGCUCAGCAACGAAGGCAUAAUCAACAGCGAGGACGAGCACCUC
CUGGCUCUGGAGAGAAAGCUGAAGAAGAUGCUCGGCCCUAGCGCAGUUGAGAUC
GGAAACGGCUGCUUCGAAACCAAGCACAAGUGCAACCAGACCUGCCUGGACAGGA
UCGCGGCAGGAACAUUCGACGCUGGGG AAUUCAGCCUCCCCACCUUCGACAGCCU
GAACAUCACAGCCGCCAGUCUGAAUGAUGACGGACUGGAUAACCAUACCAUCCUG
CUGUACUACUCUACCGCUGCUUCCUCCCUGGCCGUGACAUUGAUGAUCGCAAUCU
UUGUGGUUUAUAUGGUGAGCCGAGACAACGUCAGUUGCAGUAUCUGCCUUUAA
(SEQ ID NO: 14)
[00265] The sequence of the B/Phuket/3073/2013 antigen mRNA open reading frame
(ORF)
used here is:
AUGAAAGCCAUCAUUGUGCUGCUGAUGGUUGUUACAAGCAACGCCGACCGCAUCU
GCACCGGGAUUACAAGCAGCAAUAGCCCUCACGUGGUGAAGACAGCAACACAGGG
AGAGGUGAACGUGACCGGCGUGAUUCCACUGACAACCACCCCAACUAAAUCUUAC
UUUGCA A A CCUG A A A GGG A C A CGG A CC A G A GG A A A GCUGUGCCCUG AUUGCCUG
AAUUGCACAGACCUGGACGUGGCCCUGGGCAGACCAAUGUGCGUGGGCACUACAC
CAAGCGCCAAGGCCUCCAUCCUCCAUGAGGUGCGGCCCGUGACUUCUGGAUGUUU
CCCCAU UAUGCACGACAGAACCAAGAU UAGACAGCUGCCAAACCUGCU CCGCGGC
UACGAGAAAAUUCGCCUGUCUACACAGAAUGUGAUCGACGCCGAGAAGGCUCCAG
GAGGACCAUACAGACUGGGGACUUCUGGCAGCUGCCCUAACGCCACCUCUAAGAU
CGGGUUCUUCGCAACCAUGGCUUGGGCCGUGCCUAAAGACAAUUACAAGAAUGCC
A CCA AUCC A CUG A CUGUCG A GGUGCCAU AUAUUUGC A C A G A GGGGG A GG A CC A G
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AUCACUGUGUGGGGCUUUCAUAGCGAUAAUAAGACUCAGAUGAAGUCUCUCUAC
GGCGACUCUAACCCUCAGAAGU UCACCU CC UCUGCCAACGGGGU GACAACACACU
ACGUGUCCCAGAUCGGGGACUUUCCUGACCAGACCGAGGAUGGAGGACUGCCUCA
GUCUGGACGCAUCGUGGUGGACUAUAUGAUGCAGAAGCCUGGGAAGACCGGCAC
UAUCGUGUACCAGAGGGGCGUGCUGCUGCCCCAAAAGGUGUGGUGUGCCUCCGGA
AGAAG CAAAGUGAUUAAG G GAUCCCUG CCUCUGAUUG G G GAG GCCGAUUGCCUG
CAUGAAGAGUAUGGAGGGCUGAACAAGUCCAAGCCAUACUAUACAGGAAAGCAC
GCAAAAGCCAUCGGCAACUGUCCCAUCUGGGUCAAAACUCCUCUGAAGCUGGCCA
ACGGCACCAAAUACCGCCCUCCAGCCAAGCUGCUGAAAGAACGCGGAUUCUUCGG
CGCCAUUGCAGGGUUUCUGGAAGGAGGCUGGGAGGGCAUGAUUGCUGGAUGGCA
CGGAUAUACCUCUCACGGCGCUCACGGGGUGGCCGUGGCCGCCGAUCUGAAGUCC
ACACAGGAGGCAAUUAACAAGAUCACCAAGAAUCUGAAUUCACUGUCCGAGCUCG
AAGUGAAAAACCUG CAGCGCCUGUCCGGCGCCAUGGACGAGCUGCACAAUGAAAU
CCUGGAGCUGGACGAGAAGGUGGACGACCUGCGGGCUGACACUAUCAGCAGCCAG
AUCGAGCUGGCAGUGCUGCUGAGCA AUGA GGGC AUCAUC A ACUC A GA A GACGA A
CACCU CCU GGCAC UGGAAAGGAAACU CAAGAAGA U GC U GGGCCCCU CCGCAG U GG
AC AUUGGGA ACGGCUGUUUCGA A A CC A A GC AUA A GUGUA ACC A GACUUGUCUGG
AUAGGAUCGCAGCAGGAACCUUCAACGCCGGCGAAUUUUCUCUGCCAACAUUUGA
CUCCCUGAACAUCACAGCUGCAUCCCUGAACGACGACGGACUGGACAAUCACACC
AUCCUGCUGUACUACUCUACUG CCG CUAGCUCCCUGGCCGUGACCCUGAUGCUG G
CCAUCUUCAUCGUGUACAUGGUUUCCAGGGAUAACGUGUCUUGUAGCAUUUGCC
UGUAA (SEQ ID NO: 15)
Results
mRNA Antigen Preparation, Characterization, and Expression
[002661 mRNAs coding for the full-length codon-optimized HA and NA for the
various influenza
strains were synthesized enzymatically using unmodified ribonucleotides.
All mRNA
preparations had > 95% of 5' Cap 1 and showed a single homogenous peak on
capillary
electrophoresis. mRNA-LNP formulations were prepared by mixing the various
lipid components
with mRNA under controlled conditions and at fixed ratios. All mRNA-LNPs
exhibited >95%
encapsulation with uniform hydrodynamic radius ranging from 95-105nm and a
poly dispersity
index (PDI) of 0.060-0.136 as shown in Table 5.
Table 5. Attributes of LNP Formulations Used in Mouse Preclinical Testing
LNP Size (nm) PD! %
Encapsulation
CA09 HA 97.54 0.117 95.2
Sing16 HA 103.2 0.068 97.3
Sing16 NA 105.8 0.128 96.5
Mich15 NA 103.3 0.136 97.4
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[00267] Cryo-electron microscopy (Cryo-TEM) of the CA09 HA mRNA-LNP images
showed
uniform spherical particles with a multi-lamellar inner core structure. The
lamellarity of the solid
core structure analyzed further with Fourier Transform, indicated a 3.7 nm
periodicity between
layers. The uniform morphology of the particles seen in the micrographs arc
indicative of
homogenous LNP preparations with proper assembly of the LNPs.
[00268] Antigen expression was confirmed with flow cytometry by transiently
transfecting human
skeletal muscle cells (HskMCs) with the unencapsulated mRNA constructs of CA09
HA, Sing 1 6
HA, Sing16 NA, or Mich15 NA, and stained with protein-specific antibodies for
analysis. High
levels of HA and NA expression from HskMCs were observed, confirming proper
assembly and
trafficking of native form HA trimcrs and NA tetramers upon expression in
muscle cells. To study
the subcellular localization of expressed HA and NA proteins, HeLa cells were
transfected with
bivalent H3N2 LNP and proteins were visualized by immunostaining and confocal
microscopy.
