U.S. patent application number 16/362366 was filed with the patent office on 2019-07-11 for high potency immunogenic compositions.
This patent application is currently assigned to ModernaTX, Inc.. The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Giuseppe Ciaramella.
Application Number | 20190211065 16/362366 |
Document ID | / |
Family ID | 63169866 |
Filed Date | 2019-07-11 |
United States Patent
Application |
20190211065 |
Kind Code |
A1 |
Ciaramella; Giuseppe |
July 11, 2019 |
HIGH POTENCY IMMUNOGENIC COMPOSITIONS
Abstract
Provided herein, in some embodiments, are immunogenic
compositions that include a cationic lipid nanoparticle (LNP)
encapsulating messenger ribonucleic acid (mRNA) having an open
reading frame encoding a viral, bacterial or parasitic antigen, a
pan HLA DR-binding epitope (PADRE), and a 5' terminal cap modified
to increase mRNA translation efficiency.
Inventors: |
Ciaramella; Giuseppe;
(Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
ModernaTX, Inc.
Cambridge
MA
|
Family ID: |
63169866 |
Appl. No.: |
16/362366 |
Filed: |
March 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16031951 |
Jul 10, 2018 |
10273269 |
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16362366 |
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PCT/US2018/000026 |
Feb 16, 2018 |
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16031951 |
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62459763 |
Feb 16, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/14 20180101;
C07K 16/1081 20130101; A61K 2039/55555 20130101; C12N 2770/24123
20130101; A61K 31/7105 20130101; A61K 39/12 20130101; A61K 31/7115
20130101; C12N 2770/24034 20130101; C07K 14/1825 20130101; A61K
2039/51 20130101; A61K 39/39 20130101; C12N 2770/24134
20130101 |
International
Class: |
C07K 14/18 20060101
C07K014/18; A61K 39/12 20060101 A61K039/12; C07K 16/10 20060101
C07K016/10; A61K 31/7105 20060101 A61K031/7105; A61P 31/14 20060101
A61P031/14; A61K 39/39 20060101 A61K039/39 |
Claims
1.-30. (canceled)
31. A method of inducing an immune response in a subject, the
method comprising administering to the subject an immunogenic
composition of comprising a cationic lipid nanoparticle (LNP)
encapsulating messenger ribonucleic acid (mRNA) having an open
reading frame encoding a Zika virus (ZIKV) prM antigen, a ZIKV E
antigen, and a pan HLA DR-binding epitope (PADRE), and a 5'
terminal cap, wherein the cationic lipid nanoparticle comprises a
cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic
lipid, in an amount effective to produce in the subject an immune
response specific to ZIKV prM antigen and/or ZIKV E antigen.
32.-105. (canceled)
106. The method of claim 31, wherein the ZIKV prM antigen and the
ZIKV E antigen form a fusion antigen comprising a sequence set
forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
107. The method of claim 31, wherein the open reading frame is
codon optimized.
108. The method of claim 31, wherein at least 80% of the uracil in
the open reading frame has a chemical modification.
109. The method of claim 108, wherein the chemical modification is
N1-methylpseudouridine or N1-ethylpseudouridine.
110. The method of claim 108, wherein the chemical modification is
at the carbon 5-position of the uracil.
111. The method of claim 31, wherein the 5' terminal cap is
7mG(5')ppp(5')N1mpNp.
112. The method of claim 31, wherein the open reading frame of the
mRNA further encodes a signal sequence.
113. The method of claim 112, wherein the signal sequence is the
Japanese encephalitis prM signal sequence set forth in SEQ ID NO:
11.
114. The method of claim 31, wherein the cationic lipid
nanoparticle comprises a molar ratio of 20-60% cationic lipid,
0.5-15% PEG-modified lipid, 25-55% sterol, and 5-25% non-cationic
lipid.
115. The method of claim 31, wherein the cationic lipid is an
ionizable cationic lipid and the non-cationic lipid is a neutral
lipid, and the sterol is a cholesterol.
116. The method of claim 31, wherein the cationic lipid
nanoparticle comprises a compound of Formula (I).
117. The method of claim 116, wherein the cationic lipid
nanoparticle comprises Compound 1 or Compound 2.
118. The method of claim 31, wherein the amount administered is 5
.mu.g to 100 .mu.g of the mRNA.
119. A method of inducing an immune response in a subject, the
method comprising administering to the subject an immunogenic
composition comprising a cationic lipid nanoparticle (LNP)
encapsulating (a) a messenger ribonucleic acid (mRNA) having an
open reading frame encoding a Zika virus (ZIKV) prM antigen, a ZIKV
E antigen, and a 5' terminal cap; and (b) a mRNA having an open
reading frame encoding a pan HLA DR-binding epitope (PADRE), and a
5' terminal cap, wherein the cationic lipid nanoparticle comprises
a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid, in an amount effective to produce in the subject an immune
response specific to ZIKV prM antigen and/or ZIKV E antigen.
120. The method of claim 119, wherein the ZIKV prM antigen and the
ZIKV E antigen form a fusion antigen comprising a sequence set
forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
121. The method of claim 119, wherein the open reading frame of (a)
and/or the open reading frame of (b) are codon optimized.
122. The method of claim 119, wherein at least 80% of the uracil in
the open reading frame of (a) has a chemical modification, and/or
at least 80% of the uracil in the open reading frame of (b) has a
chemical modification.
123. The method of claim 122, wherein the chemical modification of
(a) and/or (b) is N1-methylpseudouridine or
N1-ethylpseudouridine.
124. The method of claim 122, wherein the chemical modification of
(a) and/or (b) is at the carbon 5-position of the uracil.
125. The method of claim 119, wherein the 5' terminal cap of (a)
and/or (b) is 7mG(5')ppp(5')N1mpNp.
126. The method of claim 119, wherein the open reading frame of (a)
and/or (b) further encodes a signal sequence.
127. The method of claim 126, wherein the signal sequence is the
Japanese encephalitis prM signal sequence set forth in SEQ ID NO:
11.
128. The method of claim 119, wherein the cationic lipid
nanoparticle comprises a molar ratio of 20-60% cationic lipid,
0.5-15% PEG-modified lipid, 25-55% sterol, and 5-25% non-cationic
lipid.
129. The method of claim 119, wherein the cationic lipid is an
ionizable cationic lipid and the non-cationic lipid is a neutral
lipid, and the sterol is a cholesterol.
130. The method of claim 119, wherein the cationic lipid
nanoparticle comprises a compound of Formula (I).
131. The method of claim 130, wherein the cationic lipid
nanoparticle comprises Compound 1 or Compound 2.
132. The method of claim 119, wherein the amount administered is 5
.mu.g to 100 .mu.g of the mRNA.
Description
RELATED APPLICATION
[0001] This application is a division U.S. application serial Ser.
No. 16/031,951, filed Jul. 10, 2018, which is a continuation of
international application number PCT/US2018/000026, filed Feb. 16,
2018, which claims the benefit under 35 U.S.C. .sctn. 119(e) of
U.S. provisional application No. 62/459,763, filed Feb. 16, 2017,
each of which is incorporated by reference herein in its
entirety.
BACKGROUND
[0002] Zika virus (ZIKV) was identified in 1947 from a sentinel
Rhesus monkey in the Zika Forest of Uganda. Historically, ZIKV
circulated between Aedes species mosquitoes, non-human primates in
the jungle, and episodically spilled into human populations in
Africa and parts of Southeast Asia. Infection was associated with a
mild, self-limiting febrile illness characterized by headache,
rash, conjunctivitis, myalgia, and arthralgia. Since 2010, and
especially in the context of its spread and dissemination to
countries of the Western Hemisphere, more severe clinical
consequences have been observed. Infection of fetuses in utero
during pregnancy, particularly during the first and second
trimesters, has been associated with placental insufficiency and
congenital malformations including cerebral calcifications,
microcephaly, and miscarriage. In adults, ZIKV infection is linked
to an increased incidence of Guillain-Barr6 syndrome (GBS), an
autoimmune disease characterized by paralysis and polyneuropathy.
In addition to mosquito and in utero transmission, sexual
transmission of ZIKV has been described from men-to-women,
men-to-men, and women-to-men. Persistent ZIKV infection can occur,
as viral RNA has been detected in semen, sperm, and vaginal
secretions up to 6 months following infection. Thus, ZIKV is now a
global disease with locally-acquired and travel-associated
transmission through multiple routes in the Americas. Africa, and
Asia. The emergence of Zika virus (ZIKV) infection has prompted a
global effort to develop safe and effective vaccines.
SUMMARY
[0003] Provided herein are immunogenic compositions that include
lipid nanoparticles encapsulating mRNA vaccines that enhance the
CD4.sup.+ T cell immune response. In some embodiments, the vaccines
comprise an mRNA encoding an infectious disease antigen, such as a
Zika virus (ZIKV) antigen, and an mRNA encoding an immunodominant
helper CD4 T cell epitope, such as a pan HLA DR-binding epitope
(PADRE). CD4.sup.+ T cells play an important role in the generation
of CD8.sup.+ T effector and memory T-cell immune responses. The
CD4.sup.+ T cell immune response, and thus the corresponding
antigen-specific CD8.sup.+ T cell response, can be enhanced by
administering in a single composition an mRNA vaccine encoding a
pan FILA DR-binding epitope (PADRE).
[0004] Thus, provided herein are more potent prophylactic and
therapeutic immunogenic compositions (e.g., mRNA vaccines) that
induce a stronger T cell response against viral, bacterial and/or
parasitic antigens, relative to current vaccine therapies.
[0005] Some aspects of the present disclosure provide an
immunogenic composition, comprising a cationic lipid nanoparticle
(LNP) encapsulating messenger ribonucleic acid (mRNA) having an
open reading frame encoding a Zika Virus (ZIKV) prM antigen, a ZIKV
E antigen, a pan HLA DR-binding epitope (PADRE), and a 5' terminal
cap modified to increase mRNA translation efficiency, wherein the
cationic lipid nanoparticle comprises a cationic lipid, a
PEG-modified lipid, a sterol and a non-cationic lipid.
[0006] Other aspects of the present disclosure provide an
immunogenic composition, comprising a cationic lipid nanoparticle
(LNP) encapsulating (a) a messenger ribonucleic acid (mRNA) having
an open reading frame encoding a Zika Virus (ZIKV) prM antigen, a
ZIKV E antigen, and a 5' terminal cap, and (b) a mRNA having an
open reading frame encoding a pan HLA DR-binding epitope (PADRE),
and a 5' terminal cap, wherein the 5' terminal cap of (a) and (b)
are modified to increase mRNA translation efficiency, and wherein
the cationic lipid nanoparticle comprises a cationic lipid, a
PEG-modified lipid, a sterol and a non-cationic lipid.
[0007] In some embodiments, the ZIKV prM antigen and the ZIKV E
antigen form a fusion antigen comprising a sequence identified by
SEQ ID NO: 2. In some embodiments, the ZIKV prM antigen and the
ZIKV E antigen form a fusion antigen comprising a sequence
identified by SEQ ID NO: 4.
[0008] In some embodiments, the ratio of mRNA encoding ZIKV antigen
to mRNA encoding PADRE is 0.1:1 to 10:1. In some embodiments, the
ratio of mRNA encoding ZIKV antigen to mRNA encoding PADRE is
0.1:1, 0.5:1, 1:1, 2:1, 5:1 or 10:1.
[0009] In some embodiments, the open reading frame is codon
optimized.
[0010] In some embodiments, at least 80% of the uracil in the open
reading frame have a chemical modification. In some embodiments, at
least 90% of the uracil in the open reading frame have a chemical
modification. In some embodiments, 100% of the uracil in the open
reading frame have a chemical modification.
[0011] In some embodiments, the chemical modification is
N1-methylpseudouridine or N1-ethylpseudouridine.
[0012] In some embodiments, the chemical modification is at the
5-position of the uracil.
[0013] In some embodiments, the 5' terminal cap is
7mG(5')ppp(5')N1mpNp.
[0014] In some embodiments, the open reading frame of the mRNA
further encodes a signal sequence. In some embodiments, the signal
sequence is a Japanese encephalitis prM signal sequence
(MWLVSLAPVTACAGA; SEQ ID NO: 11).
[0015] In some embodiments, the cationic lipid nanoparticle
comprises a molar ratio of about 20-60% cationic lipid, 0.5-15%
PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid.
[0016] In some embodiments, the cationic lipid is an ionizable
cationic lipid and the non-cationic lipid is a neutral lipid, and
the sterol is a cholesterol.
[0017] In some embodiments, the cationic lipid is selected from
2,2-dilinoleyl4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl4-dimethylaminobutyrate (DLin-MC3-DMA),
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butarioyl)oxy)heptadecanedioate,
(12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-2,15-dien-1-amine, and
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine.
[0018] In some embodiments, the cationic lipid nanoparticle
comprises a compound of Formula (I). In some embodiments, the
compound is selected from Compound 3, 18, 20, 25, 26, 29, 30, 60,
108-112 and 122. In some embodiments, the compound is Compound
25.
[0019] In some embodiments, the immunogenic composition is
formulated in an effective amount to induce an antigen-specific
immune response. In some embodiments, the antigen-specific immune
response comprises a B cell response and/or T cell response. In
some embodiments, the antigen-specific immune response comprises a
PADRE-specific CD4+ T cell response.
[0020] In some embodiments, the antigen-specific immune response is
at least 0.1-10 times stronger than an antigen-specific immune
response induced in a subject administered a control immunogenic
composition without an mRNA encoding a PADRE. In some embodiments,
the antigen-specific immune response is at least 0.5 times stronger
than an antigen-specific immune response induced in a subject
administered a control immunogenic composition without an mRNA
encoding a PADRE. In some embodiments, the antigen-specific immune
response is at least 2 times stronger than an antigen-specific
immune response induced in a subject administered a control
immunogenic composition without an mRNA encoding a PADRE. In sonic
embodiments, the antigen-specific immune response is at least 5
times stronger than an antigen-specific immune response induced in
a subject administered a control immunogenic composition without an
mRNA encoding a PADRE. In some embodiments, the antigen-specific
immune response is at least 10 times stronger than an
antigen-specific immune response induced in a subject administered
a control immunogenic composition without an mRNA encoding a
PADRE.
[0021] In some embodiments, the effective amount is 5 .mu.g-100
.mu.g of the mRNA.
[0022] Further aspects of the present disclosure provide a method
of inducing an immune response in a subject, the method comprising
administering to the subject the immunogenic composition in an
amount effective to produce an antigen-specific immune response in
the subject.
[0023] In some embodiments, a single dose of the immunogenic
composition is administered to the subject. In other embodiments, a
booster dose of the immunogenic composition is administered to the
subject.
[0024] In some embodiments, the efficacy of the immunogenic
composition against ZIKV infection is at least 50% following
administration of the booster dose of the immunogenic composition.
In some embodiments, the efficacy of the immunogenic composition
against ZIKV infection is at least 75% following administration of
the booster dose of the immunogenic composition.
[0025] In some embodiments, the immunogenic composition immunizes
the subject against ZIKV for more than 2 years.
[0026] In some embodiments, the anti-ZIKV antigen antibody titer
produced in the subject is increased by at least 1 log relative to
a control. In some embodiments, the anti-ZIKV antigen antibody
titer produced in the subject is increased by 1-3 log relative to a
control.
[0027] In some embodiments, the anti-ZIKV antigen antibody titer
produced in the subject is increased at least 2 times relative to a
control. In some embodiments, the anti-ZIKV antigen antibody titer
produced in the subject is increased 2-10 times relative to a
control.
[0028] In some embodiments, the control is the anti-ZIKV antigen
antibody titer produced in a subject who has not been administered
a vaccine against ZIKV; who has been administered a live attenuated
vaccine or an inactivated vaccine against ZIKV; who has been
administered a recombinant protein vaccine or purified protein
vaccine against ZIKV; or who has been administered a VLP vaccine
against ZIKV.
[0029] In some embodiments, the amount is sufficient to produce
detectable levels of the antigen as measured in serum of the
subject at 1-72 hours post administration.
[0030] In some embodiments, the amount is sufficient to produce a
1,000-10,000 neutralization titer produced by neutralizing antibody
against the antigen as measured in serum of the subject at 1-72
hours post administration.
[0031] In some embodiments, the subject is immunocompromised.
[0032] Also provided herein are uses of the immunogenic composition
in the manufacture of a medicament for use in a method of inducing
an antigen specific immune response to ZIKV in a subject, the
method comprising administering to the subject the immunogenic
composition in an amount effective to produce an antigen specific
immune response to ZIKV in the subject.
[0033] Further provided herein are pharmaceutical compositions for
use in vaccination of a subject comprising an effective dose of the
immunogenic composition, wherein the effective dose is sufficient
to produce detectable levels of ZIKV antigen as measured in serum
of the subject at 1-72 hours post administration.
[0034] Further still, provided herein are pharmaceutical
compositions for use in vaccination of a subject comprising an
effective dose of the immunogenic composition of any one of claims
1-30, wherein the effective dose is sufficient to produce a
1,000-10,000 neutralization titer produced by neutralizing antibody
against ZIKV antigen as measured in serum of the subject at 1-72
hours post administration.
[0035] Some aspects of the present disclosure provide an
immunogenic composition, comprising a cationic lipid nanoparticle
(LNP) encapsulating messenger ribonucleic acid (mRNA) having. an
open reading frame encoding at least one antigen, a pan FILA
DR-binding epitope (PADRE), and a 5' terminal cap modified to
increase mRNA translation efficiency, wherein the cationic lipid
nanoparticle comprises a cationic lipid, a PEG-modified lipid, a
sterol and a non-cationic lipid.
[0036] Other aspects of the present disclosure provide an
immunogenic composition, comprising a lipid nanoparticle (LNP)
encapsulating (a) a messenger ribonucleic acid (mRNA) having an
open reading frame encoding at least one antigen, and a 5' terminal
cap; and (h) a mRNA having an open reading frame encoding a pan HLA
DR-binding epitope (PADRE), and a 5' terminal cap, wherein the 5'
terminal cap of (a) and (b) is modified to increase mRNA
translation efficiency, wherein the cationic lipid nanoparticle
comprises a cationic lipid, a PEG-modified lipid, a sterol and a
non-cationic lipid.
[0037] In some embodiments, at least one antigen is at least one
viral antigen selected from a Betacoronavirus, Chikungunya virus,
Dengue virus, Ebola virus, Eastern Equine Encephalitis virus,
Herpes Simplex virus, Hainan Cytomegalovirus, Human
Metapneumovirus, Human Papillomavirus, influenza virus, Japanese
Encephalitis virus, Marburg virus, Measles, Parainfluenza virus,
Respiratory Syncytial virus, Sindhis virus, Varicella Zoster virus,
Venezuelan Equine Encephalitis virus, West Nile virus, Yellow Fever
virus, and Zika virus antigen. In some embodiments, at least one
viral antigen is at least one Zika virus antigen. In some
embodiments, at least one Zika virus antigen is a premembrane (prM)
protein antigen and an envelope (E) protein antigen.
[0038] In some embodiments, at least one antigen is at least one
bacterial antigen selected from a Chlamydia trachomatis antigen, a
Lyme Borrelia and a Streptococcal antigen.
[0039] In some embodiments, at least one antigen is at least one
parasitic antigen selected from Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovate, and Plasmodium malariae, and Plasmodium
knowlesi antigens.
DETAILED DESCRIPTION
[0040] Provided herein, in some aspects, are immunogenic
compositions that include messenger ribonucleic acid (mRNA) having
an open reading frame encoding at least one antigen (e.g., an
infectious disease antigen), a pan FILA DR-binding epitope (PADRE),
and a 5' terminal cap modified to increase mRNA translation
efficiency. In sonic embodiments, the antigen and the PADRE are
encoded on the same mRNA molecule, while in other embodiments, the
antigen and the PADRE are encoded on separate mRNA molecules.
[0041] The immunogenic compositions (e.g., mRNA vaccines), in some
embodiments, are formulated in a lipid nanoparticie comprising a
cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid. In some embodiments, the non-cationic lipid is Compound 25
of Formula (I). Thus, in some embodiments, an immunogenic
composition comprises a mRNA encoding an antigen (e.g., a viral,
bacterial, or parasitic antigen), a mRNA encoding a PADRE sequence
and is formulated in a lipid nanoparticle that comprises Compound
25 of Formula (I).
[0042] In some embodiments, the immunogenic compositions (e.g.,
mRNA vaccines) may be used to treat a viral, bacterial, or
parasitic infection in a subject (e.g., a human subject). The
immunogenic compositions, in some embodiments, may be used to
induce a balanced immune response, comprising both cellular and
Immoral immunity, without many of the risks associated with DNA
vaccination.
PADRE Sequences
[0043] The immunogenic compositions (e.g., mRNA vaccines) of the
present disclosure, in some embodiments, are more potent than
current immunogenic compositions due, in part, to the inclusion of
an immunodominant helper CD4 T cell epitope, referred to as a PADRE
(pan HLA DR-binding epitope: AKINAAWTLKAAA (SEQ ID NO: 1)). See,
e.g., Alexander J. et al. J of Immuno. 164(3): 1625-33,
incorporated herein by reference. This epitope is a potent
immunogen. T cell epitopes are presented on the surface of
antigen-presenting cells by MHC molecules, T cell epitopes
presented by MHC class I molecules are typically peptides between 8
and 11 amino acids in length and exhibiting MHC-specific sequence
motifs. These antigenic peptides are derived from non-structural
and structural proteins through proteolysis in the cytosolic
compartment. Peptide-MHC-I complexes are then transported to the
cell surface of antigen presenting cells and are recognized by CD8+
cytotoxic T lymphocytes (CTL). This interaction induces the
differentiation of CTLs. Activated CTL lyse the infected cell,
secrete cytokines, and proliferate. This mechanism ensures that
cells infected by viruses or intracellular bacteria or cancer cells
can be detected, since pathogen or cancer-specific MHC peptide
complexes are displayed on the cell surface. CTL can recognize such
abnormal cells and eliminate them. The genes of MHC I and II
molecules are polymorphic. Each MHC allele has a distinct peptide
binding motif which favors certain amino acid anchor residues at
defined sequence positions.
[0044] In some embodiments, a PADRE is independently encoded by a
mRNA molecule that is separate from the mRNA molecule encoding an
antigen of interest (e.g., a viral, bacterial or parasitic
antigen). In other embodiments, an antigen of interest and a PADRE
are encoded by the same mRNA molecule.
[0045] Thus, in some embodiments, the present disclosure provides
immunogenic compositions that include mRNA having an open reading
frame encoding at least one antigen, a pan HLA DR-binding epitope
(PADRE), and a 5' terminal cap that is modified to increase mRNA
translation efficiency. In other embodiments, immunogenic
compositions may include (a) a messenger ribonucleic acid (mRNA)
having an open reading frame encoding at least one antigen, and a
5' terminal cap that is modified to increase mRNA translation
efficiency and (b) a mRNA having an open reading frame encoding a
pan HLA DR-binding epitope (PADRE), and a 5' terminal cap that is
modified to increase mRNA translation efficiency.
[0046] In some embodiments, the mRNA is encapsulated in a cationic
lipid nanoparticle that comprises, for example, a cationic lipid, a
PEG-modified lipid, a sterol and a non-cationic lipid.
[0047] The ratio of mRNA encoding antigen (e.g., viral, bacterial,
or parasitic antigen) to mRNA encoding PADRE in an immunogenic
composition may vary. In some embodiments, the ratio of mRNA
encoding antigen to mRNA encoding PADRE is 0.1:1 to 10:1. For
example, the ratio of mRNA encoding antigen to mRNA encoding PADRE
may be 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1,
0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
[0048] Likewise, the ratio of antigen (e.g., viral, bacterial, or
parasitic antigen) to PADRE encoding by mRNA in an immunogenic
composition may vary. In some embodiments, the ratio of antigen to
PADRE encoded by mRNA is 1:1 to 100:1. For example, the ratio of
antigen to PADRE encoded by mRNA may be 1:1, 1:2, 1:5, 1:10, 1:20,
1:30, 1:40, 1:50, or 1:100, in some embodiments, the ratio of
antigen to PADRE encoded by mRNA is 1:1 to 100:1. For example, the
ratio of antigen to PADRE encoded by mRNA may be 1:1. 1:2, 1:5,
1:10, 1:20, 1:30, 1:40, 1:50, or 1:100.
Messenger RNA
[0049] "Messenger RNA" (mRNA) refers to any polynucleotide that
encodes a (at least one) polypeptide (a naturally-occurring,
non-naturally-occurring, or modified polymer of amino acids) and
can be translated to produce the encoded polypeptide in vitro, in
vivo, in situ or ex vivo. The skilled artisan will appreciate that,
except where otherwise noted, polynucleotide sequences set forth in
the instant application will recite "T"s in a representative DNA
sequence but where the sequence represents RNA (e.g., mRNA), the
"T"s would be substituted for "U"s. Thus, any of the mRNA encoded
by a DNA identified by a particular sequence identification number
may also comprise the corresponding mRNA sequence encoded by the
DNA, where each "T" of the DNA sequence is substituted with
"Li"
[0050] Polynucleotides of the present disclosure may function as
mRNA but can be distinguished from wild-type mRNA in their
functional and/or structural design features, which serve to
overcome existing problems of effective polypeptide expression
using nucleic-acid based therapeutics.
[0051] mRNA, for example, may be transcribed in vitro from template
DNA, referred to as an "in vitro transcription template." In some
embodiments, an in vitro transcription template encodes a 5'
untranslated (UTR) region, contains an open reading frame, and
encodes a 3' UTR and a polyA tail. The particular nucleic acid
sequence composition and length of an in vitro transcription
template will depend on the mRNA encoded by the template.
[0052] The basic components of an mRNA molecule typically include
at least one coding region, a 5' untranslated region (UTR), a 3'
UTR, a 5' cap and a poly-A tail. Both the 5'UTR and the 3'UTR are
typically transcribed from genomic DNA and are elements of the
premature mRNA. Characteristic structural features of mature mRNA,
such as the 5'-cap and the 3'-polyA tail are usually added to the
transcribed (premature) mRNA during mRNA processing.
[0053] A "5' untranslated region" (5'UTR) refers to a region of an
mRNA that is directly upstream (i.e., 5') from the start codon (the
first codon of an mRNA transcript translated by a ribosome) that
does not encode a polypeptide. In some embodiments, mRNA encoding
an antigen (e.g., viral, bacterial, or parasitic antigen) and/or a
PADRE includes a 5'UTR.
[0054] A "3' untranslated region" (3'UTR) refers to a region of an
mRNA that is directly downstream (i.e., 3') from the stop codon
(i.e., the codon of an mRNA transcript that signals a termination
of translation) that does not encode a polypeptide. In some
embodiments, mRNA encoding an antigen and/or PADRE includes a
3'UTR.
[0055] An "open reading frame" is a continuous stretch of DNA
beginning with a start codon e.g., methionine (ATG)), and ending
with a stop codon (e.g., TAA, TAG or TGA) and encodes a
polypeptide. mRNA encoding an antigen (e.g., viral, bacterial, or
parasitic antigen) and/or a PADRE includes an open reading
frame.
[0056] A "5' cap" is a specially altered nucleotide on the 5' end
of some mRNA transcripts. In some embodiments, mRNA encoding an
antigen (e.g., viral, bacterial, or parasitic antigen) and/or a
PADRE includes a 5' cap (e.g., a natural 5' cap). In some
embodiments, a 5' cap may be a 5' cap analog, such as a 5'
diguanosine cap, tetraphosphate cap analogs having a methylene -bis
(phosphonate) moiety, cap analogs having a sulfur substitution for
a non-bridging oxygen, N7-benzylated dinucleoside tetraphosphate
analogs, or anti-reverse cap analogs. In some embodiments, the 5'
cap is a 7mG(5')ppp(5')N1mpNp cap. In some embodiments, the 5' cap
is a 7mG(5')ppp(5')N1mpN2mp cap. In some embodiments, the 5' cap
analog is a 5'diguanosine cap.
[0057] A "polyA tail" or "polyadenylation signal" is a region of
mRNA that is downstream, e.g., directly downstream (i.e., 3'), from
the 3' UTR that contains multiple, consecutive adenosine
monophosphates. In some embodiments, mRNA encoding an antigen (e.
g., viral, bacterial, or parasitic antigen) and/or a PADRE includes
polyA tail. The polyA sequence or polyadenylation signal generally
should enhance the expression level of the encoded protein. A polyA
tail may contain 10 to 300 adenosine monophosphates. For example, a
polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In
some embodiments, a polyA tail comprises 50 to 250 adenosine
monophosphates. In some embodiments, a. polyA tail comprises up to
400 adenosine monophosphates. In a relevant biological setting
(e.g., in cells, in vivo) the polyA tail functions to protect mRNA
from enzymatic degradation, e.g., in the cytoplasm, and aids in
transcription termination, export of the mRNA from the nucleus and
translation. In some embodiments, the length of a 3'-polyA tail may
be an essential element with respect to the stability of the
individual mRNA.
