WO2017070620A2 - Broad spectrum influenza virus vaccine - Google Patents

Broad spectrum influenza virus vaccine Download PDF

Info

Publication number
WO2017070620A2
WO2017070620A2 PCT/US2016/058319 US2016058319W WO2017070620A2 WO 2017070620 A2 WO2017070620 A2 WO 2017070620A2 US 2016058319 W US2016058319 W US 2016058319W WO 2017070620 A2 WO2017070620 A2 WO 2017070620A2
Authority
WO
WIPO (PCT)
Prior art keywords
vaccine
protein
rna
subject
mrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2016/058319
Other languages
English (en)
French (fr)
Other versions
WO2017070620A3 (en
Inventor
Giuseppe Ciaramella
Eric Yi-Chun Huang
Kerim Babaoglu
Jessica Anne FLYNN
Lan Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ModernaTx Inc
Original Assignee
ModernaTx Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to AU2016342048A priority Critical patent/AU2016342048B2/en
Priority to JP2018541088A priority patent/JP7384512B2/ja
Priority to BR112018008078A priority patent/BR112018008078A2/pt
Priority to CN201680074920.5A priority patent/CN109310751A/zh
Priority to MX2018004916A priority patent/MX2018004916A/es
Priority to RU2018118337A priority patent/RU2018118337A/ru
Priority to CN202410855926.2A priority patent/CN118846026A/zh
Priority to KR1020187014338A priority patent/KR20180096591A/ko
Priority to CA3003103A priority patent/CA3003103A1/en
Application filed by ModernaTx Inc filed Critical ModernaTx Inc
Priority to EP16858400.1A priority patent/EP3365007A4/en
Priority to US15/767,609 priority patent/US20180311336A1/en
Publication of WO2017070620A2 publication Critical patent/WO2017070620A2/en
Publication of WO2017070620A3 publication Critical patent/WO2017070620A3/en
Anticipated expiration legal-status Critical
Priority to JP2021204270A priority patent/JP7491639B2/ja
Priority to AU2022218595A priority patent/AU2022218595A1/en
Priority to JP2024077558A priority patent/JP2024105503A/ja
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Influenza viruses are members of the orthomyxoviridae family, and are classified into three distinct types (A, B, and C), based on antigenic differences between their nucleoprotein (NP) and matrix (M) protein.
  • the orthomyxoviruses are enveloped animal viruses of approximately 100 nm in diameter.
  • the influenza virions consist of an internal
  • ribonucleoprotein core (a helical nucleocapsid) containing a single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (Ml).
  • Ml matrix protein
  • the segmented genome of influenza A virus consists of eight molecules (seven for influenza C virus) of linear, negative polarity, single-stranded RNAs, which encode several polypeptides including: the RNA-directed RNA polymerase proteins (PB2, PB 1 and PA) and
  • nucleoprotein which form the nucleocapsid
  • matrix proteins Ml, M2, which is also a surface-exposed protein embedded in the virus membrane
  • HA hemagglutinin
  • NA neuraminidase
  • NSl and NS2 nonstructural proteins
  • Hemagglutinin is the major envelope glycoprotein of influenza A and B viruses
  • hemagglutinin-esterase (HE) of influenza C viruses is a protein homologous to HA.
  • the rapid evolution of the HA protein of the influenza virus results in the constant emergence of new strains, rendering the adaptive immune response of the host only partially protective to new infections.
  • the biggest challenge for therapy and prophylaxis against influenza and other infections using traditional vaccines is the limitation of vaccines in breadth, providing protection only against closely related subtypes.
  • the length of time required to complete current standard influenza virus vaccine production processes inhibits the rapid development and production of an adapted vaccine in a pandemic situation.
  • Deoxyribonucleic acid (DNA) vaccination is one technique used to stimulate humoral and cellular immune responses to foreign antigens, such as influenza antigens.
  • the direct injection of genetically engineered DNA e.g. , naked plasmid DNA
  • this technique come potential problems, including the possibility of insertional mutagenesis, which could lead to the activation of oncogenes or the inhibition of tumor suppressor genes.
  • RNA vaccine or a composition or an immunogenic composition
  • RNA e.g. , messenger RNA (mRNA)
  • mRNA messenger RNA
  • the RNA vaccines of the present disclosure may be used to induce a balanced immune response against influenza virus, comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.
  • the RNA (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.
  • the RNA vaccines may be utilized to treat and/or prevent an influenza virus of various genotypes, strains, and isolates.
  • the RNA vaccines typically have superior properties in that they produce much larger antibody titers and produce responses earlier than commercially available anti- viral therapeutic treatments. While not wishing to be bound by theory, it is believed that the RNA vaccines, as mRNA polynucleotides, are better designed to produce the appropriate protein conformation upon translation as the RNA vaccines co-opt natural cellular machinery.
  • RNA vaccines are presented to the cellular system in a more native fashion.
  • RNA e.g., mRNA
  • therapeutic vaccines are particularly amenable to combination vaccination approaches due to a number of factors including, but not limited to, speed of manufacture, ability to rapidly tailor vaccines to accommodate perceived geographical threat, and the like.
  • the vaccines utilize the human body to produce the antigenic protein, the vaccines are amenable to the production of larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc. in the human subject.
  • a combination vaccine can be administered that includes RNA (e.g.
  • RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first influenza virus or organism and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second influenza virus or organism.
  • RNA e.g., mRNA
  • LNP lipid nanoparticle
  • influenza virus vaccines or compositions or immunogenic compositions
  • the at least one antigenic polypeptide is one of the defined antigenic subdomains of HA, termed HAl , HA2, or a combination of HAl and HA2, and at least one antigenic polypeptide selected from neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein 1 (NS 1) and nonstructural protein 2 (NS2).
  • NA neuraminidase
  • NP nucleoprotein
  • Ml matrix protein 1
  • M2 matrix protein 2
  • NS 1 non-structural protein 1
  • NS2 nonstructural protein 2
  • the at least one antigenic polypeptide is HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2, and at least one antigenic polypeptide selected from NA, NP, Ml , M2, NS 1 and NS2.
  • the at least one antigenic polypeptide is HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2 and at least two antigenic polypeptides selected from NA, NP, M l , M2, NS 1 and NS2.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza virus protein, or an immunogenic fragment thereof.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding multiple influenza virus proteins, or immunogenic fragments thereof.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one HAl , HA2, or a combination of both).
  • RNA e.g., mRNA
  • an immunogenic fragment thereof e.g. , at least one HAl , HA2, or a combination of both.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one HAl , HA2, or a combination of both, of any one of or a combination of any or all of HI , H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l , H12, H13, H14, H15, H16, H17, and/or H18) and at least one other RNA (e.g.
  • RNA e.g., mRNA
  • mRNA mRNA polynucleotide having an open reading frame encoding a protein selected from a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l , H12, H13, H14, H15, H16, H17, and/or H18) and at least two other RNAs (e.g.
  • RNA e.g. , mRNA
  • an immunogenic fragment thereof e.g. , at least one any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l , H12, H13, H14, H15, H16, H17, and/or H18
  • at least two other RNAs e.g.
  • mRNAs polynucleotides having two open reading frames encoding two proteins selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one of any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and/or H18) and at least three other RNAs (e.g.
  • RNA e.g. , mRNA
  • mRNAs polynucleotides having three open reading frames encoding three proteins selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one of any one of or a combination of any or all of HI, H2,
  • RNAs e.g. , mRNAs
  • polynucleotides having four open reading frames encoding four proteins selected from a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or an immunogenic fragment thereof (e.g. , at least one of any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and/or H18) and at least five other RNAs (e.g.
  • RNA e.g. , mRNA
  • an immunogenic fragment thereof e.g. , at least one of any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and/or H18
  • at least five other RNAs e.g.
  • mRNAs polynucleotides having five open reading frames encoding five proteins selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein or an immunogenic fragment thereof (e.g. , at least one of any one of or a combination of any or all of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and/or H18), a NP protein or an immunogenic fragment thereof, a NA protein or an immunogenic fragment thereof, a Ml protein or an immunogenic fragment thereof, a M2 protein or an immunogenic fragment thereof, a NS 1 protein or an immunogenic fragment thereof and a NS2 protein or an immunogenic fragment thereof obtained from influenza virus.
  • RNA e.g. , mRNA
  • Some embodiments of the present disclosure provide the following novel influenza virus polypeptide sequences: HlHA10-Foldon_ANglyl ; H1HA10TM-PR8 (HI A/Puerto Rico/8/34 HA); H1HA10-PR8-DS (HI A/Puerto Rico/8/34 HA; pHlHA10-Cal04-DS (HI A/California/04/2009 HA); Pandemic H1HA10 from California 04; pHlHAlO-ferritin;
  • H1HA10 from California 04; Pandemic H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04; Pandemic H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68C/R76C mutation for trimerization; H1HA10 from California 04 strain/without foldon and with K68
  • influenza virus (influenza) vaccines that include at least one RNA polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide or an immunogenic fragment of the novel influenza virus polypeptide sequences described above (e.g. , an immunogenic fragment capable of inducing an immune response to influenza).
  • an influenza vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide comprising a modified sequence that is at least 75% (e.g. , any number between 75% and 100%, inclusive, e.g.
  • the modified sequence can be at least 75% (e.g. , any number between 75% and 100%, inclusive, e.g. , 70 %, 80%, 85%, 90%, 95%, 99%, and
  • Some embodiments of the present disclosure provide an isolated nucleic acid comprising a sequence encoding the novel influenza virus polypeptide sequences described above; an expression vector comprising the nucleic acid; and a host cell comprising the nucleic acid.
  • the present disclosure also provides a method of producing a polypeptide of any of the novel influenza virus sequences described above.
  • a method may include culturing the host cell in a medium under conditions permitting nucleic acid expression of the novel influenza virus sequences described above, and purifying from the cultured cell or the medium of the cell a novel influenza virus polypeptide.
  • the present disclosure also provides antibody molecules, including full length antibodies and antibody derivatives, directed against the novel influenza virus sequences.
  • an open reading frame of a RNA (e.g. , mRNA) vaccine is codon-optimized.
  • at least one RNA polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7-13) and is codon optimized mRNA.
  • RNA e.g., mRNA
  • Tables 7-13 provide National Center for Biotechnology Information (NCBI) accession numbers of interest. It should be understood that the phrase "an amino acid sequence of Tables 7-13" refers to an amino acid sequence identified by one or more NCBI accession numbers listed in 7-13. Each of the amino acid sequences, and variants having greater than 95% identity or greater than 98% identity to each of the amino acid sequences encompassed by the accession numbers of Tables 7-13 are included within the constructs (polynucleotides/polypeptides) of the present disclosure.
  • At least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one of SEQ ID NO: 447-457, 459, 461 and having less than 80% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one SEQ ID NO: 447-457, 459, 461 and having less than 75%, 85% or 95% identity to a wild-type mRNA sequence.
  • At least one mRNA polynucleotide is encoded by nucleic acid comprising a sequence identified by any one of SEQ ID NO: 447- 457, 459, 461 and having less than 50-80%, 60- 80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identity to wild-type mRNA sequence.
  • at least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one of SEQ ID NO: 447-457, 459, 461 and having less than 40-85%, 50-85%, 60-85%, 30-85%, 70- 85%, 75-85% or 80-85% identity to wild-type mRNA sequence.
  • At least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one of SEQ ID NO: 447-457, 459, 461 and having less than 40-90%, 50- 90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.
  • At least one mRNA polynucleotide comprises a sequence identified by any one of SEQ ID NO: 491-503 and has less than 80% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one SEQ ID NO: 491-503 and has less than 75%, 85% or 95% identity to a wild-type mRNA sequence.
  • At least one mRNA polynucleotide is encoded by nucleic acid comprising a sequence identified by any one of SEQ ID NO: 491-503 and has less than 50-80%, 60- 80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identity to wild-type mRNA sequence. In some embodiments, at least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one of SEQ ID NO: 491-503 and has less than 40-85%, 50-85%, 60-85%, 30-85%, 70-85%, 75-85% or 80-85% identity to wild-type mRNA sequence.
  • At least one mRNA polynucleotide is encoded by a nucleic acid comprising a sequence identified by any one of SEQ ID NO: 491-503 and has less than 40-90%, 50- 90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.
  • At least one RNA polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and having at least 80% (e.g. , 85%, 90%, 95%, 98%, 99%) identity to wild-type mRNA sequence, but does not include wild-type mRNA sequence.
  • At least one RNA polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and has less than 95%, 90%, 85%, 80% or 75% identity to wild-type mRNA sequence.
  • at least one RNA polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and has less than 95%, 90%, 85%, 80% or 75% identity to wild-type mRNA sequence.
  • at least one RNA polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and has less than 95%, 90%, 85%, 80% or 75% identity to
  • polynucleotide encodes at least one antigenic polypeptide comprising an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and has 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 75-80% or 78-80%, 30-85%, 40-85%, 50- 805%, 60-85%, 70-85%, 75-85% or 78-85%, 30-90%, 40-90%, 50-90%, 60-90%, 70-90%, 75-90%, 80-90% or 85-90% identity to wild-type mRNA sequence.
  • At least one RNA polynucleotide encodes at least one antigenic polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13). In some embodiments, at least one RNA polynucleotide encodes at least one antigenic polypeptide having 95%-99% identity to an amino acid sequence identified by any one of 1-444, 458, 460, 462-479 (see also Tables 7- 13).
  • At least one RNA polynucleotide encodes at least one antigenic polypeptide having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) and having membrane fusion activity. In some embodiments, at least one RNA polynucleotide encodes at least one antigenic polypeptide having 95%-99% identity to amino acid sequence identified by any one of SEQ ID NO: 1- 444, 458, 460, 462-479 (see also Tables 7- 13) and having membrane fusion activity.
  • At least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that attaches to cell receptors.
  • At least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that causes fusion of viral and cellular membranes.
  • At least one RNA polynucleotide encodes at least one influenza antigenic polypeptide that is responsible for binding of the virus to a cell being infected.
  • RNA ribonucleic acid
  • mRNA ribonucleic acid
  • a 5' terminal cap is 7mG(5')ppp(5')NlmpNp.
  • At least one chemical modification is selected from
  • pseudouridine Nl-methylpseudouridine, Nl-ethylpseudouridine, 2-thiouridine, 4' - thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio- l -methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio- l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine.
  • the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a Nl-methylpseudouridine. In some embodiments, the chemical modification is a Nl-ethylpseudouridine.
  • a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
  • a cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
  • a cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4- dimethylaminoethyH 1 ,3] -dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimet ylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en- 1-yl) 9-((4-)
  • the lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • RNA e.g. , mRNA
  • a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
  • 100% of the uracil in the open reading frame have a chemical modification.
  • a chemical modification is in the 5-position of the uracil.
  • a chemical modification is a Nl-methyl pseudouridine.
  • 100% of the uracil in the open reading frame have a Nl-methyl pseudouridine in the 5-position of the uracil.
  • an open reading frame of a RNA (e.g. , mRNA) polynucleotide encodes at least two influenza antigenic polypeptides. In some embodiments, the open reading frame encodes at least five or at least ten antigenic polypeptides. In some embodiments, the open reading frame encodes at least 100 antigenic polypeptides. In some embodiments, the open reading frame encodes 2- 100 antigenic polypeptides.
  • a vaccine comprises at least two RNA (e.g. , mRNA) polynucleotides, each having an open reading frame encoding at least one influenza antigenic polypeptide.
  • the vaccine comprises at least five or at least ten RNA (e.g. , mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof.
  • the vaccine comprises at least 100 RNA (e.g. , mRNA) polynucleotides, each having an open reading frame encoding at least one antigenic polypeptide.
  • the vaccine comprises 2-100 RNA (e.g.
  • mRNA polynucleotides each having an open reading frame encoding at least one antigenic polypeptide.
  • at least one influenza antigenic polypeptide is fused to a signal peptide.
  • the signal peptide is selected from: a HuIgGk signal peptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO: 480); IgE heavy chain epsilon- 1 signal peptide (MDWTWILFLVAAATRVHS ; SEQ ID NO: 481); Japanese encephalitis PRM signal sequence (MLGSNS GQRVVFTILLLLV APA YS ; SEQ ID NO: 482), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 483) and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO: 484).
  • a HuIgGk signal peptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO: 480
  • the signal peptide is fused to the N-terminus of at least one antigenic polypeptide. In some embodiments, a signal peptide is fused to the C-terminus of at least one antigenic polypeptide.
  • At least one influenza antigenic polypeptide comprises a mutated N-linked glycosylation site.
  • influenza RNA e.g. , mRNA
  • a nanoparticle e.g. , a lipid nanoparticle
  • the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the 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. 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.
  • the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[ l ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en- l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[ l ,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethylaminobutyrate
  • L319 di((Z)-non-2-en- l-yl) 9- ((4-(dimethylamino)butan
  • the nanoparticle has a polydispersity value of less than 0.4
  • the nanoparticle has a net neutral charge at a neutral pH value.
  • the RNA (e.g. , mRNA) vaccine is multivalent.
  • Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject any of the
  • RNA (e.g. , mRNA) vaccine as provided herein in an amount effective to produce an antigen- specific immune response.
  • the RNA (e.g. , mRNA) vaccine is an influenza vaccine.
  • the RNA (e.g. , mRNA) vaccine is a combination vaccine comprising a combination of influenza vaccines (a broad spectrum influenza vaccine).
  • an antigen- specific immune response comprises a T cell response or a B cell response.
  • a method of producing an antigen-specific immune response comprises administering to a subject a single dose (no booster dose) of an influenza RNA (e.g. , mRNA) vaccine of the present disclosure.
  • a single dose no booster dose
  • an influenza RNA e.g. , mRNA
  • a method further comprises administering to the subject a second (booster) dose of an influenza RNA (e.g. , mRNA) vaccine. Additional doses of an influenza RNA (e.g. , mRNA) vaccine may be administered.
  • a second (booster) dose of an influenza RNA e.g. , mRNA
  • Additional doses of an influenza RNA (e.g. , mRNA) vaccine may be administered.
  • the subjects exhibit a seroconversion rate of at least 80% (e.g. , at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the vaccine.
  • Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, a virus can be detected in blood tests for the antibody. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.
  • an influenza RNA (e.g. , mRNA) vaccine is administered to a subject by intradermal injection, intramuscular injection, or by intranasal administration.
  • an influenza RNA (e.g. , mRNA) vaccine is administered to a subject by intramuscular injection.
  • Some embodiments, of the present disclosure provide methods of inducing an antigen specific immune response in a subject, including administering to a subject an influenza RNA (e.g. , mRNA) vaccine in an effective amount to produce an antigen specific immune response in a subject.
  • Antigen- specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titer (for titer of an antibody that binds to an influenza antigenic polypeptide) following administration to the subject of any of the influenza RNA
  • the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control.
  • the anti-antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti- antigenic polypeptide antibody titer produced in the subject is increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered a RNA (e.g. , mRNA) vaccine of the present disclosure.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated or inactivated influenza, or wherein the control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a recombinant or purified influenza protein vaccine.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered an influenza virus-like particle (VLP) vaccine (see, e.g., Cox RG et al , J Virol. 2014 Jun; 88(11): 6368-6379).
  • VLP influenza virus-like particle
  • a RNA (e.g., mRNA) vaccine of the present disclosure is administered to a subject in an effective amount (an amount effective to induce an immune response).
  • the effective amount is a dose equivalent to an at least 2-fold, at least 4-fold, at least 10-fold, at least 100-fold, at least 1000-fold reduction in the standard of care dose of a recombinant influenza protein vaccine, wherein the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant influenza protein vaccine, a purified influenza protein vaccine, a live attenuated influenza vaccine, an inactivated influenza vaccine, or an influenza VLP vaccine.
  • the effective amount is a dose equivalent to 2- 1000-fold reduction in the standard of care dose of a recombinant influenza protein vaccine, wherein the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant influenza protein vaccine, a purified influenza protein vaccine, a live attenuated influenza vaccine, an inactivated influenza vaccine, or an influenza VLP vaccine.
  • control is an anti-antigenic polypeptide antibody titer produced in a subject who has been administered a virus-like particle (VLP) vaccine comprising structural proteins of influenza.
  • VLP virus-like particle
  • the RNA (e.g., mRNA) vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • the effective amount is a total dose of 25 ⁇ g to 1000 ⁇ g, or 50 ⁇ g to 1000 ⁇ . In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a dose of 25 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 ⁇ g administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 ⁇ g administered to the subject a total of two times.
  • the efficacy (or effectiveness) of a RNA (e.g., mRNA) vaccine is greater than 60%.
  • the RNA (e.g. , mRNA) polynucleotide of the vaccine at least one Influenza antigenic polypeptide.
  • 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:
  • AR disease attack rate
  • 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.
  • 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:
  • the efficacy (or effectiveness) of a RNA (e.g. , mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.
  • the vaccine immunizes the subject against Influenza for up to 2 years. In some embodiments, the vaccine immunizes the subject against Influenza for more than 2 years, more than 3 years, more than 4 years, or for 5-10 years. In some embodiments, the subject is about 5 years old or younger. For example, the subject may be between the ages of about 1 year and about 5 years (e.g. , about 1, 2, 3, 5 or 5 years), or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months). In some embodiments, the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month). In some embodiments, the subject is about 6 months or younger.
  • the subject was born full term (e.g. , about 37-42 weeks). In some embodiments, the subject was born prematurely, for example, at about 36 weeks of gestation or earlier (e.g. , about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 weeks). For example, the subject may have been born at about 32 weeks of gestation or earlier. In some embodiments, the subject was born prematurely between about 32 weeks and about 36 weeks of gestation. In such subjects, a RNA (e.g., mRNA) vaccine may be administered later in life, for example, at the age of about 6 months to about 5 years, or older.
  • a RNA e.g., mRNA
  • the subject is a young adult between the ages of about 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or 50 years old).
  • the subject is an elderly subject about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85 or 90 years old).
  • influenza e.g., C.
  • influenza e.g., C. trachomatis
  • subject is infected with influenza (e.g., C. trachomatis); or subject is at risk of infection by influenza (e.g., C. trachomatis).
  • influenza e.g., C. trachomatis
  • subject is at risk of infection by influenza (e.g., C. trachomatis).
  • the subject is immunocompromised (has an impaired immune system, e.g. , has an immune disorder or autoimmune disorder).
  • nucleic acid vaccines described herein are chemically modified. In other embodiments the nucleic acid vaccines are unmodified.
  • compositions for and methods of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first virus antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and wherein an adjuvant is not coformulated or co-administered with the vaccine.
  • the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 ⁇ g kg and 400 ⁇ g kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA polynucleotide is 1-5 ⁇ g, 5-10 ⁇ g, 10-15 ⁇ g, 15-20 ⁇ g, 10-25 ⁇ g, 20-25 ⁇ g, 20-50 ⁇ g, 30-50 ⁇ g, 40-50 ⁇ g, 40-60 ⁇ g, 60-80 ⁇ g, 60-100 ⁇ g, 50-100 ⁇ g, 80-120 ⁇ g, 40-120 ⁇ g, 40-150 ⁇ g, 50-150 ⁇ g, 50-200 ⁇ g, 80-200 ⁇ g, 100-200 ⁇ g, 120-250 ⁇ g, 150-250 ⁇ g, 180-280 ⁇ g, 200-300 ⁇ g, 50-300 ⁇ 80-300 ⁇ g, 100- 300 ⁇ g, 40-300 ⁇ g, 50-350 ⁇ g, 100-350 ⁇ g, 200-350 ⁇ g, 300-350 ⁇ g, 320-400 ⁇ g, 40-380 ⁇ g, 40-100 ⁇ g, 100-400 ⁇
  • the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
  • a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject.
  • a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.
  • nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide does not include a stabilization element, and a pharmaceutically acceptable carrier or excipient, wherein an adjuvant is not included in the vaccine.
  • the stabilization element is a histone stem-loop.
  • the stabilization element is a nucleic acid sequence having increased GC content relative to wild type sequence.
  • nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host, which confers an antibody titer superior to the criterion for seroprotection for the first antigen for an acceptable percentage of human subjects.
  • the antibody titer produced by the mRNA vaccines of the invention is a neutralizing antibody titer. In some embodiments the neutralizing antibody titer is greater than a protein vaccine.
  • the neutralizing antibody titer produced by the mRNA vaccines of the invention is greater than an adjuvanted protein vaccine.
  • the neutralizing antibody titer produced by the mRNA vaccines of the invention is 1,000- 10,000, 1,200- 10,000, 1,400- 10,000, 1,500- 10,000, 1,000- 5,000, 1,000- 4,000, 1,800- 10,000, 2000- 10,000, 2,000- 5,000, 2,000- 3,000, 2,000- 4,000, 3,000- 5,000, 3,000- 4,000, or 2,000- 2,500.
  • a neutralization titer is typially expressed as the highest serum dilution required to achieve a 50% reduction in the number of plaques.
  • nucleic acid vaccines comprising one or more RNA
  • RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in a formulation for in vivo administration to a host for eliciting a longer lasting high antibody titer than an antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.
  • the RNA polynucleotide is formulated to produce a neutralizing antibodies within one week of a single administration.
  • the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid.
  • the cationic peptide is protamine.
  • nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotide is present in the formulation for in vivo administration to a host such that the level of antigen expression in the host significantly exceeds a level of antigen expression produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigenic polypeptide.
  • nucleic acid vaccines comprising one or more RNA
  • RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25-100 micrograms.
  • aspects of the invention also provide a unit of use vaccine, comprising between lOug and 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally no modified nucleotides, the open reading frame encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject.
  • the vaccine further comprises a cationic lipid nanoparticle.
  • aspects of the invention provide methods of creating, maintaining or restoring antigenic memory to a virus strain in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine comprising (a) at least one RNA polynucleotide, said polynucleotide comprising at least one chemical modification or optionally no modified nucleotides and two or more codon-optimized open reading frames, said open reading frames encoding a set of reference antigenic polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient.
  • the vaccine is administered to the individual via a route selected from the group consisting of intramuscular administration, intradermal administration and subcutaneous administration.
  • the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition. In some embodiments, the administering step comprises contacting a muscle tissue of the subject with a device suitable for injection of the composition in combination with electroporation.
  • aspects of the invention provide methods of vaccinating a subject comprising administering to the subject a single dosage of between 25 ug/kg and 400 ug/kg of a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide in an effective amount to vaccinate the subject.
  • nucleic acid vaccines comprising one or more RNA
  • RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25-100 micrograms.
  • nucleic acid vaccines comprising an LNP formulated RNA polynucleotide having an open reading frame comprising no nucleotide modifications (unmodified), the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 fold less RNA polynucleotide than is required for an unmodified mRNA vaccine not formulated in a LNP to produce an equivalent antibody titer.
  • the RNA polynucleotide is present in a dosage of 25-100 micrograms.
  • the invention encompasses a method of treating an elderly subject age 60 years or older comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an virus antigenic polypeptide in an effective amount to vaccinate the subject.
  • the invention encompasses a method of treating a young subject age 17 years or younger comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an virus antigenic polypeptide in an effective amount to vaccinate the subject.
  • the invention encompasses a method of treating an adult subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding an virus antigenic polypeptide in an effective amount to vaccinate the subject.
  • the invention is a method of vaccinating a subject with a
  • combination vaccine including at least two nucleic acid sequences encoding antigens wherein the dosage for the vaccine is a combined therapeutic dosage wherein the dosage of each individual nucleic acid encoding an antigen is a sub therapeutic dosage.
  • the combined dosage is 25 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 100 micrograms of the RNA polynucleotide in the nucleic acid vaccine
  • the combined dosage is 50 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 75 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject. In some embodiments, the combined dosage is 150 micrograms of the RNA polynucleotide in the nucleic acid vaccine
  • the combined dosage is 400 micrograms of the RNA polynucleotide in the nucleic acid vaccine administered to the subject.
  • the sub therapeutic dosage of each individual nucleic acid encoding an antigen is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micrograms.
  • the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not nucleotide modified.
  • RNA polynucleotide is one of SEQ ID NO: : 447-457, 459, 461 and 491-503 and includes at least one chemical modification. In other embodiments the RNA polynucleotide is one of SEQ ID NO: : 447-457, 459, 461 and 491-503 and does not include any nucleotide modifications, or is unmodified. In yet other embodiments the at least one RNA
  • polynucleotide encodes an antigenic protein of any of SEQ ID NO: 1-444, 458, 460, and 462- 479 and includes at least one chemical modification.
  • the RNA polynucleotide encodes an antigenic protein of any of SEQ ID NO: 1-444, 458, 460, and 462- 479 and does not include any nucleotide modifications, or is unmodified.
  • vaccines of the invention produce prophylactically- and/or therapeutically- efficacious levels, concentrations and/or titers of antigen- specific antibodies in the blood or serum of a vaccinated subject.
  • antibody titer refers to the amount of antigen-specific antibody produces in s subject, e.g., a human subject.
  • antibody titer is expressed as the inverse of the greatest dilution (in a serial dilution) that still gives a positive result.
  • antibody titer is determined or measured by enzyme- linked immunosorbent assay (ELISA).
  • antibody titer is determined or measured by neutralization assay, e.g., by microneutralization assay. In certain aspects, antibody titer measurement is expressed as a ratio, such as 1:40, 1: 100, etc.
  • an efficacious vaccine produces an antibody titer of greater than 1:40, greater that 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, greater than 1: 10000.
  • the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the titer is produced or reached following a single dose of vaccine administered to the subject.
  • the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antigen-specific antibodies are measured in units of ⁇ g/ml or are measured in units of IU/L (International Units per liter) or mlU/ml (milli International Units per ml).
  • an efficacious vaccine produces >0.5 ⁇ g/ml, >0.1 ⁇ g/ml, >0.2 ⁇ g/ml, >0.35 ⁇ g/ml, >0.5 ⁇ g/ml, >1 ⁇ g/ml, >2 ⁇ g/ml, >5 ⁇ g/ml or >10 ⁇ g/ml.
  • an efficacious vaccine produces >10 mlU/ml, >20 mlU/ml, >50 mlU/ml, >100 mlU/ml, >200 mlU/ml, >500 mlU/ml or > 1000 mlU/ml.
  • the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the level or concentration is produced or reached following a single dose of vaccine administered to the subject.
  • the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • neutralization assay e.g., by microneutralization assay.
  • Fig. 1 shows data obtained from an ELISA, demonstrating that vaccination with RNA encoding HA stem protein sequences from different strains induces serum antibodies that bind to diverse panel of recombinant HA (rHA) proteins.
  • Fig. 2 shows data demonstrating that serum antibody titers obtained from mice vaccinated with a second set of mRNA vaccine antigens induces serum antibodies that bind to a diverse panel of recombinant HA (rHA) proteins.
  • Fig. 3 shows combining mRNAs encoding HA stem protein from an HI strain with mRNA encoding HA stem protein from an H3 strain did not result in interference in the immune response to either HA.
  • Figs. 4A-4B depict endpoint titers of the pooled serum from animals vaccinated with the test vaccines.
  • the vaccines tested are shown on the x-axis and the binding to HA from each of the different strains of influenza is plotted as an endpoint titer.
  • the vaccines tested are shown on the x-axis, and the endpoint titer to NP protein is plotted.
  • Fig. 5 shows an examination of functional antibody response through an assessment of the ability of serum to neutralize a panel of HA-pseudotyped viruses.
  • Fig. 6 shows data plotted as fold induction (sample luminescence/background luminescence) versus serum concentration.
  • Fig. 7 is a representation of cell-mediated immune responses following mRNA vaccination. Splenocytes were harvested from vaccinated mice and stimulated with a pool of overlapping NP peptides. The % of CD4 or CD8 T cells secreting one of the three cytokines (IFN- ⁇ , IL-2, or TNF-a) is plotted.
  • cytokines IFN- ⁇ , IL-2, or TNF-a
  • Fig. 8 is a representation of cell-mediated immune responses following mRNA vaccination. Splenocytes were harvested from vaccinated mice and stimulated with a pool of overlapping HA peptides. The % of CD4 or CD8 T cells secreting one of the three cytokines (IFN- ⁇ , IL-2, or TNF-a) is plotted.
  • cytokines IFN- ⁇ , IL-2, or TNF-a
  • Fig. 9 shows murine weight loss following challenge with a lethal dose of mouse- adapted H1N1 A/Puerto Rico/8/1934.
  • the percentage of weight lost as compared to baseline was calculated for each animal and was averaged across the group. The group average was plotted over time in days. Error bars represent standard error of the mean. Efficacy of the NIHGen6HASS-foldon + NP combination vaccine was better than that of either the
  • Fig. 10 shows vaccine efficacy was similar at all vaccine doses, as well as with all co- formulation and co-delivery methods assessed.
  • the percentage of weight lost as compared to baseline was calculated for each animal and was averaged across the group. The group average was plotted over time in days. Error bars represent standard error of the mean.
  • Fig. 11 A depicts the endpoint titers of the pooled serum from animals vaccinated with the test vaccines.
  • Fig. 11B shows efficacy of the test vaccines (NIHGen6HASS-foldon and NIHGen6HASS-TM2) is similar. Following challenge with a lethal dose of mouse-adapted H1N1 A/Puerto Rico/8/1934, the percentage of group weight lost as compared to baseline was calculated and plotted over time in days.
  • Fig. 12A shows that serum from mice immunized with mRNA encoding consensus HA antigens from the HI subtype was able to detectably neutralize the PR8 luciferase virus.
  • Fig. 12B shows that serum from mice immunized with mRNA encoding HI subtype consensus HA antigens with a ferritin fusion sequence was able to detectably neutralize the PR8 luciferase virus, except for the Merck_pHl_Con_ferritin mRNA, while serum from mice vaccinated with an mRNA encoding the consensus H3 antigen with a ferritin fusion sequence was not able to neutralize the PR8 luciferase virus.
  • Figs. 13A-13B show murine weight loss following challenge with a lethal dose of mouse-adapted H1N1 A/Puerto Rico/8/1934. The percentage of group weight lost as compared to baseline was calculated and plotted over time in days..
  • Fig. 14 shows the results of neutralization assays performed on a panel of
  • pseudoviruses to assess the breadth of the serum-neutralizing activity elicited by the consensus HA antigens.
  • Fig. 15A depicts the ELISA endpoint anti-HA antibody titers of the pooled serum from animals vaccinated with the test vaccines.
  • Fig. 15B shows murine weight loss following challenge with a lethal dose of mouse-adapted B/Ann Arbor/1954. The percentage of group weight lost as compared to baseline was calculated and plotted over time in days.
  • Figs. 16A-16C show data depicting the NIHGen6HASS-foldon vaccine's robust antibody response as measured by ELISA assay (plates coated with recombinantly-expressed
  • FIG. 16A shows titers to HA stem, over time, for four rhesus macaques previously vaccinated with FLUZONE® and boosted a single time with NIHGen6HASS-foldon mRNA vaccine.
  • Fig. 16B depicts titers to HA stem, over time, from four rhesus macaques vaccinated at days 0, 28 and 56 with the same
  • Fig. 16C illustrates antibody titers to NP, over time, for four rhesus macaques vaccinated at days 0, 28 and 56 with the NP mRNA vaccine and shows that the vaccine elicited a robust antibody response to NP.
  • Figs. 17A-17B show the results of ELISAs examining the presence of antibody capable of binding to recombinant hemagglutinin (rHA) from a wide variety of influenza strains.
  • Fig. 17A shows the results of rhesus macaques previously vaccinated with
  • Fig. 17B shows the results of niave rhesus macaques vaccinated at days 0, 28 and 56 with the same NIHGen6HASS-foldon RNA vaccine.
  • Fig. 18 is a representation of cell-mediated immune responses following mRNA vaccination.
  • Peripheral blood mononuclear cells were harvested from vaccinated macaques and stimulated with a pool of overlapping NP peptides.
  • the % of CD4 or CD8 T cells secreting one of the three cytokines (IFN- ⁇ , IL-2, or TNF-a) is plotted.
  • Fig. 19 shows the results of hemagglutination inhibition (HAI) tests. Placebo subjects
  • Fig. 20 shows the HAI test kinetics per subject, including the placebo subjects (targeted to be 25% of each cohort).
  • Fig. 21 shows the results of microneutralization (MN) tests, including placebo subjects (targeted to be 25% of each cohort). The data shown is per protocol, and excludes those that did not receive a day 22 injection.
  • Fig. 22 shows the MN test kinetics per subject, including the placebo subjects (targeted to be 25% of each cohort).
  • Fig. 23 is a graph depicting the very strong correlation between HAI and MN.
  • the data includes placebo subjects (targeted to be 25% of each cohort).
  • Embodiments of the present disclosure provide RNA (e.g. , mRNA) vaccines that include polynucleotide encoding an influenza virus antigen.
  • Influenza virus RNA vaccines as provided herein may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccination.
  • the virus is a strain of Influenza A or Influenza B or combinations thereof.
  • the strain of Influenza A or Influenza B is associated with birds, pigs, horses, dogs, humans or non-human primates.
  • the antigenic polypeptide encodes a hemagglutinin protein or immunogenic fragment thereof.
  • the hemagglutinin protein is HI , H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, H18, or an immunogenic fragment thereof.
  • the hemagglutinin protein does not comprise a head domain.
  • the hemagglutinin protein comprises a portion of the head domain. In some embodiments, the hemagglutinin protein does not comprise a cytoplasmic domain. In some embodiments, the hemagglutinin protein comprises a portion of the cytoplasmic domain. In some embodiments, the truncated hemagglutinin protein comprises a portion of the transmembrane domain.
  • the amino acid sequence of the hemagglutinin protein or fragment thereof comprises at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identify with any of the amino acid sequences having an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13).
  • the virus is selected from the group consisting of HlNl , H3N2, H7N9, and H10N8.
  • the antigenic polypeptide is selected from those proteins having an amino acid sequences identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13), or immunogenic fragments thereof.
  • influenza vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a hemagglutinin protein and a pharmaceutically acceptable carrier or excipient, formulated within a cationic lipid nanoparticle.
  • the hemagglutinin protein is selected from HI , H7 and H10.
  • the RNA polynucleotide further encodes neuraminidase protein.
  • the hemagglutinin protein is derived from a strain of Influenza A virus or Influenza B virus or combinations thereof.
  • the Influenza virus is selected from H1N1 , H3N2, H7N9, and H10N8.
  • the antigen specific immune response comprises a T cell response.
  • the antigen specific immune response comprises a B cell response.
  • the antigen specific immune response comprises both a T cell response and a B cell response.
  • the method of producing an antigen specific immune response involves a single administration of the vaccine.
  • the vaccine is administered to the subject by intradermal, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.
  • the RNA (e.g., mRNA) polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof of an influenza strain as an antigen.
  • antigens include, but are not limited to, those antigens encoded by the polynucleotides or portions thereof of the polynucleotides listed in the Tables presented herein.
  • the GenBank Accession Number or GI Accession Number represents either the complete or partial CDS of the encoded antigen.
  • the RNA (e.g. , mRNA) polynucleotides may comprise a region of any of the sequences listed in the Tables or entire coding region of the mRNA listed. They may comprise hybrid or chimeric regions, or mimic s or variants.