While NA signal indicated strong colocalization in ER (about 90%), HA was
found to colocalize
moderately (25%) with ER when permeabilized cells were stained with antibodies
for
corresponding proteins and calnexin, an endoplasmic reticulum (ER) marker.
This is consistent
with the understanding that nascent NA and HA proteins arc translocatcd to ER
for assembly (Dou
et al., Front Immunol. (2018) 9:1581).
[00269] The efficiency of delivery of mRNA by LNPs and selection of optimal
formulation
parameters was evaluated using reporter mRNA expression (Thess et al.,
Molecular Therapy
(2015) 23(1):S55). A single dose of either 0.05, 0.1, 1, 5, lug of unmodified
FF-LNP formulations
was administered intramuscularly (IM) in mice. Luciferase activity, measured
by average
bioluminescence, indicated sustained expression from mRNA construct which
peaked at 6 hours
post injection and detectable beyond 72 hours at all doses (FIG. 11, panel
(a)). The high-level
mRNA-mediated protein expression was further verified with hEPO at a single
0.1 lug dose in mice
and 10 p,g in non-human primate (NHP). The study was intended to compare LNP,
using standard
LNP Dlin-MC3-DMA25 formulation as a control. Serum hEPO quantified by ELISA
demonstrated maximum expression at 6 h with approximately 12-fold higher
erythropoietin
expressed with hEPO-LNP compared to hEPO-MC3 (FIG. 11, panel (c)). Both hEPO-
LNP and
hEPO-MC3 showed similar expression kinetics in NHPs, detectable from 6 hours
to 72 hours
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(FIG. 11, panel (d)). The results confirmed the utility of the present LNP
formulation for efficient
delivery of mRNA for expression both in vitro and in vivo.
Immunogenicity of HA (HI, H3) and NA (Ni, N2) mRNA-LNP in Mice
[00270] Natural history and vaccine studies have shown that antibodies to
influenza HA and NA
have antiviral function and both antigens are considered important for
effective influenza vaccines
(Krammer et al., Nat Rev Immunol. (2019) 19(6):383-97). Unmodified CA09 HA-LNP
and Sing16
HA-LNP mRNA vaccines were evaluated in BALB/c mice (n=8) in a two-dose regimen
at 2, 0.4,
0.08, or 0.016 p,g mRNA-LNP administered at 4-week apart schedule. Recombinant
HA (rHA)
antigens of the same strain were used to evaluate the total IgG responses in
ELISAs. HA-specific
antibodies were detected in all groups after a single dose, but the titers
peaked at day 42 after the
second dose (FIG. 12). To measure functional antibodies, hcrnagglutination
inhibition (HAI)
response was evaluated against the homologous strains, CA09 and Sing16.
Although the HAT
titers after a first dose could be observed for the 2 p,g dose of CA09-LNP and
Sing16-LNP
treatment groups with GMTs of 160 and GMT 70 at day 28 respectively, a more
profound increase
in HAT titers were observed after second dose. At day 42 GMT titers were 80
and 2200 for the
0.016 pg and 0.4 pg groups respectively in the CA09 -HA-LNP and 14 and 100 for
the 0.016 p,g
and 0.4 lag groups respectively in the Sing 16 HA-LNP groups (FIG. 13).
[00271] Similarly, for testing anti-NA responses, mice were immunized with 2,
0.4, 0.08, or 0.016
1.ig of Sing16 NA-LNP or Mich15 NA-LNP. ELISA with recombinant NA antigens
were
conducted to assess the total IgG responses induced by either Mich15 NA-LNP or
Sing16
NA-
LNP formulations_ Animals developed high antibody binding responses after a
single dose, with
a marked increase in NA binding antibodies post second dose at day 42 (FIG.
14). Enzyme-linked
lectin assay (ELLA) was used as a surrogate for functional antibody titers for
Neuraminidase
inhibition (NAT) activity against H6N1 or H6N2 chimeric viruses. Although two
doses of the
vaccine substantially increased the functional antibody response as compared
to a single dose,
encouraging NAT titers with GMTs 800 and GMT 60 were recorded at day 28 after
a single dose
even with low dose of 0.016 lag of Mich15 NA-LNP and Sing16 NA-LNP,
respectively. At day
42, the GMT titers between the 0.4 p,g and 0.016 p,g, were 900 and 10200
respectively in the Sing16
NA-LNP group indicating a dose-dependent response with titers reaching above
ULOQ in case of
Mich15 NA-LNP (FIG. 15).
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Protection from Viral Challenge in Mice
[00272] To test the efficacy of the mRNA vaccine in mouse influenza virus
challenge model, we
inoculated BALB/c mice with 0.4 p,g of CA09 HA-LNP IM at week 0 and 4, along
with a negative
control group with two doses of LNP diluent buffer. HAT titers for vaccine
group serum samples
at study days 0, 14, 28, 42, 56, 92, and 107 demonstrated robust immune
response with GMT of
1660 and 1:830 at day 56 and day 92 respectively (FIG. 16A). At day 93, all
mice were challenged
intranasally with Belgium09 virus, homologous to CA09, at four times the dose
which can cause
50% lethal outcome (4xLD50). All mice in the vaccine group survived the
challenge with no
mortality, and some mild morbidity marked by transient weight loss of less
than 5% (FIG. 16B).
However, those in the diluent control group suffered significant and rapid
weight loss which led
to high mortality rate (90%) by day 9. These results demonstrated high
efficacy of HA-based
MRT formulations in a lethal mouse influenza challenge model.
[00273] To assess protective efficacy of NA-based MRT vaccines, we conducted
an analogous
challenge experiment in BALB/c mice. Since the Mich15 NA-LNP vaccine elicited
robust NAT
titers after a single immunization in naïve mice (FIG. 16A), we evaluated one
or two dosing
regimens with administrations of 0.4 or 0.016 1.1g of Mich15 NA-LNPs over a 4-
week interval.
The control groups were vaccinated at the same regimens, receiving either 0.6
pg hEPO-LNP or
diluent buffer. Robust NAT titers were observed after a single administration
with GMTs of 14,000
NAT for 0.4 and 1,800 NAT for 0.016 1.1,g of Mich15 NA-LNP recorded
at day 28 (FIG. 17A).
After the second immunization at day 42, NAT titers rose to 108,000 NAT for
0.4 lag and 37,000
NAT for 0.016 lag groups. After more than 12 weeks post vaccination regimens,
all groups were
challenged with 4xLD50 of Belgium09 H1N1 virus. Individual weight changes from
baseline over
time by treatment groups are graphed in FIG. 17B. All mice in the two control
groups suffered
significant morbidity, and all animals had to be euthanized due to >20% weight
loss by day 8 post-
infection. Remarkably, all animals except one in the vaccine groups survived
the challenge in the
single dose 0.016 p,g group, indicating high protective efficacy against death
even after a single
dose of as low as 0.016 1.(g of Mich15 NA-LNP. The higher dose (0.4 lug)
demonstrated overall
higher protection, however, in contrast to HA-immunization, NA vaccination was
not sufficient to
protect against weight loss as vaccinated animals demonstrated median weight
loss of 10 % of
initial body weight, consistent with observations reported for other NA
vaccines. Body weight
recoveries were observed for vaccinated groups resulting in an average final
weight change of
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2.7% at the low dose and 4.8% weight gain for the higher dose, as compared to
baseline. Overall,
the results demonstrated that a single low-dose MRT NA-LNP vaccination can
elicit functional
antibodies measurable for blocking influenza NA activity and sufficient to
confer protection
against lethal challenge in mice.