[0058] In some embodiments, mRNA includes, as a stabilizing
element, a histone stem-loop. The histone stem-loop is generally
derived from histone genes, and includes an intramolecular base
pairing of two neighbored partially or entirely reverse
complementary sequences separated by a spacer, including (e.g.,
consisting of) a short sequence, which forms the loop of the
structure. The unpaired loop region is typically unable to base
pair with either of the stem loop elements. It occurs more often in
RNA, as is a key component of many RNA secondary structures, but
may be present in single-stranded. DNA as well. Stability of the
stem-loop structure generally depends on the length, number of
mismatches or bulges, and base composition of the paired region. In
some embodiments, wobble base pairing (non-Watson-Crick base
pairing) may result. In some embodiments, the at least one histone
stem-loop sequence comprises a length of 15 to 45 nucleotides, in
some embodiments, mRNA comprises a 32kDa stem-loop binding protein
(SLBP), which is associated with the histone stein-loop at the
3'-end of the histone messages in both the nucleus and the
cytoplasm. Its expression level is regulated by the cell cycle; it
peaks during the S-phase, when histone mRNA levels are also
elevated. This SLBP protein has been shown to be essential for
efficient 3'-end processing of histone pre-mRNA by the U7 mRNP.
SLBP continues to be associated with the stem-loop after
processing, and then stimulates the translation of mature histone
mRNAs into histone proteins in the cytoplasm. The RNA binding
domain of SLBP is conserved through metazoa and protozoa; its
binding to the histone stem-loop depends on the structure of the
loop. The minimum binding site includes at least three nucleotides
5' and two nucleotides 3' relative to the stem-loop.
[0059] In some embodiments, mRNA encoding an antigen (e.g., viral,
bacterial, or parasitic antigen) and/or a PADRE includes both
polyadenylation signal and at least one histone stem-loop, even
though both represent alternative mechanisms in nature, acts
synergistically to increase the protein expression beyond the level
observed with either of the individual elements. It has been found
that the synergistic effect of the combination of poly(A) and at
least one histone stern-loop does not depend on the order of the
elements or the length of the poly(A) sequence.
[0060] In sonic embodiments, mRNA encoding an antigen (e.g., viral,
bacterial, or parasitic antigen) and/or a PADRE does not comprise a
histone downstream element (HDE). "Histone downstream element"
(HDE) includes a purine-rich polynucleotide stretch of
approximately 15 to 20 nucleotides 3' of naturally occurring
stem-loops, representing the binding site for the U7 mRNA, which is
involved in processing of histone pre-mRNA into mature histone
mRNA. Ideally, the inventive nucleic acid does not include an
intron.
[0061] In some embodiments, mRNA encoding an antigen (e.g., viral,
bacterial, or parasitic antigen) and/or a PADRE may or may not
contain a enhancer and/or promoter sequence, which may be modified
or unmodified or which may be activated or inactivated.
[0062] In some embodiments, mRNA encoding an antigen (e.g., viral,
bacterial, or parasitic antigen) and/or a PADRE may have one or
more AU-rich sequences removed. These sequences, sometimes referred
to as AURES are destabilizing sequences found in the 3'UTR. The
AURES may be removed from the mRNA. Alternatively, the AURES may
remain in the mRNA.
[0063] mRNA encoding an antigen (e.g., viral, bacterial, or
parasitic antigen) and/or a PADRE includes 200 to 3,000
nucleotides. For example, a mRNA may include 200 to 500, 200 to
1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to
2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500
to 3000, or 2000 to 3000 nucleotides.
[0064] Nucleic Acids
[0065] The term "nucleic acid" includes any compound and/or
substance that comprises a polymer of nucleotides (nucleotide
monomer). These polymers are referred to as polynucleotides. Thus,
the terms "nucleic acid" and "polynucleotide" are used
interchangeably.
[0066] Nucleic acids may be or may include, for example,
ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose
nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic
acids (PNAs), locked nucleic acids (LNAs, including LNA having a
.beta.-D-ribo configuration, .alpha.-LNA having an .alpha.-L-ribo
configuration (a diastereomer of LNA), 2'-amino-LNA having a
2'-amino functionalization, and 2'-amino-.alpha.-LNA having a
2'-amino functionalization), ethylene nucleic acids (ENA),
cyclohexenyl nucleic acids (CeNA) or chimeras or combinations
thereof.
[0067] In some embodiments, polynucleotides of the present
disclosure function as messenger RNA (mRNA), as discussed
above.
[0068] Polynucleotides of the present disclosure, in some
embodiments, are codon optimized. Codon optimization methods are
known in the art and may be used as provided herein. Codon
optimization, in some embodiments, may be used to match codon
frequencies in target and host organisms to ensure proper folding;
bias GC content to increase mRNA stability or reduce secondary
structures; minimize tandem repeat codons or base runs that may
impair gene construction or expression; customize transcriptional
and translational control regions; insert or remove protein
trafficking sequences; remove/add post translation modification
sites in encoded protein (e.g. glycosylation sites); add, remove or
shuffle protein domains; insert or delete restriction sites; modify
ribosome binding sites and mRNA degradation sites; adjust
translational rates to allow the various domains of the protein to
fold properly; or to reduce or eliminate problem secondary
structures within the polynucleotide. Codon optimization tools,
algorithms and services are known in the art non-limiting examples
include services from GeneArt (Life Technologies), DNA2.0 (Menlo
Park Calif.) and/or proprietary methods. In some embodiments, the
open reading frame (ORF) sequence is optimized using optimization
algorithms.
[0069] In some embodiments, a codon optimized sequence shares less
than 95% sequence identity, less than 90% sequence identity, less
than 85% sequence identity, less than 80% sequence identity, or
less than 75% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding a polypeptide or protein of interest (e.g.,
antigen)).
[0070] In some embodiments, a codon-optimized sequence shares
between 65% and 85% (e.g., between about 67% and about 85%, or
between about 67% and about 80%) sequence identity to a
naturally-occurring sequence or a wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigen)). In some
embodiments, a codon-optimized sequence shares between 65% and 75%,
or about 80% sequence identity to a naturally-occurring sequence or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding a polypeptide or protein of interest (e.g., an
antigen)).
[0071] In sonic embodiments a codon-optimized mRNA may, for
example, be one in which the levels of G/C are enhanced. The
G/C-content of nucleic acid molecules may influence the stability
of the RNA. RNA having an increased amount of guanine (G) and/or
cytosine (C) residues may be functionally more stable than nucleic
acids containing a large amount of adenine (A) and thymine (I) or
uracil (U) nucleotides. International Publication No. WO
2002/098443 discloses a pharmaceutical composition containing an
mRNA stabilized by sequence modifications in the translated region.
Due to the degeneracy of the genetic code, the modifications work
by substituting existing codons for those that promote greater RNA
stability without changing the resulting amino acid. The approach
is limited to coding regions of the RNA.
[0072] "Identity" refers to a relationship between the sequences of
two or more polypeptides or polynucleotides, as determined by
comparing the sequences. Identity also means the degree of sequence
relatedness between two sequences as determined by the number of
matches between strings of two or more amino acid residues or
nucleic acid residues. Identity measures the percent of identical
matches between the smaller of two or more sequences with gap
alignments (if any) addressed by a particular mathematical model or
computer program (e.g.,"alpritiuns"). Identity of related peptides
can be readily calculated by known methods. "Percent (%) identity"
as it applies to polypeptide or polynucleotide sequences is defined
as the percentage of residues (amino acid residues or nucleic acid
residues) in the candidate amino acid or nucleic acid sequence that
are identical with the residues in the amino acid sequence or
nucleic acid sequence of a second sequence after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent identity. Methods and computer programs for the
alignment are well known in the art. Identity depends on a
calculation of percent identity but may differ in value due to gaps
and penalties introduced in the calculation. Generally, variants of
a particular polynucleotide or polypeptide have at least 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to
that particular reference polynucleotide or polypeptide as
determined by sequence alignment programs and parameters described
herein and known to those skilled in the art. Such tools for
alignment include those of the BLAST suite (Stephen F. Altschul, et
at. (1997)." Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs," Nucleic Acids Res.
25:3389-3402). Another popular local alignment technique is based
on the Smith-Waternmn algorithm (Smith, T. F. Waterman, M. S.
(1981) "Identification of common molecular subsequences." J. Mol.
Biol. 147:195-197). A general global alignment technique based on
dynamic programming is the NeedlemanWunsch algorithm (Needleman, S.
B. Wunsch, C. D. (1970) "A general method applicable to the search
for similarities in the amino acid sequences of two proteins." J.
Mol. Biol. 48:443-453). More recently, a Fast Optimal Global
Sequence Alignment Algorithm (FOGSAA) was developed that
purportedly produces global alignment of nucleotide and protein
sequences faster than other optimal global alignment methods,
including the NeedlemanWunsch algorithm.
Stabilizing Elements
[0073] Naturally-occurring eukaryotic mRNA molecules can contain
stabilizing elements, including, but not limited to untranslated
regions (UTR) at their 5'-end (5' UTR) and/or at their 3'-end (3'
UTR), in addition to other structural features, such as a 5'-cap
structure or a 3'-poly(A) tail. Both the 5' UTR and the 3' UTR are
typically transcribed from the genomic DNA and are elements of the
premature mRNA. Characteristic structural features of mature mRNA,
such as the 5'-cap and the 3'-poly(A) tail are usually added to the
transcribed (premature) mRNA during mRNA processing.
[0074] In some embodiments, a vaccine includes at least one RNA
polynucleotide having an open reading frame encoding at least one
antigenic polypeptide having at least one modification, at least
one 5' terminal cap, and is formulated within a lipid nanoparticle.
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'-O-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 (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA may be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase, Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes may be derived from a
recombinant source.
[0075] The 3'-poly(A) tail is typically a stretch of adenine
nucleotides added to the 3'-end of the transcribed mRNA. It can, in
some instances, comprise up to about 400 adenine nucleotides. In
some embodiments, the length of the 3'-poly(A) tail may be an
essential element with respect to the stability of the individual
mRNA.
[0076] In some embodiments, the RNA vaccines may include one or
more stabilizing elements. Stabilizing elements may include for
instance a histone stem-loop. A stem-loop binding protein (SLBP), a
32 kDa protein has been identified. It is associated with the
histone stem-loop at the 3'-end of the histone messages in both the
nucleus and the cytoplasm. Its expression level is regulated by the
cell cycle; it peaks during the S-phase, when histone mRNA levels
are also elevated. The protein has been shown to be essential for
efficient 3'-end processing of histone pre-mRNA by the U7 mRNP.
SLBP continues to be associated with the stem-loop after
processing, and then stimulates the translation of mature histone
mRNAs into histone proteins in the cytoplasm. The RNA binding
domain of SLBP is conserved through metazoa and protozoa; its
binding to the histone stem-loop depends on the structure of the
loop. The minimum binding site includes at least three nucleotides
5' and two nucleotides 3' relative to the stem-loop.
[0077] In some embodiments, the RNA vaccines include a coding
region, at least one histone stem-loop, and optionally, a poly(A)
sequence or polyadenylation signal. The poly(A) sequence or
polyadenylation signal generally should enhance the expression
level of the encoded protein. The encoded protein, in some
embodiments, is not a histone protein, a reporter protein (e.g.
Luciferase, GFP, EGFP, .beta.-Galactosidase, EGFP), or a marker or
selection protein (e.g. alpha-Globin, Galactokinase and
Xanthine:guanine phosphoribosyl transferase (GPT)).
[0078] In some embodiments, the combination of a poly(A) sequence
or polyadenylation signal and at least one histone stem-loop, even
though both represent alternative mechanisms in nature, acts
synergistically to increase the protein expression beyond the level
observed with either of the individual elements. The synergistic
effect of the combination of poly(A) and at least one histone
stem-loop does not depend on the order of the elements or the
length of the poly(A) sequence.
[0079] In some embodiments, the RNA vaccines do not comprise a
histone downstream element (HDE). "Histone downstream element"
(HDE) includes a purine-rich polynucleotide stretch of
approximately 15 to 20 nucleotides 3' of naturally occurring
stem-loops, representing the binding site for the U7 mRNA, which is
involved in processing of histone pre-mRNA into mature histone
mRNA. In some embodiments, the nucleic acid does not include an
intron.
[0080] In some embodiments, the RNA vaccines may or may not contain
an enhancer and/or promoter sequence, which may be modified or
unmodified or which may be activated or inactivated. In some
embodiments, the histone stem-loop is generally derived from
histone genes, and includes an intramolecular base pairing of two
neighbored partially or entirely reverse complementary sequences
separated by a spacer, consisting of a short sequence, which forms
the loop of the structure. The unpaired loop region is typically
unable to base pair with either of the stem loop elements. It
occurs more often in RNA, as is a key component of many RNA
secondary structures, but may be present in single-stranded DNA as
well. Stability of the stem-loop structure generally depends on the
length, number of mismatches or bulges, and base composition of the
paired region. In some embodiments, wobble base pairing
(non-Watson-Crick base pairing) may result. In some embodiments,
the at least one histone stem-loop sequence comprises a length of
15 to 45 nucleotides.
[0081] In some embodiments, the RNA vaccines may have one or more
AU-rich sequences removed. These sequences, sometimes referred to
as AURES are destabilizing sequences found in the 3'UTR. The AURES
may be removed from the RNA vaccines. Alternatively the AURES may
remain in the RNA vaccine.
Antigens
[0082] An "antigen" is a product of mRNA transcription that is
capable of inducing an immune response in a subject (e.g., human
subject). Thus, in some embodiments, an antigen is a peptide or
polypeptide that induces an immune response in a subject. An
antigen (antigenic polypeptide) encoded by a mRNA of the present
disclosure may be naturally occurring or synthetic. An antigen may
be a single molecule or may be a multi-molecular complex such as a
dimer, trimer or tetramer. In some embodiments, an antigen
comprises a single chain polypeptide or multichain polypeptides and
may be associated with or linked to each other, e.g., through a
disulfide linkage. The term "polypeptide" applies to amino acid
polymers, including naturally-occurring amino acid, as well as
amino acid polymers in which at least one amino acid residue is an
artificial chemical analogue of a corresponding naturally-occurring
amino acid.
[0083] Antigens, in some embodiments, may be variants of a
naturally-occurring (native) antigen. An "antigen variant" is an
antigen that differs in its amino acid sequence relative to a
native sequence or a reference sequence. Amino acid sequence
variants may possess substitutions, deletions, insertions, or a
combination of any two or three of the foregoing, at certain
positions within the amino acid sequence, as compared to a native
sequence or a reference sequence. In some embodiments, a variant
possess at least 50% identity to a native sequence or a reference
sequence. In some embodiments, variants share at least 60%, at
least 70%, at least 80%, or at least 90% identity with a native
sequence or a reference sequence.
[0084] Thus, mRNA encoding antigens containing substitutions,
insertions, deletions, and/or covalent modifications with respect
to reference antigens (e.g., native antigens) are included within
the scope of this disclosure.
[0085] In some embodiments, sequence tags or amino acids, such as
lysine(s), can be added to peptide sequences (e.g., at the
N-terminal or C-terminal ends). Sequence tags can be used for
peptide detection, purification or localization. Lysines can be
used to increase peptide solubility or to allow for biotinylation.
Alternatively, amino acid residues located at the carboxy and amino
terminal regions of the amino acid sequence of a peptide or protein
may optionally be deleted providing for truncated sequences.
Certain amino acids (e.g., C-terminal residues or N-terminal
residues) alternatively may be deleted depending on the use of the
sequence, as for example, expression of the sequence as part of a
larger sequence that is soluble, or linked to a solid support.
[0086] "Substitutional variants" when referring to antigens are
those that have at least one amino acid residue in a native or
starting sequence removed and a different amino acid inserted in
its place at the same position. Substitutions may be single, where
only one amino acid in the molecule has been substituted, or they
may be multiple, where two or more (e.g., 3, 4 or 5) amino acids
have been substituted in the same molecule.
[0087] A "conservative amino acid substitution" refers to the
substitution of an amino acid that is normally present in the
sequence with a different amino acid of similar size, charge, or
polarity. Examples of conservative substitutions include the
substitution of a non-polar (hydrophobic) residue such as
isoleucine, valine and leucine for another non-polar residue.
Likewise, examples of conservative substitutions include the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine, and
between glycine and serine. Additionally, the substitution of a
basic residue such as lysine, arginine or histidine for another, or
the substitution of one acidic residue such as aspartic acid or
glutamic acid for another acidic residue are additional examples of
conservative substitutions. Examples of non-conservative
substitutions include the substitution of a non-polar (hydrophobic)
amino acid residue such as isoleucine, valine, leucine, alanine,
methionine for a polar (hydrophilic) residue such as cysteine,
glutamine, glutamic acid or lysine and/or a polar residue for a
non-polar residue.
[0088] "Features" when referring to polypeptides (e.g., antigens)
and polynucleotides (e.g., mRNA) are defined as distinct amino acid
sequence-based or nucleotide-based components of the molecule,
respectively. Features of polypeptides encoded by polynucleotides
include surface manifestations, local conformational shape, folds,
loops, half-loops, domains, half-domains, sites, termini and any
combination(s) thereof.
[0089] When referring to polypeptides, the term "domain" refers to
a motif of a polypeptide having at least one identifiable
structural or functional characteristics or properties (e.g.,
binding capacity, serving as a site for protein-protein
interactions).
[0090] When referring to polypeptides, the term "site" as it
pertains to amino acid based embodiments is used synonymously with
"amino acid residue" and "amino acid side chain." When referring to
polynucleotides, the term "site" as it pertains to nucleotide based
embodiments is used synonymously with "nucleotide." A site
represents a position within a polypeptide or polynucleotide that
may be modified, manipulated, altered, derivatized or varied within
the polypeptide-based or polynucleotide-based molecules.
[0091] The terms "termini" or "terminus" when referring to
polypeptides or polynucleotides refers to an extremity of a
polypeptide or polynucleotide, respectively. Such extremity is not
limited only to the first or final site of the polypeptide or
polynucleotide but may include additional amino acids or
nucleotides in the terminal regions. Polypeptide-based molecules
may be characterized as having both an N-terminus (terminated by an
amino acid with a free amino group (NH2)) and a C-terminus
(terminated by an amino acid with a free carboxyl group (COOH)).
Proteins are in some cases made up of multiple polypeptide chains
brought together by disulfide bonds or by non-covalent forces
multimers, oligomers). These proteins have multiple N- and
C-termini. Alternatively, the termini of the polypeptides may be
modified such that they begin or end, as the case may be, with a
non-polypeptide based moiety such as an organic conjugate.
[0092] Protein fragments, functional protein domains, and
homologous proteins are also considered to be within the scope of
polypeptides (e.g., antigens) of interest. For example, provided
herein is any protein fragment (meaning a polypeptide sequence at
least one amino acid residue shorter than a reference polypeptide
sequence but otherwise identical) of a reference protein having a
length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or longer than
100 amino acids. In another example, any protein that includes a
stretch of 20, 30, 40, 50, or 100 (contiguous) amino acids that are
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the
sequences described herein can be utilized in accordance with the
disclosure. In sone embodiments, a polypeptide includes 2, 3, 4, 5,
6, 7, 8, 9, 10, or more mutations as shown in any of the sequences
provided herein or referenced herein. In another example, any
protein that includes a stretch of 20, 30, 40, 50, or 100 amino
acids that are greater than 80%, 90%, 95%, or 100% identical to any
of the sequences described herein, wherein the protein has a
stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than
80%, 75%, 70%, 65% to 60% identical to any of the sequences
described herein can be utilized in accordance with the
disclosure.
Viral Antigens
[0093] In some embodiments, an antigen is a viral antigen. A "viral
antigen" is an antigen encoded by a viral genome. In some
embodiments, an immunogenic composition of the present disclosure
comprises a mRNA encoding a viral antigen. In some embodiments, an
immunogenic composition comprises a cationic lipid nanoparticle
(LNP) encapsulating mRNA having an open reading frame encoding at
least one viral antigen, a pan FHA DR-binding epitope (PADRE), and
a 5' terminal cap modified to increase mRNA translation efficiency.
In some embodiments, the cationic lipid nanoparticle comprises a
cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid. Examples of viral antigens include, but are not limited to,
Betacoronavirus, Chikungunya virus, Dengue virus, Ebola virus,
Eastern Equine Encephalitis virus, Herpes Simplex virus, Human
Cytomegalovirus, Human Papillomavirus, Human Metapneumovirus,
Influenza virus, Japanese Encephalitis virus, Marburg virus,
Measles, Parainfluenza virus, Respiratory Syncytial virus, Sindbis
virus, Varicella Zoster virus, Venezuelan Equine Encephalitis
virus, West Nile virus, Yellow Fever virus, and Zika virus
antigens.
[0094] Betacoronavirus. In some embodiments, the BetaCoV is
MERS-CoV. In some embodiments, the BetaCoV is SARS-CoV. In some
embodiments, the BetaCoV is HCoV-OC43. In some embodiments, the
BetaCoV is HCoV-229E. In some embodiments, the BetaCoV is
HCoV-NL63. In some embodiments, the BetaCoV is HCoV-HKU1. In some
embodiments, at least one antigen encoded by an mRNA of an
immunogenic composition is a Betacoronavirus structural protein.
For example, at least one antigen may be spike protein (S),
envelope protein (E), nucleocapsid protein (N), membrane protein
(M) or an immunogenic fragment thereof. In some embodiments, at
least one antigen is a spike protein (S). In some embodiments, at
least one antigen is a S1 subunit or a S2 subunit of spike protein
(S) or an immunogenic fragment thereof. In some embodiments, at
least one antigen is at least one accessory protein (e.g., protein
3, protein 4a, protein 4b, protein 5), at least one replicase
protein (e.g., protein 1a, protein 1b), or a combination of at
least one accessory protein and at least one replicase protein.
[0095] Chikungunya Virus. In some embodiments, at least one antigen
encoded by an mRNA of an immunogenic composition is a CHIKV
structural protein selected from an envelope protein (E) (e.g, E1,
E2, E3), a 6K protein, or a capsid (C) protein.
[0096] Dengue virus. In some embodiments, at least one antigen
encoded by an mRNA of an immunogenic composition is a DENV capsid
protein, a DENV membrane protein, a DENV precursor-membrane
protein, a DENY precursor membrane (pry) and envelope (E)
polypeptide (DENY prME), or a DENV non-structural protein selected
from NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. In some
embodiments, at least one antigen is from a DENY serotype selected
from DENV-1, DENY-2, DENV-3, DENY-4, and DENY-5.
[0097] Ebola virus. In some embodiments, at least one antigen
encoded by an mRNA of an immunogenic composition is EBOV
glycoprotein (GP), surface EBOV GP, wild type EBOV pro-GP, mature
EBOV GP, secreted wild type EBOV pro-GP, secreted mature EBOV GP,
EBOV nucleoprotein (NP), RNA polymerase L, and EBOV matrix protein
selected from VP35, VP40, VP24, or VP30. in some embodiments, at
least one antigen encoded by an mRNA of an immunogenic composition
is EBOV glycoprotein (GP), surface EBOV GP, wild type EBOV GP,
mature EBOV GP, secreted wild type EBOV GP, secreted mature EBOV
GP, sGP, delta peptide (A-peptide), GP1, GP1,2.DELTA.,
nucleoprotein NP, viral polymerase L, the polymerase cofactor VP35,
the transcriptional activator VP30, VP24, or the matrix protein
VP40.
[0098] Herpes Simpler virus. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is HSV
(HSV-1 or HSV-2) glycoprotein B, HSV (HSV-1 or HSV-2) glycoprotein
C, HSV (HSV-1 or HSV-2) glycoprotein D, HSV (HSV-1 or HSV-2)
glycoprotein E, or HSV (HSV-1 or HSV-2) glycoprotein I.
[0099] Human Cytomegalovirus. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is a HCMV
gH, gL, gB, gO, gN, gM, UL83, UL123, UL128, UL130, or UL131A
protein.
[0100] Human Papillomavirus. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is E1, E2,
E4, E5, E6, E7, L1, and L2, e.g., obtained from HPV serotypes 6,
11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 or
82.
[0101] Human Meiapneumovirus, Parainfluenza virus and Respiratory
Syncytial virus. In some embodiments, at least one antigen encoded
by an mRNA of an immunogenic composition is major surface
glycoprotein G or an immunogenic fragment thereof. In some
embodiments, at least one antigen is Fusion (F) glycoprotein (e.g.,
Fusion glycoprotein F0, F1 or F2) or an immunogenic fragment
thereof. In some embodiments, at least one antigen is major surface
glycoprotein G or an immunogenic fragment thereof and F
glycoprotein or an immunogenic fragment thereof In some
embodiments, at least one antigen is nucleoprotein (N) or an
immunogenic fragment thereof, phosphoprotein (P) or an immunogenic
fragment thereof, large polymerase protein (L) or an immunogenic
fragment thereof, matrix protein (M) or an immunogenic fragment
thereof, small hydrophobic protein (SH) or an immunogenic fragment
thereof, nonstructural protein 1 (NS1) or an immunogenic fragment
thereof, or nonstructural protein 2 (NS2) and an immunogenic
fragment thereof.
[0102] In, luenza virus. In some embodiments, at least one antigen
encoded by an mRNA of an immunogenic composition is an antigenic
subdomains of HA, termed HA1, HA2, or a combination of HA1 and HA2
(or a combination of both, of any one of or a combination of any or
all of H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,
H15, H16, H17, and/or H18), and at least one antigenic polypeptide
selected from neuraminidase (NA), nucleoprotein (NP), matrix
protein 1 (M1), matrix protein 2 (M2), non-structural protein 1
(NS1) and non-structural protein 2 (NS2).
[0103] Japanese Encephalitis virus. In some embodiments, at least
one antigen encoded by an mRNA of an immunogenic composition is JEV
E protein, JEV Es, JEV prM, JEV capsid, JEV NS1, or JEV prM and E
polyprotein (prME).
[0104] Marburg virus. In some embodiments, at least one antigen
encoded by an mRNA of an immunogenic composition is a MARV
glycoprotein (GP).
[0105] Measles. In some embodiments, at least one antigen encoded
by an mRNA of an immunogenic composition is a hemagglutinin (HA)
protein or an immunogenic fragment thereof The HA protein may be
from MeV strain D3 or B8, for example. In some embodiments, at
least one antigen is a Fusion (F) protein or an immunogenic
fragment thereof. The F protein may be from MeV strain D3 or B8,
for example. In some embodiments, at least one antigen comprises a
HA protein and a F protein. The HA and F proteins may be from MeV
strain D3 or B8, for example.
[0106] Varicella Zoster virus. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is a VZV
glycoprotein selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, and
gM.
[0107] West Nile virus, Eastern Equine Encephalitis virus,
Venezuelan Equine Encephalitis virus, and Sindbis virus. In some
embodiments, at least one antigen encoded by an mRNA of an
immunogenic composition is at least one Arbovirus antigen and/or at
least one Alphavirus antigen.
[0108] Yellow Fever virus. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is a YFV
polyprotein, a YFV capsid protein, a YFV premembrane/membrane
protein, a YFV envelope protein, a YFV non-structural protein 1, a
YFV non-structural protein 2A, a YFV non-structural protein 2B, a
YFV non-structural protein 3, a YFV non-structural protein 4A, a
YFV non-structural protein 4B, or a YFV non-structural protein
5.
[0109] Zika virus antigens. In some embodiments, at least one
antigen encoded by an mRNA of an immunogenic composition is a ZIKV
polyprotein, a ZIKV capsid protein, a ZIKV premembrane/membrane
protein, a ZIKV envelope protein, a ZIKV non-structural protein 1,
a ZIKV non-structural protein 2A, a ZIKV non-structural protein
213, a ZIKV non-structural protein 3, a ZIKV non-structural protein
4A, a ZIKV non-structural protein 4B, or a ZIKV non-structural
protein 5.
[0110] In some embodiments, the ZIKV antigen comprises the
following amino acid sequence:
TABLE-US-00001 AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS
YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
VMAQDKPTVDIELVTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLD
KQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPE
NLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGF
GSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGT
PHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGR
LSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA
GTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFG
DSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGS
VGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGS
ISLMCLALGGVLIFLSTAVSA (WT ZIKV prME, SEQ ID NO: 2). In some
embodiments, SEQ ID NO: 2 is fused to a PADRE sequence (e.g., SEQ
ID NO: 1).