  • the polynucleotides when referring to at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding for a specific influenza virus protein, may comprise a coding region of the specific influenza virus protein sequence or the entire coding region of the mRNA for that specific influenza virus protein sequence.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein or immunogenic fragment thereof (e.g. , at least one HA1 , HA2, or a combination of both, of H1-H18).
  • RNA e.g., mRNA
  • immunogenic fragment thereof e.g. , at least one HA1 , HA2, or a combination of both, of H1-H18.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein or immunogenic fragment thereof (e.g. , at least one HA1 , HA2, or a combination of both, of H1-H18) and at least one protein, or immunogenic fragment thereof, selected from a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein and a NS2 protein obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, (e.g. , at least one of H1 -H18) and at least two proteins, or immunogenic fragments thereof, selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a
  • NS 1 protein and a NS2 protein obtained from influenza virus are identical to NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, (e.g. , at least one of H1 -H18) and at least three proteins, or immunogenic fragments thereof, selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a
  • NS 1 protein and a NS2 protein obtained from influenza virus are identical to NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof , (e.g. , at least one of HI -HI 8) and at least four proteins, or immunogenic fragments thereof, selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a
  • NS 1 protein and a NS2 protein obtained from influenza virus are identical to NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, (e.g. , at least one of H1 -H18) and at least five proteins, or immunogenic fragments thereof, selected from a NP protein, a NA protein, a M 1 protein, a M2 protein, a
  • NS 1 protein and a NS2 protein obtained from influenza virus are identical to NS 1 protein and a NS2 protein obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein or immunogenic fragment thereof (e.g. , at least one of HI -HI 8), a NP protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, a NS 1 protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g. , mRNA polynucleotide having an open reading frame encoding a HA protein or immunogenic fragment thereof (e.g. , at least one of HI -HI 8), a NP protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, a NS 1 protein, or
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, and a NA protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g. , mRNA
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g. , mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, and a M2 protein , or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment, thereof, a NP protein and a NA protein obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, a NP protein, or immunogenic fragment, thereof and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NA protein and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, a Ml protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein, or immunogenic fragment thereof, a NS l protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, and a NA protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a NA protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a M 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NP protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a M 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NA protein and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a NA protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, and a NS l protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA1 protein, or immunogenic fragment thereof, a Ml protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a M2 protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HAl protein, or immunogenic fragment thereof, a NS l protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), and a NA protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), and a NS l protein obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NP protein, or immunogenic fragment thereof, and a NA protein, or immunogenic fragment thereof, obtained from influenza virus.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NP protein, or immunogenic fragment thereof, and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NP protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NP protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NP protein and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NA protein, or immunogenic fragment thereof, and a Ml protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HAl and/or HA2), a NA protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a NA protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a NA protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a Ml protein, or immunogenic fragment thereof, and a M2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a Ml protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a Ml protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a M2 protein, or immunogenic fragment thereof, and a NS 1 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a H HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a M2 protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a HA protein (HA or derivatives thereof comprising antigenic sequences from HA1 and/or HA2), a NS 1 protein, or immunogenic fragment thereof, and a NS2 protein, or immunogenic fragment thereof, obtained from influenza virus.
  • RNA e.g., mRNA
  • strain of influenza virus used, as provided herein, may be any strain of influenza virus. Examples of preferred strains of influenza virus and preferred influenza antigens are provided in Tables 7-13 below.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens,
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from Hi/New
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens,
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens,
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H2/Japan/305/1957.
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H9/Hong
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H3/Aichi/2/1968.
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigen
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H3/Brisbane/ 10/2007.
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H7/Anhui/1/2013.
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a M l protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza anti
  • a vaccine comprises at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from H10/Jiangxi- Donghu/346/2013.
  • an influenza antigenic polypeptide e.g. , a HA protein, a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a Ml protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza antigenic polypeptide (e.g. , a HA protein, a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigens, or any combination of two or more of the foregoing influenza antigens, variants or homologs) obtained from Hl/Vietnam/850/2009.
  • an influenza antigenic polypeptide e.g., a HA protein, a NP protein, a NA protein, a M 1 protein, a M2 protein, a NS 1 protein, a NS2 protein, an immunogenic fragment of any of the foregoing influenza antigens, a variant or homolog of any of the foregoing influenza antigen
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding influenza H7N9 HA1 protein, ferritin and a dendritic cell targeting peptide (see, e.g., Ren X et al. Emerg Infect Dis
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an avian influenza H7 HA protein.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding influenza H7 HA1 protein (see, e.g. , Steel J et al. mBio 2010; l(l):e00018-10).
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding influenza H7N9 HA1 protein and ferritin (see, e.g. , Kanekiyo M. et al. Nature 2013;499: 102-6).
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza H5N1 protein.
  • the influenza H5N1 protein is from a human strain.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza H1N1 protein.
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza protein from an influenza A strain, such as human H1N1, H5N1, H9N2 or H3N2.
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an influenza H1N1 HA having a nanoscaffold (see, e.g. , Walker A et al. Sci Rep 2011: 1(5): 1-8, incorporated herein by reference).
  • RNA e.g., mRNA
  • a vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding an aglycosylated influenza H1N1 HA (see, e.g., Chen J et al. PNAS USA 2014;111(7):2476-81, incorporated herein by reference).
  • RNA e.g., mRNA
  • An influenza vaccine may comprise, for example, at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one influenza HA2 stem antigen selected from the influenza HA2 stem antigens, provided herein, for example, those listed in Table 16, comprising an amino acid sequence identified by any one of SEQ ID NO: 394-412.
  • RNA e.g., mRNA
  • the present disclosure also encompasses an influenza vaccine comprising, for example, at least one RNA (e.g., mRNA) polynucleotide having a nucleic acid sequence selected from the influenza sequences listed in SEQ ID NO: 491-503 (see also: Mallajosyula VV et al., Front Immunol. 2015 Jun 26;6:329.; Mallajosyula VV et al., Proc Natl Acad Sci U S A. 2014 Jun 24;l l l(25):E2514-23.; Bommakanti G, et al., J Virol. 2012
  • RNA e.g., mRNA
  • the vaccines described herein are consensus sequences.
  • a "consensus sequence” as used herein refers to a polypeptide sequence based on analysis of an alignment of multiple subtypes of a particular influenza antigen. mRNA sequences that encode a consensus polypeptide sequence may be prepared and used to induce broad immunity against multiple subtypes or serotypes of a particular influenza antigen.
  • the mRNA encoding influenza antigens provided herein can be arranged as a vaccine that causes seroconversion in vaccinated mammals and provides cross -reactivity against a broad range of seasonal strains of influenza and also pandemic strains of influenza.
  • the seroconversion and broad cross -reactivity can be determined by measuring inhibiting titers against different hemagglutinin strains of influenza.
  • Preferred combinations include at least two antigens from each of the influenza antigens described herein.
  • the mRNA vaccines described herein are superior to current vaccines in several ways.
  • the lipid nanoparticle (LNP) delivery is superior to other formulations including a protamine base approach described in the literature and no additional adjuvants are to be necessary.
  • LNPs lipid nanoparticles enables the effective delivery of chemically modified or unmodified mRNA vaccines.
  • both modified and unmodified LNP formulated mRNA vaccines were superior to conventional vaccines by a significant degree.
  • the mRNA vaccines of the invention are superior to conventional vaccines by a factor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000 fold.
  • RNA vaccines including mRNA vaccines and self-replicating RNA vaccines
  • the therapeutic efficacy of these RNA vaccines have not yet been fully established.
  • the inventors have discovered, according to aspects of the invention a class of formulations for delivering mRNA vaccines in vivo that results in significantly enhanced, and in many respects synergistic, immune responses including enhanced antigen generation and functional antibody production with neutralization capability. These results can be achieved even when significantly lower doses of the mRNA are administered in comparison with mRNA doses used in other classes of lipid based formulations.
  • the formulations of the invention have demonstrated significant unexpected in vivo immune responses sufficient to establish the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents.
  • self -replicating RNA vaccines rely on viral replication pathways to deliver enough RNA to a cell to produce an immunogenic response.
  • the formulations of the invention do not require viral replication to produce enough protein to result in a strong immune response.
  • the mRNA of the invention are not self -replicating RNA and do not include components necessary for viral replication.
  • the invention involves, in some aspects, the surprising finding that lipid nanoparticle (LNP) formulations significantly enhance the effectiveness of mRNA vaccines, including chemically modified and unmodified mRNA vaccines.
  • LNP lipid nanoparticle
  • the efficacy of mRNA vaccines formulated in LNP was examined in vivo using several distinct antigens.
  • the results presented herein demonstrate the unexpected superior efficacy of the mRNA vaccines formulated in LNP over other commercially available vaccines.
  • the formulations of the invention generate a more rapid immune response with fewer doses of antigen than other vaccines tested.
  • the mRNA-LNP formulations of the invention also produce quantitatively and qualitatively better immune responses than vaccines formulated in a different carriers.
  • mRNA-LNP formulations of the invention are superior to other vaccines even when the dose of mRNA is lower than other vaccines.
  • mRNA encoding HA protein sequences such as HA stem sequences from different strains have been demonstrated to induce serum antibodies that bind to diverse panel of recombinant HA (rHA) proteins.
  • the vaccine efficacy in mice was similar at all vaccine doses, as well as with all co-formulation and co-delivery methods assessed.
  • LNP used in the studies described herein has been used previously to deliver siRNA in various animal models as well as in humans.
  • the fact that LNP is useful in vaccines is quite surprising. It has been observed that therapeutic delivery of siRNA formulated in LNP causes an undesirable inflammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and a compromised immune response.
  • the LNP-mRNA formulations of the invention are demonstrated herein to generate enhanced IgG levels, sufficient for prophylactic and therapeutic methods rather than transient IgM responses.
  • Influenza virus vaccines comprise at least one (one or more) ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one Influenza antigenic polypeptide.
  • RNA ribonucleic acid
  • 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.
  • 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 ⁇ - D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino- LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • TAAs threose nucleic acids
  • GNAs glycol nu
  • polynucleotides of the present disclosure function as messenger RNA (mRNA).
  • “Messenger RNA” 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.
  • mRNA messenger RNA
  • any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g. , mRNA) sequence encoded by the DNA, where each "T" of the DNA sequence is substituted with "U.”
  • 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.
  • 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.
  • a RNA polynucleotide of an RNA (e.g., mRNA) vaccine encodes 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 antigenic polypeptides.
  • a RNA (e.g., mRNA) polynucleotide of an influenza vaccine encodes at least 10, 20, 30, 40, 50 , 60, 70, 80, 90 or 100 antigenic polypeptides.
  • a RNA (e.g., mRNA) polynucleotide of an influenza vaccine encodes at least 100 or at least 200 antigenic polypeptides.
  • a RNA polynucleotide of an influenza vaccine encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1- 100, 2-50 or 2-100 antigenic polypeptides.
  • Polynucleotides of the present disclosure are codon optimized. Codon optimization methods are known in the art and may be used as provided herein.
  • Codon optimization 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.
  • encoded protein e.g. glycosylation sites
  • add, remove or shuffle protein domains add 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 CA) and/or proprietary methods.
  • the open reading frame (ORF) sequence is optimized using optimization algorithms.
  • 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 les 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., an antigenic protein or antigenic polypeptide)).
  • 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., an antigenic protein or antigenic polypeptide)
  • 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 antigenic protein or polypeptide)).
  • 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 antigenic protein or polypeptide)).
  • 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 antigenic protein or polypeptide)).
  • 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 antigenic protein or polypeptide)
  • a codon-optimized RNA may, for instance, 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 (T) or uracil (U) nucleotides.
  • WO02/098443 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.
  • an antigenic polypeptide (e.g. , at least one Influenza antigenic polypeptide) is longer than 25 amino acids and shorter than 50 amino acids.
  • Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
  • a polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer.
  • Polypeptides may also comprise single chain polypeptides or multichain polypeptides, such as antibodies or insulin, and may be associated or linked to each other. Most commonly, disulfide linkages are found in multichain polypeptides.
  • the term "polypeptide" may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.
  • polypeptide variant is a molecule 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.
  • variants possess at least 50% identity to a native sequence or a reference sequence.
  • variants share at least 80% identity or at least 90% identity with a native sequence or a reference sequence.
  • variant mimics are provided.
  • a “variant mimic” contains at least one amino acid that would mimic an activated sequence.
  • glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro- serine.
  • variant mimics may result in deactivation or in an inactivated product containing the mimic.
  • phenylalanine may act as an inactivating substitution for tyrosine, or alanine may act as an inactivating substitution for serine.
  • orthologs refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.
  • Analogs is meant to include polypeptide variants that differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.
  • compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives.
  • derivative is synonymous with the term “variant” and generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or a starting molecule.
  • polypeptide sequences or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein are included within the scope of this disclosure.
  • sequence tags or amino acids, such as one or more lysines 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.
  • 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.
  • substitutional variants when referring to polypeptides 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.
  • 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.
  • conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue.
  • 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.
  • 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.
  • 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.
  • Features when referring to polypeptide or polynucleotide are defined as distinct amino acid sequence-based or nucleotide-based components of a molecule respectively.
  • polypeptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini and any combination(s) thereof.
  • domain refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g. , binding capacity, serving as a site for protein-protein interactions).
  • site As used herein when referring to polypeptides the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” As used herein when referring to polynucleotides the terms “site” as it pertains to nucleotide based embodiments is used synonymously with “nucleotide.”
  • a site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide-based or polynucleotide- based molecules.
  • terminal 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.
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest.
  • any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
  • a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids.
  • 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.
  • 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.
  • 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.
  • Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g. , reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules).
  • identity refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, 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. , "algorithms"). Identity of related peptides can be readily calculated by known methods. "% 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.
  • 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.
  • tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al.
  • 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- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) "Identification of common molecular subsequences.” /. Mol. Biol. 147: 195- 197).
  • a general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, CD. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453).
  • FGSAA Fast Optimal Global Sequence Alignment Algorithm
  • homologous refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules ⁇ e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • Polymeric molecules e.g. nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide molecules
  • homologous e.g. nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues.
  • homologous is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences.
  • polymeric molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous
  • polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.
  • homolog refers to a first amino acid sequence or nucleic acid sequence (e.g. , gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence.
  • the term “homolog” may apply to the relationship between genes and/or proteins separated by the event of speciation or to the relationship between genes and/or proteins separated by the event of genetic duplication.
  • “Orthologs” are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution.
  • Polynucleotide molecules e.g. DNA molecules and/or RNA molecules
  • polypeptide molecules e.g. amino acids and amino acids
  • Calculation of the percent identity of two polynucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
  • the percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12, 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al , J. Molec. Biol , 215, 403 (1990)).
  • influenza vaccines comprising multiple RNA
  • RNA polynucleotide e.g. , mRNA
  • influenza vaccines comprising a single RNA polynucleotide encoding more than one antigenic polypeptide (e.g. , as a fusion polypeptide).
  • a vaccine composition comprising a RNA (e.g.
  • RNA polynucleotide having an open reading frame encoding a first antigenic polypeptide and a RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a second antigenic polypeptide encompasses (a) vaccines that comprise a first RNA polynucleotide encoding a first antigenic polypeptide and a second RNA
  • RNA (e.g. , mRNA) vaccines of the present disclosure in some embodiments, comprise 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), or more, RNA polynucleotides having an open reading frame, each of which encodes a different antigenic polypeptide (or a single RNA polynucleotide encoding 2-10, or more, different antigenic polypeptides).
  • the antigenic polypeptides may be selected from any of the influenza antigenic polypeptides described herein.
  • a multicomponent vaccine comprises at least one RNA (e.g., mRNA) polynucleotide encoding at least one influenza antigenic polypeptide fused to a signal peptide (e.g., SEQ ID NO: 488-490).
  • the signal peptide may be fused at the N- terminus or the C-terminus of an antigenic polypeptide.
  • antigenic polypeptides encoded by influenza RNA (e.g. , mRNA) polynucleotides comprise 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.
  • the signal peptide of a nascent precursor 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 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 environment.
  • 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.
  • Influenza vaccines of the present disclosure may comprise, for example, RNA (e.g. , mRNA) polynucleotides 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 antigenic polypeptide.
  • influenza vaccines of the present disclosure produce an antigenic polypeptide fused to a signal peptide.
  • a signal peptide is fused to the N-terminus of the antigenic polypeptide.
  • a signal peptide is fused to the C-terminus of the antigenic polypeptide.
  • the signal peptide fused to the antigenic polypeptide is an artificial signal peptide.
  • an artificial signal peptide fused to the antigenic polypeptide encoded by the RNA (e.g. , mRNA) vaccine is obtained from an immunoglobulin protein, e.g., an IgE signal peptide or an IgG signal peptide.
  • a signal peptide fused to the antigenic polypeptide encoded by a RNA (e.g. , mRNA) vaccine is an Ig heavy chain epsilon- 1 signal peptide (IgE HC SP) having the sequence of: MDWTWILFLVAA ATRVHS ; SEQ ID NO: 481.
  • a signal peptide fused to the antigenic polypeptide encoded by the (e.g. , mRNA) RNA (e.g. , mRNA) vaccine is an IgGk chain V-III region HAH signal peptide (IgGk SP) having the sequence of METPAQLLFLLLLWLPDTTG; SEQ ID NO: 480.
  • the signal peptide is selected from: lapanese encephalitis PRM signal sequence
  • MGSNSGQRVVFTILLLLVAPAYS SEQ ID NO: 482
  • VSVg protein signal sequence MKCLLYLAFLFIGVNCA; SEQ ID NO: 483
  • Japanese encephalitis JEV signal sequence MWLVS LAI VT AC AG A ; SEQ ID NO: 484
  • the antigenic polypeptide encoded by a RNA (e.g., mRNA) vaccine comprises an amino acid sequence identified by any one of SEQ ID NO: 1-444, 458, 460, 462-479 (see also Tables 7- 13) fused to a signal peptide identified by any one of SEQ ID NO: 480-484.
  • RNA e.g., mRNA
  • 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.
  • a signal peptide may have a length of 15-60 amino acids.
  • 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.
  • 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.
  • a signal peptide is typically cleaved from the nascent polypeptide at the cleavage junction during ER processing.
  • the mature antigenic polypeptide produce by an influenza RNA (e.g. , mRNA) vaccine of the present disclosure typically does not comprise a signal peptide.
  • Influenza vaccines of the present disclosure comprise at least RNA (e.g. mRNA) polynucleotide having an open reading frame encoding at least one antigenic polypeptide that comprises at least one chemical modification.
  • RNA e.g. mRNA
  • chemical modification and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids.
  • Polypeptides as provided herein, are also considered "modified" of they contain amino acid substitutions, insertions or a combination of substitutions and insertions.
  • Polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides comprise various (more than one) different modifications.
  • a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications.
  • a modified RNA polynucleotide e.g., a modified mRNA polynucleotide
  • introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide.
  • a modified RNA polynucleotide e.g. , a modified mRNA polynucleotide
  • introduced into a cell or organism may exhibit reduced
  • immunogenicity in the cell or organism e.g. , a reduced innate response
  • Polynucleotides may comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications.
  • Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g. , to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).
  • Polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides such as mRNA polynucleotides
  • polynucleotides in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties.
  • the modifications may be present on an 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 polynucleotide may be chemically modified.
  • nucleosides and nucleotides of a polynucleotide e.g. , RNA polynucleotides, such as mRNA polynucleotides.
  • 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.
  • Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodioester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • 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.
  • 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 polynucleotides of the present disclosure.
  • RNA polynucleotides such as mRNA polynucleotides
  • Modifications of polynucleotides include, but are not limited to the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio- N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6- glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6- threonylcarbamoyladenosine; l ,2'-0-dimethyladenosine; 1-methyladenosine; 2'-0- methyladenosine; 2'-0-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopen
  • alkyl adenine 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8- (alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8 -(hydroxy 1) adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7- methyladenine; 1-
  • alkyl guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7- (methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-
  • aminoalkylaminocarbonylethylenyl (aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
  • Dihydropseudouridine ( ⁇ )l-(2-Hydroxypropyl)pseudouridine TP; (2R)-l-(2- Hydroxypropyl)pseudouridine TP; (2S)-l-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2- Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara- uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; l-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-
  • Imidizopyridinyl Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6- methyl-2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl;
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of pseudouridine ( ⁇ ), Nl-methylpseudouridine 2-thiouridine, Nl-ethylpseudouridine, 4'- thiouridine, 5-methylcytosine, 2-thio- l -methyl- 1-deaza-pseudouridine, 2-thio- l-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides include a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • modified nucleobases in polynucleotides are selected from the group consisting of 1- methyl-pseudouridine ( ⁇ ' ⁇ 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine ( ⁇ ), ⁇ -thio-guanosine and a-thio-adenosine.
  • polynucleotides includes a combination of at least two (e.g. , 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise pseudouridine ( ⁇ ) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g. , RNA
  • polynucleotides such as mRNA polynucleotides
  • polynucleotides comprise 2-thiouridine (s U).
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • 2-thiouridine and 5-methyl-cytidine m 5 C
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • methoxy-uridine mithoxy-uridine
  • polynucleotides e.g.
  • RNA polynucleotides such as mRNA polynucleotides
  • RNA polynucleotides comprise 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C).
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides comprise 2' -0-methyl uridine.
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • polynucleotides e.g.
  • RNA polynucleotides such as mRNA polynucleotides
  • N6-methyl-adenosine m 6 A
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • N6-methyl-adenosine m 6 A
  • 5-methyl-cytidine m 5 C
  • polynucleotides e.g. , RNA polynucleotides, such as mRNA polynucleotides
  • RNA polynucleotides are uniformly modified (e.g. , fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methyl-cytidine (m 5 C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m 5 C).
  • m 5 C 5-methyl-cytidine
  • a polynucleotide 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.
  • nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g. , 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2- thio-5-methyl-cytidine.
  • ac4C N4- acetyl-cytidine
  • m5C 5-methyl-cytidine
  • 5-halo-cytidine e.g. , 5-iodo-cytidine
  • 5- hydroxymethyl-cytidine hm5C
  • 1-methyl-pseudoisocytidine 2-thio-cytidine (s2C)
  • 2- thio-5-methyl-cytidine 2-
  • a modified nucleobase is a modified uridine.
  • exemplary nucleobases and in some embodiments, a modified nucleobase is a modified cytosine.
  • nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.
  • a modified nucleobase is a modified adenine.
  • exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl- adenosine (mlA), 2-methyl-adenine (m2A), and N6-methyl- adenosine (m6A).
  • a modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methyl -inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m7G), 1-methyl-guanosine (ml G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
  • polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule.
  • 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
  • nucleotides X in a polynucleotide of the present disclosure are modified nucleotides, wherein X may 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.
  • the polynucleotide 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.
  • the polynucleotides 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.
  • the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine.
  • 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 polynucleotide 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), n 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 polynucleotide is replaced with a modified cytosine (e.g.
  • the RNA (e.g., mRNA) vaccines comprise a 5'UTR element, an optionally codon optimized open reading frame, and a 3 'UTR element, a poly(A) sequence and/or a polyadenylation signal wherein the RNA is not chemically modified.
  • the modified nucleobase is a modified uracil.
  • Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine ( ⁇ ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4- thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy- uridine (ho 5 U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U),
  • 5-methylaminomethyl-uridine (mnm U), 5-methylaminomethyl-2-thio-uridine (mnm s U), 5- methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5- carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine
  • methyl-pseudouridine (m ⁇ ), 5-methyl-2-thio-uridine (m s U), l-methyl-4-thio- pseudouridine (n ⁇ s 4 !]/), 4-thio- l-methyl-pseudouridine, 3-methyl-pseudouridine ( ⁇ 3 ⁇ ), 2- thio- 1 -methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- a pseudouridine, Nl
  • the modified nucleobase is a modified cytosine.
  • nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl- cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g.
  • 5- iodo-cytidine 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio- l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-meth
  • the modified nucleobase is a modified adenine.
  • exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine (e.g. , 2-amino-6-chloro-purine), 6-halo-purine (e.g.
  • the modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o 2 yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (
  • methoxy-guanosine 1 -methyl- guanosine (m'G), N2-methyl- guanosine (m'G), N2,N2- dimethyl-guanosine (m 2 2 G), N2,7-dimethyl- guanosine (m 2J G), N2, N2,7-dimethyl- guanosine (m 2A7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl- guanosine (Gm), N2-methyl-2'-0-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-0-methyl- guanosine (m 2 2 Gm), l-methyl-2'-0-methyl-guanosine (n Gm),
  • guanosine (m ' Gm), 2 r -0-methyl-inosine (Im), l,2'-0-dimethyl-inosine (m Im), 2'-0- ribosylguanosine (phosphate) (Gr(p)) , 1-thio-guanosine, 06-methyl-guanosine, 2' -F-ara- guanosine, and 2' -F-guanosine.
  • RNA e.g., mRNA
  • Influenza virus vaccines of the present disclosure comprise at least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA).
  • mRNA for example, is transcribed in vitro from template DNA, referred to as an "in vitro transcription template.”
  • 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.
  • UTR untranslated
  • a "5' untranslated region” 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a polyA tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) 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.
  • a polynucleotide includes 200 to 3,000 nucleotides.
  • a polynucleotide 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.
  • Flagellin is an approximately 500 amino acid monomeric protein that polymerizes to form the flagella associated with bacterial motion. Flagellin is expressed by a variety of flagellated bacteria (Salmonella typhimurium for example) as well as non-flagellated bacteria (such as Escherichia coli). Sensing of flagellin by cells of the innate immune system (dendritic cells, macrophages, etc.) is mediated by the Toll-like receptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified as playing a role in the activation of innate immune response and adaptive immune response. As such, flagellin provides an adjuvant effect in a vaccine.
  • TLR5 Toll-like receptor 5
  • NLRs Nod-like receptors
  • nucleotide and amino acid sequences encoding known flagellin polypeptides are publicly available in the NCBI GenBank database.
  • a flagellin polypeptide refers to a full length flagellin protein, immunogenic fragments thereof, and peptides having at least 50% sequence identify to a flagellin protein or immunogenic fragments thereof.
  • Exemplary flagellin proteins include flagellin from Salmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8), and proteins having an amino acid sequence identified by any one of SEQ ID NO 1-444, 458, 460, 462-479 (see also Tables 7-13).
  • the flagellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identify to a flagellin protein or immunogenic fragments thereof.
  • the flagellin polypeptide is an immunogenic fragment.
  • An immunogenic fragment is a portion of a flagellin protein that provokes an immune response.
  • the immune response is a TLR5 immune response.
  • An example of an immunogenic fragment is a flagellin protein in which all or a portion of a hinge region has been deleted or replaced with other amino acids.
  • an antigenic polypeptide may be inserted in the hinge region. Hinge regions are the hypervariable regions of a flagellin.
  • Hinge regions of a flagellin are also referred to as “D3 domain or region, "propeller domain or region,” “hypervariable domain or region” and “variable domain or region.” "At least a portion of a hinge region,” as used herein, refers to any part of the hinge region of the flagellin, or the entirety of the hinge region. In other embodiments an immunogenic fragment of flagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment of flagellin.
  • the flagellin monomer is formed by domains DO through D3.
  • DO and Dl which form the stem, are composed of tandem long alpha helices and are highly conserved among different bacteria.
  • the D 1 domain includes several stretches of amino acids that are useful for TLR5 activation.
  • the entire D 1 domain or one or more of the active regions within the domain are immunogenic fragments of flagellin.
  • immunogenic regions within the Dl domain include residues 88- 114 and residues 411-431 (in Salmonella typhimurium FliC flagellin. Within the 13 amino acids in the 88-100 region, at least 6 substitutions are permitted between Salmonella flagellin and other flagellins that still preserve TLR5 activation.
  • immunogenic fragments of flagellin include flagellin like sequences that activate TLR5 and contain a 13 amino acid motif that is 53% or more identical to the Salmonella sequence in 88- 100 of FliC (LQRVRELAVQS AN; SEQ ID NO: 504).
  • the RNA (e.g., mRNA) vaccine includes an RNA that encodes a fusion protein of flagellin and one or more antigenic polypeptides.
  • a carboxy-terminus of the antigenic polypeptide is fused or linked to an amino terminus of the flagellin polypeptide.
  • an amino-terminus of the antigenic polypeptide is fused or linked to a carboxy-terminus of the flagellin polypeptide.
  • the fusion protein may include, for example, one, two, three, four, five, six or more flagellin polypeptides linked to one, two, three, four, five, six or more antigenic polypeptides.
  • flagellin polypeptides and/or two or more antigenic polypeptides When two or more flagellin polypeptides and/or two or more antigenic polypeptides are linked such a construct may be referred to as a "multimer.”
  • Each of the components of a fusion protein may be directly linked to one another or they may be connected through a linker.
  • the linker may be an amino acid linker.
  • the amino acid linker encoded for by the RNA (e.g., mRNA) vaccine to link the components of the fusion protein may include, for instance, at least one member selected from the group consisting of a lysine residue, a glutamic acid residue, a serine residue and an arginine residue.
  • the linker is 1-30, 1-25, 1-25, 5- 10, 5, 15, or 5-20 amino acids in length.
  • the RNA (e.g., mRNA) vaccine includes at least two separate RNA polynucleotides, one encoding one or more antigenic polypeptides and the other encoding the flagellin polypeptide.
  • the at least two RNA polynucleotides may be co- formulated in a carrier such as a lipid nanoparticle.
  • compositions e.g., pharmaceutical compositions
  • methods, kits and reagents for prevention and/or treatment of influenza virus in humans and other mammals can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat infectious disease.
  • the influenza virus RNA vaccines of the present disclosure are used to provide prophylactic protection from influenza virus. Prophylactic protection from influenza virus can be achieved following administration of an influenza virus RNA vaccine of the present disclosure. Vaccines can be administered once, twice, three times, four times or more. 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.
  • influenza virus vaccines of the present disclosure can be used as a method of preventing an influenza virus infection in a subject, the method comprising administering to said subject at least one influenza virus vaccine as provided herein.
  • influenza virus vaccines of the present disclosure can be used as a method of inhibiting a primary influenza virus infection in a subject, the method comprising administering to said subject at least one influenza virus vaccine as provided herein.
  • influenza virus vaccines of the present disclosure can be used as a method of treating an influenza virus infection in a subject, the method comprising administering to said subject at least one influenza virus vaccine as provided herein.
  • influenza virus vaccines of the present disclosure can be used as a method of reducing an incidence of influenza virus infection in a subject, the method comprising administering to said subject at least one influenza virus vaccine as provided herein.
  • influenza virus vaccines of the present disclosure can be used as a method of inhibiting spread of influenza virus from a first subject infected with influenza virus to a second subject not infected with influenza virus, the method comprising administering to at least one of said first subject sand said second subject at least one influenza virus vaccine as provided herein.
  • a method of eliciting an immune response in a subject against an influenza virus involves administering to the subject an influenza virus RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein anti- antigenic polypeptide antibody titer in the subject is increased following vaccination relative to anti- antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the influenza virus.
  • An "anti- antigenic polypeptide antibody” is a serum antibody the binds specifically to the antigenic polypeptide.
  • a prophylactically effective dose is a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level.
  • the therapeutically effective dose is a dose listed in a package insert for the vaccine.
  • a traditional vaccine refers to a vaccine other than the mRNA vaccines of the present disclosure.
  • a traditional vaccine includes, but is not limited to, live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines, etc.
  • 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).
  • FDA Food and Drug Administration
  • EMA European Medicines Agency
  • the anti- antigenic polypeptide antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the influenza virus.
  • the anti-antigenic polypeptide antibody titer in the subject is increased 1 log, 2 log, 3 log, 5 log or 10 log following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against influenza.
  • a method of eliciting an immune response in a subject against an influenza virus involves administering to the subject an influenza virus RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one influenza virus antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to influenza virus antigenic polypeptide or an immunogenic fragment thereof, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the influenza virus at 2 times to 100 times the dosage level relative to the RNA vaccine.
  • 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 influenza vaccine.
  • 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 influenza vaccine.
  • the immune response is assessed by determining [protein] antibody titer in the subject.
  • Some embodiments provide a method of inducing an immune response in a subject by administering to the subject an influenza RNA (e.g. , mRNA) vaccine comprising at least one RNA (e.g. , mRNA) polynucleotide having an open reading frame encoding at least one influenza antigenic polypeptide, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragment thereof, 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 influenza.
  • influenza RNA e.g. , mRNA
  • RNA e.g. mRNA
  • 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 influenza RNA (e.g. , mRNA) vaccine.
  • influenza RNA e.g. , mRNA
  • 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 influenza RNA (e.g. , mRNA) vaccine.
  • influenza RNA e.g. , mRNA
  • 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.
  • 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.
  • compositions ⁇ e.g., pharmaceutical compositions
  • methods, kits and reagents for prevention, treatment or diagnosis of influenza in humans and other mammals for example.
  • Influenza 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.
  • the respiratory RNA ⁇ e.g. , mRNA) vaccines of the present disclosure are used fin 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.
  • PBMCs peripheral blood mononuclear cells
  • influenza vaccine containing RNA e.g. , mRNA