Immunogenicity of HA (H3) mRNA-LNP in NHP
[00274] To evaluate immunogenicity of the mRNA-LNP in NHP, a dose range study
covering 15,
45, 135, and 250 tig of Sing16 HA-LNP was performed in NHPs. After the first
immunization,
all vaccinated NHPs developed antibodies reactive to recombinant HA protein as
noted in ELIS A
(FIG. 18). Further boosting of titers was observed post second dose.
Surprisingly, the 15 ug dose
induced only 1.8-fold lower ELISA titers than the 135 p,g dose level (95% CI
1.0, 3.6), suggesting
a dose saturation close to 15 ug level. Robust HAI antibodies were induced in
all dose groups on
day 42 and GMTs recorded were 400 for 15 lag, 700 for 45 lag, 900 for 135 lag
and 570 for 250 lag.
At day 42, the fold increase in GMT titers with 95% CI was 2.2-fold (1.0; 5.0)
between the 135 lag
and 15 lag and was 1.3-fold (0.6; 2.8) between the 135 ug and 45 lag treatment
groups indicating
that despite the observed trend towards higher titers with increasing dose,
the difference between
groups was minimal (FIG. 19A). The neutralization potency assessed by
microneutralization
(MN) assay (FIG. 19B) showed a better trend for dose effect with GMTs on D28
of 40 for 15 jig,
180 for 45 jug, 300 and for 135 jig.
[00275] Since T cells have been shown effective in reducing viral load and
limiting disease
severity in animal models (Rimmelzwaan et al., Vaccine (2008) 26(4):D41¨D44;
Sridhar et al.,
Nat Ailed . (2013) 19(10): 1305-12; Sridliar et al., Front Immunol. (2016)
7:195), we evaluated recall
T cells in the NHPs vaccinated with 45, 135, 250 lag of Sing16 HA-LNP or with
45 jig of
recombinant HA. PBMCs collected at day 42 were evaluated in IFN-y (Thl
cytokine) and IL-13
(1h2 cytokine) ELISPOT assay with recall stimulation with pooled overlapping
peptides spanning
the entire sequence of the Singl 6 HA. All vaccinated animals except one in
250 vig group
developed IFN-y secreting cells, ranging from 28 to 1328 spot-forming cells
(SFC) per million
PBMCs (FIG. 20A). Notably, a dose-response was not observed, and the lower and
higher dose
level groups of animals showed comparable frequencies of IFN-y secreting
cells. In contrast, all
animals in the control group immunized with the recombinant Singl 6 HA protein
demonstrated
absence of IFN-y producing cells. The presence of IL-13 cytokine secreting
cells was either not
detected or very low in all the groups tested (FIG. 20B). The data suggest
that Sing16 HA-LNP
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induced strong Thl -biased cellular responses in NHPs, comparable to that seen
with MRT5500
(Kalnin et al., supra), a SARS-CoV-2 vaccine currently under development.
[00276] To investigate frequency of memory B cells (MBCs) in NHPs after
immunization with
Sing16 HA-LNP, an ELISPOT assay was developed to quantify antigen-specific
MBCs as a
readout of Immoral immune memory. On day 180, PBMCs were collected from the
NHPs
immunized with 45 lag or 15 lag of the Sing16 HA mRNA-LNP formulations or with
a recombinant
HA as a comparator at a 45 jag dose. A 4-day polyclonal stimulation of PBMCs
that is optimized
to drive memory B cells to antibody secreting cells (ASC) was performed, and
the stimulated
PBMCs were plated in an antigen-specific ELISPOT where the frequency of
antigen-specific
ASCs could be determined. Antigen-specific memory B cells were then quantified
as a percentage
of total IgG+ memory B cells. Antigen-specific memory B cells were detected in
all animals and
their frequency was ranging from 1 to 5% for the 45 ug dose group and 0.3 to
1.5% for the 15 p,g
dose group. In the rHA immunized animals, the memory B cell responses appeared
to be markedly
lower as antigen-specific memory B cells were undetectable in five out of six
animals (FIG. 21).
It was concluded that Sing16 HA-LNP, like other mRNA vaccines, elicits a
population of anti-HA
specific memory B cells that promise to prolong immunity (Lindgren et al.,
Front Immunol. (2019)
10:614).
Multivalent Influenza Virus Antigens
[00277] An advantage of mRNA-LNP platform is the flexibility of LNP
encapsulation for
multiple mRNA antigen constructs. However, this potential needs to be tested
to address the
concern of antigenic interference. To explore the combinations of influenza
antigens, co-
encapsulated HA and NA mRNA were formulated in LNPs as bivalent formulations
containing
0.2 pg each of mRNA in an H3H1, H3N2, or N1N2 combination or with the
monovalent
containing 0.2 p,g of each corresponding antigen. These formulations were
administered in mice
to determine any antigenic interference on immunogenicity by comparing the
functional titers of
the individual antigen in bivalent vs. monovalent formulations (FIG. 22,
panels (a)-(c) and Table
6).
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Table 6. Frequency of Antigen-Specific Memory B Cells in
NHPs Vaccinated with H3 mRNA-LNP Vaccine
PBMCs/ Spot # of PBMC Spot # of
% of Ag-
Animal well of Ag- Ag-Specific Total
Specific
Ani s/ mal ID Well of
group Specific IgG/million Total I gG
IgG/million IgG to
IgG PBMCs PBMCs
Total IgG
1 3 x 105 1082 5x103 21700
5.0
2 3 x 105 232 5x103 6100
3.8
H3 mRNA- 3 3 x 105 282 5x103 11700
2.4
LNP
(45 Fig) 4 3 x 105 2 5x103 100
2.0
3 x 105 283 5x103 8700 3.3
6 3 x 105 225 5x103 22800
1.0
1 3 x 105 63 5x103 21600
0.3
2 3 x 105 58 5x103 11300
0.5
H3 mRNA- 3 3 x 105 253 5x103 17300
1.5
LNP
(15 lug) 4 3 x 105 173 5x103 17300
1.0
5 3 x 105 63 5x103 9300
0.7
6 3 x 105 107 5x103 19300
0.6
1 3 x 105 2 5x103 19800
0.0
2 3 x 105 28 5x103 14300
0.2
rHA 3 3 x 105 2 5x103 17000
0.0
(45 p,g) 4 3 x 105 0 5x103 7900
0.0
5 3 x 105 0 5x103 21600
0.0
6 3 x 105 0 5x103 14600
0.0
1 3 x 105 0 5x103 30900
0.0
Diluent
2 3 x 105 0 5x103 7100
0.0
[00278] In the H1H3 combo, between the co-encapsulated and separately
administered vaccines,
5 no statistically significant difference (p= 0.2584) irrespective of the
time points was observed for
HAT titers and no significant difference (p=0.8389) at D42 was observed for H3
titers. In the case
of H3N2 combo, the NA component of the vaccine elicited high neutralizing
antibodies in
combination with the HA component, demonstrating lack of HA dominance. Between
the co-
encapsulated and separately administered vaccines, no statistically
significant difference
(p=0.2960), irrespective of the time points, was observed for H3 titers, and
no significant
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difference (p=0.0904) at D42 was observed for N2 titers. Likewise, the N1N2
combo was not
statistically significantly different (p=0.3899) for N2. Ni titers at day 42
for co-encapsulated and
separately administered vaccines were above limit of quantification.