[0111] In some embodiments, the ZIKV antigen comprises the
following amino acid sequence:
TABLE-US-00002 MWLVSLAIVTACAGAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCY
IQIMDLGRMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK
GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGF
ALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSG
GTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISD
MASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCA
KFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKV
EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEW
FHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHT
ALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKI
PAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITE
STENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRG
AKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQIL
IGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA (WT ZIKV prME, JEVprM signal;
SEQ ID NO: 3). In some embodiments, SEQ ID NO: 3 is fused to a
PADRE sequence (e.g., SEQ ID NO: 1).
[0112] In some embodiments, the ZIKV antigen comprises the
following amino acid sequence:
TABLE-US-00003 AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS
YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH
STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST
SQKVIYINMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT
VMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPREGEAYL
DKQSDTQYVCKRTLVDRGRGNGCGRFGKGSLVTCAKFACSKKMTGKSIQP
ENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGG
FGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTG
TPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKG
RLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFFKIPAETLHGTVTVEVQY
AGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPF
GDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFG
SVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNG
SISLMCLALGGVLIFLSTAVSA (Modified ZIKV prME; SEQ ID NO: 4). In some
embodiments, SEQ ID NO: 4 is fused to a PADRE sequence (e.g., SEQ
ID NO: 1).
[0113] In some embodiments, the ZIKY antigen comprises the
following amino acid sequence:
TABLE-US-00004 MWLVSLAIVTACAGAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCY
IQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK
GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLITRVENWIFRNPG
FALAAAAIAWLLGSSTSQKVIYLVMILIAAPAYSIRCIGVSNRDFVEGMS
GGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASIS
DMASDSRCPREGEAYLDKQSDTQYVCKRTLVDRGRGNGCGRFGKGSLVTC
AKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAK
VEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKE
WFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVH
TALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTK
IPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVIT
ESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVR
GAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQI
LIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA (Modified ZIKV prME, JEVprM
signal; SEQ ID NO: 5). In some embodiments, SEQ ID NO: 5 is fused
to a PADRE sequence (e.g.. SEQ ID NO: 1).
[0114] In some embodiments, the ZIKV antigen comprises the
following amino acid sequence:
TABLE-US-00005 MDWTWILFLVAAATRVHSAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMN
KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH
HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRN
PGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEG
MSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEAS
ISDMASDSRCPREGEAYLDKQSDTQYVCKRTLVDRGRGNGCGRFGKGSLV
TCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENR
AKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVH
KEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGA
VHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTF
TKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPV
ITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEAT
VRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFS
QILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA (Modified ZIKV prME, IgE;
SEQ ID NO: 6). In some embodiments, SEQ ID NO: 6 is fused to a
PADRE sequence (e.g., SEQ ID NO: 1).
[0115] Bacterial Antigens
[0116] In some embodiments, an antigen is a bacterial antigen, A
"bacterial antigen" is an antigen encoded by a bacterial genome. In
some embodiments, an immunogenic composition of the present
disclosure comprises a mRNA encoding a bacterial antigen. In some
embodiments, an immunogenic composition comprises a cationic lipid
nanoparticle (LNP) encapsulating mRNA having an open reading frame
encoding at least one bacterial antigen, a pan HLA DR-binding
epitope (PADRE), and a 5' terminal cap modified to increase mRNA
translation efficiency. In some embodiments, the cationic lipid
nanoparticle comprises a cationic lipid, a PEG-modified lipid, a
sterol and a non-cationic lipid. Examples of bacterial antigens
include, but are not limited to, Chlamydia trachomatis antigen, a
Lyme Borrelia and a Streptococcal antigen.
[0117] In some embodiments, at least one antigen encoded by an mRNA
of an immunogenic composition is a major outer membrane protein
(MOMP or OmpA), e.g., from Chlamydia trachomatis serovar (serotype)
H, F, E, D, I, G, J or K.
[0118] In some embodiments, at least one antigen encoded by an mRNA
of an immunogenic composition is a Borrelia OspA protein.
[0119] Parasitic Antigens.
[0120] In sonic embodiments, an antigen is a parasitic antigen. A
"parasitic antigen" is an antigen encoded by a parasitic genome. In
some embodiments, an immunogenic composition of the present
disclosure comprises a mRNA encoding a parasitic antigen. In some
embodiments, an immunogenic composition comprises a cationic lipid
nanoparticle (LNP) encapsulating mRNA having an open reading frame
encoding at least one parasitic antigen, a pan HLA DR-binding
epitope (PADRE), and a 5' terminal cap modified to increase mRNA
translation efficiency. In some embodiments, the cationic lipid
nanoparticle comprises a cationic lipid, a PEG-modified lipid, a
sterol and a non-cationic lipid. Examples of parasitic antigens
include, but are not limited to, Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale, and Plasmodium malariae, and Plasmodium
knowlesi antigens.
[0121] In some embodiments, at least one antigen encoded by an mRNA
of an immunogenic composition is a circumsporozoite (CS) protein or
an immunogenic fragment thereof (e.g., capable of raising an immune
response against Plasmodium). In some embodiments, at least one
antigen is RTS hybrid protein. In some embodiments, at least one
antigen is merozoite surface protein-1 (MSP1), apical membrane
antigen 1 (AMA1), or thrombospondin related adhesive protein (TRAP)
or an immunogenic fragment thereof.
[0122] Chemically Unmodified Nucleotides
[0123] In some embodiments, at least one RNA (e.g., mRNA) of the
vaccines of the present disclosure is not chemically modified and
comprises the standard ribonucleotides consisting of adenosine,
guanosine, cytosine and uridine. In some embodiments, nucleotides
and nucleosides of the present disclosure comprise standard
nucleoside residues such as those present in transcribed RNA (e.g.
A, G, C, or U). In some embodiments, nucleotides and nucleosides of
the present disclosure comprise standard deoxyribonucleosides such
as those present in DNA (e.g. dA, dG, dC, or dT).
Chemical Modifications
[0124] the RNA vaccines of the present disclosure comprise, in some
embodiments, at least one nucleic acid (e.g., RNA) having an open
reading frame encoding at least one antigen, wherein the nucleic
acid comprises nucleotides and/or nucleosides that can be standard
(unmodified) or modified as is known in the art. In some
embodiments, nucleotides and nucleosides of the present disclosure
comprise modified nucleotides or nucleosides. Such modified
nucleotides and nucleosides can be naturally-occurring modified
nucleotides and nucleosides or non-naturally occurring modified
nucleotides and nucleosides. Such modifications can include those
at the sugar, backbone, or nucleohase portion of the nucleotide
and/or nucleoside as are recognized in the art.
[0125] In some embodiments, a naturally-occurring modified
nucleotide or nucleotide of the disclosure is one as is generally
known or recognized in the art. Non-limiting examples of such
naturally occurring modified nucleotides and nucleotides can be
found, inter alia, in the widely recognized MODOMICS database.
[0126] In some embodiments, a non-naturally occurring modified
nucleotide Of nucleoside of the disclosure is one as is generally
known or recognized in the art. Non-limiting examples of such
non-naturally occurring modified nucleotides and nucleosides can he
found, inter alia, in published US application Nos.
PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US
2014/058891; PCT/US 2014/070413; PCT/US 2015/36773; PCT/US
2015/36759, PCT/US 2015/36771; or PCT/IB2017/051367 all of which
are incorporated by reference herein.
[0127] Hence, nucleic acids of the disclosure (e.g., DNA nucleic
acids and RNA nucleic acids, such as mRNA nucleic acids) can
comprise standard nucleotides and nucleosides, naturally-occurring
nucleotides and nucleosides, non-naturally-occurring nucleotides
and nucleosides, or any combination thereof.
[0128] Nucleic acids of the disclosure (e.g., DNA nucleic acids and
RNA nucleic acids, such as mRNA nucleic acids), in some
embodiments, comprise various (more than one) different types of
standard and/or modified nucleotides and nucleosides. In some
embodiments, a particular region of a nucleic acid contains one,
two or more (optionally different) types of standard and/or
modified nucleotides and nucleosides.
[0129] In some embodiments, a modified RNA nucleic acid (e.g., a
modified mRNA nucleic acid), introduced to a cell or organism,
exhibits reduced degradation in the cell or organism, respectively,
relative to an unmodified nucleic acid comprising standard
nucleotides and nucleosides.
[0130] In some embodiments, a modified RNA nucleic acid (e.g., a
modified mRNA nucleic acid), introduced into a cell or organism,
may exhibit reduced immunogenicity in the cell or organism,
respectively (e.g., a reduced innate response) relative to an
unmodified nucleic acid comprising standard nucleotides and
nucleosides.
[0131] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic
acids), in some embodiments, comprise non-natural modified
nucleotides that are introduced during synthesis or post-synthesis
of the nucleic acids to achieve desired functions or properties.
The modifications may be present on internucleotide linkages,
purine or pyrimidine bases, or sugars. The modification may be
introduced with chemical synthesis or with a polymerase enzyme at
the terminal of a chain or anywhere else in the chain. Any of the
regions of a nucleic acid may be chemically modified.
[0132] The present disclosure provides for modified nucleosides and
nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as
mRNA nucleic acids). A "nucleoside" refers to a compound containing
a sugar molecule (e.g., a pentose or ribose) or a derivative
thereof in combination with an organic base (e.g., a purine or
pyrimidine) or a derivative thereof (also referred to herein as
"nucleobase"). A "nucleotide" refers to a nucleoside, including a
phosphate group. Modified nucleotides may by synthesized by any
useful method, such as, for example, chemically, enzymatically, or
recombinantly, to include one or more modified or non-natural
nucleosides. Nucleic acids can comprise a region or regions of
linked nucleosides. Such regions may have variable backbone
linkages. The linkages can be standard phosphodiester linkages, in
which case the nucleic acids would comprise regions of
nucleotides.
[0133] Modified nucleotide base pairing encompasses not only the
standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine
base pairs, but also base pairs formed between nucleotides and/or
modified nucleotides comprising non-standard or modified bases,
wherein the arrangement of hydrogen bond donors and hydrogen bond
acceptors permits hydrogen bonding between a non-standard base and
a standard base or between two complementary non-standard base
structures, such as, for example, in those nucleic acids having at
least one chemical modification. One example of such non-standard
base pairing is the base pairing between the modified nucleotide
inosine and adenine, cytosine or uracil. Any combination of
base/sugar or linker may be incorporated into nucleic acids of the
present disclosure.
[0134] In some embodiments, modified nucleobases in nucleic acids
(e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise
1-methyl-pseudouridine (m1.psi.), 1-ethyl-pseudouridine (e1.psi.),
5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or
pseudouridine (.psi.). In some embodiments, modified nucleobases in
nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids)
comprise 5-methoxy7nethyl uridine, 5-methylthio uridine,
1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy
cytidine. In some embodiments, the polyribonucleotide includes a
combination of at least two e.g., 2, 3, 4 or more) of any of the
aforementioned modified nucleobases including but not limited to
chemical modifications.
[0135] In some embodiments, a RNA nucleic acid of the disclosure
comprises 1-methyl-pseudouridine (m1.psi.) substitutions at one or
more or all uridine positions of the nucleic acid.
[0136] In some embodiments, a RNA nucleic acid of the disclosure
comprises 1-methyl-pseudouridine (m1.psi.) substitutions at one or
more or all uridine positions of the nucleic acid and 5-methyl
cytidine substitutions at one or more or all cytidine positions of
the nucleic acid.
[0137] In some embodiments, a RNA nucleic acid of the disclosure
comprises pseudouridine (.psi.) substitutions at one or more or all
uridine positions of the nucleic acid.
[0138] In some embodiments, a RNA nucleic acid of the disclosure
comprises pseudouridine (.psi.) substitutions at one or more or all
uridine positions of the nucleic acid and 5-methyl cytidine
substitutions at one or more or all cytidine positions of the
nucleic acid.
[0139] In some embodiments, a RNA nucleic acid of the disclosure
comprises uridine at one or more or all uridine positions of the
nucleic acid.
[0140] In some embodiments, nucleic acids (e.g., RNA nucleic acids,
such as mRNA. nucleic acids) are uniformly modified (e.g., fully
modified, modified throughout the entire sequence) for a particular
modification. For example, a nucleic acid can be uniformly modified
with 1-methyl-pseudouridine, meaning that all uridine residues in
the mRNA sequence are replaced with 1-methyl-pseudouridine.
Similarly, a nucleic acid can be uniformly modified for any type of
nucleoside residue present in the sequence by replacement with a
modified residue such as those set forth above.
[0141] The nucleic acids of the present disclosure may be partially
or fully modified along the entire length of the molecule. For
example, one or more or all or a given type of nucleotide (e.g.,
purine or pyrimidine, or any one or more or all of A, G, U, C) may
be uniformly modified in a nucleic acid of the disclosure, or in a
predetermined sequence region thereof (e.g., in the mRNA including
or excluding the polyA tail). In some embodiments, all nucleotides
X in a nucleic acid of the present disclosure (or in a sequence
region thereof) are modified nucleotides, wherein X may be any one
of nucleotides A, G, U, C, or any one of the combinations A+G, A+U,
A.+-.C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[0142] The nucleic acid may contain from about 1% to about 100%
modified nucleotides (either in relation to overall nucleotide
content, or in relation to one or more types of nucleotide, i.e.,
any one or more of A, G, U or C) or any intervening percentage
(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to
60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to
95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to
60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to
95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20%
to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20%
to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from
50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,
from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to
100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90%
to 95%, from 90% to 100%, and from 95% to 100%). It will be
understood that any remaining percentage is accounted for by the
presence of unmodified A, G, U, or C.
[0143] The nucleic acids may contain at a minimum 1% and at maximum
100% modified nucleotides, or any intervening percentage, such as
at least 5% modified nucleotides, at least 10% modified
nucleotides, at least 25% modified nucleotides, at least 50%
modified nucleotides, at least 80% modified nucleotides, or at
least 90% modified nucleotides. For example, the nucleic acids may
contain a modified pyrimidine such as a modified uracil or
cytosine. In some embodiments, at least 5%, at least 10%, at least
25%, at least 50%, at least 80%, at least 90% or 100% of the uracil
in the nucleic acid is replaced with a modified uracil (e.g., a
5-substituted uracil). The modified uracil can be replaced by a
compound having a single unique structure, or can be replaced by a
plurality of compounds having different structures (e.g., 2, 3, 4
or more unique structures). In some embodiments, at least 5%, at
least 10%, at least 25%, at least 50%, at least 80%, at least 90%
or 100% of the cytosine in the nucleic acid is replaced with a
modified cytosine (e.g., a 5-substituted cytosine). The modified
cytosine can be replaced by a compound having a single unique
structure, or can be replaced by a plurality of compounds having
different structures (e.g., 2, 3, 4 or more unique structures).
Untranslated Regions (UTRs)
[0144] The nucleic acids of the present disclosure may comprise one
or more regions or parts which act or function as an untranslated
region. Where nucleic acids are designed to encode at least one
antigen of interest, the nucleic may comprise one or more of these
untranslated regions (UTRs). Wild-type untranslated regions of a
nucleic acid are transcribed but not translated. In mRNA, the 5'
UTR starts at the transcription start site and continues to the
start codon but does not include the start codon; whereas, the 3'
UTR starts immediately following the stop codon and continues until
the transcriptional termination signal. There is growing body of
evidence about the regulatory roles played by the UTRs in terms of
stability of the nucleic acid molecule and translation. The
regulatory features of a UTR can be incorporated into the
polynucleotides of the present disclosure to, among other things,
enhance the stability of the molecule. The specific features can
also be incorporated to ensure controlled down-regulation of the
transcript in case they are misdirected to undesired organs sites.
A variety of 5'UTR and 3'UTR sequences are known and available in
the art.
[0145] A 5' UTR is region of an mRNA that is directly upstream (5')
from the start codon (the first codon of an mRNA transcript
translated by a ribosome). A 5' UTR does not encode a protein (is
non-coding). Natural 5'UTRs have features that play roles in
translation initiation. They harbor signatures like Kozak sequences
which are commonly known to be involved in the process by which the
ribosome initiates translation of many genes. Kozak sequences have
the consensus CCR(A/G)CCAUGG (SEQ ID NO: 12), where R is a purine
(adenine or guanine) three bases upstream of the start codon (AUG),
which is followed by another `G`. 5'UTR also have been known to
form secondary structures which are involved in elongation factor
binding.
[0146] In some embodiments of the disclosure, a 5' UTR is a
heterologous UTR, i.e., is a UTR found in nature associated with a
different ORF. In another embodiment, a 5' UTR is a synthetic UTR,
i.e., does not occur in nature. Synthetic UTRs include UTRs that
have been mutated to improve their properties, e.g., which increase
gene expression as well as those which are completely synthetic.
Exemplary 5' UTRs include Xenopus or human derived a-globin or
b-globin (U.S. Pat. Nod. 8,278,063; 9,012,219), human cytochrome
b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and
Tobacco etch virus (U.S. Pat. Nos. 8,278,063, 9,012,219). CMV
immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the
sequence GGGAUCCUACC (SEQ ID NO: 13) (WO2014/144196) may also be
used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a
TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)
(e.g., WO2015/101414, WO2015/101415, WO2015/062738, WO2015/024667,
WO2015/024667); 5' UTR element derived from ribosomal protein Large
32 (L32) gene (WO2015/101414, WO2015/101415, WO2015/062738), 5' UTR
element derived from the 5'UTR of an hydroxysteroid (17-.beta.)
dehydrogenase 4 gene (HSD17B4) (WO2015/024667), or a 5' UTR element
derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used. In
some embodiments, an internal ribosome entry site (IRES) is used
instead of a 5' UTR.
[0147] A 3' UTR is region of an mRNA that is directly downstream
(3') from the stop codon (the codon of an mRNA transcript that
signals a termination of translation). A 3' UTR does not encode a
protein (is non-coding). Natural or wild type 3' UTRs are known to
have stretches of adenosines and uridines embedded in them. These
AU rich signatures are particularly prevalent in genes with high
rates of turnover. Based on their sequence features and functional
properties, the AU rich elements (AREs) can be separated into three
classes (Chen et al, 1995): Class I ARES contain several dispersed
copies of an AUUUA motif within U-rich regions. C-Myc and MoD
contain class I AREs. Class II AREs possess two or more overlapping
UUAUUUA(U/A)(U/A) (SEQ ID NO: 14) nonamers. Molecules containing
this type of AREs include GM-CSF and TNF-a. Class III ARES are less
well defined. These U rich regions do not contain an AUUUA motif
c-Jun and Myogenin are two well-studied examples of this class.
Most proteins binding to the AREs are known to destabilize the
messenger, whereas members of the ELAV family, most notably HuR,
have been documented to increase the stability of mRNA. HuR binds
to AREs of all the three classes. Engineering the HuR specific
binding sites into the 3' UTR of nucleic acid molecules will lead
to HuR binding and thus, stabilization of the message in vivo.
[0148] Introduction, removal or modification of 3' UTR AU rich
elements (AREs) can be used to modulate the stability of nucleic
acids (e.g., RNA) of the disclosure. When engineering specific
nucleic acids, one or more copies of an ARE can be introduced to
make nucleic acids of the disclosure less stable and thereby
curtail translation and decrease production of the resultant
protein. Likewise, AREs can be identified and removed or mutated to
increase the intracellular stability and thus increase translation
and production of the resultant protein. Transfection experiments
can be conducted in relevant cell lines, using nucleic acids of the
disclosure and protein production can be assayed at various time
points post-transfection. For example, cells can be transfected
with different ARE-engineering molecules and by using an ELISA kit
to the relevant protein and assaying protein produced at 6 hour, 12
hour, 24 hour, 48 hour, and 7 days post-transfection.
[0149] 3' UTRs may be heterologous or synthetic. With respect to 3'
UTRs, globin UTRs, including Xenopus .beta.-globin UTRs and human
.beta.-globin UTRs are known in the art (U.S. Pat. Nos. 8,278,063,
9,012,219, US2011/0086907). A modified .beta.-globin construct with
enhanced stability in some cell types by cloning two sequential
human .beta.-globin 3'UTRs head to tail has been developed and is
well known in the art (US2012/0195936, WO2014/071963). In addition
a2-globin, a1-globin, UTRs and mutants thereof are also known in
the art (WO2015/101415, WO2015/024667). Other 3' UTRs described in
the mRNA constructs in the non-patent literature include CYBA
(Ferizi et al., 2015) and albumin (Thess et al., 2015). Other
exemplary 3' UTRs include that of bovine or human growth hormone
(wild type or modified) (WO2013/185069, US2014/0206753,
WO2014/152774), rabbit .beta. globin and hepatitis B virus (HBV),
.alpha.-globin 3' UTR and Viral VEEV 3' UTR sequences are also
known in the art. In some embodiments, the sequence UUUGAAUU
(WO2014/144196) is used. In some embodiments, 3' UTRs of human and
mouse ribosomal protein are used. Other examples include rps9 3'
UTR (WO2015/101414), FIG. 4 (WO2015/101415), and human albumin 7
(WO2015/101415).
[0150] Those of ordinary skill in the art will understand that
5'UTRs that are heterologous or synthetic may be used with any
desired 3' UTR sequence. For example, a heterologous 5'UTR may be
used with a synthetic 3'UTR with a heterologous 3'' UTR.
[0151] Non-UTR sequences may also be used as regions or subregions
within a nucleic acid. For example, introns or portions of introns
sequences may be incorporated into regions of nucleic acid of the
disclosure. Incorporation of intronic sequences may increase
protein production as well as nucleic acid levels.
[0152] Combinations of features may he included in flanking regions
and may be contained within other features. For example, the ORF
may be flanked by a 5' UTR which may contain a strong Kozak
translational initiation signal and/or a 3' UTR which may include
an oligo(dT) sequence for templated addition of a poly-A tail. 5'
UTR may comprise a first polynucleotide fragment and a second
polynucleotide fragment from the same and/or different genes such
as the 5' UTRs described in US Patent Application Publication No.
2010/0293625 and PCT/US2014/069155, herein incorporated by
reference in its entirety.
[0153] It should be understood that any UTR from any gene may be
incorporated into the regions of a nucleic acid. Furthermore,
multiple wild-type UTRs of any known gene may be utilized. It is
also within the scope of the present disclosure to provide
artificial UTRs which are not variants of wild type regions. These
UTRs or portions thereof may be placed in the same orientation as
in the transcript from which they were selected or may be altered
in orientation or location. Hence a 5' or 3' UTR may he inverted,
shortened, lengthened, made with one or more other 5' UTRs or 3'
UTRs. As used herein, the term "altered" as it relates to a UTR
sequence, means that the UTR has been changed in some way in
relation to a reference sequence. For example, a 3' UTR or 5' UTR
may be altered relative to a wild-type or native UTR by the change
in orientation or location as taught above or may be altered by the
inclusion of additional nucleotides, deletion of nucleotides,
swapping or transposition of nucleotides. Any of these changes
producing an "altered" UTR (whether 3' or 5') comprise a variant
UTR.
[0154] In some embodiments, a double, triple or quadruple UTR such
as a 5' UTR or 3' UTR may be used. As used herein, a "double" UTR
is one in which two copies of the same UTR are encoded either in
series or substantially in series. For example, a double
beta-globin 3' UTR may be used as described in US Patent
publication 2010/0129877, the contents of which are incorporated
herein by reference in its entirety.
[0155] It is also within the scope of the present disclosure to
have patterned UTRs. As used herein "patterned UTRs" are those UTRs
which reflect a repeating or alternating pattern, such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than 3 times. In these patterns, each letter, A, B, or C
represent a different UTR at the nucleotide level.
[0156] In some embodiments, flanking regions are selected from a
family of transcripts whose proteins share a common function,
structure, feature or property. For example, polypeptides of
interest may belong to a family of proteins which are expressed in
a particular cell, tissue or at some time during development. The
UTRs from any of these genes may be swapped for any other UTR of
the same or different family of proteins to create a new
polynucleotide. As used herein, a "family of proteins" is used in
the broadest sense to refer to a group of two or more polypeptides
of interest which share at least one function, structure, feature,
localization, origin, or expression pattern.
[0157] The untranslated region may also include translation
enhancer elements (TEE). As a non-limiting example, the TEE may
include those described in US Patent Publication No. 2009/0226470,
herein incorporated by reference in its entirety, and those known
in the art.
In Vitro Transcription of RNA
[0158] cDNA encoding the polynucleotides described herein may be
transcribed using an in vitro transcription (IVT) system. In vitro
transcription of RNA is known in the art and is described in
International Publication WO2014/152027, which is incorporated by
reference herein in its entirety.
[0159] In some embodiments, the RNA transcript is generated using a
non-amplified, linearized DNA template in an in vitro transcription
reaction to generate the RNA transcript. In some embodiments, the
template DNA is isolated DNA. In some embodiments, the template DNA
is cDNA. In some embodiments, the cDNA is formed by reverse
transcription of a RNA polynucleotide, for example, but not limited
to infectious disease (e.g., ZIKV) RNA, e.g. mRNA. In some
embodiments, cells, e.g., bacterial cells, e.g., E. coil, e.g.,
DH-1 cells are transfected with the plasmid DNA template. In some
embodiments, the transfected cells are cultured to replicate the
plasmid DNA which is then isolated and purified. In some
embodiments, the DNA template includes a RNA polymerase promoter,
e.g., a T7 promoter located 5' to and operably linked to the gene
of interest.
[0160] In some embodiments, an in vitro transcription template
encodes a 5' untranslated (UTR) region, contains an open reading
frame, and encodes a 3' UTR and a polyA tail. The particular
nucleic acid sequence composition and length of an in vitro
transcription template will depend on the mRNA encoded by the
template.
[0161] A "5' untranslated region" (UTR) refers to a region of an
mRNA that is directly upstream (i.e., 5') from the start codon
(i.e., the first codon of an mRNA transcript translated by a
ribosome) that does not encode a polypeptide. When RNA transcripts
are being generated, the 5' UTR may comprise a promoter sequence.
Such promoter sequences are known in the art. It should be
understood that such promoter sequences will not be present in a
vaccine of the disclosure.
[0162] A "3' untranslated region'" (UTR) refers to a region of an
mRNA that is directly downstream (i.e., 3') from the stop codon
(i.e., the codon of an mRNA transcript that signals a termination
of translation) that does not encode a polypeptide.
[0163] An "open reading frame" is a continuous stretch of DNA
beginning with a start codon (e.g., methionine (ATG)), and ending
with a stop codon (e.g., TAA, TAG or TGA) and encodes a
polypeptide.
[0164] A "polyA tail" is a region of mRNA that is downstream, e.g.,
directly downstream (i.e., 3'), from the 3' UTR that contains
multiple, consecutive adenosine monophosphates. A polyA tail may
contain 10 to 300 adenosine monophosphates. For example, a polyA
tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290 or 300 adenosine monophosphates. In some
embodiments, a polyA. tail contains 50 to 250 adenosine
monophosphates. In a relevant biological setting e.g., in cells, in
vivo) the poly(A) tail functions to protect mRNA from enzymatic
degradation, e.g., in the cytoplasm, and aids in transcription
termination, and/or export of the mRNA from the nucleus and
translation.
[0165] In some embodiments, a nucleic acid includes 200 to 3,000
nucleotides. For example, a nucleic acid may include 200 to 500,
200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500,
500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000,
1500 to 3000, or 2000 to 3000 nucleotides).
[0166] An in vitro transcription system typically comprises a
transcription buffer, nucleotide triphosphates (NTPs), an RNase
inhibitor and a polymerase.
[0167] The NTPs may be manufactured in house, may be selected from
a supplier, or may be synthesized as described herein. The NTPs may
be selected from, but are not limited to, those described herein
including natural and unnatural (modified) NTPs.
[0168] Any number of RNA polymerases or variants may be used in the
method of the present disclosure. The polymerase may be selected
from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA
polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or
mutant polymerases such as, but not limited to, polymerases able to
incorporate modified nucleic acids and/or modified nucleotides,
including chemically modified nucleic acids and/or nucleotides.
Some embodiments exclude the use of DNase.
[0169] In some embodiments, the RNA transcript is capped via
enzymatic capping. In some embodiments, the RNA comprises 5'
terminal cap, for example, 7mG(5')ppp(5')N1mpNp.
Chemical Synthesis
[0170] Solid-phase chemical synthesis. Nucleic acids the present
disclosure may be manufactured in whole or in part using solid
phase techniques. Solid-phase chemical synthesis of nucleic acids
is an automated method wherein molecules are immobilized on a solid
support and synthesized step by step in a reactant solution.