  • polynucleotides as described herein can be administered to a subject ⁇ e.g. , a mammalian subject, such as a human subject), and the RNA ⁇ e.g., mRNA) polynucleotides are translated in vivo to produce an antigenic polypeptide.
  • a subject e.g. , a mammalian subject, such as a human subject
  • RNA ⁇ e.g., mRNA polynucleotides are translated in vivo to produce an antigenic polypeptide.
  • influenza RNA ⁇ e.g., mRNA vaccines may be induced for translation of a polypeptide ⁇ e.g., antigen or immunogen) in a cell, tissue or organism.
  • a polypeptide e.g., antigen or immunogen
  • such translation occurs in vivo, although such translation may occur ex vivo, in culture or in vitro.
  • the cell, tissue or organism is contacted with an effective amount of a composition containing an influenza RNA ⁇ e.g. , mRNA) vaccine that contains a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.
  • an "effective amount" of an influenza RNA ⁇ e.g. mRNA) vaccine is provided 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 vaccine, and other determinants.
  • an effective amount of the influenza RNA ⁇ e.g., mRNA) vaccine 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 RNA, e.g., mRNA, vaccine), 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.
  • RNA ⁇ e.g. mRNA vaccines in accordance with the present disclosure may be used for treatment of Influenza.
  • Influenza RNA (e.g. mRNA) 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.
  • the amount of RNA (e.g. , mRNA) vaccine of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.
  • Influenza RNA (e.g. mRNA) vaccines may be administrated with other prophylactic or therapeutic compounds.
  • a prophylactic or therapeutic compound may be an adjuvant or a booster.
  • 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
  • influenza RNA (e.g. mRNA) vaccines may be administered intramuscularly, intradermally, or intranasally, similarly to the administration of inactivated vaccines known in the art. In some embodiments, influenza RNA (e.g. mRNA) vaccines are administered intramuscularly.
  • Influenza RNA (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.
  • the RNA (e.g. , mRNA) vaccines may be utilized to treat and/or prevent a variety of influenzas.
  • RNA (e.g. , mRNA) vaccines have superior properties in that they produce much larger antibody titers and produce responses early than commercially available anti-viral agents/compositions.
  • compositions including influenza RNA (e.g. mRNA) vaccines and RNA (e.g. mRNA) vaccine compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.
  • influenza RNA e.g. mRNA
  • RNA e.g. mRNA
  • Influenza RNA e.g. mRNA
  • vaccines may be formulated or administered alone or in conjunction with one or more other components.
  • Influenza RNA e.g. , mRNA
  • vaccine compositions may comprise other components including, but not limited to, adjuvants.
  • influenza (e.g. mRNA) vaccines do not include an adjuvant
  • Influenza RNA e.g. mRNA
  • Influenza RNA vaccines may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients.
  • mRNA e.g. mRNA
  • vaccines may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients.
  • 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.
  • compositions such as vaccine compositions.
  • pharmaceutical agents such as vaccine compositions, may be found, for example, in
  • influenza RNA (e.g. mRNA) vaccines are administered to humans, human patients or subjects.
  • active ingredient generally refers to the RNA (e.g. , mRNA) vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g. , mRNA polynucleotides) encoding antigenic polypeptides.
  • Formulations of the influenza vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • 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.
  • compositions 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.
  • 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.
  • Influenza RNA (e.g. mRNA) vaccines can be formulated using one or more excipients to: increase stability; increase cell transfection; permit the sustained or delayed release (e.g. , from a depot formulation); alter the biodistribution (e.g., target to specific tissues or cell types); increase the translation of encoded protein in vivo; and/or alter the release profile of encoded protein (antigen) in vivo.
  • excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with influenza RNA (e.g. mRNA)vaccines (e.g. , for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • RNA e.g. mRNA
  • hyaluronidase e.g. , for transplantation into a subject
  • Naturally-occurring eukaryotic mRNA molecules have been found to 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.
  • UTR untranslated regions
  • 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.
  • the 3 '-poly(A) tail is typically a stretch of adenine nucleotides added to the 3 '-end of the transcribed mRNA. It can 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.
  • the RNA (e.g., mRNA) vaccine 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 is 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 snRNP.
  • 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.
  • the RNA (e.g., mRNA) 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, ⁇ -Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).
  • a reporter protein e.g. Luciferase, GFP, EGFP, ⁇ -Galactosidase, EGFP
  • a marker or selection protein e.g. alpha-Globin, Galactokinase and
  • the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop 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 stem-loop does not depend on the order of the elements or the length of the poly (A) sequence.
  • the RNA (e.g., mRNA) vaccine does not comprise a histone downstream element (HDE).
  • Histone downstream element 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 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.
  • the inventive nucleic acid does not include an intron.
  • the RNA (e.g., mRNA) vaccine may or may not contain a enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
  • 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.
  • 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.
  • the RNA ⁇ e.g., mRNA) vaccine 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 (e.g., mRNA) vaccines. Alternatively the AURES may remain in the RNA (e.g., mRNA) vaccine.
  • influenza RNA (e.g. mRNA) vaccines are formulated in a nanoparticle.
  • influenza RNA (e.g. mRNA) vaccines are formulated in a lipid nanoparticle.
  • influenza RNA (e.g. mRNA) vaccines are formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine.
  • influenza RNA (e.g., mRNA) vaccines are formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl
  • DOPE phosphatidylethanolamine
  • a lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size.
  • the lipid nanoparticle formulation is composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine,
  • lipid nanoparticle formulations may comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and/or 55% to 65% cationic lipid.
  • the ratio of lipid to RNA (e.g. , mRNA) in lipid nanoparticles may be 5: 1 to 20: 1, 10: 1 to 25: 1, 15: 1 to 30: 1 and/or at least 30: 1.
  • the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations.
  • lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(o methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol.
  • PEG-c-DOMG R-3-[(o methoxy- poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2- Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol,
  • PEG- DSG 1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol
  • PEG-DMG 1,2- Dimyristoyl-sn-glycerol
  • PEG-DPG 1,2-Dipalmitoyl-sn-glycerol
  • the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2- DMA.
  • an influenza RNA (e.g. mRNA) vaccine formulation is a nanoparticle that comprises at least one lipid.
  • the lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2- DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.
  • the amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in U.S. Patent Publication No. US20130150625, herein incorporated by reference in its entirety.
  • the cationic lipid may be 2-amino-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-2- ⁇ [(9Z,2Z)-octadeca-9, 12-dien-l-yloxy]methyl ⁇ propan- l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l-yloxy]-2- ⁇ [(9Z)- octadec-9-en-l-yloxy]methyl ⁇ propan- l-ol (Compound 2 in US20130150625); 2-amino-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)
  • Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 -DM A) , or di((Z)-non-2-en- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
  • an ionizable cationic lipid for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • a lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-
  • [l,3]-dioxolane DLin-KC2-DMA
  • DLin-MC3- DMA dilinoleyl-methyl-4-dimethylaminobutyrate
  • a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM
  • a sterol e.g. , cholesterol
  • PEG-lipid e.g.
  • a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. , 35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane
  • DLin-MC3- DMA dilinoleyl-methyl-4
  • a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g. , 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis.
  • neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM.
  • the formulation includes 5% to 50% on a molar basis of the sterol (e.g. , 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis.
  • a non- limiting example of a sterol is cholesterol.
  • a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis.
  • a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
  • a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1 ,500 Da, around 1 ,000 Da, or around 500 Da.
  • PEG- modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG- C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety).
  • PEG-DMG PEG-distearoyl glycerol
  • PEG-cDMA further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety.
  • lipid nanoparticle formulations include 25-75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% of the neutral lipid, 5- 50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-
  • lipid nanoparticle formulations include 35-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of the neutral lipid, 15- 45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl
  • lipid nanoparticle formulations include 45-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of the neutral lipid, 25- 40% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl
  • lipid nanoparticle formulations include 60% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of the neutral lipid, 31 % of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethyl
  • lipid nanoparticle formulations include 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of the neutral lipid, 38.5 % of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethyla
  • lipid nanoparticle formulations include 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of the neutral lipid, 35 % of the sterol, 4.5% or 5% of the PEG or PEG-modified lipid, and 0.5% of the targeting lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilino
  • lipid nanoparticle formulations include 40% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of the neutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modified lipid on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethylamino
  • lipid nanoparticle formulations include 57.2% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of the neutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modified lipid on a molar basis.
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-
  • lipid nanoparticle formulations include 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in their entirety), 7.5% of the neutral lipid, 31.5 % of the sterol, and 3.5% of the PEG or PEG-modified lipid on a molar basis.
  • PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in their entirety)
  • 7.5% of the neutral lipid 31.5 % of the sterol
  • 3.5% of the PEG or PEG-modified lipid on a molar basis PEG-cDMA
  • lipid nanoparticle formulations consists essentially of a lipid mixture in molar ratios of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations consists essentially of a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.
  • the molar lipid ratio is 50/10/38.5/1.5 (mol% cationic lipid/neutral lipid, e.g. , DSPC/Chol/PEG-modified lipid, e.g. , PEG-DMG, PEG-DSG or PEG- DPG), 57.2/7.1 134.3/1.4 (mol% cationic lipid/ neutral lipid, e.g. , DPPC/Chol/ PEG-modified lipid, e.g. , PEG-cDMA), 40/15/40/5 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g.
  • PEG-DMG 50/10/35/4.5/0.5 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g. , PEG-DSG), 50/10/35/5 (cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g. , PEG-DMG), 40/10/40/10 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g.
  • PEG-DMG or PEG-cDMA 35/15/40/10 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g. , PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol% cationic lipid/ neutral lipid, e.g. ,
  • DSPC/Chol/ PEG-modified lipid e.g. , PEG-DMG or PEG-cDMA.
  • Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172- 176;
  • lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid.
  • a lipid nanoparticle may comprise 40-60% of cationic lipid, 5- 15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid.
  • a lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3 -DMA and L319.
  • the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles.
  • the lipid nanoparticle may comprise a cationic lipid, a non- cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle may comprise 40-60% of cationic lipid, 5- 15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid.
  • the lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of the non- cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise 55% of the cationic lipid L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of the structural lipid cholesterol.
  • Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
  • the composition may comprise between 0.1% and 100%, e.g. , between .5 and 50%, between 1- 30%, between 5-80%, at least 80% (w/w) active ingredient.
  • influenza RNA (e.g. mRNA) vaccine composition may comprise the polynucleotide described herein, formulated in a lipid nanoparticle comprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodium citrate, sucrose and water for injection.
  • the composition comprises: 2.0 mg/mL of drug substance, 21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL of trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water for injection.
  • a nanoparticle e.g., a lipid nanoparticle
  • a nanoparticle has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm.
  • a nanoparticle e.g. , a lipid nanoparticle
  • RNA vaccines of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • pharmaceutical compositions of RNA (e.g., mRNA) vaccines include liposomes. Liposomes are artificially- prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to- batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US 20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).
  • DOXIL® DiLa2 liposomes
  • DLin-DMA 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]
  • compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid- lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281 ; Zhang et al. Gene Therapy. 1999 6: 1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al , Nat Biotechnol. 2005 2: 1002- 1007; Zimmermann et al, Nature.
  • liposomes such as those formed from the synthesis of stabilized plasmid- lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy
  • a liposome can contain, but is not limited to, 55%
  • DLPE disteroylphosphatidyl choline
  • DODMA 1,2- dioleyloxy-N,N-dimethylaminopropane
  • certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2- distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
  • DSDMA 1,2- distearloxy-N,N-dimethylaminopropane
  • DODMA 1,2- dilinolenyloxy-3-dimethylaminopropane
  • DLenDMA 1,2- dilinolenyloxy-3-dimethylaminopropane
  • liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and/or from about 48.5% cholesterol to about 60% cholesterol.
  • formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%.
  • formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.
  • the RNA (e.g., mRNA) vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SM ARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708- 1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet)
  • liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SM ARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine)
  • the cationic lipid may be a low molecular weight cationic lipid such as those described in U.S. Patent Application No. 20130090372, the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers.
  • the RNA (e.g., mRNA) vaccines may be formulated in a lipid- polycation complex.
  • the formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S . Pub. No. 20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine.
  • the RNA (e.g., mRNA) vaccines may be formulated in a lipid-polycation complex, which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C 14 to C 18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • LNP formulations may contain from about 0.5% to about 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about 3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]- l,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol.
  • PEG-c-DOMG R-3-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]- l,2-dimyristyloxypropyl-3-amine
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1 ,2- Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol,
  • the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2- DMA.
  • the RNA (e.g., mRNA) vaccines may be formulated in a lipid nanoparticle.
  • the RNA (e.g., mRNA) vaccine formulation comprising the polynucleotide is a nanoparticle which may comprise at least one lipid.
  • the lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C 12-200, DLin- MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.
  • the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.
  • the amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in U.S . Patent Publication No. US20130150625, herein incorporated by reference in its entirety.
  • the cationic lipid may be 2-amino-3-[(9Z, 12Z)-octadeca-9, 12-dien- l-yloxy]-2- ⁇ [(9Z,2Z)-octadeca-9, 12-dien- l- yloxy]methyl ⁇ propan- l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9- en- l-yloxy]-2- ⁇ [(9Z)-octadec-9-en-l-yloxy]methyl ⁇ propan- l-ol (Compound 2 in US20130150625) ; 2-amino-3 - [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy] - 2- [(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(d
  • Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.
  • an ionizable cationic lipid for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin
  • the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g.
  • PEG-lipid e.g. , PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.
  • the formulation includes from about 25% to about 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. , from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 50% or about 40% on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilin
  • the formulation includes from about 0.5% to about 15% on a molar basis of the neutral lipid e.g., from about 3 to about 12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% on a molar basis.
  • neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM.
  • the formulation includes from about 5% to about 50% on a molar basis of the sterol (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis.
  • An exemplary sterol is cholesterol.
  • the formulation includes from about 0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g. , about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis.
  • the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
  • the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da.
  • PEG-modified lipids include, but are not limited to, PEG- distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety)
  • PEG-DMG PEG- distearoyl glycerol
  • PEG-cDMA further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety
  • the formulations of the present disclosure include 25-75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5- 15% of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-
  • the formulations of the present disclosure include 35-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-d
  • the formulations of the present disclosure include 45-65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3 -DM A) , and di((Z)-non-2-en- 1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2- DMA), dilinoleyl-
  • the formulations of the present disclosure include about 60% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% of the neutral lipid, about 31 % of the sterol, and about 1.5% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4
  • the formulations of the present disclosure include about 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% of the neutral lipid, about 38.5 % of the sterol, and about 1.5% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4
  • the formulations of the present disclosure include about 50% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% of the neutral lipid, about 35 % of the sterol, about 4.5% or about 5% of the PEG or PEG- modified lipid, and about 0.5% of the targeting lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA
  • the formulations of the present disclosure include about 40% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% of the neutral lipid, about 40% of the sterol, and about 5% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-di
  • the formulations of the present disclosure include about 57.2% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1% of the neutral lipid, about 34.3% of the sterol, and about 1.4% of the PEG or PEG-modified lipid on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl
  • the formulations of the present disclosure include about 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in their entirety), about 7.5% of the neutral lipid, about 31.5 % of the sterol, and about 3.5% of the PEG or PEG-modified lipid on a molar basis.
  • PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in their entirety)
  • about 7.5% of the neutral lipid about 31.5 % of the sterol
  • about 3.5% of the PEG or PEG-modified lipid on a molar basis PEG-cDMA
  • lipid nanoparticle formulation consists essentially of a lipid mixture in molar ratios of about 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid; more preferably in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.
  • the molar lipid ratio is approximately 50/10/38.5/1.5 (mol% cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g. , PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol% cationic lipid/ neutral lipid, e.g. , DPPC/Chol/ PEG- modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/Chol/ PEG-modified lipid, e.g.
  • PEG-DMG 50/10/35/4.5/0.5 (mol% cationic lipid/ neutral lipid, e.g., DSPC/Chol/ PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic lipid/ neutral lipid, e.g., DSPC/Chol/ PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol% cationic lipid/ neutral lipid, e.g., DSPC/Chol/ PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol% cationic lipid/ neutral lipid, e.g., DSPC/Chol/ PEG- modified lipid, e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol% cationic lipid/ neutral lipid, e.g. , DSPC/
  • lipid nanoparticle compositions examples include lipid nanoparticle compositions and methods of making same, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172- 176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non- cationic lipid.
  • the lipid nanoparticle may comprise about 40-60% of cationic lipid, about 5- 15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid.
  • the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles.
  • the lipid nanoparticle may comprise a cationic lipid, a non- cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle may comprise about 40-60% of cationic lipid, about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid.
  • the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-KC2- DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 55% of the cationic lipid L319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the structural lipid cholesterol.
  • the cationic lipid may be selected from (20Z,23Z)-N,N- dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9- amine, (lZ,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)-N,N- dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa- 15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine
  • the LNP formulations of the RNA (e.g. , mRNA) vaccines may contain PEG-c-DOMG at 3% lipid molar ratio. In some embodiments, the LNP formulations of the RNA (e.g. , mRNA) vaccines may contain PEG-c-DOMG at 1.5% lipid molar ratio.
  • the pharmaceutical compositions of the RNA (e.g. , mRNA) vaccines may include at least one of the PEGylated lipids described in International
  • the LNP formulation may contain PEG-DMG 2000 (1,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
  • the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component.
  • the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g. , Geall et al, Nonviral delivery of self-amplifying RNA (e.g. , mRNA) vaccines, PNAS 2012; PMID: 22908294, the contents of each of which are herein incorporated by reference in their entirety).
  • RNA e.g. , mRNA
  • the lipid nanoparticles described herein may be made in a sterile environment.
  • the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle.
  • the lipid particle may comprise one or more active agents or therapeutic agents; one or more cationic lipids comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; one or more non- cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.
  • the nanoparticle formulations may comprise a phosphate conjugate.
  • the phosphate conjugate may increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle.
  • the phosphate conjugates may include a compound of any one of the formulas described in International Application No. WO2013033438, the contents of which are herein incorporated by reference in its entirety.
  • the nanoparticle formulation may comprise a polymer conjugate.
  • the polymer conjugate may be a water soluble conjugate.
  • the polymer conjugate may have a structure as described in U.S. Patent Application No. 20130059360, the contents of which are herein incorporated by reference in its entirety.
  • polymer conjugates with the polynucleotides of the present disclosure may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Application No. 20130072709, the contents of which are herein incorporated by reference in its entirety.
  • the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No.
  • the nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject.
  • the conjugate may be a "self peptide designed from the human membrane protein CD47 (e.g. , the "self particles described by Rodriguez et al. (Science 2013 339, 971-975), herein incorporated by reference in its entirety). As shown by Rodriguez et al, the self peptides delayed macrophage - mediated clearance of nanoparticles which enhanced delivery of the nanoparticles.
  • the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al. Science 2013 339, 971-975, herein incorporated by reference in its entirety).
  • CD47 can increase the circulating particle ratio in a subject as compared to scrambled peptides and PEG coated nanoparticles.
  • the RNA (e.g., mRNA) vaccines of the present disclosure are formulated in nanoparticles which comprise a conjugate to enhance the delivery of the nanoparticles of the present disclosure in a subject.
  • the conjugate may be the CD47 membrane or the conjugate may be derived from the CD47 membrane protein, such as the "self peptide described previously.
  • the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof.
  • nanoparticle may comprise both the "self peptide described above and the membrane protein CD47.
  • a "self peptide and/or CD47 protein may be conjugated to a virus-like particle or pseudovirion, as described herein for delivery of the RNA (e.g., mRNA) vaccines of the present disclosure.
  • RNA e.g., mRNA
  • RNA vaccine pharmaceutical compositions comprising the polynucleotides of the present disclosure and a conjugate that may have a degradable linkage.
  • conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.
  • pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine.
  • the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g. , International Publication No. WO2012109121 ; the contents of which are herein incorporated by reference in their entirety).
  • Nanoparticle formulations of the present disclosure may be coated with a surfactant or polymer in order to improve the delivery of the particle.
  • the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge.
  • the hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, RNA (e.g. , mPvNA) vaccines within the central nervous system.
  • RNA e.g. , mPvNA
  • nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in U.S. Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in their entirety.
  • the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles.
  • hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the contents of which are herein incorporated by reference in their entirety.
  • the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle
  • lonizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
  • the internal ester linkage may be located on either side of the saturated carbon.
  • an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
  • the polymer may encapsulate the nanospecies or partially encapsulate the nanospecies.
  • the immunogen may be a recombinant protein, a modified RNA and/or a polynucleotide described herein.
  • the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
  • Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g. , the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g. , stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g. , nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g. , the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g. , gastrointestinal (e.g. , stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g. , nasal, pharyngeal, trac
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104: 1482-487; Lai et al. Adv Drug Deliv Rev.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photobleaching
  • MPT high resolution multiple particle tracking
  • compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block copolymer.
  • the polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, poly aery lonitriles, and polyarylates.
  • the polymeric material may be biodegradable and/or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No.
  • the polymeric material may additionally be irradiated.
  • the polymeric material may be gamma irradiated (see e.g., International App. No.
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L- lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L- lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lys
  • polymers of acrylic acids such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), PEG-PLGA-PEG and
  • the co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
  • the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents of which are herein incorporated by reference in their entirety).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants ⁇ e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins ⁇ e.g. , bovine serum albumin), surfactants ⁇ e.g., cationic surfactants such as for example dimethyldioctadecyl- ammonium bromide), sugars or sugar derivatives ⁇ e.g., cyclodextrin), nucleic acids, polymers (e.g.
  • mucolytic agents ⁇ e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin ⁇ 4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.
  • mucolytic agents ⁇ e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine,
  • the surface altering agent may be embedded or enmeshed in the particle' s surface or disposed ⁇ e.g. , by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle. ⁇ see e.g. , U.S. Publication 20100215580 and U.S. Publication
  • the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein.
  • the polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle.
  • the polynucleotide may be covalently coupled to the lipid nanoparticle.
  • Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion, which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
  • the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating.
  • the formulation may be hypotonic for the epithelium to which it is being delivered.
  • hypotonic formulations may be found in International Patent Publication No.
  • RNA vaccine formulation in order to enhance the delivery through the mucosal barrier may comprise or be a hypotonic solution.
  • the RNA (e.g., mRNA) vaccine is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008
  • such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18: 1357-1364; Song et al, Nat Biotechnol. 2005 23:709-717; Judge et al, J Clin Invest.
  • lipid nanoparticle formulations include the DLin- DMA, DLin-KC2-DMA and DLin-MC3 -DMA-based lipid nanoparticle formulations, which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364, the contents of which are incorporated herein by reference in their entirety).
  • Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al.
  • the RNA ⁇ e.g. , mRNA) vaccine is formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al , ACS Nano, 2008, 2 , pp 1696-1702; the contents of which are herein incorporated by reference in their entirety).
  • the SLN may be the SLN described in International Patent Publication No. WO2013105101 , the contents of which are herein incorporated by reference in their entirety.
  • the SLN may be made by the methods or processes described in International Patent Publication No.
  • Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the RNA (e.g. , mRNA) vaccine; and/or increase the translation of encoded protein.
  • RNA e.g. , mRNA
  • One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al , Mol Ther. 2007 15:713-720; the contents of which are incorporated herein by reference in their entirety).
  • the liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the
  • the RNA (e.g. , mRNA) vaccines of the present disclosure can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the RNA (e.g. , mRNA) vaccines may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • the term "encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • Partially encapsulation means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and/or electron micrograph.
  • At least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.
  • the controlled release formulation may include, but is not limited to, tri-block co-polymers.
  • the formulation may include two different types of tri-block co-polymers (International Pub. No. WO2012131104 and
  • the RNA (e.g. , mRNA) vaccines may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
  • the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the RNA (e.g., mRNA) vaccine formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OPADRY®,
  • polyvinylpyrrolidone/vinyl acetate copolymer polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®,
  • EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions
  • the RNA (e.g., mRNA) vaccine controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the RNA (e.g., mRNA) vaccine controlled release and/or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and/or PEG related polymer derivatives as described in U.S . Patent No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.
  • the RNA (e.g., mRNA) vaccine controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines of the present disclosure may be encapsulated in a therapeutic nanoparticle, referred to herein as "therapeutic nanoparticle
  • RNA e.g. , mRNA vaccines.
  • Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos.
  • WO2010005740 WO2010030763, WO2010005721 , WO2010005723, WO2012054923, U.S .
  • therapeutic polymer nanoparticles may be identified by the methods described in US Pub No.
  • the therapeutic nanoparticle RNA (e.g. , mRNA) vaccine may be formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides of the present disclosure (see International Pub No. 2010075072 and US Pub No.
  • the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and/or particulate suspensions (see U.S. Patent Publication No US20130150295, the contents of each of which are incorporated herein by reference in their entirety).
  • the therapeutic nanoparticle RNA (e.g. , mRNA) vaccines may be formulated to be target specific.
  • the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO201 1084518, the contents of which are incorporated herein by reference in their entirety).
  • the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated herein by reference in their entirety.
  • the nanoparticles of the present disclosure may comprise a polymeric matrix.
  • the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxy acids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the therapeutic nanoparticle comprises a diblock copolymer.
  • the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxy acids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the diblock copolymer may be a high-X diblock copolymer such as those described in International Patent Publication No.
  • the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No 8,246,968 and
  • the therapeutic nanoparticle is a stealth nanoparticle or a target- specific stealth nanoparticle as described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g. , U.S. Pat. No. 8,263,665 and 8,287,910 and U.S. Patent Pub. No.
  • the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g. , the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-betal gene delivery vehicle in Lee et al.
  • Thermosensitive Hydrogel as a TGF- ⁇ ⁇ Gene Delivery Vehicle Enhances Diabetic Wound Healing.
  • RNA vaccines of the present disclosure may be formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (see e.g. , U.S. Pat. No. 8,263,665 and 8,287,910 and U.S. Patent Pub. No.
  • the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer, (see e.g. , U.S. Publication No. 20120076836, the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle may comprise at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer.
  • the poly(vinyl ester) polymer may be a copolymer such as a random copolymer.
  • the random copolymer may have a structure such as those described in International Application No. WO2013032829 or U.S. Patent Publication No US20130121954, the contents of each of which are herein incorporated by reference in their entirety.
  • the poly(vinyl ester) polymers may be conjugated to the polynucleotides described herein.
  • the therapeutic nanoparticle may comprise at least one diblock copolymer.
  • the diblock copolymer may be, but it not limited to, a poly(lactic) acid- poly(ethylene)glycol copolymer (see, e.g. , International Patent Publication No.
  • the therapeutic nanoparticle may be used to treat cancer (see International publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).
  • the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
  • the therapeutic nanoparticles may comprise at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,
  • poly(amidoamine) dendrimers poly(beta- amino esters) (see, e.g. , U.S. Patent No. 8,287,849, the contents of which are herein incorporated by reference in their entirety) and combinations thereof.
  • the nanoparticles described herein may comprise an amine cationic lipid such as those described in International Patent Application No.
  • the cationic lipids may have an amino-amine or an amino-amide moiety.
  • the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the synthetic nanocarriers may contain an immunostimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier.
  • the synthetic nanocarrier may comprise a Thl immunostimulatory agent, which may enhance a Thl -based response of the immune system (see International Pub No. WO2010123569 and U.S. Publication No. US20110223201, the contents of each of which are herein incorporated by reference in their entirety).
  • the synthetic nanocarriers may be formulated for targeted release.
  • the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle may be formulated to release the RNA (e.g. , mRNA) vaccines after 24 hours and/or at a pH of 4.5 (see International Publication Nos.
  • the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides described herein.
  • the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.
  • the RNA (e.g. , mRNA) vaccine may be formulated for controlled and/or sustained release wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer.
  • CYSC polymers are described in U.S. Patent No. 8,399,007, herein incorporated by reference in its entirety.
  • the synthetic nanocarrier may be formulated for use as a vaccine.
  • the synthetic nanocarrier may encapsulate at least one polynucleotide which encode at least one antigen.
  • the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Publication No. WO2011150264 and U.S. Publication No. US20110293723, the contents of each of which are herein incorporated by reference in their entirety).
  • a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Publication No.
  • the vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Publication No. WO2011150258 and U.S. Publication No. US20120027806, the contents of each of which are herein incorporated by reference in their entirety).
  • the synthetic nanocarrier may comprise at least one polynucleotide which encodes at least one adjuvant.
  • the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium- chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium- acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (see, e.g., U.S. Patent No. 8,241,610, the content of which is herein incorporated by reference in its entirety).
  • the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant.
  • the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Publication No. WO2011150240 and U.S. Publication No. US20110293700, the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarrier may encapsulate at least one polynucleotide that encodes a peptide, fragment or region from a virus.
  • the synthetic nanocarrier may include, but is not limited to, any of the nanocarriers described in International Publication No. WO2012024621, WO201202629, WO2012024632 and U.S. Publication No. US20120064110, US20120058153 and US20120058154, the contents of each of which are herein incorporated by reference in their entirety.
  • the synthetic nanocarrier may be coupled to a polynucleotide which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (see, e.g. , International Publication No. WO2013019669, the contents of which are herein incorporated by reference in their entirety).
  • CTL cytotoxic T lymphocyte
  • the RNA (e.g., mRNA) vaccine may be encapsulated in, linked to and/or associated with zwitterionic lipids.
  • zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No.
  • the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.
  • the RNA (e.g., mRNA) vaccine may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle may be optimized for oral administration.
  • the nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof.
  • the nanoparticle may be formulated by the methods described in U.S. Publication No. 20120282343, the contents of which are herein incorporated by reference in their entirety.
  • LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012/0295832, the contents of which are herein
  • LNPs comprising KL52 may be administered intravenously and/or in one or more doses.
  • administration of LNPs comprising L52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.
  • RNA (e.g. , mRNA) vaccine may be delivered using smaller LNPs.
  • Such particles may comprise a diameter from below 0.1 um up to 100 nm such as, but not limited to, less than 0.1 um, less than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 um, less than 450 um, less than 475 um, less than 500 um, less than 525 um
  • RNA e.g. , mRNA
  • vaccines may be delivered using smaller amounts of RNA (e.g. , mRNA) vaccines.
  • LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm
  • microfluidic mixers may include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom- up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N.M.
  • a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom- up design and synthesis of limit size lipid nanoparticle systems with aqueous and trigly
  • methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA).
  • MICA microstructure- induced chaotic advection
  • fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other.
  • This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004/0262223 and
  • the RNA (e.g., mRNA) vaccine of the present disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IIMM)from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany).
  • a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IIMM)from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany).
  • the RNA ⁇ e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368- 373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651 ; each of which is herein incorporated by reference in its entirety).
  • controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g. , Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, the contents of which are herein incorporated by reference in their entirety).
  • the RNA (e.g. , mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the RNA (e.g. , mRNA) vaccines of the disclosure may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, the contents of each of which are herein incorporated by reference in their entirety.
  • the microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety.
  • the amino acid, peptide, polypeptide, lipids (APPL) are useful in delivering the RNA (e.g. , mRNA) vaccines of the disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety).
  • the RNA (e.g. , mRNA) vaccines of the disclosure may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 20 to about 100
  • the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in their entirety.
  • the limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and l-palmitoyl-2- oleoyl phosphatidylcholine (POPC).
  • POPC l-palmitoyl-2- oleoyl phosphatidylcholine
  • POPC l-
  • the RNA (e.g., mRNA) vaccines may be delivered, localized and/or concentrated in a specific location using the delivery methods described in