Combination of N2N1,
H3H1, or H3N2 thus generated antibody titers equivalent to individual LNPs
separately
formulated.
[00279] Quadrivalent formulations of co-encapsulated H1, Ni, H3, and/or N2
mRNA were
further explored. These formulations were tested in NHPs in total 10 lig
composed of 2.5 lig each
of influenza antigen mRNA and filling amount of noncoding mRNA (nc mRNA) if
needed in
combinations, resulting in quadrivalent (H1N1H3N2), bivalent (H1N1 or H3N2),
or monovalent
(H1, 113, Ni, or N2) LNPs (Table 7).
Table 7. Bivalent Combination of Influenza Virus in Mouse Study
mRNA
CA09 Singl 6 Mich 1 5 Perth09
Group N mRNA1 mRNA2 LNP dose Description
HAT HAT NAT NAT
Gig)
1 8 Singl 6 P erth0 9 Coformulated
2 8 H3 N2 Separate
3 8 Sing' 6 Coformulated x
CA09 H1 Yes 0.2, 0.2
4 8 H3 Separate
5 8 Michl 5 P erth0 9 Coformulated
6 8 Ni N2 Separate
7 8 Diluent 0 single
[00280] HAT titers to H1 or H3, or NAT titers to Ni or N2 were compared
between the monovalent
formulations vs. bivalent or quadrivalent formulations (FIG. 23). On day 42,
the HAT titers to HI
of the quadrivalent group were comparable when analyzed with that of the H1
monovalent group
(p=0.9054, t-test, unpaired, two-tailed) or H1N1 bivalent group (p=0.8002).
Similarly, the H3
HAT titers of the quadrivalent group was comparable when analyzed with that of
the H3
monovalent group (p=0.2504) or H3N2 bivalent group (p=0.5894). The NAI titers
to NI were
almost identical in groups of animals vaccinated with Ni monovalent mRNA or
H1N1 bivalent
mRNA or the quadrivalent H1N1H3N2 mRNA formulations. Likewise, there was no
difference
in N2 NAT titers between the N2 monovalent mRNA (p=0.8485) or 113N2 bivalent
mRNA
(0.4545) with the quadrivalent H1N1H3N2 mRNA formulations.
[00281] Overall, these findings indicate that co-encapsulated or combination
multivalent vaccines
of HA/NA mRNA-LNPs at this dose level could efficiently deliver all four
antigens without any
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concern for antigenic interference and all antigens were as immunogenic as in
the formulation
when these antigens were delivered singularly.
Example 7: Additional LNP Formulations
[00282] Additional LNP formulations for mRNA vaccines were prepared,
designated Lipid C
(containing cationic lipid GL-HEPES-E3-E10-DS-3-E18-1), Lipid D (containing
cationic lipid
GL-HEPES-E3-E12-DS-4-E10), and Lipid E (containing cationic lipid GL-HEPES-E3-
E12-DS-
3-E14). Human erythropoietin (hEPO) mRNA was used as a test mRNA. Expression
of hEPO
was measured by ELISA from samples taken from mice injected with the LNPs.
Samples were
taken 6 hours, 24 hours, 48 hours, and 72 hours after injection. As show in
FIG. 24, hEPO
expression was consistently higher at all time points with LNP formulations
Lipid A, Lipid B,
Lipid C, Lipid D, and Lipid E, compared to a control LNP formulation
containing cationic lipid
MC3.
[00283] Table 8 below summarizes the results relative to a control LNP
containing the MC3
cationic lipid.
[00284] Table 8. Levels of hEPO from LNP formulations Lipid A-E relative to
MC3.
Fold higher hEPO at 6
LNP Formulation hours STDEV
(compared to MC3)
Lipid A 10.35 4.15
Lipid B 5.62 1.34
Lipid D 7.78 2.79
Lipid E 6.17 1.57
[00285] The same hEPO mRNA-LNP formulations were next tested in non-human
primates
(NHPs). Samples were taken at 6 hours, 48 hours, and 96 hours after injection.
As shown in FIG.
25, each LNP formulation produced levels of hEPO comparable to the MC3 control
formulation.
[00286] Influenza HA-encoding mRNA-LNP formulations were also tested in NHPs.
NHPs were
administered the LNP formulations at 10 lag via intramuscular injection and
samples were taken
at say 28 and day 42 post injection. HAT titers were measured as described
above. As shown in
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FIG. 26, each LNP formulation produced HAT titers comparable to or higher than
the MC3 control
formulation.
[00287] The same experiment as shown in FIG. 26 was performed while measuring
HAT titers
with the Ca109 H1 influenza antigen. As shown in FIG. 27, each LNP formulation
produced HAT
titers comparable to or higher than the MC3 control formulation.
[00288] As shown in FIG. 28, HAT titers with the Sing16 H3 antigen were
elevated for LNP
formulations Lipid C and Lipid D.
Example 8: Respiratory Syncytial Virus (RSV) F Protein-Encoding mRNA LNP
Formulations
[00289] The effect of different cationic lipids in thc LNP were tested for the
LNP-encapsulated
RSV F protein mRNA. Lipid formulations of Lipid A, Lipid B, Lipid C, Lipid D,
and Lipid E
were tested. Each LNP was composed of 40% of one of the five cationic lipids,
30% phospholipid
DOPE, 1.5% PEGylated lipid DMG-PEG2000, and 28.5% cholesterol. An LNP with the
cationic
lipid MC3 was also used, considered an industry benchmark (Jayaraman et al.
Angew Chem Int
Ed. 51:8529-33. 2012).
[00290] The F protein tested was designated FD3, and corresponds to a pre-
fusion RSV F protein.
The amino acid sequence for FD3 is recited below.
[00291] FD3:
MELLILK ANAITTILT AVTFCF A S GQNITEEFYQSTCS AVSKGYLS ALRTGWYTSVITIELS
NIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMGSGNVGLGGAIA SGVAVSKVLHL
EGEVNKIKSALLSTNKAVVSLSNGVSVLTEKVLDLKNYIDKQLLPILNKQSCSISNPETVI
EFQ QKNNRL LEITREF SVNAGVTTPVS TYMLTNS ELL SLINDMP ITNDQKKLM SNNVQIV
RQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKNGSNICLTRTDRGW
YCDNAGNVSFFPQAETCKVQSNRVECDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKT
DVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTESNGCDYVSNKGVDTVSVGNTLYYV
NKQEGKSLYVKGEPIINFYDPLVEPSDEFDASISQVNELINQSLAFINQSDELLHNVNAGK
STTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 16)
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[00292] The mRNA molecule described herein comprises an open reading frame
(ORF) encoding
an RSV F protein antigen, at least one 5' untranslated region (5' UTR), at
least one 3' untranslated
region (3' UTR), and at least one polyadenylation (poly(A)) sequence. The
tnRNA further
comprises a 5' cap with the following structure:
0
HHOc'HH
0- N. :**NH,
N
0 0 0
II II II
P-0-P- O-P- 0
I I
H2N N N
1;11
Fil(L4
0 0
I \
-0-P= 0 GH3
0 CH3
[00293] The nucleic acid sequence for the mRNA open reading frame (ORF)
encoding the RSV
F protein is recited below.