Solid-phase synthesis is useful in site-specific introduction of
chemical modifications in the nucleic acid sequences.
[0171] Liquid Phase Chemical Synthesis. The synthesis of nucleic
acids of the present disclosure by the sequential addition of
monomer building blocks may be carried out in a liquid phase.
[0172] Combination of Synthetic Methods. The synthetic methods
discussed above each has its own advantages and limitations.
Attempts have been conducted to combine these methods to overcome
the limitations. Such combinations of methods are within the scope
of the present disclosure. The use of solid-phase or liquid-phase
chemical synthesis in combination with enzymatic ligation provides
an efficient way to generate long chain nucleic acids that cannot
be obtained by chemical synthesis alone.
Ligation of Nucleic Acid Regions or Subregions
[0173] Assembling nucleic acids by a ligase may also be used. DNA
or RNA ligases promote intermolecular ligation of the 5' and 3'
ends of polynucleotide chains through the formation of a
phosphodiester bond. Nucleic acids such as chimeric polynucleotides
and/or circular nucleic acids may be prepared by ligation of one or
more regions or subregions. DNA fragments can be joined by a ligase
catalyzed reaction to create recombinant DNA with different
functions. Two oligodeoxynucleotides, one with a 5' phosphoryl
group and another with a free 3' hydroxyl group, serve as
substrates for a DNA ligase.
Purification
[0174] Purification of the nucleic acids described herein may
include, but is not limited to, nucleic acid clean-up, quality
assurance and quality control. Clean-up may be performed by methods
known in the arts such as, but not limited to, AGENCOURT.RTM. beads
(Beckman Coulter Genomics, Danvers, Mass.), poly-T beads. LNATM
oligo-T capture probes (EXIQON.RTM. Inc, Vedbaek, Denmark) or HPLC
based purification methods such as, but not limited to, strong
anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term
"purified" when used in relation to a nucleic acid such as a
"purified nucleic acid" refers to one that is separated from at
least one contaminant. A "contaminant" is any substance that makes
another unfit, impure or inferior. Thus, a purified nucleic acid
(e.g., DNA and RNA) is present in a form or setting different from
that in which it is found in nature, or a form or setting different
from that which existed prior to subjecting it to a treatment or
purification method.
[0175] A quality assurance and/or quality control check may be
conducted using methods such as, but not limited to, gel
electrophoresis, UV absorbance, or analytical HPLC.
[0176] In some embodiments, the nucleic acids may be sequenced by
methods including, but not limited to
reverse-transcriptase-PCR.
Quantrfication
[0177] In some embodiments, the nucleic acids of the present
invention may be quantified in exosomes or when derived from one or
more bodily fluid. Bodily fluids include peripheral blood, serum,
plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva,
bone marrow, synovial fluid, aqueous humor, amniotic fluid,
cerumen, breast milk, broncheoalveolar lavage fluid, semen,
prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat,
fecal matter, hair, tears, cyst fluid, pleural and peritoneal
fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial
fluid, menses, pus, sebtun, vomit, vaginal secretions, mucosal
secretion, stool water, pancreatic juice, lavage fluids from sinus
cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and
umbilical cord blood. Alternatively, exosomes may be retrieved from
an organ selected from the group consisting of lung, heart,
pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin,
colon, breast, prostate, brain, esophagus, liver, and placenta.
[0178] Assays may be performed using construct specific probes,
cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry,
electrophoresis, mass spectrometry, or combinations thereof while
the exosomes may be isolated using immunohistochemical methods such
as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may
also be isolated by size exclusion chromatography, density gradient
centrifugation, differential centrifugation, nanomembrane
ultrafiltration, immunoabsorbent capture, affinity purification,
microfluidic separation, or combinations thereof.
[0179] These methods afford the investigator the ability to
monitor, in real time, the level of nucleic acids remaining or
delivered. This is possible because the nucleic acids of the
present disclosure, in some embodiments, differ from the endogenous
forms due to the structural or chemical modifications.
[0180] In some embodiments, the nucleic acid may be quantified
using methods such as, but not limited to, ultraviolet visible
spectroscopy (UV/Vis). A non-limiting example of a UV/Vis
spectrometer is a NANODROP.RTM. spectrometer (Thermonsher, Waltham,
Mass.). The quantified nucleic acid may be analyzed in order to
determine if the nucleic acid may be of proper size, check that no
degradation of the nucleic acid has occurred. Degradation of the
nucleic acid may be checked by methods such as, but not limited to,
agarose gel electrophoresis, HPLC based purification methods such
as, but not limited to, strong anion exchange HPLC, weak anion
exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic
interaction HPLC (HIC-FIPLC), liquid chromatography-mass
spectrometry (LCMS), capillary electrophoresis (CE) and capillary
gel electrophoresis (CGE).
Lipid Nanoparticles (LNPs)
[0181] In some embodiments, the RNA (e.g., mRNA) vaccines of the
disclosure are formulated in a lipid nanoparticle (LNP). Lipid
nanoparticles typically comprise ionizable cationic lipid,
non-cationic lipid, sterol and PEG lipid components along with the
nucleic acid cargo of interest. The lipid nanoparticles of the
disclosure can be generated using components, compositions, and
methods as are generally known in the art, see for example
PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551;
PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129;
PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426;
PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117;
PCT/US2012/069610; PCT/US201 /027492; PCT/US 016/059575 and
PCT/US2016/069491 all of which are incorporated by reference herein
in their entirety.
[0182] Vaccines of the present disclosure are typically formulated
in lipid nanoparticle. In some embodiments, the lipid nanoparticle
comprises at least one ionizable cationic lipid, at least one
non-cationic lipid, at least one sterol, and/or at least one
polyethylene glycol (PEG)-modified lipid.
[0183] In some embodiments, the lipid nanoparticle comprises a
molar ratio of 20-60% ionizable cationic lipid. For example, the
lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%,
20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable
cationic lipid. In some embodiments, the lipid nanoparticle
comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable
cationic lipid.
[0184] In some embodiments, the lipid nanoparticle comprises a
molar ratio of 5-25% non-cationic lipid. For example, the lipid
nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%,
10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic
lipid. In some embodiments, the lipid nanoparticle comprises a
molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.
[0185] In some embodiments, the lipid nanoparticle comprises a
molar ratio of 25-55% sterol. For example, the lipid nanoparticle
may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%,
25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-3. , 35-55%, 35-50%,
35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55%
sterol. In some embodiments, the lipid nanoparticle comprises a
molar ratio of 25?, 30%, 35%, 40%, 45%, 50%, or 55% sterol.
[0186] In some embodiments, the lipid nanoparticle comprises a
molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid
nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%,
1-10%, 1-5%, 2-15%, 2-10%, 2-5?./ 5-15%, 5-10%, or 10-15%. In some
embodiments, the lipid nanoparticle comprises a molar ratio of
0.5%, 1%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or
15% PEG-modified lipid.
[0187] In sonic embodiments, the lipid nanoparticle comprises a
molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic
lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.
[0188] In some embodiments, an ionizable cationic lipid of the
disclosure comprises a compound of Formula (I):
##STR00001##
[0189] or a salt or isomer thereof, wherein.
[0190] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl --R*YR'', --YR'', and --R''M'R';
[0191] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.2, together
with the atom to which they are attached, form a heterocycle or
carbocyde;
[0192] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from carbocycle, heterocycle, --OR,
--O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R, --CX.sub.3,
--CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.4)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(--NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5;
[0193] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0194] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0195] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0196] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0197] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0198] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0199] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0200] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0201] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0202] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0203] each Y is independently a C.sub.3-6 carbocycle;
[0204] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0205] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.
[0206] In some embodiments, a subset of compounds of Formula (1)
includes those in which when R.sub.4 is --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHAR, or --CQ(R).sub.2, then (i) Q is not
--N(R).sub.2 when n is 2, 3, 4 or 5, or (ii) Q is not 5, 6, or
7-membered heterocycloalkyl when n is 1 or 2.
[0207] In some embodiments, another subset of compounds of Formula
(1) includes those in which
[0208] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0209] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0210] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2;
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(NR.sub.9)N(R).sub.2, --N(OR)C(.dbd.CHR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R, --C(O)N(R)OR,
and a 5- to 14-membered heterocycloalkyl having one or more
heteroatoms selected from N, O, and S which is substituted with one
or more substituents selected from oxo (.dbd.O), OH, amino, mono-
or di-alkylamino, and C.sub.1-3 alkyl, and each n is independently
selected from 1, 2, 3, 4, and 5;
[0211] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0212] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0213] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0214] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0215] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0216] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0217] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0218] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0219] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0220] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0221] each Y is independently a C.sub.3-6 carbocycle;
[0222] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0223] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0224] or salts or isomers thereof.
[0225] In some embodiments, another subset of compounds of Formula
(I) includes those in which
[0226] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0227] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0228] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CQ(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heterocycle having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R,
--C(O)N(R)OR, and --C(.dbd.NR.sub.9)N(R).sub.2, and each n is
independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-
to 14-membered heterocycle and (i) R.sub.4 is --(CH.sub.2).sub.nQ
in which n is 1 or 2, or (ii) R.sub.4 is --(CH2).sub.nCHQR in which
n is 1, or (iii) R.sub.4 is --CHQR, and --CQ(R).sub.2, then Q is
either a 5- to 14-membered heteroaryl or 8- to 14-membered
heterocycloalkyl;
[0229] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0230] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0231] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0232] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0233] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0234] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0235] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0236] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0237] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0238] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0239] each Y is independently a C.sub.3-6 carbocycle;
[0240] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0241] m is selected from 5, 6, 7, 8, 9, 10, 11and 13,
[0242] or salts or isomers thereof.
[0243] In some embodiments, another subset of compounds of Formula
(I) includes those in which
[0244] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R'' M'R';
[0245] R.sub.2 and R3 are independently selected from the group
consisting of H, C.sub.1-14 alkyl, C.sub.2-14 alkenyl, --R*YR'',
--YR'', and --R*OR'', or R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or
carbocycle;
[0246] R.sub.4 is selected from the group consisting of a C.sub.3-6
carbocycle, --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
--CO(R).sub.2, and unsubstituted C.sub.1-6 alkyl, where Q is
selected from a C.sub.3-6 carbocycle, a 5- to 14-membered
heteroaryl having one or more heteroatoms selected from N, O, and
S, --OR, --O(CH.sub.2).sub.nN(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --C(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR, --N(R)R.sub.8,
--O(CH.sub.2)OR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R,
--C(O)N(R)OR, and --C(.dbd.NR.sub.9)N(R).sub.2, and each n is
independently selected from 1, 2, 3, 4, and 5;
[0247] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0248] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0249] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0250] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0251] R.sub.8 is selected from the group consisting of C.sub.3-6
carbocycle and heterocycle;
[0252] R.sub.9 is selected from the group consisting of H, CN,
NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle;
[0253] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0254] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR''and H;
[0255] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0256] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.2-12 alkenyl;
[0257] each Y is independently a C.sub.3-6 carbocycle;
[0258] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0259] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0260] or salts or isomers thereof.
[0261] In some embodiments, another subset of compounds of Formula
(I) includes those in which
[0262] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R''M'R';
[0263] R.sub.2 and R.sub.3 are independently selected from the
group consisting of H, C.sub.2-14 alkyl, C.sub.2-14 alkenyl,
--R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3, together
with the atom to which they are attached, form a heterocycle or
carbocycle;
[0264] R.sub.4 is --(CH.sub.2).sub.nQ or --(Cl.sub.2).sub.nCHQR,
where Q is --N(R).sub.2, and n is selected from 3, 4, and 5;
[0265] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0266] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0267] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0268] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0269] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0270] each R' is independently selected from the group consisting
of C.sub.1-18 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0271] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0272] each R* is independently selected from the group consisting
of C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[0273] each Y is independently a C.sub.3-6 carbocycle;
[0274] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0275] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0276] or salts or isomers thereof.
[0277] In some embodiments, another subset of compounds of Formula
(I) includes those in which
[0278] R.sub.1 is selected from the group consisting of C.sub.5-30
alkyl, C.sub.5-20 alkenyl, --R*YR'', --YR'', and --R'' M'R';
[0279] R.sub.2 and R.sub.3 are independently selected from the
group consisting of C.sub.1-14 alkyl, C.sub.2-14 alkenyl, --R*YR'',
--YR'', and --R*OR'', or R.sub.2 and R.sub.3, together with the
atom to which they are attached, form a heterocycle or
carbocycle;
[0280] R.sub.4 is selected from the group consisting of
--(CH).sub.mQ, --(CH.sub.2).sub.nCHQR, --CHQR, and --CQ(R).sub.2,
where Q is --N(R).sub.2, and n is selected from 1, 2, 3, 4, and
5;
[0281] each R.sub.5 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0282] each R.sub.6 is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0283] M and M' are independently selected from --C(O)O--,
--OC(O)--, --C(O)N(R')--, --N(R')C(O)--, --C(O)--, --C(S)--,
--C(S)S--, --SC(S)--, --CH(OH)--, --P(O)(OR')O--, S(O).sub.2--,
--S--S--, an aryl group, and a heteroaryl group;
[0284] R.sub.7 is selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H;
[0285] each R is independently selected from the group consisting
of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H;
[0286] each R' is independently selected from the group consisting
of C.sub.1-8 alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and
H;
[0287] each R'' is independently selected from the group consisting
of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
[0288] each R* is independently selected from e group consisting of
C.sub.1-12 alkyl and C.sub.1-12 alkenyl;
[0289] each Y is independently a C.sub.3-6 carbocycle;
[0290] each X is independently selected from the group consisting
of F, Cl, Br, and I; and
[0291] m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
[0292] or salts or isomers thereof.
[0293] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IA):
##STR00002##
[0294] or a salt or isomer thereof, wherein 1 is selected from 1,
2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M.sub.1 is a
bond or M'; R4 is unsubstituted C.sub.1-3 alkyl, or
--(CH.sub.2).sub.nQ, in which Q is OH, --NHC(S)N(R).sub.2,
--NHC(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)R.sub.8,
--NHC(.dbd.NR.sub.9)N(R).sub.2, --NHC(.dbd.CHR.sub.9)N(R).sub.2,
--OC(0)N(R).sub.2, --N(R)C(O)OR, heteroaryl or heterocycloalkyl; M
and M' are independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group, and a
heteroaryl group; and R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.1-4 alkyl, and
C.sub.2-14 alkenyl,
[0295] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (II):
##STR00003##
or a salt or isomer thereof, wherein I is selected from 1, 2, 3, 4,
and 5; M.sub.1 is a bond or M'; R.sub.4 is unsubstituted C.sub.1-3
alkyl, or --(CH.sub.2).sub.nQ, in which n is 2, 3, or 4, and Q is
OH, --NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2, --N(R)C(O)R,
--N(R)S(O).sub.2R, --N(R)R.sub.8, --NHC(.dbd.NR.sub.9)N(R).sub.2,
--NHC(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
heteroaryl or heterocycloalkyl; M and M' are independently selected
from --C(O)O--, --OC(O)--, --C(O)N(R')--, --P(O)(OR')O--, --S--S--,
an aryl group, and a heteroaryl group; and R.sub.2 and R.sub.3 are
independently selected from the group consisting of H, C.sub.1-14
alkyl, and C.sub.2-14 alkenyl.
[0296] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IIa), (IIb), (IIc), or (IIe):
##STR00004##
[0297] or a salt or isomer thereof, wherein R.sub.4 is as described
herein.
[0298] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IId):
##STR00005##
[0299] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m,
R', R'', and R, through R.sub.6 are as described herein. For
example, each of R.sub.2 and R.sub.3 may be independently selected
from the group consisting of C.sub.5-14 alkyl and C.sub.5-14
alkenyl.
[0300] In some embodiments, an ionizable cationic lipid of the
disclosure comprises a compound having structure:
##STR00006##
[0301] In some embodiments, an ionizable cationic lipid of the
disclosure comprises a compound having structure:
##STR00007##
[0302] In some embodiments, a non-cationic lipid of the disclosure
comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC), 1-hexadecvl-sn-glycero-3-phosphocholine (C16 Lyso PC),
1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycer-
o-3-phosphocholine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,
1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt
(DOPG), sphingomyelin, and mixtures thereof.
[0303] In some embodiments, a PEG modified lipid of the disclosure
comprises a PEG-modified phosphatidylethanolamine, a PEG-modified
phosphatidic acid, a PEG-modified ceramide, a PEG-modified
dialkylamine, a PEG-modified diacylglycerol, a PEG-modified
dialkylglycerol, and mixtures thereof. In some embodiments, the
PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as
PEG-DOME), PEG-DSG and/or PEG-DPG.
[0304] In some embodiments, a sterol of the disclosure comprises
cholesterol, fecosterol, sitosterol, ergosterol, campesterol,
stigmasterol, brassicasterol, tomatidine, ursolic acid,
alpha-tocopherol, and mixtures thereof.
[0305] In some embodiments, a LNP of the disclosure comprises an
ionizable cationic lipid of Compound 1, wherein the non-cationic
lipid is DSPC, the structural lipid that is cholesterol, and the
PEG lipid is PEG-DMG.
[0306] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of from about 2:1 10 about 30:1.
[0307] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of about 6:1.
[0308] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of about 3:1.
[0309] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
from about 10:1 to about 100:1.
[0310] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
about 20:1.
[0311] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
about 10:1.
[0312] In some embodiments, a LNP of the disclosure has a mean
diameter from about 50 am to about 150 nm.
[0313] In some embodiments, a LNP of the disclosure has a mean
diameter from about 70 nm to about 120 nm.
Signal Sequences/Peptides
[0314] In some embodiments, an antigen and/or a PADRE encoded by a
mRNA of the present disclosure comprises a signal peptide. Signal
peptides, comprising the N-terminal 15-60 amino acids of proteins,
are typically needed for the translocation across the membrane on
the secretory pathway and, thus, universally control the entry of
most proteins both in eukaryotes and prokaryotes to the secretory
pathway. Signal peptides generally include three regions: an
N-terminal region of differing length, which usually comprises
positively charged amino acids; a hydrophobic region; and a short
carboxy-terminal peptide region. In eukaryotes, the signal peptide
of a nascent precursor protein (pre-protein) directs the ribosome
to the rough endoplasmic reticulum (ER) membrane and initiates the
transport of the growing peptide chain across it for processing. ER
processing produces mature proteins, wherein the signal peptide is
cleaved from precursor proteins, typically by a ER-resident signal
peptidase of the host cell, or they remain uncleaved and function
as a membrane anchor. A signal peptide may also facilitate the
targeting of the protein to the cell membrane. The signal peptide,
however, is not responsible for the final destination of the mature
protein. Secretory proteins devoid of additional address tags in
their sequence are by default secreted to the external enviromnent.
During recent years, a more advanced view of signal peptides has
evolved, showing that the functions and immunodominance of certain
signal peptides are much more versatile than previously
anticipated.
[0315] Immunogenic compositions of the present disclosure may
comprise, for example, mRNA encoding an artificial signal peptide,
wherein the signal peptide coding sequence is operably linked to
and is in frame with the coding sequence of the antigen and/or
PADRE. Thus, mRNA of the present disclosure, in some embodiments,
produce an antigen and/or a PADRE fused to a signal peptide. In
some embodiments, a signal peptide is fused to the N-terminus of
the antigen and/or a PADRE. In some embodiments, a signal peptide
is fused to the C-terminus of the antigen and/or a PADRE.
[0316] In some embodiments, the signal peptide fused to the antigen
and/or a PADRE is an artificial signal peptide. In some
embodiments, a signal peptide fused to the antigen and/or a PADRE
encoded by the mRNA is a HuIgGk signal peptide
(METPAQLLFLLLLWLPDTTG; SEQ ID NO: 7). In some embodiments, a signal
peptide fused to the antigen and/or a PADRE encoded by the mRNA is
a IgE heavy chain epsilon-1 signal peptide (MDWTWILFIVAAATRVHS; SEQ
ID NO: 8). In some embodiments, a signal peptide fused to the
antigen and/or a PADRE encoded by the mRNA is a JEV polyprotein
signal peptide (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 9). In some
embodiments, a signal peptide fused to the antigen and/or a PADRE
encoded by the mRNA is a VSVg protein signal peptide
(MKCLLYLAFLFIGVNCA; SEQ ID NO: 10. In some embodiments, a signal
peptide fused to the antigen and/or a PADRE encoded by the mRNA is
a Japanese encephalitis prM signal peptide (MWLVSLAIVTACAGA; SEQ ID
NO: 11),
[0317] The examples disclosed herein are not meant to be limiting
and any signal peptide that is known in the art to facilitate
targeting of a protein to ER for processing and/or targeting of a
protein to the cell membrane may be used in accordance with the
present disclosure.
[0318] A signal peptide may have a length of 15-60 amino acids. For
example, a signal peptide may have a length of 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a
signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60,
45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55,
45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50,
15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40,
30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30,
15-25, 20-25, or 15-20 amino acids.
[0319] A signal peptide is typically cleaved from the nascent
polypeptide at the cleavage junction during ER processing. The
mature antigen and/or a PADRE encoded by the mRNA of the present
disclosure typically does not comprise a signal peptide.
Fusion Proteins
[0320] In some embodiments, an the RNA vaccine of the present
disclosure includes an RNA encoding an antigenic fusion protein.
Thus, the encoded antigen or antigens may include two or more
proteins (e.g., protein and/or protein fragment) joined together.
Alternatively, the protein to which a protein antigen is fused does
not promote a strong immune response to itself, but rather to the
the antigen. Antigenic fusion proteins, in some embodiments, retain
the functional property from each original protein.
Scaffold Moieties
[0321] The RNA (e.g., mRNA) vaccines as provided herein, in some
embodiments, encode fusion proteins which comprise the antigens
linked to scaffold moieties. In some embodiments, such scaffold
moieties impart desired properties to an antigen encoded by a
nucleic acid of the disclosure. For example scaffold proteins may
improve the immunogenicity of an antigen, e.g., by altering* the
structure of the antigen, altering the uptake and processing of the
antigen, and/or causing the antigen to bind to a binding
partner.
[0322] In some embodiments, the scaffold moiety is protein that can
self-assemble into protein nanoparticles that are highly symmetric,
stable, and structurally organized, with diameters of 10-150 nm, a
highly suitable size range for optimal interactions with various
cells of the immune system. In some embodiments, viral proteins or
virus-like particles can be used to form stable nanoparticle
structures. Examples of such viral proteins are known in the art.
For example, in some embodiments, the scaffold moiety is a
hepatitis B surface antigen (HBsAg). HBsAg forms spherical
particles with an average diameter of .about.22 nm and which lacked
nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et
al. Computational and Structural Biotechnology Journal 14 (2016)
58-68). In some embodiments, the scaffold moiety is a hepatitis B
core antigen (HBcAg) self-assembles into particles of 24-31 nm
diameter, which resembled the viral cores obtained from
HBV-infected human liver. HBcAg produced in self-assembles into two
classes of differently sized nanoparticles of 300 .ANG. and 360
.ANG. diameter, corresponding to 180 or 240 protomers. In some
embodiments an the antigen is fused to HBsAG or HBcAG to facilitate
self-assembly of nanoparticles displaying the the antigen.
[0323] In another embodiment, bacterial protein platforms may be
used. Non-limiting examples of these self-assembling proteins
include ferritin, lumazine and encapsulin.
[0324] Ferritin is a protein whose main function is intracellular
iron storage. Ferritin is made of 24 subunits, each composed of a
four-alpha-helix bundle, that self-assemble in a quaternary
structure with octahedral symmetry (Cho K. J. et al. J Mol Biol.
2009; 390:83-98). Several high-resolution structures of ferritin
have been determined, confirming that Helicobacter pylori ferritin
is made of 24 identical protomers, whereas in animals, there are
ferritin light and heavy chains that can assemble alone or combine
with different ratios into particles of 24 subunits (Granier T. et
al. J Biol Inorg Chem. 2003; 8:105-111; Lawson D. M. et al. Nature.
1991; 349:541-544). Ferritin self-assembles into nanoparticles with
robust thermal and chemical stability. Thus, the ferritin
nanoparticle is well-suited to carry and expose antigens.
[0325] Luinazine synthase (LS) is also well-suited as a
nanoparticle platform for antigen display. LS, which is responsible
for the penultimate catalytic step in the biosynthesis of
riboflavin, is an enzyme present in a broad variety of organisms,
including archaea, bacteria, fungi, plants, and eubacteria (Weber
S. E. Flavins and Flavoproteins. Methods and Protocols, Series:
Methods in Molecular Biology. 2014). The LS monomer is 150 amino
acids long, and consists of beta-sheets along with tandem
alpha-helices flanking its sides. A number of different quaternary
structures have been reported for LS, illustrating its
morphological versatility: from homopentamers up to symmetrical
assemblies of 12 pentamers forming capsids of 150 .ANG. diameter.
Even LS cages of more than 100 subunits have been described (Zhang
X. et al. J Mol Biol. 2006; 362:753-770).
[0326] Encapsulin, a novel protein cage nanoparticle isolated from
thermophile Thermotoga maritima, may also be used as a platform to
present antigens on the surface of self-assembling nanoparticles.
Encapsulin is assembled from 60 copies of identical 31 kDa monomers
having a thin and icosahedral T=1 symmetric cage structure with
interior and exterior diameters of 20 and 24 nm, respectively
(Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). Although
the exact function of encapsulin in T. maritima is not clearly
understood yet, its crystal structure has been recently solved and
its function was postulated as a cellular compartment that
encapsulates proteins such as DyP (Dye decolorizing peroxidase) and
Flp (Ferritin like protein), which are involved in oxidative stress
responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104).
Linkers and Cleavable Peptides
[0327] In sonic embodiments, the mRNAs of the disclosure encode
more than one polypeptide, referred to herein as fusion proteins,
in some embodiments, the mRNA further encodes a linker located
between at least one or each domain of the fusion protein. The
linker can be, for example, a cleavable linker or
protease-sensitive linker. In some embodiments, the linker is
selected from the group consisting of F2A linker, P2A linker, T2A
linker, E2A linker, and combinations thereof This family of
self-cleaving peptide linkers, referred to as 24 peptides, has been
described in the art (see for example, Kim, J. H. et al. (2011)
PLoS ONE 6:e18556). In some embodiments, the linker is an FIN
linker. In some embodiments, the linker is a GGGS linker. In some
embodiments, the fusion protein contains three domains with
intervening linkers, having the structure:
domain-linker-domain-linker-domain.
[0328] Cleavable linkers known in the art may be used in connection
with the disclosure. Exemplary such linkers include: F2A linkers,
T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017/127750).
The skilled artisan will appreciate that other art-recognized
linkers may be suitable for use in the constructs of the disclosure
(e.g., encoded by the nucleic acids of the disclosure). The skilled
artisan will likewise appreciate that other polycistronic
constructs (mRNA encoding more than one antigen/polypeptide
separately within the same molecule) may be suitable for use as
provided herein.
Sequence Optimization
[0329] In some embodiments, an ORF encoding an antigen of the
disclosure is codon optimized. Codon optimization methods are known
in the art. For example, an ORF of any one or more of the sequences
provided herein may be codon optimized. Codon optimization, in some
embodiments, may be used to match codon frequencies in target and
host organisms to ensure proper folding; bias GC content to
increase mRNA stability or reduce secondary structures; minimize
tandem repeat codons or base runs that may impair gene construction
or expression; customize transcriptional and translational control
regions; insert or remove protein trafficking sequences; remove/add
post translation modification sites in encoded protein (e.g.,
glycosylation sites); add, remove or shuffle protein domains;
insert or delete restriction sites; modify ribosome binding sites
and mRNA degradation sites; adjust translational rates to allow the
various domains of the protein to fold properly; or reduce or
eliminate problem secondary structures within the polynucleotide.
Codon optimization tools, algorithms and services are known in the
art--non-limiting examples include services from GeneArt (Life
Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
In some embodiments, the open reading frame (ORF) sequence is
optimized using optimization algorithms.
[0330] In some embodiments, a codon optimized sequence shares less
than 95% sequence identity to a naturally-occurring or wild-type
sequence ORF (e.g., a naturally-occurring or wild-type mRNA
sequence encoding an antigen). In some embodiments, a codon
optimized sequence shares less than 90% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding an
antigen). In some embodiments, a codon optimized sequence shares
less than 85% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding an antigen). In some embodiments, a codon
optimized sequence shares less than 80% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding an
antigen). In some embodiments, a codon optimized sequence shares
less than 75% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding an antigen).