  • a subject may be administered an empty polymeric particle prior to, simultaneously with or after delivering the RNA (e.g. , mRNA) vaccines to the subject.
  • the empty polymeric particle undergoes a change in volume once in contact with the subject and becomes lodged, embedded, immobilized or entrapped at a specific location in the subject.
  • the RNA (e.g., mRNA) vaccines may be formulated in an active substance release system (see, e.g., U.S. Patent Publication No. US20130102545, the contents of which are herein incorporated by reference in their entirety).
  • the active substance release system may comprise 1) at least one nanoparticle bonded to an
  • RNA (e.g., mRNA) vaccines may be formulated in a nanoparticle comprising an inner core comprising a non-cellular material and an outer surface comprising a cellular membrane.
  • the cellular membrane may be derived from a cell or a membrane derived from a virus.
  • the nanoparticle may be made by the methods described in International Patent Publication No. WO2013052167, the contents of which are herein incorporated by reference in their entirety.
  • RNA e.g., mRNA
  • WO2013052167 the contents of which are herein incorporated by reference in their entirety, may be used to deliver the RNA (e.g., mRNA) vaccines described herein.
  • the RNA (e.g., mRNA) vaccines may be formulated in porous nanoparticle-supported lipid bilayers (protocells).
  • Protocells are described in International Patent Publication No. WO2013056132, the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines described herein may be formulated in polymeric nanoparticles as described in or made by the methods described in U.S. Patent Nos. 8,420, 123 and 8,518,963 and European Patent No. EP2073848B 1, the contents of each of which are herein incorporated by reference in their entirety.
  • the polymeric nanoparticle may have a high glass transition temperature such as the nanoparticles described in or nanoparticles made by the methods described in U.S. Patent No. 8,518,963, the contents of which are herein incorporated by reference in their entirety.
  • the polymer nanoparticle for oral and parenteral formulations may be made by the methods described in European Patent No. EP2073848B 1, the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g., mRNA) vaccines described herein may be formulated in nanoparticles used in imaging.
  • the nanoparticles may be liposome nanoparticles such as those described in U.S . Patent Publication No US20130129636, herein incorporated by reference in its entirety.
  • the liposome may comprise gadolinium(III)2- ⁇ 4,7-bis-carboxymethyl- 10-[(N,N-distearylarnidomethyl-N'- amido-methyl]- l,4,7, 10-tetra-azacyclododec- l-yl ⁇ -acetic acid and a neutral, fully saturated phospholipid component (see, e.g. , U.S . Patent Publication No US20130129636, the contents of which are herein incorporated by reference in their entirety).
  • the nanoparticles which may be used in the present disclosure are formed by the methods described in U.S. Patent Application No. US20130130348, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure may further include nutrients such as, but not limited to, those which deficiencies can lead to health hazards from anemia to neural tube defects (see, e.g. , the nanoparticles described in International Patent Publication No
  • the nutrient may be iron in the form of ferrous, ferric salts or elemental iron, iodine, folic acid, vitamins or micronutrients.
  • the RNA (e.g., mRNA) vaccines of the present disclosure may be formulated in a swellable nanoparticle.
  • the swellable nanoparticle may be, but is not limited to, those described in U.S. Patent No. 8,440,231, the contents of which are herein incorporated by reference in their entirety.
  • the swellable nanoparticle may be used for delivery of the RNA (e.g., mRNA) vaccines of the present disclosure to the pulmonary system (see, e.g., U.S. Patent No. 8,440,231, the contents of which are herein incorporated by reference in their entirety).
  • RNA (e.g. , mRNA) vaccines of the present disclosure may be formulated in polyanhydride nanoparticles such as, but not limited to, those described in U.S. Patent No. 8,449,916, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles and microparticles of the present disclosure may be geometrically engineered to modulate macrophage and/or the immune response.
  • the geometrically engineered particles may have varied shapes, sizes and/or surface charges in order to incorporated the polynucleotides of the present disclosure for targeted delivery such as, but not limited to, pulmonary delivery (see, e.g., International Publication No
  • WO2013082111 the contents of which are herein incorporated by reference in their entirety).
  • Other physical features the geometrically engineering particles may have include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge which can alter the interactions with cells and tissues.
  • nanoparticles of the present disclosure may be made by the methods described in International Publication No WO2013082111, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure may be water soluble nanoparticles such as, but not limited to, those described in International Publication No. WO2013090601, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles may be inorganic nanoparticles which have a compact and zwitterionic ligand in order to exhibit good water solubility.
  • the nanoparticles may also have small hydrodynamic diameters (HD), stability with respect to time, pH, and salinity and a low level of non-specific protein binding.
  • the nanoparticles of the present disclosure may be developed by the methods described in U.S . Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure are stealth nanoparticles or target- specific stealth nanoparticles such as, but not limited to, those described in U.S . Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure may be made by the methods described in U.S . Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.
  • the stealth or target-specific stealth nanoparticles may comprise a polymeric matrix.
  • the polymeric matrix may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates or combinations thereof.
  • the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high density nucleic acid layer.
  • the nanoparticle-nucleic acid hybrid structure may made by the methods described in U.S. Patent Publication No. US20130171646, the contents of which are herein incorporated by reference in their entirety.
  • the nanoparticle may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and/or known in the art.
  • At least one of the nanoparticles of the present disclosure may be embedded in in the core a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one payload within or on the surface of the nanostructure.
  • a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one payload within or on the surface of the nanostructure.
  • Non-limiting examples of the nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are herein incorporated by reference in their entirety.
  • the RNA (e.g. , mRNA) vaccine may be associated with a cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV- 1 Tat (HIV), Tat-derived peptides, Penetratin, VP 22 derived or analog peptides, Pestivirus Erns, HSV, VP 22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived
  • PEI polyethyleneimine
  • DOTMA [ 1 -(2,3 -sio ley loxy)propyl)]-N,N,N-trimethylammonium chloride
  • DMRIE di- C14-amidine
  • DOTIM SAINT
  • DC-Choi BGTC
  • CTAP CTAP
  • DOPC DODAP
  • DOPE Dioleyl phosphatidylethanol-amine
  • DOSPA DODAB
  • DOIC DOIC
  • DMEPC DOGS:
  • CLIP 1 rac- [(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride
  • CLIP6 rac- [2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium
  • CLIP9 rac-[2(2,3- dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammo- nium, oligofectamine, or cationic or polycationic polymers, e.g.
  • modified polyaminoacids such as beta-aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl-4- vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA
  • RNA [e.g., mRNA) vaccine is not associated with a cationic or polycationic compounds.
  • Influenza 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 (e.g. , mRNA) 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.
  • Influenza RNA (e.g., mRNA) vaccines compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage.
  • RNA e.g. , mRNA
  • 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.
  • influenza disease 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 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.
  • 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.
  • 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.
  • split dosing regimens such as those described herein may be used.
  • influenza 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.
  • influenza disease 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.
  • influenza disease 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,
  • influenza 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, 0.100 mg or 0.400 mg.
  • 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
  • influenza RNA (e.g. , mRNA) vaccine for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 10 g/kg and 400 ⁇ g/kg of the nucleic acid vaccine (in an effective amount to vaccinate the subject).
  • the RNA (e.g. , mRNA) vaccine for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 10 ⁇ g and 400 ⁇ g of the nucleic acid vaccine (in an effective amount to vaccinate the subject).
  • an influenza 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 ⁇ g.
  • an influenza RNA (e.g. , mRNA) 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 ⁇ g.
  • an influenza RNA (e.g., mRNA) 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 ⁇ g.
  • an influenza 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 ⁇ g of the influenza RNA (e.g. , mRNA) vaccine.
  • influenza 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, intranasal and subcutaneous).
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, intranasal and subcutaneous.
  • Influenza Virus RNA e.g., mRNA
  • vaccine formulations and methods of use are provided.
  • mRNA Influenza Virus RNA
  • RNA e.g. , mRNA
  • an effective amount is a dose of an RNA (e.g. , mRNA) vaccine effective to produce an antigen- specific immune response.
  • methods of inducing an antigen- specific immune response in a subject are also provided herein.
  • the antigen- specific immune response is characterized by measuring an anti- influenza antigenic polypeptide antibody titer produced in a subject administered an influenza 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 influenza antigenic polypeptide) 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 is a common assay for determining antibody titers, for example.
  • 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 influenza RNA (e.g. , mRNA) vaccine. In some embodiments, an anti-influenza antigenic polypeptide antibody titer produced in a subject is increased by at least 1 log relative to a control.
  • influenza RNA e.g. , mRNA
  • anti- antigenic polypeptide 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.
  • the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control.
  • the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control.
  • the anti- antigenic polypeptide 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.
  • the anti-influenza antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control.
  • the anti- antigenic polypeptide 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.
  • the anti-antigenic polypeptide antibody titer produced in the subject is increased 2, 3, 4, 5 ,6, 7, 8, 9, or 10 times relative to a control.
  • the anti-antigenic polypeptide antibody titer produced in a subject is increased 2- 10 times relative to a control.
  • the anti- antigenic polypeptide 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.
  • a control in some embodiments, is the anti-influenza antigenic polypeptide antibody titer produced in a subject who has not been administered an influenza RNA (e.g. , mRNA) vaccine of the present disclosure.
  • a control is an anti-influenza antigenic polypeptide antibody titer produced in a subject who has been administered a live attenuated influenza 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.
  • a control is an anti- influenza antigenic polypeptide antibody titer produced in a subject administered inactivated influenza vaccine.
  • a control is an anti-influenza antigenic polypeptide antibody titer produced in a subject administered a recombinant or purified influenza 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.
  • a control is an anti- influenza antigenic polypeptide antibody titer produced in a subject who has been
  • an effective amount of an influenza RNA (e.g. , mRNA) vaccine is a dose that is reduced compared to the standard of care dose of a recombinant influenza 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 influenza protein vaccine, or a live attenuated or inactivated influenza vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent influenza, or a related condition, while following the standard of care guideline for treating or preventing influenza, or a related condition.
  • the anti-influenza antigenic polypeptide antibody titer produced in a subject administered an effective amount of an influenza RNA (e.g. , mRNA) vaccine is equivalent to an anti-influenza antigenic polypeptide antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated influenza vaccine.
  • an influenza RNA e.g. , mRNA
  • an effective amount of an influenza 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 influenza protein vaccine.
  • an effective amount of an influenza RNA (e.g. , mRNA) 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 influenza protein vaccine.
  • mRNA) 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 influenza protein vaccine.
  • an effective amount of an influenza RNA (e.g. , mRNA) vaccine is a dose equivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-fold reduction in a standard of care dose of a recombinant or purified influenza protein vaccine.
  • the anti-influenza antigenic polypeptide antibody titer produced in a subject administered an effective amount of an influenza RNA (e.g. , mRNA) vaccine is equivalent to an anti-influenza antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or protein influenza protein vaccine or a live attenuated or inactivated influenza vaccine.
  • an effective amount of an influenza RNA (e.g. , mRNA) vaccine is a dose equivalent to a 2- fold to 1000-fold (e.g.
  • the effective amount of an influenza 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
  • the anti-antigenic polypeptide antibody titer produced in the subject is equivalent to an anti-antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated influenza vaccine.
  • 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
  • an anti- antigenic polypeptide antibody titer produced in the subject is equivalent to an anti- antigenic polypeptide antibody titer produced in a control subject administered the standard of care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated an influenza vaccine.
  • the effective amount of an influenza RNA (e.g. , mRNA) vaccine is a total dose of 50- 1000 ⁇ g. In some embodiments, the effective amount of an influenza 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- 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, 80-1000, 80- 900, 80-800, 80-700, 80-600, 80-500
  • the effective amount of an influenza 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 ⁇ g. In some embodiments, the effective amount is a dose of 25-500 ⁇ g administered to the subject a total of two times.
  • the effective amount of an influenza 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, 250-400, 250- 300, 300-500, 300-400, 350-500, 350-400, 400-500 or 450-500 ⁇ g administered to the subject a total of two times.
  • the effective amount of an influenza RNA (e.g. , mRNA) vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 ⁇ g administered to the subject a total of two times.
  • An influenza virus vaccine or composition or immunogenic composition comprising: at least one messenger ribonucleic acid (mRNA) polynucleotide having a 5' terminal cap, an open reading frame encoding at least one influenza antigenic polypeptide, and a 3' polyA tail.
  • mRNA messenger ribonucleic acid
  • lipid nanoparticle comprising: DLin-MC3-DMA; cholesterol; l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC); and polyethylene glycol (PEG)2000-DMG.
  • lipid nanoparticle further comprises trisodium citrate buffer, sucrose and water.
  • a influenza virus vaccine or composition or immunogenic composition comprising: at least one messenger ribonucleic acid (mRNA) polynucleotide having a 5' terminal cap 7mG(5')ppp(5')NlmpNp, a sequence identified by SEQ ID NO: 501 and a 3' polyA tail, wherein the uracil nucleotides of the sequence identified by SEQ ID NO: 501 are modified to include Nl-methyl pseudouridine at the 5-position of the uracil nucleotide.
  • mRNA messenger ribonucleic acid
  • a influenza virus vaccine comprising:
  • mRNA messenger ribonucleic acid
  • SEQ ID NO: 502 a sequence identified by SEQ ID NO: 502 and a 3' polyA tail, wherein the uracil nucleotides of the sequence identified by SEQ ID NO: 502 are modified to include Nl-methyl pseudouridine at the 5-position of the uracil nucleotide.
  • a influenza vims vaccine or composition or immunogenic composition comprising: at least one messenger ribonucleic acid (mRNA) polynucleotide having a 5' terminal cap 7mG(5')ppp(5')NlmpNp, a sequence identified by SEQ ID NO: 503 and a 3' polyA tail, wherein the uracil nucleotides of the sequence identified by SEQ ID NO: 503 are modified to include Nl-methyl pseudouridine at the 5-position of the uracil nucleotide.
  • mRNA messenger ribonucleic acid
  • the vaccine of any one of paragraphs 18-20 formulated in a lipid nanoparticle comprising DLin-MC3-DMA, cholesterol, l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and polyethylene glycol (PEG)2000-DMG.
  • the manufacture of polynucleotides and/or parts or regions thereof may be accomplished utilizing the methods taught in International Publication WO2014/152027, entitled “Manufacturing Methods for Production of RNA Transcripts,” the contents of which is incorporated herein by reference in its entirety.
  • Purification methods may include those taught in International Publication
  • Characterization of the polynucleotides of the disclosure may be accomplished using polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, detection of RNA impurities, or any combination of two or more of the foregoing.
  • RNA transcript sequence comprises determining the RNA transcript sequence, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript, for example.
  • charge heterogeneity comprises determining the charge heterogeneity of the RNA transcript, for example.
  • Example 2 Chimeric polynucleotide synthesis
  • two regions or parts of a chimeric polynucleotide may be joined or ligated using triphosphate chemistry.
  • a first region or part of 100 nucleotides or less is chemically synthesized with a 5' monophosphate and terminal 3'desOH or blocked OH, for example. If the region is longer than 80 nucleotides, it may be synthesized as two strands for ligation.
  • first region or part is synthesized as a non-positionally modified region or part using in vitro transcription (IVT)
  • IVT in vitro transcription
  • Monophosphate protecting groups may be selected from any of those known in the art.
  • the second region or part of the chimeric polynucleotide may be synthesized using either chemical synthesis or IVT methods.
  • IVT methods may include an RNA polymerase that can utilize a primer with a modified cap.
  • a cap of up to 130 nucleotides may be chemically synthesized and coupled to the IVT region or part.
  • the entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then such region or part may comprise a phosphate-sugar backbone.
  • Ligation is then performed using any known click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art.
  • the chimeric polynucleotide may be made using a series of starting segments. Such segments include:
  • a 5' triphosphate segment which may include the coding region of a polypeptide and a normal 3 ⁇ (SEG. 2)
  • segment 3 (SEG. 3) may be treated with cordycepin and then with pyrophosphatase to create the 5' monophosphate.
  • Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA ligase.
  • the ligated polynucleotide is then purified and treated with pyrophosphatase to cleave the diphosphate.
  • the treated SEG.2-SEG. 3 construct may then be purified and SEG. 1 is ligated to the 5' terminus.
  • a further purification step of the chimeric polynucleotide may be performed.
  • the ligated or joined segments may be represented as: 5'UTR (SEG. 1), open reading frame or ORF (SEG. 2) and 3'UTR+PolyA (SEG. 3).
  • PCR procedures for the preparation of cDNA may be performed using 2x KAPA HIFITM HotStart ReadyMix by Kapa Biosystems (Woburn, MA). This system includes 2x KAPA ReadyMix 12.5 ⁇ ; Forward Primer (10 ⁇ ) 0.75 ⁇ ; Reverse Primer (10 ⁇ ) 0.75 ⁇ ; Template cDNA 100 ng; and dH 2 0 diluted to 25.0 ⁇ .
  • the reaction conditions may be at 95 °C for 5 min.
  • the reaction may be performed for 25 cycles of 98 °C for 20 sec, then 58 °C for 15 sec, then 72 °C for 45 sec, then 72 °C for 5 min, then 4 °C to termination.
  • the reaction may be cleaned up using Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad, CA) per manufacturer's instructions (up to 5 ⁇ g). Larger reactions may require a cleanup using a product with a larger capacity. Following the cleanup, the cDNA may be quantified using the NANODROPTM and analyzed by agarose gel electrophoresis to confirm that the cDNA is the expected size. The cDNA may then be submitted for sequencing analysis before proceeding to the in vitro transcription reaction.
  • the in vitro transcription reaction generates RNA polynucleotides.
  • polynucleotides may comprise a region or part of the polynucleotides of the disclosure, including chemically modified RNA (e.g. , mRNA) polynucleotides.
  • the chemically modified RNA polynucleotides can be uniformly modified polynucleotides.
  • the in vitro transcription reaction utilizes a custom mix of nucleotide triphosphates (NTPs).
  • the NTPs may comprise chemically modified NTPs, or a mix of natural and chemically modified NTPs, or natural NTPs.
  • a typical in vitro transcription reaction includes the following:
  • the crude IVT mix may be stored at 4 °C overnight for cleanup the next day. 1 U of RNase-free DNase may then be used to digest the original template. After 15 minutes of incubation at 37 °C, the mRNA may be purified using Ambion's MEGACLEARTM Kit (Austin, TX) following the manufacturer' s instructions. This kit can purify up to 500 ⁇ g of RNA. Following the cleanup, the RNA polynucleotide may be quantified using the
  • polynucleotide is the proper size and that no degradation of the RNA has occurred.
  • Example 5 Enzymatic Capping
  • RNA polynucleotide Capping of a RNA polynucleotide is performed as follows where the mixture includes: IVT RNA 60 ⁇ g-180 ⁇ g and d3 ⁇ 40 up to 72 ⁇ . The mixture is incubated at 65 °C for 5 minutes to denature RNA, and then is transferred immediately to ice.
  • the protocol then involves the mixing of lOx Capping Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KC1, 12.5 mM MgCl 2 ) (10.0 ⁇ ); 20 mM GTP (5.0 ⁇ ); 20 mM S-Adenosyl Methionine (2.5 ⁇ ); RNase Inhibitor (100 U); 2'-0-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U); dH 2 0 (Up to 28 ⁇ ); and incubation at 37 °C for 30 minutes for 60 ⁇ g RNA or up to 2 hours for 180 ⁇ g of RNA.
  • lOx Capping Buffer 0.5 M Tris-HCl (pH 8.0), 60 mM KC1, 12.5 mM MgCl 2 ) (10.0 ⁇ ); 20 mM GTP (5.0 ⁇ ); 20 mM S-Adenosyl Methionine
  • RNA polynucleotide may then be purified using Ambion's MEGACLEARTM Kit (Austin, TX) following the manufacturer' s instructions. Following the cleanup, the RNA may be quantified using the NANODROPTM (ThermoFisher, Waltham, MA) and analyzed by agarose gel electrophoresis to confirm the RNA polynucleotide is the proper size and that no degradation of the RNA has occurred.
  • NANODROPTM ThermoFisher, Waltham, MA
  • the RNA polynucleotide product may also be sequenced by running a reverse-transcription- PCR to generate the cDNA for sequencing.
  • a poly-A tailing reaction must be performed before cleaning the final product. This is done by mixing capped IVT RNA (100 ⁇ ); RNase Inhibitor (20 U); lOx Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl 2 ) (12.0 ⁇ ); 20 mM ATP (6.0 ⁇ ); Poly-A Polymerase (20 U); d3 ⁇ 40 up to 123.5 ⁇ and incubation at 37 °C for 30 min.
  • Poly-A Polymerase may be a recombinant enzyme expressed in yeast.
  • polyA tails of approximately between 40-200 nucleotides, e.g., about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope of the present disclosure.
  • 5 '-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5'- guanosine cap structure according to manufacturer protocols: 3'-0-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New
  • 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, MA).
  • Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate:
  • Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl- transferase.
  • Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0- methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.
  • Enzymes are preferably derived from a recombinant source.
  • the modified mRNAs When transfected into mammalian cells, the modified mRNAs have a stability of between 12- 18 hours or more than 18 hours, e.g. , 24, 36, 48, 60, 72 or greater than 72 hours.
  • Polynucleotides encoding a polypeptide, containing any of the caps taught herein, can be transfected into cells at equal concentrations.
  • the amount of protein secreted into the culture medium can be assayed by ELISA at 6, 12, 24 and/or 36 hours post- transfection.
  • Synthetic polynucleotides that secrete higher levels of protein into the medium correspond to a synthetic polynucleotide with a higher translationally-competent cap structure.
  • RNA ⁇ e.g., mRNA polynucleotides encoding a polypeptide, containing any of the caps taught herein can be compared for purity using denaturing Agarose-Urea gel electrophoresis or HPLC analysis.
  • RNA polynucleotides with a single, consolidated band by electrophoresis correspond to the higher purity product compared to polynucleotides with multiple bands or streaking bands.
  • Chemically modified RNA polynucleotides with a single HPLC peak also correspond to a higher purity product. The capping reaction with a higher efficiency provides a more pure polynucleotide population.
  • RNA ⁇ e.g., mRNA polynucleotides encoding a polypeptide, containing any of the caps taught herein can be transfected into cells at multiple concentrations.
  • the amount of pro-inflammatory cytokines, such as TNF-alpha and IFN-beta, secreted into the culture medium can be assayed by ELISA at 6, 12, 24 and/or 36 hours post-transfection.
  • RNA polynucleotides resulting in the secretion of higher levels of pro-inflammatory cytokines into the medium correspond to a polynucleotides containing an immune- activating cap structure.
  • RNA ⁇ e.g., mRNA polynucleotides encoding a polypeptide, containing any of the caps taught herein can be analyzed for capping reaction efficiency by LC-MS after nuclease treatment.
  • Nuclease treatment of capped polynucleotides yield a mixture of free nucleotides and the capped 5'-5-triphosphate cap structure detectable by LC-MS.
  • the amount of capped product on the LC-MS spectra can be expressed as a percent of total polynucleotide from the reaction and correspond to capping reaction efficiency.
  • the cap structure with a higher capping reaction efficiency has a higher amount of capped product by LC-MS.
  • Example 9 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products
  • RNA polynucleotides 200-400 ng in a 20 ⁇ volume
  • reverse transcribed PCR products 200-400 ng
  • RNA polynucleotides 200-400 ng in a 20 ⁇ volume
  • reverse transcribed PCR products 200-400 ng
  • Example 10 NANODROPTM Modified RNA Quantification and UV Spectral Data Chemically modified RNA polynucleotides in TE buffer (1 ⁇ ) are used for
  • NANODROPTM UV absorbance readings to quantitate the yield of each polynucleotide from an chemical synthesis or in vitro transcription reaction.
  • Example 11 Formulation of Modified mRNA Using Lipidoids
  • RNA (e.g., mRNA) polynucleotides may be formulated for in vitro experiments by mixing the polynucleotides with the lipidoid at a set ratio prior to addition to cells. In vivo formulation may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA-lipidoid formulations may be used as a starting point. After formation of the particle, polynucleotide is added and allowed to integrate with the complex. The encapsulation efficiency is determined using a standard dye exclusion assays.
  • Example 12 Mouse Immunogenicity Studies
  • assays were carried out to evaluate the immune response to influenza virus vaccine antigens delivered using an mRNA/LNP platform in comparison to protein antigens.
  • the instant study was designed to test the immunogenicity in mice of candidate influenza virus vaccines comprising an mRNA polynucleotide encoding HA stem protein obtained from different strains of influenza virus. Animals tested were 6-8 week old female BALB/c mice obtained from Charles River Laboratories. Test vaccines included the following mRNAs formulated in MC3 LNP: stem of Hi/Puerto Rico/8/1934 (based on Mallajosyula V et al. /WAS 2014 Jun 24; l l l(25):E2514-23), stem of Hi/New
  • Protein vaccines tested in this study included the ⁇ -Foldon protein, as described in Mallajosyula et al. Proc Natl Acad Sci U S A. 2014;l l l(25):E2514-23.
  • mice were immunized intramuscularly with a total volume of 100 ⁇ L of each test vaccine, which was administered in a 50 ⁇ immunization to each quadricep, except for administration of the PR8 influenza virus control which was delivered intranasally in a volume of 20 ⁇ ⁇ while the animals were sedated with a mixture of Ketamine and Xylazine.
  • the group numbers for each test vaccine along with the vaccine dose are outlined in the table below: Table 1.
  • mice were immunized with two doses of the various influenza virus RNA vaccine formulations at weeks 0 and 3, and serum was collected two weeks after immunization with the second dose.
  • ELISA plates were coated with 100 ng of the following recombinant HAs obtained from Sino Biological Inc.: Influenza A H1N1 (A/New Caledonia/20/99), cat # 11683-V08H; Influenza A H3N2 (A/Aichi/2/1968), cat # 11707- V08H; Influenza A H1N1 (A/California/04/2009) cat # 11055-V08H; Influenza A H1N1 (A/Puerto Rico/8/34) cat # 11684-V08H; Influenza A H3N2 (A/Brisbane/10/2007), cat # 11056-V08H; Influenza A H2N2 (A/Japan/305/1957) cat # 11088-V08H; Influenza A H7N9 (A/Anhui/1/2013) cat # 40103-V08H; Influenza A H1N1 (A/New Caledonia/20/99), cat # 11683-V08H; Influenza A H3
  • Fig. 1 illustrates that mRNA based vaccines encoding HA-based antigens that are encapsulated in the MC3 lipid nanoparticle induced high antibody binding titers to HA.
  • Fig. 1 also illustrates that mRNA vaccines designed to express a portion of the stem domain from different H1N1 or H5N1 strains of influenza elicited high antibody titers that were capable of binding all strains of group 1 HA tested as well as several group 2 strains.
  • Fig. 1 also illustrates that mRNA vaccines designed to express a portion of the H1N1 A/Calif ornia/04/2009 stem domain induced higher titers than a protein vaccine of the same stem domain.
  • H1HA10-Foldon_delta Ngly eHIHA (ectodomain of HA from H1N1 A/Puerto Rico/8/34); eHlHA_native signal seq (eHIHA with its native signal sequence); H3N2 A Wisconsin/67/2005 stem; H3N2 A/Hong Kong/1/1968 stem (based on Mallajosyula
  • the group numbers for each test vaccine along with the vaccine dose are outlined in the table below:
  • Figs. 2 and 3 show the endpoint anti-HA antibody titers following the second immunization with the test vaccines.
  • the vaccines tested are shown on the x-axis and the binding to HA from each of the different strains of influenza is plotted. All mRNA vaccines encoding HA stem were immunogenic and elicited a robust antibody response recognizing HA from a diverse set of influenza A virus strains.
  • eHlHA_native- signal- sequence mRNAs elicited the highest overall binding titers across the panel of group 1 HAs, while the H3HA6 RNA elicited the highest overall binding titers across group 2 Has (Fig. 2).
  • Immunogenicity of combinations of stem mRNA vaccines was also tested. In this study, individual mRNAs were mixed prior to formulation with LNP (Group 9, co-form) or individual mRNAs were formulated with LNP prior to mixing (Group 10, mix-form). As shown in Fig. 3, combining HI and H3 stem-based mRNAs did not result in interference in the immune response to either antigen, regardless of the method of formulation.
  • mice tested were 6-8 week old female BALB/c mice obtained from Charles River Laboratories.
  • Test vaccines included the following mRNAs formulated in MC3 LNP: NIHGen6HASS-foldon mRNA (based on Yassine et al. Nat. Med. 2015 Sep; 21(9): 1065-70), an mRNA encoding the nucleoprotein NP from an H3N2 strain, or one of several combinations of NIHGen6HASS-foldon and NP mRNAs.
  • mice were immunized intramuscularly (IM) with a total volume of 100 ⁇ ⁇ of each test vaccine, which was administered in a 50 ⁇ ⁇ immunization to each quadricep.
  • IM intramuscularly
  • Candidate influenza virus vaccines evaluated in this study were described above and are outlined in the table below.
  • Sera were collected from all animals two weeks after the second dose.
  • ELISA plates were coated with 100 ng of the following recombinant proteins obtained from Sino Biological Inc.: Influenza A HlNl (A/New Caledonia/20/99) HA, cat # 11683-V08H; Influenza A H3N2 (A/Aichi/2/1968) HA, cat # 11707-V08H; Influenza A HlNl (A/California/04/2009) HA, cat # 11055-V08H; Influenza A HlNl (A/Puerto Rico/8/34) HA, cat # 11684-V08H; Influenza A HlNl (A/Brisbane/59/2007) HA, cat # 11052- V08H; Influenza A H2N2
  • Fig. 4 depicts the endpoint titers of the pooled serum from animals vaccinated with the test vaccines. The vaccines tested are shown on the x-axis of Fig.
  • Fig. 5 To probe the functional antibody response, the ability of serum to neutralize a panel of HA-pseudotyped viruses was assessed (Fig. 5). Briefly, 293 cells were co-transfected with a replication-defective retroviral vector containing a firefly luciferase gene, an expression vector encoding a human airway serine protease, and expression vectors encoding influenza hemagglutinin (HA) and neuraminidase (NA) proteins. The resultant pseudoviruses were harvested from the culture supernatant, filtered, and titered.
  • HA hemagglutinin
  • NA neuraminidase
  • each bar represents the IC50 for neutralization of a different virus pseudotype. While the serum from naive or NP RNA vaccinated mice was unable to inhibit pseudovirus infection, the serum from mice vaccinated with 10 ⁇ g or 5 ⁇ g of NIHGen6HASS-foldon mRNA or with a combination of NIHGen6HASS-foldon and NP mRNAs neutralized, to a similar extent, all HI and H5 virus pseudotypes tested.
  • ADCC antibody-dependent cell cytotoxicity
  • spleens were harvested from a subset of animals in each group and splenocytes from animals in the same group were pooled.
  • Splenic lymphocytes were stimulated with a pool of HA or NP peptides, and IFN- ⁇ , IL-2 or TNF-a production was measured by intracellular staining and flow cytometry.
  • Figure 7 is a representation of responses following stimulation with a pool of NP peptides
  • Figure 8 is a representation of responses following stimulation with a pool of HI HA peptides.
  • NIHGen6HASS-foldon RNA Following vaccination with NP mRNA, either in the presence or absence of NIHGen6HASS-foldon mRNA, antigen-specific CD4 and CD8 T cells were found in the spleen. Following vaccination with NIHGen6HASS-foldon RNA or delivery of
  • NIHGen6HASS-foldon and NP RNAs to distal injections sites (dist. site), only HA-specific CD4 cells were observed. However, when NIHGen6HASS-foldon and NP RNAs were co- administered to the same injection site (co-form, mix), an HA-specific CD8 T cell response was detected.
  • mice that were vaccinated with an H3N2 NP mRNA and challenged with H1N1 virus lost a significant amount (-15%) of weight prior to recovery.
  • Those vaccinated with NIHGen6HASS-foldon RNA also lost -5% body weight.
  • mice vaccinated with a combination of NIHGen6HASS-foldon and NP mRNAs appeared to be completely protected from lethal influenza virus challenge, similar to those vaccinated with mRNA expressing an HA antigen homologous to that of the challenge virus (eHIHA).
  • Vaccine efficacy was similar at all vaccine doses, as well as with all co- formulation and co-delivery methods assessed (Fig. 10). Influenza A challenge #2
  • mice tested were 6-8 week old female BALB/c mice obtained from Charles River Laboratories.
  • Test vaccines included the following mRNAs formulated in MC3 LNP: NIHGen6HASS-foldon mRNA (based on Yassine et al. Nat. Med. 2015 Sep; 21(9): 1065-70) and NIHGen6HASS-TM2 mRNA.
  • Control animals were vaccinated with an mRNA encoding the ectodomain of the HA from HlNl A/Puerto Rico/8/1934 (eHIHA, positive control) or were not vaccinated (naive).
  • mice were immunized intramuscularly (IM) with a total volume of 100 ⁇ ⁇ of each test vaccine, which was administered in a 50 ⁇ ⁇ immunization to each quadricep.
  • IM intramuscularly
  • Candidate influenza virus vaccines evaluated in this study were described above and outlined in the table below.
  • Sera were collected from all animals two weeks after the second dose.
  • all animals were challenged intranasally while sedated with a mixture of Ketamine and Xylazine with a lethal dose of mouse-adapted influenza virus strain HlNl A/Puerto Rico/8/1934. Mortality was recorded and group mouse weight was assessed daily for 20 days post-infection.
  • ELISA plates were coated with 100 ng of the following recombinant HAs obtained from Sino Biological Inc.: Influenza A HlNl (A/New Caledonia/20/99), cat # 11683-V08H; Influenza A H3N2 (A/Aichi/2/1968), cat # 11707- V08H; Influenza A HlNl (A/California/04/2009) cat # 11055-V08H; Influenza A HlNl (A/Puerto Rico/8/34) cat # 11684-V08H; Influenza A HlNl (A/Brisbane/59/2007), cat # 11052-V08H; Influenza A H2N2 (A/Japan/305/1957) cat # 11088-V08H; Influenza A H7N9 (A/Anhui/1/2013) cat # 40103-V08H and
  • Fig. 11 A depicts the endpoint titers of the pooled serum from animals vaccinated with the test vaccines.
  • the vaccines tested are shown on the x-axis and the binding to HA from each of the different strains of influenza is plotted.
  • the NIHGen6HASS-foldon mRNA vaccine elicited high titers of antibodies that bound all HI, H2 and H7 HAs tested.
  • the binding titers from NIHGen6HASS-TM2 mRNA vaccinated mice were reduced as compared to those from NIHGen6HASS-foldon mRNA vaccinated mice.
  • NIHGen6HAS S -TM2 vaccine was equivalent to that of the NIHGen6HASS-foldon vaccine.
  • HA hemagglutinin
  • influenza A serotype HI HA sequences were obtained from the NIAID Influenza Research Database (IRD) (Squires et al., Influenza Other Respir Viruses. 2012 Nov; 6(6): 404-416.) through the web site at
  • Test vaccines included the following mRNAs formulated in MC3 LNP: ConHl and ConH3 (based on Webby et al., PLoS One. 2015 Oct 15;10(10):e0140702.); Cobra_Pl and Cobra_X3 (based on Carter et al., / Virol. 2016 Apr 14;90(9):4720-34); MRK_pHl_Con and MRK_sHl_Con (pandemic and seasonal consensus sequences described above); and each of the above mentioned six antigens with a ferritin fusion sequence for potential particle formation.
  • mice were immunized intramuscularly (IM) with a total volume of 100 ⁇ L of each test vaccine, which was administered in a 50 ⁇ L ⁇ immunization to each quadricep.
  • IM intramuscularly
  • Candidate influenza virus vaccines evaluated in this study were described above and are outlined in the table below.
  • Sera were collected from all animals two weeks after the second dose (week 5).
  • the animals were challenged intranasally while sedated with a mixture of Ketamine and Xylazine with a lethal dose of mouse-adapted influenza virus strain H1N1 A/Puerto Rico/8/1934 (PR8). Mortality was recorded and group weight was assessed daily for 20 days post-infection.
  • Luminescence signal was read with a Gaussia Luciferase Glow Assay Kit (Pierce) on an En Vision reader (Perkin Elmer).
  • mice immunized with the consensus HI HA antigens survived the lethal PR8 virus challenge and showed no weight loss, except for the Merck_pHl_Con_ferritin mRNA group, while mice in the ConH3, naive and LNP only control groups rapidly lost weight upon challenge (Fig. 13).
  • Mice immunized with Merck_pHl_Con_ferritin mRNA survived the lethal PR8 virus challenge and showed 5-10% weight loss, suggesting that partial protection may be mediated by mechanism(s) other than virus neutralization.
  • mice tested were 6-8 week old female BALB/c mice obtained from Charles River Laboratories.
  • Test vaccines included the following mRNAs formulated in MC3 LNP: B/Phuket/3073/2013 sHA (soluble HA), B/Phuket/3073/2013 mHA (full-length HA with membrane anchor), B/Brisbane/60/2008 sHA, B/Victoria/02/1 87 sHA,
  • Control animals were vaccinated with a nonlethal dose of mouse-adapted B/Ann Arbor/1954 (positive control) or empty MC3 LNP (to control for effects of the LNP) or were not vaccinated (naive).
  • mice were immunized intramuscularly (IM) with a total volume of 100 ⁇ L of each test vaccine, which was administered in a 50 ⁇ L immunization to each quadricep.
  • IM intramuscularly
  • Candidate influenza virus vaccines evaluated in this study were described above and are outlined in the table below.
  • Sera were collected from all animals two weeks after the second dose.
  • sequences described herein encompasses a chemically modified sequence or an unmodified sequence which includes no nucleotide modifications.
  • Fig. 15A depicts the ELISA endpoint anti-HA antibody titers of the pooled serum from animals vaccinated with the test vaccines.
  • the vaccines tested are shown on the x-axis and the binding to HA from each of the different strains of influenza is plotted. All vaccines tested, except for those derived from B/Phuket/3073/2013 were immunogenic, and serum antibody bound to HA from both B/Yamagata/16/1988 (Yamagata lineage) and
  • B/Yamagata 16/1988 mHA RNA vaccine was able to prevent lethality and weight loss in animals challenged with a heterologous virus strain (Fig 15B).
  • Test vaccines included the following mRNAs formulated in MC3 LNP: NIHGen6HASS-foldon mRNA (based on Yassine et al. Nat. Med. 2015 Sep;
  • Animals in Group 1 had been previously vaccinated with seasonal inactivated influenza vaccine (FLUZONE ® ) and were boosted intramuscularly (IM) at day 0 with 300 ⁇ g of NIHGen6HASS-foldon mRNA.
  • Animals in Groups 2 and 3 were influenza naive at the study start and were vaccinated at days 0, 28 and 56 with 300 ⁇ g of NIHGen6HASS-foldon mRNA or 300 ⁇ g of NP mRNA, respectively.
  • Serum was collected from all animals prior to the study start (day -8) as well as at days 14, 28, 42, 56, 70, 84, 112, 140 and 168.
  • NIHGen6HASS-foldon vaccine elicited a robust antibody response as measured by ELISA assay (plates coated with recombinantly-expressed NIHGen6HASS-foldon [HA stem] or NP proteins), and the data is depicted in Fig. 16.
  • Fig. 16A shows titers to HA stem, over time, for four rhesus macaques previously vaccinated with FLUZONE ® and boosted a single time with NIHGen6HASS-foldon mRNA vaccine.
  • Fig. 16A shows titers to HA stem, over time, for four rhesus macaques previously vaccinated with FLUZONE ® and boosted a single time with NIHGen6HASS-foldon mRNA vaccine.
  • FIG. 16B depicts titers to HA stem, over time, from four rhesus macaques vaccinated at days 0, 28 and 56 with the same NIHGen6HASS-foldon RNA vaccine.
  • the NIHGen6HASS-foldon RNA vaccine was able to boost anti-HA stem antibody binding titers in animal previously vaccinated with inactivated influenza vaccine as well as elicited a robust response in naive animals.
  • HA stem titers remained elevated over baseline to at least study day 168.
  • Fig. 16C illustrates antibody titers to NP, over time, for four rhesus macaques vaccinated at days 0, 28 and 56 with the NP mRNA vaccine and shows that the vaccine elicited a robust antibody response to NP.
  • ELISA plates were coated with recombinant HAs from a diverse set of influenza strains as described above. EC 10 titers were calculated as the reciprocal of the serum dilution that reached 10% of the maximal signal.
  • a single dose of NIHGen6HASS-foldon vaccine boosted titers to HI HAs - 40 - 60 fold, and titers peaked approximately 28 days post- vaccination. Titers decreased from days 28 - 70, but day 70 titers were still ⁇ 10 - 30-fold above the titers measured prior to vaccination.
  • the NIHGen6HASS-foldon mRNA vaccine did not boost titers to HAs from H3 or H7 influenza strains.
  • antibody titers to HI and H2 HAs rose after each dose of NIHGen6HASS-foldon mRNA vaccine, and titers appeared to rise most dramatically after dose 2.
  • the NP mRNA vaccine also elicited cell- mediated immunity in rhesus.
  • PBMCs were collected from Group 3 NP mRNA vaccinated rhesus macaques. Lymphocytes were stimulated with a pool of NP peptides, and IFN- ⁇ , IL-2 or TNF-a production were measured by intracellular staining and flow cytometry.
  • Figure 18 is a representation of responses following NP peptide pool stimulation. Following vaccination with NP mRNA, antigen- specific CD4 and CD8 T cells were found in the peripheral blood, and these cells were maintained above baseline to at least study day 140.
  • Example 15 H7N9 1 mm u n ogen icity Studies
  • H7N9 immunogenicity
  • the HAI test showed a geometric mean titer (GMT) of 45 for all of the animals, including the placebo group.
  • the GMT of the responders only was 116 (Fig. 19).
  • the HAI kinetics for each individual subject are given in Fig. 20.
  • the microneutralization (MN) test showed a geometric mean titer (GMT) of 36 for all of the animals, including the placebo group.
  • the GMT of the responders only was 84 (Fig. 21).
  • the MN test kinetics for each subject are given in Fig. 22.
  • HAI and MN showed a very strong correlation (Fig. 23). Only one subject had a protective titer in one assay , but not in the other. Also, 10 subjects had no detectable HAI or MN titer at Day 43.
  • Influenza A virus (A/Bayern/ 7 / 95 (H1N1 ) ) NA 1,459 bp AJ518104.1 gene for neuraminidase, genomic RNA linear mRNA GI :31096418