[00294] FD3 mRNA ORF:
AU GGAACU GC U GAU CC U CAAAGCCAACGCAAU CACCACCAU U C U CACCGC U G U GA
CCUUCUGCUUCGCAUCGGGGCAGAACAUCACUGAAGAGUUUUACCAGAGCACUUG
CAGCGCGGUGUCAAAGGGUUACCUUUCCGCACUGCGGACCGGAUGGUACACUUCC
GUGAUCACCAUUGAGCUCAGCAACAUCA AGGAAAACAAGUGCAAUGGCACCGACG
CCAAGGUCAAGCUGAUCAAACAAGAACUGGACAAGUACAAGAACGCCGUGACAG
AAUUGCAGCUCCUGAUGGGAUCCGGAAACGUCGGUCUGGGCGGAGCCAUCGCGAG
UGGAGUGGCUGUGUCCAAGGUCUUGCACCUCGAGGGAGAAGUGAACAAGAUCAA
GUCCGCGCUGCUGUCAACGAACAAGGCCGUGGUGUCCCUGUCUAACGGCGUCAGC
GUGCUGACGUUCAAGGUCCUGGACCUGAAGAAUUACAUUGACAAGCAGCUGCUG
CCCAUCCUCAACAAGCAAUCCUGCUCCAUCUCCAACCCCGAAACCGUGAUCGAGU
UCCAGCAGAAGAACAACCGCCUGCUGGAAAUUACUCGCGAGUUCUCUGUGAAUGC
CGGCGUGACCACCCCUGUGUCCACCUACAUGCUGACCAACUCCGAGCUUCUCUCC
CUUAUCAAUGACAUGCCUAUCACGAACGACCAGAAGAAGCUGAUGUCGAACAACG
UGCAGAU UG UGCGGCAGCAGUCAUACAGCAUCAUGUCGAUCAUCAAGGAAGAAG
UGCUGGCGUACGUGGUGCAACUCCCGCUGUACGGCGUCAUCGAUACCCCGUGCUG
GAAGCUGCACACCUCGCCUUUGUGUACCACCAACACCAAGAACGGAUCCAACAUC
UGCUUAACCCGGACUGAUCGGGGUUGGUACUGCGACAACGCCGGGAAUGUUUCG
UUCUUCCCACAAGCCGAGACUUGUAAAGUGCAGUCAAACAGAGUGUUCUGUGAC
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ACCAUGAACUCGAGAACCCUGCCCAGCGAAGUGAACCUGUGUAACGUCGACAUCU
UUAACCCAAAAUACGAUUGCAAGAUUAUGACCAGCAAAACCGACGUGUCCUCCUC
CGUGAUAACAAGCCUGGGGGCGAUUGUGUCAUGCUACGGAAAGACUAAGUGCAC
CGCCUCGAACAAGAACCGCGGCAUCAUUAAGACUUUCUCGAAUGGUUGCGACUAU
GUGUCCAACAAGGGCGUGGAUACUGUGUCAGUCGGGAAUACUCUUUACUACGUG
AACAAGCAGGAGGGGAAAAGCCUCUACGUGAAGGGAGAGCCUAUUAUCAACUUU
UACGAUCCGCUGGUGUUCCCGUCCGACGAAUUCGACGCCAGCAUCAGCCAAGUCA
ACGAGCUGAUUAACCAGUCCCUCGCCUUCAUCAACCAAUCCGACGAGCUCCUGCA
UAACGUGAACGCCGGAAAGUCCACCACCAACAUCAUGAUCACUACUAUUAUCAUC
GUGAUCAUCGUCAUCCUGCUGAGCCUGAUUGCUGUGGGCCUGUUGCUGUAUUGC
AAAGCCAGGUCCACCCCGGUCACCCUGUCGAAGGAUCAGCU GUCCGGAAUCAACA
ACAUUGCCUUCUCCAACUAA (SEQ ID NO: 17)
[00295] The nucleic acid sequences for the DNA template encoding the RSV F
protein is recited
below.
[00296] FD3 DNA:
ATGGAACTGCTGATCCTCAAAGCCAACGCAATCACCACCATTCTCACCGCTGTGACC
TTCTGCTTCGCATCGGGGCAGAACATCACTGAAGAGTTTTACCAGAGCACTTGCAGC
GCGGTGTCAAAGGGTTACCTTTCCGCACTGCGGACCGGATGGTACACTTCCGTGATC
ACCATTGAGCTCAGCAACATCAAGGAAAACAAGTGCAATGGCACCGACGCCAAGGT
CAAGCTGATCAAACAAGAACTGGACAAGTACAAGAACGCCGTGACAGAATTGCAGC
TCCTGATGGGATCCGGAAACGTCGGTCTGGGCGGAGCCATCGCGAGTGGAGTGGCT
GTGTCCAAGGTCTTG CACCTCGAGGGAGAAGTGAACAAGATCAAGTCCGCGCTG CT
GTCAACGAACAAGGCCGTGGTGTCCCTGTCTAACGGCGTCAGCGTGCTGACGTTCAA
GGTCCTGGACCTGAAGAATTACATTGACAAGCAGCTGCTGCCCATCCTCAACAAGC
AATCCTGCTCCATCTCCAACCCCGAAACCGTGATCGAGTTCCAGCAGAAGAACAAC
CGCCTGCTGGAAATTACTCGCGAGTTCTCTGTGAATGCCGGCGTGACCACCCCIGTG
TCCACCTACATGCTGACCAACTCCGAGCTTCTCTCCCTTATCAATGACATGCCTATCA
CGA A CGA CC A GA AGA A GCTGATGTCGA A C A A CGTGC A GATTGTGCGGCA GC A GTCA
TACAGCATCATGTCGATCATCAAGGAAGAAGTGCTGGCGTACGTGGTGCAACTCCC
GCTGTACGGCGTCATCGATACCCCGTGCTGGAAGCTGCACACCTCGCCTTTGTGTAC
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CACCAACACCAAGAACGGATCCAACATCTGCTTAACCCGGACTGATCGGGGTTGGT
ACTGCGACAACGCCGGGAATGTTTCGTTCTTCCCACAAGCCGAGACTTGTAAAGTGC
AGTCAAACAGAGTGTTC TGTGACACCATGAACTCGAGAACCCTGCCCAGCGAAGTG
AACCTGTGTAACGTCGACATCTTTAACCCAAAATACGATTGCAAGATTATGACCAGC
AAAACCGACGTGTCCTCCTCCGTGATAACAAGCCTGGGGGCGATTGTGTCATGCTAC
GGAAAGACTAAGTGCACCGCCTCGAACAAGAACCGCGGCATCATTAAGACTTTCTC
GAATGGTTGCGACTATGTGTCCAACAAGGGCGTGGATACTGTGTCAGTCGGGAATA
CTCTTTACTACGTGAACAAGCAGGAGGGGAAAAGCCTCTACGTGAAGGGAGAGCCT
ATTATCAACTTTTACGATCCGCTGGTG TTCCCGTCCGACGAATTCGACG CCAGCATC
AGCCAAGTCAACGAGCTGATTAACCAGTCCCTCGCCTTCATCAACCAATCCGACGAG
CTCCTGCATAACGTGAACGCCGGAAAGTCCACCACCAACATCATGATCACTACTATT
ATCATCGTGATCATCGTCATCCTGCTGAGCCTGATTGCTGTGGGCCTGTTGCTGTATT
GC A A A GCCA GGTCC ACCCCGGTC ACCCTGTCGA A GGATCA GCTGTCCGG A A TC A AC
AACATTGCCTTCTCCAACTAA (SEQ ID NO: 18)
[00297] The nucleic acid sequences for the 5 'UTR and 3 'UTR are recited
below.