[0331] In some embodiments, a codon optimized sequence shares
between 65% and 85% (e.g., between about 67% and about 85% or
between about 67% and about 80%) sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding an
antigen). In some embodiments, a codon optimized sequence shares
between 65% and 75% or about 80% sequence identity to a
naturally-occurring or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding an
antigen).
[0332] In some embodiments, a codon-optimized sequence encodes an
antigen that is as immunogenic as, or more immunogenic than (e.g.,
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 100%, or at least 200% more), than an antigen encoded
by a non-codon-optimized sequence.
[0333] When transfected into mammalian host cells, the modified
mRNAs have a stability of between 12-18 hours, or greater than 18
hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are
capable of being expressed by the mammalian host cells.
[0334] In some embodiments, a codon optimized RNA may be one in
which the levels of G/C are enhanced. The G/C-content of nucleic
acid molecules (e.g., mRNA) may influence the stability of the RNA.
RNA having an increased amount of guanine (G) and/or cytosine (C)
residues may be functionally more stable than RNA containing a
large amount of adenine (A) and thymine (T) or uracil (U)
nucleotides. As an example, WO2002/098443 discloses a
pharmaceutical composition containing an mRNA stabilized by
sequence modifications in the translated region. Due to the
degeneracy of the genetic code, the modifications work by
substituting existing codons for those that promote greater RNA
stability without changing the resulting amino acid. The approach
is limited to coding regions of the RNA.
Pharmaceutical Formulations
[0335] Provided herein are compositions e.g., pharmaceutical
compositions), methods, kits and reagents for prevention or
treatment of an infectious disease (e.g., ZIKV) in humans and other
mammals, for example. The RNA (e.g., mRNA) vaccines can be used as
therapeutic or prophylactic agents. They may be used in medicine to
prevent and/or treat infectious disease.
[0336] In some embodiments, a vaccine containing RNA
polynucleotides as described herein can be administered to a
subject (e.g., a mammalian subject, such as a human subject), and
the RNA polynucleotides are translated in vivo to produce an
antigenic polypeptide (antigen).
[0337] An "effective amount" of a vaccine is based, at least in
part, on the target tissue, target cell type, means of
administration, physical characteristics of the RNA (e.g., length,
nucleotide composition, and/or extent of modified nucleosides),
other components of the vaccine, and other determinants, such as
age, body weight, height, sex and general health of the subject.
Typically, an effective amount of a vaccine provides an induced or
boosted immune response as a function of antigen production in the
cells of the subject. In some embodiments, an effective amount of
the RNA vaccine containing RNA polynucleotides having at least one
chemical modifications are more efficient than a composition
containing a corresponding unmodified polynucleotide encoding the
same antigen or a peptide antigen. Increased antigen production may
be demonstrated by increased cell transfection (the percentage of
cells transfected with the RNA vaccine), increased protein
translation and/or expression from the polynucleotide, decreased
nucleic acid degradation (as demonstrated, for example, by
increased duration of protein translation from a modified
polynucleotide), or altered antigen specific immune response of the
host cell.
[0338] The term "pharmaceutical composition" refers to the
combination of an active agent with a carrier, inert or active,
making the composition especially suitable for diagnostic or
therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable
carrier," after administered to or upon a subject, does not cause
undesirable physiological effects. The carrier in the
pharmaceutical composition must be "acceptable" also in the sense
that it is compatible with the active ingredient and can be capable
of stabilizing it. One or more solubilizing agents can be utilized
as pharmaceutical carriers for delivery of an active agent.
Examples of a pharmaceutically acceptable carrier include, but are
not limited to, biocompatible vehicles, adjuvants, additives, and
diluents to achieve a composition usable as a dosage form. Examples
of other carriers include colloidal silicon oxide, magnesium
stearate, cellulose, and sodium lauryl sulfate. Additional suitable
pharmaceutical carriers and diluents, as well as pharmaceutical
necessities for their use, are described in Remington's
Pharmaceutical Sciences.
[0339] In sonic embodiments, RNA vaccines (including
polynucleotides and their encoded polypeptides) in accordance with
the present disclosure may be used for treatment or prevention of
infectious disease (e.g., ZIKV), the RNA vaccines may be
administered prophylactically or therapeutically as part of an
active immunization scheme to healthy individuals or early in
infection during the incubation phase or during active infection
after onset of symptoms. In some embodiments, the amount of RNA
vaccines of the present disclosure provided to a cell, a tissue or
a subject may be an amount effective for immune prophylaxis.
[0340] The RNA (e.g., mRNA) vaccines may be administered with other
prophylactic or therapeutic compounds. As a non-limiting example, a
prophylactic or therapeutic compound may be an adjuvant or a
booster. As used herein, when referring to a prophylactic
composition, such as a vaccine, the term "booster" refers to an
extra administration of the prophylactic (vaccine) composition. A
booster (or booster vaccine) may be given after an earlier
administration of the prophylactic composition. The time of
administration between the initial administration of the
prophylactic composition and the booster may be, but is not limited
to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes,
20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years,
17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35
years, 40 years, 45 years, 50 years, 55 years, 60 years, 6.5 years,
70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more
than 99 years. In exemplary embodiments, the time of administration
between the initial administration of the prophylactic composition
and the booster may be, but is not limited to, 1 week, 2
.sup.-weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1
year.
[0341] In some embodiments, the RNA vaccines may be administered
intramuscularly, intranasally or intradermally, similarly to the
administration of inactivated vaccines known in the art.
[0342] The the RNA vaccines may be utilized in various settings
depending on the prevalence of the infection or the degree or level
of unmet medical need. As a non-limiting example, the RNA vaccines
may be utilized to treat and/or prevent a variety of infectious
disease. RNA vaccines have superior properties in that they produce
much larger antibody titers, better neutralizing immunity, produce
more durable immune responses, and/or produce responses earlier
than commercially available vaccines.
[0343] Provided herein are pharmaceutical compositions including
the RNA vaccines and RNA vaccine compositions and/or complexes
optionally in combination with one or more pharmaceutically
acceptable excipients.
[0344] The RNA (e.g., mRNA) vaccines may be formulated or
administered alone or in conjunction with one or more other
components. For instance, the RNA vaccines (vaccine compositions)
may comprise other components including, but not limited to,
adjuvants.
[0345] In some embodiments, the RNA vaccines do not include an
adjuvant (they are adjuvant free).
[0346] The RNA (e.g., mRNA) vaccines may be formulated or
administered in combination with one or more
pharmaceutically-acceptable excipients. In some embodiments,
vaccine compositions comprise at least one additional active
substances, such as, for example, a therapeutically-active
substance, a prophylactically-active substance, or a combination of
both. Vaccine compositions may be sterile, pyrogen-free or both
sterile and pyrogen-free. General considerations in the formulation
and/or manufacture of pharmaceutical agents, such as vaccine
compositions, may be found, for example, in Remington: The Science
and Practice of Pharmacy 21st ed., Lippincott Williams &
Wilkins, 2005 (incorporated herein by reference in its
entirety).
[0347] In sonic embodiments, the RNA vaccines are administered to
humans, human patients or subjects. For the purposes of the present
disclosure, the phrase "active ingredient" generally refers to the
RNA vaccines or the polynucleotides contained therein, for example,
RNA polynucleotides (e.g., mRNA polynucleotides) encoding
antigens.
[0348] Formulations of the vaccine compositions described herein
may be prepared by any method known or hereafter developed in the
art of pharmacology. In general, such preparatory methods include
the step of bringing the active ingredient (e.g., mRNA
polynucleotide) into association with an excipient and/or one or
more other accessory ingredients, and then, if necessary and/or
desirable, dividing, shaping and/or packaging the product into a
desired single- or multi-dose unit.
[0349] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
disclosure will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
[0350] In some embodiments, the RNA vaccines are formulated using
one or more excipients to: (1) increase stability; (2) increase
cell transfection; (3) permit the sustained or delayed release
(e.g., from a depot formulation); (4) alter the biodistribution
(e.g., target to specific tissues or cell types); (5) increase the
translation of encoded protein in vivo; and/or (6) alter the
release profile of encoded protein (antigen) in vivo. In addition
to traditional excipients such as any and all solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, excipients can include, without
limitation, lipidoids, liposomes, lipid nanoparticles, polymers,
lipoplexes, core-shell nanoparticles, peptides, proteins, cells
transfected with the RNA vaccines e.g., for transplantation into a
subject), hyaluronidase, nanoparticle mimics and combinations
thereof.
Therapeutic and Prophylactic Compositions
[0351] Provided herein are compositions (e.g., pharmaceutical
compositions), kits and reagents for prevention, treatment or
diagnosis of an infection disease in humans and other mammals, for
example. The immunogenic compositions of the present disclosure can
be used as therapeutic agents or prophylactic agents. They may be
used in medicine to prevent and/or treat infectious disease. In
some embodiments, the immunogenic compositions are used in the
priming of immune effector cells, for example, to activate
peripheral blood mononuclear cells (PBMCs) ex vivo, which are then
infused (re-infused) into a subject. In some embodiments, the
immunogenic compositions are administered prophylactically or
therapeutically as part of an active immunization scheme to healthy
individuals or early in infection during the incubation phase or
during active infection after onset of symptoms.
[0352] Typically, an immunogenic composition comprising mRNA
encoding an antigen and/or PADRE is administered to a subject
(e.g., a mammalian subject, such as a human subject), and the mRNA
is translated in vivo (e.g., in a cell, tissue or organism) to
produce the antigen and/or PADRE, although such translation may
occur ex vivo, in culture or in vitro. In some embodiments, the
cell, tissue or organism is contacted with an effective amount of
an immunogenic composition containing mRNA that has at least one a
translatable region encoding an antigen and/or PADRE.
[0353] An "effective amount" is a dose of an immunogenic
composition e.g., mRNA vaccine) effective to produce an
antigen-specific immune response. An effective amount is based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the polynucleotide
(e.g., size, and extent of modified nucleosides) and other
components of the composition, and other determinants. In general,
an effective amount of an immunogenic composition provides an
induced or boosted immune response as a function of antigen
production in the cell, preferably more efficient than a
composition containing a corresponding unmodified polynucleotide
encoding the same antigen or a peptide antigen. Increased antigen
production may be demonstrated by increased cell transfection (the
percentage of cells transfected with the mRNA), increased protein
translation from the polynucleotide, decreased nucleic acid
degradation (as demonstrated, for example, by increased duration of
protein translation from a modified polynucleotide), or altered
antigen-specific immune response of the host cell. In some
embodiments, the amount of an immunogenic composition provided to a
cell, a tissue or a subject may be an amount effective for immune
prophylaxis. In sonic embodiments, immunogenic compositions in
accordance with the present disclosure may be used for treatment of
infectious diseases, such as Zika virus.
[0354] In some embodiments, an effective amount of an immunogenic
composition is a dose that is reduced compared to the standard of
care dose of a recombinant protein vaccine. A "standard of care,"
as provided herein, refers to a medical or psychological treatment
guideline and can be general or specific. "Standard of care"
specifies appropriate treatment based on scientific evidence and
collaboration between medical professionals involved in the
treatment of a given condition. It is the diagnostic and treatment
process that a physician/clinician should follow for a certain type
of patient, illness or clinical circumstance. A "standard of care
dose," as provided herein, refers to the dose of a recombinant or
purified protein vaccine, or a live attenuated or inactivated viral
vaccine, that a physician/clinician or other medical professional
would administer to a subject to treat or prevent an infectious
disease, while following the standard of care guideline for
treating or preventing the infectious disease.
[0355] A "prophylactically effective amount" or a "prophylactically
effective dose" is an effective amount that prevents infection with
a virus, bacteria or parasite at a clinically acceptable level.
[0356] In some embodiments, an effective close is a dose listed in
a package insert for the vaccine. A traditional vaccine, as used
herein, refers to a vaccine other than the immunogenic composition
(e.g., mRNA vaccine) of the present disclosure. For instance, a
traditional vaccine includes, but is not limited to,
live/attenuated microorganism vaccines, killed/inactivated
microorganism vaccines, subunit vaccines, protein antigen vaccines,
DNA vaccines, and VLP vaccines. In some embodiments, a traditional
vaccine is a vaccine that has achieved regulatory approval and/or
is registered by a national drug regulatory body, for example the
Food and Drug Administration (FDA) in the United States or the
European Medicines Agency (EMA).
[0357] In some embodiments, the effective amount of immunogenic,
composition is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to
800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to
200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-,
2 to 40-, 2 to 30-, 2 to 20-, 2 to 10-2 to 9-, 2 to 8-, 2 to 7-, 2
to 6-, 2 to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-,
3 to 70 3 to 600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-,
3 to 100-, 3 to 90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to
40-, 3 to 30-, 3 to 20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to
6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4 to 900-, 4 to 800-, 4 to 700-,
4 to 600-, 4 to 500-, 4 to 400-, 4 to 4 to 00-, 4 to 200-, 4 to
100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4 to 50-, 4 to 40-, 4
to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to 7-, 4 to 6-, 4
to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, to 800-, 5 to 700-, 5 to
600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-, 5 to
90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-, 5
to 20-, 5 to 10-, 5 to 9-, 5 to 8-, 5 to 7-, 5 to 6-, 6 to 1000-, 6
to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6
to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to
60-, 6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6
to 8-, 6 to 7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to
600-, 7 to 500-, 7 to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to
90-, 7 to 80-, 7 to 70-, 7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7
to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to 1000-, 8 to 900-, 8 to
800-, 8 to 700-, 8 to 600-, 8 to 500-, 8 to 400-, 8 to 300-, 8 to
200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-, 8 to 50-,
8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9 to
900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to
300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-,
9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10
to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to
400-, 10 to 30 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to
70-, 10 to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to
1000-, 20 to 900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to 500-,
20 to 400-, 20 to 300-, 20 to 200-, 20 to 100-, 20 to 90-, 20 to
80-, 20 to 70 20 to 60-, 20 to 50-, 20 to 40-, 20 to 30-, 30 to
1000-, 30 to 900-, 30 to 800-, 30 to 700-, 30 to 600-, 30 to 500-,
30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-, 30 to 90-, 30 to
80-, 30 to 70 30 to 60-, 30 to 50-, 30 to 40-, 40 to 1000-, 40 to
900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to 400-,
40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40 to
70-, 40 to 60-, 40 to 50-. 50 to 1000-. 50 to 900-, 50 to 800-, 50
to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to
200-, 50 to 100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to
1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-,
60 to 400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to
80-, 60 to 70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70
to 600-, 70 to 500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to
100-, 70 to 90-, 70 to 80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80
to 700-, 80 to 600-, 80 to 500-, 80 to 400-, 80 to 300-, 80 to
200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to 900-, 90 to 800-,
90 to 700-, 90 to 600-, 90 to 500-, 90 to 400-, 90 to 300-, 90 to
200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-, 100 to
700-, 100 to 600-, 100 to 500-100 to 400-, 100 to 300-, 100 to
200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to
600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to
900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-. 300 to
400-. 400 to 1000-. 400 to 900-, 400 to 800-, 400 to 700-, 400 to
600-, 400 to 500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to
700-, 500 to 600-, 600 to 1000-, 600 to 900-, 600 to 800-, 600 to
700-, 700 to 1000-, 700 to 900-, 700 to 800-, 800 to 1000-, 800 to
900-, or 900 to 1000-fold reduction in the standard of care dose of
a recombinant protein vaccine.
[0358] In some embodiments, the anti-antigen antibody titer
produced in the subject is equivalent to an anti-antigen antibody
titer produced in a control subject administered the standard of
care dose of a recombinant or purified protein vaccine, or a live
attenuated or inactivated viral vaccine. In some embodiments, the
effective amount is a dose equivalent to (or equivalent to an at
least) 2-, 3 -,4 -,5 -,6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-,
60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-,
1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-, 270-, 280-,
290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-, 390-,
400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-,
510-, 520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-. 600-, 610-,
620-, 630-, 640-, 650-, 660-. 670-, 680-, 690-, 700-, 710-, 720-,
730-, 740-, 750-, 760-, 770-, 780-, 790-. 800-, 810-, 820--, 830-,
840-, 850-, 860-. 870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-,
950-, 960-, 970-, 980-, 990-, or 1000-fold reduction in the
standard of care dose of a recombinant protein vaccine. In some
embodiments, an anti-antigen antibody titer produced in the subject
is equivalent to an anti-antigen antibody titer produced in a
control subject administered the standard of care dose of a
recombinant or purified protein vaccine, or a live attenuated or
inactivated viral vaccine.
[0359] In some embodiments, the effective amount is 5 .mu.g-100
.mu.g of the mRNA encoding the antigen and/or the PADRE. For
example, the effective amount may be 5 .mu.g-10 .mu.g, 5 .mu.g-20
.mu.g, 5 .mu.g-30 .mu.g, 5 .mu.g-40 .mu.g, 5 .mu.g-50 .mu.g, 5
.mu.g-60 .mu.g, 5 .mu.g-70 .mu.g, 5 .mu.g-80 .mu.g, 5.mu.g-90
.mu.g, 5 .mu.g-100 .mu.g, 10 .mu.g-20 .mu.g, 10 .mu.g-30 .mu.g, 10
.mu.g-40 .mu.g, 10 .mu.g-50 .mu.g, 10 .mu.g-60 .mu.g, 10 .mu.g-70
.mu.g, 10 .mu.g-80 .mu.g, 10 .mu.g-90 .mu.g, 10 .mu.g-100 .mu.g, 25
.mu.g-30 .mu.g, 25 .mu.g-40 .mu.g, 25 .mu.g-50 .mu.g, 25 .mu.g-60
.mu.g, 25 .mu.g-70 .mu.g, 25 .mu.g-80 .mu.g, 25 .mu.g-90 .mu.g, 25
.mu.g-100 .mu.g, 50 .mu.g-60 .mu.g, 50 .mu.g-70 .mu.g, 50 .mu.g-80
.mu.g, 50 .mu.g-90 .mu.g, or 50 .mu.g-100 .mu.g of the mRNA In some
embodiments, the effective amount is 5 .mu.g, 10 .mu.g, 12.5 .mu.g,
20 .mu.g 25 .mu.g, 30 .mu.g, 40 .mu.g, 50 .mu.g, 60 .mu.g, 70
.mu.g, 80 .mu.g, 90 .mu.g, 100 .mu.g of the mRNA.
[0360] In some embodiments, a composition for or method of
vaccinating a subject comprises administering to the subject a
nucleic acid vaccine comprising at least one mRNA having an open
reading frame encoding an infectious disease antigen (e.g., a
viral, bacterial, and/or parasitic antigen) and/or a PADRE, wherein
a dosage of between 10 .mu.g/kg and 400 .mu.g/kg of the nucleic
acid vaccine is administered to the subject. In some embodiments
the dosage of the mRNA encoding the infectious disease antigen is
1-5 .mu.g, 5-10 .mu.g, 10-15 .mu.g, 15-20 .mu.g, 10-25 .mu.g, 20-25
.mu.g, 20-50 .mu.g, 30-50 .mu.g, 40-50 .mu.g, 40-60 .mu.g, 60-80
.mu.g, 60-100 .mu.g, 50-100 .mu.g, 80-120 .mu.g, 40-120 .mu.g,
40-150 .mu.g, 50-150 .mu.g, 50-200 .mu.g, 80-200 .mu.g, 100-200
.mu.g, 120-250 .mu.g, 150-250 .mu.g, 180-280 .mu.g, 200-300 .mu.g,
50-300 .mu.g, 80-300 .mu.g, 100-300 .mu.g, 40-300 .mu.g, 50-350
.mu.g, 100-350 .mu.g, 200-350 .mu.g, 300-350 .mu.g, 320-400 .mu.g,
40-380 .mu.g, 40-100 .mu.g, 100-400 .mu.g, 200-400 .mu.g, or
300-400 .mu.g per dose. In some embodiments the dosage of the mRNA
encoding the PADRE is 1-5 .mu.g, 5-10 .mu.g, 10-15 .mu.g, 15-20
.mu.g, 10-25 .mu.g, 20-25 .mu.g, 20-50 .mu.g, 30-50 .mu.g, 40-50
.mu.g, 40-60 .mu.g, 60-80 .mu.g, 60-100 .mu.g, 50-100 .mu.g, 80-120
.mu.g, 40-120 .mu.g, 40-150 .mu.g, 50-150 .mu.g, 50-200 .mu.g,
80-200 .mu.g, 100-200 .mu.g, 120-250 .mu.g, 150-250 .mu.g, 180-280
.mu.g, 200-300 .mu.g, 50-300 .mu.g, 80-300 .mu.g, 100-300 .mu.g,
40-300 .mu.g, 50-350 .mu.g, 100-350 .mu.g, 200-350 .mu.g, 300-350
.mu.g, 320-400 .mu.g, 40-380 .mu.g, 40-100 .mu.g, 100-400 .mu.g,
200-400 .mu.g, or 300-400 .mu.g per dose. In some embodiments the
dosage of the mRNA encoding the infectious disease antigen and the
PADRE is 1-5 .mu.g, 5-10 .mu.g, 10-15 .mu.g, 15-20 .mu.g, 10-25
.mu.g, 20-25 .mu.g, 20-50 .mu.g, 30-50 .mu.g, 40-50 .mu.g, 40-60
.mu.g, 60-80 .mu.g, 60-100 .mu.g, 50-100 .mu.g, 80-120 .mu.g,
40-120 .mu.g, 40-150 .mu.g, 50-150 .mu.g, 50-200 .mu.g, 80-200
.mu.g, 100-200 .mu.g, 120-250 .mu.g, 150-250 .mu.g, 180-280 .mu.g,
200-300 .mu.g, 50-300 .mu.g, 80-300 .mu.g, 100-300 .mu.g, 40-300
.mu.g, 50-350 .mu.g, 100-350 .mu.g, 200-350 .mu.g, 300-350 .mu.g,
320-400 .mu.g, 40-380, 40-100 .mu.g, 100-400 .mu.g, 200-400 .mu.g,
or 300-400 .mu.g per dose.
[0361] In some embodiments, a dosage of 25 micrograms of the mRNA
(e.g., mRNA encoding infectious disease antigen, encoding PADRE, or
encoding both the infectious disease antigen and PADRE) is included
in the nucleic acid vaccine administered to the subject. In some
embodiments, a dosage of 100 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In some
embodiments, a dosage of 50 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In some
embodiments, a dosage of 75 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In some
embodiments, a dosage of 150 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In some
embodiments, a dosage of 400 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In sonic
embodiments, a dosage of 200 micrograms of the mRNA (e.g., mRNA
encoding infectious disease antigen, encoding, PADRE, or encoding
both the infectious disease antigen and PADRE) is included in the
nucleic acid vaccine administered to the subject. In some
embodiments, the mRNA accumulates at a 100 fold higher level in the
local lymph node in comparison with the distal lymph node. In some
embodiments, the nucleic acid vaccine is chemically modified, and
in other embodiments the nucleic acid vaccine is not chemically
modified.
[0362] In sonic embodiments, the effective amount is a total dose
of 1-100 .mu.g. In some embodiments, the effective amount is a
total dose of 100 .mu.g. In some embodiments, the effective amount
is a dose of 25 .mu.g administered to the subject a total of one or
two times. In some embodiments, the effective amount is a dose of
100 .mu.g administered to the subject a total of two times. In some
embodiments, the effective amount is a dose of 1 .mu.g-10 .mu.g, 1
.mu.g-20 .mu.g, 1 .mu.g-30 .mu.g, 5 .mu.g-10 .mu.g, 5 .mu.g-20
.mu.g, 5 .mu.g-30 .mu.g, 5 .mu.g-40 .mu.g, 5 .mu.g-50 .mu.g, 10
.mu.g-15 .mu.g, 10 .mu.g-20 .mu.g, 10 .mu.g-25 .mu.g, 10 .mu.g-30
.mu.g, 10 .mu.g-40 .mu.g, 10 .mu.g-50 .mu.g, 10 .mu.g-60 .mu.g, 15
.mu.g-20 .mu.g, 15 .mu.g-25 .mu.g, 15 .mu.g-30 .mu.g, 15 .mu.g-40
.mu.g, 15 .mu.g-50 .mu.g, 20 .mu.g-25 .mu.g, 20 .mu.g-30 .mu.g, 20
.mu.g-40 .mu.g 20 .mu.g-50 .mu.g, 20 .mu.g-60 .mu.g, 20 .mu.g-70
.mu.g, 20 .mu.g-75.mu.g, 30 .mu.g-35 .mu.g, 30 .mu.g-40 .mu.g, 30
.mu.g-45 .mu.g 30 .mu.g-50 .mu.g, 30 .mu.g-60 .mu.g, 30 .mu.g-70
.mu.g, 30 .mu.g-75.mu.g which may be administered to the subject a
total of one or two times or more.
[0363] The immunogenic compositions, in some embodiments, may be
used in combination with one or more pharmaceutically acceptable
excipients and/or adjuvants (in addition to the PADRE).
[0364] Immunogenic compositions may be sterile, pyrogen-free or
both sterile and pyrogen-free. General considerations in the
formulation and/or manufacture of pharmaceutical agents, such as
immunogenic compositions (e.g., vaccines), may be found, for
example, in Remington: The Science and Practice of Pharmacy 21st
ed., Lippincott Williams &. Wilkins, 2005 (incorporated herein
by reference in its entirety).
[0365] Herein, the term "active ingredient" generally refers to
mRNA encoding an antigen and/or PADRE of the immunogenic
compositions. By way of example, an immunogenic composition may
comprise between 0.1% and 100%. e.g., between 0.5 and 50%, between
1-30%, between 5-80%, at least 80% (w/w) active ingredient.
Relative amounts of the active ingredient, the pharmaceutically
acceptable excipient, and/or any additional ingredients in an
immunogenic composition in accordance with the disclosure will
vary, depending upon the identity, size, and/or condition of the
subject treated and further depending upon the route by which the
composition is to be administered.
[0366] Immunogenic compositions can be formulated using one or more
excipients to: (1) increase stability; (2) increase cell
transfection; (3) permit the sustained or delayed release (e.g.,
from a depot formulation); (4) alter the biodistribution (e.g.,
target to specific tissues or cell types); (5) increase the
translation of encoded protein in vivo; and/or (6) alter the
release profile of encoded protein (antigen) in vivo. In addition
to traditional excipients such as any and all solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, excipients can include, without
limitation, lipidoids, liposomes, lipid nanoparticles, polymers,
lipoplexes, core-shell nanoparticles, peptides, proteins, cells
transfected the mRNA (e.g., for transplantation into a subject),
hyaluronidase, nanoparticle mimics and combinations thereof.
[0367] Immunogenic compositions may be prepared by any method known
or hereafter developed in the art of pharmacology. In general, such
preparatory methods include the step of bringing the active
ingredient (e.g., mRNA) into association with an excipient and/or
one or more other accessory ingredients, and then, if necessary
and/or desirable, dividing, shaping and/or packaging the product
into a desired single-dose or multi-dose unit.
Dosing/Administration
[0368] Provided herein are compositions e.g., pharmaceutical
compositions), methods, kits and reagents for prevention and/or
treatment of infectious disease (e.g., ZIKV) in humans and other
mammals, the RNA vaccines can be used as therapeutic or
prophylactic agents. In some aspects, the RNA vaccines of the
disclosure are used to provide prophylactic protection from an
infectious disease (e.g, ZIKV). In some aspects, the RNA vaccines
of the disclosure are used to treat an infection. In some
embodiments, the vaccines of the present disclosure are used in the
priming of immune effector cells, for example, to activate
peripheral blood mononuclear cells (PBMCs) ex vivo, which are then
infused (re-infused) into a subject.
[0369] A subject may be any mammal, including non-human primate and
human subjects. Typically, a subject is a human subject.
[0370] In some embodiments, the vaccines are administered to a
subject (e.g., a mammalian subject, such as a human subject) in an
effective amount to induce an antigen-specific immune response. The
RNA encoding the antigen is expressed and translated in vivo to
produce the antigen, which then stimulates an immune response in
the subject.
[0371] Prophylactic protection from infectious disease (e.g., ZIKV)
can be achieved following administration of an RNA vaccine of the
present disclosure. Vaccines can be administered once, twice, three
times, four times or more but it is likely sufficient to administer
the vaccine once (optionally followed by a single booster). It is
possible, although less desirable, to administer the vaccine to an
infected individual to achieve a therapeutic response. Dosing may
need to be adjusted accordingly.