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pulmonology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/US2016/058319 2015-10-22 2016-10-21 Broad spectrum influenza virus vaccine Ceased WO2017070620A2 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
CA3003103A CA3003103A1 (en) 2015-10-22 2016-10-21 Broad spectrum influenza virus vaccine
BR112018008078A BR112018008078A2 (pt) 2015-10-22 2016-10-21 vacina de vírus influenza de amplo espectro
EP16858400.1A EP3365007A4 (en) 2015-10-22 2016-10-21 WIDE SPECTRUM VACCINE AGAINST INFLUENZA VIRUS
MX2018004916A MX2018004916A (es) 2015-10-22 2016-10-21 Vacuna contra el virus de la influenza de amplio espectro.
RU2018118337A RU2018118337A (ru) 2015-10-22 2016-10-21 Вакцина против вируса гриппа широкого спектра действия
CN202410855926.2A CN118846026A (zh) 2015-10-22 2016-10-21 广谱流感病毒疫苗
KR1020187014338A KR20180096591A (ko) 2015-10-22 2016-10-21 광범위 인플루엔자 바이러스 백신
AU2016342048A AU2016342048B2 (en) 2015-10-22 2016-10-21 Broad spectrum influenza virus vaccine
CN201680074920.5A CN109310751A (zh) 2015-10-22 2016-10-21 广谱流感病毒疫苗
JP2018541088A JP7384512B2 (ja) 2015-10-22 2016-10-21 広域インフルエンザウイルスワクチン
US15/767,609 US20180311336A1 (en) 2015-10-22 2016-10-21 Broad spectrum influenza virus vaccine
JP2021204270A JP7491639B2 (ja) 2015-10-22 2021-12-16 広域インフルエンザウイルスワクチン
AU2022218595A AU2022218595A1 (en) 2015-10-22 2022-08-19 Broad spectrum influenza virus vaccine
JP2024077558A JP2024105503A (ja) 2015-10-22 2024-05-10 広域インフルエンザウイルスワクチン