[00298] 5'UTR:
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC
GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG
UGCCAAGAGUGACUCACCGUCCUUGACACG_(SEQ ID NO: 19)
[00299] 3 'UTR:
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGA A GULJGCC
ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 20)
[00300] The nucleic acid sequences for the full-length mRNA encoding the RSV F
protein is
recited below.
[00301] FD3 mRNA:
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACC
GGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCG
UGCCAAGAGUGACUCACCGUCCUUGACACGAUGGAACUGCUGAUCCUCAAAGCCA
ACGCAAUCACCACCAUUCUCACCGCUGUGACCUUCUGCUUCGCAUCGGGGCAGAA
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CAUCACUGAAGAGUUUUACCAGAGCACUUGCAGCGCGGUGUCAAAGGGUUACCU
UUCCGCACUGCGGACCGGAUGGUACACUUCCGUGAUCACCAUUGAGCUCAGCAAC
AUCAAGGAAAACAAGUGCAAUGGCACCGACGCCAAGGUCAAGCUGAUCAAACAA
GAACUGGACAAGUACAAGAACGCCGUGACAGAAUUGCAGCUCCUGAUGGGAUCC
GGAAACGUCGGUCUGGGCGGAGCCAUCGCGAGUGGAGUGGCUGUGUCCAAGGUC
UUGCACCUCGAGGGAGAAGUGAACAAGAUCAAGUCCGCGCUGCUGUCAACGAACA
AGGCCGUGGUGUCCCUGUCUAACGGCGUCAGCGUGCUGACGUUCAAGGUCCUGGA
CCUGAAGAAUUACAUUGACAAGCAGCUGCUGCCCAUCCUCAACAAGCAAUCCUGC
UCCAUCUCCAACCCCGAAACCGUGAUCGAGUUCCAGCAGAAGAACAACCGCCUGC
UGGAAAUUACUCGCGAGUUCUCUGUGAAUGCCGGCGUGACCACCCCUGUGUCCAC
CUACAUGCUGACCAACUCCGAGCUUCUCUCCCUUAUCAAUGACAUGCCUAUCACG
AACGACCAGAAGAAGCUGAUGUCGAACAACGUGCAGAUUGUGCGGCAGCAGUCA
UACAGCAUCAUGUCGAUCAUCAAGGAAGAAGUGCUGGCGUACGUGGUGCAACUC
CCGCUGUACGGCGUCAUCGAUACCCCGUGCUGGAAGCUGCACACCUCGCCUUUGU
GUACCACCAACACCAAGAACGGAUCCAACAUCUGCUUAACCCGGACUGAUCGGGG
UUGGUACUGCGACAACGCCGGGAAUGUUUCGUUCUUCCCACAAGCCGAGACUUGU
AAAGUGCAGUCAAACAGAGUGUUCUGUGACACCAUGAACUCGAGAACCCUGCCCA
GCGAAGUGAACCUGUGUAACGUCGACAUCUUUAACCCAAAAUACGAUUGCAAGA
UUAUGACCAGCAAAACCGACGUGUCCUCCUCCGUGAUAACAAGCCUGGGGGCGAU
UGUGUCAUGCUACGGAAAGACUAAGUGCACCGCCUCGAACAAGAACCGCGGCAUC
AUUAAGACUUUCUCGAAUGGUUGCGACUAUGUGUCCAACAAGGGCGUGGAUACU
GUGUCAGUCGGGAAUACUCUUUACUACGUGAACAAGCAGGAGGGGAAAAGCCUC
UACGUGAAGGGAGAGCCUAUUAUCAACUUUUACGAUCCGCUGGUGUUCCCGUCCG
ACGAAUUCGACGCCAGCAUCAGCCAAGUCAACGAGCUGAUUAACCAGUCCCUCGC
CUUCAUCAACCAAUCCGACGAGCUCCUGCAUAACGUGAACGCCGGAAAGUCCACC
ACCAACAUCAUGAUCACUACUAUUAUCAUCGUGAUCAUCGUCAUCCUGCUGAGCC
UGAUUGCUGUGGGCCUGUUGCUGUAUUGCAAAGCCAGGUCCACCCCGGUCACCCU
GUCGAAGGAUCAGCUGUCCGGAAUCAACAACAUUGCCUUCUCCAACUAACGGGUG
GCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCA
GUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 21)
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[00302] LNP-RSV FD3 mRNA compositions were administered to NHPs. Groups of 6
cynomolgus macaques were administered a 5 tg dose of mRNA encapsulated with
the above
LNPs, or a 10 pig dose of an RSV Pre-F NP subunit control vaccine adjuvanted
with Al(OH)3, by
intramuscular (IM) injection on DO and D21. Monkeys were bled prior to each
vaccine
administration as well as at two weeks post-last vaccination (D35). As shown
in FIG. 29, all tested
cationic lipids effectively induced the production of anti-RSV F protein
antibodies to a similar
level as a Pre-F NP with an aluminum adjuvant.
[00303] As shown in FIG. 30, all tested cationic lipids generated effective
RSV neutralization
titers to a similar level as a Pre-F NP with an aluminum adjuvant.
[00304] The cumulative results of FIG. 29 and FIG. 30 are shown below in Table
9 and Table
10.
Table 9. Magnitude of immune response
LNP Neutralization
Fold vs. MC3
Formulation Titer
LIPID A 9.86 23.43
LIPID B 10.03 26.35
LIPID C 8.509 9.18
LIPID E 6.929 3.07
LIPID D 8.894 11.99
MC3 5.308 1.00
Pre-F NP 10.97 50.56
Table 10. Quality of immune response
Antibody Titer /
Neutralization
Cationic Lipid Antibody Titer Neutralization Titer
Titer
ratio
LIPID A 15.58 9.86 52.71
LIPID B 15.56 10.03 46.21
LIPID C 14.67 8.51 71.51
LIPID E 13.27 6.93 81.01
LIPID D 14.71 8.89 56.49
MC3 11.3 5.31 63.56
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Prc-F NP 17.59 10.97 98.36
[00305] A better quality of an immune response is demonstrated with a lower
value for the
antibody titer / neutralization titer ratio. Here, the LNP formulation Lipid B
demonstrated the best
quality of immune response, while all LNP formulations demonstrated a superior
quality of
immune response compared to the non-mRNA vaccine, Pre-F NP, and several were
better than the
industry benchmark LNP formulation of MC3.