[0372] A method of eliciting an immune response in a subject
against infectious disease (e.g., ZIKV) is provided in aspects of
the present disclosure. The method involves administering to the
subject an RNA vaccine comprising at least one RNA (e.g., mRNA)
having an open reading frame encoding at least one antigen, thereby
inducing in the subject an immune response specific to the antigen,
wherein anti-antigen antibody titer in the subject is increased
following vaccination relative to anti-antigen antibody titer in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine against the pathogen. An "anti-antigen
antibody" is a serum antibody the binds specifically to the
antigen.
[0373] A prophylactically effective dose is an effective dose that
prevents infection with the virus at a clinically acceptable level.
In some embodiments, the effective dose is a dose listed in a
package insert for the vaccine. A traditional vaccine, as used
herein, refers to a vaccine other than the mRNA vaccines of the
present disclosure. For instance, a traditional vaccine includes,
but is not limited, to live microorganism vaccines, killed
microorganism vaccines, subunit vaccines, protein antigen vaccines,
DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary
embodiments, a traditional vaccine is a vaccine that has achieved
regulatory approval and/or is registered by a national drug
regulatory body, for example the Food and Drug Administration (FDA)
in the United States or the European Medicines Agency (EMA).
[0374] In some embodiments, the anti-antigen antibody titer in the
subject is increased 1 log to 10 log following vaccination relative
to anti-antigen antibody titer in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
the pathogen or an unvaccinated subject. In some embodiments, the
anti-antigen antibody titer in the subject is increased 1 log, 2
log, 3 log, 4 log, 5 log, or 10 log following vaccination relative
to anti-antigen antibody titer in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
the pathogen or an unvaccinated subject.
[0375] A method of eliciting an immune response in a subject
against an infectious disease (e.g., ZIKV) is provided in other
aspects of the disclosure. The method involves administering to the
subject the RNA vaccine comprising at least one RNA polynucleotide
having an open reading frame encoding at least one infectious
disease (e.g., ZIKV) antigen, thereby inducing in the subject an
immune response specific to the antigen, wherein the immune
response in the subject is equivalent to an immune response in a
subject vaccinated with a traditional vaccine against the
infectious disease (e.g., ZIKV) at 2 times to 100 times the dosage
level relative to the RNA vaccine.
[0376] In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at twice the dosage level relative to the RNA
vaccine. In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at three times the dosage level relative to the
RNA vaccine. In some embodiments, the immune response in the
subject is equivalent to an immune response in a subject vaccinated
with a traditional vaccine at 4 times, 5 times, 10 times, 50 times,
or 100 times the dosage level relative to the RNA vaccine. In some
embodiments, the immune response in the subject is equivalent to an
immune response in a subject vaccinated with a traditional vaccine
at 10 times to 1000 times the dosage level relative to the RNA
vaccine. In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 100 times to 1000 times the dosage level
relative to the RNA vaccine.
[0377] In other embodiments, the immune response is assessed by
determining [protein] antibody titer in the subject. In other
embodiments, the ability of serum or antibody from an immunized
subject is tested for its ability to neutralize viral uptake or
reduce viral transformation of human B lymphocytes. In other
embodiments, the ability to promote a robust T cell response is
measured using art recognized techniques.
[0378] Other aspects the disclosure provide methods of eliciting an
immune response in a subject against an infectious disease (e.g.,
ZIKV) by administering to the subject an RNA vaccine comprising at
least one RNA polynucleotide having an open reading frame encoding
at least one antigen, thereby inducing in the subject an immune
response specific to the antigen, wherein the immune response in
the subject is induced 2 days to 10 weeks earlier relative to an
immune response induced in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
the pathogen. In some embodiments, the immune response in the
subject is induced in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine at 2 times to 100 times the
dosage level relative to the vaccine.
[0379] In some embodiments, the immune response in the subject is
induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks. 5 weeks, or 10
weeks earlier relative to an immune response induced in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine.
[0380] Also provided herein are methods of eliciting an immune
response in a subject against an infectious disease (e.g., ZIKV) by
administering to the subject an RNA vaccine having an open reading
frame encoding a first antigen, wherein the RNA polynucleotide does
not include a stabilization element, and wherein an adjuvant is not
co-formulated or co-administered with the vaccine.
[0381] The RNA (e.g., mRNA) vaccines may be administered by any
route which results in a therapeutically effective outcome. These
include, but are not limited, to intradermal, intramuscular,
intranasal, and/or subcutaneous administration. The present
disclosure provides methods comprising administering RNA vaccines
to a subject in need thereof. The exact amount required will vary
from subject to subject, depending on the species, age, and general
condition of the subject, the severity of the disease, the
particular composition, its mode of administration, its mode of
activity, and the like. The RNA (e.g., mRNA) vaccines compositions
are typically formulated in dosage unit form for ease of
administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the RNA (e.g., mRNA)
vaccines compositions may be decided by the attending physician
within the scope of sound medical judgment. The specific
therapeutically effective, prophylactically effective, or
appropriate imaging dose level for any particular patient will
depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed; and like factors well known in the
medical arts.
[0382] The effective amount of a vaccine, as provided herein, may
be as low as 20 .mu.g, administered for example as a single dose or
as two 10 .mu.g doses. In some embodiments, the effective amount is
a total dose of 20 .mu.g-200 .mu.g. For example, the effective
amount may be a total dose of 20 .mu.g, 25 .mu.g, 30 .mu.g, 35
.mu.g, 40 .mu.g, 45 .mu.g, 50 .mu.g, 55 .mu.g, 60 .mu.g, 65 .mu.g,
70 .mu.g, 75 .mu.g, 80 .mu.g, 85 .mu.g, 90 .mu.g, 95 .mu.g, 100
.mu.g, 110 .mu.s, 120 .mu.g, 130 .mu.g, 140 .mu.g, 150 .mu.g, 160
.mu.g, 170 .mu.g. 180 .mu.g, 190 .mu.g or 200 .mu.g. In some
embodiments, the effective amount is a total dose of 25 .mu.g-200
.mu.g. In some embodiments, the effective amount is a total dose of
50 .mu.g-200 .mu.g.
[0383] In some embodiments, the RNA (e.g., mRNA) vaccines
compositions may be administered at dosage levels sufficient to
deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005
mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5
mg/kg, 0.01 mg/kg to 50 mg/kg, 0,1 mg/kg to 40 mg/kg, 0.5 mg/kg to
30 mg/kg, 0.01 mg/kg to 10 ma/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg
to 25 mg/kg, of subject body weight per day, one or more times a
day, per week, per month, etc. to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect (see e.g., the range of
unit doses described in International Publication No. WO2013078199.
herein incorporated by reference in its entirety). The desired
dosage may be delivered three times a day, two times a day, once a
day, every other day, every third day, every week, every two weeks,
every three weeks, every four weeks, every 2 months, every three
months, every 6 months, etc. In certain embodiments, the desired
dosage may be delivered using multiple administrations (e.g., two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, or more administrations). When multiple
administrations are employed, split dosing regimens such as those
described herein may be used. In exemplary embodiments, the RNA
(e.g., mRNA) vaccines compositions may be administered at dosage
levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g.,
about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg,
about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about
0.004 mg/kg or about 0.005 mg/kg.
[0384] In some embodiments, the RNA (e.g., mRNA) vaccine
compositions may be administered once or twice (or more) at dosage
levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025
mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to
1.0 mg/kg.
[0385] In sonic embodiments, the RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050
mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,
0,225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375
mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg,
0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700
mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg,
0.875 mg. 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher
and lower dosages and frequency of administration are encompassed
by the present disclosure. For example, an RNA (e.g., mRNA) vaccine
composition may be administered three or four times.
[0386] In sonic embodiments, the RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.1.00 mg
or 0.400 mg.
[0387] In sonic embodiments, the RNA (e.g., mRNA) vaccine for use
in a method of vaccinating a subject is administered the subject a
single dosage of between 10 .mu.g/kg and 400 jig/kg of the nucleic
acid vaccine in an effective amount to vaccinate the subject. In
some embodiments, the RNA vaccine for use in a method of
vaccinating a subject is administered the subject a single dosage
of between 10 .mu.g and 400 .mu.g of the nucleic acid vaccine in an
effective amount to vaccinate the subject. In some embodiments, an
RNA (e.g., mRNA) vaccine for use in a method of vaccinating a
subject is administered to the subject as a single dosage of
25-1000 .mu.g (e.g., a single dosage of mRNA encoding an antigen).
In some embodiments, an RNA vaccine is administered to the subject
as a single dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000
.mu.g. For example, an RNA vaccine may be administered to a subject
as a single dose of 25-100, 25-500, 50-100, 50-500, 50-1000,
100-500, 100-1000, 250-500, 250-1000, or 500-1000 .mu.g. In some
embodiments, an RNA (e.g., mRNA) vaccine for use in a method of
vaccinating a subject is administered to the subject as two
dosages, the combination of which equals 25-1000 .mu.g of the RNA
(e.g., mRNA) vaccine.
[0388] An RNA (e.g., mRNA) vaccine pharmaceutical composition
described herein can be formulated into a dosage form described
herein, such as an intranasal, intratracheal, or injectable (e.g.,
intravenous, intraocular, intravitreal, intramuscular, intradermal,
intracardiac, intraperitoneal, and subcutaneous).
Methods of Treatment/Vaccination
[0389] Provided herein are methods of using the immunogenic
compositions (e.g., mRNA vaccines) for prevention and/or treatment
of an infectious disease (e.g., Zika virus). The immunogenic
compositions can be used as therapeutic or prophylactic agents,
alone or in combination with other vaccine(s). They may be used in
medicine to prevent and/or treat an infectious disease. In some
embodiments, the immunogenic compositions of the present disclosure
are used to provide prophylactic protection from an infectious
disease. Prophylactic protection can be achieved following
administration of an immunogenic compositions of the present
disclosure. Immunogenic compositions may be used to treat or
prevent "co-infections" containing two or more viral, bacterial
and/or parasitic infections. Immunogenic compositions can be
administered once, twice, three times, four times or more, but it
is likely sufficient to administer the composition once (optionally
followed by a single booster). It is possible, although less
desirable, to administer the composition to an infected individual
to achieve a therapeutic response. Dosing may need to be adjusted
accordingly.
[0390] Immunogenic compositions (e.g., mRNA vaccines) may be
utilized in various settings depending on the prevalence of the
infection or the degree or level of unmet medical need. As a
non-limiting example, immunogenic compositions may be utilized to
treat and/or prevent a variety of infectious diseases. Immunogenic
compositions (e.g., mRNA vaccines) of the present disclosure have
superior properties in that they produce much larger antibody
titers and produce responses early than commercially available
anti-viral/anti-bacterial agents/compositions.
[0391] A subject may be a mammalian subject, such as a human
subject. In some embodiments, a subject is an elderly human subject
(e.g., an individual that is at least 65 years of age). For
example, an elderly human subject may be 65, 66, 67, 68, 70, 71,72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 years of age or older.
In some embodiments, a subject is a child (e.g., an individual that
is 5 years of age or younger). For example, a child may be 5, 4, 3,
2, 1 years of age or younger. In some embodiments, a child may be
6-12 months of age. For example, a child may be 6-7, 6-8, 6-9,
6-10, 6-11, 6-12, 7-8, 7-9, 7-10, 7-11, 7-12, 8-9, 8-10, 8-11,
8-12, 9-10, 9-11, 9-1 10-11, 10-12, or 11-12 months of age. In some
embodiments, a child may be 6 months, 7 months, 8 months, 9 months,
10 months, 11 months, or 12 months of age. In some embodiments, a
subject is immunocompromised.
[0392] In some embodiments, an immunogenic composition capable of
eliciting an immune response is administered intramuscularly and
includes a compound according to Formula (I), (IA), (II), (IIa),
(IIb), (IIc), (IId) or (IIe) (e.g., Compound 3, 18, 20, 25, 26, 29,
30, 60, 108-112, or 122).
[0393] A method of eliciting an immune response in a subject
against an infectious disease is provided in aspects of the present
disclosure. In some embodiments, the method involves administering
to the subject an effective amount of an immunogenic composition
(e.g., mRNA vaccine) to induce in the subject an immune response
specific to an infectious disease antigen, wherein antibody titer
of antibodies against an infectious disease antigen in the subject
is increased following administration of the immunogenic
composition relative to antibody titer in a subject vaccinated with
a prophylactically effective dose of a traditional vaccine against
the infectious disease.
[0394] In some embodiments, the antigen-specific immune response is
characterized by measuring an anti-antigen antibody titer produced
in a subject administered an immunogenic composition (e.g., mRNA
vaccine) as provided herein. An antibody titer is a measurement of
the amount of antibodies within a subject, for example, antibodies
that are specific to a particular antigen (e.g., an anti-ZIKV F
protein) or epitope of an antigen. Antibody titer is typically
expressed as the inverse of the greatest dilution that provides a
positive result. Enzyme-linked immunosorbent assay (ELISA) is a
common assay for determining antibody titers, for example.
[0395] In some embodiments, an antibody titer is used to assess
whether a subject has had an infection or to determine whether
immunizations are required. In some embodiments, an antibody titer
is used to determine the strength of an autoimmune response, to
determine whether a booster immunization is needed, to determine
whether a previous vaccine was effective, and to identify any
recent or prior infections. In accordance with the present
disclosure, an antibody titer may be used to determine the strength
of an immune response induced in a subject by the immunogenic
composition (e.g., mRNA vaccine)
[0396] In some embodiments, an antibody induced by an immunogenic
composition is a neutralizing antibody against the infectious
disease antigen. A neutralizing titer is produced by a neutralizing
antibody against a viral, bacterial or parasitic antigen, for
example, as measured in serum of the subject. In some embodiments,
an effective dose of the immunogenic composition is sufficient to
produce more than a 500 neutralization titer. For example, an
effective dose of the immunogenic composition is sufficient to
produce a 1000-10,000 neutralization titer. In some embodiments, an
effective dose of the immunogenic composition is sufficient to
produce a 1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-6000,
1000-7000, 1000-8000, 1000-9000, 1000-10,000, 2000-3000, 2000-4000,
2000-5000, 2000-6000, 2000-7000, 2000-8000, 2000-9000, 2000-10,000,
3000-4000, 3000-5000, 3000-6000, 3000-7000, 3000-8000, 3000-9000,
3000-10,000, 4000-5000, 4000-6000, 4000-7000, 4000-8000, 4000-9000,
4000-10,000, 5000-6000, 5000-7000, 5000-8000, 5000-9000;
5000-10,000, 6000-7000, 6000-8000, 6000-9000, 6000-10,000,
7000-8000, 7000-9000, 7000-10,000, 8000-9000, 8000-10,000, or a
9000-10,000 neutralization titer. In some embodiments, an effective
dose of the immunogenic composition is sufficient to produce a 500,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000,
6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11000, 12,000,
13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19000, 20,000 or
higher neutralizing titer. In some embodiments, neutralizing titer
is produced 1-72 hours post administration. For example,
neutralizing titers may be produced 1-10, 1-20, 1-30, 1-40, 1-50,
1-60, 1-70, 1-72, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-72,
20-30, 20-40, 20-50, 20-60, 20-70, 20-72, 30-40, 30-50, 30-60,
30-70, 30-72, 40-50, 40-60, 40-70, 40-72, 50-60, 50-70, 50-72,
60-70, 60-72, or 70-72 hours post administration. In some
embodiments, neutralizing titers may be produced 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 56, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours
post administration.
[0397] In some embodiments, the anti-antigen antibody titer in the
subject is increased 1 log to 10 log following administration of
the immunogenic composition relative to anti-antigen antibody titer
in a subject vaccinated with a prophylactically effective dose of a
traditional vaccine against the antigen. In some embodiments, the
anti-antigen antibody titer in the subject is increased 1 log, 2
log, 3 log, 5 log or 10 log following administration of the
immunogenic composition relative to anti-antigen antibody titer in
a subject vaccinated with a prophylactically effective dose of a
traditional vaccine against the antigen.
[0398] In some embodiments, the anti-antigen antibody titer
produced in a subject is increased at least 2 times relative to a
control. For example, the anti-antigen antibody titer produced in a
subject may be increased at least 3 times, at least 4 times, at
least 5 times, at least 6 times, at least 7 times, at least 8
times, at least 9 times, or at least 10 times relative to a
control. In some embodiments, the anti-antigen antibody titer
produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10
times relative to a control. In some embodiments, the anti-antigen
antibody titer produced in a subject is increased 2-10 times
relative to a control. For example, the anti-antigen antibody titer
produced in a subject may be increased 2-10, 2-9, 2-8, 2-7. 2-6,
2-5, 2-4, 2-3. 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8,
4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,
7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.
[0399] A control, in some embodiments, is the anti-antigen antibody
titer produced in a subject who has not been administered
immunogenic composition of the present disclosure. In some
embodiments, a control is the anti-antigen antibody titer produced
in a subject who has been administered a live attenuated viral
vaccine. An attenuated vaccine is a vaccine produced by reducing
the virulence of a viable (live). An attenuated virus is altered in
a manner that renders it harmless or less virulent relative to
live, unmodified virus. In some embodiments, a control is an
anti-antigen antibody titer produced in a subject administered
inactivated viral vaccine. In some embodiments, a control is an
anti-antigen antibody titer produced in a subject administered a
recombinant or purified protein vaccine. Recombinant protein
vaccines typically include protein antigens that either have been
produced in a heterologous expression system (e.g., bacteria or
yeast) or purified from large amounts of the pathogenic organism.
In some embodiments, a control is an antibody titer produced in a
subject who has been administered a virus-like particle (VLP)
vaccine.
[0400] In some embodiments, the method involves administering to
the subject an effective amount of an immunogenic composition
(e.g., mRNA vaccine) to induce in the subject an immune response
specific to an infectious disease, wherein the immune response in
the subject is equivalent to an immune response in a subject
vaccinated with a traditional vaccine against the infectious
disease antigen at 2 times to 100 times the dosage level relative
to the immunogenic composition. In some embodiments, the immune
response in the subject is equivalent to an immune response in a
subject vaccinated with a traditional vaccine at 2, 3, 4, 5, 10,
50, 100 times the dosage level relative to the immunogenic
composition (e.g., mRNA vaccine). In some embodiments, the immune
response in the subject is equivalent to an immune response in a
subject vaccinated with a traditional vaccine at 10-100 times, or
100-1000 times, the dosage level relative to the immunogenic
composition (e.g., mRNA vaccine). In some embodiments, the immune
response is assessed by determining [protein] antibody titer in the
subject.
[0401] In some embodiments, the immune response in the subject is
induced 2 days earlier, or 3 days earlier, relative to an immune
response induced in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine. In some embodiments, the
immune response in the subject is induced 1 week, 2 weeks, 3 weeks,
5 weeks, or 10 weeks earlier relative to an immune response induced
in a subject vaccinated with a prophylactically effective dose of a
traditional vaccine.
[0402] In sonic embodiments, the method involves administering to
the subject an effective amount of an immunogenic composition(e.g.,
mRNA vaccine) to produce an anti-antigen antibody titer equivalent
to an anti-antigen antibody titer produced in a control subject
administered a standard of care dose of a recombinant or purified
protein vaccine or a live attenuated or inactivated viral
vaccine.
[0403] In some embodiments, an effective amount of an immunogenic
composition (e.g., mRNA vaccine) is a dose equivalent to an at
least 2-fold reduction in a standard of care dose of a recombinant
or purified protein vaccine. For example, an effective amount of an
immunogenic composition may be a dose equivalent to an at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, or at least 10-fold
reduction in a standard of care dose of a recombinant or purified
protein vaccine. In sonic embodiments, an effective amount of an
immunogenic composition is a dose equivalent to an at least at
least 100-fold, at least 500-fold, or at least 1000-fold reduction
in a standard of care dose of a recombinant or purified protein
vaccine. In some embodiments, an effective amount of an immunogenic
composition is a dose equivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-,
9-, 10-, 20-, 50-, 100-, 500-, or 1000-fold reduction in a standard
of care dose of a recombinant or purified protein vaccine.
[0404] In some embodiments, the anti-antigen antibody titer
produced in a subject administered an effective amount of an
immunogenic composition (e.g., mRNA vaccine) is equivalent to an
anti-antigen antibody titer produced in a control subject
administered the standard of care dose of a recombinant or protein
vaccine, or a live attenuated or inactivated viral vaccine. In
sonic embodiments, an effective amount of an immunogenic
composition is a dose equivalent to a 2-fold to 1000-fold (e.g.,
2-fold to 100-fold, 10-fold to 1000-fold) reduction in the standard
of care dose of a recombinant or purified protein vaccine, wherein
die anti-antigen antibody titer produced in the subject is
equivalent to an anti-antigen antibody titer produced in a control
subject administered the standard of care dose of a recombinant or
purified protein vaccine, or a live attenuated or inactivated viral
vaccine.
[0405] In some embodiments, an effective dose of an immunogenic
composition (e.g., mRNA vaccine) described herein is sufficient to
produce detectable levels of viral, bacterial or parasitic as
measured in serum of the subject at 1-72 hours post administration.
For example, antigen may be detected at 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 72 hours
post administration. In some embodiments, the cut off index of the
antigen is 1-2 (e.g., 1, 1.5, or 2). In some embodiments, wherein
the effective dose is sufficient to produce a 1,000-10,000
neutralization titer produced by neutralizing antibody against
antigen as measured in serum of the subject at 1-72 hours post
administration. In some embodiments, the cut-off index of antigen
is 1-2.
Vaccine Efficacy
[0406] Some aspects of the present disclosure provide formulations
of the RNA (e.g., mRNA) vaccine, wherein the RNA vaccine is
formulated in an effective amount to produce an antigen specific
immune response in a subject (e.g., production of antibodies
specific to an antigen). "An effective amount" is a dose of an RNA
(e.g., mRNA) vaccine effective to produce an antigen-specific
immune response. Also provided herein are methods of inducing an
antigen-specific immune response in a subject.
[0407] As used herein, an immune response to a vaccine or LNP of
the present invention is the development in a subject of a humoral
and/or a cellular immune response to a (one or more) protein(s)
present in the vaccine. For purposes of the present invention, a
"humoral" immune response refers to an immune response mediated by
antibody molecules, including, e.g., secretory (IgA) or IgG
molecules, while a "cellular" immune response is one mediated by
T-lymphocytes (e.g., CD4+ helper and/or CDS+ T cells (e.g., CTLs)
and/or other white blood cells. One important aspect of cellular
immunity involves an antigen-specific response by cytolytic T-cells
(CTLs). CTLs have specificity for peptide antigens that are
presented in association with proteins encoded by the major
histocompatibility complex (MHC) and expressed on the surfaces of
cells. CTLs help induce and promote the destruction of
intracellular microbes or the lysis of cells infected with such
microbes. Another aspect of cellular immunity involves and
antigen-specific response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity nonspecific
effector cells against cells displaying peptide antigens in
association with MHC molecules on their surface. A cellular immune
response also leads to the production of cytokines, chemokines, and
other such molecules produced by activated T-cells and/or other
white blood cells including those derived from CD4+ and CD8+
T-cells.
[0408] In some embodiments, the antigen-specific immune response is
characterized by measuring an anti-antigen antibody titer produced
in a subject administered an RNA (e.g., mRNA) vaccine as provided
herein. An antibody titer is a measurement of the amount of
antibodies within a subject, for example, antibodies that are
specific to a particular antigen (e.g., an anti-antigen) or epitope
of an antigen. Antibody titer is typically expressed as the inverse
of the greatest dilution that provides a positive result.
Enzyme-linked immunosorbent assay (ELISA) is a common assay for
determining antibody titers, for example.
[0409] In some embodiments, an antibody titer is used to assess
whether a subject has had an infection or to determine whether
immunizations are required. In some embodiments, an antibody titer
is used to determine the strength of an autoimmune response, to
determine whether a booster immunization is needed, to determine
whether a previous vaccine was effective, and to identify any
recent or prior infections. In accordance with the present
disclosure, an antibody titer may be used to determine the strength
of an immune response induced in a subject by the RNA (e.g., mRNA)
vaccine.
[0410] In some embodiments, an anti-antigen antibody titer produced
in a subject is increased by at least 1 log relative to a control.
For example, anti-antigen antibody titer produced in a subject may
be increased by at least 1.5, at least 2, at least 2.5, or at least
3 log relative to a control. In some embodiments, the anti-antigen
antibody titer produced in the subject is increased by 1, 1.5, 2,
2.5 or 3 log relative to a control. In some embodiments, the
anti-antigen antibody titer produced in the subject is increased by
1-3 log relative to a control. For example, the anti-antigen
antibody titer produced in a subject may be increased by 1-1.5,
1-2, 1-2,5, 1-3, 1,5-2, 1.5-2.5, 1,5-3, 2-2,5, 2-3, or 2.5-3 log
relative to a control.
[0411] In some embodiments, the anti-antigen antibody titer
produced in a subject is increased at least 2 times relative to a
control. For example, the anti-antigen antibody titer produced in a
subject may be increased at least 3 times, at least 4 times, at
least 5 times, at least 6 times, at least 7 times, at least 8
times, at least 9 times, or at least 10 times relative to a
control. In some embodiments, the anti-antigen antibody titer
produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10
times relative to a control. In some embodiments, the anti-antigen
antibody titer produced in a subject is increased 2-10 times
relative to a control. For example, the anti-antigen antibody titer
produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6,
2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8,
4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,
7-9, 8-10, 8-9, or 9-10 times relative to a control.
[0412] A control, in some embodiments, is the anti-antigen antibody
titer produced in a subject who has not been administered an RNA
(e.g., mRNA) vaccine. In some embodiments, a control is an
anti-antigen antibody titer produced in a subject administered a
recombinant or purified protein vaccine. Recombinant protein
vaccines typically include protein antigens that either have been
produced in a heterologous expression system bacteria or yeast) or
purified from large amounts of the pathogenic organism.
[0413] In some embodiments, the ability of a vaccine to be
effective is measured in a murine model. For example, the vaccines
may be administered to a murine model and the murine model assayed
for induction of neutralizing antibody titers. Viral challenge
studies may also be used to assess the efficacy of a vaccine of the
present disclosure. For example, the vaccines may be administered
to a murine model, the murine model challenged with the pathogen
(e.g., ZIKV) and the murine model assayed for survival and/or
immune response (e.g., neutralizing antibody response, T cell
response (e.g., cytokine response)).
[0414] In some embodiments, an effective amount of an RNA (e.g.,
mRNA) vaccine is a dose that is reduced compared to the standard of
care dose of a recombinant protein vaccine. A "standard of care,"
as provided herein, refers to a medical or psychological treatment
guideline and can be general or specific. "Standard of care"
specifies appropriate treatment based on scientific evidence and
collaboration between medical professionals involved in the
treatment of a given condition. It is the diagnostic and treatment
process that a physician/clinician should follow for a certain type
of patient, illness or clinical circumstance. A "standard of care
dose," as provided herein, refers to the dose of a recombinant or
purified protein vaccine, or a live attenuated or inactivated
vaccine, or a VLP vaccine, that a physician/clinician or other
medical professional would administer to a subject to treat or
prevent, or an infectious disease-related condition, while
following the standard of care guideline for treating or preventing
infectious disease (e.g., ZIKV), or related condition.
[0415] In some embodiments, the anti-antigen antibody titer
produced in a subject administered an effective amount of an RNA
vaccine is equivalent to an anti-antigen antibody titer produced in
a control subject administered a standard of care dose of a
recombinant or purified protein vaccine, or a live attenuated or
inactivated vaccine, or an VLP vaccine.
[0416] In some embodiments, an effective amount of an RNA (e.g.,
mRNA) vaccine is a dose equivalent to an at least 2-fold reduction
in a standard of care dose of a recombinant or purified protein
vaccine. For example, an effective amount of an RNA vaccine may be
a dose equivalent to an at least 3-fold, at least 4-fold, at least
5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least
9-fold, or at least 10-fold reduction in a standard of care dose of
a recombinant or purified protein vaccine. In some embodiments, an
effective amount of an RNA vaccine is a dose equivalent to an at
least at least 100-fold, at least 500-fold, or at least 1000-fold
reduction in a standard of care dose of a recombinant or purified
protein vaccine. In some embodiments, an effective amount of an RNA
vaccine is a dose equivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 10-,
20-, 50-, 100-, 250-, 500-, or 1000-fold reduction in a standard of
care dose of a recombinant or purified protein vaccine. In some
embodiments, the anti-antigen antibody titer produced in a subject
administered an effective amount of RNA vaccine is equivalent to an
anti-antigen antibody titer produced in a control subject
administered the standard of care dose of a recombinant or protein
vaccine, or a live attenuated or inactivated vaccine, or an VLP
vaccine. In some embodiments, an effective amount of an RNA (e.g.,
mRNA) vaccine is a dose equivalent to a 2-fold to 1000-fold (e.g.,
2-fold to 100-fold, 10-fold to 1000-fold) reduction in the standard
of care dose of a recombinant or purified protein vaccine, wherein
the anti-antigen antibody titer produced in the subject is
equivalent to an anti-antigen antibody titer produced in a control
subject administered the standard of care dose of a recombinant or
purified protein vaccine, or a live attenuated or inactivated
vaccine, or an VLP vaccine.