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US201562245225P 2015-10-22 2015-10-22
US201562245031P 2015-10-22 2015-10-22
US62/245,225 2015-10-22
US62/245,031 2015-10-22
US201562247501P 2015-10-28 2015-10-28
US62/247,501 2015-10-28
US201562248248P 2015-10-29 2015-10-29
US62/248,248 2015-10-29

Publications (2)

Publication Number Publication Date
WO2017070620A2 true WO2017070620A2 (en) 2017-04-27
WO2017070620A3 WO2017070620A3 (en) 2017-07-13

Family

ID=58558155

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/058319 Ceased WO2017070620A2 (en) 2015-10-22 2016-10-21 Broad spectrum influenza virus vaccine

Country Status (12)

Country Link
US (1) US20180311336A1 (enExample)
EP (1) EP3365007A4 (enExample)
JP (3) JP7384512B2 (enExample)
KR (1) KR20180096591A (enExample)
CN (2) CN109310751A (enExample)
AU (2) AU2016342048B2 (enExample)
BR (1) BR112018008078A2 (enExample)
CA (1) CA3003103A1 (enExample)
MA (1) MA46023A (enExample)
MX (2) MX2018004916A (enExample)
RU (1) RU2018118337A (enExample)
WO (1) WO2017070620A2 (enExample)

Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018075592A1 (en) * 2016-10-21 2018-04-26 Merck Sharp & Dohme Corp. Influenza hemagglutinin protein vaccines
WO2018089851A3 (en) * 2016-11-11 2018-06-21 Modernatx, Inc. Influenza vaccine
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
WO2018167320A1 (en) 2017-03-17 2018-09-20 Curevac Ag Rna vaccine and immune checkpoint inhibitors for combined anticancer therapy
WO2018170245A1 (en) * 2017-03-15 2018-09-20 Modernatx, Inc. Broad spectrum influenza virus vaccine
US10207010B2 (en) 2015-12-10 2019-02-19 Modernatx, Inc. Compositions and methods for delivery of agents
WO2019038332A1 (en) 2017-08-22 2019-02-28 Curevac Ag VACCINE AGAINST BUNYAVIRUS
US10272150B2 (en) 2015-10-22 2019-04-30 Modernatx, Inc. Combination PIV3/hMPV RNA vaccines
US10273269B2 (en) 2017-02-16 2019-04-30 Modernatx, Inc. High potency immunogenic zika virus compositions
WO2019193183A2 (en) 2018-04-05 2019-10-10 Curevac Ag Novel yellow fever nucleic acid molecules for vaccination
US10449244B2 (en) 2015-07-21 2019-10-22 Modernatx, Inc. Zika RNA vaccines
US10493143B2 (en) 2015-10-22 2019-12-03 Modernatx, Inc. Sexually transmitted disease vaccines
US10517940B2 (en) 2015-10-22 2019-12-31 Modernatx, Inc. Zika virus RNA vaccines
WO2020002525A1 (en) 2018-06-27 2020-01-02 Curevac Ag Novel lassa virus rna molecules and compositions for vaccination
US10653712B2 (en) 2016-09-14 2020-05-19 Modernatx, Inc. High purity RNA compositions and methods for preparation thereof
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
US10709779B2 (en) 2014-04-23 2020-07-14 Modernatx, Inc. Nucleic acid vaccines
US10730924B2 (en) 2016-05-18 2020-08-04 Modernatx, Inc. Polynucleotides encoding relaxin
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
CN111971393A (zh) * 2018-03-14 2020-11-20 仁济大学校产学协力团 使用重组腺病毒的多价活流感疫苗平台
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021123332A1 (en) 2019-12-20 2021-06-24 Curevac Ag Lipid nanoparticles for delivery of nucleic acids
US11045540B2 (en) 2017-03-15 2021-06-29 Modernatx, Inc. Varicella zoster virus (VZV) vaccine
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
US11103578B2 (en) 2016-12-08 2021-08-31 Modernatx, Inc. Respiratory virus nucleic acid vaccines
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
WO2022002783A1 (en) 2020-06-29 2022-01-06 Glaxosmithkline Biologicals Sa Adjuvants
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
WO2022099003A1 (en) * 2020-11-06 2022-05-12 Sanofi Lipid nanoparticles for delivering mrna vaccines
US11351242B1 (en) 2019-02-12 2022-06-07 Modernatx, Inc. HMPV/hPIV3 mRNA vaccine composition
US11364292B2 (en) 2015-07-21 2022-06-21 Modernatx, Inc. CHIKV RNA vaccines
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
WO2022200574A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200582A1 (en) * 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
US11464848B2 (en) 2017-03-15 2022-10-11 Modernatx, Inc. Respiratory syncytial virus vaccine
WO2022233880A1 (en) 2021-05-03 2022-11-10 Curevac Ag Improved nucleic acid sequence for cell type specific expression
WO2022248353A1 (en) 2021-05-24 2022-12-01 Glaxosmithkline Biologicals Sa Adjuvants
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
WO2022264109A1 (en) * 2021-06-18 2022-12-22 Sanofi Multivalent influenza vaccines
WO2023021427A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Freeze-drying of lipid nanoparticles (lnps) encapsulating rna and formulations thereof
WO2023020992A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023021421A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Low-dose lyophilized rna vaccines and methods for preparing and using the same
WO2023020993A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023020994A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023057930A1 (en) * 2021-10-08 2023-04-13 Pfizer Inc. Immunogenic lnp compositions and methods thereof
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
US11643441B1 (en) 2015-10-22 2023-05-09 Modernatx, Inc. Nucleic acid vaccines for varicella zoster virus (VZV)
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
EP4227319A1 (en) 2018-04-17 2023-08-16 CureVac SE Novel rsv rna molecules and compositions for vaccination
US11752206B2 (en) 2017-03-15 2023-09-12 Modernatx, Inc. Herpes simplex virus vaccine
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
US11897944B2 (en) 2017-01-17 2024-02-13 Ablynx N.V. Immunoglobulin single variable domain (ISVD) capable of binding to serum albumin
US11911453B2 (en) 2018-01-29 2024-02-27 Modernatx, Inc. RSV RNA vaccines
DE202023106198U1 (de) 2022-10-28 2024-03-21 CureVac SE Impfstoff auf Nukleinsäurebasis
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
RU2819143C2 (ru) * 2018-03-12 2024-05-14 ЭсКьюЗед БАЙОТЕКНОЛОДЖИЗ КОМПАНИ Внутриклеточная доставка биомолекул для модуляции иммунного ответа
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation
WO2024126809A1 (en) * 2022-12-15 2024-06-20 Sanofi Mrna encoding influenza virus-like particle
WO2024133160A1 (en) 2022-12-19 2024-06-27 Glaxosmithkline Biologicals Sa Hepatitis b compositions
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
US12070495B2 (en) 2019-03-15 2024-08-27 Modernatx, Inc. HIV RNA vaccines
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
US12098166B2 (en) 2018-11-06 2024-09-24 Boehringer Ingelheim Vetmedica Gmbh Immunogenic composition against avian influenza virus H5 subtype
WO2024223724A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024223728A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024230934A1 (en) 2023-05-11 2024-11-14 CureVac SE Therapeutic nucleic acid for the treatment of ophthalmic diseases
US12151029B2 (en) 2018-09-19 2024-11-26 Modernatx, Inc. PEG lipids and uses thereof
WO2025012461A1 (en) 2023-07-13 2025-01-16 Sanofi Methods and compositions for analyzing messenger rna
WO2025011529A2 (en) 2023-07-07 2025-01-16 Shanghai Circode Biomed Co., Ltd. Circular rna vaccines for seasonal flu and methods of uses
EP4494652A2 (en) 2017-07-04 2025-01-22 CureVac SE Cancer rna-vaccine
WO2025045142A1 (en) 2023-08-29 2025-03-06 Shanghai Circode Biomed Co., Ltd. Circular rna encoding vegf polypeptides, formulations, and methods of uses
EP4342490A4 (en) * 2021-05-19 2025-06-04 Daiichi Sankyo Company, Limited Influenza virus nucleic acid lipid particle vaccine
US12329811B2 (en) 2021-01-11 2025-06-17 Modernatx, Inc. Seasonal RNA influenza virus vaccines
WO2025132839A1 (en) 2023-12-21 2025-06-26 Glaxosmithkline Biologicals Sa Influenza virus vaccines
US20250228788A1 (en) * 2018-09-04 2025-07-17 The Board Of Regents Of The University Of Texas System Compositions and methods for organ specific delivery of nucleic acids
US12383508B2 (en) 2018-09-19 2025-08-12 Modernatx, Inc. High-purity peg lipids and uses thereof
US12390523B2 (en) 2020-02-04 2025-08-19 CureVac SE Coronavirus vaccine
US12460259B2 (en) 2019-03-11 2025-11-04 Modernatx, Inc. Fed-batch in vitro transcription process