Example 9: SARS-CoV-2 Spike (S) protein-encoding mRNA LNP Formulations
[00306] LNP Formulations with SARS-CoV-2 Spike (S) protein-encoding mRNA:
[00307] An LNP formulation containing a SARS-CoV-2 S protein-encoding mRNA was
administered to human subjects. The subjects were administered an LNP of
formulation Lipid B.
The unmodified mRNA encoded a SARS-CoV-2 S protein mutated to remove the furin
cleavage
site and to mutate residues 986 and 987 to prolinc. The subjects were
administered the LNP-
SARS-CoV-2 vaccine under clinical trial protocol for NCT04798027, described
below.
[00308] This was a sequential group prevention study consisting of a sentinel
cohort followed by
the Full Enrollment Cohort. There were 3 dose levels (up to 25 participants 18-
49 years of age for
each dose level) in the Sentinel Cohort, which was done in an open-label
fashion with stepwise
safety evaluation for each dose level and each vaccination. All sentinel
participants received 2
vaccinations, 21 days apart. For the Full Enrollment Cohort, participants were
stratified into 2 age
groups based on age at enrollment: the younger adult age group (140
participants 18-49 years of
age) and the older adult age group (168 participants? 50 years of age). The
Full Enrollment Cohort
1 (Groups 1 to 4) received a single injection of study intervention while
participants in Cohort 2
(Groups 5 to 8) received 2 vaccinations (to be given 21 days apart). The route
of administration
for all groups was intramuscular (IM).
[00309] Experimental: Group 1 ¨ 1 injection of SARS-CoV-2 mRNA vaccine
formulation 1 at
Day 1.
[00310] Experimental: Group 2 ¨ 1 injection of SARS-CoV-2 mRNA vaccine
formulation 2 at
Day 1.
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[00311] Experimental: Group 3 ¨ 1 injection of SARS-CoV-2 mRNA vaccine
formulation 3 at
Day 1.
[00312] Placebo Comparator: Group 4 ¨ 1 injection of placebo (0.9% normal
saline) at Day 1.
[00313] Experimental: Group 5 ¨ 2 injections of SARS-CoV-2 mRNA vaccine
formulation 1 at
Day 1 and Day 22.
[00314] Experimental: Group 6 ¨ 2 injections of SARS-CoV-2 mRNA vaccine
formulation 2 at
Day 1 and Day 22.
[00315] Experimental: Group 7 ¨ 2 injections of SARS-CoV-2 mRNA vaccine
formulation 3 at
Day land Day 22.
[00316] Placebo Comparator: Group 8 ¨ 2 injections of placebo (0.9% normal
saline) at Day 1
and Day 22.
[00317] Results from the study showed neutralizing antibody seroconversion
(defined as 4-fold
increase vs baseline) in 91% to 100% of study participants, two weeks after a
second injection,
across all 3 dosages tested. No safety concern has been observed and the
tolerability profile is
comparable to that of other unmodified mRNA SARS-CoV-2 vaccines.
Example 10: Further Studies on Quadrivalent or Octavalent Influenza Vaccine
LNP
Formulations
[00318] HAT titers and NAT titers were measured from mice administered various
multivalent
LNP-influenza mRNA vaccines. HAT titers were measured against influenza
strains
A/Michigan/45/2015, A/SINGAPORE/INFIMH160019/2016, B/Maryland/15/2016 BX69A,
and
B/Phuket/3073/2013. NAT titers were measured against influenza strains
A/Michigan/45/2015,
A/SINGAPORE/INFIMH160019/2016, B/Colorado/06/201, and B/Phuket/3073/2013.
[00319] The HAT titers and NAT titers were compared against mice receiving
mono- or
quadrivalent HA or NA mRNA vaccines.
[00320] Mice were injected with a prime vaccine on day 0 and a booster vaccine
of the same
dosage on Day 21. Blood was collected on days 1, 20, 22, and 35. For
monovalent compositions
containing mRNA encoding HA or NA antigens, mRNA encoding each of the
following
individually was used: H1, H3, HA from a B/Victoria lineage, and HA from a
B/Yamagata lineage
(specifically from strains A/Michigan/45/2015; A/Singapore/Intimh160019/2016;
B/Maryland/15/2016; and B/Phuket/3037/2013). Quadrivalent vaccine compositions
containing
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mRNA encoding each of Ni, N2, NA from a BNictoria lineage and NA from a
B/Yamagata
lineage, and each of H1, H3, HA from a B/Victoria lineage and HA from a
B/Yamagata lineage
(specifically from strains A/Michigan/45/2015; A/Singapore/Infimh160019/2016;
B/Colorado/06/2017; and B/Phuket/3037/2013) were also prepared. Finally, an
octavalent vaccine
composition containing mRNA encoding each of H1, H3, HA from a B/Victoria
lineage, HA from
a B/Yamagata lineage, each of N1, N2, NA from a B/Victoria lineage and NA from
a B/Yamagata
lineage (specifically from strains A/Michigan/45/2015;
A/Singapore/Infimh160019/2016;
B/Colorado/06/2017; and B/Pliuket/3037/2013) was prepared and administered as
an octavalent
vaccine. Each mRNA for all compositions was added in an amount of 0.4
lug/strain. For each
group, n=6 mice.
[00321] An overview of each experimental group is recited below in Table 11.
Table 11. Overview of experimental groups for multivalent influenza vaccines
in mice
Gro Prime (D0)/boost Dose mRNA NA Prime (D0)/boost (D21) -
Dose rHA (ag Adjuvan
up # N (D21) - NA mRNA (lig per strain) HA
(together with NA) per strain) t (rHA)
1 6 LNP diluent
NA mRNA-LNP (N2
3 6 Perth) 0.4 -
NA mRNA-LNP
4 6 (N1) 0.4 -
NA mRNA-LNP
5 6 (N2) 0.4 -
NA mRNA-LNP
6 6 (NV) 0.4 -
NA mRNA-LNP
7 6 (NY) 0.4 -
NA mRNA-LNP
8 6 (N1, N2, BV, BY) 0.4 -
9 6 - HA mRNA-LNP (H1) 0 4 -
10 6 - HA mRNA-LNP (H3) 0.4 -
11 6 - HA mRNA-LNP (BV) 0.4 -
12 6 - HA mRNA-LNP (BY) 0.4 -
HA mRNA-LNP (H1, H3,
13 6 - BV, BY) 0.4 -
NA mRNA-LNP HA mRNA-LNP (H1, H3,
14 6 (N1, N2, BV, BY) 0.4 BV, BY) 0.4
[00322] As shown in FIG. 31, octavalent mRNA-LNP formulations led to HAT
titers within 4-
fold of the quadrivalent for 3 out of 4 influenza strains.
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[00323] An overview of the NAT titer results for each of the groups above is
shown in FIG. 33.