[0417] In some embodiments, the effective amount of an RNA (e.g.,
mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to
800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to
200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-,
2 to 40-, 2 to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-,
2 to 6-, 2 to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to
800-, 3 to 700-, 3 to 600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3
to 200-, 3 to 100-, 3 to 90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to
50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to
7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4 to 900-, 4 to 800-, 4
to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to 4 to 00-, 4 to 200-,
4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4 to 50, 4 to
40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to 7-, 4 to
6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5 to 700-,
5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-, 5
to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-,
5 to 20-, 5 to 10-, 5 to 9-, 5 to 8, 5 to 7-, 5 to 6-, 6 to 1000-,
6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6
to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to
60-, 6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6
to 8-, 6 to 7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to
600-, 7 to 500-, 7 to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to
90-, 7 to 80-, 7 to 70-, 7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7
to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to 1000-, 8 to 900-, 8 to
800-, 8 to 700-, 8 to 600-, 8 to 500-, 8 to 400-, 8 to 300-, 8 to
200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-, 8 to 50-,
8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9 to
900-, 9 to 800-, 9 to 00-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to
300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-,
9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10
to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to
400-, 10 to 300-, 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10
to 70-, 10 to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20
to 1000-, 20 to 900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to
500-, 20 to 400-, 20 to 300-, 20 to 200-, 20 to 100-, 20 to 90-, 20
to 80-, 20 to 70-, 20 to 60-, 20 to 50-, 20 to 40-, 20 to 30-, 30
to 1000-, 30 to 900-, 30 to 800-, 30 to 700-, 30 to 600-, 30 to
500-, 30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-, 30 to 90-, 30
to 80-, 30 to 70-, 30 to 60-, 30 to 50-, 30 to 40-, 40 to 1000-, 40
to 900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to
400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40
to 70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50 to 800-,
50 to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to
200-, 50 to 100-, 50 to 90-, 50 to 80-. 50 to 70-, 50 to 60-, 60 to
1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-,
60 to 400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to
80-, 60 to 70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70
to 600-, 70 to 500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to
100-, 70 to 90-, 70 to 80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80
to 700-, 80 to 600-, 80 to 500-, 80 to 400-, 80 to 300-, 80 to
200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to 900-, 90 to 800-,
90 to 700-, 90 to 600-, 90 to 500-90 to 400-, 90 to 300-, 90 to
200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-, 100 to
700-, 100 to 6W-, 100 to 500-, 100 to 400-, 100 to 300-, 100 to
200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to
600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to
900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to
400-, 400 to 1000-, 400 to 900-, 400 to 800-, 400 to 700-, 400 to
600-, 400 to 500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to
700-, 500 to 600-, 600 to 1000-, 600 to 900-, 600 to 800-, 600 to
700-, 700 to 1000-, 700 to 900-, 700 to 800-, 800 to 1000-, 800 to
900-, or 900 to 1000-fold reduction in the standard of care dose of
a recombinant protein vaccine. In sonic embodiments, such as the
foregoing, the anti-antigen antibody titer produced in the subject
is equivalent to an anti-antigen antibody titer produced in a
control subject administered the standard of care dose of a
recombinant or purified protein vaccine, or a live attenuated or
inactivated vaccine, or an VLP vaccine. In some embodiments, the
effective amount is a dose equivalent to (or equivalent to an at
least) 2-, 3-, 4 -,5 -,6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-,
60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-,
1280-, 190-, 200-, 210-, 220-, 230-, 240-. 250-, 260-, 270-, 280-,
290-, 300-, 310-, 320-, 330-, 340-, 3 360-, 370-, 380-, 390-, 400-,
410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-, 510-,
520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-,
630-, 640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-,
740-, 750-, 760-, 770-, 780-, 790-, 800-, 810-, 820--, 830-, 840-,
850-, 860-, 870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-,
960-, 970-, 980-, 990-, or 1000-fold reduction in the standard of
care dose of a recombinant protein vaccine. In some embodiments,
such as the foregoing, an anti-antigen antibody titer produced in
the subject is equivalent to an anti-antigen antibody titer
produced in a control subject administered the standard of care
dose of a recombinant or purified protein vaccine, or a live
attenuated or inactivated vaccine, or an VLP vaccine.
[0418] In some embodiments, the effective amount of an RNA (e.g.,
mRNA) vaccine is a total dose of 50-1000 .mu.g. In some
embodiments, the effective amount of an RNA (e.g, mRNA) vaccine is
a total dose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500,
50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60,
60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300,
60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900, 70-800,
70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90,
70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400,
80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700,
90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900,
100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200,
200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400,
200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500,
300-4(X), 400-1000, 400-9(X), 400-800, 400-700, 400-600, 400-500,
500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900,
600-900, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or
900-1000 .mu.g. In some embodiments, the effective amount of an RNA
(e.g., mRNA) vaccine is a total dose of 50, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950 or 1000 .mu.g. In some embodiments, the effective amount is a
dose of 25-500 .mu.g administered to the subject a total of two
times. In some embodiments, the effective amount of an RNA (e.g.,
mRNA) vaccine is a dose of 25-500, 25-400, 25-300, 25-200, 25-100,
25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400,
100-300, 100-200, 150-500, 150-400, 150-300, 150-200, 200-500,
200-400, 200-300, 250-500, 250-40) 250-300, 300-500, 300-400,
350-500, 350-400, 400-500 or 450-5001 .mu.g administered to the
subject a total of two times. In some embodiments, the effective
amount of an RNA (e.g., mRNA) vaccine is a total dose of 25, 50,
100, 150, 200, 250, 300, 350, 400, 450, or 500 .mu.g administered
to the subject a total of two times.
[0419] Vaccine efficacy may be assessed using standard analyses
(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;
201(11):1607-10). For example, vaccine efficacy may be measured by
double-blind, randomized, clinical controlled trials. Vaccine
efficacy may be expressed as a proportionate reduction in disease
attack rate (AR) between the unvaccinated (ARU) and vaccinated
(ARV) study cohorts and can be calculated from the relative risk
(RR) of disease among the vaccinated group with use of the
following formulas:
Efficacy=(ARU-ARV)/ARU.times.100; and
Efficacy=(1.times.RR).times.100.
[0420] Likewise, vaccine effectiveness may be assessed using
standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010
Jun. 1; 201(11):1607-10). Vaccine effectiveness is an assessment of
how a vaccine (which may have already proven to have high vaccine
efficacy) reduces disease in a population. This measure can assess
the net balance of benefits and adverse effects of a vaccination
program, not just the vaccine itself, under natural field
conditions rather than in a controlled clinical trial. Vaccine
effectiveness is proportional to vaccine efficacy (potency) but is
also affected by how well target groups in the population are
immunized, as well as by other non-vaccine-related factors that
influence the `real-world` outcomes of hospitalizations, ambulatory
visits, or costs. For example, a retrospective case control
analysis may be used, in which the rates of vaccination among a set
of infected cases and appropriate controls are compared. Vaccine
effectiveness may be expressed as a rate difference, with use of
the odds ratio (OR) for developing infection despite
vaccination:
Effectiveness=(1-OR).times.100.
[0421] In some embodiments, efficacy of the vaccine is at least 60%
relative to unvaccinated control subjects. For example, efficacy of
the vaccine may be at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 95%, at least 98%, or 100%
relative to unvaccinated control subjects.
[0422] Sterilizing Immunity. Sterilizing immunity refers to a
unique immune status that prevents effective pathogen infection
into the host. In some embodiments, the effective amount of a
vaccine of the present disclosure is sufficient to provide
sterilizing immunity in the subject for at least 1 year. For
example, the effective amount of a vaccine of the present
disclosure is sufficient to provide sterilizing immunity in the
subject for at least 2 years, at least 3 years, at least 4 years,
or at least 5 years. In some embodiments, the effective amount of a
vaccine of the present disclosure is sufficient to provide
sterilizing immunity in the subject at an at least 5-fold lower
dose relative to control. For example, the effective amount may be
sufficient to provide sterilizing immunity in the subject at an at
least 10-fold lower, 15-fold, or 20-fold lower dose relative to a
control.
[0423] Detectable Antigen. In some embodiments, the effective
amount of a vaccine of the present disclosure is sufficient to
produce detectable levels of antigen as measured in serum of the
subject at 1-72 hours post administration.
[0424] Titer. An antibody titer is a measurement of the amount of
antibodies within a subject, for example, antibodies that are
specific to a particular antigen (e.g., an anti-antigen) Antibody
titer is typically expressed as the inverse of the greatest
dilution that provides a positive result. Enzyme-linked
immunosorbent assay (ELISA) is a common assay for determining
antibody titers, for example.
[0425] In some embodiments, the effective amount of a vaccine of
the present disclosure is sufficient to produce a 1,000-10,000
neutralizing antibody titer produced by neutralizing antibody
against the antigen as measured in serum of the subject at 1-72
hours post administration. In some embodiments, the effective
amount is sufficient to produce a 1,000-5,000 neutralizing antibody
titer produced by neutralizing antibody against the antigen as
measured in serum of the subject at 1-72 hours post administration.
In some embodiments, the effective amount is sufficient to produce
a 5,000-10,000 neutralizing antibody titer produced by neutralizing
antibody against the antigen as measured in serum of the subject at
1-72 hours post administration.
[0426] In some embodiments, the neutralizing antibody titer is at
least 100 NT.sub.50. For example, the neutralizing antibody titer
may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000
NT50. In some embodiments, the neutralizing antibody titer is at
least 10,000 NT.sub.50.
[0427] In some embodiments, the neutralizing antibody titer is at
least 100 neutralizing units per milliliter (NU/mL). For example,
the neutralizing antibody titer may be at least 200, 300, 400, 500,
600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the
neutralizing antibody titer is at least 10,000 NU/mL.
[0428] In some embodiments, an anti-antigen antibody titer produced
in the subject is increased by at least 1 log relative to a
control. For example, an anti-antigen antibody titer produced in
the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or
10 log relative to a control.
[0429] In some embodiments, an anti-antigen antibody titer produced
in the subject is increased at least 2 times relative to a control.
For example, an anti-antigen antibody titer produced in the subject
is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative
to a control.
[0430] In some embodiments, a geometric mean, which is the nth root
of the product of n numbers, is generally used to describe
proportional growth. Geometric mean, in some embodiments, is used
to characterize antibody titer produced in a subject.
[0431] A control may be, for example, an unvaccinated subject, or a
subject administered a live attenuated vaccine, an inactivated
vaccine, or a protein subunit vaccine.
[0432] In some embodiments, the immunogenic composition immunizes
the subject against infection for up to 2 years (e.g., 6 months. 12
months, 18 months, or 24 months). I n some embodiments the
immunogenic composition immunizes the subject against infection for
more than 2 years (e.g., 3, 4, 5, 6, 7, 8, 9, 10 years or
more).
[0433] In some embodiments, the antigen-specific immune response
comprises a B cell response. In some embodiments, the
antigen-specific immune response comprises a T cell response.
[0434] In some embodiments, the antigen-specific immune response
comprises a PADRE-specific CD4+ T cell response. A PADRE-specific
CD4+ T cell response may be an increase in an antigen-specific
immune response in a subject administered an immunogenic
composition of the present disclosure (comprising mRNA encoding
PADRE), relative to an antigen-specific immune response (to the
same antigen) in the absence of PADRE. The level of an
antigen-specific immune response may be assessed by measuring
levels of B cell activation, antibody production, cytotoxic T cell
activation or helper I cell activation, for example.
[0435] In some embodiments, the antigen-specific immune response is
0.1 to 10 times stronger than an antigen-specific immune response
induced in a subject administered a control immunogenic composition
without an mRNA encoding a PADRE. In some embodiments, the
antigen-specific immune response is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times stronger. In
some embodiments, the antigen-specific immune response is at least
2 times stronger than an antigen-specific immune response induced
in a subject administered a control immunogenic composition without
an mRNA encoding a PADRE. In some embodiments, the antigen-specific
immune response is at least 5 times stronger than an
antigen-specific immune response induced in a subject administered
a control immunogenic composition without an mRNA encoding a PADRE.
In some embodiments, the antigen-specific immune response is at
least 10 times stronger than an antigen-specific immune response
induced in a subject administered a control immunogenic composition
without an mRNA encoding a PADRE.
[0436] Immunogenic compositions described herein may be
administered by any route which results in a therapeutically
effective outcome. These include, but are not limited, to
intradermal, intramuscular, and/or subcutaneous administration.
Immunogenic compositions described herein can be formulated into a
dosage form described herein, such as an intranasal, intratracheal,
or injectable (e.g., intravenous, intraocular, intravitreal,
intramuscular, intradermal, intracardiac, intraperitoneal, and
subcutaneous). In some embodiments, immunogenic compositions may be
administered intramuscularly or intradermally, similarly to the
administration of inactivated vaccines known in the art. Other
modes of administration are encompassed by the present
disclosure.
[0437] Immunogenic compositions may be administrated with other
prophylactic or therapeutic compounds. As a non-limiting example, a
prophylactic or therapeutic compound may be an adjuvant or a
booster. As used herein, when referring to a prophylactic
composition, such as a vaccine, the term "booster" refers to an
extra administration of the prophylactic (vaccine) composition. A
booster (or booster vaccine) may be given after an earlier
administration of the prophylactic composition. The time of
administration between the initial administration of the
prophylactic composition and the booster may be, but is not limited
to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes,
20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years,
17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35
years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years,
70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more
than 99 years. In some embodiments, the time of administration
between the initial administration of the prophylactic composition
and the booster may be, but is not limited to, 1 week, 2 weeks, 3
weeks, 1 month, 2 months, 3 months, 6 months or 1 year.
[0438] The exact amount of an immunogenic composition (e.g., mRNA
vaccine) required will vary from subject to subject, depending on
the species, age, and general condition of the subject, the
severity of the disease, the particular composition, its mode of
administration, its mode of activity, and the like. Immunogenic
compositions are typically formulated in dosage unit form for ease
of administration and uniformity of dosage. It will be understood,
however, that the total daily usage of the immunogenic compositions
may be decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective,
prophylactically effective, or appropriate imaging dose level for
any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts.
[0439] In some embodiments, the immunogenic compositions may be
administered at dosage levels sufficient to deliver the active
(e.g., mRNA) at 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05
mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05
mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0,1 mg/kg to 40 mg/kg,
0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10
mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one
or more times a day, per week, per month, etc. to obtain the
desired therapeutic, diagnostic, prophylactic, or imaging effect
(see, e.g., the range of unit doses described in International
Publication No WO2013078199, the contents of which are herein
incorporated by reference in their entirety). The desired dosage
may be delivered three times a day, two times a day, once a day,
every other day, every third day, every week, every two weeks,
every three weeks, every four weeks, every 2 months, every three
months, every 6 months, etc. In some embodiments, the desired
dosage may be delivered using multiple administrations (e.g., two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, or more administrations). When multiple
administrations are employed, split dosing regimens such as those
described herein may be used. In some embodiments, immunogenic
compositions (e.g., mRNA vaccines) may be administered at dosage
levels sufficient to deliver the active (e.g., mRNA) at 0.0005
mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075
mg/kg, e.g., about 0.0005 mg/kg, about 0,001 mg/kg, about 0.002
mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005
mg/kg.
[0440] In some embodiments, immunogenic compositions may be
administered once or twice (or more) at dosage levels sufficient to
deliver the active (e.g., mRNA) at 0.025 mg/kg to 0.250 mg/kg,
0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025
mg/kg to 1.0 mg/kg.
[0441] In some embodiments, immunogenic compositions (e.g., mRNA
vaccines) may be administered twice (e.g., Day 0 and Day 7, Day 0
and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050
mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,
0,225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375
mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg,
0,550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700
mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg,
0.875 mg. 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher
and lower dosages and frequency of administration are encompassed
by the present disclosure. For example, an immunogenic composition
(e.g., mRNA vaccine) may be administered three or four times.
[0442] In some embodiments, the immunogenic compositions may be
administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0
and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90,
Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and
3 months later, Day 0 and 6 months later, Day 0 and 9 months later,
Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2
years later, Day 0 and 5 years later, or Day 0 and 10 years later)
at a total dose of or at dosage levels sufficient to deliver a
total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.
[0443] In sonic embodiments, an immunogenic compositions for use in
a method of vaccinating a subject is administered to the subject as
a single dosage of between 10 .mu.g/kg and 400 .mu.g/kg of the mRNA
(in an effective amount to vaccinate the subject). In some
embodiments the immunogenic composition for use in a method of
vaccinating a subject is administered to the subject as a single
dosage of between 10 .mu.g and 400 .mu.g of the mRNA (in an
effective amount to vaccinate the subject).
[0444] In some embodiments, an immunogenic composition for use in a
method of vaccinating a subject is administered to the subject as a
single dosage of 2-1000 .mu.g (e.g., a single dosage of mRNA
encoding antigen and/or PADRE). In some embodiments, an immunogenic
composition for use in a method of vaccinating a subject is
administered to the subject as a single dosage of 5-100 .mu.g
(e.g., a single dosage of mRNA encoding antigen and/or PADRE). In
some embodiments, an immunogenic composition is administered to the
subject as a single dosage of 2, 5, 10, 15, 20 25, 50, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550. 600, 650, 700, 750. 800,
850, 900, 950 or 1000 .mu.g. For example, an immunogenic
composition may be administered to a subject as a single dose of
5-100, 10-100, 15-100, 20-100, 25-100, 25-500, 50-100, 50-500,
50-1000, 100-500, 100-1000, 250-500, 250-1000, or 500-1000 .mu.g.
In some embodiments, an immunogenic composition for use in a method
of vaccinating a subject is administered to the subject as two
dosages, the combination of which equals 10-1000 .mu.g of the mRNA
encoding antigen and/or PADRE.
EXAMPLES
Example 1
An mRNA Vaccine Platform for ZIKV
[0445] A vaccine platform is developed for generating multiple
lipid nanoparticles that encapsulate modified mRNA for
intramuscular delivery to induce high levels of protein expression
in vivo. The flexibility of platform allows for the inclusion of
multiple mRNA encoding any flavivirus proteins (e.g., NS1, prME)
that could augment protective responses (Costa et al., 2007; Costa
et al., 2006), or immunodominant helper CD4 T cell epi topes (e.g.,
a PAR epitope as described in Hung et al., 2007, Mol Ther. 2007
June; 15(6): 1211-1219, incorporated herein by reference).
[0446] A modified mRNA was designed, which encodes the prM and E
proteins from an Asian ZIKV strain (Mirconesia 2007, GenBank
accession number EL 545988 (Lanciotti et al., 2008)), which has
>99% amino acid sequence identity relative to strains from the
Americas. A second modified mRNA is synthesized to encode a Pan
HLA-DR reactive epitope (PADRE), which is a CD4+ T-helper epitope.
Alternatively, a third modified mRNA is designed to encode the prM
and E genes from an Asian ZIKV strain (GenBank accession number
EU545988) fused to a PADRE epitope. The amino acid sequence of the
PADRE epitope is AKFVAAWTLKAAA (SEQ ID NO: 1).
[0447] The modified mRNAs are designed to include a type 1
(N7mGpppAm) cap, proprietary 5' and 3' untranslated sequences, and
a coding sequence for the signal sequence from human IgE. The
modified mRNAs are synthesized chemically. The prM-E-encoding mRNA
is packaged alone or with the PADRE-encoding mRNAs are into one
lipid nanoparticle (LNP), and the mRNA encoding the prM-E-Ii-PADRE
fusion is packaged into one LNP. Incubation of LNPs containing IgE
signal encoding mRNA (IgE.sub.sig-prM-E, with or without PADRE or
IgE.sub.sig-prM-E-PADRE fusion) with 293T or HeLa cells results in
efficient expression and secretion of 30 nm SVPs, as judged by
electron microscopy and negative staining, Western blotting and
mass spectrometry for ZIKV structural proteins in the cell
supernatants.
Example 2
Vaccine Efficacy in Immunocompromised AG129 Mice
[0448] The immunogenicity and protective activity of the prM-E
encoding LNPs are assessed in immunocompromised mice lacking type I
and II interferon (IFN) signaling responses. Eight week-old
Ifnar1-/-Ifngr-/- AG129 male and female mice are divided into seven
groups, which receive an intramuscular inoculation of 2 or 10 .mu.g
of the IgEsig-prM-E, IgE.sub.sig-prM-E+PADRE, or
IgE.sub.sig-prM-E-PADRE fusion LNPs or 10 .mu.g of a
non-translating RNA LNP. Groups 1, 3, 5, and 7 are boosted with the
same dose at 21 days after vaccination, whereas Groups 2, 4, and
bare not boosted. At day 42 after immunization, mice are
phlebotomized for serum neutralizing antibody analysis. Mice
receiving the IgE.sub.sig-prM-E+PADRE or IgE.sub.sig-prM-E-PADRE
fusion LNPs are expected to have stronger T-cell response against
ZIKV than mice receiving the IgE.sub.sig-prM-E LNPs alone. Mice
receiving only a single immunization have lower neutralizing titers
than mice receiving a boost dose. At 42 days after vaccination,
AG129 mice are challenged with the ZIKV strain P6-740 (Malaysia,
1966). AG129 mice receiving the negative control LNP vaccine
succumbed to ZIKV infection as expected (Aliota et al., 2016).
Recipients of the 2 or 10 .mu.g LNP containing any of the
IgEsig-prM-E-encoding mRNA vaccines with or without a boost are all
expected to survive the infection. Weight measurements correlates
with lethality, as mice receiving the IgE.sub.sig-prM-E,
IgE.sub.sig-prM-E+PADRE, or IgE.sub.sig-prM-E-PADRE fusion LNP
vaccines are protected, whereas the negative controls lose weight
beginning at approximately day 10 after challenge to the time of
death.
Example 3
Vaccine Efficacy in Wild-Type C57BL/6 Mice
[0449] To test the immunogenicity and efficacy of the
IgE.sub.sig-prM-E, gE.sub.sig-prM-E+PADRE, or
IgE.sub.sig-prM-E-PADRE fusion LNP vaccine efficacy in an
immunocompetent mouse strain, 8 week-old male C57BL/6 WT mice are
inoculated via intramuscular injection with 10 .mu.g of any of the
LNPs. These animals are phlebotomized prior to a single boost at
day 28 (4 weeks) and before challenge at either day 56 (8 weeks) or
day 126 (18 weeks), Serum neutralization titers are expected to be
relatively low prior to boosting. However, titers peak at 4 weeks
after boosting and remain elevated 18 weeks post initial
vaccination. To create a lethal challenge model, 2 mg of a blocking
anti-Ifnar1 antibody are passively transferred one day prior to
infection with 106 focus-forming units (FFU) of a mouse-adapted
African Z1KV strain (Dakar 41519) (Sapparapu et al., 2016; Zhao et
al., 2016). All mice immunized with IgE.sub.sig-prM-E,
IgE.sub.sig-prM-E+PADRE, or IgE.sub.sig-prM-E-PADRE fission LNPs,
are protected against lethal ZIKV infection compared to the control
group, which has a less than 30% survival rate. Mice vaccinated
with any of the prM-E mRNA LNPs do not display any loss in weight
nor have measurable viremia in serum at 5 days after challenge.
Example 4
Modified Vaccines Lacking the Immunodominant E-DII-FL Epitope
Generate a Protective Anti-ZIKV Response
[0450] The highly conserved FL epitope in DII of the flavivirus E
protein is immunodominant in humans (Beltramello et al., 2010;
Crill et al., 2007; Dejnirattisai et 2010; Oliphant et al., 2007;
Sapparapu et al., 2016; Stettler et al., 2016). As such, infection
with ZIKV or vaccination with ZIKV structural proteins could induce
cross-reactive antibodies, which might enhance DENV infection and
disease through ADE (Morens, 1994; Stettler et al., 2016). To
minimize this possibility, modified mRNA vaccines by engineering
four mutations (I76R, Q77E, W101R, and L107R) in or near the FL
(IgE.sub.sig-prM-E-FL, IgE.sub.sig-prM-E-FL+PADRE, and
IgE.sub.sig-prM-E-FL-PADRE fusion) are generated that abolish
antibody reactivity of FL-specific antibodies (Chabierski et al.,
2014; Crill et al., 2012; Oliphant et al., 2007). Thus, a separate
series of mRNA LNPs as described in Example 1 (prM-E, prM-E+PADRE,
prM-E-PADRE fusion, prM-E-FL, prM-E-FL+PADRE, and prM-E-FL-PADRE
fusion) are generated by replacing the IgE leader sequence with one
from Japanese encephalitis virus (JEV.sub.sig), a feature included
in other flavivirus prM-E DNA vaccines to increase the efficiency
of host signalise cleavage (Davis et al., 2001; Dowd et al.,
2016b), and by further optimizing codon usage. Western blotting
analysis showed similar levels of SVP expression in HeLa cell
supernatants after transfection of WT and FL mutant mRNA, and a
loss of FL reactivity is confirmed for the prM-E-FL mRNAs by an
absence of binding of a mAb (WNV E60) that recognizes this epitope
(Oliphant et al., 2006).
[0451] Immunocompetent 8 week-old female BALB/c mice are immunized
with 2 .mu.g or 10 .mu.g of IgE.sub.sig-prM-E or JEV.sub.sig-prM-E
(WT or FL mutant) LNPs and boosted with the same LNPs 4 weeks
later. At 8 weeks after initial vaccination, serum is analyzed for
neutralizing activity using ZIKV reporter virus particles (RVPs)
(Dowd et al., 2016a). Mice receiving 2 or 10 .mu.g doses of the
IgE.sub.sig-prM-E WI and FL mutant LNPs show similar neutralization
titers, with mean EC50 values of .about.1/5,000. The 2 and 10 .mu.g
dose of the WT JEV.sub.sig-prM-E LNPs induces stronger inhibitory
responses with remarkable EC50 values of .about.1/100,000 and EC90
values of .about.1/10,000. The mutant JEV.sub.sig-prM-E-FL LNPs,
however, induces antibody responses with lower EC50 and EC90
values, which still approach 1/10,000 and 1/500, respectively. At
13 weeks after initial vaccination, mice are challenged with ZIKV
Dakar 41519 after pre-administration of anti-Ifnar1 blocking
antibody. At day 3 after infection, serum is analyzed for viremia.
Consistent with their high neutralizing titers, all mice immunized
with 2 or 10 .mu.g doses of JEV.sub.sig-prM-E LNPs lack measurable
viremia. All other vaccine groups (IgE.sub.sig-prM E LNPs (WT or
FL) or JEV.sub.sigprM-E-FL LNPs) have breakthrough viremia in some
animals, although levels are 10 to 100-fold lower than observed
with placebo LNPs. Mice are sacrificed at day 7 after infection and
spleen, uterus, and brain are analyzed for ZIKV RNA levels. Most
mice vaccinated with prM-E LNPs show markedly reduced levels of
viral RNA in the spleen (>100-fold) and virtually no detectable
viral RNA in the uterus or brain, whereas the placebo immunized
animals have mean levels of 105 to 106 FFU equivalents per gram of
tissue. Mice vaccinated with the 2 .mu.g dose of
IgE.sub.sig-prM-E-FL or JEV.sub.sig-prM-E-FL LNPs, with or without
PARDE, show slightly less protection, as viral RNA is detectable in
some animals in the spleen and brain, albeit at much lower levels
than those immunized with placebo LNPs. To begin to establish an
immune correlate of complete protection against ZIKV in this model,
the EC50 values from all mRNA LNP vaccines are compared with the
levels of viral RNA recovered in the serum and spleen from
individual mice. This analysis reveal an expected inverse
relationship between neutralizing titers and levels of ZIKV RNA in
serum and tissues, and establish a cut-off EC50 value of
.about.1/10,000 to completely prevent viremia and tissue
dissemination. Next, the JEV.sub.sigprM-E mRNA LNP vaccines, with
or without PADRE, which protect almost completely against viremia
or infection in tissues, are evaluated for conferred sterilizing
immunity. The vast majority of animals (80 to 90%) receiving 2 or
10 .mu.g doses of the JEV.sub.sigprM-E mRNA vaccines failed to
boost their neutralizing titers one week after challenge with
infectious ZIKV, consistent with sterilizing immunity. In
comparison, most animals (82-100%) showing breakthrough viremia or
tissue burden with the IgE.sub.sig-prM-E or JEV.sub.sig-prM-E-FL
mRNA vaccines sustain marked increases in EC50 and EC90 values
after challenge, consistent with the induction of an anamnestic
response.