Families Citing this family (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12150980B2 (en) 2015-07-30 2024-11-26 Modernatx, Inc. Concatemeric peptide epitope RNAs
US11564893B2 (en) 2015-08-17 2023-01-31 Modernatx, Inc. Methods for preparing particles and related compositions
MA46316A (fr) 2015-10-22 2021-03-24 Modernatx Inc Vaccin contre le cytomégalovirus humain
US10465190B1 (en) 2015-12-23 2019-11-05 Modernatx, Inc. In vitro transcription methods and constructs
WO2017201342A1 (en) 2016-05-18 2017-11-23 Modernatx, Inc. Polynucleotides encoding jagged1 for the treatment of alagille syndrome
WO2018075980A1 (en) 2016-10-21 2018-04-26 Modernatx, Inc. Human cytomegalovirus vaccine
US11384352B2 (en) 2016-12-13 2022-07-12 Modernatx, Inc. RNA affinity purification
US20200030432A1 (en) 2017-03-17 2020-01-30 Modernatx, Inc. Zoonotic disease rna vaccines
CA3056819A1 (en) 2017-03-24 2018-09-27 Biontech Rna Pharmaceuticals Gmbh Methods and compositions for stimulating immune response
EP3607074A4 (en) 2017-04-05 2021-07-07 Modernatx, Inc. REDUCTION OR ELIMINATION OF IMMUNE RESPONSES TO NON-INTRAVENOUS THERAPEUTIC PROTEINS, FOR EXAMPLE SUBCUTANEOUSLY
US11786607B2 (en) 2017-06-15 2023-10-17 Modernatx, Inc. RNA formulations
US11866696B2 (en) 2017-08-18 2024-01-09 Modernatx, Inc. Analytical HPLC methods
WO2019036682A1 (en) 2017-08-18 2019-02-21 Modernatx, Inc. RNA VARIANTS POLYMERASE
US11912982B2 (en) 2017-08-18 2024-02-27 Modernatx, Inc. Methods for HPLC analysis
MX2020002348A (es) 2017-08-31 2020-10-08 Modernatx Inc Métodos de elaboración de nanopartículas lipídicas.
MA50253A (fr) 2017-09-14 2020-07-22 Modernatx Inc Vaccins à arn contre le virus zika
BR112020015308A8 (pt) 2018-01-29 2023-02-07 Merck Sharp & Dohme Proteínas f de rsv estabilizadas e usos das mesmas
US12090235B2 (en) 2018-09-20 2024-09-17 Modernatx, Inc. Preparation of lipid nanoparticles and methods of administration thereof
KR102370100B1 (ko) * 2019-02-15 2022-03-07 아이디바이오 주식회사 이종 인플루엔자 a 바이러스에 대한 면역/치료반응을 형성하는 신규한 재조합 인플루엔자 바이러스 및 이를 포함하는 유전자 전달체 및 치료백신
JP2022521094A (ja) 2019-02-20 2022-04-05 モデルナティエックス インコーポレイテッド 共転写キャッピング用ｒｎａポリメラーゼバリアント
US11851694B1 (en) 2019-02-20 2023-12-26 Modernatx, Inc. High fidelity in vitro transcription
WO2020232426A1 (en) * 2019-05-16 2020-11-19 Vanderbilt University Peptide vaccine based on a new universal influenza a hemagglutinin head domain epitope and human monoclonal antibodies binding thereto
CA3157859A1 (en) * 2019-10-15 2021-04-22 Modernatx, Inc. Mrnas encoding immune modulating polypeptides and uses thereof
GB2605538B (en) 2020-03-23 2024-06-26 Hdt Bio Corp Compositions and methods for delivery of RNA
MX2022012251A (es) * 2020-03-31 2023-03-13 Univ Pennsylvania Vacuna universal contra la influenza usando arnm modificado con nucleósidos.
RU2742336C1 (ru) * 2020-04-06 2021-02-04 Общество с ограниченной ответственностью "ВиЭй Фарма" Кросс-реактивная рекомбинантная вакцина против вируса гриппа а человека
JP2023522249A (ja) 2020-04-22 2023-05-29 ビオンテック・ソシエタス・エウロパエア コロナウイルスワクチン
WO2021222830A1 (en) * 2020-05-01 2021-11-04 Meso Scale Technologies, Llc. Viral serology assays
CN115803333A (zh) 2020-07-02 2023-03-14 生命技术公司 三核苷酸帽类似物、其制备和用途
GB202011367D0 (en) * 2020-07-22 2020-09-02 Micropore Tech Limited Method of preparing liposomes
KR20230167008A (ko) * 2020-08-06 2023-12-07 모더나티엑스, 인크. 지질 나노입자의 제조 방법
US11406703B2 (en) 2020-08-25 2022-08-09 Modernatx, Inc. Human cytomegalovirus vaccine
US20240024456A1 (en) * 2020-09-07 2024-01-25 Intervet Inc. Ha stem vaccine for ha antibody-positive targets
US20220218622A1 (en) 2020-10-14 2022-07-14 George Mason Research Foundation, Inc. Ionizable lipids and methods of manufacture and use thereof
JP2023549112A (ja) 2020-11-05 2023-11-22 ネオイミューンテック， インコーポレイテッド Ｉｌ－７タンパク質とヌクレオチドワクチンの組み合わせで腫瘍を治療する方法
EP4277655A1 (en) * 2021-01-15 2023-11-22 ModernaTX, Inc. Variant strain-based coronavirus vaccines
AU2022230446A1 (en) * 2021-03-05 2023-09-21 Modernatx, Inc. Vlp enteroviral vaccines
US20220363937A1 (en) 2021-05-14 2022-11-17 Armstrong World Industries, Inc. Stabilization of antimicrobial coatings
EP4366768A4 (en) * 2021-07-09 2025-05-21 ModernaTX, Inc. Pan-human coronavirus vaccines
CN118510549A (zh) * 2021-09-22 2024-08-16 圣诺制药公司 具有减小的纳米颗粒尺寸和改善的多分散性指数的纳米颗粒药物组合物
JP2024535353A (ja) 2021-09-22 2024-09-30 エイチディーティー バイオ コーポレーション 感染性疾患に対するｒｎａワクチン
CA3232725A1 (en) * 2021-09-22 2023-03-30 Steven Gregory REED Cancer therapy compositions and uses thereof
CN113827714B (zh) * 2021-09-26 2023-07-14 华南农业大学 一种h7n9亚型禽流感病毒样颗粒疫苗制剂及制备和应用
CA3237139A1 (en) * 2021-11-05 2023-05-11 Sanofi Hybrid multivalent influenza vaccines comprising hemagglutinin and neuraminidase and methods of using the same
US12186387B2 (en) 2021-11-29 2025-01-07 BioNTech SE Coronavirus vaccine
WO2023125889A1 (en) * 2021-12-31 2023-07-06 Suzhou Abogen Biosciences Co., Ltd. Quadrivalent mrna vaccines for influenza viruses
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine
EP4634388A1 (en) 2022-12-14 2025-10-22 Providence Therapeutics Holdings Inc. Compositions and methods for infectious diseases
CN118256522A (zh) * 2022-12-19 2024-06-28 斯微(上海)生物科技股份有限公司 一种广谱流感mRNA疫苗
WO2025092866A1 (zh) * 2023-10-31 2025-05-08 仁景(苏州)生物科技有限公司 一种多价流感mRNA疫苗
WO2025102071A1 (en) * 2023-11-09 2025-05-15 The General Hospital Corporation Hemagglutinin polypeptides and uses thereof

Citations (63)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3051957B2 (ja) 1997-08-28 2000-06-12 榮太郎 清水 融雪機
WO2002098443A2 (de) 2001-06-05 2002-12-12 Curevac Gmbh Stabilisierte mrna mit erhöhtem g/ c- gehalt und otimierter codon usage für die gentherapie
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US20080166414A1 (en) 2004-01-28 2008-07-10 Johns Hopkins University Drugs And Gene Carrier Particles That Rapidly Move Through Mucous Barriers
WO2008121949A1 (en) 2007-03-30 2008-10-09 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
US20100003337A1 (en) 2004-12-10 2010-01-07 Justin Hanes Functionalized poly(ether-anhydride) block copolymers
WO2010005740A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles
WO2010005726A2 (en) 2008-06-16 2010-01-14 Bind Biosciences Inc. Therapeutic polymeric nanoparticles with mtor inhibitors and methods of making and using same
WO2010005725A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising vinca alkaloids and methods of making and using same
WO2010005723A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
WO2010030763A2 (en) 2008-09-10 2010-03-18 Bind Biosciences, Inc. High throughput fabrication of nanoparticles
WO2010075072A2 (en) 2008-12-15 2010-07-01 Bind Biosciences Long circulating nanoparticles for sustained release of therapeutic agents
US20100215580A1 (en) 2006-09-08 2010-08-26 The Johns Hopkins University Compositions and methods for enhancing transport through mucus
WO2011084518A2 (en) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising corticosteroids and methods of making and using same
WO2011084521A2 (en) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising epothilone and methods of making and using same
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
US20120076836A1 (en) 2009-03-31 2012-03-29 The University Of Tokyo Polyion complex of double-stranded ribonucleic acid
WO2012054923A2 (en) 2010-10-22 2012-04-26 Bind Biosciences, Inc. Therapeutic nanoparticles with high molecular weight copolymers
US20120121718A1 (en) 2010-11-05 2012-05-17 The Johns Hopkins University Compositions and methods relating to reduced mucoadhesion
US20120140790A1 (en) 2009-12-15 2012-06-07 Ali Mir M Therapeutic Polymeric Nanoparticle Compositions with High Glass Transition Termperature or High Molecular Weight Copolymers
WO2012082165A1 (en) 2010-01-24 2012-06-21 Novartis Ag Irradiated biodegradable polymer microparticles
US20120189700A1 (en) 2011-01-19 2012-07-26 Zoraida Aguilar Nanoparticle Based Immunological Stimulation
WO2012099755A1 (en) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Pegylated lipids and their use for drug delivery
US20120201859A1 (en) 2002-05-02 2012-08-09 Carrasquillo Karen G Drug Delivery Systems and Use Thereof
US8241670B2 (en) 2004-04-15 2012-08-14 Chiasma Inc. Compositions capable of facilitating penetration across a biological barrier
WO2012109121A1 (en) 2011-02-07 2012-08-16 Purdue Research Foundation Carbohydrate nanoparticles for prolonged efficacy of antimicrobial peptide
US8263665B2 (en) 2005-04-01 2012-09-11 Intezyne Technologies, Inc. Polymeric micelles for drug delivery
WO2012131104A2 (en) 2011-03-31 2012-10-04 Ingell Technologies Holding B.V. Biodegradable compositions suitable for controlled release
WO2012131106A1 (en) 2011-03-31 2012-10-04 Ingell Technologies Holding B.V. Biodegradable compositions suitable for controlled release
US8287849B2 (en) 2000-10-10 2012-10-16 Massachusetts Institute Of Technology Biodegradable poly(beta-amino esters) and uses thereof
US8287910B2 (en) 2009-04-30 2012-10-16 Intezyne Technologies, Inc. Polymeric micelles for polynucleotide encapsulation
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
US20120282343A1 (en) 2001-10-03 2012-11-08 Johns Hopkins University Compositions for oral gene therapy and methods of using same
US20120295832A1 (en) 2011-05-17 2012-11-22 Arrowhead Research Corporation Novel Lipids and Compositions for Intracellular Delivery of Biologically Active Compounds
WO2012166923A2 (en) 2011-05-31 2012-12-06 Bind Biosciences Drug loaded polymeric nanoparticles and methods of making and using same
WO2013012476A2 (en) 2011-07-21 2013-01-24 Arizona Chemical Company, Llc Branched polyether-polyamide block copolymers and methods of making and using the same
WO2013032829A1 (en) 2011-08-26 2013-03-07 Arrowhead Research Corporation Poly(vinyl ester) polymers for in vivo nucleic acid delivery
US20130059360A1 (en) 2005-04-12 2013-03-07 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
WO2013033438A2 (en) 2011-08-31 2013-03-07 Mallinckrodt Llc Nanoparticle peg modification with h-phosphonates
US20130072709A1 (en) 2006-02-21 2013-03-21 Nektar Therapeutics Segmented Degradable Polymers and Conjugates Made Therefrom
US8404222B2 (en) 1996-09-26 2013-03-26 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
WO2013044219A1 (en) 2011-09-22 2013-03-28 Bind Biosciences Methods of treating cancers with therapeutic nanoparticles
WO2013059496A1 (en) 2011-10-18 2013-04-25 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
WO2013063468A1 (en) 2011-10-27 2013-05-02 Massachusetts Institute Of Technology Amino acid derivates functionalized on the n- terminal capable of forming drug incapsulating microspheres
US8440614B2 (en) 2000-12-29 2013-05-14 Aphios Corporation Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein
US20130130348A1 (en) 2006-05-15 2013-05-23 The Brigham And Women's Hospital, Inc. Polymers for Functional Particles
WO2013072929A2 (en) 2011-09-23 2013-05-23 Indian Institute Of Technology Nanop article based cosmetic composition
WO2013078199A2 (en) 2011-11-23 2013-05-30 Children's Medical Center Corporation Methods for enhanced in vivo delivery of synthetic, modified rnas
US20130150625A1 (en) 2010-05-24 2013-06-13 Brian W. Budzik Novel Amino Alcohol Cationic Lipids for Oligonucleotide Delivery
US20130150295A1 (en) 2006-12-21 2013-06-13 Stryker Corporation Sustained-Release Formulations Comprising Crystals, Macromolecular Gels, and Particulate Suspensions of Biologic Agents
US20130184443A1 (en) 2005-06-16 2013-07-18 Nektar Therapeutics Methods for Preparing Conjugates
US20130183244A1 (en) 2010-09-10 2013-07-18 The Johns Hopkins University Rapid Diffusion of Large Polymeric Nanoparticles in the Mammalian Brain
WO2013110028A1 (en) 2012-01-19 2013-07-25 The Johns Hopkins University Nanoparticle formulations with enhanced mucosal penetration
US20130196948A1 (en) 2010-06-25 2013-08-01 Massachusetts Insitute Of Technology Polymers for biomaterials and therapeutics
WO2013116804A2 (en) 2012-02-03 2013-08-08 Rutgers, The State Of University Of New Jersey Polymeric biomaterials derived from phenolic monomers and their medical uses
WO2013120052A1 (en) 2012-02-10 2013-08-15 E. I. Du Pont De Nemours And Company Preparation, purification and use of high-x diblock copolymers
WO2014144039A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Characterization of mrna molecules
WO2014144711A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Analysis of mrna heterogeneity and stability
WO2014144767A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Ion exchange purification of mrna
WO2014152027A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Manufacturing methods for production of rna transcripts
WO2014152030A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Removal of dna fragments in mrna production process
WO2014152031A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Ribonucleic acid purification
WO2015164674A1 (en) 2014-04-23 2015-10-29 Moderna Therapeutics, Inc. Nucleic acid vaccines

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5703055A (en) * 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
FR2676072B1 (fr) * 1991-05-03 1994-11-18 Transgene Sa Vecteur de delivrance d'arn.
PL2358386T3 (pl) * 2008-11-28 2017-04-28 Statens Serum Institut Zoptymalizowane szczepionki na grypę
PL2590676T3 (pl) * 2010-07-06 2017-02-28 Glaxosmithkline Biologicals Sa Wirionopodobne cząstki dostarczające dla autoreplikujących się cząsteczek rna
EP2591114B1 (en) * 2010-07-06 2016-06-08 GlaxoSmithKline Biologicals SA Immunisation of large mammals with low doses of rna
US20120058154A1 (en) * 2010-08-20 2012-03-08 Selecta Biosciences, Inc. Synthetic nanocarrier vaccines comprising peptides obtained or derived from human influenza a virus m2e
EP2670430B1 (en) * 2011-01-31 2015-04-01 Baxter International Inc Recombinant viral vectors and methods for inducing a heterosubtypic immune response to influenza a viruses
WO2012116715A1 (en) * 2011-03-02 2012-09-07 Curevac Gmbh Vaccination in newborns and infants
WO2012135805A2 (en) * 2011-03-31 2012-10-04 modeRNA Therapeutics Delivery and formulation of engineered nucleic acids
US20140206753A1 (en) * 2011-06-08 2014-07-24 Shire Human Genetic Therapies, Inc. Lipid nanoparticle compositions and methods for mrna delivery
JP6113155B2 (ja) * 2011-06-20 2017-04-12 ユニバーシティ オブ ピッツバーグ − オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション 計算で最適化した広い反応性を示すｈ１ｎ１インフルエンザの抗原
EP4115875A1 (en) * 2011-07-06 2023-01-11 GlaxoSmithKline Biologicals S.A. Liposomes having useful n:p ratio for delivery of rna molecules
CA2849822C (en) * 2011-09-23 2021-05-11 Gary J. Nabel, M.D. Novel influenza hemagglutinin protein-based vaccines
KR20140102759A (ko) * 2011-12-16 2014-08-22 모더나 세라퓨틱스, 인코포레이티드 변형된 뉴클레오사이드, 뉴클레오타이드 및 핵산 조성물
WO2013143555A1 (en) * 2012-03-26 2013-10-03 Biontech Ag Rna formulation for immunotherapy
MX2014015041A (es) * 2012-06-08 2015-06-17 Shire Human Genetic Therapies Administración pulmonar de arnm a células objetivo no pulmonares.
US20150366997A1 (en) * 2012-12-07 2015-12-24 Shire Human Genetics Therapies, Inc. COMPOSITIONS AND METHODS FOR mRNA DELIVERY
AU2014204826A1 (en) * 2013-01-10 2015-07-09 Seqirus UK Limited Influenza virus immunogenic compositions and uses thereof
LT2970456T (lt) * 2013-03-14 2021-08-10 Translate Bio, Inc. Būdai ir kompozicijos, skirti mrnr koduojamų antikūnų pristatymui
PL4332576T3 (pl) * 2013-03-15 2025-09-08 Translate Bio, Inc. Synergistyczne wzmocnienie dostarczania kwasów nukleinowych poprzez formulacje zmieszane
TW201534578A (zh) * 2013-07-08 2015-09-16 Daiichi Sankyo Co Ltd 新穎脂質
CN105473157A (zh) * 2013-08-21 2016-04-06 库瑞瓦格股份公司 组合疫苗
WO2015149944A2 (en) * 2014-04-01 2015-10-08 Curevac Gmbh Polymeric carrier cargo complex for use as an immunostimulating agent or as an adjuvant
SG11201705264WA (en) * 2014-12-31 2017-07-28 The Usa As Represented By The Secretary Detp Of Health And Human Services Novel multivalent nanoparticle-based vaccines
EP3247363A4 (en) * 2015-01-21 2018-10-03 Moderna Therapeutics, Inc. Lipid nanoparticle compositions

Patent Citations (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8404222B2 (en) 1996-09-26 2013-03-26 Nektar Therapeutics Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution
JP3051957B2 (ja) 1997-08-28 2000-06-12 榮太郎 清水 融雪機
US8287849B2 (en) 2000-10-10 2012-10-16 Massachusetts Institute Of Technology Biodegradable poly(beta-amino esters) and uses thereof
US8440614B2 (en) 2000-12-29 2013-05-14 Aphios Corporation Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein
WO2002098443A2 (de) 2001-06-05 2002-12-12 Curevac Gmbh Stabilisierte mrna mit erhöhtem g/ c- gehalt und otimierter codon usage für die gentherapie
US20040262223A1 (en) 2001-07-27 2004-12-30 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US20120282343A1 (en) 2001-10-03 2012-11-08 Johns Hopkins University Compositions for oral gene therapy and methods of using same
US20120201859A1 (en) 2002-05-02 2012-08-09 Carrasquillo Karen G Drug Delivery Systems and Use Thereof
US20080166414A1 (en) 2004-01-28 2008-07-10 Johns Hopkins University Drugs And Gene Carrier Particles That Rapidly Move Through Mucous Barriers
US8241670B2 (en) 2004-04-15 2012-08-14 Chiasma Inc. Compositions capable of facilitating penetration across a biological barrier
US20100003337A1 (en) 2004-12-10 2010-01-07 Justin Hanes Functionalized poly(ether-anhydride) block copolymers
US8263665B2 (en) 2005-04-01 2012-09-11 Intezyne Technologies, Inc. Polymeric micelles for drug delivery
US20130195987A1 (en) 2005-04-01 2013-08-01 Intezyne Technologies, Inc. Polymeric micelles for drug delivery
US20130059360A1 (en) 2005-04-12 2013-03-07 Nektar Therapeutics Polymer-based compositions and conjugates of antimicrobial agents
US20130184443A1 (en) 2005-06-16 2013-07-18 Nektar Therapeutics Methods for Preparing Conjugates
US20130072709A1 (en) 2006-02-21 2013-03-21 Nektar Therapeutics Segmented Degradable Polymers and Conjugates Made Therefrom
US20130130348A1 (en) 2006-05-15 2013-05-23 The Brigham And Women's Hospital, Inc. Polymers for Functional Particles
US20100215580A1 (en) 2006-09-08 2010-08-26 The Johns Hopkins University Compositions and methods for enhancing transport through mucus
US20130164343A1 (en) 2006-09-08 2013-06-27 The Johns Hopkins University Compositions and methods for enhancing transport through mucus
US20130150295A1 (en) 2006-12-21 2013-06-13 Stryker Corporation Sustained-Release Formulations Comprising Crystals, Macromolecular Gels, and Particulate Suspensions of Biologic Agents
US8246968B2 (en) 2007-03-30 2012-08-21 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
WO2008121949A1 (en) 2007-03-30 2008-10-09 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
US20130172406A1 (en) 2007-09-28 2013-07-04 Bind Biosciences, Inc. Cancer Cell Targeting Using Nanoparticles
US8236330B2 (en) 2007-09-28 2012-08-07 Bind Biosciences, Inc. Cancer cell targeting using nanoparticles
US20120004293A1 (en) 2007-09-28 2012-01-05 Zale Stephen E Cancer Cell Targeting Using Nanoparticles
US20100068286A1 (en) 2008-06-16 2010-03-18 Greg Troiano Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20100069426A1 (en) 2008-06-16 2010-03-18 Zale Stephen E Therapeutic polymeric nanoparticles with mTor inhibitors and methods of making and using same
US8318211B2 (en) 2008-06-16 2012-11-27 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising vinca alkaloids and methods of making and using same
US20120288541A1 (en) 2008-06-16 2012-11-15 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US20130230567A1 (en) 2008-06-16 2013-09-05 Bind Therapeutics, Inc. Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
WO2010005740A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles
US8293276B2 (en) 2008-06-16 2012-10-23 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US8206747B2 (en) 2008-06-16 2012-06-26 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US20100104645A1 (en) 2008-06-16 2010-04-29 Bind Biosciences, Inc. Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles
WO2010005726A2 (en) 2008-06-16 2010-01-14 Bind Biosciences Inc. Therapeutic polymeric nanoparticles with mtor inhibitors and methods of making and using same
US20100104655A1 (en) 2008-06-16 2010-04-29 Zale Stephen E Therapeutic Polymeric Nanoparticles Comprising Vinca Alkaloids and Methods of Making and Using Same
WO2010005725A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising vinca alkaloids and methods of making and using same
WO2010005723A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US20110274759A1 (en) 2008-06-16 2011-11-10 Greg Troiano Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
US8318208B1 (en) 2008-06-16 2012-11-27 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
US20100068285A1 (en) 2008-06-16 2010-03-18 Zale Stephen E Drug Loaded Polymeric Nanoparticles and Methods of Making and Using Same
WO2010005721A2 (en) 2008-06-16 2010-01-14 Bind Biosciences, Inc. Drug loaded polymeric nanoparticles and methods of making and using same
WO2010030763A2 (en) 2008-09-10 2010-03-18 Bind Biosciences, Inc. High throughput fabrication of nanoparticles
US20100087337A1 (en) 2008-09-10 2010-04-08 Bind Biosciences, Inc. High Throughput Fabrication of Nanoparticles
US20130123351A1 (en) 2008-09-10 2013-05-16 Bind Biosciences, Inc. High throughput fabrication of nanoparticles
WO2010075072A2 (en) 2008-12-15 2010-07-01 Bind Biosciences Long circulating nanoparticles for sustained release of therapeutic agents
US20100216804A1 (en) 2008-12-15 2010-08-26 Zale Stephen E Long Circulating Nanoparticles for Sustained Release of Therapeutic Agents
US20110217377A1 (en) 2008-12-15 2011-09-08 Zale Stephen E Long Circulating Nanoparticles for Sustained Release of Therapeutic Agents
US20120076836A1 (en) 2009-03-31 2012-03-29 The University Of Tokyo Polyion complex of double-stranded ribonucleic acid
US8287910B2 (en) 2009-04-30 2012-10-16 Intezyne Technologies, Inc. Polymeric micelles for polynucleotide encapsulation
US20120276209A1 (en) 2009-11-04 2012-11-01 The University Of British Columbia Nucleic acid-containing lipid particles and related methods
WO2011084518A2 (en) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising corticosteroids and methods of making and using same
WO2011084521A2 (en) 2009-12-15 2011-07-14 Bind Biosciences, Inc. Therapeutic polymeric nanoparticles comprising epothilone and methods of making and using same
US20120140790A1 (en) 2009-12-15 2012-06-07 Ali Mir M Therapeutic Polymeric Nanoparticle Compositions with High Glass Transition Termperature or High Molecular Weight Copolymers
WO2012082165A1 (en) 2010-01-24 2012-06-21 Novartis Ag Irradiated biodegradable polymer microparticles
US20110262491A1 (en) 2010-04-12 2011-10-27 Selecta Biosciences, Inc. Emulsions and methods of making nanocarriers
US20130150625A1 (en) 2010-05-24 2013-06-13 Brian W. Budzik Novel Amino Alcohol Cationic Lipids for Oligonucleotide Delivery
US20130196948A1 (en) 2010-06-25 2013-08-01 Massachusetts Insitute Of Technology Polymers for biomaterials and therapeutics
US20130183244A1 (en) 2010-09-10 2013-07-18 The Johns Hopkins University Rapid Diffusion of Large Polymeric Nanoparticles in the Mammalian Brain
WO2012054923A2 (en) 2010-10-22 2012-04-26 Bind Biosciences, Inc. Therapeutic nanoparticles with high molecular weight copolymers
US20120121718A1 (en) 2010-11-05 2012-05-17 The Johns Hopkins University Compositions and methods relating to reduced mucoadhesion
WO2012099755A1 (en) 2011-01-11 2012-07-26 Alnylam Pharmaceuticals, Inc. Pegylated lipids and their use for drug delivery
WO2012099805A2 (en) 2011-01-19 2012-07-26 Ocean Nanotech, Llc Nanoparticle based immunological stimulation
US20120189700A1 (en) 2011-01-19 2012-07-26 Zoraida Aguilar Nanoparticle Based Immunological Stimulation
WO2012109121A1 (en) 2011-02-07 2012-08-16 Purdue Research Foundation Carbohydrate nanoparticles for prolonged efficacy of antimicrobial peptide
WO2012131104A2 (en) 2011-03-31 2012-10-04 Ingell Technologies Holding B.V. Biodegradable compositions suitable for controlled release
WO2012131106A1 (en) 2011-03-31 2012-10-04 Ingell Technologies Holding B.V. Biodegradable compositions suitable for controlled release
US20120295832A1 (en) 2011-05-17 2012-11-22 Arrowhead Research Corporation Novel Lipids and Compositions for Intracellular Delivery of Biologically Active Compounds
WO2012166923A2 (en) 2011-05-31 2012-12-06 Bind Biosciences Drug loaded polymeric nanoparticles and methods of making and using same
WO2013012476A2 (en) 2011-07-21 2013-01-24 Arizona Chemical Company, Llc Branched polyether-polyamide block copolymers and methods of making and using the same
US20130121954A1 (en) 2011-08-26 2013-05-16 Arrowhead Madison Inc. Poly(vinyl ester) Polymers for In Vivo Nucleic Acid Delivery
WO2013032829A1 (en) 2011-08-26 2013-03-07 Arrowhead Research Corporation Poly(vinyl ester) polymers for in vivo nucleic acid delivery
WO2013033438A2 (en) 2011-08-31 2013-03-07 Mallinckrodt Llc Nanoparticle peg modification with h-phosphonates
WO2013044219A1 (en) 2011-09-22 2013-03-28 Bind Biosciences Methods of treating cancers with therapeutic nanoparticles
WO2013072929A2 (en) 2011-09-23 2013-05-23 Indian Institute Of Technology Nanop article based cosmetic composition
WO2013059496A1 (en) 2011-10-18 2013-04-25 Dicerna Pharmaceuticals, Inc. Amine cationic lipids and uses thereof
WO2013063468A1 (en) 2011-10-27 2013-05-02 Massachusetts Institute Of Technology Amino acid derivates functionalized on the n- terminal capable of forming drug incapsulating microspheres
WO2013078199A2 (en) 2011-11-23 2013-05-30 Children's Medical Center Corporation Methods for enhanced in vivo delivery of synthetic, modified rnas
WO2013110028A1 (en) 2012-01-19 2013-07-25 The Johns Hopkins University Nanoparticle formulations with enhanced mucosal penetration
WO2013116804A2 (en) 2012-02-03 2013-08-08 Rutgers, The State Of University Of New Jersey Polymeric biomaterials derived from phenolic monomers and their medical uses
WO2013120052A1 (en) 2012-02-10 2013-08-15 E. I. Du Pont De Nemours And Company Preparation, purification and use of high-x diblock copolymers
WO2014144039A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Characterization of mrna molecules
WO2014144711A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Analysis of mrna heterogeneity and stability
WO2014144767A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Ion exchange purification of mrna
WO2014152027A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Manufacturing methods for production of rna transcripts
WO2014152030A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Removal of dna fragments in mrna production process
WO2014152031A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Ribonucleic acid purification
WO2015164674A1 (en) 2014-04-23 2015-10-29 Moderna Therapeutics, Inc. Nucleic acid vaccines