The octavalent mRNA-LNP formulations led to NAT titers comparable to the
quadrivalent mRNA-
LNP formulations.
[00324] Thus, the data demonstrate that an octavalent vaccine was capable of
inducing robust HA
and NA immune responses and that the presence of the immunodominant HA from
four different
influenza strains does not appear to suppress or interfere with the anti-NA
immune response.
[00325] High content imaging-based neutralization test (HINT) titers for HA
and NAT titers were
additionally measured from ferrets administered various multivalent LNP-
influenza mRNA
vaccines. The HINT assay is described in further detail in Jorquera et al.
(Scientific Reports. 9:
2676. 2019), incorporated herein by reference. HINT titers were measured
against influenza strains
A/Michigan/45/2015, A/SINGAPORE/INFIMH160019/2016, B/IOWA/06/2017, and
B/Phuket/3073/2013. NAT titers were measured against influenza strains
A/Michigan/45/2015,
A/S INGAP ORE/INFIMH160019/2016, B/Colorado/06/201, and B/Phuket/3073/2013.
[00326] Ferrets used to assess multivalent vaccine immunogenicity were
vaccinated twice 21 days
apart with (1) a mixture of four mRNAs encoding NA antigens (Ni, N2, BvNA, and
ByNA), (2)
a mixture of four mRNAs encoding HA antigens (H1, H3, BvHA, and ByHA), or (3)
a mixture of
four mRNAs encoding NA antigens (Ni, N2, BvNA, and ByNA) and four mRNAs
encoding HA
antigens (H1, H3, BvHA, and ByHA), as shown below in Table 12. Each HA
includes HA from
one of the following four strains: A/Michigan/45/2015 (H1); A/S
ingapore/Infimh-16-0019/2016
(H3); B/Towa/06/2017 (B/Victoria lineage); and B/Pfiuket/3073/2013 (BNamagata
lineage). All
antigens were administered at a 1:1 ratio.
[00327] An overview of each experimental group is recited below in Table 12.
[00328] All ferrets were bled under sedation (isoflurane) at baseline, one day
before or just before
booster, at booster vaccination, and two weeks after challenge as required.
Sera samples (stored
at ¨20 C until required) were tested by ELLA to assess NAT activity.
Additionally, the
hemagglutinin inhibition assay (HAT) was undertaken to assess antibody
responses to
hemagglutinin antigens following multivalent vaccination.
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Table 12. Overview of experimental groups for multivalent influenza vaccines
in ferrets
Group Dose (lug per
Adjuva
N Prime (D0)/boost (D21) - NA Prime
(D0)/boost (D21) - HA strain) nt
1 6 PBS PBS
0 -
NA mRNA-LNP (Ni, N2, By,
11 6 BY) - 1 -
NA mRNA-LNP (Ni, N2, By,
12 6 BY)
15 -
HA mRNA-LNP (H1, H3, BY,
13 6 - BY)
1 -
HA mRNA-LNP (H1, H3, By,
14 6 - BY)
15 -
NA mRNA-LNP (Ni, N2, By, HA mRNA-LNP (H1, H3, By,
15 6 BY) BY)
1 -
NA mRNA-LNP (Ni, N2, By, HA mRNA-LNP (H1, H3, By,
16 6 BY) BY)
15 -
[00329] An overview of the HINT results for each of the groups above is shown
in FIG. 32. The
octavalent mRNA-LNP formulations led to HINT titers comparable to the
quadrivalent mRNA-
LNP formulations.
[00330] An overview of the NAT titer results for each of the groups above is
shown in FIG. 34
(day 20) and FIG. 35 (day 42). The octavalent mRNA-LNP formulations led to NAT
titers
comparable to the quadrivalent mRNA-LNP formulations. This was true from the
day 20 and day
42 samples.
Example 11: Functional antibody titers to influenza heterologous subtype
strains recorded
with mRNA in Lipid A or Lipid B LNP Formulations:
[00331] To evaluate immunogenicity of the mRNA-LNP in NHP, 0, 15, 45, ug of
Sing16 HA-
encoding mRNA encapsulated in either Lipid A or Lipid B LNP formulation
(encoding for HA
A/Singapore/INFIMH-16-0019/2016) were immunized. Naïve male and female
Mauritius origin
Cynomolgus macaques (Macaca fascicularis) were used. Animals weighed >2kg and
were >2
years of age at the start of the studies. Groups consisted of up to 6 animals
per treatment group
and were vaccinated in 0.5 mL of their respected vaccine dose or diluent via
the IM route in one
forelimb of each animal, targeting the deltoid, on Study Day 0. Twenty-eight
days after the first
immunization took place, a second immunization was given to the animals in the
contralateral
limb. A quadrivalent egg-derived inactivated influenza vaccine (IIV)
containing the
A/Singaporc/1NFIMH-16-0019/2016 (H3 N2 ) strain was uscd as a comparator.
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[00332] Influenza assays were performed using the A/Singapore/INFIMH-16-
0019/2016 (H3N2)
virus stocks from BIOQUAL, Inc. Additional breadth testing by HAT was
performed using the
following H3N1 virus stocks: A/Shandoglaicheng/1763/2016, A/Louisiana/13/2017,
A/Kenya/105/2017, ANictoria/746/2017, and A/Michigan/84/2016,
A/Aksaray/4048/2016.
These include strains from both 3c.2a and 3c.3a clades, as well as a very
distant swine-like H3
sequence (A/Michigan/84/2016) based on bioinformatics analysis to select a set
of maximally
diverse H3N2 sequences from the same timeframe as A/Singapore/INFIMH-16-
0019/2016.
[00333] For micron eutralization (MN) assays, sera samples were diluted in
receptor-destroying
enzyme (Denka Seiken, 370013) and incubated overnight in a 37 C water bath.
Samples were
heat-inactivated for 30-minutes at 56 C then two-fold serial dilutions were
run in duplicate in 96-
well plates. An equal volume of virus at 100TC1D50 was added to the plates
followed by a 1-hour
incubation at 37 C. One-hundred microliters of sample/virus mixture was
transferred to 96-well
flat-bottom plates of MDCK cells (ATCC#CCL-34) containing TPCK-treated media
and
incubated for 48-hours at 37 C with 5% CO2. Plates were fixed with cold
acetone then stained
with biotin-conjugated anti-Influenza A NP (Millipore, MAB8258B) followed by
incubation with
DELFIA Europium-labeled streptavidin in Delfia assay buffer. Fluorescence was
measured and
endpoint titers reported.
[00334] At day 43, after the second immunization, NHPs vaccinated with
Singl6HA-CL-059 and
Singl6HA-CL017 developed neutralizing antibodies to homologous virus,
A/Singapore/INFIMH-
16-0019/2016 (H3N2), as noted in MN assay (FIG. 36 and FIG. 37). Further, in
this model, MN
titers were observed to lietero subtype viral panel including
A/Sliandoglaiclieng/1763/2016,
A/Louisiana/13/2017, A/Kenya/105/2017, A/Victoria/746/2017, and
A/Aksaray/4048/2016
contrary to the IW vaccine. The data indicates the said mRNA formulations have
potential to
provide greater breadth than IW covering for hetero subtype strains of
influenza.
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