Example 5
Mutation of the DII-Fusion Loop Epitope Diminishes ADE in Cells and
Mice
[0452] The FL mutant LNPs are engineered to reduce the production
of cross-reactive antibodies and the possibility of immune
enhancement of DENV. To evaluate whether the FL mutant LNP vaccines
diminished induction of cross-reactive enhancing antibodies,
dilutions of serum obtained at 8 weeks after immunization are
incubated with DENY serotype 1 (DENV-1) RVPs and assessed infection
in K562 cells, which express the activating human Fc-.gamma.
receptor HA (CD32A). Whereas sera from mice vaccinated with WT
IgE.sub.sig-prM-E or JEV.sub.sig-prM-E mRNA LNPs all show
bell-shaped, canonical antibody enhancement curves, many of the
sera from mice immunized with serum from IgE.sub.sig-prM-E-FL or
JEV.sub.sig-prM-E-FL LNPs (with or without PADRE) support
enhancement at only high concentrations and with considerably
reduced efficiency. Indeed, the peak serum enhancement titer (PET
(Boonnak et al., 2008)) is .about.100-fold lower in mice immunized
with mutant forms of the fusion loop. A similar reduction in
enhancing power, or the relative fraction of infected cells at the
PET assayed in parallel, is observed. Thus, introduction of
mutations in the FL of ZIKV E reduces the production of enhancing
antibodies against DENV, as judged by cell culture assays. To
determine the physiological significance of these results, an
established passive transfer model of ADE for DENY in AG129 mice is
used with serum from the IgE.sub.sig-prM-E and IgE.sub.sig-prM-E-FL
(with or without PADRE) vaccinated mice. First, the relative
neutralizing activity of DENV-2 of pooled ZIKV serum is evaluated
using an established DENV-2 RVP assay in Raji-DCSIGNR cells.
Consistent with studies using FL-specific mAbs, the pooled sera
from IgE.sub.sig-prM-E but not IgE.sub.sig-prM-E-FL vaccinated
animals inhibit DENV-2 infection in cell culture, indicating a
cross-reactive antibody (likely FL specific) is produced only in
mice receiving any of the IgE.sub.sig-prM-E vaccines, and this
antibody can neutralize infection in Raji-DCSIGNR cells lacking
activating Fc-.gamma. receptors. Next, pooled sera are transferred
to AG129 mice one day prior to challenge with a non-lethal dose of
DENV-2. Whereas administration of serum from mice vaccinated with
WT IgE.sub.sig-prM-E LNPs or a positive control anti-prM mAb (2H2)
results in lethal infection and severe disease due to antibody
enhancement, transfer of equivalent amounts of sera from mice
vaccinated with IgE.sub.sig-prM-E-FL mutant LNPs results in no
mortality and manifested only mild clinical signs of disease.
[0453] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0454] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0455] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0456] in the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
14113PRTArtificial SequenceSynthetic Polypeptide 1Ala Lys Phe Val
Ala Ala Trp Thr Leu Lys Ala Ala Ala1 5 102672PRTZika virus 2Ala Glu
Val Thr Arg Arg Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg1 5 10 15Asn
Asp Ala Gly Glu Ala Ile Ser Phe Pro Thr Thr Leu Gly Met Asn 20 25
30Lys Cys Tyr Ile Gln Ile Met Asp Leu Gly His Met Cys Asp Ala Thr
35 40 45Met Ser Tyr Glu Cys Pro Met Leu Asp Glu Gly Val Glu Pro Asp
Asp 50 55 60Val Asp Cys Trp Cys Asn Thr Thr Ser Thr Trp Val Val Tyr
Gly Thr65 70 75 80Cys His His Lys Lys Gly Glu Ala Arg Arg Ser Arg
Arg Ala Val Thr 85 90 95Leu Pro Ser His Ser Thr Arg Lys Leu Gln Thr
Arg Ser Gln Thr Trp 100 105 110Leu Glu Ser Arg Glu Tyr Thr Lys His
Leu Ile Arg Val Glu Asn Trp 115 120 125Ile Phe Arg Asn Pro Gly Phe
Ala Leu Ala Ala Ala Ala Ile Ala Trp 130 135 140Leu Leu Gly Ser Ser
Thr Ser Gln Lys Val Ile Tyr Leu Val Met Ile145 150 155 160Leu Leu
Ile Ala Pro Ala Tyr Ser Ile Arg Cys Ile Gly Val Ser Asn 165 170
175Arg Asp Phe Val Glu Gly Met Ser Gly Gly Thr Trp Val Asp Val Val
180 185 190Leu Glu His Gly Gly Cys Val Thr Val Met Ala Gln Asp Lys
Pro Thr 195 200 205Val Asp Ile Glu Leu Val Thr Thr Thr Val Ser Asn
Met Ala Glu Val 210 215 220Arg Ser Tyr Cys Tyr Glu Ala Ser Ile Ser
Asp Met Ala Ser Asp Ser225 230 235 240Arg Cys Pro Thr Gln Gly Glu
Ala Tyr Leu Asp Lys Gln Ser Asp Thr 245 250 255Gln Tyr Val Cys Lys
Arg Thr Leu Val Asp Arg Gly Trp Gly Asn Gly 260 265 270Cys Gly Leu
Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Ala 275 280 285Cys
Ser Lys Lys Met Thr Gly Lys Ser Ile Gln Pro Glu Asn Leu Glu 290 295
300Tyr Arg Ile Met Leu Ser Val His Gly Ser Gln His Ser Gly Met
Ile305 310 315 320Val Asn Asp Thr Gly His Glu Thr Asp Glu Asn Arg
Ala Lys Val Glu 325 330 335Ile Thr Pro Asn Ser Pro Arg Ala Glu Ala
Thr Leu Gly Gly Phe Gly 340 345 350Ser Leu Gly Leu Asp Cys Glu Pro
Arg Thr Gly Leu Asp Phe Ser Asp 355 360 365Leu Tyr Tyr Leu Thr Met
Asn Asn Lys His Trp Leu Val His Lys Glu 370 375 380Trp Phe His Asp
Ile Pro Leu Pro Trp His Ala Gly Ala Asp Thr Gly385 390 395 400Thr
Pro His Trp Asn Asn Lys Glu Ala Leu Val Glu Phe Lys Asp Ala 405 410
415His Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser Gln Glu Gly Ala
420 425 430Val His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu Met Asp
Gly Ala 435 440 445Lys Gly Arg Leu Ser Ser Gly His Leu Lys Cys Arg
Leu Lys Met Asp 450 455 460Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser
Leu Cys Thr Ala Ala Phe465 470 475 480Thr Phe Thr Lys Ile Pro Ala
Glu Thr Leu His Gly Thr Val Thr Val 485 490 495Glu Val Gln Tyr Ala
Gly Thr Asp Gly Pro Cys Lys Val Pro Ala Gln 500 505 510Met Ala Val
Asp Met Gln Thr Leu Thr Pro Val Gly Arg Leu Ile Thr 515 520 525Ala
Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys Met Met Leu 530 535
540Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val
Gly545 550 555 560Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly
Ser Thr Ile Gly 565 570 575Lys Ala Phe Glu Ala Thr Val Arg Gly Ala
Lys Arg Met Ala Val Leu 580 585 590Gly Asp Thr Ala Trp Asp Phe Gly
Ser Val Gly Gly Ala Leu Asn Ser 595 600 605Leu Gly Lys Gly Ile His
Gln Ile Phe Gly Ala Ala Phe Lys Ser Leu 610 615 620Phe Gly Gly Met
Ser Trp Phe Ser Gln Ile Leu Ile Gly Thr Leu Leu625 630 635 640Met
Trp Leu Gly Leu Asn Thr Lys Asn Gly Ser Ile Ser Leu Met Cys 645 650
655Leu Ala Leu Gly Gly Val Leu Ile Phe Leu Ser Thr Ala Val Ser Ala
660 665 6703687PRTZika virus 3Met Trp Leu Val Ser Leu Ala Ile Val
Thr Ala Cys Ala Gly Ala Ala1 5 10 15Glu Val Thr Arg Arg Gly Ser Ala
Tyr Tyr Met Tyr Leu Asp Arg Asn 20 25 30Asp Ala Gly Glu Ala Ile Ser
Phe Pro Thr Thr Leu Gly Met Asn Lys 35 40 45Cys Tyr Ile Gln Ile Met
Asp Leu Gly His Met Cys Asp Ala Thr Met 50 55 60Ser Tyr Glu Cys Pro
Met Leu Asp Glu Gly Val Glu Pro Asp Asp Val65 70 75 80Asp Cys Trp
Cys Asn Thr Thr Ser Thr Trp Val Val Tyr Gly Thr Cys 85 90 95His His
Lys Lys Gly Glu Ala Arg Arg Ser Arg Arg Ala Val Thr Leu 100 105
110Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser Gln Thr Trp Leu
115 120 125Glu Ser Arg Glu Tyr Thr Lys His Leu Ile Arg Val Glu Asn
Trp Ile 130 135 140Phe Arg Asn Pro Gly Phe Ala Leu Ala Ala Ala Ala
Ile Ala Trp Leu145 150 155 160Leu Gly Ser Ser Thr Ser Gln Lys Val
Ile Tyr Leu Val Met Ile Leu 165 170 175Leu Ile Ala Pro Ala Tyr Ser
Ile Arg Cys Ile Gly Val Ser Asn Arg 180 185 190Asp Phe Val Glu Gly
Met Ser Gly Gly Thr Trp Val Asp Val Val Leu 195 200 205Glu His Gly
Gly Cys Val Thr Val Met Ala Gln Asp Lys Pro Thr Val 210 215 220Asp
Ile Glu Leu Val Thr Thr Thr Val Ser Asn Met Ala Glu Val Arg225 230
235 240Ser Tyr Cys Tyr Glu Ala Ser Ile Ser Asp Met Ala Ser Asp Ser
Arg 245 250 255Cys Pro Thr Gln Gly Glu Ala Tyr Leu Asp Lys Gln Ser
Asp Thr Gln 260 265 270Tyr Val Cys Lys Arg Thr Leu Val Asp Arg Gly
Trp Gly Asn Gly Cys 275 280 285Gly Leu Phe Gly Lys Gly Ser Leu Val
Thr Cys Ala Lys Phe Ala Cys 290 295 300Ser Lys Lys Met Thr Gly Lys
Ser Ile Gln Pro Glu Asn Leu Glu Tyr305 310 315 320Arg Ile Met Leu
Ser Val His Gly Ser Gln His Ser Gly Met Ile Val 325 330 335Asn Asp
Thr Gly His Glu Thr Asp Glu Asn Arg Ala Lys Val Glu Ile 340 345
350Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu Gly Gly Phe Gly Ser
355 360 365Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly Leu Asp Phe Ser
Asp Leu 370 375 380Tyr Tyr Leu Thr Met Asn Asn Lys His Trp Leu Val
His Lys Glu Trp385 390 395 400Phe His Asp Ile Pro Leu Pro Trp His
Ala Gly Ala Asp Thr Gly Thr 405 410 415Pro His Trp Asn Asn Lys Glu
Ala Leu Val Glu Phe Lys Asp Ala His 420 425 430Ala Lys Arg Gln Thr
Val Val Val Leu Gly Ser Gln Glu Gly Ala Val 435 440 445His Thr Ala
Leu Ala Gly Ala Leu Glu Ala Glu Met Asp Gly Ala Lys 450 455 460Gly
Arg Leu Ser Ser Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys465 470
475 480Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys Thr Ala Ala Phe
Thr 485 490 495Phe Thr Lys Ile Pro Ala Glu Thr Leu His Gly Thr Val
Thr Val Glu 500 505 510Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys
Val Pro Ala Gln Met 515 520 525Ala Val Asp Met Gln Thr Leu Thr Pro
Val Gly Arg Leu Ile Thr Ala 530 535 540Asn Pro Val Ile Thr Glu Ser
Thr Glu Asn Ser Lys Met Met Leu Glu545 550 555 560Leu Asp Pro Pro
Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly Glu 565 570 575Lys Lys
Ile Thr His His Trp His Arg Ser Gly Ser Thr Ile Gly Lys 580 585
590Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg Met Ala Val Leu Gly
595 600 605Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly Ala Leu Asn
Ser Leu 610 615 620Gly Lys Gly Ile His Gln Ile Phe Gly Ala Ala Phe
Lys Ser Leu Phe625 630 635 640Gly Gly Met Ser Trp Phe Ser Gln Ile
Leu Ile Gly Thr Leu Leu Met 645 650 655Trp Leu Gly Leu Asn Thr Lys
Asn Gly Ser Ile Ser Leu Met Cys Leu 660 665 670Ala Leu Gly Gly Val
Leu Ile Phe Leu Ser Thr Ala Val Ser Ala 675 680
6854672PRTArtificial SequenceSynthetic Polypeptide 4Ala Glu Val Thr
Arg Arg Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg1 5 10 15Asn Asp Ala
Gly Glu Ala Ile Ser Phe Pro Thr Thr Leu Gly Met Asn 20 25 30Lys Cys
Tyr Ile Gln Ile Met Asp Leu Gly His Met Cys Asp Ala Thr 35 40 45Met
Ser Tyr Glu Cys Pro Met Leu Asp Glu Gly Val Glu Pro Asp Asp 50 55
60Val Asp Cys Trp Cys Asn Thr Thr Ser Thr Trp Val Val Tyr Gly Thr65
70 75 80Cys His His Lys Lys Gly Glu Ala Arg Arg Ser Arg Arg Ala Val
Thr 85 90 95Leu Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser Gln
Thr Trp 100 105 110Leu Glu Ser Arg Glu Tyr Thr Lys His Leu Ile Arg
Val Glu Asn Trp 115 120 125Ile Phe Arg Asn Pro Gly Phe Ala Leu Ala
Ala Ala Ala Ile Ala Trp 130 135 140Leu Leu Gly Ser Ser Thr Ser Gln
Lys Val Ile Tyr Leu Val Met Ile145 150 155 160Leu Leu Ile Ala Pro
Ala Tyr Ser Ile Arg Cys Ile Gly Val Ser Asn 165 170 175Arg Asp Phe
Val Glu Gly Met Ser Gly Gly Thr Trp Val Asp Val Val 180 185 190Leu
Glu His Gly Gly Cys Val Thr Val Met Ala Gln Asp Lys Pro Thr 195 200
205Val Asp Ile Glu Leu Val Thr Thr Thr Val Ser Asn Met Ala Glu Val
210 215 220Arg Ser Tyr Cys Tyr Glu Ala Ser Ile Ser Asp Met Ala Ser
Asp Ser225 230 235 240Arg Cys Pro Arg Glu Gly Glu Ala Tyr Leu Asp
Lys Gln Ser Asp Thr 245 250 255Gln Tyr Val Cys Lys Arg Thr Leu Val
Asp Arg Gly Arg Gly Asn Gly 260 265 270Cys Gly Arg Phe Gly Lys Gly
Ser Leu Val Thr Cys Ala Lys Phe Ala 275 280 285Cys Ser Lys Lys Met
Thr Gly Lys Ser Ile Gln Pro Glu Asn Leu Glu 290 295 300Tyr Arg Ile
Met Leu Ser Val His Gly Ser Gln His Ser Gly Met Ile305 310 315
320Val Asn Asp Thr Gly His Glu Thr Asp Glu Asn Arg Ala Lys Val Glu
325 330 335Ile Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu Gly Gly
Phe Gly 340 345 350Ser Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly Leu
Asp Phe Ser Asp 355 360 365Leu Tyr Tyr Leu Thr Met Asn Asn Lys His
Trp Leu Val His Lys Glu 370 375 380Trp Phe His Asp Ile Pro Leu Pro
Trp His Ala Gly Ala Asp Thr Gly385 390 395 400Thr Pro His Trp Asn
Asn Lys Glu Ala Leu Val Glu Phe Lys Asp Ala 405 410 415His Ala Lys
Arg Gln Thr Val Val Val Leu Gly Ser Gln Glu Gly Ala 420 425 430Val
His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu Met Asp Gly Ala 435 440
445Lys Gly Arg Leu Ser Ser Gly His Leu Lys Cys Arg Leu Lys Met Asp
450 455 460Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys Thr Ala
Ala Phe465 470 475 480Thr Phe Thr Lys Ile Pro Ala Glu Thr Leu His
Gly Thr Val Thr Val 485 490 495Glu Val Gln Tyr Ala Gly Thr Asp Gly
Pro Cys Lys Val Pro Ala Gln 500 505 510Met Ala Val Asp Met Gln Thr
Leu Thr Pro Val Gly Arg Leu Ile Thr 515 520 525Ala Asn Pro Val Ile
Thr Glu Ser Thr Glu Asn Ser Lys Met Met Leu 530 535 540Glu Leu Asp
Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly545 550 555
560Glu Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser Thr Ile Gly
565 570 575Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg Met Ala
Val Leu 580 585 590Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
Ala Leu Asn Ser 595 600 605Leu Gly Lys Gly Ile His Gln Ile Phe Gly
Ala Ala Phe Lys Ser Leu 610 615 620Phe Gly Gly Met Ser Trp Phe Ser
Gln Ile Leu Ile Gly Thr Leu Leu625 630 635 640Met Trp Leu Gly Leu
Asn Thr Lys Asn Gly Ser Ile Ser Leu Met Cys 645 650 655Leu Ala Leu
Gly Gly Val Leu Ile Phe Leu Ser Thr Ala Val Ser Ala 660 665
6705687PRTArtificial SequenceSynthetic Polypeptide 5Met Trp Leu Val
Ser Leu Ala Ile Val Thr Ala Cys Ala Gly Ala Ala1 5 10 15Glu Val Thr
Arg Arg Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg Asn 20 25 30Asp Ala
Gly Glu Ala Ile Ser Phe Pro Thr Thr Leu Gly Met Asn Lys 35 40 45Cys
Tyr Ile Gln Ile Met Asp Leu Gly His Met Cys Asp Ala Thr Met 50 55
60Ser Tyr Glu Cys Pro Met Leu Asp Glu Gly Val Glu Pro Asp Asp Val65
70 75 80Asp Cys Trp Cys Asn Thr Thr Ser Thr Trp Val Val Tyr Gly Thr
Cys 85 90 95His His Lys Lys Gly Glu Ala Arg Arg Ser Arg Arg Ala Val
Thr Leu 100 105 110Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser
Gln Thr Trp Leu 115 120 125Glu Ser Arg Glu Tyr Thr Lys His Leu Ile
Arg Val Glu Asn Trp Ile 130 135 140Phe Arg Asn Pro Gly Phe Ala Leu
Ala Ala Ala Ala Ile Ala Trp Leu145 150 155 160Leu Gly Ser Ser Thr
Ser Gln Lys Val Ile Tyr Leu Val Met Ile Leu 165 170 175Leu Ile Ala
Pro Ala Tyr Ser Ile Arg Cys Ile Gly Val Ser Asn Arg 180 185 190Asp
Phe Val Glu Gly Met Ser Gly Gly Thr Trp Val Asp Val Val Leu 195 200
205Glu His Gly Gly Cys Val Thr Val Met Ala Gln Asp Lys Pro Thr Val
210 215 220Asp Ile Glu Leu Val Thr Thr Thr Val Ser Asn Met Ala Glu
Val Arg225 230 235 240Ser Tyr Cys Tyr Glu Ala Ser Ile Ser Asp Met
Ala Ser Asp Ser Arg 245 250 255Cys Pro Arg Glu Gly Glu Ala Tyr Leu
Asp Lys Gln Ser Asp Thr Gln 260 265 270Tyr Val Cys Lys Arg Thr Leu
Val Asp Arg Gly Arg Gly Asn Gly Cys 275 280 285Gly Arg Phe Gly Lys
Gly Ser Leu Val Thr Cys Ala Lys Phe Ala Cys 290 295 300Ser Lys Lys
Met Thr Gly Lys Ser Ile Gln Pro Glu Asn Leu Glu Tyr305 310 315
320Arg Ile Met Leu Ser Val His Gly Ser Gln His Ser Gly Met Ile Val
325 330 335Asn Asp Thr Gly His Glu Thr Asp Glu Asn Arg Ala Lys Val
Glu Ile 340 345 350Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu Gly
Gly Phe Gly Ser 355 360 365Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly
Leu Asp Phe Ser Asp Leu 370 375 380Tyr Tyr Leu Thr Met Asn Asn Lys
His Trp Leu Val His Lys Glu Trp385 390 395 400Phe His Asp Ile
Pro Leu Pro Trp His Ala Gly Ala Asp Thr Gly Thr 405 410 415Pro His
Trp Asn Asn Lys Glu Ala Leu Val Glu Phe Lys Asp Ala His 420 425
430Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser Gln Glu Gly Ala Val
435 440 445His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu Met Asp Gly
Ala Lys 450 455 460Gly Arg Leu Ser Ser Gly His Leu Lys Cys Arg Leu
Lys Met Asp Lys465 470 475 480Leu Arg Leu Lys Gly Val Ser Tyr Ser
Leu Cys Thr Ala Ala Phe Thr 485 490 495Phe Thr Lys Ile Pro Ala Glu
Thr Leu His Gly Thr Val Thr Val Glu 500 505 510Val Gln Tyr Ala Gly
Thr Asp Gly Pro Cys Lys Val Pro Ala Gln Met 515 520 525Ala Val Asp
Met Gln Thr Leu Thr Pro Val Gly Arg Leu Ile Thr Ala 530 535 540Asn
Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys Met Met Leu Glu545 550
555 560Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile Gly Val Gly
Glu 565 570 575Lys Lys Ile Thr His His Trp His Arg Ser Gly Ser Thr
Ile Gly Lys 580 585 590Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg
Met Ala Val Leu Gly 595 600 605Asp Thr Ala Trp Asp Phe Gly Ser Val
Gly Gly Ala Leu Asn Ser Leu 610 615 620Gly Lys Gly Ile His Gln Ile
Phe Gly Ala Ala Phe Lys Ser Leu Phe625 630 635 640Gly Gly Met Ser
Trp Phe Ser Gln Ile Leu Ile Gly Thr Leu Leu Met 645 650 655Trp Leu
Gly Leu Asn Thr Lys Asn Gly Ser Ile Ser Leu Met Cys Leu 660 665
670Ala Leu Gly Gly Val Leu Ile Phe Leu Ser Thr Ala Val Ser Ala 675
680 6856690PRTArtificial SequenceSynthetic Polypeptide 6Met Asp Trp
Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val1 5 10 15His Ser
Ala Glu Val Thr Arg Arg Gly Ser Ala Tyr Tyr Met Tyr Leu 20 25 30Asp
Arg Asn Asp Ala Gly Glu Ala Ile Ser Phe Pro Thr Thr Leu Gly 35 40
45Met Asn Lys Cys Tyr Ile Gln Ile Met Asp Leu Gly His Met Cys Asp
50 55 60Ala Thr Met Ser Tyr Glu Cys Pro Met Leu Asp Glu Gly Val Glu
Pro65 70 75 80Asp Asp Val Asp Cys Trp Cys Asn Thr Thr Ser Thr Trp
Val Val Tyr 85 90 95Gly Thr Cys His His Lys Lys Gly Glu Ala Arg Arg
Ser Arg Arg Ala 100 105 110Val Thr Leu Pro Ser His Ser Thr Arg Lys
Leu Gln Thr Arg Ser Gln 115 120 125Thr Trp Leu Glu Ser Arg Glu Tyr
Thr Lys His Leu Ile Arg Val Glu 130 135 140Asn Trp Ile Phe Arg Asn
Pro Gly Phe Ala Leu Ala Ala Ala Ala Ile145 150 155 160Ala Trp Leu
Leu Gly Ser Ser Thr Ser Gln Lys Val Ile Tyr Leu Val 165 170 175Met
Ile Leu Leu Ile Ala Pro Ala Tyr Ser Ile Arg Cys Ile Gly Val 180 185
190Ser Asn Arg Asp Phe Val Glu Gly Met Ser Gly Gly Thr Trp Val Asp
195 200 205Val Val Leu Glu His Gly Gly Cys Val Thr Val Met Ala Gln
Asp Lys 210 215 220Pro Thr Val Asp Ile Glu Leu Val Thr Thr Thr Val
Ser Asn Met Ala225 230 235 240Glu Val Arg Ser Tyr Cys Tyr Glu Ala
Ser Ile Ser Asp Met Ala Ser 245 250 255Asp Ser Arg Cys Pro Arg Glu
Gly Glu Ala Tyr Leu Asp Lys Gln Ser 260 265 270Asp Thr Gln Tyr Val
Cys Lys Arg Thr Leu Val Asp Arg Gly Arg Gly 275 280 285Asn Gly Cys
Gly Arg Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys 290 295 300Phe
Ala Cys Ser Lys Lys Met Thr Gly Lys Ser Ile Gln Pro Glu Asn305 310
315 320Leu Glu Tyr Arg Ile Met Leu Ser Val His Gly Ser Gln His Ser
Gly 325 330 335Met Ile Val Asn Asp Thr Gly His Glu Thr Asp Glu Asn
Arg Ala Lys 340 345 350Val Glu Ile Thr Pro Asn Ser Pro Arg Ala Glu
Ala Thr Leu Gly Gly 355 360 365Phe Gly Ser Leu Gly Leu Asp Cys Glu
Pro Arg Thr Gly Leu Asp Phe 370 375 380Ser Asp Leu Tyr Tyr Leu Thr
Met Asn Asn Lys His Trp Leu Val His385 390 395 400Lys Glu Trp Phe
His Asp Ile Pro Leu Pro Trp His Ala Gly Ala Asp 405 410 415Thr Gly
Thr Pro His Trp Asn Asn Lys Glu Ala Leu Val Glu Phe Lys 420 425
430Asp Ala His Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser Gln Glu
435 440 445Gly Ala Val His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu
Met Asp 450 455 460Gly Ala Lys Gly Arg Leu Ser Ser Gly His Leu Lys
Cys Arg Leu Lys465 470 475 480Met Asp Lys Leu Arg Leu Lys Gly Val
Ser Tyr Ser Leu Cys Thr Ala 485 490 495Ala Phe Thr Phe Thr Lys Ile
Pro Ala Glu Thr Leu His Gly Thr Val 500 505 510Thr Val Glu Val Gln
Tyr Ala Gly Thr Asp Gly Pro Cys Lys Val Pro 515 520 525Ala Gln Met
Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly Arg Leu 530 535 540Ile
Thr Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser Lys Met545 550
555 560Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val Ile
Gly 565 570 575Val Gly Glu Lys Lys Ile Thr His His Trp His Arg Ser
Gly Ser Thr 580 585 590Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly
Ala Lys Arg Met Ala 595 600 605Val Leu Gly Asp Thr Ala Trp Asp Phe
Gly Ser Val Gly Gly Ala Leu 610 615 620Asn Ser Leu Gly Lys Gly Ile
His Gln Ile Phe Gly Ala Ala Phe Lys625 630 635 640Ser Leu Phe Gly
Gly Met Ser Trp Phe Ser Gln Ile Leu Ile Gly Thr 645 650 655Leu Leu
Met Trp Leu Gly Leu Asn Thr Lys Asn Gly Ser Ile Ser Leu 660 665
670Met Cys Leu Ala Leu Gly Gly Val Leu Ile Phe Leu Ser Thr Ala Val
675 680 685Ser Ala 690720PRTArtificial SequenceSynthetic
Polypeptide 7Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu
Trp Leu Pro1 5 10 15Asp Thr Thr Gly 20818PRTArtificial
SequenceSynthetic Polypeptide 8Met Asp Trp Thr Trp Ile Leu Phe Leu
Val Ala Ala Ala Thr Arg Val1 5 10 15His Ser924PRTArtificial
SequenceSynthetic Polypeptide 9Met Leu Gly Ser Asn Ser Gly Gln Arg
Val Val Phe Thr Ile Leu Leu1 5 10 15Leu Leu Val Ala Pro Ala Tyr Ser
201017PRTArtificial SequenceSynthetic Polypeptide 10Met Lys Cys Leu
Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys1 5 10
15Ala1115PRTArtificial SequenceSynthetic Polypeptide 11Met Trp Leu
Val Ser Leu Ala Ile Val Thr Ala Cys Ala Gly Ala1 5 10
15129RNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(3)..(3)n is a or g 12ccnccaugg
91311RNAArtificial SequenceSynthetic Polynucleotide 13gggauccuac c
11149RNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(8)..(9)n is a or u 14uuauuuann 9
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