Non-Patent Citations (73)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computer Analysis of Sequence Data, Part I", 1994, HUMANA PRESS
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
ABRAHAM ET AL.: "Chaotic Mixer for Microchannels", SCIENCE, vol. 295, 2002, pages 647 - 651
ALTSCHUL, S. F. ET AL., J. MOLEC. BIOL., vol. 215, 1990, pages 403
BASHA ET AL., MOL THER., vol. 19, 2011, pages 2186 - 2200
BASHA ET AL., MOL. THER., vol. 19, 2011, pages 2186 - 2200
BELLIVEAU, N.M. ET AL.: "Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA", MOLECULAR THERAPY-NUCLEIC ACIDS., vol. 1, 2012, pages e37, XP002715253, DOI: 10.1038/mtna.2012.28
BENOIT ET AL., BIOMACROMOLECULES, vol. 12, 2011, pages 2708 - 2714
BOMMAKANTI G ET AL., J VIROL., vol. 86, no. 24, December 2012 (2012-12-01), pages 13434 - 44
BOMMAKANTI G ET AL., PNAS, vol. 107, no. 31, 3 August 2010 (2010-08-03), pages 13701 - 6
BOMMAKANTI G ET AL., PROC NATL ACAD SCI USA., vol. 107, no. 31, 3 August 2010 (2010-08-03), pages 13701 - 6
CARILLO, H.LIPMAN, D., SIAM J APPLIED MATH., vol. 48, 1988, pages 1073
CARTER ET AL., J VIROL., vol. 90, no. 9, 14 April 2016 (2016-04-14), pages 4720 - 34
CHANG: " Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle.", J CONTROLLED RELEASE., vol. 118, 2007, pages 245 - 253, XP005912153, DOI: 10.1016/j.jconrel.2006.11.025
CHEN J ET AL., PNAS USA, vol. 111, no. 7, 2014, pages 2476 - 81
CHEN, D. ET AL.: "Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation", J AM CHEM SOC., vol. 134, no. 16, 2012, pages 6948 - 51, XP002715254, DOI: 10.1021/ja301621z
COX RG ET AL., J VIROL, vol. 88, no. 11, June 2014 (2014-06-01), pages 6368 - 6379
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387
ENSIGN ET AL., BIOMATERIALS, vol. 34, no. 28, 2013, pages 6922 - 9
FENSKECULLIS, EXPERT OPIN DRUG DELIV., vol. 5, 2008, pages 309 - 319
GEALL ET AL.: "Nonviral delivery of self-amplifying RNA (e.g., mRNA) vaccines", PNAS, 2012
GUTBIER ET AL., PULM PHARMACOL. THER., vol. 23, 2010, pages 334 - 344
IMPAGLIAZZO ET AL., SCIENCE, vol. 349, 18 September 2015 (2015-09-18), pages 6254
JAYARAMA ET AL., ANGEW. CHEM. INT. ED., vol. 51, 2012, pages 8529 - 8533
JUDGE ET AL., J CLIN INVEST., vol. 119, 2009, pages 661 - 673
KANEKIYO M. ET AL., NATURE, vol. 499, 2013, pages 102 - 6
KAUFMANN ET AL., MICROVASC RES, vol. 80, 2010, pages 286 - 293
KIM ET AL., METHODS MOL BIOL., vol. 721, 2011, pages 339 - 353
KOLHATKAR ET AL., CURR DRUG DISCOV TECHNOL., vol. 8, 2011, pages 197 - 206
LAI ET AL., ADV DRUG DELIV REV., vol. 61, 2009, pages 158 - 171
LAI ET AL., PNAS, vol. 104, 2007, pages 1482 - 487
LANGMUIR., vol. 28, 2012, pages 3633 - 40
LEE ET AL.: "Thermosensitive Hydrogel as a TGF-ø 1 Gene Delivery Vehicle Enhances Diabetic Wound Healing", PHARMACEUTICAL RESEARCH, vol. 20, no. 12, 2003, pages 1995 - 2000, XP055716493, DOI: 10.1023/B:PHAM.0000008048.58777.da
LI ET AL.: "Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel", PHARMACEUTICAL RESEARCH, vol. 20, 2003, pages 884 - 888
MAIER ET AL., MOLECULAR THERAPY, vol. 21, 2013, pages 1570 - 1578
MALLAJOSYULA ET AL., PROC NATL ACAD SCI USA., vol. 111, no. 25, 2014, pages E2514 - 23
MALLAJOSYULA V ET AL., PNAS, vol. 11 1, no. 25, 24 June 2014 (2014-06-24), pages E2514 - 23
MALLAJOSYULA VV ET AL., FRONT IMMUNOL., vol. 6, 26 June 2015 (2015-06-26), pages 329
MALLAJOSYULA VV ET AL., PROC NATL ACAD SCI U S A., vol. 111, no. 25, 24 June 2014 (2014-06-24), pages E2514 - 23
MEYERSMILLER, CABIOS, vol. 4, 1989, pages 11 - 17
MUSACCHIOTORCHILIN, FRONT BIOSCI., vol. 16, 2011, pages 1388 - 1412
NEEDLEMAN, S.BWUNSCH, C.D.: "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
PAN ET AL., NAT COMMUN., vol. 4, 2013, pages 2369
PATIL ET AL., CRIT REV THER DRUG CARRIER SYST., vol. 25, 2008, pages 1 - 61
PEER ET AL., PROC NATL ACAD SCI USA., vol. 104, 2007, pages 4095 - 4100
PEER ET AL., SCIENCE, vol. 319, 2008, pages 627 - 630
PEER, J CONTROL RELEASE., vol. 20, 2010, pages 63 - 68
PEERLIEBERMAN, GENE THER., vol. 18, 2011, pages 1127 - 1133
REN X ET AL., EMERG INFECT DIS, vol. 19, no. 11, 2013, pages 1881 - 84
REYES ET AL., J. CONTROLLED RELEASE, vol. 107, 2005, pages 276 - 287
RODRIGUEZ ET AL., SCIENCE, vol. 339, 2013, pages 971 - 975
SANTEL ET AL., GENE THER, vol. 13, 2006, pages 1360 - 1370
SEMPLE ET AL., NAT. BIOTECHNOL., vol. 28, 2010, pages 172 - 176
SEMPLE ET AL., NATURE BIOTECH., vol. 28, 2010, pages 172 - 176
SMITH, T.F.WATERMAN, M.S.: "Identification of common molecular subsequences", J. MOL. BIOL., vol. 147, 1981, pages 195 - 197, XP024015032, DOI: 10.1016/0022-2836(81)90087-5
SONG ET AL., NAT BIOTECHNOL., vol. 23, 2005, pages 709 - 717
SQUIRES ET AL., INFLUENZA OTHER RESPIR VIRUSES., vol. 6, no. 6, November 2012 (2012-11-01), pages 404 - 416
SRINIVASAN ET AL., METHODS MOL BIOL., vol. 757, 2012, pages 497 - 507
STEEL J ET AL., MBIO, vol. 1, no. 1, 2010, pages e00018 - 10
STEPHEN F. ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
SUBRAMANYA ET AL., MOL THER., vol. 18, 2010, pages 2028 - 2037
THOMSEN ET AL., NUCLEIC ACIDS RES., vol. 40, July 2012 (2012-07-01), pages W281 - 7
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
WALKER A ET AL., SCI REP, vol. 1, no. 5, 2011, pages 1 - 8
WEBBY ET AL., PLOS ONE., vol. 10, no. 10, 15 October 2015 (2015-10-15), pages e0140702
WEINBERG ET AL., J INFECT DIS., vol. 201, no. 11, 1 June 2010 (2010-06-01), pages 1607 - 10
WHITESIDES, GEORGE M.: "The Origins and the Future of Microfluidics", NATURE, vol. 442, 2006, pages 368 - 373, XP055123139, DOI: 10.1038/nature05058
XU ET AL., J CONTROL RELEASE, vol. 170, 2013, pages 279 - 86
YANG ET AL., ANGEW. CHEM. INT. ED., vol. 50, 2011, pages 2597 - 2600
YASSINE ET AL., NAT MED., vol. 21, no. 9, September 2015 (2015-09-01), pages 1065 - 70
YASSINE ET AL., NAT. MED., vol. 21, no. 9, September 2015 (2015-09-01), pages 1065 - 70
YU ET AL., MOL MEMBR BIOL., vol. 27, 2010, pages 286 - 298

Cited By (137)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US12329812B2 (en) 2014-04-23 2025-06-17 Modernatx, Inc. Nucleic acid vaccines
US10709779B2 (en) 2014-04-23 2020-07-14 Modernatx, Inc. Nucleic acid vaccines
US12274743B2 (en) 2014-04-23 2025-04-15 Modernatx, Inc. Nucleic acid vaccines
US10702597B2 (en) 2015-07-21 2020-07-07 Modernatx, Inc. CHIKV RNA vaccines
US11364292B2 (en) 2015-07-21 2022-06-21 Modernatx, Inc. CHIKV RNA vaccines
US11007260B2 (en) 2015-07-21 2021-05-18 Modernatx, Inc. Infectious disease vaccines
US10449244B2 (en) 2015-07-21 2019-10-22 Modernatx, Inc. Zika RNA vaccines
US12403336B2 (en) 2015-10-22 2025-09-02 Modernatx, Inc. Betacorona virus mRNA vaccines
US10543269B2 (en) 2015-10-22 2020-01-28 Modernatx, Inc. hMPV RNA vaccines
US10272150B2 (en) 2015-10-22 2019-04-30 Modernatx, Inc. Combination PIV3/hMPV RNA vaccines
US11278611B2 (en) 2015-10-22 2022-03-22 Modernatx, Inc. Zika virus RNA vaccines
US10493143B2 (en) 2015-10-22 2019-12-03 Modernatx, Inc. Sexually transmitted disease vaccines
US10517940B2 (en) 2015-10-22 2019-12-31 Modernatx, Inc. Zika virus RNA vaccines
US12403335B2 (en) 2015-10-22 2025-09-02 Modernatx, Inc. Betacoronavirus MRNA vaccines
US11235052B2 (en) 2015-10-22 2022-02-01 Modernatx, Inc. Chikungunya virus RNA vaccines
US12208288B2 (en) 2015-10-22 2025-01-28 Modernatx, Inc. Betacoronavirus RNA vaccines
US10933127B2 (en) 2015-10-22 2021-03-02 Modernatx, Inc. Betacoronavirus mRNA vaccine
US10675342B2 (en) 2015-10-22 2020-06-09 Modernatx, Inc. Chikungunya virus RNA vaccines
US12409347B2 (en) 2015-10-22 2025-09-09 Modernatx, Inc. Betacoronavirus mRNA vaccines
US10702600B1 (en) 2015-10-22 2020-07-07 Modernatx, Inc. Betacoronavirus mRNA vaccine
US11872278B2 (en) 2015-10-22 2024-01-16 Modernatx, Inc. Combination HMPV/RSV RNA vaccines
US10702599B2 (en) 2015-10-22 2020-07-07 Modernatx, Inc. HPIV3 RNA vaccines
US11643441B1 (en) 2015-10-22 2023-05-09 Modernatx, Inc. Nucleic acid vaccines for varicella zoster virus (VZV)
US10556018B2 (en) 2015-12-10 2020-02-11 Modernatx, Inc. Compositions and methods for delivery of agents
US10485885B2 (en) 2015-12-10 2019-11-26 Modernatx, Inc. Compositions and methods for delivery of agents
US10207010B2 (en) 2015-12-10 2019-02-19 Modernatx, Inc. Compositions and methods for delivery of agents
US12491260B2 (en) 2015-12-10 2025-12-09 Modernatx, Inc. Compositions and methods for delivery of agents
US11285222B2 (en) 2015-12-10 2022-03-29 Modernatx, Inc. Compositions and methods for delivery of agents
US10730924B2 (en) 2016-05-18 2020-08-04 Modernatx, Inc. Polynucleotides encoding relaxin
US12103955B2 (en) 2016-05-18 2024-10-01 Modernatx, Inc. Polynucleotides encoding relaxin
US12233084B2 (en) 2016-09-14 2025-02-25 Modernatx, Inc. High purity RNA compositions and methods for preparation thereof
US10653712B2 (en) 2016-09-14 2020-05-19 Modernatx, Inc. High purity RNA compositions and methods for preparation thereof
US12246029B2 (en) 2016-09-14 2025-03-11 Modernatx, Inc. High purity RNA compositions and methods for preparation thereof
US11202793B2 (en) 2016-09-14 2021-12-21 Modernatx, Inc. High purity RNA compositions and methods for preparation thereof
WO2018075592A1 (en) * 2016-10-21 2018-04-26 Merck Sharp & Dohme Corp. Influenza hemagglutinin protein vaccines
EP3939604A3 (en) * 2016-10-21 2022-06-22 Merck Sharp & Dohme Corp. Influenza hemagglutinin protein vaccines
US10925958B2 (en) 2016-11-11 2021-02-23 Modernatx, Inc. Influenza vaccine
WO2018089851A3 (en) * 2016-11-11 2018-06-21 Modernatx, Inc. Influenza vaccine
US11696946B2 (en) 2016-11-11 2023-07-11 Modernatx, Inc. Influenza vaccine
EP3538146A4 (en) * 2016-11-11 2020-07-15 ModernaTX, Inc. INFLUENZA VACCINE
US12318443B2 (en) 2016-11-11 2025-06-03 Modernatx, Inc. Influenza vaccine
CN110167587A (zh) * 2016-11-11 2019-08-23 摩登纳特斯有限公司 流感疫苗
US12409218B2 (en) 2016-11-11 2025-09-09 Modernatx, Inc. Influenza vaccine
US11103578B2 (en) 2016-12-08 2021-08-31 Modernatx, Inc. Respiratory virus nucleic acid vaccines
US11897944B2 (en) 2017-01-17 2024-02-13 Ablynx N.V. Immunoglobulin single variable domain (ISVD) capable of binding to serum albumin
US10273269B2 (en) 2017-02-16 2019-04-30 Modernatx, Inc. High potency immunogenic zika virus compositions
WO2018170245A1 (en) * 2017-03-15 2018-09-20 Modernatx, Inc. Broad spectrum influenza virus vaccine
US11752206B2 (en) 2017-03-15 2023-09-12 Modernatx, Inc. Herpes simplex virus vaccine
US11045540B2 (en) 2017-03-15 2021-06-29 Modernatx, Inc. Varicella zoster virus (VZV) vaccine
US11576961B2 (en) 2017-03-15 2023-02-14 Modernatx, Inc. Broad spectrum influenza virus vaccine
US11464848B2 (en) 2017-03-15 2022-10-11 Modernatx, Inc. Respiratory syncytial virus vaccine
US11918644B2 (en) 2017-03-15 2024-03-05 Modernatx, Inc. Varicella zoster virus (VZV) vaccine
WO2018167320A1 (en) 2017-03-17 2018-09-20 Curevac Ag Rna vaccine and immune checkpoint inhibitors for combined anticancer therapy
EP4494652A2 (en) 2017-07-04 2025-01-22 CureVac SE Cancer rna-vaccine
WO2019038332A1 (en) 2017-08-22 2019-02-28 Curevac Ag VACCINE AGAINST BUNYAVIRUS
US11911453B2 (en) 2018-01-29 2024-02-27 Modernatx, Inc. RSV RNA vaccines
US12453766B2 (en) 2018-01-29 2025-10-28 Modernatx, Inc. RSV RNA vaccines
RU2819143C2 (ru) * 2018-03-12 2024-05-14 ЭсКьюЗед БАЙОТЕКНОЛОДЖИЗ КОМПАНИ Внутриклеточная доставка биомолекул для модуляции иммунного ответа
CN111971393A (zh) * 2018-03-14 2020-11-20 仁济大学校产学协力团 使用重组腺病毒的多价活流感疫苗平台
WO2019193183A2 (en) 2018-04-05 2019-10-10 Curevac Ag Novel yellow fever nucleic acid molecules for vaccination
EP4227319A1 (en) 2018-04-17 2023-08-16 CureVac SE Novel rsv rna molecules and compositions for vaccination
WO2020002525A1 (en) 2018-06-27 2020-01-02 Curevac Ag Novel lassa virus rna molecules and compositions for vaccination
US20250228788A1 (en) * 2018-09-04 2025-07-17 The Board Of Regents Of The University Of Texas System Compositions and methods for organ specific delivery of nucleic acids
US12151029B2 (en) 2018-09-19 2024-11-26 Modernatx, Inc. PEG lipids and uses thereof
US12383508B2 (en) 2018-09-19 2025-08-12 Modernatx, Inc. High-purity peg lipids and uses thereof
US12098166B2 (en) 2018-11-06 2024-09-24 Boehringer Ingelheim Vetmedica Gmbh Immunogenic composition against avian influenza virus H5 subtype
WO2020128031A2 (en) 2018-12-21 2020-06-25 Curevac Ag Rna for malaria vaccines
WO2020161342A1 (en) 2019-02-08 2020-08-13 Curevac Ag Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
EP4491229A2 (en) 2019-02-08 2025-01-15 CureVac SE Coding rna administered into the suprachoroidal space in the treatment of ophtalmic diseases
US11351242B1 (en) 2019-02-12 2022-06-07 Modernatx, Inc. HMPV/hPIV3 mRNA vaccine composition
US12460259B2 (en) 2019-03-11 2025-11-04 Modernatx, Inc. Fed-batch in vitro transcription process
US12070495B2 (en) 2019-03-15 2024-08-27 Modernatx, Inc. HIV RNA vaccines
WO2020254535A1 (en) 2019-06-18 2020-12-24 Curevac Ag Rotavirus mrna vaccine
WO2021028439A1 (en) 2019-08-14 2021-02-18 Curevac Ag Rna combinations and compositions with decreased immunostimulatory properties
WO2021123332A1 (en) 2019-12-20 2021-06-24 Curevac Ag Lipid nanoparticles for delivery of nucleic acids
DE112021000012T5 (de) 2020-02-04 2021-11-18 Curevac Ag Coronavirus-Vakzine
EP4147717A1 (en) 2020-02-04 2023-03-15 CureVac SE Coronavirus vaccine
DE112021000012C5 (de) 2020-02-04 2024-11-14 CureVac SE Coronavirus-Vakzine
DE202021004123U1 (de) 2020-02-04 2022-10-26 Curevac Ag Coronavirus-Vakzine
DE202021003575U1 (de) 2020-02-04 2022-01-17 Curevac Ag Coronavirus-Vakzine
DE202021004130U1 (de) 2020-02-04 2022-10-26 Curevac Ag Coronavirus-Vakzine
DE112021000012B4 (de) 2020-02-04 2023-08-31 CureVac SE Coronavirus-Vakzine
US12390523B2 (en) 2020-02-04 2025-08-19 CureVac SE Coronavirus vaccine
DE112021000012C9 (de) 2020-02-04 2025-02-06 CureVac SE Coronavirus-Vakzine
WO2021156267A1 (en) 2020-02-04 2021-08-12 Curevac Ag Coronavirus vaccine
WO2021239880A1 (en) 2020-05-29 2021-12-02 Curevac Ag Nucleic acid based combination vaccines
WO2022002783A1 (en) 2020-06-29 2022-01-06 Glaxosmithkline Biologicals Sa Adjuvants
WO2022023559A1 (en) 2020-07-31 2022-02-03 Curevac Ag Nucleic acid encoded antibody mixtures
WO2022043551A2 (en) 2020-08-31 2022-03-03 Curevac Ag Multivalent nucleic acid based coronavirus vaccines
US11771653B2 (en) 2020-11-06 2023-10-03 Sanofi Lipid nanoparticles for delivering mRNA vaccines
US12239735B2 (en) 2020-11-06 2025-03-04 Sanofi Lipid nanoparticles for delivering mRNA vaccines
WO2022099003A1 (en) * 2020-11-06 2022-05-12 Sanofi Lipid nanoparticles for delivering mrna vaccines
US11771652B2 (en) 2020-11-06 2023-10-03 Sanofi Lipid nanoparticles for delivering mRNA vaccines
WO2022135993A2 (en) 2020-12-22 2022-06-30 Curevac Ag Pharmaceutical composition comprising lipid-based carriers encapsulating rna for multidose administration
WO2022137133A1 (en) 2020-12-22 2022-06-30 Curevac Ag Rna vaccine against sars-cov-2 variants
US12329811B2 (en) 2021-01-11 2025-06-17 Modernatx, Inc. Seasonal RNA influenza virus vaccines
WO2022162027A2 (en) 2021-01-27 2022-08-04 Curevac Ag Method of reducing the immunostimulatory properties of in vitro transcribed rna
US11622972B2 (en) 2021-02-19 2023-04-11 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
WO2022200575A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200574A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022200582A1 (en) * 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
WO2022207862A2 (en) 2021-03-31 2022-10-06 Curevac Ag Syringes containing pharmaceutical compositions comprising rna
WO2022233880A1 (en) 2021-05-03 2022-11-10 Curevac Ag Improved nucleic acid sequence for cell type specific expression
EP4342490A4 (en) * 2021-05-19 2025-06-04 Daiichi Sankyo Company, Limited Influenza virus nucleic acid lipid particle vaccine
WO2022248353A1 (en) 2021-05-24 2022-12-01 Glaxosmithkline Biologicals Sa Adjuvants
WO2022264109A1 (en) * 2021-06-18 2022-12-22 Sanofi Multivalent influenza vaccines
WO2023020993A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023020994A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023020992A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023021421A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Low-dose lyophilized rna vaccines and methods for preparing and using the same
WO2023021427A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Freeze-drying of lipid nanoparticles (lnps) encapsulating rna and formulations thereof
WO2023031392A2 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids comprising phosphatidylserine
WO2023031394A1 (en) 2021-09-03 2023-03-09 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
WO2023057930A1 (en) * 2021-10-08 2023-04-13 Pfizer Inc. Immunogenic lnp compositions and methods thereof
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2024068545A1 (en) 2022-09-26 2024-04-04 Glaxosmithkline Biologicals Sa Influenza virus vaccines
DE202023106198U1 (de) 2022-10-28 2024-03-21 CureVac SE Impfstoff auf Nukleinsäurebasis
WO2024126809A1 (en) * 2022-12-15 2024-06-20 Sanofi Mrna encoding influenza virus-like particle
WO2024133160A1 (en) 2022-12-19 2024-06-27 Glaxosmithkline Biologicals Sa Hepatitis b compositions
WO2024160936A1 (en) 2023-02-03 2024-08-08 Glaxosmithkline Biologicals Sa Rna formulation
WO2024171052A1 (en) 2023-02-14 2024-08-22 Glaxosmithkline Biologicals Sa Analytical method
WO2024184500A1 (en) 2023-03-08 2024-09-12 CureVac SE Novel lipid nanoparticle formulations for delivery of nucleic acids
WO2024223728A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024223724A1 (en) 2023-04-27 2024-10-31 Glaxosmithkline Biologicals Sa Influenza virus vaccines
WO2024230934A1 (en) 2023-05-11 2024-11-14 CureVac SE Therapeutic nucleic acid for the treatment of ophthalmic diseases
WO2025011529A2 (en) 2023-07-07 2025-01-16 Shanghai Circode Biomed Co., Ltd. Circular rna vaccines for seasonal flu and methods of uses
WO2025012461A1 (en) 2023-07-13 2025-01-16 Sanofi Methods and compositions for analyzing messenger rna
WO2025045142A1 (en) 2023-08-29 2025-03-06 Shanghai Circode Biomed Co., Ltd. Circular rna encoding vegf polypeptides, formulations, and methods of uses
WO2025132839A1 (en) 2023-12-21 2025-06-26 Glaxosmithkline Biologicals Sa Influenza virus vaccines
GB202404607D0 (en) 2024-03-29 2024-05-15 Glaxosmithkline Biologicals Sa RNA formulation
WO2025202360A1 (en) 2024-03-29 2025-10-02 Glaxosmithkline Biologicals Sa Rna formulation
RU2838904C1 (ru) * 2024-10-15 2025-04-23 Федеральное Государственное Бюджетное Учреждение "Национальный Исследовательский Центр Эпидемиологии И Микробиологии Имени Почетного Академика Н.Ф.Гамалеи" Министерства Здравоохранения Российской Федерации Иммунобиологическое средство для индукции иммунного ответа против вируса гриппа и его применение

Also Published As

Publication number Publication date
JP2024105503A (ja) 2024-08-06
JP2018537521A (ja) 2018-12-20
WO2017070620A3 (en) 2017-07-13
AU2016342048A1 (en) 2018-06-07
JP2022031942A (ja) 2022-02-22
AU2016342048B2 (en) 2022-09-08
MA46023A (fr) 2019-07-03
JP7491639B2 (ja) 2024-05-28
EP3365007A2 (en) 2018-08-29
CA3003103A1 (en) 2017-04-27
CN118846026A (zh) 2024-10-29
KR20180096591A (ko) 2018-08-29
BR112018008078A2 (pt) 2018-11-13
US20180311336A1 (en) 2018-11-01
AU2022218595A1 (en) 2022-09-29
MX2022006603A (es) 2022-07-11
RU2018118337A (ru) 2019-11-25
EP3365007A4 (en) 2019-07-03
CN109310751A (zh) 2019-02-05
JP7384512B2 (ja) 2023-11-21
MX2018004916A (es) 2019-07-04

Similar Documents

Publication Publication Date Title
US20230310576A1 (en) Broad spectrum influenza virus vaccine
JP7491639B2 (ja) 広域インフルエンザウイルスワクチン
US12208288B2 (en) Betacoronavirus RNA vaccines
US20250099569A1 (en) Respiratory syncytial virus vaccine
US20230390379A1 (en) Respiratory syncytial virus vaccine
WO2017015457A1 (en) Ebola vaccine
CA3002922A1 (en) Human cytomegalovirus vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16858400

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 15767609

Country of ref document: US

ENP Entry into the national phase

Ref document number: 3003103

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: MX/A/2018/004916

Country of ref document: MX

Ref document number: 2018541088

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112018008078

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20187014338

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2018118337

Country of ref document: RU

Ref document number: 2016858400

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2016342048

Country of ref document: AU

Date of ref document: 20161021

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112018008078

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20180420